Escherichia coli and Klebsiella pneumoniae, which are commensals and pathogens for humans and animals, have become increasingly resistant to third-generation cephalosporins. Moreover, in certain countries, they are also resistant to carbapenems and therefore susceptible only to tigecycline and colistin. Resistance is primarily attributed to the production of beta-lactamase genes located on mobile genetic elements, which facilitate their transfer between different species. In some rare cases, Gram-negative rods are resistant to virtually all known antibiotics. The causes are numerous, but the role of the overuse of antibiotics in both humans and animals is essential, as well as the transmission of these bacteria in both the hospital and the community, notably via the food chain, contaminated hands, and between animals and humans. In addition, there are very few new antibiotics in the pipeline, particularly for Gram-negative bacilli. The situation is slightly better for Gram-positive cocci as some potent and novel antibiotics have been made available in recent years. A strong and coordinated international programme is urgently needed. To meet this challenge, 70 internationally recognized experts met for a two-day meeting in June 2011 in Annecy (France) and endorsed a global call to action . Bundles of measures that must be implemented simultaneously and worldwide are presented in this document. In particular, antibiotics, which represent a treasure for humanity, must be protected and considered as a special class of drugs.Resistance to antibiotics has increased dramatically over the past few years and has now reached a level that places future patients in real danger. Microorganisms such as  Escherichia coli, Salmonella spp, Klebsiella spp, Pseudomonas aeruginosa, Acinetobacter spp), which are resistant to almost all currently available antibiotics in some settings. Resistance can be combined with virulence, acting as a potentially deadly duo, as observed in the recent large epidemic outbreak of E. coli 0104:H4 in Europe, notably in Germany [In the golden age of the discovery of antibiotics, these potent "miracle" drugs saved millions of lives. In contrast, we are entering an era where bacterial infections, such as bloodstream infections and ventilator-associated pneumonia, might no longer be successfully treated with antibiotics . We now  Germany . Second, Germany . Several Germany ,4. We card World Healthcare-Associated Infections (HAI) Forum held in June 2011. The meeting gathered together 70 leading world experts and opinion leaders in the domain of antimicrobial resistance (AMR) from 33 countries to discuss the challenges and possible options to tackle the problem. The main objectives were to structure and propose a hierarchy of the various measures reported in the recent literature and to collect information on the experiences of the many countries represented to discuss if some may be transposable to other nations.In this position paper, we summarize important messages and conclusions from the 3Many alarming facts regarding AMR have accumulated, particularly over the last few years.E. coli, Salmonella spp and Shigella spp), Pseudomonas spp, Acinetobacter spp, and Mycobacterium tuberculosis) [-- An increase in global resistance rates in many bacterial species responsible for both community- and healthcare-related infections, e.g., staphylococci, enterococci, gonococci, and enterobacteria (including culosis) ,5-7.E. coli, one of the most common human pathogens, is increasing in Europe, mainly due (but not only) to resistant strains [-- The burden of bacteremias due to  strains .Enterobacteriacae is alarming because these "superbugs" are resistant to most available antibiotics and can disseminate worldwide very rapidly, in particular as a consequence of medical tourism [-- Emergence and dissemination of new mechanisms of resistance, e.g., novel extended-spectrum beta-lactamases (ESBL) and carbapenemases -12. The Staphylococcus aureus (MRSA) rates following the implementation of successful infection control programmes in several high-income countries, such as Belgium, France, United Kingdom (UK), and the USA [-- The rapid increase in the multiresistance of Gram-negative rods stands in contrast to a steady decrease in methicillin-resistant  the USA -15. In sEnterobacteriaceae, despite the fact that these antibiotics should be preserved as our last weapons against multiresistant Gram-negative bacteria.-- Propensity to use last-line therapy  to treat healthcare-related and community-acquired infections triggered by a fear of infections caused by ESBL-producing -- Re-use of old drugs with poor safety and efficacy profiles and uncertain pharmacokinetic/pharmacodynamic characteristics  due to a lack of alternative drugs .-- High morbidity and mortality attributable to multiresistant bacteria in critically ill patients.• In Europe, the European Centre for Disease Prevention and Control (ECDC) reported that 25,000 people die each year from antibiotic-resistant bacteria .• In the USA, MRSA is associated with a staggering 90,000 infections and an estimated 19,000 deaths annually .-- Serious financial consequences of bacterial resistance.• Multidrug-resistant organisms (MDROs) result in massive extra healthcare costs and productivity losses of at least 1.5 billion euros each year in Europe .• In the USA, the annual cost of AMR in hospitals is estimated at more than US$ 20 billion with an even wider clinical impact than human immunodeficiency virus (HIV)-related disease .However, these data on morbidity, mortality, and cost must be considered with caution and may be over- or underestimated because of a lack of in-depth adjustment for risk factors or evaluation of the indirect costs of AMR. Moreover, these figures were calculated before the pandemic with multiresistant Gram-negative rods. Therefore, morbidity, mortality, and the associated economic burden are very likely to increase dramatically during the next decade . FurtherThe most important cause is that there has been a massive overuse of antibiotics worldwide across all ecosystems over the past decades, including humans, animals, aquaculture, and agriculture  facilitate intra- and interspecies transfer.In particular, the spread of antibiotic-resistant Many countries and healthcare facilities still lack effective antibiotic stewardship programmes . AntibioSimultaneously, the antibiotic pipeline is drying up for two reasons  and the ECDC http://www.ecdc.europa.eu, supports and organizes comprehensive and well-validated surveillance networks for AMR and antibiotic consumption, which has allowed to monitor the impact of these interventions [http://www.ema.europa.eu) have jointly organized a meeting and urged pharmaceutical companies to accelerate the search for new antibiotics [http://ecdc.europa.eu/en/activities/diseaseprogrammes/tatfar/pages/index.aspx?MasterPage=1) was established during the Swedish EU presidency to promote a mutual understanding of US and European activities and programmes related to AMR issues [http://ecdc.europa.eu/en/activities/diseaseprogrammes/tatfar/documents/210911_tatfar_report.pdf.Europe, in particular through the European Union (EU) Directorate General for Health and Consumers , Alliance for the Prudent Use of Antibiotics , the European Society for Clinical Microbiology and Infectious Diseases (ESCMID) Study Group on Antibiotic Policies  [http://http//:www.who.int/world-health-day 2011/en/index.html. Hopefully, this will be the starting point for tangible and sustained efforts by WHO through a worldwide campaign.Regional and international networks or alliances have been developed also with various actions proposed, i.e., Action on Antibiotic Resistance  , and the/esgap/) . FinallyS. aureus and Clostridium difficile. Without any doubt, antibiotics remain the cornerstone of antibacterial management and they are still acutely needed for the next generations. It is our duty to protect them.The answer is clearly no! Today, antibiotics are critical to treat bacterial infections. Indeed, there are very few therapeutic compounds, if any, able to modulate the inflammatory burst during severe sepsis . Anti-tohttp://www.who.int/patientsafety/patients_for_patient/en/. The EU has established a strategy against AMR to encourage the prudent use of these agents in human medicine. Several countries have launched national campaigns to educate physicians and patients about antimicrobial misuse and the threat of resistance.The answer is clearly yes, but the examples are few. Education, legislation, and improved diagnosis can reduce antibiotic consumption. Several clinical trials at the community level, have shown that patient education can result in the decrease of the use of antibiotics . The PatThe French campaign, often considered as a model, exceeded expectations with a 23% reduction in the number of antibiotic prescriptions over the first five years . HoweverStreptococcus pneumoniae and S. pyogenes. The hand hygiene campaigns, launched in 2005 and targeting patients admitted to hospital and healthcare workers, resulted in an increase in hand hygiene compliance and alcohol-based handrub use in hospitals and decreased hospital-acquired (HA)-MRSA.The Belgian Antibiotic Policy Coordination Committee (BAPCOC) organized several national campaigns, financially supported by the government. These multimedia campaigns, launched in 1999 and targeting the general public, resulted in a 36% decrease in antibiotic prescription in the community between 1999 and 2007  and decrEnterobacteriaceae [However, some national campaigns, e.g., Australia, England, Greece, and Spain, have failed to show a major impact on antibiotic prescriptions . In the eriaceae . In Israeriaceae  and the eriaceae . To conteriaceae . Furthereriaceae .Nevertheless, despite targeted information and awareness-raising campaigns, the general public has still preconceived ideas concerning antibiotics and their effects. For example, according to a Pan-European survey published in 2010, 53% of Europeans still believe that antibiotics kill viruses and 47% that they are effective against colds and influenza. Large variations between countries were observed and knowledge increased in countries with targeted media campaigns, such as Belgium. Education remains an immense challenge .In response to this global public health threat, 70 leading international experts formulated "The Pensières Antibiotic Resistance Call to Action" during a two-day meeting held in Annecy (France) in June 2011. Lectures were given on a wide range of topics with extensive and in-depth discussion. Each participant presented data and the results of country-specific intervention programmes targeted at the control of AMR and healthcare-associated infection, such as infection prevention and control and antibiotic stewardship strategies. Thirty-four posters provided for the first time a unique overview of actions and policies in place worldwide in 29 countries with an evaluation of their degree of efficacy. At the end of the meeting, participants were asked to rank a series of 25 actions linked to the topics highlighted using a multi-voting system .A coordinated programme based on six main lines of action was defined as follows: 1) a worldwide upgrade in infection control practices to limit resistant bacteria cross-transmission; 2) a worldwide antibiotic stewardship strategy to decrease antibiotic pressure on bacteria; 3) the improved use of diagnostic techniques; 4) an acceleration in the discovery and development of new antibiotics, particularly targeting Gram-negative bacteria; 5) the acceleration of vaccine development programmes, and 6) a strong educational programme for both healthcare practitioners, consumers, and children.The programme is conceived as a "bundle" whose different components should be implemented simultaneously. Implementing only one line of action or selecting only some components will very likely lead to failure. Such a multifaceted programme looks easy to implement, but is in fact a serious challenge. Although the multiple actors to involve have different backgrounds and interests, such as the hospital, community, and human and animal medicine, and may not work spontaneously together, cooperation between all is the key to success.A strong political commitment at international, national, and local levels is of paramount importance to trigger such an ambitious programme. This is absolutely essential. Recent national or international programmes should be evaluated. Healthcare professionals will need the strong involvement of policy makers through to hospital managers to ensure its adoption. It will take many years to obtain significant results and we will certainly never return to the pre-antibiotic era where all pathogens were fully susceptible to antibiotics. But we have no choice and must preserve antibiotics for the next generations. We must realize also that such a programme is not aiming at simply saving money--and could even increase healthcare costs initially--but it will become eventually cost-effective when taking a long-range perspective.http://www.imi-europa.edu) is investing 15 million euros in the RAPP-ID project  to develop new diagnostic tools for bloodstream infections, lower respiratory tract infections, and tuberculosis. It is to be hoped that some promising ongoing EU-funded projects will help to find new solutions, e.g., "Mastering hOSpital Antimicrobial Resistance" ; "Impact of Specific Antibiotic Therapies on the prevalence of hUman host ResistaNt bacteria" ; and "Resistance in Gram-Negative Organisms: Studying Intervention Strategies" .The importance of a coordinated programme combining infection control with other actions in a rational and sustainable manner, e.g., antibiotic stewardship, must be strongly emphasized. Prevention of cross-transmission and epidemics must be based on a multifaceted strategy that should include appropriate screening policies, use of universal precautions, improved hand hygiene, particularly through the systematic recourse to alcohol-based handrub formulations (ABHRs), and specific contact precautions when appropriate . However, several of these measures remain controversial and costly. For instance, it remains unknown if specific isolation precautions are better than standard precautions if the latter are strictly and permanently applied, which so far is hard to obtain . Rapid dSuccessful measures for controlling MRSA are probably not sufficient to prevent the spread of ESBL or carbapenemases for several reasons: far higher bacterial load in the gut for Gram-negative rods; fecal excretion; dissemination through waste; transferable resistance genes on plasmid or transposons; lack of effective decolonization regimens; or the substantial role of antibiotic selection pressure by commonly misused drugs. Although MRSA bloodstream infections are decreasing in many European countries, infections due to ESBL-producing Gram-negative rods are increasing in these same countries. To succeed in combating these Gram-negative rods, there is a need to upgrade and tailor the prevention of cross-transmission outside hospitals  and to take into account environmental aspects. Moreover, those actors with an important role to play, such as specialists in infection control and healthcare managers, have been somewhat paralyzed in front of the ESBL invasion, although sometimes simultaneously very active against MRSA or VRE.The WHO Global Patient Safety Challenge "Clean Care is Safer Care" is a striking example of a programme that could provide guidance, boost hand hygiene promotion initiatives worldwide, including in developing countries. Proof of effectiveness of additional actions will hopefully be provided by ongoing studies . QualityAntibiotics are natural gifts belonging to humanity and strategies for their active protection must be developed in a philosophy of "sustainable development" . A worldhttp://www.whc.unesco.org. Finally, it is of paramount importance to realize that in many countries there is a very limited access to antibiotics, which impairs safety of care. A balance between appropriate usage and access to antibiotics is needed. These two actions are not mutually exclusive, but complementary.A concerted international programme should induce a marked decrease in the overall consumption of antibiotics in every sector of human and animal medicine, aquaculture, and agriculture. There is no specific culprit and all antibiotic prescribers must work together. A strong and sustained cooperation between healthcare professionals and consumers (antibiotic users) in an ecological and civic attitude is pivotal for the success of these programmes. Antibiotics must be considered as a specific class of drugs ,57, a ceth century, and many of their culture methods are still used today in our routine clinical diagnostic laboratories.Rapid diagnostic tests should be urgently developed to help physicians to target the organisms causing the infection. Physicians should not rely only on fever, which is very often due to non-bacterial infections, to prescribe antibiotics. Unfortunately, microbiology diagnostic techniques have not evolved much since Pasteur and others were able to grow bacteria at the end of the 19C. difficile and MRSA. Simple tests are available to detect Streptococcus pyogenes in the throat, but often not used by general practitioners  are currently discussing mechanisms to jointly develop new antibiotics.WHO, TATFAR, the Infectious Diseases Society of America (IDSA), and European institutions, as well as healthcare professionals, have proposed measures and incentives to fix the broken antibiotic pipeline and encourage biotechnology and pharmaceutical companies to invest in the development of new antibacterial agents, particularly against Gram-negative bacteria. In 2010, IDSA launched a new initiative entitled "10 × 20" to mobilize key leaders, research institutions, and scientific associations to create an antibacterial research and development enterprise powerful enough to produce 10 new antibiotics by the year 2020 rg/10x20/. Fast-trrg/10x20/. The IMIhttp://www.e-bug-edu[It is of paramount importance that both professionals and consumers understand that the two main causes of antibiotic resistance are their overuse in both humans, animals, and agriculture. A complicity between these two groups is key to the success of such a programme. We need also to provide information to children and to promote and establish large programmes such as the Pan-European e-Bug project e-bug-edu,65. In tWe have overused and abused antibiotics in both humans and animals with huge variations between countries . Today, Staphylococcus aureus; CDC: Centers for Disease Prevention and Control; DDD: defined daily dose; DG-SANCO: Directorate General for Health and Consumers; ECDC: European Centre for Disease Prevention and Control; EFPIA: European Federation of Pharmaceutical Industries and Associations; EMEA: European Medicines Agency; ESGAP: European Society for Clinical Microbiology and Infectious Diseases (ESCMID) Study Group on Antibiotic Policies; EU: European Union; ESBL: extended-spectrum beta-lactamase; FDA: Food and Drug Administration; HA-MRSA: hospital-acquired methicillin-resistant Staphylococcus aureus; HIV: human immunodeficiency virus; IDSA: Infectious Diseases Society of America; IMI: Innovative Medicines Initiative; MDROs: multidrug-resistant organisms; MOSAR: Mastering; hOSpital: Antimicrobial Resistance; MRSA: methicillin-resistant Staphylococcus aureus; NDM-1: New Delhi metallo-beta-lactamase 1; RAPP-ID: Development of Rapid Point-of-Care Test Platforms for Infectious Diseases; REACT: Action on Antibiotic Resistance; R-GNOSIS: Resistance in Gram-Negative Organisms: Studying Intervention Strategies; SATURN: Impact of Specific Antibiotic Therapies on the prevalence of hUman host ResistaNt bacteria; TATFAR: Transatlantic Taskforce for Antimicrobial Resistance; UK: United Kingdom; UNESCO: United Nations Educational, Scientific and Cultural Organization; USA: United States of America; VRE: vancomycin-resistant enterococci; WHO: World Health OrganizationABHRs: alcohol-based handrubs; AMR: antimicrobial resistance; APUA: Alliance for the Prudent Use of Antibiotics; BABCOC: Belgian Antibiotic Policy Coordination Committee; CA-MRSA: community-acquired methicillin-resistant C. difficile colitis. He has also received speaker's honoraria related to new antibacterial agents from Janssen-Ortho, Pfizer, and Astellas Pharma during the past five years. All other authors declare no competing interests.Jean Carlet has served as a consultant for Biomerieux, Astra-Zeneca, Astellas, Thermo-Fisher, Da Voltera, and Aromatechnologies. John Conly has received honoraria from the Canadian Agency for Drugs and Technologies in Health for work as an expert reviewer and clinical expert, respectively, for projects on the role of rapid polymerase chain reaction (PCR) testing for MRSA in hospitalized patients, and the use of vancomycin or metronidazole for the treatment of JC conceived and drafted the manuscript and produced the different versions, in particular the reference list. VJ and DP provided an extensive review of the first draft of the manuscript. DP, SH, HG, and AV reviewed the manuscript and provided important intellectual content. All attendees to the meeting were given the possibility to comment on the content. All authors have read and approved the final version of the manuscript.The content of this paper expresses the views of the experts co-authoring it and in no way represents the position of their affiliations.In Europe (29 countries), the overall human consumption of antimicrobials was 3350 tons in 2007 [In the USA, 3300 tons of antibiotics were sold [Half of all antibiotic consumption may be unnecessary and greatly contributes to increasing bacterial resistance . In Euro in 2007 . In the ere sold .• But they are often prescribed for self-limiting illnesses, such as colds and influenza, caused by viruses that will not respond to antibacterial drugs.• Diagnostic uncertainty is a key driver of drug misuse and overuse. Since classical laboratory methods, based on culture of the pathogenic agent, require 36-48 hours to provide results, few infections are accurately diagnosed.• In the absence of a clear diagnosis, physicians often prescribe antibiotics just "to be on the safe side" or to prevent possible secondary bacterial infections.• In addition, patients often put pressure on physicians. In a survey conducted in the USA, nearly half (48%) of respondents indicated that they expected an antibiotic when they visit a doctor  In anoth• According to the US Centers for Disease Control and Prevention (CDC), an estimated 150,000 cases per year present to US emergency departments for antimicrobial-related adverse events .• Incorrect use of antibiotics accelerates AMR. In this respect, AMR is like pollution: it has so little immediately perceptible effect that in the absence of regulation, nothing changes .Patient compliance with recommended treatment is another major problem• Patients forget to take medication or may be unable to afford a full course. They tend to consider antibiotics as antipyretics that treat symptoms and stop taking them as soon as they feel better.Self-medication is also an important driver of antimicrobial overuse• It has been observed in the USA  and Euro• It is especially prevalent in developing countries where antibiotics can be bought over the counter in pharmacies or even in the local market place.• Sales via the internet drive self-medication; they are on the rise and difficult to control .Resistance to antibiotics is increasing both in commensal and pathogenic bacteria, raising an emerging threat to public health and the environment. Antimicrobial administration to food animals is among the most important factors contributing to the selection of antimicrobial-resistant bacteria that can be transmitted from animals to humans. More than half of all antibiotics produced globally are used in animals . In the Antimicrobials are used by veterinary practitioners for the treatment and control of infectious diseases in a wide variety of farm and companion animal species. Antibiotic treatment of sick animals is common practice. When a certain percentage of farm animals or certain species  are affected, the entire group is treated, including animals that are not infected. Sub-therapeutic levels of antibiotics are also administered to animals for the prevention of bacterial infections to compensate poor production practices, often without prescription.Low levels of antibiotic agents are frequently added to animal feed for growth promotion in livestock  . This isOn January 1 2006, the EU banned the feeding of all antibiotics and related drugs to livestock for growth promotion purposes . The USAWidespread use of antimicrobials for disease control and growth promotion in animals has been paralleled by an increase in resistance in those bacteria in animals. Resistant bacteria then spread among groups of animals, including fish, or to the local environment  through the spreading of manure.Through long-term survival and transfer of resistant genes to the resident flora [-- nt flora  Studies nt flora .Through direct contact between farm animals and humans  The same strains of MRSA have been found in livestock and livestock workers in The Netherlands, Italy, Canada and the USA [--  the USA -91.Through contaminated food-- E. coli strains that cause urinary tract and bloodstream infections in humans appear likely to have been acquired from contaminated retail meat.Although correct cooking kills bacteria, contamination can occur through improper handling before cooking. Many of the antimicrobial-resistant E. coli isolates, of which 39% were also found in human clinical samples tested in 31 microbiological laboratories [Salmonella and Campylobacter in animals and humans has been observed in several countries [Salmonella Schwarzengrund from chickens to humans in Thailand and from imported Thai food products to humans in Denmark and the USA [In The Netherlands, 94% of a representative sample of chicken retail meat was contaminated with ESBL-producing ratories ,93. An aountries . Reports the USA .• Consumption of antibiotic residues represents a potential threat to human health, through direct toxicity, allergic reactions, or alteration of the bacterial flora present in the human digestive tract .• To safeguard humans from exposure to antibiotic-added food, a withholding period must be observed until the residues are no longer detected before the animal or animal products can be processed. Heavy responsibility is placed on the veterinarian and livestock producer to observe the period of withdrawal. In Europe, rapid tests are regularly performed to check the absence of antibiotic residues in food.• Eliminating the unnecessary usage of antibiotics implies a change in mindset, integrating both long-term public health concerns and productivity. This involves everyone -- from governments to producers to the consumer. To stem the rising threat of resistant bacteria to human health, there is an urgent need for regulation of antibiotic usage in animals at the global level.In the past, the discovery of potent new classes of antimicrobials allowed to provide therapeutic options for newly emerging AMR. During the 30 years following the introduction of penicillin, scientists discovered a wide range of antimicrobials to treat bacterial diseases. By the early 1970s, 11 distinct antibiotic classes and more than 270 antibiotics had been brought into clinical use .The process of novel antimicrobial discovery has slowed to a virtual standstill. Most antimicrobials introduced since the early 1970s have been chemical modifications of previously discovered classes of drugs . The pro• Of the 15 companies with previous had antibiotic discovery programmes, only 5 still maintain an active research and development capacity in antibiotics .• According to two recent reports from IDSA  and the • Only 15 antibiotics under development (mostly in the early phases) present a new mechanism of action with the potential to meet the challenge of multidrug resistance. Of these, only two, both in the early development phase, may be active against multidrug- resistant Gram-negative bacteria, a group of bacteria causing serious therapeutic concerns due to their increasingly high resistance to antibiotics.The discovery and development of new antimicrobials is an expensive and time-consuming process. Pharmaceutical companies must prioritize competing projects and antibiotic development has a lower priority than other competing drugs in the portfolio.• In the late 1960s, infectious diseases were thought to be conquered, opening the way for a shift in resources to chronic conditions, such as cancer and cardiovascular diseases.• The limited duration of antibiotic treatments makes them less profitable than other drugs prescribed for years to treat chronic conditions, such as hypertension and diabetes.• There is strong competition with other drugs already on the market. While resistance is an emerging problem, low-priced generic antibiotics on the market are still effective in treating most infections and are used as first-line therapy.• New antibiotics may be kept as last-resort treatments, resulting in low sales for companies.• New antimicrobials can also have a limited lifespan because of the development of resistance.versus those already registered within tighter statistical parameters, i.e., shifting from "non-inferiority" to "superiority" trials [• Modifications in regulatory procedures have been perceived as having created an "unfriendly" environment. Regulators have been demanding demonstrations of the relative efficacy of new antibiotics " trials ,100.Antibiotic use, misuse, and abuse. Supplementary list of the main issues discussed (references [ferences -78).Click here for fileAntibiotic use in animals: a major concern for public health and the environment. Supplementary list of the main issues discussed (references [ferences -98).Click here for fileThe antibiotic pipeline is running dry. Supplementary list of the main issues discussed .Click here for file


The IgG4-RD lesions were also detected in the mesentery of the sigmoid colon, retroperitoneal soft tissue, abdominal aorta, liver, extrahepatic bile duct, bilateral lungs, bilateral kidneys, urinary bladder, prostate, epicardium, bilateral coronary arteries, and lymph nodes. Interestingly, infiltration into the lesions was most notable around the peripheral nerves in every organ. Thus, this case describes an IgG4-RD that progressed from PTGC-type IgG4-related lymphadenopathy to systemic IgG4-RD, suggesting that IgG4-RD may affect many organs through peripheral nerve involvement.A 77-year-old man, with a lengthy medical history of chronic dysuria, constipation, hypertension, myocardial infarction, and a submandibular lymphadenopathy that was excised 3 years ago, was hospitalized due to elevated liver enzyme levels. He demonstrated hypergammaglobulinemia, hyperproteinemia, high levels of IgG and IgG4, eosinophilia, sclerosing cholangitis, and retroperitoneal fibrosis. He was diagnosed with IgG4-related disease (IgG4-RD). While hospitalized, he had several episodes of syncope while standing and was diagnosed with autonomic nerve dysfunction. Thirty days after hospitalization, he died of nonocclusive mesenteric ischemia (NOMI). Post-mortem, his submandibular lymphadenopathy lesion was diagnosed with progressively transformed germinal center (PTGC)-type IgG4-related lymphadenopathy. At autopsy, small and large intestines showed mucosal necrosis and the wall muscles of the transverse to sigmoid colon were necrotic. The sigmoid colon was fibrotic and infiltrated with numerous IgG4http://www.diagnosticpathology.diagnomx.eu/vs/9995992971155224.The virtual slides for this article can be found here:  Generally, if the histological IgG4+/IgG+ cell ratio is greater than 40%, the diagnostic possibility of IgG4-RD is high, but there are also many “non-IgG4-related” diseases such as multicentric Castleman’s disease, rheumatoid arthritis, or other autoimmune diseases that need to be included in the differential diagnosis [Immunoglobulin G4-related disease (IgG4-RD) comprises a recently recognized systemic syndrome that is characterized by mass-forming lesions, mainly in exocrine tissues, which consist of lymphoplasmacytic infiltrations and sclerosing fibrosis ,2. IgG4-iagnosis ,3,4.The lymph node lesions of IgG4-RD can be subdivided into at least 5 histological subtypes, including the multicentric Castleman’s disease-like, reactive follicular hyperplasia-type, interfollicular expansion and immunoblastosis, progressively transformed germinal center (PTGC)-type, and inflammatory pseudotumor (IPT)-like ,4. The PWe present a rare autopsy case of IgG4-RD that began as PTGC-type IgG4-related lymphadenopathy and progressed into a systemic disease by infiltration around the peripheral nerves of many organs.A 77-year-old man, with a lengthy history of chronic dysuria, constipation, hypertension, and a previous myocardial infarction that was treated with a coronary stent, developed a right submandibular mass 3 years prior to his most recent admission and underwent an excisional biopsy. As a result, he was diagnosed with reactive follicular hyperplasia with PTGC. During the intervening period, the patient had slight fatigue, loss of appetite, and lightheadedness. At a regular check-up, serological tests showed high levels of liver enzymes and he was admitted to a local hospital. The serological examination results revealed that serum IgG, IgG4, and IgE levels were notably elevated; we also noted eosinophilia, hyperproteinemia, hypoalbuminemia, low albumin/globulin ratio, hyponatremia, and elevations of liver enzymes Table .As part of the differential diagnosis of high gammaglobulinemia, M-proteins were not detected in the globulin fraction and the immunoglobulin exhibited a polyclonal pattern, excluding hematopoietic diseases such as multiple myeloma. The hyponatremia was considered to be a syndrome of inappropriate antidiuretic hormone secretion, considering the patient’s low osmotic blood pressure, his high urinary osmotic pressure, and the lack of serum renin elevation. After hospitalization, the patient’s dysuria and constipation were exacerbated. After 2 weeks of hospitalization, he experienced 3 episodes of syncope while standing up. The cause of the syncope was determined to be orthostatic hypotension, and no hormonal abnormalities were detected in his catecholamine, thyroid hormone, and renin-aldosterone-angiotensin levels. Using cardiac metaiodobenzylguanidine scintigraphy, he was diagnosed with severe autonomic nerve dysfunction.+ plasma cells were seen, immunohistologically, within the germinal centers. There was no evidence of kappa or lambda immunoglobulin light chain restriction by in situ hybridization. This finding led us to re-diagnose his previous lymphadenopathy as PTGC-type IgG4-related lymphadenopathy  indicated inflammation of the pituitary gland, and a magnetic resonance cholangiopancreatography revealed mild narrowing of the extrahepatic and intrahepatic bile ducts in the portal area. This was considered as evidence of sclerosing cholangitis. Abdominal MRI also showed a retroperitoneal mass that was 2 cm in diameter. These lesions were also considered as indications of IgG4-RD.Following this diagnosis, the patient was scheduled to begin steroid therapy. However, prior to starting the therapy, 30 days after hospitalization, the patient had abdominal distention, nausea, and vague abdominal tenderness that developed into a sharp abdominal pain. He also exhibited low blood pressure, tachycardia, and marked metabolic acidosis, eventually going into cardiac arrest. The patient was resuscitated and, upon suspicion of ileus or non-occlusive intestinal ischemia, he underwent emergent abdominal surgery. The patient was subsequently determined to have inoperable, massive necrosis of the small and large intestines. Some hours later, he succumbed to shock and an exacerbation of metabolic acidosis.An autopsy was performed 8 h after death. A central nervous system examination was not permitted by the family; thus, the autopsy was restricted to the chest, abdomen, and spinal cord. The peritoneal examination revealed the presence of approximately 550 mL of sanguineous ascites. The small and large intestines were diffusely dark red to black, but no mechanical obstruction was observed; the intestinal tract was filled with bloody stool. Histologically, the intestinal mucosa was diffusely necrotic, and the muscle layers were also necrotic. The necrosis of the colonic muscle layers was coagulative, focal, and patchy along the transverse and descending colon, whereas there was diffuse necrosis in the sigmoid colon. In addition to necrosis, the sigmoid colon wall was infiltrated by numerous plasma cells and eosinophils. These lesions were primarily located in the smooth muscle of the colon wall and in the intramuscular nerve plexus of Auerbach; plasma cell infiltration, through the epineurium, into the plexus was also observed Figure . Similar+/IgG+ cell ratio of more than 40%, histologically consistent with IgG4-RD. Within this lesion, some of the small veins in the sigmoid colon mesentery showed obliterative phlebitis and a few of the accompanying arteries were organized , or “intestinal gangrene in the presence of a patent arterial tree”, is an uncommon condition in which severe, unrelenting, microvascular vasoconstriction results in bowel ischemia in the presence of pulsatile macroscopic arterial blood flow. NOMI is manifested by abdominal pain, usually occurring in patients with known atherosclerotic heart diseases ,13. In tIn the liver, although IgG4-RD caused secondary sclerosing cholangitis, bile stasis was minimal. Therefore, obliterative phlebitis of the portal veins, rather than cholangitis, may explain the elevated liver enzyme levels. In contrast to the hepatic or the upper and middle common bile ductular lesions, the lower common bile duct and pancreas were spared, which is not typical in cases of IgG4-RD.In summary, different morphological types of IgG4-related lymphadenopathy were seen in a single patient. These lesions included a PTGC-type feature that developed 3 years prior to the determination of an IPT-like feature, suggesting that one type of IgG4-related lymphadenopathy may progress into another morphological type. Obliterative phlebitis was also noticed within the patient’s lymph nodes at autopsy. Another important feature was the presence of IgG4-related lesions around the peripheral nerves in several organs, which may be considered as IgG4-related perineural disease. The intestinal lesions of this patient may have been responsible for his end-stage constipation and ischemia. Moreover, the infiltration of plasma cells through the epineurium into the nerve fascicles has not been previously reported in the English literature. The reasons why inflammtory cells preferentially distribute around and into the peripheral nerves, and how often and to what extent IgG4-RD can cause neurological symptoms, remain to be elucidated.We encountered an autopsy case of systemic IgG4-RD with extensive involvement of the peripheral nerves. This case indicated that IgG4-RD may affect systemic organs through extensive peripheral nerve involvement.Written informed consent was obtained from the patient's family for publication of this Case Report and any accompanying images.IgG: Immunoglobulin G; IgG4: Immunoglobulin G4; PTGC: Progressively transformed germinal centers; NOMI: Non-occlusive mesenteric ischemia; IPT: Inflammatory pseudotumor.The authors declare that they have no competing interests.YS conceived and designed the study. YS, MF, NO, and TY made the pathological diagnosis and wrote the paper. KH, HK, and YK collected clinical information. All authors read and approved the final manuscript.


MET is a proto-oncogene with its copy number (CN) alterations been reported in some cancers, but not in primary intestinal diffuse large B-cell lymphoma (PI-DLBL) yet.MET CN alterations on 28 surgically resected PI-DLBLs.In this retrospective study, we performed histology and chart reviews, immunohistochemistry and quantitative polymerase chain reaction for MET CN was 2.20 . CN gain was observed in 11 cases, including 5 with CN greater than 3. Nine patients (32%) had diploid CN and eight (29%) with CN loss. Patients with gain or diploid CN showed significantly worse prognosis (P = 0.046) than those with CN loss. Furthermore, MET CN greater than 3 was associated with an adverse outcome (P = 0.003). Intestinal perforation at presentation was the sole clinicopathological factor associated with a poor prognosis (P = 0.004) and perforation was correlated with CN greater than 3 (P = 0.002).There were 12 men and 16 women with a median age of 70 and a mean follow-up of 32 months. The median MET CN gain as a poor prognostic factor in PI-DLBL patients might serve as the rationale for targeting MET signaling pathway in the treatment of these patients.Our finding of  MET gene and ligand-independent kinase activation by extracellular proteins, autocrine overexpression of HGF ligand, and increased expression of HGF activator [MET CNAs in gastric cancer and non-small cell lung cancer (NSCLC) [MET CNA in the survival of patients with these hematological malignancies.Copy number alterations (CNAs) of genes are indications of genomic instability that are common in cancers and have been known to be associated with the development of malignancy ,2. CNAs ctivator ,7. Recen (NSCLC) -10. Alth (NSCLC) -18, it iMET CNAs by quantitative polymerase chain reaction (qPCR) method and found that in addition to perforation, MET CNAs were also significantly associated with prognosis.In a previous study, we identified perforation as the sole clinicopathological prognostic factor in patients with primary intestinal DLBL (PI-DLBL) . In thisStrict inclusion and diagnostic criteria for PI-DLBL were applied as previously reported . MedicalTo avoid the contamination by normal cells, we reviewed the HE sections of each case and chose those containing greater than 90% tumor cells without or with only minimal tumor necrosis for DNA extraction. Three to five 10 μm thick paraffin sections of each case were cut into an eppendorf tube from a representative block with disposable blades. The paraffin rolls were de-paraffinized with xylene followed by three washings with ethanol. Paraffin-free tissue was dried in a heating box for 15 min at 37°C and then subjected to the DNA extraction procedures using QIAamp DNA mini kit . The quality and DNA concentration were measured by NanoPhotometer . DNA with 260/280 ratios of 1.6–2.0 with a minimal concentration of 25 ng/μl DNA were required for qPCR assay.MET gene by using the commercially available 6-carboxy-fluorescine (FAM)-labeled MET probe . Ribonuclease P (RNase P), which is known to exist in two copies, was used as the endogenous control with 4,7,2-trichloro-7-phenyl-6-carboxyfluorescein (VIC)-labeled probe (Applied Biosystems Part no. 4403326). The peripheral blood mononuclear cells from six healthy individuals served as control samples. Each experiment was performed using a 96-well optical PCR plate and the StepOnePlus real-time PCR machine (Applied Biosystems) with default cycling conditions. According to manufacturer’s instruction, a total of 10 ng of genomic DNA was added to the PCR reaction mixture containing 1× final concentration TaqMan Gene Expression PCR Master Mix (Applied Biosystems) and 1x final pre-designed primer/probe Mix (Applied Biosystems) in a final volume of 10μL. Each sample was run in triplicate. After amplification, the Ct values for both target and reference genes were imported into the CopyCaller Software (Applied Biosystems) for data analysis. The CN of the target gene was determined by comparative quantitative threshold cycle (ΔΔCt) method where ΔΔCt =  −  and then using the formula 2 × 2− ΔΔCt[MET CN, gain, or loss.We performed qPCR assay to determine the CNAs of × 2− ΔΔCt. The cutMET CN and various clinical and immunophenotypical parameters were assessed by χ2 test or Fisher’s exact test. Kaplan–Meier survival curves were drawn and the log rank test was used to compare the differences between survival curves. All statistical analyses were performed using SPSS for windows 11.0 software . A P value of 0.05 or less was considered statistically significant.The associations between P = 0.004; Figure These 28 patients included 12 males and 16 females with a median age of 70 years . Six patients 21%) presented with intestinal perforation. The staging results were available in 27 patients including 9 (33%) at stage IE, 14 (52%) at stage IIE and 4 (15%) at stage IVE. Nineteen patients (70%) received chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP) or similar regimens. Among these, 12 patients received combined Rituximab therapy and others additional radiotherapy. Eight patients (30%) received only supportive treatment without chemo- or radiotherapy due to poor general condition. The mean follow-up time was 32 months . The overall 1-, 2- and 5-year survival rates were 50, 43 and 18%, respectively. Immunohistochemically, 22 tumors (79%) expressed bcl-2, bcl-6 and MUM1/IRF4; while only 6 tumors (21%) expressed CD10. Nineteen tumors (68%) had concurrent expression of bcl-2 and MUM1. All tumors were negative for cyclin D1. Seven tumors (25%) were classified as germinal centre B-cell (GCB) phenotype and 21 non-GCB (75%) according to the algorithm of Hans et al. [% presentMET CN and the clinicopathologic features. Of these 28 cases the median MET CN was 2.20  had MET CN gain , 8 (29%) with CN loss , and 9 (32%) with normal/diploid CN . Patients with MET CN gain or normal/diploid MET CN showed a significant worse prognosis than those with MET CN loss .Table MET CNAs in surgically resected PI-DLBL and found that tumors with MET CN gain greater than 3 copies were associated with an unfavorable prognosis and also with perforation at disease presentation. Normal or lower level MET CN gain also denoted a trend toward poor prognosis, although the statistical significance was low. We speculate that the clinical impact of MET CN on prognosis might be amplification dosage dependent. Furthermore, we re-confirmed our previous findings that tumor perforation was a poor prognostic factor in PI-DLBL and that the relative frequency of GCB phenotype was low in PI-DLBL, although the latter was not significantly related to prognosis [In this study, we investigated the prognostic role of the rognosis .MET gene would result in MET protein over-expression and constitutive kinase activation [MET CNAs have been investigated in solid cancer such as gastric carcinoma and NSCLC [MET CNAs, MET amplification has been identified in 4-21% of gastric carcinomas [MET CN was associated with higher tumor stage at presentation and poorer survival in patients with gastric cancer [MET amplification is one of the most frequently involved mechanisms [MET gene has been shown to be amplified in 21-24% of tumors and the impact of MET CN on prognosis is histological subtype dependent: MET amplification with a poorer prognosis in patients with adenocarcinoma but not in those with squamous cell carcinoma [HGF/MET pathway could be activated in both benign and malignant B-cells. Amplification of the tivation . MET CNAnd NSCLC -10. Depercinomas ,24. An ic cancer . For a schanisms . In NSCLarcinoma ,27.P = 0.0025) [MET gene CN has not been investigated in malignant lymphoma yet. To the best of our knowledge, our study is the first report describing the prognostic significance of MET CNAs in patients with PI-DLBL. Our results suggested that higher MET CN, possibly leading to MET over-expression, might be a poor prognostic indicator for PI-DLBL.Among the various entities of non-Hodgkin lymphomas, MET is frequently over-expressed in DLBL ,15,17. O 0.0025) . A study 0.0025) . So far MET CN gain in PI-DLBL patients with poor prognosis suggested that blocking HGF/MET pathway might be a promising adjuvant therapeutic option.High serum HGF levels have been found in patients with DLBL and MM, and they were associated with an unfavorable prognosis ,30-32. FCN: Copy number;PI-DLBL: Primary intestinal diffuse large B-cell lymphoma;CNA: Copy number alteration;HGF: Hepatocyte growth factor;NSCLC: Non-small cell lung cancer;DLBL: Diffuse large B-cell lymphoma;MM: Multiple myelomaThe authors declare that they have no competing interests.WTH did the experiment, analyzed the data and drafted the manuscript. SSC conceived and coordinated the study, analyzed the data and wrote the manuscript. Both authors read and approved the final manuscript.


To investigate on the expressions and the clinical significances of hepatocyte growth factor receptor (c-MET), phosphorylated c-MET (p-MET) and e2f-1 transcription factor in primary lesion of gastric adenocarcinoma (GC).2/D3 radical gastrectomy with R0/R1 resection were stained by immunohistochemistry of c-MET, p-MET, e2f-1 and Ki-67. The univariate and the multivariate analyses involving in clinicopathological parameters and prognostic factors were evaluated.Tissue samples from the primary lesion of GC in patients who accepted Dχ2 = 13.437, P = 0.001). However, in the cases with Stage III-IV, no significant difference could be identified in the prognostic comparison between positive and negative expressions of e2f-1.The positivity rates for c-MET , p-MET , e2f-1  and Ki-67  in primary lesion of GC was significantly higher than that in non-cancerous tissue at 5 cm places far from the margin of primary lesion . The deeper tumor invasion, the severer lymph node metastasis, the later stage of TNM and the higher expression of Ki-67 was respectively an independent risk factor for the higher expression of c-MET or p-MET, but the younger age and the shorter survival time was an independent risk factor for the higher expression of e2f-1 respectively. Survival analysis showed that the worse prognosis could be observed in the patients with the combination of both c-MET-positive and e2f-1-negative (P = 0.038) or both p-MET-positive and e2f-1-negative (P = 0.042). Cox analysis demonstrated that the severer lymphatic node metastasis and the higher positivity rate of c-MET, p-MET or e2f-1 were an independent prognosis factor respectively. The higher expression of e2f-1 was identified in patients with Stage I-II, which correlated with a shorter survival time. Survival analysis also revealed that the prognosis of patients with positive expression of e2f-1 at Stage I-II was significantly worse than that in patients with negative expression of e2f-1 (The expression of c-MET or p-MET is an independent prognosis factor. It has been observed that the higher expression of e2f-1 occurred in the early stages while the lower expression of it in the later stages in GC. This initiates or shuts down the corresponding gene transcription, leading to an increase or a decrease in the level of the cell cycle protein Cyclin D , the severer lymph node metastasis , the later stage of TNM  and the higher expression of Ki-67  were the independent risk factors for the higher expression of c-MET respectively.At the same time, the multivariate analysis showed that the deeper tumor invasion , the severer lymph node metastasis , the later stage of TNM [P = 0.011 95.0% CI. for Exp (B) = 3.390 ~ 302.37] and the higher expression of Ki-67  were the independent risk factors for the higher expression of p-MET respectively.However, the younger age  and the shorter survival time  were the independent risk factors for the higher expression of e2f-1 respectively as suggested by the multivariate analysis.As shown in Table Analysis on subgroup of either c-MET positive expression or p-MET positive expression revealed that the average survival month of the strongly positive group  was significantly shorter than that in the moderately [(22.1 ± 3.3) mo.] and weakly [(29.8 ± 3.8) mo.] positive groups, and simultaneously, that the average survival month of the strongly positive group  was significantly shorter than that in the moderately [(23.0 ± 2.9) mo.] and weakly [(30.1 ± 4.1) mo.] positive groups. However, the analysis of e2f-1 expression subgroup demonstrated that the average survival month of the strongly positive group  was also significantly shorter than that in the moderately [(22.5 ± 2.9) mo.] and weakly [(26.2 ± 2.8) mo.] positive groups.a, Ib, II, IIIa, IIIb and IV were 62.5% (10 cases/16 cases), 47.1% (8 cases/17 cases), 55% (11 cases/20 cases), 40% (8 cases/20 cases), 27.3% (6 cases/22 cases) and 26.9% (7 cases/26 cases), respectively. These results showed that e2f-1 was expressed at a higher level in patients with Stage I-II, and that the higher expression of e2f-1 correlated with a shorter survival time in the early stage of GC. On the other hand, lower expression of e2f-1 was observed in patients with Stage III-IV, but no connection between e2f-1 expression and survival time was observed in patients with Stage III-IV based on statistical analysis . However, such difference for the prognosis of patients between the positive and negative for the expression of e2f-1 was not significant among patients during Stage III-IV  , and the positivity rates for Stage ITo choose the optimal treatment in order to improve the prognosis of GC, it is necessary to seek more sensitive indicators for the early diagnosis and much more exquisite choice for the optimal treatment. Such indicators as tumor invasion depth, the number of regional lymph node metastases, distant metastasis and histological grade were used in UICC classification , but the1 phase, the cell cycle will stop at the restriction point and the cell will pass into the quiescent state, namely the G0 phase. G0 phase cells enter G1 phase when stimulated by growth factors. The binding of a growth factor with its receptor induces a signal which activates Cyclin D-Cdk4/6 and phosphorylates Rb, thereby releasing the e2f transcription factor, which is mainly made up of e2f-1. This process in turn gives rise to the transcription of several genes, driving the cell cycle to continue and mediating the transition from phase G1 to phase S [Tumor cells are characterized by abnormal cell cycle initiation coupled with unrestricted proliferation. The start of the cell cycle is inseparable from growth factors. If growth factors are lacking during the G phase S .The c-MET binding to HGF causes the growth, the proliferation and the differentiation of the target cell. The incremental expression of c-MET has been identified in hepatocellular carcinoma, gastric carcinoma and colon carcinoma ,19,20. ERecently, the relationship between e2f-1 and apoptosis of tumor cells, which plays a complicated role in the generation and progression of many kinds of tumors, has received more attention as revealed by experimental study . E2f-1 iE2f-1 can induce apoptosis in many types of tumor cells, which has been demonstrated by in vitro gene transfection experiments . EnhanceBased on classical signaling pathways, c-MET is an upstream molecule to e2f-1. E2f-1 should be regulated by p-MET as the active status of c-MET, but we could not find such connection either between c-MET and e2f-1  or between p-MET and e2f-1 . This indicates that in addition to being a target molecule of c-MET, e2f-1 may be regulated by some other signaling pathways, which is worthy of making the further investigations on this regulation of signaling pathway. Nevertheless, when the expression of e2f-1 was lower during the later stages of advanced GC, c-MET and p-MET were still highly expressed. Therefore, further investigation for the relationship of p-MET with e2f-1 is required to verify this hypothesis in clinical and experimental studies.The expression of either c-MET or its activity status, i.e. p-MET, is one of the markers reflecting the degree of malignancy of GC, and either c-MET or p-MET is an effective prognosis factor because the significantly shorter survival time of patients positive for expression of c-MET and p-MET. E2f-1 was highly to express in relatively earlier stages of GC, while the lower expression of e2f-1 was observed in later periods of GC. As such, e2f-1 could play different roles at different stages of cancer; that is, it might promote the formation of the cancer in the early stages but inhibit the progression of cancer in the intermediate and advanced stages.GC: Gastric adenocarcinoma; HGF: Hepatocyte growth factor; p-MET: Phosphorylated c-MET; PBS: Phosphorylation buffer solution; SABC: Streptococcus avidin-biotin-peroxidase complex; UICC: The United States Joint Committee on Cancer; JGCA: The Japanese Gastric Cancer Association.The authors declare that they have no competing interests.JGW and JWY contributed in study design, editing and revision of manuscript. HBW and JGW contributed in study concepts, carried out study design, definition of intellectual content, literature research, experimental studies, data acquisition, data analysis, statistical analysis and manuscript preparation. JWY, JGW, LHZ, XCN and BJJ contributed in clinical managements. XQL and GYD contributed in pathological studies. BJJ contributed in guarantor of integrity of the entire study, study concepts, study design, manuscript review and revised version correction. BJJ and JWY contributed in the grants for this study. All authors read and approved the final manuscript.JWY and LHZ are associated professors of surgery and JWY is acting chairman of 1st Department of General Surgery in our hospital. JGW and XCN are doctor-in-charges of 1st Department of General Surgery in our hospital. HBW was a postgraduate student in our hospital and works as doctor-in-charge of surgery in Wu-gang People’s Hospital, Hubei Province now. XQL is associated professor of pathology and GYD is lecturer of pathology. BJJ is a professor of surgery and vise chairman of Department of General Surgery in our hospital.


From the travel ban on people living with HIV (PLHIV) to resistance to needle exchange programmes, there are many examples where policy responses to HIV/AIDS in the United States seem divorced from behavioural, public health and sociological evidence. At its root, however, the unknowns about HIV/AIDS lie at biomedical science, and scientific researchers have made tremendous progress over the past 30 years of the epidemic by using antiretroviral therapy to increase the life expectancy of PLHIV almost to the same level as non-infected individuals; but a relationship between biomedical science discoveries and congressional responses to HIV/AIDS has not been studied. Using quantitative approaches, we directly examine the hypothesis that progress in HIV/AIDS biomedical science discoveries would have a correlative relationship with congressional response to HIV/AIDS from 1981 to 2010.This study used original data on every bill introduced, hearing held and law passed by the US Congress relating to HIV/AIDS over 30 years (1981–2010). We combined congressional data with the most cited and impactful biomedical research scientific publications over the same time period as a metric of biomedical science breakthroughs. Correlations between congressional policy and biomedical research were then analyzed at the aggregate and individual levels.Biomedical research advancements helped shape both the level and content of bill sponsorship on HIV/AIDS, but they had no effect on other stages of the legislative process. Examination of the content of bills and biomedical research indicated that science helped transform HIV/AIDS bill sponsorship from a niche concern of liberal Democrats to a bipartisan coalition when Republicans became the majority party. The trade-off for that expansion has been an emphasis on the global epidemic to the detriment of domestic policies and programmes.Breakthroughs in biomedical science did associate with the number and types of HIV/AIDS bills introduced in Congress, but that relationship did not extend to the passage of laws or to hearings. When science matters, it cannot be separated from political considerations. An important implication of our work has been the depoliticizing role that science can play. Scientific breakthroughs helped to transform HIV/AIDS policy from a niche of liberal Democrats into bipartisan support for the global fight against the disease. On 30 October 2009, President Barack Obama announced that his administration was ending the travel ban against people living with HIV (PLHIV). Obama described the travel ban as “a decision rooted in fear rather than fact” . In the The travel ban is an example of the US Congress responding to public fear and giving it priority over scientific knowledge. This incident is not isolated from other congressional decisions regarding HIV/AIDS. Behavioural, public health and sociological studies have been disregarded: examples include laws disallowing federal funds for needle exchange programs despite their well-documented efficacy for reducing HIV transmission ,15,16, lAt its core, this is a study of how and whether science interacts with politics. The science for policy literature suggests that climate change, like HIV/AIDS, is a case where science has been politicized . JasanofThere are important differences between climate change and HIV/AIDS policy. Firstly, detached HIV/AIDS activists are not possible because the need for treatment ties them to the biomedical community . SecondlDespite these apparent differences between climate change and HIV/AIDS, their policy fates may be inevitably similar. Guston and colleagues posit that scientific knowledge is not separable from ideology, but has inherent partisanship . A comprThis is a statistical analysis of how congressional policy making – bills introduced, hearings held and laws passed – correlates with the annual number of scientific, biomedical breakthroughs published from 1981 to 2010.HIV/AIDS scientific breakthroughs were defined using the most-cited biomedical articles and journals with the highest impact factors . Using tAs a supplement, a list of important articles was compiled based on our own literature review. This supplemental list allowed the inclusion of influential articles that were published before the HIV/AIDS nomenclature was used or that were not among the top cited but are referenced at international conferences. This included the earliest studies by Montagnier and Gallo from 1981 to 1987, which were nicely summarized . Articlehearings was the yearly count of HIV/AIDS hearings in Congress. Data on bill sponsorship and laws passed were also collected. Using the THOMAS database maintained by the US Library of Congress, searches for “AIDS (Disease),” “human immunodeficiency virus” and “HIV/AIDS” were completed. Bills identified from these searches were then cross-referenced with the Congressional Bills Project database. The bills variable was the yearly count of HIV/AIDS bills introduced in Congress, and the laws variable was the yearly count of HIV/AIDS bills enacted into law. Bills on HIV/AIDS were also coded by subject for consistent comparison to scientific breakthroughs.The Congressional Hearing Digital Archive maintained by LexisNexis was searched using “acquired immune deficiency syndrome” to identify hearings related to HIV/AIDS from 1981 to 2010. Our dependent variable for New York Times in LexisNexis, and the news variable was the yearly count of those identified articles. We operationalized partisanship to take the value of 1 when Democrats held a majority in both houses of Congress, 0 when control was split and −1 when Republicans were the majority [Major events was a dummy variable that took the value of 1 for years involving major non-scientific HIV/AIDS events and zero for all others. Major-event years were 1983, when the US Centers for Disease Control and Prevention officially acknowledged AIDS; 1985–1987, when Ryan White was in the press, President Ronald Reagan spoke about AIDS for the first time and azidothymidine was introduced; 1991–1992, when Magic Johnson announced his status of living with HIV; and 1994, when AIDS became the leading killer of men aged 25–44 in the United States.Media coverage , partisamajority . Major eStatistical software R was used for statistical analysis. Augmented Dickey–Fuller tests showed that none of the five time trends – bills, hearings, laws, scientific breakthroughs or newspaper articles – followed stationary processes. Vector auto-regression (VAR) allowed estimation of the relationship between scientific breakthroughs and congressional response to HIV/AIDS while controlling for reciprocal feedback between media coverage and congressional policy making. The “vars” package was used to run separate models for bills, hearings and laws. The congressional measures and media attention were treated as endogenous variables in a VAR(1) process, and breakthroughs, partisanship and major events were treated as exogenous variables. The breakthrough variable was lagged by one year to allow time for scientific breakthroughs to work through the political system. For example, the following equation was used for bill sponsorship:An analysis of bill sponsorship at the individual level supplemented the aggregate. The dependent variable was whether or not a given member of Congress introduced any HIV/AIDS bills in a given year. Standard political science variables of partisanship, ideology, race, gender and institutional position were included with aggregate measures of scientific breakthroughs, media attention and major events. Partisanship was binary: a value of 1 for Democrats and 0 for Republicans. Ideology was measured as the common space score from NOMINATE . These sNew York Times articles is shown as the number of each by year, from 1981 to 2010 , resulting in a decade of bipartisan HIV/AIDS bill sponsorship .In the aggregate and at the individual level, science had a real – although limited – role to play in congressional policy making concerning HIV/AIDS. For HIV/AIDS, major scientific papers correlated with bill sponsorship by members of Congress. Scientific breakthroughs did not impact legislative hearings or the passage of bills into law. Sponsoring bills requires lower thresholds of attention, and given this, it makes sense that biomedical scientific advances matter for bill sponsorship rather than hearings or laws.We interpret these results through the lens of the agenda-setting literature in political science. Accordingly, policy entrepreneurs define or redefine issues for appeal to broader audiences, and then those audiences help to break established policy monopolies that are keeping new issues off the agenda ,29,62–65Kingdon  argues tIt is interesting to postulate what role the latest biomedical breakthroughs will play in congressional attention and domestic policy going forward, with the success of the CAPRISA (Centre for the AIDS Programme of Research in South Africa) trial, in which a vaginal microbicide was shown to reduce transmission to women through heterosexual contact , and theWe have shown that breakthroughs in biomedical research did associate with the number and types of HIV/AIDS bills introduced in Congress, but that relationship did not extend to passage of laws or to congressional hearings. We began by asking whether science could shape policy without itself being shaped by politics. This study makes three contributions to the literature around that question. Firstly, our findings are slightly counterintuitive because we show that science can matter for policy making. The agenda-setting literature suggests that complex information, like biomedical research, would not impact bill sponsorship. Secondly, we provide a more direct quantitative test of the hypothesized relationship between science and policy. Thirdly, we provide additional nuance to the science-for-policy debate. We have argued that biomedical breakthroughs created opportunities for policy entrepreneurs – that, in effect, science opened a way for politics. However, the ultimate consequence has been a depoliticization of HIV/AIDS policy at the congressional level. Scientific breakthroughs helped to transform HIV/AIDS policy from a niche of liberal Democrats to bipartisan support to fight the disease globally. In that regard, these findings bring us closer to Guston's position. Science matters when it suits the politics.


In France, the 2009 A(H1N1) influenza epidemic occurred between September 2009 and January 2010. Sparking widespread controversy, it was intensely reported in the media. Despite therapeutic inefficacy, antibiotic consumption and viral respiratory infections are positively correlated, particularly in France, where antibiotic overconsumption is well-known. We first determined the period when media coverage was high, and then compared, during this period, observed outpatient antibiotic consumption to estimated outpatient antibiotic consumption “without media attention”.To evaluate media coverage, two online databases were consulted: Factiva and Europresse. To quantify outpatient antibiotic consumption, we used data on reimbursements of outpatient systemic antibiotics from the computerized databases of the two main National Health Insurance agencies. Influenza-like syndromes data came from the French GPs Sentinelles Network. Weekly time-series of antibiotic consumption were modeled by autoregressive moving-average models with exogenous inputs and interventions. Analyses were computed for the entire series and by age group .Media coverage was intense between April 2009 and January 2010. No effect on total outpatient antibiotic consumption was observed during the whole mediatic period. However, during the epidemic in France (September 2009-January 2010), we found an antibiotic underconsumption for the entire series, 0–5 and >60 years. Additionally, at the beginning of the pandemic, when cases were still outside France (June 2009-August 2009), we found an antibiotic overconsumption for patients >16 years.The early period of A(H1N1) virus circulation compared with seasonal influenza or an overdeclaration of ILS cases might explain the antibiotic underconsumption observed during the period of active A(H1N1) virus transmission in France. At the pandemic onset, when uncertainty was high, the overconsumption observed for individuals >16 years might have been caused by alarmist media reporting. Additional analyses are needed to understand the determinants of antibiotic consumption during this period. Streptococcus pneumoniae is one of the best-known bacteria that has spread during the past 20 years, and, more recently, Enterobacteria producing extended-spectrum β-lactamase has become a major worry S. pneumoniae antibiotic resistance was observed in France, which also has one of the highest rates of community antibiotic consumption, more than three-fold higher than the Netherlands During the last few decades, the emergence and dissemination of multidrug-resistant bacterial strains have become a major public health issue worldwide In April 2009, the A(H1N1) virus emerged in the USA and Mexico. It spread throughout the world very quickly, resulting in outbreaks in more than 200 countries and claimed the lives of more than 18,000 persons http://www.insee.fr). The results are presented as weekly rates of antibiotic prescriptions per 1,000 inhabitants.The French National Health Insurance (NHI) program covers all medical care provided by outpatient and private practice physicians and pharmacies. We obtained aggregated 2000–2010 data on all antibiotics prescribed, dispensed by outpatient and private pharmacies, and reimbursed computed from anonymous computerized individual files in the databases of the two main NHI agencies that cover salaried workers and the self-employed (>85% of the French population). Each file contains drug-related information, prescription date, patient’s sex, year of birth and region of residence. The study concerned only systemic antibiotics  used in the community. Demographic data were obtained from the French National Institute for Statistics and Economic Studies  Weekly influenza-like syndrome (ILS) incidence was provided by the French Sentinelles Network  and Europresse (http://www.europresse.com). These online databases provide access to newspaper articles of many French and international newspapers. We conducted a search on articles published in two major national daily newspapers, Le Monde and Le Figaro, between January 2009 and August 2010, in order to retrieve and count the number of A(H1N1) influenza-related articles published in these two newspapers. Results are presented as the number of articles published in these two newspapers per week.Two online databases were used to assess media coverage of the A(H1N1) pandemic: Factiva  influenza would have been without media coverage 1 to c7) and six for the rest of the time (r1 to r6)) to the ARMA model identified in step 1. The ILS incidence was added to that model using a simple transfer function (linear function). This allows estimating an average effect of the ILS incidence during the entire period. To select the models, we first tested the independence of the residual series (Ljung & Box test) and its Gaussian distribution (Shapiro-Wilk test). When two or more models were identified, we used Schwartz information criterion and choose the one which minimize that criterion. Every step of model identification is detailed in §1 and 2 in file S1.The intervention ARMAX models were built in two steps. The first concerned the 2000–2002 period, before the first public health campaign, considering antibiotic consumption during it as the baseline. Because of seasonal fluctuations, a trigonometric function had to be estimated and removed to render the residual series in a stationary mode. We then fitted an autoregressive moving-average (ARMA) model to the observed data. The second step evaluated the impact of the public health campaigns on antibiotic consumption between October 2002 and March 2009. As those campaigns were held every year at the same period of the year since 2002, we assumed that they did not modify seasonal fluctuations or change the ARMA model structure, but affected only the means. We added 13 dummy variables   influenza media coverage had to be known  . We then® 9.1, R2.13.1 and Excel® 2010 softwares.Analyses of the entire series and by age group  were computed with SASA total of 530 articles on the A(H1N1) virus were released between January 2009 and August 2010. According to our criteria, media coverage was intense during the 42 weeks beginning in April 2009 (week 18) and ending in February 2010 (week 8) , with 50The link between viral infection and antibiotic consumption has now been very well-documented in numerous studies The data used in this study were provided by two different sources. Antibiotic-prescription reimbursements came from NHI agencies, assuring reliable and thorough data recording during the pandemic. ILS-incidence data was provided by the Sentinelles Network. This network estimates ILS incidence relying on declaration of general practitioners, without virological confirmation. During the A(H1N1) pandemic, they declared that the epidemic threshold was crossed week 36–2009. Another surveillance network (GROG), relying on samples to estimate virologically confirmed influenza number of cases, declared the start of the influenza epidemic in France week 47–2009. This discrepancy suggests there might have been an overdeclaration of ILS by general practitioners in September and October 2009, leading to an overestimation of ILS incidence by Sentinelles Network. Some studies suggest that the increase in ILS in September and October 2009 was caused by respiratory viruses other than influenza (eg rhinovirus) http://inatheque.ina.fr) devoted to A(H1N1) indicated the same media period (data not shown).To determine the period of A(H1N1)-influenza–related media coverage in France, we only examined articles from two national newspapers available in two online databases, but no local newspaper coverage, thereby probably underestimating the overall coverage of this period. However, first, because our objective was not to quantify media coverage but to determine the trend and identify the period of most intense media coverage, we considered that examination of only two national daily newspapers was sufficient; second, the comparison with weekly television-news time . The observed impact of the campaigns between 2002 and 2009 were very different depending on the series; however, another analysis of time series until June 2010 yielded estimated values in accordance with our predicted ones . For our predictions, we tried to adhere closely to the observed tendency, and used two scenarios to estimate forecasts and their 95% CI limits to avoid falsely significant results.Media attention was strong for almost 10 months (42 weeks). During that long period, no change in overall antibiotic consumption was observed, which might reflect a loss of power because prediction 95% CI, and thus the final used, widened with the time. When considering a shorter media-coverage period (18 weeks), corresponding to high A(H1N1)-virus circulation in France, underconsumption was estimated for the global antibiotic consumption, and for subjects 0–5 and >60 years old. This observed underconsumption reached −14%. Considering the antibiotic-consumption profile during 2009–2010, no antibiotic-prescription peak was seen when the ILS incidence was at its maximum, which had not been observed in any previous years. Several hypotheses can be made to explain this model-estimated result. First, as previously mentioned, the ILS incidence might have been overestimated; then adjusting to it led to an overestimation of expected antibiotic use, and consequently an observed “underconsumption” as the predicted number was higher than the observed one. Second, due to the early arrival of the pandemic influenza virus in France (September 2009) compared to seasonal influenza (December–January), general practitioners’ offices might have been less saturated than usual during the A(H1N1) period of activity as classical winter viruses were not circulating yet, generating more careful antibiotic prescription Finally, over the 6-week period without A(H1N1) virus circulating in France, overconsumption which varies from 1% to 20%, was observed for individuals >16 years. We here only assessed what would have been the antibiotic consumption “without media attention” and thererefore cannot draw any formal conclusion on the link between media attention and the observed overconsumption for individuals >16 years during this period. However, subjects 16–60 years old might have been affected by the omnipresent media coverage, because unlike seasonal influenza, it corresponds to the age category for which deaths of people without any risk factors were seen. It has already been previously documented that media coverage can influence medical care and drug consumption In conclusion, despite media attention and uncertainty about health effects, no upsurge of antibiotic prescriptions was observed for the whole study period, and in particular when the pandemic virus circulated in France. However, early during the pandemic, when the uncertainty was high, increased antibiotic use for individuals >16 years was observed. Alarmist media reporting might have heightened the worries of those individuals, resulting in increased antibiotic use.Figure S1# for age-group series.Weekly number of antibiotics prescriptions*: fit of the model and predictions for both scenarios *per 1,000 inhabitants. #Scenario 1 corresponds to the “stationary” hypothesis and scenario 2 to the “evolutionary” one.(TIFF)Click here for additional data file.Figure S2# of intervention functions for age-group series.Estimated and predicted changes for both scenarios# Scenario 1 corresponds to the “stationary” hypothesis and scenario 2 to the “evolutionary” one.(TIFF)Click here for additional data file.Text S1Identification of intervention ARMAX models.(PDF)Click here for additional data file.


Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). MetO is a promising physiological marker of oxidative stress and its inefficient repair by MetO reductases (Msrs) has been linked to neurodegeneration and aging. Conventional methods of assaying MetO formation and reduction rely on chromatographic or mass spectrometry procedures, but the use of Met-rich proteins (MRPs) may offer a more streamlined alternative.We carried out a computational search of completely sequenced genomes for MRPs deficient in cysteine (Cys) residues and identified several proteins containing 20% or more Met residues. We used these MRPs to examine Met oxidation and MetO reduction by in-gel shift assays and immunoblot assays with antibodies generated against various oxidized MRPs. The oxidation of Cys-free MRPs by hydrogen peroxide could be conveniently monitored by SDS-PAGE and was specific for Met, as evidenced by quantitative reduction of these proteins with Msrs in DTT- and thioredoxin-dependent assays. We found that hypochlorite was especially efficient in oxidizing MRPs. Finally, we further developed a procedure wherein antibodies made against oxidized MRPs were isolated on affinity resins containing same or other oxidized or reduced MRPs. This procedure yielded reagents specific for MetO in these proteins, but proved to be ineffective in developing antibodies with broad MetO specificity.Our data show that MRPs provide a convenient tool for characterization of Met oxidation, MetO reduction and Msr activities, and could be used for various aspects of redox biology involving reversible Met oxidation. Methionine (Met) is a sulfur-containing amino acid that is particularly susceptible to oxidation by reactive oxygen species (ROS). Methionine oxidation results in the formation of a mixture of two diastereomeric forms of methionine sulfoxide (MetO), which can be reduced back to Met by methionine sulfoxide reductases (Msrs) A (MsrA) and B (MsrB). These enzymes play an important role in protection of proteins against oxidative stress and have been implicated in regulation of the aging process. Insufficient repair of oxidized Met by these enzymes may alter protein structure and cause protein aggregation and/or loss of function, accelerating age-associated diseases -10. HighConventional assays for detection of MetO and monitoring its reduction rely on HPLC and mass-spectrometry procedures ,15. In aIn this regard, an important approach in developing tools for MetO research is the use of Met-rich proteins (MRPs). Previously, we identified and characterized three such proteins and developed methods to monitor their Met oxidation and MetO reduction by SDS-PAGE and immunoblot assays . In the Materials. Genomic DNA of Idiomarina loihiensis L2TR and Saccharophagus degradans 2–40 was kindly provided by Dr. Maqsudul Alam and Dr. Steven Hutcheson, respectively. Pseudomonas putida W619 cells [19 cells ,20,21. M19 cells . Reagentftp://ftp.ncbi.nih.gov) was scanned for Met content and protein length. Sequences with Met content above 20%, length more than 50 amino acids and pronounced secondary structures, were selected. Subsequently, Cys content was analyzed for each MRP. Proteins with more than 20% Met and low number of Cys (or lacking Cys) were further considered. The MRP candidates deemed more likely to be soluble were then selected by manual analysis for further experimental studies.In an initial step of the search procedure, we adopted a strategy similar to that previously used for identification of MRPs . SpecifiZea mays. MRP7 coding region was amplified from mouse liver mRNA. PCR products of MRP3-MRP6 were sequenced, digested with either NdeI and NotI or Nhe1 and Not1  and cloned into pET21b vector to generate the constructs coding for proteins with His-tags at the C-terminus. The PCR product of MRP7 was cloned into Nde1/Not1 sites of pET28a+ to generate the construct coding a protein with a His-tag at both N- and C-terminus. Soluble  and insoluble (MRP3) recombinant MRPs were purified on Talon columns following expression in E. coli using a previous procedure [Sequences coding for MRP4 (from codon 23 to stop codon), MRP5 (from codon 21 to stop codon), and MRP6 (from codon 21 to stop codon) were PCR-amplified from the corresponding genomic DNA. MRP3 coding region starting from amino acid 22 was amplified from mRNA isolated from rocedure . Mass-sprocedure .2O2 or other indicated oxidants, wherein oxidation was monitored by the mobility of proteins on SDS-PAGE gels. To verify that the shift in mobility was due to Met oxidation, the oxidized proteins were subjected to reduction with Msrs in the presence of DTT or thioredoxin (Trx) as described below.Purified MRPs were subjected to controlled oxidation by various concentrations of H2O2 at room temperature for 12 h, the oxidized proteins subjected to SDS-PAGE analysis to determine the extent of oxidation, and ESI-MS analyses were carried out to measure the number of oxidized Met residues. The conditions that resulted in oxidation of all Met in proteins (these were slightly different for various MRPs) were used to prepare oxidized MRPs at large scale for immunization. Rabbit polyclonal antibodies were produced by Covance, Inc.Purified MRP3-MRP7 were oxidized with 10–50 mM HAffinity columns containing reduced or oxidized MRP were used to enrich polyclonal antibodies specific for oxidized MRPs. To prepare an MRP affinity column, 2–6 mg of a reduced or oxidized MRP (the same preparation as that used for immunization) were coupled to NHS-activated Sepharose  according to manufacturer’s instructions. The antibody purification procedure included the following two alternatives: (i) One-step enrichment. The antisera were first purified on a protein G Sepharose column , and then the resulting IgG fraction (10–20 mg) was dialyzed against PBS and further applied to a column containing a reduced MRP at room temperature for 2 h, with shaking. The unbound fraction was collected and the Sepharose column was washed with 5 volumes of PBS, followed by elution of the bound antibodies. The unbound and wash fractions were re-purified on the column with reduced MRP twice using the same procedure. The final unbound and wash fractions were used for immunoblot analyses to examine specificity of the resulting antibodies. (ii) Two-step enrichment. The final unbound and wash fractions obtained by the procedure described above were further purified on the affinity columns containing oxidized MRPs and the bound antibodies were eluted and used for further immunoblot analyses.The DDT-dependent assay was carried out as following: 2–4 μg of an oxidized MRP were incubated with 5 mM DTT and 0.5-4 μM Msrs at 37°C for 2 h, and the reaction mixture was mixed with SDS-PAGE sample buffer and subjected to SDS-PAGE analysis. The Trx-dependent assay was as follows: 2–4 μg of an oxidized MRP were incubated with 6.8 μM Trx, 0.2 μM NADPH, and 0.4 μM Trx reductase at 37°C for 2 h, and the reaction was subjected to SDS-PAGE analysis.The three first-generation MRPs characterized in our previous work  containeNostoc sp, and LegP (herein designated MRP2) from Legionella. Recombinant MRP1 contained 15 Cys residues and MRP2 a single Cys residue. Both proteins were prepared as His-tag forms and found to be soluble. The new MRPs included DZS18 (here designated MRP3), a previously characterized storage protein from Zea mays[Pseudomonas putida W619 (designated MRP4), Idiomarina loihiensis L2TR (MRP5), and Saccharophagus degradans 2–40 (MRP6). In addition, we analyzed calmodulin, a calcium sensor protein that contains regulatory Met residues [For experimental analyses of MRPs, we selected two proteins from a previous study, including FeNos (herein designated MRP1) from  Zea mays, and thrresidues . Mouse c2O2 and for various periods of time. The as-isolated and oxidized forms of MRP1 were resolved on 10% SDS-PAGE gels and visualized by Coomassie blue staining . However, calmodulin (it contained fewer Met than other MRPs examined in our study) required approximately 50 mM H2O2 for full oxidation. These data are also consistent with the observations in a previous report [Although our data suggested that all Met residues could be oxidized by peroxide and targeted for reduction by Msrs, we observed important differences with regard to efficiency of these processes. For example, the use of Hs report  and sugg4 and FeCl3, and the mobility of MRP2 also did not change when it was treated with 25 μM CuCl2 and 25 mM ascorbate for 1 h. With regard to two-electron oxidants, H2O2 being a milder oxidant was more specific than NaOCl in oxidizing Met, whereas a NaOCl oxidized Met at lower concentrations. NaOCl generates HOCl, which is a strong microbicidal agent, capable of killing bacteria, fungi and viruses, and it can modify various residues in proteins, especially Cys, Met, Trp, and Tyr [The conversion of Met to MetO involves a two-electron oxidation process ,31. We f and Tyr ,33. Prot and Tyr . We founAnother application of MRPs is the use of these proteins as substrates in Msr activity assays. We previously found that oxidized MRPs can be reduced by Msrs in a DTT-dependent assay. In the current work, we utilized Trx-dependent reactions involving MsrA, MsrB and a fusion protein MsrBA and found that this assay is equally amenable to the analysis of MetO reduction in MRPs.Protein state- and modification-specific antibodies, both monoclonal and polyclonal, have been a great instrument in protein analysis, and this especially applies to phosphorylation state-specific antibodies . These r2O2. Since this protein was weakly detected by the same batch of antibodies in the sample not treated with H2O2, we reasoned that it is sensitive to oxidation. Previously developed antibodies against MRP3 [2O2. However, in our hands, this band was not regulated by MsrA/MsrB status and was not increased by treatment with higher concentration of peroxide. More generally, none of the antibodies we developed were able to generally recognize MetO in proteins. This is also consistent with a recent report [We tested a potential application of detecting MetO residues in proteins with the antibodies against various MRPs. MRP3 antibodies enriched on the resin with the oxidized MRP4 could recognize a 62 kDa protein band in yeast lysates treated with Hnst MRP3  also dett report .MRPs represent a convenient tool to study Met oxidation and MetO reduction. These proteins and antibodies against them could be used in a variety of applications, several of which are highlighted in our study. MetO can be formed under physiological conditions in cells, but due to low abundance of Met residues and their random oxidation, the analyses of Met oxidation and MetO reduction have been difficult. With the second-generation MRP tools, some of the outstanding questions in Met oxidation and MetO reduction processes can be addressed.Met: Methionine; MRP: Met-rich protein; MetO: Methionine sulfoxide; Msr: Methionine sulfoxide reductase; Cys: Cysteine; DTT: Dithiothreitol; Trx: Thioredoxin.The authors declare they have no competing interests.Designed experiments: XL, YZ, VNG. Carried out research: XL, AK, YZ, DTL, DH. Wrote paper: XL, VNG. All authors read and approved the final manuscript.