Announced at the end of 2014 during the final phase of research and development, the rapid diagnostic test for Ebola virus disease developed by the French Atomic Energy Commission (CEA), with support from the Inserm Jean Mérieux P4 high-level biological containment laboratory (Lyon), has been assessed in the field, including in Guinea, over a period of several months. This assessment now allows the test to be CE marked, leading to its authorisation for use as a diagnostic tool. The eZYSCREEN^® test will thus help to enhance the available means of control, particularly by detecting residual sporadic cases among symptomatic patients, or by post-mortem identification.

The rapid diagnostic test for Ebola, developed in the public health emergency of 2014 and 2015 by a team from the Life Sciences Division (DSV) of CEA, with support from the Inserm Jean Mérieux P4 high-level biological containment laboratory (Lyon), allows diagnosis of the disease within 15 minutes, using a few drops of blood or serum. An agreement has been drawn up with the Vedalab company (Alençon), which manufactures it.

In contrast to laboratory tests, this easy to use field test does not require sophisticated instrumentation or even electricity, and can be used by non-specialist staff. The result is read directly and visually. Storage for eight and a half months at 30°C, and for 14 days at 45°C, did not affect its performance, proving its high robustness and stability.

The eZYSCREEN^® test was assessed under real conditions, including in the Ebola Treatment Centres in Coyah and Forécariah in Guinea (French Red Cross), and part of the analyses were also conducted in Donka National Hospital (Conakry, Guinea). These studies ended in August 2015. 

The rapid test shows excellent specificity, eliminating almost any risk of false positive results, and an appropriate degree of sensitivity for its intended use, i.e. to very rapidly identify the maximum number of cases possible in situ with a view to preventing epidemics.

These results enabled CE marking of the eZYSCREEN^® test for Ebola, which is essential for its use during the sporadic cases that continue to occur in Guinea and Sierra Leone. The objective is to be able to avoid the start of a new large-scale epidemic outbreak.

Discussions are underway between CEA and diagnostics industries in order to allow the widest possible distribution of the test.

For this body of work, the Technological Innovations for Detection and Diagnosis (Li2D/DSV) Laboratory in the CEA Marcoule Centre has just received the Societal Resilience Trophies Technological Innovation Prize for 2015, awarded by the French High Committee for Civil Defence.

The efforts of the international community for the past ten years in the fight against malaria have reduced the number of disease-related deaths. The emerging resistance to standard therapies threatening South-East Asia, and new research carried out by the team led by Françoise Benoit-Vical, Inserm Research Director in the CNRS (French National Centre for Scientific Research) Coordination Chemistry Laboratory in collaboration with the Pasteur Institute and Inserm, are not reassuring factors. The in vitro examination of a strain of parasites solely exposed to artemisinin (the base compound for standard therapy) demonstrates the development of a widespread resistance to most other anti-malarial drugs. This new resistance cannot be detected by tests currently used and represents an additional threat to antimalarial treatments in the field.This research is published in the journal Emerging Infectious Diseases.

Malaria is caused by a parasite propagated through bites from infected mosquitoes from the Anopheles genus. This disease is most prevalent in tropical areas and still responsible for over 600,000 deaths each year. Policies to combat this disease have led to a 60% decrease in mortality over 15 years. However, artemisinin, the pharmaceutical base compound for antimalarial therapies, is encountering increasing clinical failure due to emerging resistance throughout South-East Asia. As of yet, this resistance has not been observed on the African continent.

Artemisinin is an active ingredient from a Chinese plant whose benefits have been known for over 2,000 years. It is used in combination with other antimalarial agents. The value of these combination therapies lies in the assurance that even if the parasite develops a resistance to one molecule, it is less likely to develop a resistance to both molecules.

Scientists must nevertheless stay one step ahead of the parasite, given the recent and rapid development of resistance to artemisinin. It is against this background that the team led by Françoise Benoit-Vical, Inserm Research Director in the CNRS Coordination Chemistry Laboratory in Toulouse, in collaboration with Inserm in Toulouse and the Pasteur Institute in Paris, is studying the resistance mechanisms developed by Plasmodium falciparum, the parasite responsible for malaria, and researching new antimalarial drugs.

Researchers have shown that parasites who survive in vitro in the presence of only artemisinin for five years develop a widespread resistance to most other artemisinin-based or non-artemisinin-based antimalarial therapies, including partner molecules present in combination therapies used in endemic areas.

Scientists have demonstrated that these parasites do not exhibit any known mutation in resistance genes. However, they circumvent the toxic effect of the drugs by a phenomenon known as quiescence. Parasites are capable of suspending their development during exposure to antimalarial agents. As soon as they are no longer subjected to antimalarial therapy, the parasites “wake-up” and proliferate once again.

The new multi-drug resistance based on this quiescence phenomenon cannot be detected by tests currently carried out to analyse parasitic resistance. “In vitro tests carried out using the patient’s blood predict high sensitivity and, therefore, the treatment’s effectiveness, while parasites are resistant because they are quiescent. As such, it is essential to conduct research with relevant and appropriate tests in the field if the multi-resistant phenomenon that we identified in vitro is also present, in order to design therapeutic strategies accordingly.” explains Françoise Benoit-Vical.

Our bodies have ten times the amount of microbes than human cells. This set of bacteria is called microbiota. In some instances, bacteria known as pathogens can cause infectious diseases. However, these micro-organisms can also protect us from certain diseases. Researchers from Inserm, Paris Descartes University and the CNRS (French National Centre for Scientific Research), through collaboration with teams from China and Sweden, have recently shown how microbiota protects against the development of type 1 diabetes. This research is published in the Immunity journal, 4 August 2015.

A pancreatic islet of Langerhans expressing the immunoregulator antimicrobial peptide CRAM (in red). The insulin-producting beta-cells are in green and the glucagon-producting alpha-cells are in blue. © Julien Diana

To combat pathogens, the immune system has developed various mechanisms to detect, defend against and even destroy micro-organisms that are harmful to the body. This includes antimicrobial peptides and natural proteins that destroy pathogenic bacteria by disrupting their cellular membrane. Not only are they produced by immune cells, they are also produced by cells whose functions are not immune-related.

A research team coordinated by Julien Diana, an Inserm Research Fellow at Inserm Unit 1151 “Institut Necker-Enfant Malades” [Necker Institute for Sick Children] (Inserm/CNRS/Université Paris Descartes), is focussing on a category of antimicrobial peptides, i.e. cathelicidins. Apart from their protective function, these peptides have also exhibited immunoregulatory abilities against several autoimmune diseases. As such, scientists hypothesise that cathelicidins may be involved in the control of type 1 diabetes, an autoimmune disease where certain cells in the immune system attack beta cells in the pancreas which secrete insulin.

Firstly, they observed that beta pancreatic cells in non-diseased mice produce cathelicidins and that, interestingly, this production is impaired in diabetic mice.

To test this hypothesis, they are injecting diabetic mice with cathelicidins where production is defective.

“Injecting cathelicidins inhibits the development of pancreatic inflammation and, as such, suppresses the development of autoimmune disease in these mice” states Julien Diana.

Given that the production of cathelicidins is controlled by short-chain fatty acids produced by gut bacteria, Julien Diana’s team are studying the possibility that this may by the cause of the cathelicidin deficiency associated with diabetes. Indeed, researchers have observed that diabetic mice have a lower level of short-chain fatty acids than that found in healthy mice.

By transferring part of the gut bacteria from healthy mice to diabetic mice, they are re-establishing a normal level of cathelicidin. Meanwhile, the transfer of micro-organisms reduces the occurrence of diabetes.

For the authors, “this research is further evidence of the undeniable role microbiota plays in autoimmune diseases, particularly in controlling the development of autoimmune diabetes”. 

A large-scale collaborative study, conducted by a French team comprising of the Infectious and Tropical Disease departments of the AP-HP, the hospital bacteriology and hygiene laboratories, the Research Unit of the Bichat – Claude Bernard hospital, the Paris-Diderot University, Inserm, the Pasteur institute and InVS, revealed the acquisition of multi-resistant enterobacteria (MRE) in one in two travellers returning from a stay in a country situated in a tropical region. These results were published in the international journal Clinical Infectious Diseases on 22^nd April, 2015.

©Fotolia

Enterobacteria are microorganisms naturally present in our digestive tract.  While some are completely harmless, others can be responsible for serious infections. In such cases, the patients are treated with antibiotics. Unfortunately, some of these bacteria are resistant, making treatment of these diseases difficult.

The teams coordinated by Sophie Matheron and Etienne Ruppé carried out a study on 824 healthy people before and after a stay in a tropical region (sub-Saharan Africa, Asia, or South America). These people were asked to answer a medical questionnaire and to provide a stool sample in the week before their departure and within three days of their return. This step was repeated for travellers carrying multi-resistant enterobacteria (MRE) until the samples were negative.

The study revealed an overall acquisition frequency of MRE in the digestive tract during the trip of 51%, i.e. one in two travellers. This increased frequency differs depending on the geographical regions visited: on returning from Asia, over 72% of travellers were carriers, more than 47% of those returning from sub-Saharan Africa were carriers, and a little more than 31% of those returning from South America were carriers.

The acquisition of these microorganisms is favoured by the taking of antibiotics (which alters the natural “barrier” protection effect of the bacteria normally present in the digestive tract), the occurrence of diarrhoea during the trip, and the type of trip.  The open stays (organised trips, the family or the backpacker type) present a greater risk than closed stays in hotel-clubs. The prolonged follow-up showed that 95% of the travellers spontaneously eliminate the MRE in the three months after their return.

This study reminds us of the importance of the prudent use of antibiotics (often consumed in an excessive way in cases of simple diarrhoea) and the importance of respecting the preventative and basic hygiene rules during the trip (wash food, wash hands, drink water from a bottle, etc.). Thanks to dedicated consultations, the international vaccine and advice centres provide this information to travellers. They also give other advice and prescriptions depending on the destination (vaccination, prevention of malaria).

Finally, these results highlight the necessity to study the involvement of MRE in patients reporting an infection in the three months following their return from a tropical region. Thus, the health professionals in towns and hospitals may prescribe a treatment adapted to a possible antibiotic resistance.

It should be noted that travellers carrying MRE are not ill and, on this ground alone, have no reason to consult a doctor.

Researchers at INRA, INSERM, AP-HP, and UPMC have just discovered a new protein with anti-inflammatory properties, which has been named MAM (microbial anti-inflammatory molecule). It is secreted by the bacterium Faecalibacterium prausnitzii and helps fight intestinal inflammation. This discovery, recently published in the journal Gut, is a major advance in efforts to develop new means of treating inflammatory bowel disease. Consequently, the results of this research have great potential future applications.

A few years ago, researchers discovered that the gut bacterium Faecalibacterium prausnitzii was present at lower levels in individuals affected by inflammatory bowel diseases (IBDs), such as Crohn’s disease or ulcerative colitis.They also found that this species secretes one or more molecules with anti-inflammatory properties that help in the fight against IBDs. The identification of the molecules produced by F. prausnitzii is a major step forward, which has occurred thanks to the efforts of a team of researchers from INRA, INSERM, AP-HP (Greater Paris University Hospitals), and UPMC (Pierre-and-Marie-Curie University).

Using biochemistry and mass spectrometry, the researchers discovered the existence of several peptides that were all derived from a single molecule produced by F. prausnitzii, a protein that they called MAM for microbial anti-inflammatory molecule. First, they showed that the addition of MAM to intestinal cells reduced inflammation. Second, they demonstrated that MAM helped alleviate an IBD in a mouse model. More specifically, mice that received MAM lost less weight than control mice. The researchers also showed that MAM’s effect was due to its ability to reduce the levels of certain immune system compounds in the intestinal mucosa that cause inflammation. It therefore seems that the bacteria that live in our guts protect us using the same tools as medical professionals.

When F. prausnitzii is present at low levels, IBDs are exacerbated, creating a vicious cycle. To break the cycle of chronic intestinal inflammation, researchers are working on ways to restore F. prausnitzii’s presence in the gut. These include providing nutritional supplements that contain the bacterium (i.e., probiotics) and/or molecules that promote the bacterium’s growth (prebiotics). The discovery and characterization of MAM also has relevance for the pharmaceutical industry: the protein could eventually be manufactured and serve as an active ingredient in medications. These results, which add to the growing base of knowledge regarding intestinal bacteria, also clearly have practical industrial or medical applications.

Image en microscopie électronique à balayage de F. prausnitzii © Inra, plateforme MIMA 2, T. Meylheuc

By increasing the stiffness of erythrocytes infected by the causal agent of malaria, Viagra favors their elimination from the blood circulation and may therefore reduce transmission of the parasite from humans to mosquitoes. This astonishing discovery, made by scientists from the CNRS, INSERM, Université Paris Descartes – at the Institut Cochin – and the Institut Pasteur, working in collaboration with a team from the London School of Hygiene and Tropical Medicine, could lead to a treatment to reduce the spread of malaria within a population. Their work is published in PLOS Pathogens on 7 May 2015.

Plasmodium falciparum, the parasite that causes malaria, has a complex developmental cycle that is partially completed in humans and partially in the anopheline mosquito. Treatments for malaria target the asexual forms of this parasite that cause symptoms, but not the sexual forms transmitted from a human to a mosquito when it bites. Eradication of this disease thus necessitates the development of new types of treatments against sexual forms of the parasite in order to block transmission and thus prevent dissemination of the disease within the population.

The sexual forms of the parasite develop in human erythrocytes sequestered in the bone marrow before they are released into the blood. They are then accessible to mosquitoes, which can ingest them when they bite (see the top of the image on page 2). But circulating erythrocytes — whether they are gametocyte-infected or not — are deformable, thus preventing their clearance via the spleen, which constantly filters the blood and only retains stiff, old or abnormal erythrocytes. However, gametocyte-infected erythrocytes can easily pass through the spleen and persist for several days in the blood circulation.

During a new study, the scientists thus sought to stiffen the infected erythrocytes. They showed that the deformability of gametocyte-infected erythrocytes is regulated by a signaling pathway that involves cAMP. When the cAMP molecules accumulate, the erythrocyte becomes stiffer. cAMP is degraded by the enzyme phosphodiesterase, whose action thus promotes erythrocyte deformability.

Using an in vitro model reproducing filtration by the spleen, the scientists were able to identify several pharmacological agents that inhibit phosophodiesterases and can therefore increase the stiffness of infected erythrocytes. One of these agents is sildenafil citrate, better known under its brand name of Viagra. The authors showed that this agent, used at a standard dose, had the potential to increase the stiffness of sexual forms of the parasite and thus favor the elimination of infected erythrocytes by the spleen.

The deformability of gametocyte-infected erythrocytes, facilitated when the cAMP pathway is inhibited, allows them to circulate freely through the spleen and remain accessible to mosquitoes in the blood circulation (top). The action of Viagra increases the stiffness of infected erythrocytes by activating the cAMP pathway, preventing their passage through the spleen endothelium and clearing the parasites from the blood circulation (bottom). © Catherine Lavazec

This discovery could help find new ways to stop the spread of malaria in a population. 

Modifying the active substance in Viagra to block its erectile effect, or testing similar agents devoid of this adverse effect, could indeed result in a treatment to prevent transmission of the parasite from humans to mosquitoes.

Several years ago, INRA researchers in Jouy-en-Josas showed that levels of the gut bacterium Faecalibacterium prausnitzii tended to decline in the human gut at the onset of chronic inflammatory bowel disease (IBD). Is this disappearance of F. prausnitzii one of the causes of inflammation, or is its disappearance a consequence of the disease? Today, the same INRA team, in collaboration with an American team (Berkeley), AgroParisTech, lnserm, AP-HP and UPMC, are answering these questions. Not only do their results show that F. prausnitzii plays an active role in protecting against intestinal inflammation, they also propose explanations regarding mechanisms of action. This research is published in the journal mBio on 21 April 2015.

Seven years ago, researchers at INRA (French National Institute for Agricultural Research) drew the attention of scientists and the public to a specific bacterium found in our gut. This bacterium, Faecalibacterium prausnitzii, is abundant in the gut of healthy humans, but as soon as a chronic inflammatory bowel disease (IBD) occurs, it tends to decline. One nagging question has been raised by the scientific community: is the disappearance of F. prausnitzii one of the causes of inflammation, or is its disappearance a consequence of the disease? In other words, is F. prausnitzii a bacterium that might protect our digestive tract from an inflammatory disease?

To find an answer, the researchers used mice harbouring only two types of bacteria in their digestive tract, in contrast to several billion under normal circumstances. Following a treatment that generates inflammation, the presence of F. prausnitzii on its own protects from the development of intestinal inflammation. This demonstrates the anti-inflammatory potential of F. prausnitzii.

The INRA researchers and their colleagues from Berkeley, in collaboration with AgroParisTech, the French National Institute of Health and Medical Research (Inserm), the Paris public hospitals (AP-HP), and Pierre and Marie Curie University (UPMC), also propose new approaches to explaining how this bacterium could protect us. The presence of this bacterium is actually associated with many anti-inflammatory molecules in the gut and bloodstream of animals. F. prausnitzii may be able to provide protection to our digestive tract by a varied arsenal of metabolic activities. For example, salicylic acid, a precursor in the synthesis of drugs used to treat patients with IBD, is found in the gut of mice carrying F. prausnitzii. The bacteria that we harbour may well play an active role in our health via the same strategies that are used in the medical arena.

[exergue]Once there is intestinal inflammation, the decline in the presence of the bacterium F. prausnitzii therefore aggravates the disease. In order to break this vicious cycle leading to chronic inflammation of the digestive tract, the scientists plan to restore the presence of F. prausnitzii using new food supplements containing the bacterium (probiotics) and/or favouring development of the bacterium (prebiotics). [/exergue]This study, which has added to our fundamental knowledge in the area of microbiology, is also at the interface of new industrial and medical applications.Scanning electron micrographs of F. prausnitzii © MIMA 2 Platform, T. Meylheuc)

French teams from CIC Biothérapie (AP-HP/Inserm), from pediatric hematology department of Necker Hospital for Children (AP-HP), led by Marina Cavazzana, Salima Hacein Bey Albina and Alain Fischer and from Genethon led by Anne Galy (Genethon/Inserm UMR-S951), and English teams from UCL Institute of Child Health and Great Ormond Street Hospital in London led by Adrian Thrasher and Bobby Gaspar demonstrated the efficacy of gene therapy treatment for Wiskott-Aldrich Syndrome (WAS). Six children that were treated and followed for at least 9 months had their immune system restored and clinical condition improved. This work, which was published today in the Journal of the American Medical Association (JAMA), was carried out with support from the AFM-Telethon.

Wiskott-Aldrich syndrome is a rare congenital immune and platelet deficiency which is X-linked and has an estimated prevalence of 1/250 000. It is caused by mutations in the gene encoding the WAS protein (WASp) expressed in hematopoietic cells. This disease, which primarily affects boys, causes bleeding, severe and recurrent infections, severe eczema and in some patients autoimmune reactions and the development of cancer. The only treatment available today is bone marrow transplantation, which requires a compatible donor and can itself cause serious complications.

The Phase I / II study, with Genethon as the promoter, was launched in December 2010 and conducted in Paris and London to treat severely ill patients without a compatible donor. This study, which is ongoing, assesses the feasibility and efficacy of gene therapy in this indication. The article published in JAMA reports the results for the first six patients, aged 8 months to 16 years, where the monitoring period allowed assessment of the initial effects of the treatment.

The treatment involves collected blood stem cells carrying the genetic anomaly of patients and corrected them in the laboratory by introducing a healthy WAS gene using a lentiviral vector developed and produced by Genethon. The corrected cells were reinjected into patients who in parallel were treated with chemotherapy to suppress their defective stem cells and autoimmune cells to make room for new corrected cells. After reinjection, these cells were then differentiated into the various cell lines that make up the blood (red and white cells, platelets).

To date treated patients showed significant clinical improvement. Severe eczema and severe infection disappeared in all cases. Arthritis was eliminated in one patient and another saw major improvement in vasculitis of the lower limbs and was able to return to normal physical activity without a wheelchair. However, the rate of corrected platelets varies from one patient to another.

Fulvio Mavilio, Chief Scientific Officer Genethon: “We are all very happy and encouraged by the results of this study. It is the first time that a gene therapy based on genetically modified stem cells is tested in a multicenter, international clinical trial that shows a reproducible and robust therapeutic effect in different centers and different countries. For very rare diseases such as WAS, multicenter clinical trials are the only effective way of proving the safety and efficacy of gene therapy and having it rapidly approuved and made available to all patients. We are following the same approach for other rare and less rare blood diseases.”

Frédéric Revah, CEO of Genethon, the laboratory of the AFM-Telethon and the trial sponsor, said “These first results of our clinical trial for the treatment of Wiskott Aldrich syndrome are very encouraging. They illustrate not only the ability of Genethon to carry out the upstream research to develop treatments for these rare and complex diseases, but also to construct and conduct international clinical trials, to produce these advanced therapy products, to work with international teams and to manage the regulatory aspects of the trials in France and abroad. These are skills that we implement for other international trials of gene therapy for rare genetic diseases of the immune system, blood, muscle, vision or liver… We will continue the current study with the objective of providing treatment for patients.”

Marina Cavazzana: “The results obtained in this multicenter clinical trial constitute an important therapeutic advance (overhang) because they concern a complex pathology which affects almost all of blood cells with dramatic clinical consequences. After transfer of gene, the patients showed a significant clinical improvement due to the reexpression of the protein WASp in the cells of the immune system. The efficiency of the treatment of such a deficit for which a high level of correction of hematopoietic stem cells is required, indicates that it is from now on justifiable to hope to treat other complex genetic diseases as those affecting red blood cells.”

Professor Thrasher says: “This is a very powerful example of how gene therapy can offer highly effective treatment for patients with complex and serious genetic disease. It also excitingly demonstrates the potential for treatment of a large number of other diseases for which existing therapies are either unsatisfactory or unavailable.”

While most of us recover from influenza after a week, it can be a very severe disease, and even fatal in rare cases, with no reason for physicians to have expected such an outcome. By analysing the genome of a little girl who contracted a severe form of influenza at the age of two and a half years, researchers at the Laboratory of Human Genetics of Infectious Diseases (a joint French-American international laboratory), which brings together researchers from Inserm, Paris Descartes University, and physicians from the Paris public hospitals (AP-HP; Necker Hospital for Sick Children), working at the Imagine Institute, and from The Rockefeller University in New York, have discovered that she has a genetic mutation, unknown until now, that causes a subtle dysfunction in her immune system. More generally, these results show that genetic mutations could be the root cause of some severe forms of influenza in children, and indicate in any event that immune mechanisms missing in this little girl are needed for protection against this virus in humans. These results are published in the journal Science Express.

©fotolia

Seasonal influenza is an acute viral infection caused by the influenza virus. It is characterised by high fever, headaches, sore muscles, etc. Apart from vaccination, there is no treatment for it other than symptomatic (pain) treatment. In most cases, patients recover after a week, but in more vulnerable people influenza can cause acute respiratory distress, which is potentially fatal.

The main known risk factors for severe forms of influenza are some acquired comorbidities, such as chronic lung disease. However, the cause of most fatal cases remains unexplained, especially in children.

The absence of cases of severe influenza in patients with known acquired immunodeficiencies, which usually increase susceptibility to infections, is also surprising.

Given these different observations, the researchers at Jean-Laurent Cassanova’s and Laurent Abel’s laboratory, in Paris and New York, therefore formulated a hypothesis whereby severe influenza in healthy children might be the result of genetic errors.

To test this hypothesis, they sequenced the entire genome of a 7-year-old child who had contracted a severe form of influenza (influenza A virus strain H1N1), requiring her admission to a paediatric intensive care unit in January 2011, at the age of two and a half years. At the time, she showed no other known pathology that might have suggested greater vulnerability to the virus than that of other children.

This analysis, combined with analysis of her parents’ genomes, made it possible to show that the little girl had inherited a mutated allele of the gene encoding interferon regulatory factor (IRF7) from both of her parents. The latter is a transcription factor known to amplify the production of interferons in response to viral infection in mice and humans.

In contrast to her parents, in whom the mutation of a single allele of the gene is of no consequence, in the little girl, mutation of both alleles of the gene encoding IRF7 has led to its inactivation. The result: failure to produce interferons, disrupting her system of defence against influenza virus infection in a cascading manner.

By carrying out a comprehensive series of experiments on blood cells, particularly dendritic cells, and by generating lung cells from stem cells taken from the young girl, the researchers provided proof that the mutations observed in this little girl explain the development of severe influenza. Furthermore, this discovery demonstrates that interferon amplification dependent on IRF7 expression is needed for protection against influenza virus in humans. They now need to search for mutations in this or other genes in other children recruited following an episode of unexplained severe influenza.

Based on these initial observations, the researchers at Inserm believe that therapeutic strategies based on recombinant interferons, available in the pharmacopoeia, could help to combat severe forms of influenza in children.

This multidisciplinary study required multiple collaborations in Europe and the United States.