2eefde0ef2d54748b892970703c15b798192274e	new epidemics of infectious diseases often involve health care workers in this short communication we present a case report of a health care professional who became the fi rst case of infl uenza h1n1 virus to be notifi ed in the united arab emirates there are several issues related to workplace considerations and general public health including preventive measures the need for isolation of the patient dealing with contacts return to work and communication with the workforce	"in recent years influenza viruses have circulated in seasonal h3n2 h1n1 and avian including h5n1 forms there has been concern that influenza a h5n1 a worldwide cause of large poultry outbreaks which by december 2009 had affected 467 persons 282 deaths would drift or shift to become the next pandemic strain [1]  however in april 2009 swine flu caused by a new strain of influenza a pandemic h1n1 2009 emerged
this has now become the dominant strain producing an illness that is transmitted in the same way as seasonal influen
this is an openaccess article distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonsorglicensesbync30 which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited za which in most cases is mild which can be effectively treated with antiviral drugs and for which a vaccine is now available by the end of 2009 many countries were still reporting disease activity and an impact on healthcare services [2] 
in the early days of the h1n1 pandemic when there was uncertainty about the infectivity and virulence of the new virus a more precautionary approach to management was advocated this included laboratory testing of suspected cases contact tracing isolation of cases and contacts antiviral medication for treatment and prophylaxis and clinical surveillance and followup
in this short case report we describe the personal experience and management of the first case of h1n1 reported in the united arab emirates uae
the patient was a 48 yearold male academic public health physician who had just returned to the middle east after
wwweshaworg spending a week with his family in saskatoon in canada his journey to the uae was via calgary and heathrow airport in london uk he started feeling lethargic and developed a sore throat with cough and high fever for around 10 hours since the night of his arrival in dubai uae this led him to consult the onduty infectious disease consultant at the emergency department of a local hospital at around 800 am the following day
the consultation included a discussion of any possible exposure to h1n1 since canada was recognized then as experiencing a large number of cases of the infection a combined influenza ab antigen screen on a nasopharyngeal swab was positive and an additional swab and a blood sample were then sent for further confirmatory testing he was prescribed oseltamivir 75 mg orally twice daily for five days azithromycin 500 mg daily and paracetamol 500 mg three times daily for three days and advised to remain at home until the confirmatory test results were available
by the next morning the patients fever and sore throat had subsided and he was feeling better despite the very low but nevertheless real risk of having swine flu the patient had to make some important difficult decisions regarding his state of health and his work deadlines his work place was a university campus and as he had no lectures that day he had no need to be in contact with any students all scheduled appointments on his calendar for the day were cancelled but he decided to proceed with a tenminute scheduled presentation to six of his peers regarding a large research grant proposal a mask was not worn during the presentation and he returned home immediately after the event the patient was alone at home but one of his relatives came to visit him unannounced accompanied by his wife and a ten year old child from a neighboring town they stayed at his home for that night as the distance for return travel was considerable
on the next day the patient received a call from the health authority confirming influenza a h1n1 infection and he was therefore in the unenviable although historical position of being the first reported case of h1n1 infection in the uae the patient was admitted to hospital with airborne and contact isolation where he completed the rest of the maximum recommended 10 days quarantine period the visiting couple and child also had to stay at the patients home for 10 days of quarantine and all also received prophylactic medicine oseltamivir no lab tests were advised
as a public health physician the index case had considered the h1n1 situation before commencing his travels to canada at that time may 8 2009 the world health organization who did not recommend restricting travel although some individual national authorities were advising against nonessential travel the advice on the various websites seemed very pragmatic observe basic hygiene handwashing and cough etiquette do not travel when ill and seek medical advice if you become ill after your return the patients route to canada took him through london 34 cases reported in the uk at that time and toronto 15 cases in ontario to saskatoon 2 cases by the time he was due to return to the uae from saskatoon via calgary the number of cases in canada had increased from 242 to 496 with 19 in saskatchewan and 67 in alberta during his stay in saskatoon he did not recall meeting anyone with respiratory symptoms and he was quite well on his journey back to dubai he was therefore not certain where and from whom he caught the infection
this case raised several issues related to workplace and general public health measures taken by the uae government to prevent an influenza epidemic include the installation of thermal scanners at dubai sharjah and abu dhabi airports three major international airports in the united arab emirates the individual was afebrile and symptomfree on arrival at the airport and so was not detained for further enquiry the thermal scanners will detect individuals with fever from whatever cause but will not necessarily detect those with early h1n1 infection especially if they are afebrile [3] 
effective and timely communication is essential to allay unwarranted concerns from the public and at the workplace queries from the media were channeled to a senior member of the administration from the office of the dean to ensure consistency in the information provided he was briefed by public health physicians occupational health physicians and hospital clinicians dealing directly with the case a central news release was provided to staff and students on h1n1 reiterating the importance of hygiene in regards to limitation of transmission the workplace was a university campus this case did not have any lectures or meetings with students contact with a few coworkers was transient not more than 15 minutes in the same area these contacts were counselled on the low likelihood of acquiring the infection they were informed about seeking medical advice if they had any other reasons for concern or if they developed h1n1 symptoms doctors nurses and ancillary healthcare workers looking after the case while in hospital were briefed on hygiene and infection control procedures n95 masks gloves and gowns were provided to healthcare staff
the health department took prompt action family members with close contact were quarantined at home they were given a prophylactic course of oseltamivir adequate supplies of food and provisions and maintenance of phone communiwwweshaworg cation was confirmed the public health department dealt with general queries from the public official release of information and contact with the who was through the federal ministry of health the airline that transported the case from canada to the uae sought to contact passengers in the rows adjacent to the passengers allocated seat none of those who were traced developed any flulike illness within the incubation period following the timing of the flight
where new epidemics of infectious diseases appear history has shown that the cases have often included healthcare workers and their family members [4]  the index case for ebola infection was a hospital laboratory worker and secondary cases occurred in other healthcare workers and within the family twothirds of the deaths from the early outbreaks of ebola infection occurred in healthcare staff the early cases of sars and h5n1 infection included doctors and nurses [5] [6] [7]  the likelihood of healthcare staff being affected in such infections is high especially in the absence of adequate preventive measures or if there is poor compliance with recommended precautions in this particular first reported case of h1n1 infection in the uae prompt and appropriate action resulted in the individual being treated the risk of transmission being reduced and the provision of information being timely and adequate none of the known contacts developed signs and symptoms of the disease it was not possible to contact the taxi driver who shared the same vehicle with the case during the hour long journey from the airport home but there were no reports of infection in any dubai taxi driver in the 2 weeks following the journey
we now believe that even if they are infectious clinicians who practice good respiratory and hand hygiene will limit the risk of transmission to others standard and droplet precautions should be in place [8]  standard precautions minimize exposure to potentially infected blood and body fluids and include hand hygiene and the use of appropriate personal protective equipment droplet precautions require that a medical mask is worn when working within one meter of the patient and that when performing aerosolgenerating procedures further measures are taken including the use of eye protection n95 masks or other equivalent or more effective respirators and other personal protective equipment in addition respiratory or cough etiquette should be observed so that all persons cover their mouth and nose with a disposable tissue when coughing or sneezing and then disposing the used tissue promptly within the healthcare setting administrative environmental and engineering controls such as frequent cleaning of work areas should also be in place
generally it will not be appropriate to conduct contact tracing of patients or to provide antiviral prophylaxis however if there has been a particular type of contact between a healthcare worker and a patient for example intubation or a patient is at high risk of severe or complicated infection then further risk assessment is indicated with a view to offering prophylaxis an alternative approach if practical is to monitor exposed persons and administer antiviral treatment when symptoms develop when a vaccine becomes available the first priority should be to immunize healthcare staff
when pandemic influenza is widespread in a community it will inevitably have consequences for the workplace not least because that is a setting where transmission can occur in these circumstances occupational health practitioners should be prepared to lead a consistent and proportionate response staff with influenza will be diagnosed on the basis of symptoms the clinical diagnostic criteria are fever  38 o c or a history of fever and two or more symptoms of an influenzalike illness ie cough sore throat headache etc those who satisfy this case definition should be sent home and advised not to work until fully recovered a risk assessment should be carried out and the risk of transmission to other staff members should be considered in terms of the excess risk compared to acquiring the infection from other community sources
stories about the new h1n1 case in town appeared daily and reflected public anxiety the media can play an important role in allaying the fears of the community by providing adequate and accurate information the installation of thermal scanners at points of entry has their limitations and is not recommended by the who studies indicate many of its drawbacks including a low positive predictive value of 35 [2] 
an unpublished population study carried out in the uae during october 2009 by medical students investigated the impact of the recent h1n1 pandemic on the parents of primary school children they found that while the majority of parents had good knowledge of h1n1 and its mode of transmission many had mistaken beliefs about the origin of the virus for example thinking that it had been genetically engineered parents reported changing their behaviour because of h1n1 taking measures such as cancelling travel plans and restricting socializing also while most had confidence in the way in which the authorities had managed the pandemic they continued to worry that their families were at risk of infection and were not persuaded of the safety of available vaccines
in conclusions as for many epidemics dealing with initial cases is often a key to successful subsequent management of further outbreaks this case documents the experience of a public health physician as a patient in an infectious disease epidemic with lessons for occupational and public health management the lack of further transmission from this first case in the uae may be a combination of good and effective public health intervention or serendipity even though h1n1 has high infectivity with low case fatality rates the number of cases globally declined and who declared the end of the pandemic on 10 th august 2010"	"Shah, Syed M.. Aw, Tar-Ching..."	" Personal, Occupational, and Public Health<br>Perspectives on Dealing with the First Case of Influenza A<br>(H1N1) in the United Arab Emirates"	Saf Health Work	" New epidemics of infectious diseases often<br>involve health care workers. In this short<br>communication we present a case report of a health care<br>professional who became the fi rst case of infl uenza H1N1<br>virus to be notifi ed in the United Arab Emirates.<br>There are several issues related to workplace<br>considerations and general public health, including<br>preventive measures, the need for isolation of the<br>patient, dealing with contacts, return to work, and<br>communication with the workforce."	77	2139
b09fe466052873919115ea1da6510a01704c6c3a	background chronic cough in children is a diagnostic challenge objective to discover the utility of nasal dipsticks and polymerase chain reaction pcrdna analysis in differentiating bacterial sinusitis from other causes of chronic cough and identifying pathogens from the nasal cavity method we recruited 22 patients under 15 years of age with cough lasting longer than 4 weeks group 1 7 controls with allergic rhinitis group 2 and 10 controls without respiratory symptoms group 3 based on symptoms the results of nasal secretion assays and nasal endoscopy a diagnosis of clinical bacterial sinusitis was made we identified potential pathogens by quantitative pcr of nasal secretions results group 1a cough with clinical bacterial sinusitis n  10 eight 80 patients had bacterial sinusitis associated with dominant potential pathogenic bacteria ppb streptococcus pneumoniae haemophilus influenzae and moraxella catarrhalis group 1b cough without clinical bacterial sinusitis n  12 none had dominant ppb group 2 allergic rhinitis n  7 none had dominant ppb group 3 asymptomatic n  10 none had dominant ppb twenty to 57 of all groups were colonized with staphylococcus aureus fifty to 70 were colonized with staphylococcus epidermidis corynebacterium pseudodiphtheriticum and dolosigranulum pigrum conclusion in children with chronic cough clinicians can utilize a simple and inexpensive nasal secretion dipstick assay for rapid diagnosis of sinusitis and identify ppb by dnapcr test for specific antibiotic treatment	"chronic cough is defined as daily cough lasting 4 weeks or longer 1 in children under 15 years of age the causes of chronic cough include congenital defects asthma foreign body aspiration infections allergic diseases gastroesophageal reflux tumors and other rare causes 2 among these the most common etiologies include asthma upper airway cough syndrome protracted bacterial bronchitis and nonspecific cough 3 4 the diagnosis of asthma or cough variant asthma can be made on the basis of naepp epr3 criteria and response to a bronchodilator 5 upper airway cough syndrome is a common entity 3 4 most likely triggered by postnasal drip due to either allergic and nonallergic rhinitis or bacterial and nonbacterial sinusitis protracted bacterial bronchitis is now recognized as one of the most common causes of chronic wet cough especially in young children under 5 years of age 6 7 once asthma and other conditions listed above are ruled out upper airway cough syndrome or protracted bacterial bronchitis need to be considered as the most likely diagnoses in approaching these clinical entities our goal was to promptly detect the patients with bacterial sinusitis and the involved pathogens and to treat them with appropriate antibiotics to this end we decided to employ a novel method described by huang and small 8 who reported that nasal secretion dipstick assay was highly correlated with sinus imaging studies and could be utilized for the diagnosis of sinusitis we employed this simple approach for obvious reasons speed costeffectiveness and avoidance of radiation risk associated with sinus imaging studies we also sought an equally simple and rapid method for identification of the pathogens for the suspected sinusitis positive findings in imaging studies of sinuses such as opacities and thickened lining indicate the presence of inflammation possibly infection but may have poor correlation with clinical disease 910 and do not reveal any information on etiologies although sinus puncture would yield such information 11 this cannot be performed in most nonsurgical office settings studies have documented that nasal culture is a poor reflection of the microbes in the sinus cavity 12 therefore we analyzed the microbes from the osteomeatal area utilizing a quantitative pcr method in an attempt to detect the pathogens that may cohabit both sinus and nasal cavities
this prospective study was conducted between 2017 and 2018 with the approval of the institutional review board and human subject committee at la biomedharborucla medical center informed consent was obtained from all patients
we recruited 39 children from 1 to 14 years of age composed of 3 groups
patients with chronic cough group 1a and 1b we studied 22 children who had cough for longer than 4 weeks among these 10 patients who met the clinical diagnostic criteria of bacterial sinusitis listed below were subclassified as group 1a cough with clinical bacterial sinusitis and the other 12 as group 1b cough without clinical bacterial sinusitis
patients with allergic rhinitis group 2 we studied 7 patients with rhinitis and positive skin tests for respiratory allergens but without cough and lower respiratory symptoms
subjects without respiratory symptoms group 3 we studied 10 children without current or past respiratory complaints who were seen for routine physical checkups
all patients were interviewed for pertinent history and had nasal secretion assays and quantitative pcr analysis of nasal secretions the patients with chronic cough group 1 and allergic rhinitis group 2 had additional procedures nasal endoscopy and allergy skin testing
the clinical diagnosis of bacterial sinusitis was made when a patient met 2 out of 3 criteria consisting of positive clinical history of sinusitis nasal endoscopy and nasal secretion score as explained below
clinical history the complaint of facial pain sinus pressure or purulent nasal drainage was considered as positive history
nasal endoscopy jedmed flexible fiberoptic nasopharyngoscope st louis mo was used after the application of oxymetazoline nasal spray and 2 lidocaine spray to each nostril 3 times the presence of purulent discharge in the middle meatus or in the nasopharynx was considered to be a strong indicator for bacterial sinusitis
nasal secretion assay nasal discharge was obtained by swabbing with a wet cotton applicator 1 to 2 cm into the nasal antrum laterally and aiming at the middle meatus the applicator tip was then smeared over a urine dip strip the strip was scored in 4 components protein nitrite ph at 60 s and leukocytes at 120 s the scoring was done according to the protocol published by huang et al 8 as follows
the total score was the sum of individual scores of protein ph leukocyte esterase and nitrite a score above 3 was considered as an indicator of bacterial sinusitis
nasal secretion samples were collected by inserting the swab into the nostrils and rubbing while rotating the swab from a depth of 2 to 3 cm and sent to microgen diagnostics lubbock tx for dna analysis of nasal microbes for rapid identification of ppb potential pathogenic bacteria and ppv potential pathogenic virus
this report generated within 24 hours of sample collection utilized the rapid quantitative pcr method using a roche lightcycler 480 ii instrument that detects 7 types of bacteria and the bacterial load 1 fungus 19 resistance genes for the following antibiotics were included vancomycin methicillins betalactams carbapenems macrolides aminoglycosides tetracyclines and quinolones once the presence of bacteria was identified relative quantity abundance of these was reported in percentage of total bacterial load subsequently by using ngs nextgeneration sequencing machine the method utilized universal 16s and internal transcribed space amplicon sequencing on the ion torrant personal genome machine a bacterium with abundance greater than 50 was considered to be a dominant species
streptococcus pneumonia sp hi and moraxella catarrhalis mc were classified as ppb and staphylococcus epidermidis se corynebacterium pseudodiphtheriticum cp and dolosigranulum pigrum dp were classified as pnpb sa was classified separately rhinovirus and parainfluenza virus were classified as potentially pathogenic viruses ppv these classifications will be elaborated in the discussion section
allergy skin tests were employed to identify patients with allergic rhinitis group 2 tests were performed according to a standard procedure using 16 to 40 inhalant allergens stallergenes greer london uk applied to the skin by multitest applicator lincoln diagnostics decatur il the allergens included dust mites cat dog molds and pollens of grass trees and weeds specific to southern california
means or medians and standard deviations or interquartile ranges were computed for continuous variables frequencies and percentages were compared for categorical variables analysis of variance was used if the outcome followed a normal distribution the kruskalwallis test was used for nonnormal continuous outcomes chisquare or fisher exact tests were used to compare differences in proportions among groups sas 94 software cary nc was used for all data analyses
as stated in the methods section subjects who met 2 out of 3 sinusitis criteria were classified as group 1a nasal endoscopic findings p  04 and nasal secretion scores p  0001 were highly sensitive and could have been used as the selection criteria by themselves as shown in table 1  tables 2 and 3  and figure 1 
the group with clinical diagnosis of bacterial sinusitis group 1a carried ppb in their nasal cavity in higher percentage nppb sa and ppv were present in various degrees in all 3 groups but there were no statistically significant patterns discernible group 1b twelve patients were classified into a nonbacterial cough group since they did not meet the above bacterial sinusitis criteria two patients 17 demonstrated the presence of ppb but none of which were dominant three 25 patients demonstrated the presence of ppv antigens and 1 8 had concomitant ppb and ppv group 2 among 7 patients with allergic rhinitis 1 patient had nondominant ppb one 14 patient carried ppv 
all patients in group 1a reported improvement of cough after 2 to 8 weeks of antibiotic treatment nasal secretion assay score also decreased below 4 in all patients in group 1b were symptomatically managed with varying success without the use of antibiotics
all patients in group 1a 1b and 2 were tested for the sensitization to aeroallergens allergic sensitization was present in 5 out of 10 patients 50 in group 1a and 11 out of 12 patients 91 in group 1b
in children acute sinusitis is usually defined as symptoms lasting less than 30 days chronic sinusitis longer than 90 days and subacute sinusitis in between these 2 ranges 13 acute sinusitis is associated with sinus pressure purulent nasal drainage cough and the presence of ppb 13 14 in chronic and subacute sinusitis protracted cough may play a more prominent role than nasal symptoms 13 15 the pathology of subacute sinusitis may resemble acute sinusitis 14 while chronic sinusitis may involve inflammatory processes in association with various microbes including sa especially in patients with nasal polyposis 1416 among group 1 patients 9 patients had chronic and 13 patients subacute symptoms data not shown once we made the clinical diagnosis of bacterial sinusitis based on the previously mentioned criteria group 1a we sought to identify the predominant bacteria associated with the condition although asymptomatic children may be colonized by ppb in the middle meatus 17 the presence of these ppb in the nasal cavity of patients diagnosed with clinical sinusitis may reveal the pathogens lee et al reported a study of 31 adults with acute sinusitis in which 61 of nasopharyngeal and 48 of middle meatal samples grew ppb and that the concordance rate between the 2 sites was 84 18 we wanted to increase the sensitivity of microbial identification by employing dnabased molecular diagnostics recent studies indicate that molecular diagnostic approaches yield much more accurate results with regard to diversity and quantities of microbes present in the sinus and nasal cavities [19] [20] [21] [22] [23] once the pcr results were reported we classified these bacteria into different categories depending on their pathogenic potential sp hi and mc were classified as ppb since these have been established as predominant pathogens for acute and subacute sinusitis by previous studies 12 24 se is reported to be a commensal bacterium in the nasal cavity 25 although cp was characterized as pathogenic in some studies 26 it was reported to be part of natural microbiota of the nares and throat 27 dp has not been reported to cause sinusitis 28 sa was classified separately because of its presence in the nasal and sinus cavities of asymptomatic subjects [29] [30] [31] and patient with sinusitis 32 33 rhinovirus and parainfluenza virus were the only viruses identified and classified as potentially pathogenic viruses ppv since it is well known to cause upper respiratory symptoms as seen in tables 2 and 3  the presence of ppb as a dominant species in the nasal cavities correlated with the clinical diagnosis of bacterial sinusitis p  002 indicating that dominance of microbes over others is an important element of pathogenesis 34 the presence of a ppb in group 1b 2 and 3 is not surprising because it is known to inhabit the nasal cavities of asymptomatic subjects 17 the nondominance of these ppb along with other clinical features should enable the clinicians to separate these groups from the ones with bacterial sinusitis 1a group 1a may represent a combination of recurrent acute subacute and chronic sinusitis with bacterial infections these ppb are also reported to be the most common pathogens associated with acute otitis media 33 and protracted bacterial bronchitis 6 7 it is possible that these patients may indeed have simultaneous sinusitis and protracted bacterial bronchitis since antibiotic treatment stops coughing in both entities such coexistence via one airway needs to be explored in the future in group 1b chronic cough was most likely due to postnasal drip resulting from nonbacterial pathologies such as allergy 91 of the patients had allergen sensitization as shown in table 1  viral infection or chronic irritation although sa was present as dominant species in 2 patients in this group its pathogenic role is doubtful due to their presence in control groups 2 and 3 staphylococci were present in the sinus aspirates of acute 29 chronic sinusitis 14 20 21 and in the nasal cavities of those with crs with nasal polyps 16 and may play a role in the pathogenesis of sinusitis especially in adults 32 33 but they were also shown to be present in the nasal cavities of 25 to 30 of asymptomatic subjects 30 in a competing relationship with pnpb 22 and their presence may be a transient colonization rather than infection 36 the presence of pnpb in all groups table 2 and figure 1  is consistent with the recent studies revealing asymptomatic cohorts also carry diverse commensal bacteria in the nasal cavities including sa se dp and cp and they maintain competing relationship among themselves 20 22 an intriguing finding was the presence of rhinovirus in all groups including asymptomatic controls indicating that its colonization was not necessarily associated with clinical symptoms in group 1a 60 of the patients carried ppv and 40 carried both ppb and ppv indicating that both viruses and bacteria were coexistent rhinovirus the most common viral pathogen in our study was shown to increase adhesion of ppb to nasal epithelial cells at least 2fold compared to uninfected controls 37 38 conclusion we demonstrated as others have in children with chronic cough with sinusitis microbial balance of the sinus cavity is disturbed and ppb takes the dominance this can be detected by simple methods dipstick assay and quantitative dnapcr test of the nasal secretion therefore we recommend that clinicians utilize these methods as an alternative to imaging studies to make a diagnosis of bacterial sinusitis and offer a specific therapy for the pathogens in a rapid and accurate manner further studies are indicated to replicate these findings and refine the potential diagnostic capabilities of dnabased methods for bacterial sinusitis and bronchitis
the editors gratefully acknowledge the assistance of margaret keller md lynn smith md and joaquin madrenas md phd who reviewed the manuscript
the authors declared no potential conflicts of interest with respect to the research authorship andor publication of this article
this study was approved by los angeles biomed clinical trial registration httpsimedrislabiomedorgdatabase3090701
the authors disclosed receipt of the following financial support for the research authorship andor publication of this article dnabased analysis was provided to the authors free of charge by microgen diagnostics lubbock tx
approved by institutional review board
informed consents were obtained from all subjects who participated in this project
this article does not contain any studies with animal subjects"	"Song, Charles. Chorath, Jeena..."	 Use of Dipstick Assay and Rapid PCR-DNA<br>Analysis of Nasal Secretions for Diagnosis of Bacterial<br>Sinusitis in Children With Chronic Cough	Allergy Rhinol (Providence)	" Background: Chronic cough in children is a<br>diagnostic challenge. Objective: To discover the utility<br>of nasal dipsticks and polymerase chain reaction<br>(PCR)-DNA analysis in differentiating bacterial<br>sinusitis from other causes of chronic cough and<br>identifying pathogens from the nasal cavity. Method: We<br>recruited 22 patients under 15 years of age with cough<br>lasting longer than 4 weeks (group 1), 7 controls with<br>allergic rhinitis (group 2), and 10 controls without<br>respiratory symptoms (group 3). Based on symptoms, the<br>results of nasal secretion assays, and nasal<br>endoscopy, a diagnosis of clinical bacterial sinusitis<br>was made. We identified potential pathogens by<br>quantitative..."	226	2421
165792449dc650fba4a923f3a94a851754a7bcb7		"the international health regulations ihr 2005 as the overarching instrument for global health security are designed to prevent and cope with major international public health threats but poor implementation in countries hampers their effectiveness in the wake of a number of major international health crises such as the 2014 ebola and 2016 zika outbreaks and the findings of a number of highlevel assessments of the global response to these crises it has become clear that there is a need for more joinedup thinking between health system strengthening activities and health security efforts for prevention alert and response who is working directly with its member states to promote this approach more specifically around how to better embed the ihr 2005 core capacities into the main health system functions this paper looks at how and where the intersections between the ihr and the health system can be best leveraged towards developing greater health system resilience this merging of approaches is a key component in pursuit of universal health coverage and strengthened global health security as two mutually reinforcing agendas
background in todays increasingly interconnected and interdependent world where people goods and services move easily across borders it is more important than ever to ensure that countries are able to respond in timely and effective fashion to contain and indeed prevent threats to public health [1] [2] [3] recent global health crises including h1n1 influenza 2009 ebola 2014 and zika 2016 have resulted in pointed criticisms of the international health communitys ability to deal with such threats but crises also offer opportunities for learning and improvement an important result of such criticism has been an incremental strengthening of international resolve and knowhow to promote and improve global health security covering both individual and collective health security at globalinternational level 4 
as the leading global organisation with responsibility for health governance who has bore the brunt of the criticism [5] [6] [7] depending on the crisis accusations have ranged from responding too slowly or in ad hoc fashion to overreacting and fearmongering as well as not learning lessons and not making necessary structural and organisational reforms proposals for taking the health security agenda forward have thus included reaffirming and strengthening whos central role and the need to better resource the organisation to removing emergency response from whos purview and even setting up a new body entirely 8 9 against the backdrop of such debate who continues to implement a wider reform process which since ebola includes emergency capacities and work in promoting global health security i 
central to these discussions are the international health regulations ihr which have been at the heart of the global health security agenda since 1969 preceded by the international sanitary regulations from 1951 the ihr aim to prevent protect against control and provide a response to public health threats through improved surveillance reporting and international cooperation and to do so in ways which avoid unnecessary interference with international traffic and trade 10 
enforceability has long been seen as a concern 11 who works directly with countries to make the ihr 2005 obligations easier to implement and maintain moreover a concerted effort is underway to ensure that the ihr requirements are an integral part of essential public health operations and to better embed them into whos health systems strengthening work this is to ensure that the ihr 2005 core capacity requirements are integral to national health systems rather than seen as a topdown set of externally imposed stipulations
in making the case for better embedding the ihr into national health systems in pursuit of universal health coverage uhc this paper outlines the need for more joinedup thinking between the ihr core capacities and health system functions it provides a brief outline of the ihr before focusing on a number of important intersections with health systems and showing where they can be built on in closing we touch on actions that who is taking to increase its effectiveness in this area and stress the importance of strong health systems for delivering ihr commitments the aim is to identify a number of key issues in order to prompt discussion about health systems and global health security in general as well as whos role and the ihr
the international health regulations working for global health security following the severe acute respiratory syndrome crisis of 2003 the international community agreed to improve the detection reporting and response to potential public health emergencies worldwide this required reevaluating the existing ihr 1969 which was a framework for reporting only three infectious diseases cholera plague and yellow fever smallpox was removed in 1981 following its official eradication in 1980 the result was a new articulation of the ihr in 2005 that widened the scope of coverage to include all events including chemical and nuclear hazards that could lead to public health emergencies of international concern pheic the revised ihr 2005 came into force in 2007
in 2009 in the aftermath of the h1n1 influenza pandemic whos executive board convened an independent review of the effectiveness of the ihr 2005 12 the review highlighted a number of positives but concluded that more was required for the world to respond adequately to sustained public health emergencies and delivered a series of recommendations including lessons for future pheics what are the new findings  that health system strengthening and health security efforts for prevention alert and response need to be pursued in tandem as part of the same mutually reinforcing approach to developing resilient health systems is a new understanding  there is now a demonstrated need to embed the ihr 2005 core capacities into health systems across the six health system functions where the leadership and governance function is probably the most important to improving ihr implementation and pursuing universal health coverage uhc  uhc supports health security eg preventing outbreaks through high immunisation coverage providing early alert by rapid access of all patients to healthcare better response thanks to reliable infrastructure and healthcare workforce for case management etc while health security investment supports uhc by avoiding health crises that prevent patients accessing healthcare eg a health workforce diverted from regular care to focus on crisis response or is itself victim of the crisis as seen during severe acute respiratory syndrome influenza pandemics ebola etc or patients fear of contamination sees them avoid regular care seeking  understanding this mutual reinforcement and the urgent need for joint work and synergy between health system strengthening and health security efforts is a new concept
recommendations for policy  close coordination between the health system and health security is a new approach which is gaining momentum as major donors as well as the g7 and g20 want to see systematic coordination between uhc and global health security  things are already changing for instance through the joint external evaluations jee for country health emergency preparedness and the subsequently developed national action plans which embed health security functions within the national health system strategy and budget  in future it is expected that the bridge between health systems and global health security will become stronger given their shared objective of creating resilient health systems
 the ability to detect and assess events ensure that surveillance systems and laboratories can detect potential threats and understand the nature and potential severity and impact of the event in order to be able to make decisions in public health emergencies  notify and report events report specific diseases plus any potential public health emergencies to who through a network of national ihr focal points  verify and respond countries are expected to be able to implement preliminary control measures immediately and respond appropriately to public health risks and emergencies 10 the ihr 2005 also require some core capacity for designated airports ports and ground crossingspoints of entryat all times as well as responding to pheics in order to limit the international spread of public health risks and to prevent unwarranted travel and trade restrictions there are further expectations around countries capacity for coordination multisectoral action eg between health transport food agriculture the environment etc and ability to mutually support each other in the event of a public health emergency
once an event is reported who reviews the situation declaring the event a pheic if it is thought to constitute a public health risk to other countries through the international spread of disease and if it potentially requires a coordinated international response to date despite increasing numbers of potential events being reported and hundreds of updates and announcements posted on the ihr event information site for national ihr focal points who has declared just four pheics influenza a h1n1 pandemic 2009 intersections with health systems who supports assessments of countries ihr 2005 core capacities to date these have been selfreported and involve states parties returning annually a completed questionnaire to who implementation and reporting has not been consistent across countries 11 13 and the information does not necessarily indicate how the ihr 2005 capacity requirements are actually implemented in the country ii  to improve the quality of reporting countries have been recommended to move from exclusive selfevaluation to approaches that combine selfevaluation peer review and voluntary external evaluations involving a combination of domestic and independent experts this has been addressed by the newly proposed ihr monitoring and evaluation framework which includes in addition to the selfevaluation voluntary joint external evaluation jee simulation exercises and afteraction ii the commission on creating a global health risk framework for the future ghrf noted in its report that only a third of countries had so far complied with the ihr 2005 requirements reviews the jee and the other assessment instruments help assess gaps to develop a national action plan to strengthen country ihr capacity including through multisectoral action iii 
much of the data and feedback can also be related to how well the health system itself is functioning as the ihr 2005 address a subset of health systems strengthening and coordination challenges 14 a countrys ability to detect report and respond to health threats requires strong relationships between for example clinical laboratories and health information systems and medical technologies and between numbers of emergency personnel and training of the public health workforce moreover emergency responses to health threats involve coordination financing incident management systems public awareness and community engagement underpinned by strong government commitment and resources 15 these are all system issues and are reflected in the who health systems framework iv  which comprises six independent but interrelated building blocks working in tandem
1 service delivery 2 health workforce 3 health information systems 4 medical products vaccines and health technologies 5 health financing and 6 leadership and governance 16 a recent systematic review of the building blocks relevance to the ebola outbreak underlines their importance in practice and as an evaluative framework 17 while all of these components are necessary for organising a systemwide response this paper focuses primarily on two areas at the backbone of any response to a public health emergency and where the ihrhealth system intersections can be particularly strengthened and better institutionalised in countries leadershipgovernance and health information systems these blocks are broader functional domains requiring more crosscutting policy responses and longterm strategic planning
of all of the health system building blocks leadership and governance is probably the most important in improving ihr implementation and in countering outbreaks in general it underpins the other health system components and constitutes the cornerstone of any effort to strengthen health security this is true at both national and global level
at national level where compliance with ihr 2005 remains patchy despite a whoissued series of guidance for implementation in national legislation v  a stronger legal basis to overcome the lack of a formal enforcement mechanism and to ensure coordinated and rapid action through the health system could help to address iii see httpappswhointirishandle10665204368 iv who defines a health system as consisting of all organisations people and actions whose primary intent is to promote restore or maintain health its goals are improved health and health equity towards universal health coverage uhc v see httpwwwwhointihrlegal_issueslegislationen
some of the implementation gaps and failings already identified for instance the usa employs a public health legal preparedness phlp framework which represents a legal imperative for multisectoral action in emergencies 18 while the us framework was borne of the need to serve a federal structure there is a need for something similar in countries in order to formally mandate obligatory multisectoral responses in support of health system emergency preparedness and the ihr 2005 and while this cannot necessarily eliminate the potential for domestic political factors to impede ihr 2005 complianceas was the case with both the h1n1 pandemic and ebola outbreaksuch a mesolevel bottomup approach can help to ensure an adequate response and make the case for greater compliance this is in line with calls from civil society for a socialisation of the ihr 2005 19 the need for strong intervention at and with community level 20 and the need to confer national ownership to countries a stronger implementation of the ihr 2005 both in terms of its embedding into the fabric of health systems and into national law potentially supported via an external funding source 21 could facilitate improved and timely detection and response to health threats and governance more widely
regarding the global level whos strengthening of the ihr 2005 is not just normative but constructive in a global health environment characterised by an increasing number of actors and agencies who is the de facto steward facilitating action and collaboration within the global health system at large 22 this involves priority setting at global level and ensuring that ihr 2005 and health system strengthening activities are part of wider international frameworks and directions such as the move towards uhc and the sustainable development agenda 2030 strong health systems resilient to health crises and with robust emergency policies are central to uhc and research has highlighted that a resilient health system is indeed one that is moving towards uhc 23 24 who can help to ensure that countries work towards meeting the sustainable development goals in line with global emergency preparedness activities eg in health financing and human resources for health collaboration with relevant international initiatives such as the global health security agenda vi  support global health security as an international priority and global public good requiring full implementation of the ihr 2005
additionally there are longstanding calls for who to work more closely with nonstate actors such as the private sector and civil society vii  such engagement is necessary to institutionalise the ihr 2005 requirements and build up health systems emergency response capacity 20 as animal health transport education finance civil defence and security towards such an objective article 44 of the ihr 2005 on collaboration and assistance requires who to the extent possible to work with other international bodies and networks and this could be further leveraged in a more proactive manner
finally messaging is crucial in a global health climate characterised by the need to demonstrate outcomes it is difficult to sell prevention and preparedness governments should acknowledge that health security has a cost with no immediate apparent outcome but that such investment is irreplaceable in the face of an imminent health emergency when the health system is capable of preventing detecting or effectively addressing a public health threat the greatest beneficiary is society at large at the same time many actors of the national economy eg transport tourism and trade and the private sector also benefit thus the messaging around investing in health security needs to be less on the tools and procedures and more on the destination for example a safer world such that public health emergencies do not spread globally and have limited if any impact on international travel trade and the economy
surveillance and monitoring is another central pillar of the ihr 2005 yet many countries continue to lack the required capabilities 13 25 from a health systems perspective this is a concern but perhaps not surprising a recent review of a number of leading health system frameworks found that surveillance capacity was in general insufficiently integrated and in some cases even nonexistent as a dedicated function who unpublished report 2015 where surveillance was included it was indicatorbased in turn highlighting the need for more eventbased surveillance for quicker risk and event detection as called for under the ihr 2005
national health information systems need to have the ability to detect verify and track events as soon as possible and to ensure the flow of health data among a variety of national and international stakeholders including who moreover they need to be able to rapidly transform such data into information for realtime decisionmaking all of this implies a good integration of data sources and systems involving surveillance clinical and laboratory services alert functions evidence synthesis and communication activities census results observational data and health system resources data continuing improvement of incident management systems requires the integration and standardisation of information and reporting requirements so that they are in place during emergency responses most countries already have some type of public health surveillance system that measures disease burden and mortalitymorbidity trends in order to guide programmes and resources along with an early warning and response system for public health threats integrating the ihr 2005 requirements into such bmj global health systems and creating or strengthening them where they are weak or nonexistent is a necessity
but the ihr 2005 also have more specific surveillance requirements such those as relating to points of entry in these jurisdictions for example customs immigration shipping and conveyance authorities etc collecting public health data is rarely seen as a priority addressing this is complex it would require changing protocols to ensure that more and relevant data are collected by such systems and services on an ongoing basis as well as training officials and including public health medical personnel in such settings this is equally the case for veterinary public health and agriculture as per the ihr 2005 given the potential threats stemming from the movement of animals and livestock and food production and distribution national health information systems need to be able to speak to and have interoperability with other sectors in terms of data exchange this includes being able to capture local specificities and connect with affected communities and actors an aspect of core capacitybuilding that is not explicitly covered in the ihr 2005 and which was clearly lacking in the countries affected by the ebola outbreak in west africa 26 mobilising other health system components for health emergency preparedness and response while leadership and health information systems require longterm strategic thinking the ability to quickly activate other health system building blocks are priorities both during emergencies and for securing the health system itself fulfilment of the ihr 2005 requires contributions from all parts of the health system encompassing service delivery as well as human financial and technological resources
with regard to services how these are organised managed and delivered is the most visible demonstration of the overall functioning and efficiency of the health systemespecially during a crisisand a core component of the uhc agenda the provision and maintenance of safe healthcare services ie with infection isolation procedures in place together with other infection control services that health professionals provide is the frontline of outbreak response with respect to the ihr 2005 there is a need to improve the coordination of delivery systems for public health and clinical care around emergenciessystems need to be flexible with plans developed and functions articulated collaboration with other stakeholders most notably the private sector for improved logistics in emergencies is also needed local healthcare service providers and local communities along with civil society must be involved as well indeed community awareness can boost surveillance 13 and all can play a crucial role in the rapid delivery of key services
a related health system building block is medical products vaccines and health technologies which are central to delivering emergency response under the ihr 2005 plans for their bulk purchase stockpiling and distribution need to be in place moreover stockpiles need to be real rather than simply pledged close relations with the private sector to help with drug development and vaccine delivery in emergency situations are also required
another crucial issue for emergency preparedness and response is human resources for healthin terms of numbers and availability relevant expertise and training and deployment for ihr 2005 purposes there is a raft of profiles required from the health workforce this includes epidemiologists clinicians public health specialists laboratory personnel health information experts and biostatisticians risk communication professionals sociologists and anthropologists as well as doctors nurses and veterinarians close collaboration with the health system can help to understand the optimal size skillmix and distribution of the health workforce required and can help in the design of appropriate training curricula for instance given the centrality of laboratory systems and services to the ihr 2005 designing field epidemiology and laboratory training programmes for staff are essential as is linking them to the health system
finally the importance of financing cannot be understated in estimating the economic cost of the ebola crisis on the economies of guinea liberia and sierra leone the world bank stresses how important investment in surveillance detection and treatment capacity is would have been 27 countries need to invest in their public health institutions and infrastructure such as local laboratory and diagnostic services to identify the hazards and events which can lead to emergencies and potential pheics as well as in specialist personnel and supplies additionally being able to mobilise health system finances in an emergency situation is key a health financing component should therefore be a central element of a countrys ihr 2005 planning
in terms of more concrete actions who is further supporting ihr 2005 training and capacity development in countries promoting the effectiveness of surveillance systems and supporting timely communication and informationsharing through the global network of national ihr focal points to complement the voluntary jee under the ihr monitoring and evaluation framework who is promoting and supporting public health threat simulation exercises and afteraction review whose results reflect the actual operational capacity of the alert and response system
additionally the organisation is heeding calls for housekeeping 28 the implementation of the ihr 2005 is often done in a vertical manner outside the health system strengthening effort at national level this situation traditionally reflects a similar issue within who where the ihr programme is seen as a vertical one even though it overlaps with other frameworks eg uhc the sustainable development goals essential public health functionsoperations with individual departments bmj global health and with other programmes responsible for delivering in ihrrelated areas eg the antimicrobial resistance and vaccine preventable diseases programmes the imperative for improving internal coherence and joint working has led to the creation of the new who health emergencies programme whe designed to build up whos effective operational role in emergency preparedness and during health crises its establishment reflects a key recommendation of the ebola interim assessment panel report 8 the new programme has one workforce one budget one line of accountability one set of processes systems and one set of benchmarks and maintains a standing interdepartmental task force at headquarters and regional office levels
changes are also required in terms of more immediate programmatic and daytoday activities one proposal is for the establishment of a who crosscutting task force comprising staff from health systems whe including ihr and other relevant programmes for it is clear that there are a number of very practical questions in relation to embedding the ihr capacity requirements within health systems viii  such a who crosscutting task force and interdisciplinary group would provide guidance where technical and operational details need to be developed the group is already looking to develop a matrix crossreferencing ihr 2005 capacitiesspecifically coordination surveillance response preparedness and laboratory capabilitieswith the six health system building blocks in order to draw out areas of synergy promoting a systems approach as well as for the jee areas of work moreover there are key interlays with public health functionsall who regions have their own frameworks 29 which need to be developed such a group through its interregional composition would minimise silos and will introduce the ihr 2005 at all levels of who
stronger and more resilient health systems to improve global health security this paper has made an initial case for better embedding the ihr 2005 into health systems also highlighting whos crucial role in supporting this but what the discussion has also underlinedfor the ihr 2005 and for global health security more widelyis the importance of investing in health systems and activities to strengthen them both as an end of their own and so that they become resilient to health emergencies and can deliver health services in times when they are most needed this is also key in the pursuit of uhc the message from the us institute of medicine is that as health threats require the deployment of the same skills and infrastructure that support routine healthcare investing in strong viii this proposal developed out of an interregional meeting hosted by the who european regional office in copenhagen in april 2016 httpwwweurowhointenhealthtopicshealthsystemspages newsnews201604whotoembedinternationalhealthregulationsinhealthsystemsstrengtheningprocess and resilient health systems facilitates their emergency response capacity 30 likewise the ghrf commission stresses the need to invest in national health systems to ensure a robust global health risk framework 13 and civil society too has pressed home this point 20 additionally it should not be forgotten that public health crises also carry economic development and social consequences that could be mitigated by better health system investment upfront the world bank estimated the economic impact of ebola in guinea liberia and sierra leone through 2015 at us22 billion ix  the majority of which were economic impacts that disproportionately affected the poor who itself has consistently stated that health systems are at the heart of how countries respond to new disease threats and sustained investment to keep them strong is required 16 ultimately investing in stronger and more resilient health systems is investing in health security and towards uhc 31 32 this is not a new message and while its reiteration is important given recent public health emergencies it needs to be more nuanced and mindful of different national settings simply calling on countries such as those in west africa to invest more in order to contribute to global health security through the ihr is not helpful as a way forward strategies and policies at regional and global level to help lowerincome countries strengthen their systems will be crucial in respect of future preparedness in this regard the need for a global strategy for local investment in core capacity to detect report and respond rapidly to outbreaks is the first recommendation of the harvardlshtm independent panel on the global response to ebola 7 and others have further noted the need for a new funding source entirely 21 equally clear is that governments need to see the ihr as theirs and as part of the national health system such that investment can be sustained and activities institutionalised as ebola and other global crises have shown health systems and global health security are only as strong as their weakest linkthis points to the most fragile and unprepared states and our collective need to work together to strengthen not just their ihr 2005 capacities but more fundamentally their health systems insofar as the military provides an appropriate metaphor it is important to plan build and test our health systems capacities and responsiveness during peacetime remaining attentive to the potential for war through the sudden emergence of health threats when war erupts it is too late to begin planning for it working towards a closer embedded relationship between the ihr 2005 and national health systems is an important step in this direction and who will need to play a leading role
contributors hk provided strategic guidance gp developed the concept and undertook the purposive literature review and gp and hk drafted the manuscript ix httpsreliefwebintsitesreliefwebintfilesresources958040wp0ouo900e0april150box385458bpdf"	"Kluge, Hans. Martín-Moreno, Jose Maria..."	 Strengthening global health security by<br>embedding the International Health Regulations<br>requirements into national health systems	BMJ Glob Health	Not provided.	0	4683
d2c536058f4f78ea00836ff72187c4c1b43e47da	"the 19771978 influenza epidemic was probably not a natural event as the genetic sequence of the virus was nearly identical to the sequences of decadesold strains while there are several hypotheses that could explain its origin the possibility that the 1977 epidemic resulted from a laboratory accident has recently gained popularity in discussions about the biosafety risks of gainoffunction gof influenza virus research as an argument for why this research should not be performed there is now a moratorium in the united states on funding gof research while the benefits and risks including the potential for accident are analyzed given the importance of this historical epidemic to ongoing policy debates we revisit the evidence that the 1977 epidemic was not natural and examine three potential origins a laboratory accident a livevaccine trial escape or deliberate release as a biological weapon based on available evidence the 1977 strain was indeed too closely matched to decadesold strains to likely be a natural occurrence while the origin of the outbreak cannot be conclusively determined without additional evidence there are very plausible alternatives to the laboratory accident hypothesis diminishing the relevance of the 1977 experience to the modern gof debate
citation rozo m gronvall gk 2015 the reemergent 1977 h1n1 strain and the gainoffunction debate mbio 64e0101315"	"i n 1977 an h1n1 influenza virus appeared and circled the globe
colloquially referred to as the russian flu as the ussr was the first to report the outbreak to the world health organization who the 1977 strain was actually isolated in tientsin liaoning and jilin china almost simultaneously in may of that year 1  it was atypically mild for a new epidemic strain the influenza mortality rate imr of the 1977 flu was calculated to be 5 out of 100000 less than typical seasonal influenza infections imr of 6100000 people 2  in addition the 1977 strain appeared to affect only those 26 years of age and younger 3  these odd characteristics turned out to have a simple scientific explanation the virus was not novel the 1977 strain was virtually identical to an h1n1 influenza strain that was prevalent in the 1950s but had since dropped out of circulation 4 
the first researchers to point out the unusual characteristics of the 1977 strain suggested multiple theories to explain the remarkable preservation of the genetic information in the resurgent strain these possibilities included sequential passage in an animal reservoir in which influenza viruses replicate without rapid genetic change or perhaps a frozen [reservoir] in nature or elsewhere 4  however given the extensive experience with typical influenza strain evolution a natural origin for the 1977 strain is not likely
there are multiple potential explanations that may explain the viral resurgence but the possibility that the epidemic was the result of a laboratory accident has recently gained currency in discussions about the biosafety risks of gainoffunction gof influenza virus research and has been used as an argument for why this research should not be performed gof studies aim to better understand disease pathways but they have been controversial because they involve enhancing viral traits such as pathogenicity or transmissibility prompting biosafety concerns there is now a moratorium in the united states on funding gof research while the risks and benefits are being analyzed and the possibility that a laboratory escape could lead to an epidemic will be considered and quantified 5  given the importance of this historical epidemic to modern policy debates we compared the sequences of 1977 strains to earlier strains and examined available evidence that could explain the 1977 h1n1 resurgence we summarize these possible hypotheses discuss informal evidence and examine the trends of how the most popular explanation has changed over time in relation to political and world events explanations for the 1977 h1n1 reemergence include the deliberate release of the virus a vaccine trial or challenge mishap or a laboratory accident
confirmation that the 1977 strain was derived from a 1950s strain in 1978 researchers demonstrated that an h1n1 influenza virus strain from 1950 and another strain from 1977 fort warren [fw] and ussr90 respectively were unusually closely related although they were isolated 27 years apart 4 6 7  using the ncbi influenza virus resource database we analyzed the hemagglutinin ha sequences of all the late 1950s h1n1 strains 1947 to 1957 and compared them to the ha sequences of the 1977 isolates table 1  we found that the 1977 cluster has the closest degree of genetic similarity to strains isolated in albany ny in 1948 and 1950 strains isolated in rome italy in 1949 and strains isolated in fort leonard wood mo in 1951 instead of the fw 1950 strain examined previously fig 1  these strains are 984 identical table 2  containing only four differences among the 566 amino acids that make up the protein evidence that the 1977 h1n1 epidemic strain is derived from a 1950s virus
possible origin i deliberate release there are historical and epidemiological aspects of the 1977 influenza epidemic that can be considered suspicious during that time the soviet union employed tens of thousands of scientists to make biological weapons and as the 1979 release of aerosolized anthrax in sverdlovsk soviet union demonstrated the safety record for the weapons program was not perfect 8  in addition influenza was considered to be an incapacitating agent especially to those without previous exposure to a specific virus strain the lack of immunity to the resurgent strain was clearly evident by the affected population individuals who were 26 years of age or younger were especially vulnerable to infection as this is the predominant age range of the activeduty military population influenza virus could have been used as a biological weapon to target this group
indeed outbreaks of aussr9077h1n1 in military academies were described in official memos as explosive 9 10 the royal air force in upper heyford england was first affected in january 1978 followed by the us air force academy usafa in colorado in february the outbreak at the usafa was so severeover the course of 9 days 76 or 3280 cadets became illthat all academic and military training was suspended this was the first such interruption in training due to influenza illness in the cadet population 9  an epidemiological investigation at usafa revealed no link to other outbreaks nor a temporal association between the onset of cases and athletic competitions with other institutions with influenza cases it should be noted however that the investigations of illness at military academies were likely better investigated and documented than similar outbreaks at other universities and colleges
while it is possible that the 1977 influenza was caused by deliberate release of the virus the soviet bioweapons program biopreparat tended to use influenza preparedness as a cover story for some of the more nefarious work that was being performed 11  for example the omutninsk chemical factory manufactured large amounts of influenza vaccine and crop production bacteria aboveground while plague and tularemia were researched in heavily guarded underground facilities the omutninsk chemical factorys capacity to mass produce viruses and bacteria allowed the production of 100 tons of each weapon annually 11  while biopreparat has not been judged by experts to have seriously investigated influenza as a bioweapon there were documented attempts to find the 1918 pandemic h1n1 strain in old icehouses where victims were buried and studies were performed attempting to create radiationresistant and aerosolized influenza virus 11  thus the likelihood of a biological weapons explanation for the 1977 epidemic cannot be completely ruled out though it may not be considered likely
ii vaccine trial or challenge there are two factors that point to the 1977 epidemic as resulting from vaccine challenge or trials i live attenuated influenza virus laiv research was extensive at the time and ii a 1976 h1n1 swine flu outbreak was feared to have pandemic potential and led to a resurgent interest in h1n1 protection and research 12 
between 1962 and 1973 almost 40000 children participated in eight laiv trials in the ussr 13  scientists at the peking vaccine and serum institute in china also carried out clinical trials using live vaccines during the same time period 1  additionally there are records of the mass production of a live h1n1 vaccine in odessa ussr in 1977 14 15  in the early days of research in the 1940s laivs were often able to regain virulence upon administration to humans and cause disease 16  in addition many strains isolated from the 1977 outbreak for example the atientsin7877 isolate were temperature sensitive ts meaning that the virus could not replicate at higher temperatures temperature sensitivity generally occurs only after a series of laboratory manipulations typical in generation of laivs and is used as a biological marker of attenuation while not all of the 19771978 strains were temperature sensitive a comparison of all 1977 strains shows a higher prevalence of the ts phenotype than in 1950 strains supporting the claim that the outbreak may have resulted from attempts at attenuation for vaccine purposes 1 17  the possibility that the 19771978 strain could have resulted from a laiv trial was also mentioned in a personal communication from c m chu renowned virologist and the former director of the chinese academy of medical sciences to peter palese who described the introduction of this 1977 virus [as] the result of vaccine trials in the far east involving the challenge of several thousand military recruits with live h1n1 virus 18  whether this involved an ineffectively attenuated vaccine or a laboratorycultivated challenge strain the deliberate infection of several thousand people with h1n1 would be a plausible spark for the outbreak
the timing is probably not coincidental in 1976 the swine h1n1 epizootic influenza virus infected 230 soldiers at fort dix nj causing severe respiratory illness in 13 and one death 12  edwin kilbourne and others led a campaign that resulted in president gerald ford announcing a program to inoculate everyone in the united states against swine flu and the concomitant production of 150 million doses of influenza vaccine however the program was halted soon after as it became clear that anew jersey 1976 was not spreading outside the basic training group it is possible that an archival h1n1 strain from the early 1950s was used as a challenge virus to evaluate the efficacy of the h1n1 vaccines prepared in response to the 1976 swine flu outbreak if this virus were not attenuated properly it may have been able to spread and cause a global epidemic iii laboratory accident a biosafety lapse in a research laboratory is now most often cited as the cause of the 19771978 reemergence of the h1n1 influenza virus strain fig 2  the evidence in favor of this possibility is the clear unnatural origin of the virus and its temperature sensitivity suggesting laboratory manipulations at the time of the epidemic however the world health organization excluded the lab accident possibility after discussions with influenza virus laboratory researchers in the soviet union and china finding that the laboratories concerned either had never kept h1n1 virus or had not worked with it for a long time 1  it is likely that the swine flu scare the previous year prompted the international community to reexamine their stocks of the latest previously circulating h1n1 strains to attempt to develop a vaccine however the tripartite origin of the outbreak in northeast china that produced almost identical isolates is not supportive of the conclusion that this was a single laboratory accident
it is more likely that either the vaccines produced from these stocks or the viruses themselves used in tests of vaccine development were virulent enough to spark the 1977 epidemic the bulk of the evidence rests with this possibility the unnatural origin mildness of presentation of the virus widespread dissemination of cases in a short amount of time temperature sensitivity of the samples contemporary observations and existence of livevirus vaccine trials which were occurring at that time
explanations been influenced by political considerations fig 2  in 1991 in the last days of the soviet union researchers suggested that the virus was potentially frozen in nature until its reemergence an unsatisfying explanation that places no blame on china or russia for the incident see reference 13 in the appendix in 2008 it was suggested that the epidemic was probably the result of an influenza vaccine trial 19 20  the 2009 h1n1 flu pandemic h1n109 virus brought the 1977 epidemic back to the forefront as there were soondiscredited rumors that it was the result of a lab accident 21 22  this reenergized the discussion on the origin of the epidemic with explanations now assum[ing] that the virus was kept frozen in a yet unidentified laboratory although how it was released was left in doubt 23 24  however morerecent publications focusing on the gof debate have strengthened this stance concluding that it was almost certainly due to an escape from a virology lab 25 26 also see references 34 to 41 in the appendix additionally proponents against gof research continue to use the potential reemergence from a laboratory accident in their slides and presentations at debates and public forums as a cautionary tale some examples include the risks and benefits of gof studies are performed with the aim to better understand disease pathways but have been controversial because they involve enhancing viral traits such as pathogenicity or transmissibility prompting biosafety concerns coupled with recent laboratory accidents at the us centers for disease control and prevention cdc the controversy over the potential risks of gof research led to the recent decision by the us government to pause federal funding for gof influenza research and severe acute respiratory syndrome sars research until an assessment can be made of its risks and benefits 30  the moratorium for mers and other coronavirus research was lifted but the evaluation of gof influenza research risks and benefits is expected to take nearly a year 31 32 
while the use of the 1977 influenza epidemic as a cautionary tale for potential laboratory accidents is expedient the relevance to gof research is greatly diminished if the 1977 epidemic was the result of a vaccine trial or vaccine development gone awry these are both more plausible explanations than a single laboratory accident in addition in 1977 influenza research was performed without modern biosafety regulations and protective equipment making the lab accident hypothesis much less relevant to the modern gof debate while the events that led to the 1977 influenza epidemic cannot preclude a future consequential accident stemming from the laboratory it remains likely that to this date there has been no realworld example of a laboratory accident that has led to a global epidemic 29 "	"Rozo, Michelle. Gronvall, Gigi Kwik"	 The Reemergent 1977 H1N1 Strain and the<br>Gain-of-Function Debate	mBio	" The 1977-1978 influenza epidemic was probably<br>not a natural event, as the genetic sequence of the<br>virus was nearly identical to the sequences of<br>decades-old strains. While there are several hypotheses<br>that could explain its origin, the possibility that<br>the 1977 epidemic resulted from a laboratory<br>accident has recently gained popularity in discussions<br>about the biosafety risks of gain-of-function (GOF)<br>influenza virus research, as an argument for why this<br>research should not be performed. There is now a<br>moratorium in the United States on funding GOF research<br>while the benefits and risks, including the<br>potential for accident, are analyzed. Given..."	213	2256
a7dea443868b1327fef754465f3792ea33a224ca		"being sessile organisms plants are constantly challenged by their environment and their situation is compounded by biotic stresses a number of plant pathogens such as fungi oomycetes bacteria viruses nematodes etc pose serious threats to the plant wellbeing nonetheless over the course of evolution plants have acquired a refined twolayered immune system to respond to pathogen attack 1 the first line of plant immunity thought to be the most ancient relies on the recognition of evolutionarilyconserved pathogen molecules known as pamps pathogenassociated molecular patterns and is therefore referred to as pamptriggered immunity pti [2] [3] [4] pattern recognition receptors prrs are plant components responsible for the detection of pamps 5 and for activating the immune machinery of plants one of the best characterized prrs in plants is flagellin sensitive 2 fls2 a receptor kinase that activates pti upon perception of flagellin a conserved protein found in bacterial flagellum 6 7 to gain greater access to plant resources for subsequent colonization plant pathogens just like their animal equivalents deploy an arsenal of highlysophisticated molecules known as effectors these molecules greatly augment the pathogens capacity to propagate on its host by interfering with various cellular processes including pti fortunately plants monitor the presence of some effectors through their resistance rproteins which constitutes the second line of defense also known as effectortriggered immunity eti 1 eti typically results in a strong hypersensitive response characterized by cell death which shows some mechanistical similarities with apoptosis in animals 8 it is regulated by direct physical interaction between a rprotein and its corresponding effector ligandreceptor model or between a rprotein and a hostprotein modified by an effector guard model resistance thus depends on the presence of both the rprotein and its corresponding effector a situation depicted by flors geneforgene model 9 10 for pathogens to succeed proper delivery of these effectors is as crucial as the molecule itself the bacterial type three secretion system t3ss one of many secretion systems deployed by pseudomonas syringae is wellcharacterized and has been studied in great detail the syringelike t3ss provides bacteria with a robust mechanical structure which enables it to inject key molecules involved in pathogenicity directly into host cells 11 obligate biotrophic filamentous pathogens such as many fungi and oomycetes are devoid of such secretion systems instead they invaginate within host cells to form particular infection structures called haustoria 12 13 to accommodate haustoria host cells are forced to greatly expand their plasma membrane and it is plausible that pathogens drive this process for their own benefit
filamentous pathogens have a large suite of predicted secreted proteins which could act early during infection to suppress pti as the pathogens are establishing themselves and at later stages to rewire host cellular activities to meet the pathogens metabolic needs it has been proposed that protein trafficking from haustoria allows pathogens to hijack host cells for their own purposes however the precise mechanism governing effector translocation from the extrahaustorial space to host cells has eluded scientists thus far 14 for the purpose of this review we have classified effectors into three types based on the subcellular compartment they target apoplastic effectors cytoplasmic effectors and nuclear effectors apoplastic effectors can be secreted by appressoria and or hyphae invading the intercellular space where they remain outside the cells this class of effectors includes proteins with inhibitory functions interfering with plant proteases and peroxidases for example the avr2 effector from the biotrophic fungal pathogen cladosporium fulvum suppresses basal defense through inhibition of specific host proteases [15] [16] [17] on the other side cytoplasmic and nuclear effectors affect host defense mechanisms by several obligate biotrophic phytopathogens namely oomycetes and fungi invade and feed on living plant cells through specialized structures known as haustoria deploying an arsenal of secreted proteins called effectors these pathogens balance their parasitic propagation by subverting plant immunity without sacrificing host cells such secreted proteins which are thought to be delivered by haustoria conceivably reprogram host cells and instigate structural modifications in addition to the modulation of various cellular processes as effectors represent tools to assist disease resistance breeding this short review provides a birds eye view on the relationship between the virulence function of effectors and their subcellular localization in host cells
targeting proteins involved in plant immune signaling cascades moreover they also manipulate various plant processes further predisposing the host cellular machinery to act in a pathogenconducive manner 18 19 as their names suggest cytoplasmic effectors target cytosolic components or are redirected to other organelles while nuclear effectors transit via the cytosol but have a different purpose than the other two effector types described in subsequent sections the biology of infection of obligate biotrophic pathogens is rather unique due to the establishment of haustoria the different strategies deployed by intracellular biotrophic hyphae produced by various pathogens to secrete their effectors are beautifully illustrated by giraldo and valent 13 in this minireview we offer a retrospective of the molecular interactions between obligate biotrophic pathogens and their hosts speculating on this rather intimate relationship at the molecular level and focusing on cellular components representing potential effector targets
it is pertinent to demystify the terminological ambiguity around effectors since until recently their nomenclature was contingent upon host reactions when a molecule from a particular pathogen modulates the hosts defensive cover to increase the pathogens fitness it is called a virulence factor however when the same molecule is recognized by host immunoreceptors thereby failing to augment pathogenicity and instead triggering a defense response it is referred to as an avirulence factor this variation in pathogenicity is a commonlyoccurring phenomenon a particular effector may be a virulence factor on one host and an avirulence factor on another a situation observed even within a single plant species where interactions are racespecific because of this inconsistency terms such as virulence and avirulence have their limitations since they are dependent on the specific host system in which they have been observed the above discussed terminology in plant pathology is thus rather different from that employed in the medical field in plant immunity the terms virulence and avirulence are mainly related to the plants ability to resist or succumb to the pathogen thus depending on plant genotype 9 in the medical field avirulence refers to the loss of a virulence component belonging to the pathogen consequently an inclusive and neutral term such as effector is preferred 20 as it accounts for all the molecules secreted by a pathogen during infection that alter host cell structure or function 21 as mentioned earlier flors work was instrumental in establishing the geneforgene concept 9 10 flor was quite foresighted when he noted that for each gene conditioning a reaction in the host there is a corresponding gene that conditions pathogenicity in the pathogen 9 his deduction came from studies on the inheritance of pathogenicity in flax rust melampsora lini and on the inheritance of resistance in flax linum usitatissimum 10 many years later the flaxflax rust pathosystem remains instrumental in our understanding of the molecular aspects of geneforgene interactions this pathosystem enabled inroads to be made in the molecular interaction between rand avrprotein mainly through studies of l and m resistance genes and their corresponding avr loci flax rust avrl567 genes whose products are recognized by the l5 l6 and l7 rproteins of flax are highly diverse and under diversifying selection pressure with 12 sequence variants identified from six rust strains 22 ravensdale et al 23 studied direct molecular interactions between l5 and l6 two alleles of l and their avirulence targets in detail sitedirected mutagenesis in avrl567 and the construction of chimeric lproteins revealed that the recognition specificities of l5 and l6 are conditioned by their leucinerich repeat regions their study indicated that mutations in the tir or nbarc domain also affect recognition which prompted the authors to suggest that interaction with the avr ligand directly competes with intramolecular interactions causing rprotein activation 23 the avrm effector from flax rust also interacts directly with the flax rprotein m and this interaction can also be observed in yeast twohybrid assays catanzariti et al showed that the cterminal domain of avrm is required for mdependent celldeath consistent with the fact that it interacts with mprotein in yeast 24 furthermore these authors demonstrated that cterminal 34 amino acids formed a structured domain unlike the nterminal part of the protein and gel filtration revealed that avrma can dimerize 22 recently ve et al resolved the structure of avrm and avrma and showed that both possess an lshaped fold and form a dimer with an unusual nonglobular shape 25 the avirulence properties of avrm and avrl have been described but yield no clues with regard to their targets and their potential virulence functions few rust effectors have been shown to be expressed during infection and translocated to host cells one of these effectors is rusttransferred protein 1 rtp1 which belongs to a family of effector proteins specific to the order pucciniales 26 rtp1 from uromyces fabae was the first rust effector demonstrated to localize in host cells and it was also observed that the transfer of the protein was dependent on the developmental stage of haustoria 27 rtp1 translocates from the extrahaustorial matrix where it first accumulates transits through the cytoplasm then further moves to the nucleus 27 unlike most localization studies cited herein which are mainly based on green fluorescent protein gfp fusion and transient expression rtp1 localization was assessed by immunolocalization during uromyces fabae infection using four independentlyraised polyclonal antibodies 27 rtp1 sequence analyses indicated that the cterminal domain exhibited similarities to cysteine protease inhibitors and rtp1 was indeed shown to inhibit proteolytic activity 26
when dealing with a subject as broad as effectors it is worthwhile to classify them to the extent that current knowledge in this domain will allow therefore in an attempt to draw clear lines they can be largely divided into three major groups based on their localization and site of activity apoplastic cytoplasmic and nuclearnucleolar effectors
as the name suggests apoplastic effectors are localized to plant extracellular spaces this class of effectors includes but is not restricted to small and cysteinerich proteins which function primarily by inhibiting host proteases hydrolases glucanases and other lytic enzymes 13 recent models suggest that these could be the first effectors to potentially activate the plant defense response pti 13 the architecture of these effectors often having a signal peptide and a cysteinerich cterminus is highly reminiscent of plant small signaling peptides 28 which may reflect the prototypic structure that a protein must harbor to survive its passage in apoplastic space however apoplastic effectors may have a much more refined mechanism and could exert a longlasting action in protection of the pathogen cell wall or in chelatingneutralizing antimicrobial compounds being secreted by the host
on the other hand cytoplasmic effectors have the duty of dealing with host cells at a much more intricate level cytoplasmic effectors are active once they reach the plant cytoplasm and tend to target plant defense signaling components effectors from p syringae have been shown to target antipathogenic vesicle trafficking and kinasebased recognition activity of the host a prime defense component 29 some effectors may also transit through the cytoplasm to reach their final destination eg organelles
nuclear effectors are seemingly ultimate weapons in the inventory of pathogens since they are thought to suppress the immune response from upstream nuclear effectors could potentially shut off master switches of the immune machinery or reprogram host transcription to the benefit of pathogens a recent investigation of 49 putative effectors from h arabidopsidis revealed that 33 localized strictly to the nucleus and an additional 33 were nucleocytoplasmic 30 since several effectors tend to migrate toward the nucleus it would be logical to assume that some rproteins act in the nucleus indeed several rproteins such as snc1 n and rps4 were found to localize to the nucleus [31] [32] [33] [34] tobacco tirnblrr rprotein n localizes to the nucleus in the absence of its elicitor the tobacco mosaic virus p50 helicase fragment 32 lending support to a default presence of rproteins in the nucleus to monitor their corresponding effectors rather than being relocalized upon effector binding however snc1 and n nuclear accumulation is reduced at elevated temperatures making their mode of action temperaturedependent 35 it was demonstrated recently that eti is more active at low temperatures 1023 c while pti takes over at higher temperatures 2332 c  36 it has also been shown that bacterial pathogens strive and multiply at higher temperatures but secrete their effectors more actively at lower temperatures 37 38 these observations suggest that the immune system of plants is adapted to pathogen physiology however some pathogens prefer more temperate environments around 18 c for optimal growth 3940
computer software such as nod psort ii and wolf psort can predict the subcellular localization of various proteins but that of very few candidate effectors has been verified experimentally [41] [42] [43] relative to the wealth of those from all plant pathogens a number of plant pathogensecreted effector proteins have been reported to localize in the nucleus but most localization studies have been conducted with gfptagged assays it should be noted that gfp fusion may abrogate proper effector localization either by hiding a sorting signal or by inducing change in the 3d structure of native effectors which could prevent interaction with a protein involved in true effector localization in addition most of these experiments are transient assays and do not examine localization during infection therefore although gfp represents a very powerful tool at our disposal to identify subcellular effector localization care should be taken when analyzing the results however since gfp does not diffuse to the nucleolus it is safe to assume that nucleolar localization is effectordriven rxlr effectors such as harxll3b haatr13 emoy2 and harxl44 from hyaloperonospora arabidopsidis localize to the nucleolus of plant cells 30 in phytophtora capsici crn effectors all localize to the nucleus and at least two have been found to accumulate in the nucleolus suggesting that there might be subnuclear localization domains 44 the nucleolus is a multifunctional subcellular organelle critically involved in ribosome biogenesis and protein synthesis 45 several dna viruses and retroviruses are known to target the nucleolus umbravirus orf3 potato leafroll virus capsid protein and influenza virus nucleoprotein are some examples of viral proteins localizing to the nucleolus [46] [47] [48] [49] given that viruses are entirely dependent on the host machinery to translate their genome into proteins they are expected to target the nucleolus however one can wonder why biotrophic filamentous pathogens would target this subnuclear compartment the effector harxl44 from the obligate biotrophic pathogen h arabidopsidis was recently shown to target nucleolar and nuclear mediator subunit 19a med19a this interaction results in med19a degradation in a proteasomedependent manner med19a degradation appears to shift transcription from salicylic acidresponsive defense to jasmonic acid and ethyleneresponsive transcription thereby conning the host to enhance its susceptibility 50
what happens once a pathogen gets access to its host how does the host respond to the pathogens demands and what are the overall cellular dynamics in play answering such questions becomes a lot more imperative when dealing with obligate biotrophs because of their intimate relationship with the host and since they can only survive in living cells obligate biotrophic pathogens thus have to be subtle when dealing with their host after invasion first of all they have to keep host immunity in check at all times by suppressing pti second they have to continuously feed from plant cells finally they need to steadily propagate and multiply
fungal spores grow on plant surfaces upon germination it has been shown that the rust fungus uromyces appendiculatus uses topographical cues for orientation and the formation of infection structures 51 once u appendiculatus detects a 05m ridge which it interprets as the presence of the stomatal lip its entry point into tissue it starts producing its infection structure 51 when the pathogen has forced its way into plant tissue nutrient acquisition and defense suppression occur primarily through haustoria although effectors are also released from growing hyphae support for such a mechanism is lent by deep sequencing of the biotrophic growth phase of colletotrichum higginsianum during a thaliana infection 52 in this pathosystem effector genes are expressed in consecutive waves associated with pathogenic transition and some are expressed before host invasion at the appressorial stage 52 in fact multistage transcriptome analysis of melampsora laricipopulina the causative agent of the poplar leaf rust obligate biotroph revealed that a number of smallsecreted proteins were even expressed in resting urediniospores 53 therefore we can infer that suppression of plant immunity starts prior to the formation of haustorial structures in host tissue while our understanding of molecular partners at play is progressing we have made few inroads into the establishment of planthaustoria interactions and postinvasion events dynamic interplay could be mainly driven by the invader and as we progress in this review we will examine some important phenomena that may hold clues to these questions
it should not be difficult to conceptualize massive host cellular reprogramming occurring in response to the development of haustoria haustoria are found to be surrounded by endoplasmic reticulum actin cytoskeleton and cytoplasm along with the accumulation of golgi bodies and mitochondria 54 it has also been observed that a significant amount of tonoplast is present around these complexes 54 to host such critical appendages cells have to expand their plasma membrane tremendously haustoria are separated from the host cytoplasm by an extrahaustorial matrix ehm the ehm has been speculated to be mostly of host origin sealed from haustoria by a hautorial neck band 55 56 however it differs from the plasma membrane in both cytological and biochemical properties 55 57 the ehm also appears to vary in composition over time 58 59 recently lu et al 60 reported that some plasma membrane resident proteins relocalize to the extrahaustorial membrane during infection for example the aquaporin pip14 and the calcium atpase aca8 remained at the plasma membrane during infection with either h arabidopsidis or phytophtora infestans while the syntaxin pen1 penetration deficient 1 the synaptotagmin syt1 and the remorin strem13 were present in the extrahaustorial membrane around p infestans haustoria interestingly this relocalization appears to be pathogendependent since prr fls2 localized in the ehm of p infestans but remained at the plasma membrane and was excluded from the ehm in h arabidopsidis however the most remarkable feature of this cellular rearrangement is the position of the nucleus studies have shown that the arabidopsis nucleus stays close to h arabidopsidis haustoria 30 and this is presumably driven by the actin cytoskeleton 61 62 it is possible that proximity of haustoria to the nucleus enables pathogens to deliver their effectors more quickly to the nucleus for cell reprogramming proximity of the nucleus to the intruder would thus be driven by the pathogen per se but one cannot exclude that host plants could steer this process autonomously to respond quickly to pathogen attack
pathogens are known to target host vesicular trafficking a key element of plant defense 30 in h arabidopsidis 26 of examined effectors have been found to localize to membranes the majority of them 18 associating with the endoplasmic reticulum 63 arabidopsis cells hosting h arabidopsidis haustoria develop bulging vesicular structures compared with noninfected cells 30 the occurrence of such vesicles being attributed to presence of the pathogen it is possible that the formation of these vesicles is driven by a particular effector or effectors to upset vesicular movement and disrupt any organized defense response they may also be pathogendriven and provide the extraphospholipid bilayer required at the plasma membrane to accommodate fastexpanding haustoria regardless support for the fact that these are vacuolar structures comes from the observations of very similar structures in cotyledons of transgenic arabidopsis tipgfp plants 64 other types of membrane structures have been shown to differentially localize around haustoria formed by h arabidopsidis and p infestans 60 harxl17 localizes to the ehm during infection by h arabidopsidis however in the absence of the pathogen it localizes to the tonoplast where its ability to enhance plant susceptibility is possibly linked with a task in plant cell membrane trafficking 30 since tonoplast is located close to the ehm along with the effector harxl17 in the event of infection the effector may be interfering with plant cell membrane trafficking and interestingly this also suggests a role for tonoplast in ehm formation however no single effector has been reported to cause the bulblike vesicular structures observed in the presence of growing pathogens 29 and it is not clear whether it is a plant defense response or an effectordriven process surprisingly our understanding of the detailed mechanism of vacuolar biogenesis is still limited justifying the need to push the investigation further into such peculiar vesicular structures it is difficult to elucidate possible pathways being targeted by pathogens to hinder vesicular trafficking and eventually give rise to these bulblike structures in a thaliana a point mutation in the deubiquitinating enzyme amsh3 renders cells incapable of forming central lytic vacuoles in addition amsh3 mutant cells accumulate autophagosomes and incorrectly sort their vacuolar protein cargo 65 vacuoles are important in various plant defense mechanisms and two vacuolemediated mechanisms have been postulated to affect programmed cell death 66 in one of them vacuolarprocessing enzymes mediate vacuolar membrane disruption thus releasing vacuolar content into the cell cytoplasm demonstrated for viral infection 67 in the second proposed mechanism vacuole fusion with the plasma membrane enables the extracellular release of vacuolar content demonstrated in bacterial infection 68 interestingly and coincidentally phenotypic similarity between vesicular structures from amsh3 mutants and cells hosting haustoria can be noticed 60 65 this concurring vesicular signature suggests that pathogens could be targeting amsh3 or similar components to alter the vesicular pathway
octomericexocyst complexes could also be targeted by pathogens given that the exocyst architecture plays an important role in vesicular tethering and redefining cell polarity which are integral to plant defense responses 69 targeted exocytosis occurs during infection and freshlysynthesized defenserelated compounds are delivered to infection foci which eventually leads to asymmetrical plasma membrane development small gtpases from the rab and rho families are known to be essential in this process which involves delivery anchoring and integration of secretory vesicles to the plasma membrane 70 71 whereas the exocyst complex works as a scaffold in tethering operations 72 73 the final process of attachment is mediated by the integral membrane proteins vsnare and tsnare where plasma membrane and vesicle bilayers are fused together to complete the process 74 75 it has already been demonstrated that upon mutating two exocyst subunitsexo70b and exo70h1 from arabidopsis plantsare more susceptible to infection validating their importance in plant immunity 69 pen1 is a classic example of proteins preventing penetration by pathogens pen1 encodes a syntaxin known to interact with the snare proteins snap33 and vamp72 76 and regulates papillae formation in cells under attack 77 papillae are bellshaped cell wall appositions deposited in epidermal cells within papillae various secondary antimicrobial metabolites accumulate along with lytic enzymes and reactive oxygen species which stops the pathogen penetration peg in arabidopsis pen1 is found in significant amounts when the nonhost fungus blumeria graminis f sp hordei endeavors an unsuccessful invasion however when the host fungus erysiphe cichoracearum successfully penetrates arabidopsis cells pen1 is then downregulated 77 the pen1 single mutant allows increased penetration of the nonhost fungus b graminis f sp hordei thereby showing that pen1 helps in procuring an effective penetration barrier 77 thus pen1 could participate actively in polarizing secretion events that lead to papillae formation 77
obligate biotrophic phytopathogens have evolved a robust and elaborate offensive strategy to invade their host by deploying numerous effector proteins it appears that the effectors inventory of pathogens is organized around different types of molecules which have unique capabilities and functions therefore most socalled effectors should be considered candidate effectors a crude way to envision effector deployment is to see apoplastic effectors at the onset of attack performing all the bullwork and setting the stage for more sophisticated weaponry true cytoplasmic effectors could act at the intermediate stage by deactivating local surveillance paving the way for nuclear effectors to enter the nucleus taking over the entire defensive network and stalling the complete immune setup nucleolar effectors from various pathogens are increasingly being reported 44 78 79 and it is likely that they have an important function in pathogenesis many cellular processes including plant defenses depend on the formation of new proteins thus further study needs to be undertaken to understand the task of nucleolar effectors some effectors are also involved in disrupting vesicle trafficking and as such they may be compromising vacuolar integrity which is believed to play a significant role in plant defense plant cells hosting haustoria experience unique cellular rearrangements that are likely influenced by haustoria themselves and driven by secreted effectors
as genomesequencing costs are falling the full sequences of many more genomes are becoming available despite the dazzling speed at which effector catalogs can be assembled functional study of effectors remains a relatively slow and strenuous process in obligate biotrophs functional studies of effectors by virulence assays are hindered by the lack of molecular genetic approaches as a result alternative tactics with heterologous systems are increasingly being adopted given the very large repertoire of effectors observed in obligate biotrophic fungi such as rusts that encode over 1000 small secreted proteins 80 81 one could propose that the outcome of each effector may be a lot more subtle than the bacterial effectors of pseudomonas syringae that have roughly 30 or so effectors 82 and a direct quantifiable impact on virulence may prove difficult to observe since the cumulative result of many effectors may be required alternatively redundancy could explain the huge number of effectors in filamentous pathogens in either case deciphering the interactions of these effectors will likely reveal many unknown components of various plant processes with these issues in mind localization remains one of the first aspects to consider when assessing effector functions in addition combination of genetic evidence and proteinprotein interaction approaches either yeast twohybrid assay coimmunoprecipitation or bimolecular fluorescence complexes may prove to be the best ways of investigating effectors from biotrophic pathogens
no potential conflicts of interest were disclosed"	"Chaudhari, Prateek. Ahmed, Bulbul..."	 Effector biology during biotrophic invasion<br>of plant cells	Virulence	Not provided.	0	4371
9a8ffb71928a37a550e689f36fa3196493e036fe	it has been proposed that cholesterol in host cell membranes plays a pivotal role for cell entry of hiv however it remains largely unknown why virions prefer cholesterolrich heterogeneous membranes to uniformly fluid membranes for membrane fusion using giant plasma membrane vesicles containing cholesterolrich ordered and cholesterolpoor fluid lipid domains we demonstrate that the hiv receptor cd4 is substantially sequestered into ordered domains whereas the coreceptor ccr5 localizes preferentially at ordereddisordered domain boundaries we also show that hiv does not fuse from within ordered regions of the plasma membrane but rather at their boundaries ordereddisordered lipid domain coexistence is not required for hiv attachment but is a prerequisite for successful fusion we propose that hiv virions sense and exploit membrane discontinuities to gain entry into cells this study provides surprising answers to the longstanding question about the roles of cholesterol and ordered lipid domains in cell entry of hiv and perhaps other enveloped viruses	"cell membranes consist of numerous proteins and lipids exhibiting complex behavior that includes organization into dynamic nanodomains enriched in sphingolipids and cholesterol that are sometimes referred to as lipid rafts 1 2  accumulating evidence indicates that membrane domain organization plays a vital role in cell signaling and adaptive immune responses to combat infections by many pathogens 3 4 5  for example hiv has evolved to exploit organized membrane domains to gain entry into host cells 6 7 8 9  it is well established that recognition and attachment of hiv to host cells are mediated by the binding of the viral envelope glycoprotein gp120 to the cellsurface receptor cd4 and a coreceptor ccr5 or cxcr4 10 11  after attachment membrane fusion mediated by subunit gp41 of the envelope glycoprotein leads to cell entry because cd4 has been found to associate with detergentresistant fractions of the cell membrane it has been assumed that cholesteroland sphingomyelinrich rafts are platforms for hiv entry 12 13  however because lipids in cholesterolrich regions are more tightly packed and more ordered than those in the surrounding membrane these sites may seem energetically unfavorable for membrane fusion thereby raising doubts about the involvement of ordered lipid regions in the fusion step of hiv entry
because ordered lipid domains are thought to be dynamically fluctuating nanoscopic assemblies of lipids and resident proteins in living cells visualization of the potential involvement of these membrane regions upon entry of viral particles into intact cells remains technically challenging 14 15  in a first step toward solving this problem we showed recently that model membranes with coexisting microscopic ordered and fluid lipid domains were useful in proving that the fusion peptide of gp41 interacts preferentially with ordereddisordered lipid domain boundaries and that these boundaries are also the preferred sites for fusion peptidemediated membrane fusion 16  however these discoveries raised the important question of whether the results obtained in this highly artificial model system could be translated to biological membranes that contain the hiv receptor and coreceptor and to virus particles bearing the full trimeric gp120gp41 complex that is whether membrane domain boundaries would still be the preferred sites for virus attachment andor membrane fusion at the plasma membranes of appropriate target cells therefore we developed a new approach to measuring binding and fusion of hiv particles with plasma membranes to assess the role of membrane heterogeneity in these processes
we used giant plasma membrane vesicles gpmvs derived from hela cells that stably express the cd4 receptor and ccr5 coreceptor cd4  ccr5   investigated the lateral partitioning of both receptors in these membranes and imaged the preferred regions of hiv binding and fusion at the singleparticle level fig 1a  gpmvs show temperaturedependent micrometerscale liquidorderedliquiddisordered lold phase separation and have been extremely useful to study the dynamics and lateral distribution of resident membrane proteins 17 18  using this approach we found that lipid order discontinuities in heterogeneous receptorand coreceptorcontaining plasma membranes are important for gp120mediated receptorcoreceptor targeting and gp41mediated membrane fusion
a number of studies have been conducted to localize cd4 and ccr5 in lymphocyte cell membranes although the association of cd4 with lipid rafts is generally accepted divergent conclusions have been reached regarding the raft localization of ccr5 6 12  confirming previous results we observed by immunofluorescence that cd4 colocalized with raftresident ganglioside gm1 in cd4  ccr5  hela cells whereas ccr5 colocalized only partially with gm1  fig s1  when we prepared gpmvs from cd4  ccr5  hela cells by gentle formaldehyde and dithiothreitol dtt treatment movie s1 and fig s2 a and b which forms membrane blebs by releasing otherwise intact cell membranes from the cytoskeleton 17 18  we found that most gpmvs showed microscopic lold phase separation below 25c movie s2 and fig s2  c to e as expected from immunostaining of intact cells cd4 and gm1 were substantially excluded from the ld region in gpmvs indicating the partitioning of cd4 into the lo region  fig 1  b and c preferential localization of ccr5 at the boundaries between lo and ld phases occurred and was evident in cellattached blebs and celldetached gpmvs fig 1 b and c  this result might explain the debate about the controversial association of ccr5 with lipid rafts ccr5 is not associated with lo or with ld lipid phases but accumulates at the boundaries between them very similar results were obtained with an alternative procedure to induce membrane blebs by the application of small concentrations of nethylmaleimide nem  fig s3  suggesting that the results are independent of the blebbing agent and that the resulting gpmvs do not have serious membrane defects
recognition of lold membrane boundaries by hiv next we examined whether and how hiv envelope env particles interact with gpmvs murine leukemia viruses mlvs pseudotyped with hiv gp120gp41 and fluorescently labeled with mkogag were incubated with cd4  ccr5  gpmvs we visualized bound particles by epifluorescence microscopy and found that they migrate along the preparation of largescale phaseseparated gpmvs facilitates the study of the lateral distribution of cd4 and ccr5 and the role of ordered lipid domains in hiv entry lo and ld phases on gpmvs are indicated with red and blue lines respectively b and c partitioning of cd4 and ccr5 in cellattached b or celldetached c gpmvs gpmvs were first stained with 11didodecyl3333tetramethylindodicarbocyanine perchlorate dii at 4c for 60 min and then incubated with fluorescentlabeled ctxb alexa fluor 555 anticd4 alexa fluor 488 or anticcr5 alexa fluor 647 antibodies at 4c for 60 min epifluorescence images of gpmvs performed at room temperature 22c show largescale lold phase coexistence in which dii and ctxb are used as markers of ld and lo phases respectively the selected rectangles are enlarged and shown below d hiv binding to cellattached gpmvs cellattached gpmvs left and viruses labeled with mkogag center are visualized by brightfield and epifluorescence microscopy respectively image overlay right shows that some bright dots hiv env particles indicated by arrows are sitting on the gpmv surface top time series of images from the selected box region shows that the particles bound to gpmv are free to move laterally along the gpmv surface bottom a movie version of this figure is available movie s3 e and f hiv binding to the boundaries between lo and ld phases in cellattached e or celldetached f gpmvs gpmvs left and hiv env particles center were labeled with dio and mkogag respectively g quantification of virions bound to three different regions lo ld and lold boundary of the gpmvs data are means  sd of triplicates a total of 120 virions were analyzed for their distribution between the three compartments the largest fraction of virions was found in lold boundary regions scale bars 10 mm the gpmv surfaces  fig 1d and movie s3  staining the gpmvs with the ld marker 33dilinoleyloxacarbocyanine perchlorate dio showed that most bound particles localize to lold boundary regions in cellattached fig 1e movie s4 and fig s4  and celldetached gpmvs  fig 1f and movie s5 we quantified the virions bound to three different regions lo ld and lold boundaries as shown in fig 1g  to assess whether the targeting of the virions to the lold interface is specific to hiv env we also performed similar experiments with mlvs pseudotyped with the env g protein glycoprotein from vesicular stomatitis virus vsvg vsvg virions preferentially bound to the ld phase in gpmvs  fig s5  indicating that the lold boundary interaction is not a general feature of all viral particles and requires hiv env given that cd4 is initially localized in lo phase regions of the membrane it seems reasonable to propose that hiv virions bind to cd4rich lo phase membrane regions and then move to domain boundaries where they also engage with ccr5 ccr5 binding and the preferential targeting of the gp41 fusion peptide to lipid phase boundaries 16 likely combine to direct the particles for fusion at these boundaries
to determine whether hiv env particles indeed fuse with gpmvs at lipid phase boundaries we first examined whether they fuse with gpmvs in a bulk lipid mixing assay virions were labeled with selfquenching concentrations of the fluorescent lipid probe r18 and mixed with unlabeled gpmvs lipid mixing was only observed when the gpmvs contained both cd4 and ccr5 but was negligible with gpmvs that were derived from hela cells lacking receptors and coreceptors  fig 2a  demonstrating the specificity of the hivgpmv fusion system we found that lipid mixing is strongly suppressed by the wellknown hiv entry inhibitors maraviroc and enfuvirtide fig 2b  maraviroc inhibits binding to the coreceptor ccr5 19  and enfuvirtide inhibits fusion by blocking the required conformational change of gp41 20  maraviroc reduced the targeting of hiv virions to phase boundaries fig 2c  further supporting the notion that ccr5 recognition occurs at these boundaries by contrast enfuvirtide had little effect on the boundary preference of hiv binding while still blocking fusion fig 2d  suggesting that boundary binding alone is not sufficient for fusion it requires the energy from refolding gp41 into the sixhelix bundle structure as well
we also observed fusion with gpmvs at the singleparticle level the fluorescence of many hiv env particles that were bound at lold phase boundaries spread over time indicating that the particles fused with the gpmvs fig 2e  in addition we carried out electron cryomicroscopy cryoem in an attempt to directly visualize the process of fusion of viral particles with gpmvs as previously observed for bare mlvs containing only gag and gagpol 21 the spherical or tennis racketshaped particles showed a characteristic morphology with a capsid surrounded by the viral envelope fig 2f  representative electron micrographs obtained from four different incubations with two separate viral and two separate gpmv preparations show hiv env particles in intimate contact with the gpmv surfaces  fig 2g  and fig s6  we also observed membrane perturbations in very confined areas of the gpmv lipid bilayer that may represent fusion intermediates fig 2g  to ensure that the observed contacts were specific controls with gpmvs lacking cd4 and ccr5 were mixed with hiv env particles fifteen to 30 randomly selected gpmvs per grid were imaged for three different samples and no virusgpmv fusion events were observed however the present resolution of the cryoem images does not permit a distinction between lo and ld phases and is thus not sufficient to determine whether fusion occurs at the lold phase boundaries
next we examined whether the lold phase coexistence on gpmvs is required for hiv env particle binding andor fusion to disrupt lo phase domains we treated gpmvs with methylbcyclodextrin mbcd which depletes cholesterol from the membranes mbcd treatment of gpmvs led to a marked change from approximately circular lo domains to corrugated shapes that are characteristic for bilayers in the gel phase  fig 3a and movie s6 however cholesterol extraction did not significantly alter the overall surface expression levels of cd4 or ccr5 on gpmvs fig 3b  virion binding was quantified by directly counting the number of particles attached to gpmvs fig 3c  or by a centrifugationbased assay  fig s7  regardless of the method particle binding was high and not diminished by cholesterol depletion of the gpmvs indicating that the presence of lo domains is not required for the binding of virions however hiv env particles did not bind to gpmvs lacking cd4 and ccr5 further confirming that their attachment is mediated by these receptors rather than lipid phase separation
contrary to virion attachment disruption of the lo phase domains in gpmvs by mbcd significantly decreased the efficiency of fusion of hiv env virions with gpmvs  fig 3d  this result agrees with a previous report demonstrating a decrease of hiv cell entry after depletion of cholesterol from the host cell membrane 22  the inhibitory effect on fusion by the disruption of lo phases and lold phase boundaries in gpmvs suggests that the unique properties of these boundaries are responsible for attracting receptors and coreceptors and the high fusogenicity of hiv env virions at these sites thus the same physical principles that promote fusion peptidemediated fusion in lipid model membranes namely line tension and lipid packing defects at lold phase boundaries 23 also provide a significant driving force for membrane fusion of hiv env viral particles with biological membranes
lysolipids which adopt an invertedcone molecular shape have been reported to inhibit fusion in a wide variety of systems owing to their propensity to induce positive intrinsic membrane curvature 24 25  here we tested whether lysolipids affect the lipid phase behavior of gpmvs and the ability of hiv to fuse with them lysosphingomyelin lysosm partially dissolved the lo domains of gpmvs into much smaller domains whereas lysophosphatidylcholine lysopc had little effect on lo domains  fig 4a and movie s7 although both lysosm and lysopc share a choline headgroup and a similar molecular shape they may differently affect line tension at phase boundaries lysosm is a linactant that appears to reduce line tension whereas lysopc probably partitions more uniformly into the ld phase with a lesser effect on line tension hiv env virion binding was not affected by lysosm or lysopc fig 4b  however lysosm significantly reduced the fusion efficiency of hiv particles whereas lysopc had only a moderate effect fig 4b  together these results again support the notion that the site of fusion is the domain boundary region where inhibitory lysosm is enriched but lysopc is not enriched the same effect could be triggered enzymatically treatment of gpmvs with phospholipase a2 pla2 which hydrolyzes glycerophospholipids including pc to lysopc had little effect on the lipid phase behavior of gpmvs and hiv fusion whereas sphingolipid ceramide ndeacylase scdase which converts sm into lysosm dispersed the lo domains which eventually spread over the entire gpmv surface and significantly inhibited fusion fig 4  c and d note that the fluorescent lipid probe dii always marks the ld phases in these images the apparent reversal of contrast in some images is due to different levels of cholesterol that can change the connectivity of lo phases as previously demonstrated in model systems 26 
to further probe the influence of lysolipids on lipid phases and hiv fusion in welldefined model membranes we used giant unilamellar vesicles guvs and large unilamellar vesicles luvs composed of bsmbpcbpscholesterol 2111 as models 16  only lysosm significantly changed the lo domains in guvs fig 4e  and inhibited the fusion of luvs with hiv fusion peptidedecorated virosomes whereas lysopc had only a small effect at the highest concentration fig 4f  the domain edges first undulated before larger invaginations formed eventually dispersing the domains into much smaller ones movie s8 regulating the size of lipid rafts by using lysosphingolipids has significant consequences not only on hiv entry but also on cell signaling for example it is well known that sphingosine1phosphate is a potent mediator of numerous signaling pathways 27 and the finding that these signaling mechanisms could be potentiated by raft regulation may be an interesting topic for future investigations as previously reported the recruitment of some proteins such as ras and rac1 to domain boundaries may be more general and may contribute to the regulation of signaling pathways by regulating the nature and size of lipid rafts in these systems 28 29 
supported lipid bilayers generated from synthetic and natural lipids have been widely applied since their original introduction 30  including more recently to study fusion of single particles 16 31  the planar geometry of supported membranes has the advantage over guvs that a large extended membrane can be observed by total internal reflection fluorescence tirf microscopy and therefore that large numbers of single events can be simultaneously recorded with high signaltonoise ratio in each experiment leading to greatly improved statistics 32  to exploit this advantage for biological membranes as well we have developed supported planar plasma membranes sppms for singleparticle fusion studies fig 5a  our strategy for preparing sppms is to fuse gpmvs onto polymersupported lipid monolayers that have been transferred from a langmuir trough 33  which is different from previous methods 34 and preferentially orients membrane receptors with their binding sites accessible from solution similar to plain supported lipid bilayers 26 35  sppms prepared in this fashion display micrometersized domains in which raft and nonraft lipid components are laterally mobile  fig s8  in agreement with the observations with gpmvs the overlay of cd4 and ccr5 images in sppms shows that cd4 was substantially associated with lo domains whereas ccr5 was preferentially located at the edges of the domains fig 5b and fig s9  we observed by tirf microscopy that single hiv env particles bound predominantly to domain boundaries whereas most vsvg particles bound to ld phases on sppms fig 5  c and d in addition these experiments recapitulate the observation made with gpmvs that the location of hiv binding to sppms is determined primarily by the location of the receptor and coreceptor in these membranes and not by the lipid phases fig 5e 
we observed large numbers of singlemembrane fusion events of hiv env particles with sppms  fig 5f and movie s9 and classified them as docking hemifusion or full fusion fig 5g in lo ld and lold compartments of the sppm we observed no direct full fusion and very little hemifusion in ld phases and only 11  3 direct full fusion and 13  4 hemifusion of hiv virions in the lo phase domains fig 5h  in contrast hiv virions fused very efficiently 56  3 of all particles at the lold domain boundaries and an additional 9  4 hemifused in these locations fig 5h  these results are qualitatively similar to but quantitatively more robust than those obtained in gpmvs and therefore validate sppms as a useful additional system to quantitatively analyze fusion in heterogeneous biological membranes at the singleparticle level
membrane fusion in hiv entry is a highly cooperative process that must overcome several energy barriers associated with different fusion intermediates this energy must be provided and released at the right time and at the right place to drive the merger between the viral and target membranes although cholesterolrich lipid domains have been implicated in cell entry their proposed involvement was puzzling because ordered lipids are thought to be detrimental to membrane bending and fusion however we recently discovered that lold phase boundaries facilitate fusion in model systems and demonstrated that line tension at these boundaries might provide an additional previously unrecognized driving force for fusion 23  this previous work was limited because no receptors or coreceptors were present and because it could be argued that pure lipid model membranes oversimplify biological membranes to overcome these limitations we demonstrated in this work that a very similar mechanism governs hiv fusion with biological host membranes containing its natural receptors we directly observed that lipid domain boundaries are the portals for hiv binding and membrane fusion presumably because they present the weakest points in the host membrane on the basis of these results we propose the following model hiv gp120 binds to cd4 receptors located in cholesterolrich lipid domains which exist transiently in the plasma membranes of living cells if located near ccr5 coreceptors at domain boundaries this initial binding leads to structural changes of gp120 and binding to the ccr5 coreceptor enriching bound virions at the boundaries fig 6a  after cd4 and ccr5 binding gp41 changes its conformation to expose the fusion peptide to lipids in the boundary region 16  the predisposed preference of ccr5 for membrane domain boundaries greatly facilitates the targeting of the fusion peptide to these same fusionpromoting areas of the host membrane although we did not examine the other hiv coreceptor cxcr4 in this study it might function equivalently to ccr5 in recruiting virions to domain boundary regions for more efficient fusion because receptors cooperate with membrane domain boundaries to form hiv entry sites it would be interesting to examine in future studies the binding and fusion efficiencies of hiv particles with gpmvs or sppms where cd4 and ccr5 are distributed differently or randomly
in contrast to many other enveloped viruses hiv displays only a few 14 trimeric glycoprotein spikes on its envelope 36  this small number is certainly inefficient for hiv entry and fusion however the cooperativity of hiv recognition and fusion at membrane domain boundaries may be a unique way to overcome this deficiency and may provide enough energy to drive fusion through its transition states with low env copy numbers just as in the model systems 23  line tension at the boundary likely serves as an additional driving force for fusion it is well known that cd4  t cells play a central role in the immune response following t cell antigen receptor engagement which triggers t cell activation and proliferation against invading pathogens 37 38  t cell activation elicits the recruitment of a number of proteins to ordered lipid regions 39 and results in the formation of larger membrane domains which can be exploited by hiv to facilitate entry fig 6 b and c  hiv exhibits infection at a much lower frequency in nave cd4  t cells than in activated cd4  t cells 40 41  suggesting that hiv infection may be associated with the coalescence of cholesterolrich lipid domains just as in model systems we have shown here that line tension may also be a dominant force that promotes membrane fusion in biological membranes the boundaries of the larger lipid domains in activated t cells could contribute to the elevated fusogenic capacity of activated t cells versus resting t cells it is possible that activation of the immune system against other invading pathogens makes the cells more prone to hiv entry through the described mechanism therefore enhanced domain activation could lead to an elevated production of hiv in people infected by other pathogens and thus an acceleration of the progression of aids fig 6d 
finally this work also showed that gpmvs and a novel procedure to produce supported planar gpmvderived membranes that we call sppms are very useful to study the selected targeting and membrane fusion of hiv virions we anticipate that these methods will also be useful to study the binding fusion and properties of many other particles on cellderived plasma membranes the new approaches are versatile and should lead to many new discoveries regarding the role of lipid and protein heterogeneity of cell membranes they may also serve as novel platforms for screening of viral entry inhibitors 
most lipids and fluorescent probes were from avanti polar lipids and dio dii did and octadecyl rhodamine b chloride r18 were from invitrogen mbcd nem and fluorescein isothiocyanate alexa 647 or alexa 555labeled ctxb were purchased from sigmaaldrich formaldehyde and dtt were purchased from avantor and rpi respectively alexa 488labeled cd4 antibody and alexa 647labeled ccr5 antibody were purchased from novus biologicals hiv entry inhibitors maraviroc and enfuvirtide scdase and pla2 were purchased from sigmaaldrich hiv fusion peptide was customsynthesized by the yale wm keck biotechnology resource laboratory
hiv pseudovirus production hiv env particles pseudotyped with envelope gp160 protein and containing mlv core proteins were prepared by cotransfection of 293t cells with 3 mg of pfbluc mlv vector plasmid 2 mg of phit60 packaging construct 1 mg of mlv mkogag and 3 mg of hiv jrfl env plasmids using the polyfect reagent qiagen 16  the viruscontaining medium was collected at 48 hours after transfection and centrifuged at 2500 rpm for 10 min at 4c to clear debris to prepare didlabeled hiv particles 293t medium was replaced with optimem i gibco life technologies containing 10 mm did at 16 to 20 hours after transfection after 4 hours of incubation at 37c unincorporated dye was removed by washing and cells were returned to regular growth medium after 24 hours of incubation the extracellular medium was collected centrifuged at low speed to remove cell debris filtered through a 045mm syringe filter and stored at 80c until use for control experiments mlvs pseudotyped with vsvg and mlv core proteins were also prepared for bulk binding and lipid mixing assays membranes of viral particles were labeled with 20 ml of octadecyl rhodamine b chloride 1 mgml r18 in 50 ml of virus for 60 min at room temperature and then free r18 was removed on a sephadex pd10 desalting column ge healthcare the infectious titer was determined by a luciferase assay as previously described 42  in cd4  ccr5  hela cells for hiv env pseudotyped viruses
preparation of gpmvs derived from cd4  ccr5  hela cells cd4  ccr5  hela cells were provided by d m rekosh university of virginia gpmvs were produced and isolated from the cells as previously described 17 18  with slight modifications briefly cd4  ccr5  hela cells were cultured in iscoves modified dulbeccos medium with 10 fetal bovine serum fbs g418 02 mgml 1 penicillinstreptomycin penstrep and puromycin 1 mgml in an incubator at 37c with 5 co 2  plain cd4  ccr5   hela cells which lack cd4 and ccr5 were grown in dulbeccos modified eagles medium 10 fbs and 1 penstrep in the same incubator after growing the cells to a confluence of 70 to 90 in culture dishes cells were washed three times with blebbing buffer [10 mm hepes 150 mm nacl and 2 mm cacl 2 ph 74] membrane vesiculation was then induced by adding 25 mm formaldehyde and 2 mm dtt or 2 mm nem in ordered membrane domains in resting t cell plasma membranes are nanoscopic and shortlived but small dynamic domains can coalesce to create larger ones to function as signaling platforms upon pathogen invasion helper cd4  t cells recognize the pathogenderived antigens on the surface of antigenpresenting cells apc and become activated by coalescence of membrane domains the activation of cd4  t cells stimulates the ability of b cells and cd8  t cells to defend against the invading pathogens in this model we propose that cd4  t cells that are challenged by pathogens are more prone to hiv infection than resting t cells as indicated by the red arrows the hiv infection leads to apoptotic t cell death and ultimately results in the progression to aids
blebbing buffer for 1 hour at 37c following shaking solutions containing celldetached gpmvs were gently decanted into a 50ml tube the gpmvs were placed on ice for 30 min to settle relatively large gpmvs 10 mm in diameter which were then transferred from the bottom of the tube to coverslips for observation on an epifluorescence microscope
lateral distribution and relative amount of cd4 and ccr5 on gpmvs we observed the lateral distributions of cd4 and ccr5 on cellattached and isolated gpmvs for the preparation of cellattached gpmvs cd4  ccr5  hela cells were seeded on quartz slides or 18mm glass coverslips and grown to about 40 confluence after inducing gpmvs from the cells as described above they were rinsed extensively with phosphatebuffered saline pbs to remove gpmvinducing chemicals cellattached gpmvs or isolated gpmvs were blocked with 01 bovine serum albumin in pbs and then the vesicles were stained with the ld marker dii 02 mm or dio 05 mm andor the lo marker alexa 555conjugated ctxb 1 mgml for 60 min on ice after labeling the gpmvs were incubated with an anticd4 antibody andor an anticcr5 antibody conjugated with alexa 488 andor alexa 647 respectively at a concentration of 5 mgml at 4c for 60 min the lateral distribution of fluorescent lipids and proteins on cells or gpmvs was visualized using an epifluorescence microscope the immunofluorescence staining was also used to evaluate the amount of cd4 and ccr5 on the surface of gpmvs for quantification isolated gpmvs were incubated with antibodies at 4c for 60 min and then unbound antibodies were removed by centrifugation 16000g for 30 min at 4c gpmv pellets were resuspended in 400 ml of hepes buffer relative amounts of cd4 and ccr5 were measured at room temperature by fluorescence emission intensities of alexa 488 at 520 nm and alexa 647 at 667 nm in a jobinyvon fluorolog3 spectrofluorometer jobinyvon with excitation wavelength at 495 and 650 nm respectively gpmvs from cd4  ccr5  hela cells were used as negative controls and displayed only low or negligible fluorescence intensity
single and bulk binding assay of hiv env particles to gpmvs for the binding of single hiv env particles to gpmvs gpmvs were induced from cd4  ccr5  hela cells on quartz or glass slides after staining with dio or 7nitro213benzoxadiazol4yl nbddope gpmvs were rinsed with buffer in a fabricated flowthrough chamber hiv particles labeled with mko mkogag were injected into the chamber containing the cellattached or isolated gpmvs and incubated at 4c for 60 min binding of the particles to gpmvs was visualized by epifluorescence microscopy for binding sites of virions and the number of virions bound to gpmvs was counted for binding efficiency binding sites of vsvg env particles on gpmvs were also performed for control experiments to quantify bulk binding efficiency of hiv r18labeled viral particles were added to gpmvs labeled in a tube with nbddope and incubated at 4c for 60 min unbound particles were removed by centrifugation 16000g for 30 min at 4c the pellets containing virusbound gpmvs were resuspended in 400 ml of 05 vv triton x100 fluorescence emission intensities of r18 at 590 nm and nbd at 535 nm were recorded at room temperature in a jobinyvon fluorolog3 spectrofluorometer jobinyvon with excitation wavelength at 560 and 460 nm respectively the ratio of fluorescence intensity at 590 nm535 nm was used to measure relative binding efficiencies of hiv env particles to gpmvs
fusion between r18labeled virus particles and unlabeled gpmvs was measured by the dequenching of r18 fluorescence unlabeled gpmvs were added to the r18labeled virions 1  10 8 particles in hepes buffer at room temperature concentration of gpmvs was estimated by total protein concentration in gpmvs using a bicinchoninic acid assay protein concentrations of gpmvs derived from cd4  ccr5   mbcdtreated cd4  ccr5   and plain cd4  ccr5  hela cells were 101 87 and 62 mgml respectively fluorescence intensities were measured under constant stirring using a fluorolog3 spectrofluorometer jobinyvon with excitation and emission wavelengths of 555 and 590 nm respectively dequenching of r18 fluorescence was normalized to the initial fluorescence ff 0 
to prepare the samples for electron cryomicroscopy cryoem hiv env particles were incubated with gpmvs for 60 s at room temperature to capture early stages of membrane fusion between hiv particles and gpmvs samples 35 ml were applied to either cflat or quantifoil holey carbon grids manually blotted to near dryness with filter paper and rapidly plunged into a slurry of liquid ethane the grids were then transferred under liquid nitrogen to a tecnai f20 cyroem philipsfei operating at 120 kv images were recorded at magnifications of 11000 or 30000 under lowelectron dose conditions 20 e   2  using a 4k  4k chargecoupled device ccd camera gatan
for cholesterol depletion isolated gpmvs from cd4  ccr5  hela cells were incubated with 5 mm mbcd in hepes buffer for 30 min at room temperature other gpmvs were incubated with 5 mm lysosm or 20 mm lysopc in hepes buffer for 30 min at room temperature to examine the effect of lysolipids on lipid phases yet other gpmvs were added to 10 u of pla2 in 10 mm trishcl buffer ph 85 or 10 mu of scdase in 50 mm sodium acetate buffer ph 60 which was adjusted to an osmolality of 300 mmolkg by the addition of nacl and incubated at room temperature for 30 min after incubation gpmvs were isolated by centrifugation 16000g for 30 min at 4c and were used for measurements of hiv binding and fusion
fluorescence images were recorded on a zeiss axiovert 200 fluorescence microscope carl zeiss wi"						
h a mercury lamp as a light source 40 or 63 water immersion objectives carl zeiss numerical aperture 095 and an electronmultiplying ccd cooled to 70c ixon dv887escbv andor as a detector images were acquired using homemade software written in labview national instruments membranes stained with dio or nbd were epiilluminated through a 480nm bandpass filter d48030 chroma and via a dichroic mirror 505dclp chroma fluorescence was observed through a 535nm bandpass filter d53540 chroma diior rhodaminestained membranes and mkolabeled viral particles were epiilluminated through a 540nm bandpass filter d54025 chroma and via a dichroic mirror 565dclp chroma fluorescence was observed through a 605nm bandpass filter d60555 chroma viral particles or luvs stained with did were illuminated through a 620nm bandpass filter et62060 chroma and observed through a 665nm bandpass filter hq66560 chroma all fluorescence imaging was performed at room temperature 22c images were processed and assessed for colocalization using imagej								
guvs were prepared via the electroformation technique briefly 25 ml of a 10 mm lipid mixture composed of bsmbpcbpscholesterol 2111 containing the fluorescent lipid probe rhpe [01 mole percent mol ] was deposited on the conducting surfaces of two indium tin oxidecoated glass slides after the slides were placed in a vacuum desiccator for 90 min to remove residual solvent a fabricated chamber composed of two conducting slides facing each other separated by a 05mm spacer was filled with 300 mm sucrose in h 2 o guvs were generated by applying alternating electric current 3 v 10 hz through a function generator for 120 min at 60c and transferred into a 300 mm glucose solution to settle by gravity on the microscope slide								
lipid mixing of luvs mediated by hiv fusion peptide luvs were prepared by extrusion phospholipids composed of bsm bpcbpscholesterol 2111 were dissolved in a mixture of chloroform and methanol and the solvent was evaporated under a stream of nitrogen gas in a glass test tube the thin lipid film was further dried overnight under vacuum and hydrated with 05 ml of hepes buffer [10 mm hepes and 120 mm nacl ph 72] the suspension was vigorously vortexed for 5 min was subjected to 10 freezethaw cycles and was then extruded 21 times through two stacked polycarbonate filters with a pore size of 100 nm avestin the lipid mixing assay was based on fluorescence resonance energy transfer between nbdpe and rhodaminepe the hiv fusion peptide was added to 50 mm luvs with a ratio of 19 of labeled 1 mol  of nbdpe and rhodaminepe each to unlabeled luvs in hepes buffer at room temperature the fluorescence was recorded under constant stirring in a fluorolog3 spectrofluorometer jobinyvon with the excitation and emission wavelengths set at 460 and 535 nm respectively the value for 0 lipid mixing was the fluorescence intensity of the luv suspension before the fusion peptide was added whereas the value for 100 lipid mixing was obtained by adding triton x100 [final concentration 05 vv] to the suspension at the end of each experiment								
quartz slides were cleaned by boiling in contrad detergent for 15 min and by sonication in a hot bath for 30 min after rinsing with deionized water and ethanol the slides were immersed in piranha solution 31 mixture of 95 sulfuric acid and 30 hydrogen peroxide to remove remaining organic residues followed by extensive rinsing in pure water the first leaflet of the sppm was prepared by langmuirblodgett lb transfer directly onto the quartz slide lipid mixtures composed of bsm bpccholesterol 221 with 3 mol  of dmpe 12dimyristoylsnglycero3phosphoethanolaminepolyethyleneglycoltriethoxysilane 43 were spread onto a pure water surface in a nima 611 lb trough ksv nima after allowing the solvent to evaporate for 10 min the monolayer was compressed at a rate of 10 cm 2 min to reach a surface pressure of 32 mnm immediately before use quartz slides were further cleaned for 10 min in an argon plasma sterilizer harrick scientific and then dipped into the trough at a speed of 200 mmmin and withdrawn at 5 mmmin while keeping the monolayer surface pressure constant at 32 mnm the transferred lipid monolayer was dried in a vacuum desiccator at room temperature overnight and cured in a 70c oven for 40 min to covalently link the polymer to the sio 2 slide surface after equilibration in the desiccator to room temperature the slide with the tethered polymersupported lb monolayer was placed in a custombuilt flowthrough chamber a suspension of gpmvs in hepes buffer was injected into the chamber and incubated for at least 2 hours at room temperature to form the distal leaflet of the sppm bilayer excess unfused gpmvs were then washed out by extensive rinsing with hepes buffer								
the integrity of the sppms and the diffusion of the lipids within them were examined by fluorescence recovery after photobleaching frap bilayers were bleached in a pattern of parallel stripes and the data were fit to the model ft  f   f 0  f  expda 2 t where f 0 and f  are the initial and final fluorescence intensities after bleaching respectively a  2pp p is the stripe period 127 or 32 mm and d is the lateral diffusion coefficient the mobile fraction mf which reflects the percentage of observed fluorescence recovery within the time frame of a frap experiment 1 min is given by mf  ff0 f pre f 0  200 where f pre is the fluorescence intensity before photobleaching ten regions on three independently prepared bilayers were sampled to determine the reported average values								
binding and fusion of single hiv env particles on sppms by tirf microscopy to measure binding to sppms mkolabeled mkogag hiv env particles were injected into a chamber with unlabeled sppms and the fluorescence increase by hiv binding to sppms was monitored over time by prismbased tirf microscopy the prismquartz interface was lubricated with glycerol to allow easy translocation of the sample cell on the microscope stage the light source was an argon ion laser innova 90c coherent emitting light at 514 nm and fluorescence was observed through a 610nm bandpass filter d61060 chroma the beam was totally internally reflected at an angle of 72from the normal to produce an evanescent wave the intensity of the laser beam was computercontrolled through an acoustooptic modulator isomet or could be blocked entirely by a computercontrolled shutter the laser intensity shutter and camera were controlled by a homemade program written in labview national instruments to investigate the targeting of hiv particles to different regions of ldlo phaseseparated sppms mkolabeled particles were incubated with sppms that were stained with dio for 60 min at room temperature after unbound particles were washed away with hepes buffer phaseseparated sppms were visualized by epifluorescence microscopy and bound particles were visualized by tirf microscopy to analyze and quantify the distribution of sppmbound particles we distinguished three regions of the sppm lo domains ld phase areas and lold boundary regions with a 075mm width centered on the perimeter of each lo domain custombuilt particletracking software 35 was used to automatically detect the position x and y coordinates of each particle to monitor the fusion of single hiv particles with the sppm didlabeled hiv env particles were injected into a chamber one large window wall of which was formed by the sppm the light source for tirf illumination was a diode laser cube640 coherent to excite the lipid dye did through a 620nm filter et62060 chroma and via a dichroic mirror 660dclp chroma did fluorescence was observed through a 665nm bandpass filter hq66560 chroma data acquisition and image analysis were accomplished through custombuilt software written in labview national instruments 35  single events that included docking hemifusion and full fusion were measured by analyzing the peak fluorescence intensities from each bound particle as a function of time events with no fluorescence change over time were classified as docking those with decays to around onehalf of the original peak intensity were classified as hemifusion and those with complete decays were classified as direct full fusion events 44 								
"supplementary material for this article is available at httpadvancessciencemagorgcgi contentfull36e1700338dc1 fig s1  association of cd4 and ccr5 with lipid rafts in cd4  ccr5  hela cells with stably expressed cd4 and ccr5 fig s2  formation of gpmvs by treatment of cd4  ccr5  hela cells with small amounts of formaldehyde and dtt fig s3  partitioning of cd4 and ccr5 in gpmvs induced by nem instead of formaldehyde and dtt from cd4  ccr5  cells fig s4  hiv env pseudovirus particles bind preferentially at boundaries between coexisting lo and ld domains in gpmvs fig s5  vsvg pseudovirus particles bind to ld membrane regions in gpmvs fig s6  electron cryomicrographs of hiv env particles boundfused to gpmvs fig s7  modulation of lipid phases does not affect binding of hiv to gpmvs fig s8  preparation of sppms with gpmvs fig s9  lateral distribution of cd4ccr5 in sppms movie s1 growing gpmvs on cell surfaces """	"Yang, Sung-Tae. Kreutzberger, Alex J. B...."	 HIV virions sense plasma membrane<br>heterogeneity for cell entry	Sci Adv	" It has been proposed that cholesterol in host<br>cell membranes plays a pivotal role for cell entry of<br>HIV. However, it remains largely unknown why<br>virions prefer cholesterol-rich heterogeneous<br>membranes to uniformly fluid membranes for membrane<br>fusion. Using giant plasma membrane vesicles<br>containing cholesterol-rich ordered and<br>cholesterol-poor fluid lipid domains, we demonstrate that the<br>HIV receptor CD4 is substantially sequestered<br>into ordered domains, whereas the co-receptor CCR5<br>localizes preferentially at ordered/disordered domain<br>boundaries. We also show that HIV does not fuse from within<br>ordered regions of the plasma membrane but rather at<br>their boundaries. Ordered/disordered lipid domain<br>coexistence is not..."	154	6872		
92142e072745fbab996c5861e5974aa03805398a	macrophages found in circulating blood as well as integrated into several tissues and organs throughout the body represent an important first line of defense against disease and a necessary component of healthy tissue homeostasis additionally macrophages that arise from the differentiation of monocytes recruited from the blood to inflamed tissues play a central role in regulating local inflammation studies of macrophage activation in the last decade or so have revealed that these cells adopt a staggering range of phenotypes that are finely tuned responses to a variety of different stimuli and that the resulting subsets of activated macrophages play critical roles in both progression and resolution of disease this review summarizes the current understanding of the contributions of differentially polarized macrophages to various infectious and inflammatory diseases and the ongoing effort to develop novel therapies that target this key aspect of macrophage biology	"1 macrophages m represent a ubiquitous yet complex and nuanced population of immune cells that play essential roles in both disease and homeostasis throughout the body hume 2008  in addition to monocytes and m circulating throughout the bloodstream specialized tissueresident m can be found in most major organs including kupffer cells in the liver langerhans cells in the skin microglia in the brain splenic red pulp m lung alveolar m adipose tissue m and bone osteoclasts to name a few davies et al 2013 gautier et al 2012 ji et al 2013 you et al 2013  while some identify these populations as the endpoint of bone marrow monocyte maturation several lines of evidence indicate that tissue resident m originate during embryogenesis in association with their specific tissue independently from blood monocytes and monocytesm recruited to sites of inflammation davies et al 2013 gomez and geissmann 2013 schulz et al 2012  regardless of their location m are responsible for the maintenance of healthy tissues through phagocytic clearance of apoptotic cells and foreign materials and through tissue repair and remodeling during wound healing duffield 2005 ghavami et al 2014 majai et al 2014 mantovani et al 2013  m are also major regulators of the inflammatory response to disease and infection acting as a bridge between innate and adaptive immunity by monitoring the microenvironment through an array of surface receptors and secreting appropriate cytokines and chemokines heydtmann 2009 
depending on the stimuli they encounter tissue resident and circulatory m populations can be directed to distinct phenotypic programs in a process known as m polarization fig 1  table  1  the diverse properties of different m subsets can have drastic effects on health and disease within the tissues where they reside while the induction of a particular subset can be protective during homeostasis or disease this process can be altered or subverted to enhance pathogenesis and disease progression by for example inappropriately dampening the immune response or exacerbating harmful inflammation murray and wynn 2011  therefore this review aims to summarize recent findings regarding the identity properties and roles of polarized m in various disease models and the development of therapeutic strategies that target both the process of m polarization and individual m subsets
the most welldescribed and commonly reported paradigm of m polarization is the m1m2 polarization axis mantovani et al 2004 martinez et al 2009 sica et al 2013  originally named to reflect relationships to th1th2 polarization of immune responses m1 and m2 m are also referred to as classically or alternatively activated m respectively gordon 2003 mills et al 2000 
classical activation is stimulated by microbial products and proinflammatory cytokines ifn andor lps or tnf and the resulting m1 m are characterized by high antigen presentation high production of il12 and il23 and high production of nitric oxide no and reactive oxygen intermediates roi verreck et al 2004  m1 m have been shown to produce several other inflammatory cytokines like tnf il1 6 and 12 type i ifn cxcl13 5 and 810 ccl25 and 11 cxcl16 and cx3cl1 mantovani et al 2004 sica and mantovani 2012 
by contrast alternativem2 activation is mediated by il4 il table 1  sr scavenger receptor mr mannose receptor ho1 heme oxygenase1 vegf vascular endothelial growth factor sd1 sulfiredocin1 trreductase thioredoxinreductase kadl et al 2010 leitinger and schulman 2013 murray and wynn 2011 polarization state  gene expression  cytokines  chemokines   m1  cd80 cd86 mhc iii il1r i tlr2  tlr4 inos   tnf il1 il6 il12 il15 il23  ros inos type i ifn   cxcl13 cxcl5 cxcl810  cxcl16 ccl25 ccl8 ccl11  ccl15 ccl20 cx3cl1   m2a  cd163 mhc ii sr cd206 mr  il1r ii ym1 fizz1 arg1  il10 tgf il12 il1ra  ccl1 ccl2 ccl24 10 and il13 which were initially thought to produce deactivated m martinez et al 2009  m2 m are marked by the upregulation of several surface molecules including dectin1 dcsign mannose receptor mrc1cd206 scavenger receptor a cd204 scavenger receptor b1 cd163 ccr2 cxcr1 and cxcr2 gordon 2003 mantovani et al 2004 martinez et al 2009  m2 m exhibit altered cytokine and chemokine production and typically produce high levels of il10 and low levels of il12 mosser 2003  ccl1 ccl2 ccl17 ccl18 ccl22 ccl24 and il1ra are also made by alternatively activated m mantovani et al 2004  genetic studies of m2 m in mouse models have identified additional signatures of alternative activation including arginase 1 arg1 ym1 a member of the chitinase family and fizz1 found in inflammatory zone 1 retnla raes et al 2002 2005  generally the m2 polarization state is characterized by little to no secretion of proinflammatory cytokines increased secretion of antiinflammatory cytokines enhanced scavenging of cellular debris promotion of tissue remodeling and repair and in some cases increased capacity to fight parasitic infections  additionally the concept of resolution of inflammation has evolved and is no longer perceived as a passive process that simply occurs when the insult disappears but rather as a highly orchestrated response coordinated by a complex regulatory network of cells and antim1 mediators called proresolving mediators rius et al 2012  m2 m can be further divided into subtypes according to their inductive stimuli and secreted chemokines martinez et al 2008  m2a m are stimulated by il4 and il13 and produce ccl24 ccl22 ccl17 and ccl18 which are recognized by ccr3 ccr4 and ccr8 and promote recruitment of eosinophils basophils and th2 cells m2b m result from activation with immune complexes and tlr agonists like lps and produce ccl1 which recruits tregs il10 drives m polarization to m2c cells which produce ccl16 and 18 recruiting eosinophils and nave t cells respectively m2d m accumulate in the tumor microenvironment and present an il10 hi vegf hi m2 profile but also exhibit some m1 characteristics such as expression of infinducible chemokines ccl5 cxcl10 and cxcl16 duluc et al 2009  a full understanding of the m1m2 paradigm of m polarization however contains some caveats first m1 and m2 m as defined in the foundational literature most likely do not exist as distinct categories but rather should be considered as extremes at either end of a continuum of overlapping functional states mosser and edwards 2008  indeed m with combinations of m1 and m2 markers can be found in atherosclerotic plaques and some murine tumors kadl et al 2010 umemura et al 2008  second unlike the irreversible phenotypic changes seen in lymphocytes after exposure to polarizing cytokines m polarization is both transient and plastic biswas and mantovani 2010 biswas et al 2008 sica et al 2013 stout and suttles 2004  for example m2 m can be reprogrammed to express m1 genes following exposure to tlr ligands or ifn mylonas et al 2009 stout et al 2005  finally while there is partial overlap of m1and m2identifying markers in murine and human studies there are still markers in each system that fail to translate to the other the chitinaselike proteins ym1 and ym2 along with fizz1 are markers of murine m2 polarization which lack human orthologs while ccl14 ccl18 and ccl23 are humanrestricted m2 markers with no murine orthologs chang et al 2001 martinez et al 2009 raes et al 2002  finally there are other specially activated m m4 mhem and mox that have been described in atherosclerosis and may lie on a separate activation axis from m1m2 m fig 1  these atherosclerotic m subsets have been discussed in recent reviews fenyo and gafencu 2013 leitinger and schulman 2013  but will not be examined in detail here
the network of molecular mediators that regulate m1m2 polarization in response to various stimuli is incompletely understood but several signaling pathways have been implicated in this process fig 2  one of the major pathways identified is the jakstat pathway which mediates responses to a collection of different cyotkines and growth factors and regulates processses from hematopoiesis and immune development to lactation and adipogenesis rawlings et al 2004  binding of ifn to its cell surface receptor leads to activation of receptorassociated jaks which in turn cause stat1 to dimerize and translocate to the nucleus where it initiates transcription of genes that promote m1associated functions like enhanced microbicidal activity and proinflammatory cytokine production hu et al 2007 rauch et al 2013  mspecific deletion of socs3 an inhibitor httpmolcellsorg
mol cells 277 of cytokine and jakstat signaling was found to increase levels of the m1 genes il1 il6 il12 il23 and inos qin et al 2012a and increase phosphorylation of stat1 and stat3 qin et al 2012b  in contrast stat6 is activated by il4 and il13 to induce m2 polarization daley et al 2009 moreno et al 2003 stolfi et al 2011  cjun nterminal kinase jnk a mitogenactivated protein kinase mapk involved in cell proliferation transformation differentiation and apoptosis is likely involved in this pathway  upon activation jnk can phosphorylate serine 707 on stat6 thereby deactivating it shirakawa et al 2011  a study of m polarization in obesity showed that mice lacking the jnk activator mlk3 were also deficient in m1 m polarization gadang et al 2013  the transcription factors ppar and ppar are activated by stat6 and necessary for m2 polarization and ppar m exhibit enhanced activation of jnk following treatment with adipocyteconditioned medium which contains the m2 cytokines il4 and il13 kang et al 2008 odegaard et al 2007  the zincfinger transcriptional regulator krppellike factor 4 klf4 is involved in this pathway as well and cooperates with stat6 to skew polarization towards m2 by sequestering coactivators of nfb liao et al 2011 
furthermore the phosphoinositol3kinase pi3k signaling pathway which activates multiple kinase cascades through the production of the second messenger pip3 regulates m survival and gene expression via activation of the akt family of serinethreonine protein kinases luyendyk et al 2008  knockout studies have demonstrated that m1 polarization depends on the activation of akt2 while m2 polarization requires akt1 arranz et al 2012  in addition the pi3kakt signaling pathway controls the activation of mtor which promotes m2 polarization byles et al 2013 mercalli et al 2013 weichhart and semann 2008 
interferonregulatory factor irf proteins are also regulators of m polarization irf5 is associated with m1 polarization and promotes the transcription of genes encoding il12 while repressing the gene that encodes il10 krausgruber et al 2011  notch signaling through the nuclear transducer rbpj controls expression of irf8 which induces m1 gene expression xu et al 2012  irf4 is highly expressed in adipose tissue m atm and its deletion leads to increased production of il1 and tnf and expression of m1 markers indicating that irf4 activation contributes to m2 polarization eguchi et al 2013  the irfs also underlie the ability of gmcsf and mcsf to induce polarization gmcsf leads to downstream activation of irf5 m1 while mcsf leads to irf4 m2 activation lawrence and natoli 2011 
given the ability of m to acquire enhanced microbicidal abilities following stimulation with microbial products and the preeminent roles of m in both innate and adaptive immune responses one might predict that pathogens would evolve strategies to redirect and alter m activation in their favor several transcriptome analysis studies have established that innate immune cells particularly m engage in a common response to pathogen challenge that involves a shared pattern of gene expression jenner and young 2005 nau et al 2002  a multistudy review of transcriptional responses of mononuclear phagocytes to bacteria and bacterial components focusing specifically on genes involved in m polarization identified a common response program that mainly involved the upregulation of m1associated genes including the cytokines tnf il6 il12 il1 the cytokine receptors il7r and il15ra the chemokines ccl2 ccl5 and cxcl8 and the chemokine receptor ccr7 benoit et al 2008  this m1 activation program is typically associated with protection against disease and m1 polarization has been shown to aid host control of several bacteria including listeria monocytogenes salmonella typhimurium mycobacterium tuberculosis mycobacterium ulcerans and chlamydia infections benoit et al 2008 chacnsalinas et al 2005 jouanguy et al 1999 kiszewski et al 2006 rottenberg et al consequently several pathogenic bacteria especially intracellular species have developed mechanisms to interfere with m polarization in order to enhance their own survival some species accomplish this by blunting m1 polarization to reduce inflammation and microbicidal functions of m the intracellular form of the enteropathogen shigella flexneri produces an altered hypoacetylated form of lps that evades recognition by tlr4 and elicits decreased production of proinflammatory cytokines from murine bone marrow derived m bmdm paciello et al 2013  during pulmonary infection in mice staphylococcus aureus induces akt1 signaling to enhance socs1 activity and inhibit nfb activity shifting m from an antimicrobial m1 phenotype to a functionally inert one  m tuberculosis secretes the virulence factors lipoarabinomannan and early secretory antigenic target6 esat6 which inhibit m1 activation by inhibiting phagosome maturation and nfb activation respectively lugovillarino et al 2011  m tuberculosis also subverts the inflammatory response by stimulating wnt6 signaling in infected m in granulomatous lesions in the lung driving m2like polarization schaale et al 2013  s aureus biofilms are resistant to m invasion but those m that do successfully penetrate catheterassociated biofilms in vitro display decreased expression of il1 tnf and inos expression but robust arg1 expression signifying an m2 profile thurlow et al 2011  s typhimurium has been shown to preferentially associate with m2 m and ppar expression is upregulated in salmonellainfected m while ppar deficiency severely inhibits bacterial replication and persistence eisele et al 2013  interestingly the dependency of s typhimurium on ppar expression was shown to be due to its metabolic effects rather than its ability to reduce production of antimicrobial mediators by promoting m2 polarization and it remains to be determined whether s typhimurium directly augments ppar activity to promote persistence
similar to evasion strategies employed by bacterial pathogens many viruses take advantage of the m polarization system to enhance their own growth and virulence however unlike bacterial pathogens which generally tend to thrive within and encourage production of m2polarized m viral pathogens more commonly activate m1 polarization this inflammatory phenotype is often correlated with disease severity hepatitis c virus preferentially infects hepatocytes and establishes a chronic inflammatory infection often leading to fibrotic cirrhosis and hepatocellular carcinoma hcc lavanchy 2011  it has been demonstrated that the viral protein ns3 enhances il12 and tnf production by thp1 m implicating m1 polarization in sustaining inflammation hajizadeh et al 2013  furthermore activation of m with tlr agonists triggers the secretion of tnf which promotes hcv entry into polarized hepatoma cells by relocalizing the tight junction protein and hcv entry factor occludin fletcher et al 2013  of the three common clades of avian h5n1 influenza virus circulating in poultry in china 232 234 and 7 clade 234 is the most successful at infecting replicating within and inducing cytopathic effects in human monocytederived m mdm  h5n1 clade 234 also stimulated the highest expression of il1 il6 il8 tnf ifn and mcp1 in mdms  m2 m polarization by s aureus which is commonly present among the airway mucosal microbiota inhibits influenzamediated lung injury implying that m1 m exacerbate flu infection 
nonetheless some viruses do benefit by skewing m polarization towards an m2 phenotype during infection by severe acute respiratory syndrome coronavirus sarscov lung damage resulting from both intrinsic viral infection and dysregulation of the host immune response rapidly progresses to diffuse alveolar damage resulting in acute respiratory distress syndrome and pulmonary fibrosis franks et al 2003 peiris et al 2003  a recent study revealed that sarscovinfected mice lacking hematopoeitic stat1 expression have greater weight loss and lung pathology associated with upregulation of the m2 indicators ym1 fizz1 il4 and il13 page et al 2012  absence of lung disease and prefibrotic lesions in infected stat1stat6 doubleknockout mice also supported the notion that m2 m contribute to sarscov pathogenesis human cytomegalovirus hcmv has a more complex relationship with m polarization the hcmv gene ul111a encodes a homolog of human il10 that is capable of polarizing monocytes towards an antiinflammatory m2c phenotype including high expression of the scavenger receptor cd163 suppression of mhc expression and exppression of heme oxygenase 1 which suppresses tnf and il1 avdic et al 2013  additionally hcmv optimally infects m2but not m1polarized m and latephase hcmv infection is dependent on the m2promoting activation of mtor poglitsch et al 2012  despite this hcmvactivated m have been shown to adopt an m1 transcriptome profile chan et al 2008  hiv1 similarly seems to benefit from m2 polarization hiv1 displays impaired viral dna synthesis delayed proviral integration and reduced proviral transcription in m1 m while the m2a surface receptor dcsign facilitates hiv1 entry dna synthesis and transmission from infected m to cd4  t cells cassol et al 2010  notably clathrinmediated endocytosis of hiv1 is increased in m1 and decreased in m2 m but this method of endocytosis leads to increased viral degradation and is unlikely to result in productive infection gobeil et al 2012  yet like hcmv hiv1 infection of mdms drives them toward m1 polarization and the viral protein nef is preferentially taken up by m2 m and stimulates an m2tom1 transition cassol et al 2010 chihara et al 2012 lugovillarino et al 2011  these contradictions may be explained by a viral survival strategy that takes advantage of both m1 and m2 m as means to different ends m2 m as a reservoir of replication and m1 m to recruit fresh immune cells to spread the infection this can also be inferred from the ability of proinflammatory cytokines and chemokines from hcmvinfected m to enhance virus replication and dissemination smith and bentz 2004a 2004b 
type 1 diabetes is an autoimmune disease that results in high blood sugar following the destruction of insulinproducing pancreatic beta cells via activation of innate immunity and expansion of autoreactive t cells and autoantibodyproducing b cells monocytesm from patients with type 1 diabetes present a proinflammatory profile high levels of tnf il6 and il1 when compared to normal subjects bradshaw et al 2009 devaraj et al 2006 shanmugam et al 2004  moreover elevated levels of glucose and islet amyloid polypeptide iapp deposition lead to the activation of tlrs and inflammasomes resulting in beta cell death and decreased insulin secretion henaomejia et al 2013  recently it has been suggested that m1 m may contribute to diabetesrelated complications such as cardiovascular diseases by altering the immune system of type 1 diabetics burke and kolodgie 2004  furthermore the sustained increase of growth hormone in murine models of type 1 diabetes leads to a reduction of diabetes symptoms by attenuating the apoptosis and increasing the expansion of beta cells villares et al 2013  growth hormone also triggers m2 polarization of m via modulation of the cytokine milieu stimulating the activity of suppressor t cells and limiting th17 cell activation villares et al 2013 
obesity is a major health problem in western countries and a risk factor for insulin resistance type 2 diabetes hepatic steatosis and artherosclerosis obesity is closely associated with chronic inflammation in adipose tissues suggesting that the chronic excess of nutrients triggers an immune response in adipose tissues goh et al 2011 hotamisligil 2006 kanneganti and dixit 2012  white adipose tissues store energy in the form of fat and regulate systemic metabolism through the release of adipokines by adipocytes that control insulin sensitivity in the liver and skeletal muscle sun et al 2011 tateya et al 2013  in lean subjects and mice adipose tissue m atm present an m2 phenotype and are critical to maintaining insulin sensitivity in adipocytes through il10 production liao et al 2011 lumeng et al 2007a 2007b  in metabolic homeostasis m2 atms are maintained by il4 and il13 secreted by adipocytes in a pparand klf4dependent manner wynn et al 2013 zhou et al 2013  in obese subjects and mice adipocytes release proinflammatory mediators ie ccl2mcp1 tnf ccl5 ccl8 and free fatty acid promoting the infiltration of ly6c hi inflammatory monocytes which differentiate into m1polarized atms that express high levels of tnf inos il6 and il1 cinti et al 2005 lumeng et al 2007a olefsky and glass 2010 tateya et al 2013 weisberg et al 2003 weisberg et al  2006  therefore the severity of obesityrelated metabolic dysfunctions correlates with m1 atm infiltration whereas chronic inflammation in adipose tissue inhibits the production of adiponectin thus contributing to the development of insulin resistance in surrounding adipocytes lumeng et al 2007a weisberg et al 2003 2006 
recently nafld nonalcoholic fatty liver disease has emerged as an obesityrelated health problem characterized by steatosis accumulation of lipids in hepatocytes hepatic steatosis can evolve to nonalcoholic steatohepatitis nash when accompanied by liver injury ballooning hepatocytes and hepatic inflammation which may be associated with fibrosis and eventually culminates in cirrhosis and hcc the development of the complex pathology of nash involves a variety of liver cells including hepatocytes hepatic m and stellate cells inflammatory mediators especially those derived from adipose tissues the gut and the liver have recently been reported to play a major role in initiating and controlling the progression of nash by regulating lipid metabolism day and james 1998 racanelli and rehermann 2006 tilg and moschen 2010  in particular the activation of innate immune cells such as kupffer cells and infiltrating bloodderived monocytes is a major event of nash development in homeostatic conditions kupffer cells perform immune surveillance by removing pathogens and toxins from the circulation and maintain liver tolerance through il10 secretion thomson and knolle 2010  kupffer cells communicate with a variety of hepatic immune cells and interact directly with hepatocytes passing through the space of disse racanelli and rehermann 2006  in early mouse models of dietinduced steatohepatitis kupffer cells are the first innate cells responding to injured hepatocytes and differentiate toward m1 m promoting the recruitment of bloodderived cd11b int ly6c hi monocytes through secretion of tnf and chemokines mcp1 and ip10 tosellotrampont et al 2012  the recruitment of these inflammatory m1polarized ly6c  bloodderived monocytes is dependent of ccr2 and mcp1 karlmark et al 2009 klein et al 2007 miura et al 2012 obstfeld et al 2010  the hallmarks of nash ie steatosis lowgrade inflammation and hepatic recruitment of m1polarized m are reduceddelayed following specific depletion of kupffer cells or by silencing of tnf in myeloid cells tosellotrampont et al 2012  moreover m1polarized kupffer cells also produce inflammatory mediators such as il1 and ros which induce hepatic steatosis and fibrosis miura et al 2012  nfb and jnk activation in kupffer cells may contribute to the development of hepatic inflammation by promoting m1like m polarization 
liver m are also implicated in the severity of nash via the expression of tolllike receptors tlr2 tlr4 tlr9 myd88 and scavenger receptors scavenger receptor a and cd36 bieghs et al 2010 miura et al 2010  tlrs and scavenger receptors trigger proinflammatory responses following recognition of hepatic free fatty acids damageassociated molecular pattern damps expressed by steatotic hepatocytes andor bacterial products derived from the gut farrell et al 2012 roh and seki 2013  nash patients show increased intestinal permeability resulting in greater hepatic abundance of bacterial products and other tlr ligands derived from the gut via portal vein circulation  the imbalance of gut flora may influence liver disease by activating tlrs expressed on liver cells and leading to the activation of nlpr3 csak et al 2011 farrell et al 2012 henaomejia et al 2013 roh and seki 2013  in models of dietinduced nash and obesity inflammasomedeficient mice develop more severe nash which is fully transferable to wt mice upon prolonged cohousing suggesting that commensal bacteria in the gi tract play an important role in nash disease progression csak et al 2011 henaomejia et al 2012 weisberg et al 2003 
m are a highly influential cell type in most varieties of cancer and are recruited to all solid tumors cassetta et al 2011  the contributions of different subsets of polarized m to the tumor microenvironment and cancer progression are therefore a subject of great interest m1 m are generally considered to be beneficial to the host and peritumoral m that express m1 cytokines like ifn il1 and il6 have been shown to have antitumoral effects and are associated with improved prognoses dumont et al 2008 klimp et al 2002 niino et al 2010 berg et al 2002 zhou et al 2010  m1 m may have the opposite effect in virally induced cancers however administration of ifn or tnf to patients infected with kaposi sarcoma virus enhances disease progression monini et al 1999  proinflammatory m are also harmful in intraocular tumors where tnfand inosdependent antitumor responses lead to necrosis of bystander cells and destruction of the eye coursey et al 2012 
m2polarized tumorassociated m tam on the other hand have been repeatedly and consistently associated with unfavorable effects like tumor growth angiogenesis and metastasis in malignant cancers  the m2 cytokines il4 il13 and il10 are present within the tumor microenvironment and tams from various cancer models have been shown to express an m2 activation profile that includes enhanced expression of cd163 mrc1 ctype lectins il10 and arg1 and decreased production of il12 beck et al 2009 biswas et al 2006 schmieder et al 2012 sica et al 2002  the small secretory lectin reg3 is an important inhibitor of inflammation in pancreatic and intestinal tissues and deficiency of reg3 an activator of the stat3 pathway drastically impairs pancreatic tumor growth by skewing m polarization away from m2 and towards m1 gironella et al 2013  m2 tams have also been shown to increase fibroblastic morphology vimentin and snail expression metalloproteinase activity and proliferation and migration of pancreatic cancer cells implicating them in the development of eptihelialmesenchymal transition and metastasis  in hcc high expression of the heparinsulfate proteoglycan glypican3 gpc3 on the surface of cancer cells is associated with increased m infiltration in human patients and humanmouse xenograft transplantation with a gpc3overexpressing cell line leads to infiltration by m expressing m2specific markers takai et al 2009a 2009b  m2 tams worsen hcc both by promoting tumor growth and angiogenesis and by encouraging liver fibrosis through il13 and tgf secretion 
given that m play important roles in maintaining tissue homeostasis and fighting disease polarized m subsets that specifically contribute to the pathogenesis or amelioration of various diseases present themselves as attractive targets for therapeutic intervention different therapeutic strategies include either targeting the polarized m themselves or manipulating the signaling pathways involved in the process of m polarization to a desirable outcome
bacterial biofilms that form on body surfaces or on surgical implants lead to chronic and recurrent infections and are difficult to treat with antibiotics donlan and costerton 2002 otto 2008  early local administration of m1 m or the c5a receptor agonist ep67 which stimulates m1 polarization significantly attenuated biofilm formation in a mouse model hanke et al 2013  furthermore treatment of established biofilms significantly reduced bacterial burden compared to antibiotic treatment suggesting the potential of a therapeutic alternative hanke et al 2013  microbes themselves may also prove to be useful sources of therapeutics that modulate m polarization in vitro application of extracellular polysaccharide secreted by an oligotrophic bacteria found in lop nur desert bacillus sp lbp32 was found to limit lpsinduced inflammation in the m cell line raw 2647 by inhibiting nfb and jnk activation and may prove useful in diseases characterized by excessive m1 polarization  similarly the smallmolecule compound bisnnorgliovictin isolated from the marinederived endophytic fungus s31c inhibits lpsinduced m1 polarization of raw 2647 cells and murine peritoneal m and improves survival in mouse models of sepsis song et al 2013  as a proof of concept for treating inflammatory gastrointestinal diseases a lab strain of e coli was created that secretes a herpes virus homolog of il10 via a secdependent transporter construct viral il10 delivered in this manner was shown to activate stat3 and suppress tnf production in the j7741 murine m cell line frster et al 2013 
ikk a downstream mediator of insulin resistance and activator of the nfb pathway and therefore of m1 polarization is inhibited by antiinflammatory salicylates like aspirin which attenuates hyperglycemia and hyperinsulinemia in obese rodents yin et al 1998 yuan et al 2001  several small trials in patients with type 2 diabetes have demonstrated that treatment with salicylates results in a marked reduction of diabetic metabolic parameters and improved glycemic control tateya et al 2013 
apolipoprotein ai mimetics are a class of therapeutic molecules that attempt to modulate hdl to treat atherosclerosis and are the subject of extensive clinical and mechanistic study as reviewed in leman et al 2013  interestingly the mimetic d4f also has potential for cancer therapy d4f inhibits the m2associated scavenger receptor cd204sra on tams preventing metastatic spread neyen et al 2013  anticancer therapies also seek to convert protumoral m2 m into m1 m m2 m generated by il6 and prostaglandin e 2 secreted by cervical cancer cells can be repolarized to m1 by coculture with th1 cells and this interaction could possibly be reproduced by activation with cd40l and ifn heusinkveld et al 2011  moreover ifn was shown to successfully switch m2 tams purified from human ovarian tumors to an m1 phenotype and the addition of ifn skewed de novo tumorinduced m2 differentiation of monocytes to favor m1 polarization duluc et al 2009  other potentially therapeutic molecules found to repolarize tams from an m2 to an m1 phenotype include zoledronic acid cpg oligonucleotide and histidinerich glycoprotein coscia et al 2010 huang et al 2012 rolny et al 2011 
the integral importance of m in the maintenance of nearly every tissue throughout the body and their position as the first line of defense against many diseases guarantees that they play critical roles in both disease progression and in resolution and that altering the behavior of these cells can mean the difference between healthy recovery and severe illness m polarization itself is an extremely nuanced and finetuned process and can produce nearly infinite variations of endpoint phenotypes each of which has the potential to affect various diseases in different ways while polarized m subsets and the polarization process itself are attractive and novel therapeutic targets in both infectious and inflammatory disease better understanding of how polarization is controlled and how polarized m modulate specific diseases is necessary to fully harness the potential of these strategies
this work was supported by nih grants r01ai098126 and u19ai066328"	"Labonte, Adam C.. Tosello-Trampont, Annie-Carole..."	 The Role of Macrophage Polarization in<br>Infectious and Inflammatory Diseases	Mol Cells	" Macrophages, found in circulating blood as<br>well as integrated into several tissues and organs<br>throughout the body, represent an important first line of<br>defense against disease and a necessary component of<br>healthy tissue homeostasis. Additionally,<br>macrophages that arise from the differentiation of<br>monocytes recruited from the blood to inflamed tissues<br>play a central role in regulating local<br>inflammation. Studies of macrophage activation in the last<br>decade or so have revealed that these cells adopt a<br>staggering range of phenotypes that are finely tuned<br>responses to a variety of different stimuli, and that the<br>resulting subsets of activated macrophages play<br>critical..."	143	5112