̽��ֱ�� of Cambridge - National Centre for the Replacement Refinement and Reduction of Animals in Research (NC3Rs)

Effectiveness of antibiotics significantly reduced when multiple bugs present

jg533 — Sat, 19 Mar 2022 00:01:00 +0000

In the study, published today in ̽��ֱ��ISME Journal, researchers say that even a low level of one type of microbe in the airways can have a profound effect on the way other microbes respond to antibiotics.

̽��ֱ��results highlight the need to consider the interaction between different species of microbe when treating infections with antibiotics - and to adjust dosage accordingly.

“People with chronic infections often have co-infection with several pathogens, but the problem is we don’t take that into account in deciding how much of a particular antibiotic to treat them with. Our results might help explain why, in these people, the antibiotics just don’t work as well as they should,” said Thomas O’Brien, who carried out the research for his PhD in the ̽��ֱ�� of Cambridge’s Department of Biochemistry and is joint first author of the paper.

Chronic bacterial infections such as those in the human airways are very difficult to cure using antibiotics. Although these types of infection are often associated with a single pathogenic species, the infection site is frequently co-colonised by a number of other microbes, most of which are not usually pathogenic in their own right.

Treatment options usually revolve around targeting the pathogen, and take little account of the co-habiting species. However, these treatments often fail to resolve the infection. Until now scientists have had little insight into why this is.

To get their results the team developed a simplified model of the human airways, containing artificial sputum (‘phlegm‘) designed to chemically resemble the real phlegm coughed up during an infection, packed with bacteria.

̽��ֱ��model allowed them to grow a mixture of different microbes, including pathogens, in a stable way for weeks at a time. This is novel, because usually one pathogen will outgrow the others very quickly and spoil the experiment. It enabled the researchers to replicate and study infections with multiple species of microbe, called ‘poly-microbial infections’, in the laboratory.

̽��ֱ��three microbes used in the experiment were the bacteria Pseudomonas aeruginosa and Staphylococcus aureus, and the fungus Candida albicans – a combination commonly present in the airways of people with cystic fibrosis.

̽��ֱ��researchers treated this microbial mix with an antibiotic called colistin, which is very effective in killing Pseudomonas aeruginosa. But when the other pathogens were present alongside Pseudomonas aeruginosa, the antibiotic didn’t work.

“We were surprised to find that an antibiotic that we know should clear an infection of Pseudomonas effectively just didn’t work in our lab model when other bugs were present,” said Wendy Figueroa-Chavez in the ̽��ֱ�� of Cambridge’s Department of Biochemistry, joint first author of the paper.

̽��ֱ��same effect happened when the microbial mix was treated with fusidic acid – an antibiotic that specifically targets Staphylococcus aureus, and with fluconazole - an antibiotic that specifically targets Candida albicans.

̽��ֱ��researchers found that significantly higher doses of each antibiotic were needed to kill bacteria when it was part of poly-microbial infection, compared to when no other pathogens were present.

“All three species-specific antibiotics were less effective against their target when three pathogens were present together,” said Martin Welch, Professor of Microbial Physiology and Metabolism in the ̽��ֱ�� of Cambridge’s Department of Biochemistry and senior author of the paper.

At present antibiotics are usually only laboratory tested against the main pathogen they are designed to target, to determine the lowest effective dose. But when the same dose is used to treat infection in a person it often doesn’t work, and this study helps to explain why. ̽��ֱ��new model system will enable the effectiveness of potential new antibiotics to be tested against a mixture of microbe species together.

Poly-microbial infections are common in the airways of people with cystic fibrosis. Despite treatment with strong doses of antibiotics, these infections often persist long-term. Chronic infections of the airways in people with asthma and chronic obstructive pulmonary disorder (COPD) are also often poly-microbial.

By looking at the genetic code of the Pseudomonas bacteria in their lab-grown mix, the researchers were able to pinpoint specific mutations that give rise to this antibiotic resistance. ̽��ֱ��mutations were found to arise more frequently when other pathogens were also present.

Comparison with the genetic code of 800 samples of Pseudomonas from around the world revealed that these mutations have also occurred in human patients who had been infected with Pseudomonas and treated with colistin.

“ ̽��ֱ��problem is that as soon as you use an antibiotic to treat a microbial infection, the microbe will start to evolve resistance to that antibiotic. That’s what has happened since colistin started to be used in the early 1990’s. This is another reminder of the vital need to find new antibiotics to treat human infections,” said Welch.

This research was funded by the British Lung Foundation, the Cystic Fibrosis Trust, and the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs).

Reference

O’Brien, T et al: ‘Decreased efficacy of antimicrobial agents in a polymicrobial environment.’ ̽��ֱ��ISME Journal, March 2022. DOI: 10.1038/s41396-022-01218-7

A study has found that much higher doses of antibiotics are needed to eliminate a bacterial infection of the airways when other microbes are present. It helps explain why respiratory infections often persist in people with lung diseases such as cystic fibrosis despite treatment.

People with chronic infections often have co-infection with several pathogens, but the problem is we don’t take that into account in deciding how much of a particular antibiotic to treat them with

Thomas O’Brien

Science Photo Library

Woman coughing

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Scientists develop mouse ‘embryo-like structures’ with organisation along body’s major axes

cjb250 — Wed, 03 Oct 2018 17:00:21 +0000

̽��ֱ��definitive architecture of the mammalian body is established shortly after the embryo implants into the uterus. This body plan has spatial references, or axes, that guide the emergence of tissues and organs: an antero-posterior axis defined by the head at one end and the tail at the other, an orthogonal dorso-ventral axis and a medio-lateral axis, which orientates the arrangement of internal organs like the liver, pancreas or the heart.

Studying the processes orchestrating the formation of early mammalian embryos is hampered by the difficulty in obtaining them. Earlier findings from the Cambridge group had shown that embryonic stem cells could self-organise in culture into structures with an antero-posterior polarity.

Now, in collaboration with researchers from the ̽��ֱ�� of Geneva and the Swiss Federal Institute of Technology Lausanne (EPFL), they have extended the cultures to reveal a capacity of mouse stem cells to produce ‘pseudo-embryos’ that display some of the important characteristics of a normal mouse embryo. Established from only 300 embryonic stem cells, these structures, called ‘gastruloids’, exhibit developmental features and organisation comparable to the posterior part of a six to ten day-old embryo.

̽��ֱ��study shows that gastruloids organise themselves with regard to the three main body axes, as they do in embryos, and follow similar patterns of gene expression. One example of this is the pattern of expression of Hox genes, an ensemble of genes that are expressed in a precise sequential order in the embryo and act as landmarks for different aspects of the body, including the position of different vertebrae or of limbs. This degree of organisation makes gastruloids a remarkable system for the study of the early stages of normal or abnormal embryonic development in mammals.

“These results significantly extend our earlier findings. We were surprised to see how far gastruloids develop, their complex organisation and the presence of early-stage tissues and organ,” says Professor Alfonso Martinez Arias, leader of the ̽��ֱ�� of Cambridge team, at its Department of Genetics.

Professor Denis Duboule from the ̽��ֱ�� of Geneva and at the EPFL explained, “To determine whether gastruloids organise themselves into bona fide embryonic structures, we characterised their level of genetic activity at different stages of development”.

̽��ֱ��researchers identified and quantified the RNA transcribed from gastruloids and compared the expressed genes with those of mouse embryos at comparable stages of development, which showed there was a high degree of similarity.

“Gastruloids form structures similar to the posterior part of the embryo, from the base of the brain to the tail, whose development program is somewhat different from that of the head,” says Dr Leonardo Beccari, co-first author of the study, from the ̽��ֱ�� of Geneva.

These embryo-like structures express genes characteristic of the various types of progenitor cells necessary for the constitution of future tissues.

“ ̽��ֱ��complexity of gene expression profiles increases over time, with the appearance of markers from different embryonic cell lineages, much like the profiles observed in control embryos,” adds Dr Naomi Moris from the Cambridge team, co-first author of the article.

“ ̽��ֱ��implementation of the Hox gene network over time, which mimics that of the embryo, particularly confirms the remarkably high level of self-organisation of gastruloids,” explains Mehmet Girgin, co-first author of the study and PhD student at the Institute of Bioengineering at EPFL.

̽��ֱ��researchers say that these pseudo-embryos will allow an alternative method to animal research, in accordance with the principle of the ‘3Rs’ (the reduction, replacement and refinement of the use of animals in research). ̽��ֱ��finding that so much of the development of an embryo can be recapitulated using stem cells will also increase researchers’ ability to study the genetic mechanisms underlying normal development and disease.

Earlier in the year, the group led by Professor Magdalena Zernicka-Goetz at the Department of Physiology, Development and Neuroscience at the ̽��ֱ�� of Cambridge reported embryo-like structures capable of generating an anteroposterior axis but which required additional, extra-embryonic, stem cells to generate anteroposterior polarity. ̽��ֱ��new work shows surprisingly that stem cells can self-organise the three axes independently of the extra-embryonic tissues.

“It makes things much simpler for research,” says Professor Martinez Arias. “Not only do gastruloids self-organise to generate the three axes, but they also mimic the spatial and temporal patterns of embryos, without extra-embryonic tissue. This suggests that gastruloids can become a useful tool, particularly in understanding gene expression during development.”

This work was largely funded by the Biotechnology and Biological Sciences Research Council (BBSRC), the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), the Engineering and Physical Sciences Research Council (EPSRC) and the European Research Council.

Adapted from a press release from the ̽��ֱ�� of Geneva.

Reference
Beccari, L, Moris, N, Girgin, M, et al. Multi-axial self-organisation properties of mouse embryonic stem cells into gastruloids. Nature; 3 Oct 2018; DOI: 10.1038/s41586-018-0578-0

Image caption
Seven-day old gastruloid. ̽��ֱ��cell nuclei are marked in blue. Neural progenitor cells (green) are distributed along the antero-posterior axis. Progenitor cells of the tail bud (pink) are confined to the posterior extremity of the gastruloid and indicate the direction of its elongation. © Mehmet Girgin, EPFL

A team of scientists at the ̽��ֱ�� of Cambridge has developed an artificial mouse embryo-like structure capable of forming the three major axes of the body. ̽��ֱ��technique, reported today in the journal Nature, could reduce the use of mammalian embryos in research.

We were surprised to see how far gastruloids develop, their complex organisation and the presence of early-stage tissues and organ

Alfonso Martinez Arias

Mehmet Girgin, EPFL

Seven-day old gastruloid

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̽��ֱ��body in miniature

cjb250 — Tue, 20 Mar 2018 15:22:55 +0000

In Cambridge alone, there are groups growing mini-livers, mini-brains, mini-oesophaguses,mini-bile ducts, mini-lungs, mini-intestines, mini-wombs, mini-pancreases… Almost the whole body in miniature, it seems.

Read more about how these remarkable 'organoids' are helping transform biomedical research - including helping reduce the number of animals used in research.

̽��ֱ��past few years has seen an explosion in the number of studies using organoids – so-called ‘mini organs’. While they can help scientists understand human biology and disease, some in the field have questioned their usefulness. But as the field matures, we could see their increasing use in personalised and regenerative medicine.

David Turner

Confocal microscope image of gastruloid

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‘Mini liver tumours’ created in a dish for the first time

cjb250 — Thu, 16 Nov 2017 08:25:55 +0000

Primary liver cancer is the second most lethal cancer worldwide. To better understand the biology of the disease and develop potential treatments, researchers need models that can grow in the lab and accurately reflect how the tumours behave in patients. Previously, cultures of cells had been used but these are hard to maintain and fail to recreate the 3D structure and tissue architecture of human tumours.

̽��ֱ��researchers created the mini tumours (up to 0.5mm) – termed ‘tumouroids’ – to mimic the three most common forms of primary liver cancer. ̽��ֱ��tumour cells were surgically removed from eight patients and grown in a solution containing specific nutrients and substances which prevent healthy cells out-competing the tumour cells.

̽��ֱ��team, from the Wellcome/Cancer Research UK Gurdon Institute in Cambridge, used the tumouroids to test the efficacy of 29 different drugs, including those currently used in treatment and drugs in development. One compound, a type of protein inhibitor, was found to inhibit the activation of a protein called ERK in two of the three types of tumouroids, a crucial step in the development of liver cancer.

̽��ֱ��researchers then tested this compound in vivo, transplanting two types of tumouroids into mice and treating them with the drug. A marked reduction in tumour growth was seen in mice treated with the drug, identifying a potential novel treatment for some types of primary liver cancer.

̽��ֱ��tumouroids were able to preserve tissue structure as well as the gene expression patterns of the original human tumours from which they were derived. ̽��ֱ��individual subtypes of three different types of liver cancer, as well as the different tumour tissues which they came from, were all still distinguishable even after they had been grown in a dish for a long time. As the tumouroids retain the biological features of their parent tumour, they could play an important role in developing personalised medicine for patients.

̽��ֱ��creation of biologically accurate models of tumours will also reduce the number of animals needed in certain experiments. Animal studies will still be required to validate findings, but the tumouroids will allow scientists to explore key questions about the biology of liver cancer in cultures rather than mice.

Lead researcher Dr Meritxell Huch, a Wellcome Sir Henry Dale Fellow from the Gurdon Institute, said: “We had previously created organoids from healthy liver tissue, but the creation of liver tumouroids is a big step forward for cancer research. They will allow us to understand much more about the biology of liver cancer and, with further work, could be used to test drugs for individual patients to create personalised treatment plans.”

Dr Andrew Chisholm, Head of Cellular and Developmental Sciences at Wellcome said: “This work shows the power of organoid cultures to model human cancers. It is impressive to see just how well the organoids are able to mimic the biology of different liver tumour types, giving researchers a new way of investigating this disease. These models are vital for the next generation of cancer research, and should allow scientists to minimise the numbers of animals used in research.”

Dr Vicky Robinson, Chief Executive of the NC3Rs which partially funded the work, said: “We are pleased to see that the funds from our annual 3Rs prize, sponsored by GlaxoSmithKline, have furthered Dr Huch's research. Each year the prize recognises exceptional science which furthers the 3Rs, and the work being conducted by Meri and her team is continuing to make progress in this area. This new breakthrough involving liver cancer organoids has the potential to reduce the number of animals required in the early stages of liver cancer research, and provide more biologically accurate models of human tumours.”

This work was funded by a National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) research prize, Wellcome and Cancer Research UK Cambridge Centre.

Reference
Broutier, L et al. Human primary liver cancer–derived organoid cultures for disease modelling and drug screening. Nature Medicine; 13 Nov 2017; DOI: 10.1038/nm.4438

Press release from Wellcome.

Scientists have created mini biological models of human primary liver cancers, known as organoids, in the lab for the first time. In a paper published in Nature Medicine, the tiny laboratory models of tumours were used to identify a new drug that could potentially treat certain types of liver cancer.

Learning from the liver how to regenerate

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Fruit fly model of deadly brain diseases could lead to blood test for vCJD

cjb250 — Wed, 12 Oct 2016 23:33:41 +0000

Currently, methods to detect vCJD-infected human blood samples that involve experimental animals, such as mice, are time consuming and expensive. This new test could potentially be used on blood samples collected during pre-clinical disease and would be able to give a result in a matter of days or weeks rather than months or years.

In the late eighties, the UK saw an outbreak of bovine spongiform encephalopathy (BSE), a fatal brain condition in cattle, often referred to as ‘mad cow disease.’ BSE is a type of neurodegenerative brain condition known as a prion disease, caused by the build-up of a rogue form of a normal protein found in neurons. This aggregated form of the normal protein is referred to as a prion and is infectious. Following the BSE outbreak, a number of people were diagnosed with vCJD, a fatal human prion disease, believed to have occurred through the consumption of BSE-contaminated beef. vCJD causes changes in mood and behaviour, followed by difficulty in walking, and eventually leads to loss of movement and speech before death.

Other cases of vCJD have occurred in patients who received blood products prepared from donors who themselves later developed the disease; hence, blood-borne transmission of vCJD is a major concern for blood transfusion banks, manufacturers of blood plasma-derived products and public health authorities.

Although the number of people known to have died from vCJD is small – less than 180 cases in the UK – recent research has suggested that, within a certain age group of people in the UK, the number of individuals infected with vCJD, but who have not developed clinical signs of the condition, could be as high as one person in 2,000. Whether these individuals will go on to develop the clinical form of the disease during their natural life span remains uncertain.

At the moment, the only reliable way to detect infectious prions in blood is through a test known as a bioassay. This involves injecting suspected infected samples into experimental animals and waiting to see if these recipients develop prion disease. This is usually carried out by injecting potentially prion-infected samples into the brains of mice. These assays are slow and cumbersome, since the incubation time for prion disease may be over a year. This means that very few blood samples are routinely screened for prion infectivity.

Now, in a study published today in the Biochemical Journal, scientists at the ̽��ֱ�� of Cambridge, UK, and the Ecole Nationale Veterinaire de Toulouse, France, report the development of a genetically-modified fruit fly (Drosophila melanogaster) into which a gene has been inserted to make the fly capable of producing the rogue protein that aggregates in the brain of sheep with the prion disease scrapie.

When the researchers fed these transgenic flies plasma from sheep known to have prions in their blood, they found that this caused prion disease in the flies. This response to prion-infected blood was evident within only a few weeks after exposure to the material.

Dr Raymond Bujdoso from the Department of Veterinary Medicine at the ̽��ֱ�� of Cambridge, who led the research, says: “We have found the fruit flies respond so quickly to infected blood that it means we can develop a faster, more versatile and more sensitive test to detect infectious prions in blood than currently exists.

“At the moment, screening blood products for vCJD prion infectivity is just not practical – it is expensive and time consuming, and would require the use of a large number of animals, which is ethically unacceptable. ̽��ֱ��development of a vCJD blood test that could easily and reliably screen for prion-infectivity would represent an ideal solution for identifying donors and blood donations that might present a risk of causing the disease.”

Fruit flies are relatively easy and economical to work with, and widely accepted to be an ethical alternative to higher organisms such as mice. Dr Bujdoso and colleagues say that their fruit fly model will help contribute to the so-called 3Rs – the replacement, refinement and reduction of the use of animals in research.

Professor David Carling, Chair of the Biochemical Journal, adds: “ ̽��ֱ��paper from Dr Bujdoso and colleagues provides a proof-of-principle study demonstrating that the fruit fly can be used to detect the infectious agent responsible for a type of neurodegenerative disease. Although the work is at a preliminary stage, it offers the exciting possibility of developing a quick and reliable screen for early diagnosis of a devastating disease.”

̽��ֱ��research was supported by the Isaac Newton Trust and the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs).

Reference
Thackray, AM, Andréoletti, O and Bujdoso, R. Bioassay of prion-infected blood plasma in PrP transgenic Drosophila. Biochem Journal; 13 Oct 2016; 10.1042/BCJ20160417

A new model of fatal brain diseases is being developed in the fruit fly by a team led by Dr Raymond Bujdoso at the ̽��ֱ�� of Cambridge, and could lead to a low cost, fast and efficient blood test to diagnose – and prevent possible transmission of – variant Creutzfeldt-Jakob disease (vCJD).

We have found the fruit flies respond so quickly to infected blood that it means we can develop a faster, more versatile and more sensitive test to detect infectious prions in blood than currently exists

Raymond Bujdoso

Veljo Runnel

Drosophila

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Mini-livers show promise to reduce animal use in science

sjr81 — Wed, 26 Feb 2014 10:18:48 +0000

Dr Meritxell Huch from the Gurdon Institute, who tonight receives the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 3Rs Prize, has developed a method that enables adult mouse stem cells to grow and expand into fully functioning three-dimensional liver tissue.

Using this method, cells from one mouse could be used to test 1000 drug compounds to treat liver disease, and reduce animal use by up to 50,000.

Growing hepatocytes (liver cells) in the laboratory has been attempted by liver biologists for many years, since it would reduce their reliance on using mice to study liver disease and would open up new opportunities in medical research and drug safety testing. Until now no laboratory has been successful in deciphering how to isolate and grow these cells.

Liver stem cells are typically found in a dormant state in the liver, only becoming active following injury to produce new liver cells and bile ducts. Dr Huch and colleagues at the Netherlands’ Hubrecht Institute located the specific type of stem cells responsible for this regeneration, which are recognised by a key surface protein (Lgr5+) that they share with similar stem cells in the intestine, stomach and hair follicles.

By isolating these cells and placing them in a culture medium with the right conditions, the researchers were able to grow small liver organoids, which survive and expand for over a year in a laboratory environment. When implanted back into mice with liver disease they continued to grow, ameliorating the disease and extending the survival of the mice.

Having further refined the process using cells from rats and dogs, Dr Huch is now moving onto testing it with human cells, which could potentially translate to the development of a patient’s own liver tissue for transplantation.

Commenting on the new method’s potential to reduce animal use in liver research, Dr Huch said: “Typically a study to investigate one potential drug compound to treat one form of liver disease would require up to 50 live animals per experiment, so testing 1000 compounds would need 50,000 mice. By using the liver culture system I developed, we can test 1000 compounds using cells that come from only one mouse, resulting in a significant reduction in animal use.

“If other laboratories adopt this method then the impact on animal use in the liver research field would be immediate. A vast library of potential drug compounds could be narrowed down to just one or two very quickly and cheaply, which can then be tested further in an animal study.”

Dr Vicky Robinson, Chief Executive of the NC3Rs said: “Growing functioning liver cells in culture has been the Holy Grail for liver biologists for many years, so a limitless supply of hepatocytes could have a huge 3Rs impact both on basic research to understand liver disease and for the screening and safety testing of pharmaceuticals. Researchers need to utilise this alternative technology as soon as possible to ensure the benefits to animals and human health are fully realised.”

Professor Kevin Shakesheff, Director of the UK Regenerative Medicine Hub in Acellular Materials, said: “ ̽��ֱ��work of Dr Huch and team demonstrates how three-dimensional culture and molecular biology combine to open new possibilities in the regeneration of complex tissues. ̽��ֱ��liver is an excellent target for this work as the human body has an ability to regenerate liver tissue that is very hard to replicate in the lab. Unlocking new mechanisms to generate functional liver creates therapeutic approaches for patients with liver disease or injury and could offer a route to high quality human liver models that enhance drug development.”

Cambridge research that has for the first time successfully grown “mini-livers” from adult mouse stem cells has won the UK’s international prize for the scientific and technological advance with the most potential to replace, reduce or refine the use of animals in science (the 3Rs).

If other laboratories adopt this method then the impact on animal use in the liver research field would be immediate.

Meritxell Huch

Mini-liver research to reduce animal use in science

NC3Rs

Meritxell Huch

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