Anaphylactic shock, an exacerbated allergic reaction that can prove fatal, is sometimes caused by the use of drugs during surgery. In most of these extreme reactions, evidence can be provided that patients have anti-drug antibodies of the IgE class. In 10 to 20% of anaphylactic cases evidence for the involvement of anti-drug IgE is lacking. Anti-drug IgE enable activation of mast cells and basophils that release histamine, a potent mediator involved in anaphylaxis. Teams from the Institut Pasteur, Inserm, the Paris Public Hospital Network (AP-HP), the CNRS, Paris-Sud University and Sorbonne University have successfully identified a new pathological mechanism responsible for these previously unexplained cases, involving neutrophils activated by antibodies of the IgG class. These findings, published on July 10 in the journal Science Translational Medicine, will help improve diagnosis and treatment for patients with this type of shock.
Anaphylaxis is a hyperacute allergic reaction caused by an inappropriate immune response following the introduction of a usually harmless antigen into the body. When this antigen binds to antibodies already in the body, it triggers the secretion of large quantities of potent vasodilating mediators, sending the patient into a state of shock that may result in multiple organ failure and even death.
Anaphylaxis may be brought about by various substances, including drugs (antibiotics or neuromuscular blocking agents), food and insect venom. In this study, the scientists focused on allergic reactions to neuromuscular blocking agents (NMBAs), drugs used during general anesthesia to induce muscle relaxation.
The incidence of anaphylactic shock caused by NMBAs is one case in every 10,000 to 20,000 surgeries, representing approximately 3 to 5 cases each week in the Greater Paris area.
Although it was already known that IgE antibodies could cause anaphylaxis, teams from the Institut Pasteur, Inserm, the Paris Public Hospital Network (AP-HP), the CNRS, Paris-Sud University and Sorbonne University have demonstrated in a clinical study that IgG antibodies can also be involved in drug-induced anaphylactic shocks. This unexpected role of IgG antibodies had already been identified in mice in 2011 by some of the same authors.[1]
This multicenter study known as “NASA”, led by Bichat Hospital (part of the Paris Public Hospital Network), was launched in 2012 by a consortium of scientists, clinical biologists and anesthetists. The consortium monitored 86 patients with perioperative anaphylactic shock and 86 control patients in 11 hospitals in the Greater Paris area, coordinated at Bichat Hospital by immunologist Professor Sylvie Chollet-Martin (Paris-Sud University) and anesthetist Professor Dan Longrois. Blood samples were taken as soon as an anaphylactic shock occurred in the operating room, enabling the scientists to identify the alternative IgG-dependent mechanism. They demonstrated that IgG antibodies activate neutrophils (50-70% of our white blood cells), releasing high doses of harmful vasodilating mediators. Neutrophil activation was more pronounced in cases of severe shock than in cases of moderate shock. Interestingly, the IgG-neutrophil pathway was also identified in most cases of shock where the traditional IgE-dependent mechanism was observed, suggesting that IgGs and neutrophils may contribute to the severity of most cases of shock via an additive effect.
“These findings elucidate 10 to 20% of cases of anaphylactic shock that previously had no biological explanation. They will be extremely valuable in refining diagnosis in these patients and avoiding any future exposure with the drug that triggered the allergic reaction,” explains Professor Sylvie Chollet-Martin (Paris-Sud University), joint last author of the study and Head of the Immunology laboratory on Autoimmunity and Hypersensitivity at Bichat Hospital.
“Although IgG antibodies are known to protect the body from infection and to act as aggressors in some autoimmune diseases, this study reveals that they may be involved in humans in another reaction that is harmful for the body, anaphylaxis.We are currently carrying out experimental research to explore how we might block this new activation pathway for IgG antibodies so that we can propose a therapeutic solution,” comments Pierre Bruhns, joint last author of the study, Inserm Research Director and Head of the Institut Pasteur’s Antibodies in Therapy and Pathology Unit.
This research was funded by the organizations mentioned in the first paragraph and by the European Research Council (ERC – FP7; MyeloSHOCK project).
[1] Mouse and Human Neutrophils induce Anaphylaxis, The Journal of Clinical Investigation, March 23, 2011. doi: 10.1172/JCI45232
Not only are they effective against Gram-positive and negative multi-resistant bacteria, they also appear not to trigger resistance when used to treat infection in mice. Such are the promises of the two new antibiotics created by Prof. Brice Felden and his team at the Inserm and Université de Rennes 1 ‘Bacterial Regulatory RNAs and Medicine’ joint laboratory (U1230), in conjunction with a team from the Rennes Institute of Chemical Sciences (CNRS/Université Rennes 1/ENSCR/INSA Rennes). This French advance could bring both fresh impetus and new possibilities for fighting antibiotic resistance worldwide. Details on this research will be published July 9 in Plos Biology.
Antibiotics have saved so many lives over the previous century of their use in humans that they are considered to be one of the major breakthroughs of contemporary medicine. Unfortunately, growing resistance is gradually rendering them ineffective, with the threat of catastrophic public health consequences should this trend continue much longer. The few new antibiotics being brought to market essentially consist of so-called me-too drugs – meaning that they are derived from existing classes of antibiotics.
Researchers from Inserm and Université de Rennes recently identified a new bacterial toxin which they transformed into potent antibiotics active against various bacteria responsible for human infections, whether Gram-positive or negative.
“It all started with a fundamental discovery made in 2011”, explains Brice Felden, Director of the Bacterial Regulatory RNAs and Medicine laboratory in Rennes. “We realized that a toxin produced by Staphylococcus aureus whose role is to facilitate infection is also capable of killing other bacteria present in our body. What we had identified was a molecule with dual toxic and antibiotic properties. We thought that if we could separate these activities, we would be able to create a new antibiotic non-toxic to the body. A challenge that we accepted!”.
In conjunction with the team of ISCR chemist Michèle Baudy Floc’h, a new family of so-called peptidomimetics was synthesized. As their name suggests, these peptides are inspired by the existing natural bacterial peptides but have been shortened and modified. Out of the twenty molecules created, two proved effective against resistant Staphylococcus aureus and Pseudomonas aeruginosa in mouse models of severe sepsis or skin infection. In addition, no toxicity to the other cells and organs, whether in animals or human cells was observed. These new compounds are well tolerated at their active doses – and even beyond – and are devoid of the renal toxicity issues often encountered with this type of compound. “We tested them at doses 10 to 50 times higher than the effective dose without seeing toxicity” specifies Felden, adding that “the participation and imagination of the team and our chemist colleagues was needed to devise the most active molecules possible”.
Little resistance observed under experimental conditions
Important to note was that the bacteria that the researchers had left in contact for several days in the animals with these antibiotics showed no signs of resistance. In order to go further, the researchers created conditions favorable to the development of resistance in vitro and in vivo – with nothing happening. However, caution is still required here given the short experimental time periods (up to 15 days).
The antibacterial activity of these peptidomimetics is partially due to the capacity of its non-natural amino acids to reinforce the association of these compounds with the membranes of the infectious bacteria. This strong binding leads to membrane permeability and the death of the bacteria. “We think these new molecules represent promising candidates for the development of new antibiotics that can provide alternative treatments to antimicrobial resistance.”
The next step involves launching phase I clinical trials in humans. The patent has been licensed and a start-up created.
Why do eczema patches caused by skin contact with an allergen reappear in the same areas despite having had time to heal? This is what an International Center for Infectiology Research team with members from Inserm, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon and CNRS were keen to find out. The researchers discovered that not only do the allergens persist in the skin for several weeks but also that they are not alone in doing so. Indeed, immune cells – known as tissue-resident memory T cells – proliferate at the lesion sites and remain there for long periods, reactivating the onset of eczema patches in the event of re-exposure to the allergen. This research, published inThe Journal of Allergy and Clinical Immunology, opens up new perspectives when it comes to understanding the mechanism and treatment of allergic contact dermatitis.
Allergic contact dermatitis (ACD) (a type of eczema) is a skin reaction triggered by exposure to allergens. The resulting inflammation of the upper layers of the skin can last for several days, persists for as long as the area remains in contact with the allergen in question and can even become chronic. It manifests as localized skin rashes (eczema patches) accompanied by itching and burning, and reappears if the healed areas are re-exposed to the allergen.
Tissue-resident memory T (TRM) cells are immune cells that persist in peripheral tissues, such as the skin, over the long term. They contribute to the secondary immune response that – while especially rapid and effective against pathogens encountered previously – can cause the exacerbation of some inflammatory diseases, such as ACD. In the eczema patches caused by ACD, a build-up of TRMs is indeed observed.
A research team at the International Center for Infectiology Research (CIRI), with members from Inserm, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon and CNRS, studied the contribution of TRMs to the severity and chronicity of ACD in mice. They observed that the TRMs proliferate locally in the areas of the skin in contact with the allergen.
When ACD-induced inflammation resorbs, TRMs gradually accumulate in the epidermis and persist there for several weeks.
If the eczema lesion is re-exposed to the allergen – even if it appears to be healed – these cells are then responsible for the appearance of eczema patches.
The team, in its desire to find out why TRMs persist in the skin, observed that the allergens can remain in the epidermis for much longer than was previously thought (at least one month).
This persistence of the allergens in the healed areas could explain the stimulation over several weeks of the proliferation of the TRMs that are specific to them, as well as their persistence in the eczema lesion.
Finally, the researchers observed that the reactivation of the TRMs responsible for the eczema patches was subject to a retro-control enabled by a specific set of inhibitory receptors carried by the TRMs. When re-exposed to a low dose of allergen, these receptors are activated and suppress the activity of the TRMs, thereby preventing an excessive immune reaction.
This research helps to elucidate the role played by the TRMs in the local reappearance of eczema patches. It also shows that the development of therapeutic strategies to prevent the local reactivation of the TRMs through their inhibitory receptors should open up new perspectives in the treatment of ACD.
Today, around 1 in 8 couples seek help because they are struggling to conceive. This is probably linked to the fact that couples are starting families later in life than before, or because they are setting aside the taboos linked to infertility and are more willing to seek help. Infertility has therefore become a public health problem, and the scientific community is rallying in response.
Where are we with research into this area, which lies at the heart of current societal problems? What are the prospects for the transfer of such research into clinical practice? Fertility research covers many different areas. The aim of this press kit is not to tackle them exhaustively, but to highlight the sectors in which research is making progress.
When research makes progress, everyone’s health benefits.
Research into combating infertility
The term infertility is used when a couple are unable to conceive a child naturally after 12 months of trying. This term covers cases of total sterility, where there is no hope of natural conception, and subfertility, the majority of cases, in which couples have a reduced – but not zero – chance of achieving a pregnancy.
Cases of infertility are divided into 4 categories based on their cause:
– 30% are female-related;
– 30% are male-related. In men, azoospermia and oligospermia are the two leading causes of infertility identified to date;
– 30% are combined, meaning that they are caused by reduced fertility in both partners;
– 10% are unexplained.
In women, with the exception of mechanical causes affecting the fallopian tubes – when they are impaired or blocked (usually following an infection) – or uterus, endometriosis and abnormal ovulation are the most common causes of infertility.
Causes of abnormal ovulation include polycystic ovary syndrome (which affects around 10% of women around the world), hyperprolactinemia, and primary ovarian insufficiency (which may also be a side effect of chemotherapy).
Current research seeks to both improve understanding of the causes of infertility, and also to study new therapies or management methods that aim to increase the chances of conception.
Improving understanding of the causes
The genetic approach
Many researchers are studying the genetic causes of ovarian insufficiency. Several fertility problems are caused by certain genes not working, or not working properly. One rapidly growing area of research, due in particular to the improvement in high-throughput screening methods, is the study of genetic variants.
The Inserm laboratory led by Nadine Binart, for example, is working on primary ovarian insufficiency (POI), which is characterized by the inability of ovarian follicles to mature or by diminished ovarian reserve. Based on DNA analysis of women with POI, researchers are working to isolate the genes that are involved or altered in their genetic make-up. This approach is helping to improve understanding of the disease, but does not make it possible to provide specific treatment to these women, as sterility becomes definitive once there are no more eggs left in their ovaries. Preventive management can however be introduced if the genetic abnormality is found before the ovarian reserve is entirely depleted – for example, during family testing. This is the role of clinical research, which makes it possible to lessen the impact of these diseases when mutations are identified in affected families, to inform young patients about the risk of losing their eggs over time, and to introduce fertility preservation methods if appropriate.
The hormonal approach: the example of kisspeptin and prolactin
It is well-established that breastfeeding results in increased secretion of prolactin (PRL) by the pituitary gland, inhibiting a woman’s ability to ovulate. This prevents the onset of a new pregnancy. Some diseases also lead to an increase in PRL, including tumors of the pituitary gland from which this hormone is secreted. These cases of hyperprolactinemia, which result in period problems and infertility, are a leading cause of anovulation. In 2011, the Inserm team led by Jacques Young and Nadine Binart revealed the underlying mechanism that blocks ovarian function. Using a mouse model of the disease, the researchers showed that PRL inhibits secretion of a neurohormone called kisspeptin, which is the starting point for the entire hormone cascade responsible for ovarian cyclicity. In a mouse model, administration of kisspeptin made it possible to restore ovarian cyclicity despite the hyperprolactinemia.
This pathophysiological discovery explains the link between infertility and hyperprolactinemia for the first time, and points the way to developing innovative therapies. The basic concept has recently been validated in women,[1] which will make it possible to offer a therapeutic alternative for patients who do not respond to the drugs currently used.
1.2. Preserving fertility: areas of research and latest findings
Specialist “oncofertility” consultations have developed extensively in recent years and should now be an integral part of the care pathway for all young female patients with cancer. Several “fertility preservation” techniques designed to cryopreserve gametes, or preserve reproductive capacity, are now available, and others are currently in development. In France, since 1994, these methods have been included in various pieces of bioethics legislation. Article L.2141 11, modified by law 2011-814 of July 7, 2011, states that “All persons whose fertility is likely to be impaired by their medical care, or whose fertility risks being prematurely impaired, may have their gametes or reproductive tissue collected and preserved with a view to their later use of assisted reproductive technology, or with a view to preserving and restoring their fertility.” Fertility preservation methods are also included in the 2014-2019 Cancer Plan, which stipulates that “all patients must have access to cancer treatments, and innovative treatments in particular.”
Improving gamete preservation
Several techniques for cryopreserving female gametes are currently available. The standard method involves freezing mature eggs or embryos obtained from these eggs. It is not however suitable for prepubescent girls, who need to begin treatment urgently, and can also present problems in patients with hormone-sensitive cancers. Therefore, other techniques, although still considered experimental, may be offered in these situations.
Improving the available methods and developing new strategies is currently a major focus for oncofertility. This is one of the areas of research on which the Inserm team led by Nadine Binard and Charlotte Sonigo is working, in collaboration with Prof. Michael Grynberg.
Using anti-Müllerian hormone
Chemotherapy reduces fertility through a direct toxic effect on the ovaries. Cyclophosphamide, which is commonly used in cancer treatment, causes massive destruction of the germ cells contained in the ovarian follicles. In a mouse model, researchers have recently shown that treatment with anti-Müllerian hormone, which is normally secreted by the ovaries, can limit reduction of follicular reserve during chemotherapy. Use of anti-Müllerian hormone is therefore a promising approach to fertility preservation.
1.3. The role of new technologies: Using artificial intelligence in reproductive research
The store of germ cells contained in the follicles constitutes the ovarian reserve. Assessing the quantity of these germ cells is a common way of providing information on ovarian physiology and of measuring the impact of the environment on the ovaries. The standard method used in mice is time-consuming and tedious. In conjunction with a company specializing in artificial intelligence, Inserm researchers have recently developed an automated artificial intelligence method for follicle counting that uses a deep learning approach.[2] This new tool will be made available to the fertility research scientific community, saving a great deal of time and enabling better reproducibility of data.
Research into combating endometriosis
Endometriosis is a complex disorder characterized by chronic inflammation due to the presence of tissue resembling the uterine lining outside the uterus. This “ectopic uterus” continues to respond to ovarian hormones, which in some women can cause severe pain and sometimes infertility. In response to increased visibility of the disease in the media, notably due to the work of patient organizations, the French health minister has announced an action plan to improve management of endometriosis. In terms of research, there has been a surge in studies of endometriosis over the last 5 years. Around 1,200 articles per year are being produced by researchers around the world, helping to advance understanding of this disorder.
An estimated 1 in 10 women have some form of endometriosis.
The locations of endometriosis lesions vary.
Endometrial cell reflux during periods occurs in 90% of women, but only 10% of them develop disease.
The disease is typically described as having 4 stages, based on the extent and depth of lesions; however, there is no correlation between disease symptoms and severity.
There are 3 forms of endometriosis: superficial peritoneal endometriosis, ovarian endometriosis (or endometrial cyst, or endometrioma), and deep endometriosis.
2.1. Improving understanding of the causes
The epidemiological approach
At present, little is known about the causes of endometriosis, its natural history, and the factors affecting its progression. Epidemiological research plays a crucial role in advancing knowledge in this area. There are only a few large epidemiological cohorts around the world in which these aspects can be explored. The largest cohort for exploring endometriosis risk factors is currently a cohort of 116,430 American female nurses who were between 25 and 42 years old in 1989. The risk factors identified in the literature and confirmed in this cohort include: low birth weight, early menstruation, low body mass index, and short menstrual cycles (under 24 days).[3] However, beyond these factors, little information is available on the causes of the disease, and its natural history is largely unknown. The following table is based on a review of the literature published in August 2018:
*The positive association between smoking and reduced risk of endometriosis may be explained by the antiestrogenic effect of tobacco. This would confirm the therapeutic interest of estrogen blockers, which are available in drug form: far more suitable than cigarettes, whose harmful effects have been widely documented.
In a bid to improve understanding of this disease, several epidemiological studies are being launched in France by the team led by Marina Kvaskoff, Inserm epidemiologist and researcher. These include a recently formed patient cohort dedicated to the study of endometriosis: the ComPaRe-Endometriosis cohort. The study team’s objective is to have enough women in the cohort to obtain robust findings in relation to the many questions that are still unanswered about this disease. In less than 6 months, over 8,000 women have already taken part in the study. The team aims to recruit 15,000 to 20,000 participants, and a broad call for participation has gone out to women with endometriosis or adenomyosis (a form of endometriosis limited to the muscle wall of the uterus) to help speed up research into these disorders simply by completing online questionnaires about their experience of the disease (https://compare.aphp.fr/). The study initially looks to explore the natural history of the disease (change in the symptoms and characteristics of the disease over time), and to identify the factors that determine its progression and result in better response to treatment. This research will also make it possible to describe the circumstances of diagnosis and the patient care pathway, and to assess the impact of the disease on patients’ daily lives.
Endometriosis is also being studied within large French cohorts, such as the CONSTANCES cohort, a prospective study of 200,000 men and women (105,000 women) representative of the French population. Marina Kvaskoff’s team has developed an epidemiological research study to determine the prevalence and incidence of the disease in France, and to explore its risk factors within this cohort. Other studies are currently in development and will be conducted in other cohorts in due course.
The environmental approach
Several epidemiological studies have explored the associations between organochlorine chemicals (solvents, pesticides, insecticides, fungicides, etc.) and endometriosis, but their results have been inconsistent. A French meta-analysis of 17 studies[4] published in February 2019 attempted to draw more robust findings. The risk of developing endometriosis was 1.65 times higher in women exposed to dioxins, 1.70 times higher for those exposed to polychlorinated biphenyls (PCB), and 1.23 times higher for organochlorine pesticides. Although statistically significant, these estimates should be considered with caution due to the significant heterogeneity of the studies and the small estimated effect size. The level of evidence was judged to be “moderate” with a serious risk of bias, supporting the need to conduct further well-designed epidemiological research in order to fill the persistent data gaps.
Using the genetic and epigenetic approach for early detection
Detecting endometriosis in the early stages, before patients experience symptoms, would make it possible to improve patient care. Although the heritability of endometriosis has been estimated at 50%, it is highly complex and clearly highly polygenic. Numerous candidate genes have been studied from this perspective in analyses of disease predisposition. Initial results have shown that there is no gene for endometriosis, but that the existence of genetic variants characteristic of the disease could enable it to be diagnosed and to improve patient care. In 2017, efforts by the international community made it possible to identify a total of 14 variants (located on the genes WNT4, GREB1, ETAA1, IL1A, KDR, ID4, CDKN2B-AS1, VEZT, FN1, CCDC170, SYNE1, FSHB, and in the chromosomal regions 7p15.2 and 7p12.3). These 14 genes are involved in proliferation and the cell cycle, adhesion and the extracellular matrix, andinflammation, which makes sense in relation to endometriosis. However, each of the variants identified explain only a small part of the genetic variation in endometriosis. In future, the combination of high-risk alleles in a patient might provide a probability of being affected that could be used to diagnose patients and categorize them based on endometriosis type and severity.
The existence of specific epigenetic markers for endometriosis could also theoretically be used for early detection, with endometrial cells presenting specific epigenetic abnormalities that modify expression of the main transcription factors. However, it is not known how the interactions between the defective epigenomic cells and mutated epithelial cell genes contribute to the pathogenesis of endometriosis.
The microRNA approach
The full complexity of endometriosis cannot however be understood through genetics alone. Genes only influence phenotype through their expression. This expression is regulated by epigenetic molecular mechanisms. As such, most research focuses on studying the microRNA that could be “markers” for the disease. Several have been identified in patients’ plasma thus far, but with very poor reproducibility from one research team to the next. For example, a study published in 2013[5] identified just four miRNA (miR-199a, miR122, miR145*, and miR-542-3p) as enough to categorize patients, with very few errors. Confirmation of this article’s findings in independent cohorts has however been slow. One possible explanation for this is the fact that extraction of circulating RNA remains very heterogeneous from one study to the next, perhaps due to the technical tools used in extraction. In future, new, more comprehensive approaches could provide more consistent results.
The cellular approach: oxidative stress
Several studies have shown increased oxidative stress in the serum of women with endometriosis. Oxidative stress is a highly general mechanism that induces and is caused by inflammation. It would seem logical to find changes linked to oxidative stress in the context of a painful disease like endometriosis. In mouse models, treatment with antioxidants (N-acetylcysteine) has been seen to reduce endometrial lesions.
Research led by a team from the Institut Cochin has also identified several genes linked to glutathione metabolism within the gene cascades that are deregulated in endometriosis lesions. Glutathione is a peptide that plays a key role in detoxification of hydrogen peroxide, a central molecule in oxidative stress. Down-regulation, particularly of the GCLM and GCLC genes crucial to glutathione synthesis, could explain increased oxidative stress in endometriosis lesions.
The dysfunctional immune system: a possible approach?
The survival of endometrial cells outside the uterus could be linked to poor function of the immune system causing chronic local inflammation. Studies have shown an increase in some immune cells around endometriosis lesions.
2.2. Treatment: areas of research and latest findings
Changing diagnostic methods: phasing out surgery
Before considering treatment, the first stage is to reduce the diagnosis time for endometriosis, which is currently around 7 to 10 years after onset of the initial symptoms. With this in mind, doctors and researchers are working to develop a diagnostic score, based on a dozen questions, from which doctors will be able to provide a diagnosis with 85-90% reliability. This score may be accompanied if necessary by imaging, which can inform endometriosis diagnosis if carried out and interpreted by trained medical personnel.
Doctors and researchers agree that diagnostic surgery is contraindicated for endometriosis.
The 3 pillars of treatment
Drug therapy, surgery, and assisted reproductive technology (ART) are currently the only 3 methods for treating the symptoms of endometriosis and its potential impact on fertility. In the absence of new treatments, the key is to understand the role played by each component in this therapeutic arsenal, so that they can be used effectively.
Drug therapy is based on blocking ovarian function to bring about artificial menopause via continuous administration of contraceptives. These therapies (the combined pill, estrogen pill, or GnRH agonists) must be personalized and adapted to the needs of the patient. These therapies should be prescribed as the first-line treatment for women who are not seeking to become pregnant, in order to reduce the pain caused by the disease.
For patients who want to conceive, ART and surgery may be considered. ART should be used routinely before all surgical procedures in order to maximize the chance of conceiving a child for couples who want to do so. Surgery must not be used in women who do not want to conceive in whom drug therapy is effective. Endometriosis surgery can be highly invasive and debilitating (removing some parts of the colon, with a high risk of ovarian reserve depletion if ovarian cysts are removed, etc.), and does not prevent the disease from recurring, as it does not treat the cause. Doctors and researchers also agree that women who undergo surgery at a young age have a high risk of their endometriosis lesions recurring, and encounter further difficulties if they decide they want to become pregnant.
All efforts must therefore be made to move away from using surgery as the standard treatment for endometriosis, as has too often been the case in the past.
Some forms of endometriosis – particularly those affecting the ovaries – are now an indication for providing women with access to various fertility preservation techniques.
The role of new technologies: the example of high-intensity focused ultrasound
In Lyon, teams of research clinicians led by Prof. Gil Dubernard (Hospices Civils de Lyon and Inserm unit 1032 LabTAU) have developed an ultrasound-based treatment for bowel endometriosis. When endometriosis infiltrates the rectal wall, it causes debilitating rectal pain that may affect quality of life. After failure of medical treatment, a surgical procedure is often proposed that consists of removing all or part of the rectum and sometimes requires a temporary colostomy (artificial anus).
A phase I clinical trial carried out in 11 patients in 2017 demonstrated that high-intensity focused ultrasound may be a useful alternative to surgery. An ultrasound probe inserted into the rectal passage is able to “desensitize” the lesions within a few minutes. A follow-up trial of 12 patients seeking to confirm these initial results was completed on April 1, 2019. Data analysis is ongoing and will be available shortly.
Meanwhile, in collaboration with the company EDAP TMS (the clinical trial sponsor), the therapeutic ultrasound Inserm laboratory led by Cyril Lafon, LabTAU (Université Claude Bernard Lyon 1/Inserm), is working on optimizing the conditions of ultrasound delivery (insonification) and improving the ergonomics of the probe in order to increase the number of patients eligible for this new treatment.
It is highly likely that this innovative therapy will replace many of the rectal surgeries carried out in this functional disorder that resolves upon menopause.
Millar RP, Sonigo C, Anderson RA, George J, Maione L, Brailly-Tabard S, Chanson P, Binart N, Young J.
[2]Sonigo C, Jankowski S, Yoo O, Trassard O, Bousquet N, Grynberg M, Beau I, Binart N. High-throughput ovarian follicle counting by an innovative deep learning approach. Sci Rep. 2018 Sep 10;8(1):13499. doi: 10.1038/s41598-018-31883-8.
Are there ways to improve the efficacy of flu vaccines? Are there markers that could, at the time of vaccination, predict the quality of the immune response several weeks down the line? Thanks to the work of Inserm Research Director Béhazine Combadière’s team at Unit 1135 “Center for Immunology and Infectious Diseases”, the answer to these two major questions is “yes”.
Their findings were published on April 8, 2019 in JCI.
While flu continues to claim lives every year[1], a vaccine exists to protect the populations. And while this vaccine is the best means of preventing the disease and reducing the risk of severe complications and death, it is not 100 % effective. This is due to the fact that its formulation is determined each year by the WHO several months before the epidemic peak and that it is based only on the probability that such and such a flu strain will be in circulation during the coming winter. Flu viruses are highly unpredictable, and the vaccine formulation must change from one year to the next. However, given that 5 to 6 months are needed to develop it, the vaccine does not always target all of the circulating strains.
The team of Béhazine Combadière, Inserm Research Director at Unit 1135 “Center for Immunology and Infectious Diseases”, has been working for years on the impact of vaccine administration routes on the quality of immune responses. The vaccines are usually administered by the muscular route and are effective in inducing humoral responses (production of antibodies), whereas the other immune response component, the cytotoxic response (production of T cells that directly destroy the infected cells) is poorly-induced by this route of administration.
The team studied the utility of administration via the skin – either by intradermal injection or transcutaneous application (via the hair follicles) – in inducing cytotoxic responses during flu vaccination. This involved conducting a phase I/II clinical trial on 60 people between 18 and 45 years of age in collaboration with the Vaccinology CIC led by Dr. Odile Launay. The study, published in JCI, demonstrates that in some subjects the cutaneous routes induce a cytotoxic response following flu vaccination. “This finding argues in favor of considering this vaccine injection route given that it triggers an immune reaction additional to that obtained with a standard vaccination. These cytotoxic responses would be particularly protective in elderly people following flu vaccination. ” explains Combadière.
In addition to these findings, the team brings new elements to the table concerning the specific imprints left by these injection routes in the body. For this, the researchers studied the gene signature of innate immunity, i.e. the expression of the messenger RNA of the genes in the blood the day after vaccination for each administration route. “Since previous findings showed that each administration route had its own innate response, we expected to have three signatures of innate immunity corresponding to the three administration routes, yet our findings only show two. These two signatures are correlated with the immune response of the individual: those that respond to the vaccine by increasing their humoral response and those that respond by inducing a cytotoxic response. “
Among these signatures, a certain number of biomarkers expressed the day after vaccination are thought to be predictive of the quality of immune response three weeks later. “However, these latest findings require other studies to validate the utility of these biomarkers and their future use”, conclude the researchers.
[1] For the winter of 2018-2019, over 2,000 deaths were attributed to the flu according to French Public Health Agency data.
Researchers are now able to edit the genome with precision using the “gene editing scissors” of CRISPR-Cas9, which is a highly promising tool for gene therapy.The technical challenge now is to get this tool into the genome of certain cells.With this in mind, a joint team from Inserm, the CNRS, the Université Claude Bernard Lyon 1, and the École Normale Supérieure de Lyon, working within the International Center for Infectiology Research (CIRI), have developed capsules that allow CRISPR-Cas9 to reach the target DNA: Nanoblades.Described in a recent article in Nature Communications, they open up avenues of research for genome editing in human stem cells.
Since 2012, the scientific community has had access to a revolutionary method for highly precise genome “surgery”: the CRISPR-Cas9 system. These molecular scissors are able to cut DNA at a precise place in a wide variety of cell types. The technique therefore offers significant prospects for research and human health. However, getting these “gene editing scissors” to their target—including the genome of certain stem cells—remains technically challenging.
Tackling this problem has been the focus for research teams from Inserm, the CNRS, the Université Claude Bernard Lyon 1, and the École Normale Supérieure de Lyon, who have developed Nanoblades,[1] particles that enable CRISPR-Cas9 to be delivered into numerous different cells, including human cells.
The scientists had the idea of encapsulating the CRISPR-Cas9 system in structures that strongly resemble viruses as a way to deliver it into target cells, by fusing with the target cell membrane.
In developing Nanoblades, researchers exploited the properties of the retroviral Gag protein, which is able to produce viral particles that have no genome and are therefore non-infectious. The research team fused the Gag protein from a mouse retrovirus with the Cas9 protein—the scissor component of the CRISPR system. This new “fusion” protein is what makes Nanoblades original.
As a result, and unlike classic genome modification techniques, Nanoblades encapsulate a CRISPR/Cas9 complex that is immediately functional rather than delivering a nucleic acid coding for the CRISPR-Cas9 system in the treated cells. “The action of CRISPR-Cas9 on the cells is therefore temporary.It is also more precise and preserves the non-target regions of the genome, which is a particularly important feature in the context of therapeutic applications”, explain the authors.
Legend:
Représentation schématique d’une particule Nanoblades livrant CRISPR CAS9
Schematic diagram of a Nanoblades particle delivering CRISPR-Cas9
La protéine GAG tapissant l’intérieur des particules rétrovirales
The Gag protein internally lining the retroviral particles
La protéine CAS9, ciseau effecteur du système CRISPR, pouvant cliver l’ADN
The Cas9 protein, the scissor component of the CRISPR system, is able to cleave DNA
L’ARN guide, qui va placer CAS9 sur la région ADN cible. Il a une affinité naturelle pour CAS9
The RNA guides Cas9, then positions it at the target DNA region. It has a natural affinity for Cas9
Les deux enveloppes virales conférant un tropisme large aux particules
The two viral envelopes give the particles a broad tropism
La bicouche lipidique qui entoure la particule
The lipid bilayer surrounding the particle
Finally, researchers used an original combination of two viral envelope proteins on the surface of Nanoblades to enable them to enter a wide range of target cells.
The scientists have demonstrated the efficacy of Nanoblades in vivo, in mouse embryos, for a broad range of applications and in a broad panel of target cells for which other methods have had limited success. “Nanoblades have turned out to be particularly effective for editing the genome of human stem cells. These cells are of major therapeutic interest (particularly in tissue regeneration), but remain difficult to manipulate using standard methods”, explain the study authors.
[1] Nanoblades have been tested in mice and were patented by Inserm Transfert in 2016.
1.2 million people in the world are co-infected by Mycobacterium tuberculosis, the bacteria which causes tuberculosis, and AIDS (HIV-1). This combination is deadly: it makes patient diagnosis and treatment difficult, and increases the pathogenicity of these two infectious agents. An international team led by researchers at the CNRS and Inserm have revealed that in the presence of tuberculosis, HIV-1 moves from one cell to the next via nanotubes which form between macrophages, drastically increasing the percentage of infected cells. These findings appear in the 26 March 2019 edition of Cell Reports.
Researchers at the Institute of Pharmacology and Structural biology (CNRS/Université Toulouse III – Paul Sabatier) and the IM-TB/HIV international laboratory, a consortium between the CNRS and the National Scientific and Technical Research Council (Conicet) (Argentina), together with the Center for Pathophysiology of Toulouse Purpan (CNRS/INSERM/Université Toulouse III – Paul Sabatier), have shown that macrophages – which act as host cells for tuberculosis and HIV-1, join to form nanotubes when exposed to Interleukin-10, a molecule secreted in the presence of tuberculosis. The abundance of these specific M(IL-10) macrophages in the lungs is correlated with the severity of the disease. HIV-1 particles travel through these tunnel-like nanotubes to infect neighbouring cells and multiply. Using different approaches to inhibit their formation, scientists successfully reduced viral transfer between macrophages, leading to a drop in HIV-1 production.
In a case of severe TB, the development of nanotubes between macrophages accelerates, increasing the spread of the AIDS virus and viral production as a result. Because the presence of this specific type of macrophage can be measured, diagnosis and treatment of patients suffering from both illnesses could be made easier. This research paves the way to new therapeutic approaches aimed at limiting viral load increases in tuberculosis patients.
Les microbes colonisent l’ensemble des surfaces de notre corps et participent au bon équilibre de notre système immunitaire. Chez les nouveau-nés, le microbiote intestinal est d’abord formaté par les composants du lait maternel. Lors de la diversification alimentaire, il se développe et de nombreuses bactéries prolifèrent. Des chercheurs de l’Institut Pasteur et de l’Inserm montrent chez la souris qu’une réponse immunitaire importante se produit lors de l’introduction de nourriture solide et du développement du microbiote. Mais surtout, ils ont montré que cette réaction immunitaire était essentielle car elle participe à l’éducation du système immunitaire, et permet, à l’âge adulte, une faible susceptibilité aux maladies inflammatoires (allergies, colites, maladies auto-immunes, cancer). Ces résultats ont été publiés dans la revue Immunity, le 19 mars 2019.
L’introduction d’une hygiène de qualité au milieu du XIXe a drastiquement fait diminuer la mortalité due aux maladies causées par des micro-organismes. Dans nos sociétés industrielles actuelles, l’hypothèse hygiéniste affirme désormais que la réduction de l’exposition en bas âge aux microbes entraînerait une augmentation de la sensibilité aux maladies allergiques ou auto-immunes. De précédentes études ont montré que la perturbation du microbiote, notamment par l’exposition aux antibiotiques, peut se traduire par des réponses allergiques[1].
Chez le nouveau-né, la constitution du microbiote se fait lors de l’accouchement par l’acquisition des bactéries de la mère mais aussi, grâce à la composition du lait maternel. Il est alors majoritairement composé de bifidobacteria et de lactobacilles. A l’introduction de nouveaux aliments dans le régime, le microbiote prolifère et le nombre de bactéries augmente de 10 à 100 fois. Des chercheurs (Ziad Al Nabhani et ses collègues) de l’unité Microenvironnement et Immunité (Institut Pasteur/Inserm) dirigée par Gérard Eberl, ont découvert chez la souris que ce phénomène était accompagné d’une réponse immunitaire intense. « Nous avons pu montrer que ce mécanisme se produisait dans une fenêtre de temps très spécifique : entre 2 et 4 semaines chez la souris ce qui correspondrait à 3 et 6 mois chez l’homme » explique Gérard Eberl, principal auteur de l’étude.
« Nous avons ensuite supposé que l’existence d’une fenêtre de temps déterminée indique que la réponse immunitaire est programmée dans le temps et possède de ce fait une fonction unique dans le développement du système immunitaire » poursuit Gérard Eberl. En effet, les chercheurs ont pu démontrer qu’en traitant les souris par antibiotiques sur la fenêtre critique de la réponse immunitaire, elles étaient par la suite plus sujettes à développer certaines maladies inflammatoires : les allergies intestinales, le cancer colorectal et les colites. Ainsi, le microbiote une fois détruit par les antibiotiques, on constate que la réaction immunitaire ne se produit pas. « C’est ce que l’on appelle l’empreinte pathogénique » explique Gérard Eberl, « c’est-à-dire que des évènements se produisant dans la prime enfance déterminent une future susceptibilité aux maladies inflammatoires ».
Les chercheurs ont également pu mettre en évidence la présence des cellules spécifiques au moment de cette réaction et qui participent au bon fonctionnement des réponses immunitaires : les cellules T régulatrices (Tregs), des modulateurs clés sans lesquelles les réponses immunitaires sont exacerbées, entraînant par la suite des maladies inflammatoires.
L’ensemble de ces données montre l’importance d’une exposition précoce au microbiote, ciblée dans le temps, pour le développement d’un système immunitaire équilibré. « Nous aimerions maintenant valider ces résultats sur l’influence du microbiote au moment de la diversification alimentaire sur l’apparition d’autres types de pathologies comme les maladies neurodégénératives par exemple » conclut Gérard Eberl.
Ces travaux ont été financés par l’Institut Pasteur et l’Inserm, mais également par l’Association François Aupetit, la Crohn’s Colitis Foundation of America, l’European Crohn’s and Colitis Organisation, la Fondation pour la Recherche Médicale, Janssen, et la Fondation Kenneth Rainin.
Ces travaux ont été menés dans le cadre du programme transversal “Microbiote” mis en place en 2016 dans le cadre du plan stratégique de l’Inserm.
A mechanism of tolerance towards intestinal flora is thought to be implicated in the onset of a rare familial autoinflammatory disease induced by cold temperatures. This is the finding of researchers from the Center for Infection and Immunity of Lille (Inserm/Université de Lille/CNRS/University Hospital Lille/Institut Pasteur de Lille), the Pathophysiology of Pediatric Genetic Diseases laboratory (Inserm/Sorbonne Université) and the Department of Immunology at the University of Hohenheim. Their research, published in Nature Communications, reveals the implication in its onset of an exacerbated inflammatory response against the gut flora, making for a more effective immune response against certain pathogens. Findings which open up new avenues when it comes to treating patients.
Familial cold autoinflammatory syndrome (or familial cold urticaria) is characterized by episodes of fever triggered by cold temperatures, accompanied by hives and gastrointestinal and joint pain. The patients – with some twenty cases identified to date – carry an NLRP12 gene mutation that is inherited in an autosomal dominant manner (the presence of a single mutated allele is enough to bring on the disease). Until now, the pathophysiological mechanisms behind the disease were unknown.
A team led by Mathias Chamaillard, Inserm researcher at the Center for Infection and Immunity of Lille (Inserm/Université de Lille/CNRS/University Hospital Lille/Institut Pasteur de Lille) along with his coworkers at the Pathophysiology of Pediatric Genetic Diseases laboratory (Inserm/Sorbonne Université) and Department of Immunology at the University of Hohenheim, sought to elucidate the development of this syndrome through human and mouse studies.
The researchers saw that while inactivating the NLRP12 gene in mice triggered inflammation in the gut, it made it resistant to certain pathogenic bacteria, suggesting that NLRP12 could play a key role in immune tolerance towards intestinal flora.
However, they observed that another molecule, NOD2, also played a role in intestinal immunity by promoting the defense against these same bacterial pathogens.
In addition, a NOD2 gene mutation predisposes to Crohn’s disease, which presents disturbing similarities with the syndrome being studied here: intestinal pain and a higher prevalence in cold countries. Finally, the researchers observed the existence of a physical interaction between the proteins NOD2 and NLRP12.
Reduced tolerance of intestinal flora bacteria
In individuals with familial cold autoinflammatory syndrome, the production of protein NLRP12 is reduced. When reproduced in mice, this phenomenon modifies NOD2 activity and reduces tolerance of commensal bacteria with an intensified recruitment of inflammatory cells in the digestive tract. However, the efficacy of pathogen elimination is improved. In other words, under normal conditions, NLRP12 suppresses NOD2 activity and improves tolerance of intestinal bacteria. These findings suggest that an inhibitor of the NOD2 pathway could attenuate these patients’ symptoms.
The reduced tolerance in subjects with familial cold autoinflammatory syndrome generates chronic inflammation which could be the reason behind their intestinal pain. But why does the cold trigger additional symptoms outside the digestive tract? The researchers suspect increased intestinal permeability in the presence of low temperatures, a phenomenon which would be of no consequence in healthy subjects. However, in those with the disease, numerous pro-inflammatory molecules and bacterial debris could pass into the blood en masse, with secondary local inflammation thereby partially explaining the other symptoms, such as fever, headache and joint pain. A new research avenue that Mathias Chamaillard and his colleagues are now exploring, with the help of mice.
Successful In Utero Hematopoietic Stem Cell Transplantation in a Fetus with Severe Combined Immunodeficiency
Teams from the Biological Therapy Department and Pediatric Immunohematology Unit at Necker-Enfants Malades Hospital AP-HP, the Fetal Medicine Department at Trousseau Hospital AP-HP, Inserm, the Imagine Institute, Université Paris Descartes and Sorbonne Université have achieved the in utero transplantation of hematopoietic stem cells in a fetus with X-linked severe combined immunodeficiency.
The transplantation was performed in July 2015 in response to a risk of maternofetal transmission of a parasite, Toxoplasma gondii, whose consequences on fetal brain development can be serious and more particularly so here in a fetus with no T-cells.
The graft taken from the patient’s sister, whose immune system was compatible, comprised a mixture of hematopoietic stem cells (able to restore normal immune system development over the long-term) and mature T-cells (able to rapidly defend the fetus from infection with the parasite).
The graft was prepared in the Cell and Gene Therapy Laboratory of the Biological Therapy Department at Necker-Enfants Malades Hospital AP-HP and then infused into the fetus via the umbilical vein under ultrasound guidance in the Fetal Medicine Department at Trousseau Hospital AP-HP.
The transplantation and remainder of the pregnancy were uneventful, with the baby born at full term in the Maternity Unit at Trousseau with a functional immune system. The child is now over three years old and in good health, having needed no hospitalizations or therapeutic interventions since birth.
With such a procedure, the fetus was able to heal during pregnancy without the practical constraints or emotional burden inherent in standard hematopoietic stem cell transplantation, which involves a lengthy hospital stay in a highly protected environment for these very vulnerable young patients.
The indication for in utero transplantation continues to remain very restricted due to its potential risks and the possibility of performing the transplantation very soon after birth.
Nevertheless, the success of this transplantation opens up new therapeutic prospects for fetuses with severe immunodeficiency, where a compatible donor exists and where there is a manifest infectious risk during pregnancy.
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