Association of Increased Pathogenicity of Asian H5N1 Highly Pathogenic Avian Influenza Viruses in Chickens with Highly Efficient Viral Replication Accompanied by Early Destruction of Innate Immune Responses

The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.Since the first detection of the Asian lineage of highly pathogenic avian influenza (HPAI) virus (H5N1) in southern China in 1996, H5N1 virus infection in birds has continued for 13 years in Asia, acquiring pathogenicity not only in birds but also in mammals. In 1997, the H5N1 Hong Kong isolates caused illness and death in a variety of terrestrial birds and even in humans (9, 37, 48, 49). In 2001, emerging H5N1 Hong Kong isolates were more pathogenic to chickens and the mean death time (MDT) was 2 days without any prior clinical signs (12). In 2003 to 2004, the H5N1 epizootic occurred simultaneously in East Asian countries (22, 30). The 2003/2004 H5N1 isolates caused death in taxonomically diverse avian species, including domestic ducks (46, 47, 51), and humans (7, 55). Furthermore, the first indication of wild aquatic bird involvement occurred at recreational parks in Hong Kong in late 2002 to 2003 (46), and then migratory aquatic bird die-off occurred in 2005 at Qinghai Lake in China (6, 24). The broad host spectrum and increased pathogenicity of H5N1 viruses to diverse bird species raise serious concerns about the worldwide spread of the virus by migratory birds.According to the international criteria, HPAI viruses are defined by over 75% mortality in 4- to 8-week-old chickens following an intravenous pathogenicity test or an intravenous pathogenicity index (IVPI) of more than 1.2 in 6-week-old chickens (34); however, there are some variations in pathogenicity intensity among the HPAI viruses in chickens (1, 3, 5, 12, 15, 28, 31, 48, 50-52, 57). Most of the HPAI viruses that were isolated before 1996 cause severe clinical signs (e.g., ruffled feathers, depression, labored breathing, and neurological signs) and severe gross lesions (e.g., head and face edema, cyanosis, subcutaneous hemorrhages in combs and leg shanks, and necrosis of combs and wattles) in chickens (1, 3, 15, 31, 50, 52, 57). These viruses usually kill chickens 3 to 6 days after intranasal inoculation. On the other hand, the recently emerged Asian H5N1 HPAI viruses are more virulent and kill chickens within 1 to 2 days without causing typical clinical signs and gross lesions (5, 12, 27, 33, 48, 51), although some Asian H5N1 viruses, such as A/Goose/Guangdong/2/96 (23), A/goose/Hong Kong/437-10/99 (17), and A/chicken/Indonesia/7/03 (58), were less virulent. To successfully control HPAI in poultry, it is important to better understand the mechanisms of increased pathogenicity of recent H5N1 HPAI viruses in chickens.The Asian H5N1 HPAI virus has another important characteristic, which is its capability of crossing host-species barriers. It was reported that the H5N1 virus can infect and cause death in mammals such as mice (5, 9, 12, 14, 29), cats (21), tigers (2), ferrets (11, 26), monkeys (40), and humans (7, 49, 55). High-level inductions of proinflammatory cytokines in mammals infected with the H5N1 viruses, referred to as “cytokine storms,” have been hypothesized to contribute to the severity of pathological changes and ultimate death (4, 7, 13, 45, 55). Cytokine and chemokine dysregulation was detected in clinical cases of H5N1-infected humans (8, 13, 36) and also in monkeys experimentally infected with the H1N1 Spanish flu strain (20). In a mouse model, lymphocyte apoptosis and cytokine dysregulation have been proposed to contribute to the severity of the disease caused by H5N1 (56). Investigations with transgenic mice deficient in each cytokine gene suggest that tumor necrosis factor alpha (TNF-α) may contribute to morbidity and interleukin-1 (IL-1) may be important for virus clearance (53). However, mice deficient in TNF-α or IL-6 succumb to infection with H5N1, and cytokine inhibition treatment does not prevent death (42), suggesting that therapies targeting the virus rather than cytokines may be preferable. Thus, the significance of elevated proinflammatory cytokine responses in the pathogenesis of H5N1-infected mammals requires further studies.In contrast, little is known about proinflammatory cytokine responses and their roles in pathogenicity in chickens infected with HPAI viruses, including the recent Asian H5N1 viruses. It was reported that infection with an HPAI virus results in upregulation of gene expression of gamma interferon (IFN-γ) and inducible nitric oxide synthase (58). However, the roles of proinflammatory cytokines in disease severity and outcomes in chickens infected systemically with HPAI viruses are largely unknown. The less-virulent Asian H5N1 virus, which causes severe clinical signs and gross lesions in chickens (17, 23, 27, 58), would be a valuable tool for investigating the role of proinflammatory cytokines in chickens infected with HPAI viruses, as well as for exploring the pathogenesis of the more-virulent Asian H5N1 HPAI virus, because of the antigenic and molecular similarities between them.In this study, we compared the pathogenicities in chickens of the less-virulent and more-virulent Asian H5N1 HPAI viruses based on MDT, fever, cytokine responses, and viral replication. Our results suggest that the shift in the Asian H5N1 virus to increased virulence may be associated with efficient and rapid replication of the virus in chickens, accompanied by early destruction of host immune responses and followed by peracute death before fever can be induced. Finally, we discuss candidate genes that may account for the high pathogenicity of Asian H5N1 HPAI viruses in chickens.     

Effect of ambient air pollution on the incidence of appendicitis

Background

The pathogenesis of appendicitis is unclear. We evaluated whether exposure to air pollution was associated with an increased incidence of appendicitis.

Methods

We identified 5191 adults who had been admitted to hospital with appendicitis between Apr. 1, 1999, and Dec. 31, 2006. The air pollutants studied were ozone, nitrogen dioxide, sulfur dioxide, carbon monoxide, and suspended particulate matter of less than 10 μ and less than 2.5 μ in diameter. We estimated the odds of appendicitis relative to short-term increases in concentrations of selected pollutants, alone and in combination, after controlling for temperature and relative humidity as well as the effects of age, sex and season.

Results

An increase in the interquartile range of the 5-day average of ozone was associated with appendicitis (odds ratio [OR] 1.14, 95% confidence interval [CI] 1.03–1.25). In summer (July–August), the effects were most pronounced for ozone (OR 1.32, 95% CI 1.10–1.57), sulfur dioxide (OR 1.30, 95% CI 1.03–1.63), nitrogen dioxide (OR 1.76, 95% CI 1.20–2.58), carbon monoxide (OR 1.35, 95% CI 1.01–1.80) and particulate matter less than 10 μ in diameter (OR 1.20, 95% CI 1.05–1.38). We observed a significant effect of the air pollutants in the summer months among men but not among women (e.g., OR for increase in the 5-day average of nitrogen dioxide 2.05, 95% CI 1.21–3.47, among men and 1.48, 95% CI 0.85–2.59, among women). The double-pollutant model of exposure to ozone and nitrogen dioxide in the summer months was associated with attenuation of the effects of ozone (OR 1.22, 95% CI 1.01–1.48) and nitrogen dioxide (OR 1.48, 95% CI 0.97–2.24).

Interpretation

Our findings suggest that some cases of appendicitis may be triggered by short-term exposure to air pollution. If these findings are confirmed, measures to improve air quality may help to decrease rates of appendicitis.Appendicitis was introduced into the medical vernacular in 1886.¹ Since then, the prevailing theory of its pathogenesis implicated an obstruction of the appendiceal orifice by a fecalith or lymphoid hyperplasia.² However, this notion does not completely account for variations in incidence observed by age,^3,4 sex,^3,4 ethnic background,^3,4 family history,⁵ temporal–spatial clustering⁶ and seasonality,^3,4 nor does it completely explain the trends in incidence of appendicitis in developed and developing nations.^3,7,8The incidence of appendicitis increased dramatically in industrialized nations in the 19th century and in the early part of the 20th century.¹ Without explanation, it decreased in the middle and latter part of the 20th century.³ The decrease coincided with legislation to improve air quality. For example, after the United States Clean Air Act was passed in 1970,⁹ the incidence of appendicitis decreased by 14.6% from 1970 to 1984.³ Likewise, a 36% drop in incidence was reported in the United Kingdom between 1975 and 1994¹⁰ after legislation was passed in 1956 and 1968 to improve air quality and in the 1970s to control industrial sources of air pollution. Furthermore, appendicitis is less common in developing nations; however, as these countries become more industrialized, the incidence of appendicitis has been increasing.⁷Air pollution is known to be a risk factor for multiple conditions, to exacerbate disease states and to increase all-cause mortality.¹¹ It has a direct effect on pulmonary diseases such as asthma¹¹ and on nonpulmonary diseases including myocardial infarction, stroke and cancer.^11–13 Inflammation induced by exposure to air pollution contributes to some adverse health effects.^14–17 Similar to the effects of air pollution, a proinflammatory response has been associated with appendicitis.^18–20We conducted a case–crossover study involving a population-based cohort of patients admitted to hospital with appendicitis to determine whether short-term increases in concentrations of selected air pollutants were associated with hospital admission because of appendicitis.     

Gut microbiota of healthy Canadian infants: profiles by mode of delivery and infant diet at 4 months

Background:

The gut microbiota is essential to human health throughout life, yet the acquisition and development of this microbial community during infancy remains poorly understood. Meanwhile, there is increasing concern over rising rates of cesarean delivery and insufficient exclusive breastfeeding of infants in developed countries. In this article, we characterize the gut microbiota of healthy Canadian infants and describe the influence of cesarean delivery and formula feeding.

Methods:

We included a subset of 24 term infants from the Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort. Mode of delivery was obtained from medical records, and mothers were asked to report on infant diet and medication use. Fecal samples were collected at 4 months of age, and we characterized the microbiota composition using high-throughput DNA sequencing.

Results:

We observed high variability in the profiles of fecal microbiota among the infants. The profiles were generally dominated by Actinobacteria (mainly the genus Bifidobacterium) and Firmicutes (with diverse representation from numerous genera). Compared with breastfed infants, formula-fed infants had increased richness of species, with overrepresentation of Clostridium difficile. Escherichia–Shigella and Bacteroides species were underrepresented in infants born by cesarean delivery. Infants born by elective cesarean delivery had particularly low bacterial richness and diversity.

Interpretation:

These findings advance our understanding of the gut microbiota in healthy infants. They also provide new evidence for the effects of delivery mode and infant diet as determinants of this essential microbial community in early life.The human body harbours trillions of microbes, known collectively as the “human microbiome.” By far the highest density of commensal bacteria is found in the digestive tract, where resident microbes outnumber host cells by at least 10 to 1. Gut bacteria play a fundamental role in human health by promoting intestinal homeostasis, stimulating development of the immune system, providing protection against pathogens, and contributing to the processing of nutrients and harvesting of energy.^1,2 The disruption of the gut microbiota has been linked to an increasing number of diseases, including inflammatory bowel disease, necrotizing enterocolitis, diabetes, obesity, cancer, allergies and asthma.¹ Despite this evidence and a growing appreciation for the integral role of the gut microbiota in lifelong health, relatively little is known about the acquisition and development of this complex microbial community during infancy.³Two of the best-studied determinants of the gut microbiota during infancy are mode of delivery and exposure to breast milk.^4,5 Cesarean delivery perturbs normal colonization of the infant gut by preventing exposure to maternal microbes, whereas breastfeeding promotes a “healthy” gut microbiota by providing selective metabolic substrates for beneficial bacteria.^3,5 Despite recommendations from the World Health Organization,⁶ the rate of cesarean delivery has continued to rise in developed countries and rates of breastfeeding decrease substantially within the first few months of life.^7,8 In Canada, more than 1 in 4 newborns are born by cesarean delivery, and less than 15% of infants are exclusively breastfed for the recommended duration of 6 months.^9,10 In some parts of the world, elective cesarean deliveries are performed by maternal request, often because of apprehension about pain during childbirth, and sometimes for patient–physician convenience.¹¹The potential long-term consequences of decisions regarding mode of delivery and infant diet are not to be underestimated. Infants born by cesarean delivery are at increased risk of asthma, obesity and type 1 diabetes,¹² whereas breastfeeding is variably protective against these and other disorders.¹³ These long-term health consequences may be partially attributable to disruption of the gut microbiota.^12,14Historically, the gut microbiota has been studied with the use of culture-based methodologies to examine individual organisms. However, up to 80% of intestinal microbes cannot be grown in culture.^3,15 New technology using culture-independent DNA sequencing enables comprehensive detection of intestinal microbes and permits simultaneous characterization of entire microbial communities. Multinational consortia have been established to characterize the “normal” adult microbiome using these exciting new methods;¹⁶ however, these methods have been underused in infant studies. Because early colonization may have long-lasting effects on health, infant studies are vital.^3,4 Among the few studies of infant gut microbiota using DNA sequencing, most were conducted in restricted populations, such as infants delivered vaginally,¹⁷ infants born by cesarean delivery who were formula-fed¹⁸ or preterm infants with necrotizing enterocolitis.¹⁹Thus, the gut microbiota is essential to human health, yet the acquisition and development of this microbial community during infancy remains poorly understood.³ In the current study, we address this gap in knowledge using new sequencing technology and detailed exposure assessments²⁰ of healthy Canadian infants selected from a national birth cohort to provide representative, comprehensive profiles of gut microbiota according to mode of delivery and infant diet.     

A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998

Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.Wild birds, including wild waterfowls, gulls, and shorebirds, are the natural reservoirs for influenza A viruses, in which they are thought to be in evolutionary stasis (2, 33). However, when avian influenza viruses are transmitted to new hosts such as terrestrial poultry or mammals, they evolve rapidly and may cause occasional severe systemic infection with high morbidity (20, 29). Despite the fact that avian influenza virus infection occurs commonly in chickens, it is unable to persist for a long period of time due to control efforts and/or a failure of the virus to adapt to new hosts (29). In the past 20 years, greater numbers of outbreaks in poultry have occurred, suggesting that the avian influenza virus can infect and spread in aberrant hosts for an extended period of time (5, 14-16, 18, 32).During the past 10 years, H9N2 influenza viruses have become panzootic in Eurasia and have been isolated from outbreaks in poultry worldwide (3, 5, 11, 14-16, 18, 24). A great deal of previous studies demonstrated that H9N2 influenza viruses have become established in terrestrial poultry in different Asian countries (5, 11, 13, 14, 18, 21, 24, 35). In 1994, H9N2 viruses were isolated from diseased chickens in Guangdong province, China, for the first time (4), and later in domestic poultry in other provinces in China (11, 16, 18, 35). Two distinct H9N2 virus lineages represented by A/Chicken/Beijing/1/94 (H9N2) and A/Quail/Hong Kong/G1/98 (H9N2), respectively, have been circulating in terrestrial poultry of southern China (9). Occasionally these viruses expand their host range to other mammals, including pigs and humans (6, 17, 22, 34). Increasing epidemiological and laboratory findings suggest that chickens may play an important role in expanding the host range for avian influenza virus. Our systematic surveillance of influenza viruses in chickens in China showed that H9N2 subtype influenza viruses continued to be prevalent in chickens in mainland China from 1994 to 2008 (18, 19, 36).Eastern China contains one metropolitan city (Shanghai) and five provinces (Jiangsu, Zhejiang, Anhui, Shandong, and Jiangxi), where domestic poultry account for approximately 50% of the total poultry population in China. Since 1996, H9N2 influenza viruses have been isolated regularly from both chickens and other minor poultry species in our surveillance program in the eastern China region, but their genetic diversity and the interrelationships between H9N2 influenza viruses and different types of poultry have not been determined. Therefore, it is imperative to explore the evolution and properties of these viruses. The current report provides insight into the genesis and evolution of H9N2 influenza viruses in eastern China and presents new evidence for the potential crossover between H9N2 and H5N1 influenza viruses in this region.     

Characterization of the H5N1 Highly Pathogenic Avian Influenza Virus Derived from Wild Pikas in China

The highly pathogenic H5N1 avian influenza virus emerged from China in 1996 and has spread across Eurasia and Africa, with a continuous stream of new cases of human infection appearing since the first large-scale outbreak among migratory birds at Qinghai Lake. The role of wild birds, which are the natural reservoirs for the virus, in the epidemiology of the H5N1 virus has raised great public health concern, but their role in the spread of the virus within the natural ecosystem of free-ranging terrestrial wild mammals remains unclear. In this study, we investigated H5N1 virus infection in wild pikas in an attempt to trace the circulation of the virus. Seroepidemiological surveys confirmed a natural H5N1 virus infection of wild pikas in their native environment. The hemagglutination gene of the H5N1 virus isolated from pikas reveals two distinct evolutionary clades, a mixed/Vietnam H5N1 virus sublineage (MV-like pika virus) and a wild bird Qinghai (QH)-like H5N1 virus sublineage (QH-like pika virus). The amino acid residue (glutamic acid) at position 627 encoded by the PB2 gene of the MV-like pika virus was different from that of the QH-like pika virus; the residue of the MV-like pika virus was the same as that of the goose H5N1 virus (A/GS/Guangdong [GD]/1/96). Further, we discovered that in contrast to the MV-like pika virus, which is nonpathogenic to mice, the QH-like pika virus is highly pathogenic. To mimic the virus infection of pikas, we intranasally inoculated rabbits, a species closely related to pikas, with the H5N1 virus of pika origin. Our findings first demonstrate that wild pikas are mammalian hosts exposed to H5N1 subtype avian influenza viruses in the natural ecosystem and also imply a potential transmission of highly pathogenic avian influenza virus from wild mammals into domestic mammalian hosts and humans.Highly pathogenic avian influenza (HPAI) is an extremely infectious, systemic viral disease that causes a high rate of mortality in birds. HPAI H5N1 viruses are now endemic in avian populations in Southeast Asia and have repeatedly been transmitted to humans (9, 14, 27). Since 2003, the H5N1 subtype has been reported in 391 human cases of influenza and has caused 247 human deaths in 15 countries, leading to greater than 60% mortality among infected individuals (38). Although currently incapable of sustained human-to-human transmission, H5N1 viruses undoubtedly pose a serious threat to public health, as well as to the global economy. Hence, preparedness for such a threat is a global priority (36).Wild birds are considered to be natural reservoirs for influenza A viruses (6, 18, 21, 35, 37). Of the 144 type A influenza virus hemagglutinin-neuraminidase (HA-NA) combinations, 103 have been found in wild birds (5, 7, 17, 37). Since the first HPAI outbreak among migratory wild birds appeared at Qinghai Lake in western China in May 2005 (3, 16, 25, 34, 41), HPAI viruses of the H5N1 subtype have been isolated from poultry throughout Eurasia and Africa. The continued occurrence of human cases has created a situation that could facilitate a pandemic emergence. There is heightened concern that wild birds are a reservoir for influenza A viruses that switch hosts and stably adapt to mammals, including horses, swine, and humans (11, 19, 22, 37).Despite the recent expansion of avian influenza virus (AIV) surveillance and genomic data (5, 17, 20, 21, 33, 40), fundamental questions remain concerning the ecology and evolution of these viruses. Little is known about how terrestrial wild mammals within their natural ecological systems affect HPAI H5N1 epidemiology or about the virus''s effects on public health, though there are many reports of natural and experimental H5N1 virus infection in animals belonging to the taxonomic orders Carnivora (12, 13, 15, 28, 29) and Artiodactyla (15). Herein, we provide the results of our investigation into H5N1 virus infection in wild pikas (Ochotona curzoniae of the order Lagomorpha) within their natural ecological setting. We describe our attempt to trace the circulation of H5N1 viruses and to characterize pika H5N1 influenza virus (PK virus).     

Reassortment between Avian H5N1 and Human H3N2 Influenza Viruses in Ferrets: a Public Health Risk Assessment

This study investigated whether transmissible H5 subtype human-avian reassortant viruses could be generated in vivo. To this end, ferrets were coinfected with recent avian H5N1 (A/Thailand/16/04) and human H3N2 (A/Wyoming/3/03) viruses. Genotype analyses of plaque-purified viruses from nasal secretions of coinfected ferrets revealed that approximately 9% of recovered viruses contained genes from both progenitor viruses. H5 and H3 subtype viruses, including reassortants, were found in airways extending toward and in the upper respiratory tract of ferrets. However, only parental H5N1 genotype viruses were found in lung tissue. Approximately 34% of the recovered reassortant viruses possessed the H5 hemagglutinin (HA) gene, with five unique H5 subtypes recovered. These H5 reassortants were selected for further studies to examine their growth and transmissibility characteristics. Five H5 viruses with representative reassortant genotypes showed reduced titers in nasal secretions of infected ferrets compared to the parental H5N1 virus. No transmission by direct contact between infected and naïve ferrets was observed. These studies indicate that reassortment between H5N1 avian influenza and H3N2 human viruses occurred readily in vivo and furthermore that reassortment between these two viral subtypes is likely to occur in ferret upper airways. Given the relatively high incidence of reassortant viruses from tissues of the ferret upper airway, it is reasonable to conclude that continued exposure of humans and animals to H5N1 alongside seasonal influenza viruses increases the risk of generating H5 subtype reassortant viruses that may be shed from upper airway secretions.Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused devastating outbreaks in avian species during the past decade. After emerging in the Guangdong province of China in 1996, H5N1 viruses have extended their geographic distribution from Asia into Europe and Africa (45, 51). Sporadic transmission of H5N1 viruses from infected birds to humans has resulted in over 380 laboratory-confirmed infections and a case fatality rate of ∼60% since 2003 (48). Currently circulating H5N1 viruses lack the ability to undergo efficient and sustained transmission among humans although instances of limited human-to-human transmission have been reported (13, 41). If H5N1 viruses were to acquire genetic changes that confer efficient transmissibility among humans, then another pandemic would likely occur.The pandemics of 1957 and 1968 highlight the importance of genetic reassortment between avian and human influenza viruses as a mechanism for the generation of human pandemic strains (15, 46, 47). The structural separation of the influenza virus genome into eight independent genes allows formation of hybrid progeny viruses during coinfections. The 1957 H2N2 and 1968 H3N2 pandemic viruses acquired the hemagglutinin (HA) and PB1 genes, with or without the neuraminidase (NA) gene, respectively, from an avian virus progenitor (14, 33). The remaining genes of these pandemic reassortants were derived from a contemporary human virus (14, 33). The host species in which such human pandemic strains were generated by reassortment between human and avian viruses is not known. However, coinfection of the same cell with both human and avian viruses must have occurred, even though human and avian influenza viruses have preferences for different sialic acid receptor structures present on cell surface glycoproteins and glycolipids (20, 30). The HA of human viruses preferentially binds α(2,6)-linked sialic acids while that of avian viruses preferentially bind α(2,3)-linked sialic acids (3, 12). Cells possessing both of these receptors could support coinfection of avian and human viruses, leading to reassortment.Human respiratory tract epithelial cells can possess surface glycans with α(2,3)- and α(2,6)-linked sialic acids and as such represent a potential host for the generation of avian-human reassortant viruses (24, 35). The general distribution of surface α(2,3)- and α(2,6)-linked sialic acids varies among cells of the human upper and lower respiratory tracts, which are anatomically separated by the larynx. Recent studies have shown that α(2,3)-linked sialic acids are present in tissues of the human lower respiratory tract (i.e., lung alveolar cells) (24, 35) as well as tissues of the human upper respiratory tract (24). Consistent with these findings, HPAI H5N1 viruses have been shown to attach to and infect tissues belonging to the lower respiratory tract (i.e., trachea, bronchi, and lung) (5, 25, 35, 40, 42, 43) as well as tissues belonging to the upper respiratory tract (i.e., nasopharyngeal, adenoid, and tonsillar) (25). Glycans with α(2,6)-linked sialic acids are more widespread on epithelial cells of the upper airways than lung alveoli (24, 35). In accordance, human seasonal influenza viruses preferentially attach to and infect cells of the upper respiratory tract (6, 25, 35, 43). If cells with both types of receptors are present in the human respiratory tract, simultaneous infection of a person with both human and avian viruses could generate reassortant viruses.Although viruses derived by reassortment between avian H5N1 and human H3N2 progenitors have been generated in vitro (17), reassortment between these avian and human strains in a coinfected mammalian host has not been shown. Furthermore, our knowledge of the genetic and phenotypic repertoire of such reassortants generated in vivo and their potential for transmission to uninfected hosts is limited (2, 17). In the present study, we used the ferret model to better understand the generation of reassortant viruses in a host coinfected with contemporary avian (H5N1) and human (H3N2) viruses and the extent to which such reassortants replicate and transmit from animal to animal. The domestic ferret (Mustela putoris) serves as an ideal small-animal model for influenza because ferrets are susceptible to human and avian influenza viruses, including HPAI H5N1 viruses, and reflect the relative transmissibility of human and avian influenza viruses in humans (9, 17, 18, 31, 36, 39, 53). Our study revealed that coinfection of ferrets reproducibly generated reassortant viruses that could be recovered from tissues within and extending toward the upper respiratory tract. Although H5 reassortant viruses were recovered from the upper airways, they displayed no transmissibility to contact ferrets, suggesting that additional functional changes are required for these viral subtypes to become pandemic within human populations.     

Sirtuins and ageing—new findings

Sirtuins are a promising avenue for orally administered drugs that might deliver the anti-aging benefits normally provided by calorie restriction.Calorie (or dietary) restriction was first shown to extend rodent lifespan almost 80 years ago, and remains the most robust longevity-promoting intervention in mammals, genetic or dietary. Sirtuins are NAD-dependent deacylases homologous to yeast Sir2p and were first shown to extend replicative lifespan in budding yeast [1]. Because of their NAD requirement, sirtuins were proposed as mediators of the anti-ageing effects of calorie restriction [1]. Indeed, many studies in yeast, Caenorhabditis elegans, Drosophila melanogaster and mice have supported these ideas [2]. However, a 2011 paper posed a challenge: transgenic strains of C. elegans and Drosophila that overexpress SIR2 were found not to be long-lived [3].Rather than review the extensive sirtuin literature previous to that paper, I focus on a few key studies that have followed it, which underscore a conserved role of sirtuins in slowing ageing. In the first study, two highly divergent budding yeast strains—a lab strain and a clinical isolate—were crossed. A genome-wide quantitative trait locus analysis was then performed to map genes that determine differences in replicative lifespan [4]. The top hit was SIR2, explaining more than one-half of the difference in replicative lifespan between the two strains (due to five codon differences between the SIR2 alleles). In Drosophila, overexpression of dSIR2 in the fat body extended the lifespan of flies on the normal diet, whereas deletion of dSIR2 in the fat body abolished the extension of lifespan by a calorie-restriction-like protocol [5]. This example illustrates the key role of dSIR2 in lifespan determination and its central role in mediating dietary effects on longevity, discussed further below. Another study showed that two transgenic mouse lines that overexpress the mammalian SIRT6—mammals have seven sirtuins—had significantly extended lifespans [6]. Finally, a recent study clearly showed that worm sir2.1 could extend lifespan by regulating two distinct longevity pathways involving insulin-like signalling and the mitochondrial unfolded protein response [7]. All told, this body of work supports the original proposal that sirtuins are conserved mediators of longevity.Many other studies also illustrate that sirtuins can mediate the effects of diet. As an example, calorie restriction completely protected against ageing-induced hearing loss in wild type but not SIRT3^−/− mice [8]. The mitochondrial sirtuin SIRT3 thus helps to protect the neurons of the inner ear against oxidative damage during calorie restriction. Of course, these studies do not imply that sirtuins are the only mediators of calorie restriction effects, but they do indicate that they must be central components.Finally, what about the translational potential of this research, namely using putative SIRT1-activating compounds—resveratrol and newer, synthetic STACs? Two new studies provide strong evidence that the effects of these compounds really do occur through SIRT1. First, acute deletion of SIRT1 in adult mice prevented many of the physiological effects of resveratrol and other STACs [9]. Second, a single mutation adjacent to the SIRT1 catalytic domain abolished the ability of STACs to activate the enzyme in vitro, or to promote the canonical physiological changes in vivo [10].In summary, sirtuins seem to represent a promising avenue by which orally available drugs might deliver anti-ageing benefits normally triggered by calorie restriction. Indeed, the biology of sirtuins is complex and diverse, but this is an indication of their deep reach into key disease processes. Connections between sirtuins and cancer metabolism are but one new example of this. The future path of discovery promises to be exciting and might lead to new drugs that maintain robust health.     

Risk of vascular events in patients with polymyalgia rheumatica

Background:

Polymyalgia rheumatica is one of the most common inflammatory rheumatologic conditions in older adults. Other inflammatory rheumatologic disorders are associated with an excess risk of vascular disease. We investigated whether polymyalgia rheumatica is associated with an increased risk of vascular events.

Methods:

We used the General Practice Research Database to identify patients with a diagnosis of incident polymyalgia rheumatica between Jan. 1, 1987, and Dec. 31, 1999. Patients were matched by age, sex and practice with up to 5 patients without polymyalgia rheumatica. Patients were followed until their first vascular event (cardiovascular, cerebrovascular, peripheral vascular) or the end of available records (May 2011). All participants were free of vascular disease before the diagnosis of polymyalgia rheumatica (or matched date). We used Cox regression models to compare time to first vascular event in patients with and without polymyalgia rheumatica.

Results:

A total of 3249 patients with polymyalgia rheumatica and 12 735 patients without were included in the final sample. Over a median follow-up period of 7.8 (interquartile range 3.3–12.4) years, the rate of vascular events was higher among patients with polymyalgia rheumatica than among those without (36.1 v. 12.2 per 1000 person-years; adjusted hazard ratio 2.6, 95% confidence interval 2.4–2.9). The increased risk of a vascular event was similar for each vascular disease end point. The magnitude of risk was higher in early disease and in patients younger than 60 years at diagnosis.

Interpretation:

Patients with polymyalgia rheumatica have an increased risk of vascular events. This risk is greatest in the youngest age groups. As with other forms of inflammatory arthritis, patients with polymyalgia rheumatica should have their vascular risk factors identified and actively managed to reduce this excess risk.Inflammatory rheumatologic disorders such as rheumatoid arthritis,^1,2 systemic lupus erythematosus,^2,3 gout,⁴ psoriatic arthritis^2,5 and ankylosing spondylitis^2,6 are associated with an increased risk of vascular disease, especially cardiovascular disease, leading to substantial morbidity and premature death.^2–6 Recognition of this excess vascular risk has led to management guidelines advocating screening for and management of vascular risk factors.^7–9Polymyalgia rheumatica is one of the most common inflammatory rheumatologic conditions in older adults,¹⁰ with a lifetime risk of 2.4% for women and 1.7% for men.¹¹ To date, evidence regarding the risk of vascular disease in patients with polymyalgia rheumatica is unclear. There are a number of biologically plausible mechanisms between polymyalgia rheumatica and vascular disease. These include the inflammatory burden of the disease,^12,13 the association of the disease with giant cell arteritis (causing an inflammatory vasculopathy, which may lead to subclinical arteritis, stenosis or aneurysms),¹⁴ and the adverse effects of long-term corticosteroid treatment (e.g., diabetes, hypertension and dyslipidemia).^15,16 Paradoxically, however, use of corticosteroids in patients with polymyalgia rheumatica may actually decrease vascular risk by controlling inflammation.¹⁷ A recent systematic review concluded that although some evidence exists to support an association between vascular disease and polymyalgia rheumatica,¹⁸ the existing literature presents conflicting results, with some studies reporting an excess risk of vascular disease^19,20 and vascular death,^21,22 and others reporting no association.^23–26 Most current studies are limited by poor methodologic quality and small samples, and are based on secondary care cohorts, who may have more severe disease, yet most patients with polymyalgia rheumatica receive treatment exclusively in primary care.²⁷The General Practice Research Database (GPRD), based in the United Kingdom, is a large electronic system for primary care records. It has been used as a data source for previous studies,²⁸ including studies on the association of inflammatory conditions with vascular disease²⁹ and on the epidemiology of polymyalgia rheumatica in the UK.³⁰ The aim of the current study was to examine the association between polymyalgia rheumatica and vascular disease in a primary care population.     

The health risks of ART

Assisted reproductive technologies enable subfertile couples to have children. But there are health risks attached for both mothers and children that need to be properly understood and managed.Assisted reproductive technology (ART) has become a standard intervention for couples with infertility problems, especially as ART is highly successful and overall carries low risks [1,2]. The number of infants born following ART has increased steadily worldwide, with more than 5,000,000 so far [3]. In industrialized countries, 1–4% of newborns have been conceived by using ART [4,5], probably owing to the fact that couples frequently delay childbearing until their late 30s, when fertility decreases in both men and women [2]. Considering the possibility that male fertility might be declining, as Richard Sharpe has discussed in this series [6], it is likely that ART will be even more widely used in the future. Yet, as the rate of ART and the total number of pregnancies has increased, it has become apparent that ART is associated with potential risks to the mother and fetus. The most commonly cited health problems pertain to multiple gestation pregnancies and multiple births. More recently, however, concerns about the risks of birth defects and genetic disorders have been raised. There are questions about whether the required manipulations and the artificial environments of gametes and embryos are potentially creating short- and long-term health risks in mothers and children by interfering with epigenetic reprogramming.Notwithstanding, ART represents a tremendous achievement in human reproductive medicine. The birth of Louise Brown, the first ‘test tube baby'' in 1978, was the result of the collaborative work of embryologist Robert Edwards and gynaecologist Patrick Steptoe [7]. This success was a culmination of many years of work at universities and clinics worldwide. An initial lack of support, as well as criticism from ethicists and the church, delayed the opening of the first in vitro fertilization (IVF) clinic in Bourn Hall near Cambridge until 1980. By 1986, 1,000 children conceived by IVF at Bourn Hall had been born [8]. In 2010, Edwards received the Nobel Prize in Medicine for the development of IVF. Regrettably, Steptoe had passed away in 1988 and could not share the honour.…as the rate of ART and the total number of pregnancies has increased, it has become apparent that ART is associated with potential risks to mother and fetusOver the next decades, many improvements in IVF procedures were made to reduce the risks of adverse effects and increase success rates, including controlled ovarian stimulation, timed ovulation induction, ultrasound-guided egg retrieval, cryopreservation of embryos and intracytoplasmic sperm injection (ICSI)—a technique in which a single sperm cell is injected into an oocyte using a microneedle. In addition, there were further improvements such as assisted hatching and in media composition, such as sequential media, which allow the in vitro culture of the embryo to reach the blastocyst stage [8].Current IVF procedures involve multiple steps including ovarian stimulation and monitoring, oocyte retrieval from the ovary, fertilization in vitro and embryo transfer to the womb. Whereas the first IVF cycles, including the conception of Louise Brown, used natural ovulatory cycles, which result in the retrieval of one or two oocytes, most IVF cycles performed today rely on controlled ovarian stimulation using injectable gonadotropins—follicle stimulating hormone and luteinizing hormone—in supraphysiological concentrations for 10–14 days, followed by injection of human chorionic gonadotropin (hCG) 38–40 h before egg retrieval to trigger ovulation. This updated protocol makes it possible to grow multiple follicles and to retrieve 10–20 oocytes in one IVF cycle, thereby increasing the number of eggs available for fertilization.Post-retrieval, the embryologist places an egg and sperm together in a test tube for fertilization. Alternatively, a single sperm cell can be injected into an egg by using ICSI. This procedure was initially developed for couples with poor sperm quality [9], but has become the predominant fertilization technique used in many IVF clinics worldwide [8]. The developing embryos are monitored by microscopy, and viable embryos are transferred into the woman''s womb for implantation. Louise Brown, as with many embryos today, was transferred three days after egg retrieval, at approximately the eight-cell stage. However, using sequential media, many clinics advocate culturing embryos until day five when they reach the blastocyst stage. The prolonged culture period allows self-selection of the most viable embryos for transfer and increases the chance of a viable pregnancy. Excess embryos can be cryopreserved and transferred at a later date by using a procedure known as frozen embryo transfer (FET). In this article we use the term ART to refer to IVF procedures with or without ICSI and FET.

Science & Society Series on Sex and Science

Sex is the greatest invention of all time: not only has sexual reproduction facilitated the evolution of higher life forms, it has had a profound influence on human history, culture and society. This series explores our attempts to understand the influence of sex in the natural world, and the biological, medical and cultural aspects of sexual reproduction, gender and sexual pleasure.Embryos can also be screened for chromosomal aneuploidies—missing or extra chromosomes—by preimplantation genetic diagnosis (PGD) when indicated and when available. PGD can also be used to test fertile couples at increased risk of genetic disorders. To perform PGD, a single cell is obtained from three-day-old embryos for molecular testing, for example sequencing for inherited monogenic disorders or fluorescent in situ hybridization for chromosomal abnormalities [8]. Only embryos with a normal chromosomal constitution, and without the genetic disorder in question, would then be transferred into the woman''s womb.Despite tremendous progress during the past three decades, people undertaking ART still face a considerable risk of failure to achieve parenthood. The rate of clinical pregnancies in Bourn Hall between 1980 and 1985 was 24% and 14% in women younger and older than 40 years, respectively [10]. The reported rates for clinical pregnancies and live births vary by country; the average delivery rate is 22.4%, 23.3% and 17.1% for IVF, ICSI and FET cycles, respectively [11]. According to the last Centers for Disease Control and Prevention report in 2009, the average live-birth rate was 35% per fresh ART cycle, although it sharply declines with age, from 45% among women younger than 35 years to 7% among women older than 42 years [5]. The reasons include poor response to ovarian stimulation, ovarian hyperstimulation syndrome and failure of eggs to fertilize. However, these failures occur in only a minority of patients and the success rate of egg retrieval and fertilization leading to embryo transfer is a remarkable 90% [12].Implantation remains the least understood process and is a key rate-limiting step in ART. Poor embryo quality is considered to be the main cause of implantation failure and it reflects a high incidence of chromosomal aneuploidies, which increases with maternal age [13]. One obvious solution to improve implantation rates is to transfer more embryos. However, this also increases the risk of multiple births, and related morbidity and mortality in newborns. An alternative approach is to select for good-quality embryos by culturing them to the blastocyst stage, because it seems that aneuploid embryos arrest by this stage and that blactocysts are more likely to have a normal chromosomal complement. There is ongoing research aimed at identifying viable embryos through PGD and metabolic profiling [13].Despite tremendous progress during the past three decades, people undertaking ART still face a considerable risk of failure to achieve parenthoodIt has also been suggested that failure to implant could be caused by the inability of the embryo to hatch out of a glycoprotein layer surrounding the embryo, known as the ‘zona pellucida''; this layer hardens if the embryo is cultured or frozen. Assisted hatching by rupturing the zona pellucida before embryo transfer does increase clinical pregnancy rates, especially for thawed embryos [13]. Another factor linked to the failure of implantation is endometrial receptivity. The endometrium consists of multi-layered mucosa cells in the inner wall of the uterus, which undergoes coordinated remodelling during the menstrual cycle and there is a specific time window when it is receptive to embryo implantation. Several research studies have identified molecular biomarkers of poor endometrial receptivity, showing that prostaglandins, cell adhesion molecules, mucins and cytokines are important [13].When it comes to health risks for mothers and infants, the use of ART increases the risk of multiple births, including higher rates of caesarian sections, prematurity, low birth weight, infant death and disability. More recently, concerns regarding elevated risks of birth defects, genetic abnormalities, neurodevelopmental disorders and imprinting disorders have been reported; however, not all are substantiated. There are still many unanswered questions regarding the potential short- and long-term health risks of ART for women and children, and there are tremendous challenges in studying the safety of ART procedures. Apart from the subset of individuals undergoing ART for social reasons—single parents or same sex couples—most patients are subfertile couples. Subfertility, defined as a failure to conceive naturally after 12 months of unprotected intercourse, affects 8–20% of couples [2], and it can occur for a variety of unknown or known reasons including maternal factors—endocrine, hormonal, endometriosis and blocked fallopian tubes—and paternal factors such as spermatogenesis abnormalities.Most studies have assessed the risks of ART by comparing the outcomes of ART-conceived pregnancies to naturally conceived pregnancies. There is emerging evidence that underlying maternal or paternal subfertility might be an important factor in obstetric, neonatal and childhood outcomes in the ART population. Therefore, to determine the specific health risks associated with the ART process itself, the outcomes of ART-conceived pregnancies should be assessed in comparison with naturally conceived pregnancies in subfertile parents, which is methodologically difficult. Alternatively, studying the health risks of ART in fertile couples—for instance, same-sex couples and couples at risk of genetic disorders—would be informative, but the number of such couples is relatively small.Women who undergo ART are at risk of ovarian hyperstimulation syndrome (OHSS). OHSS is a complication of ovulation induction resulting in enlargement of ovaries and retention of fluids leading to various secondary complications, which normally resolve within two weeks, but can persist if pregnancy occurs. Patients with OHSS can be offered embryo cryopreservation and frozen embryo transfer when symptoms resolve. Moderate forms of OHSS occur in 5% of patients undergoing ART; 2% of patients require hospitalization. Death occurs with an incidence of approximately 3 per 100,000 ART cycles [14]. OHSS is predominantly caused by human chorionic gonadotropin injection used for inducing final oocyte maturation and ovulation. Research is focused on optimizing alternative stimulation protocols [14].The use of supraphysiological concentrations of hormones during ovarian stimulation has also raised concerns that ART can increase cancer risks linked to hormonal fluctuations. These include breast, ovarian, endometrial, cervical and colon cancers, as well as melanoma. Studies evaluating the risks of cervical cancers, colon cancers and melanoma have not demonstrated increased risks for women undergoing ART [1]. The data for breast, ovarian and endometrial cancer is more complex, however, and more research is required to conclusively determine whether there is an increased risk.The perinatal and obstetric risks of ART are most significantly influenced by multiple pregnancies. These are at a more than 60% risk of low birth weight or premature delivery [2], and related risks of pregnancy complications such as gestational diabetes, abnormal placentation and hypertensive disorders [1]. Multiple pregnancies occur in 1% of naturally conceived pregnancies and 25–50% of ART pregnancies, owing to multiple embryo transfer. In the Western world, about 30–50% of all twin pregnancies result from ART [2]. Whilst double or triple embryo transfer is still common, the development of cryopreservation techniques and extended blastocyst culture has increased the use of single embryo transfer (SET), especially for younger women. Many European countries and the province of Quebec, in Canada, where ART is publicly funded, have adopted a policy of SET, which has dramatically decreased the incidence of multiple pregnancies. In Belgium and Quebec, SET policies have reduced multiple pregnancies from 19% to 3% and from 27% to 6%, respectively. It has been argued that SET results in a lower live-birth rate than a double-embryo transfer, but this is almost completely overcome by an additional single frozen embryo cycle [2].…there are tremendous challenges in studying the safety of ART proceduresThe question of whether ART increases the risks of pregnancy complications, including prematurity and low birth weight in singletons, remains unresolved; several studies have found an increased risk, but others have not replicated these findings [1,2]. It has been suggested that the fertility history of patients undergoing ART is an important factor, as there is an association between the length of time to conception and prematurity and birth weight [15]. Prematurity and low birth weight are also known to be associated with long-term health effects, including adult onset coronary artery disease, hypertension, obesity and type 2 diabetes [16,17].Various studies have also reported a higher incidence of congenital anomalies in ART-conceived children, with a suggested 30% increase of malformations [2]. However, this is another risk that might be attributable to parental subfertility, as a study comparing children conceived by ART to subfertile parents and children conceived naturally to subfertile parents did not find any significant difference in the congenital anomaly rate [2]. Findings from another study of the risks of birth defects in children conceived naturally to women with and without a history of subfertility compared with children conceived with the assistance of ART also suggest that it is subfertility, rather than ART, that is associated with an increased risk of birth defects [18].Several studies reported an increased risk of cerebral palsy and other neurological abnormalities in children conceived by ART [2]. But again, these findings are mainly attributed to complications resulting from multiple pregnancies including prematurity and low birth weight. The increased utilization of SET is therefore expected to result in fewer multiple pregnancies, which should result in a concomitant decrease in neurological complications. Further evidence that neurological complications in ART children are not exclusively related to ART came from studies that have assessed neurodevelopmental outcomes, such as locomotion, cognition, language and behavioural development of ART children in comparison with naturally conceived children. These analyses did not reveal any differences when adjusted for confounding factors of low birth weight and prematurity. In a similar vein, numerous studies have investigated whether there is an increased incidence of autism in ART-conceived children, but these have been inconclusive [19].There are potential concerns regarding the fertility of ART children. However, this requires future studies as most of this population is younger than 30 years of age. There is some evidence that boys conceived through ICSI have an increased rate of genital anomalies [2] and that males with severe infertility, such as low sperm counts, are more likely to carry chromosomal abnormalities, which could be passed on to their children conceived through ICSI [15].It has also been suggested that there might be an increased risk of cancers in ART-conceived offspring. Although multiple studies have identified no such risk, a large Swedish study reported a marginally increased risk of cancer, including haematologic, eye, nervous system, solid tumours and histiocytosis [2]. Similarly to other ART-related adverse health outcomes, it has been suggested that the increased risk of cancer could be attributed to prematurity, a recognized risk factor for cancer, rather than to the ART procedure itself. Further long-term studies are required to determine if there is truly an increased risk of adult cancers in ART offspring.…there remain unanswered questions about both the health risks associated with ART and the potential mechanisms that could account for these findingsOne thing is clear from the available evidence to date: there remain unanswered questions about both the health risks associated with ART and the potential mechanisms that could account for these findings. One possible explanation is that the exposure of gametes and preimplantation embryos to the various steps of ART might affect growth and development of offspring through dysregulation of epigenetic pathways [20]. In addition, there is evidence that genetic and epigenetic alterations might be inherited from the gametes of subfertile parents, which would reinforce assertions that subfertility itself might play a role in ART-related health outcomes [1,20].Epigenetics refers to heritable changes in gene expression without alterations to the underlying DNA sequence. DNA methylation and modifications of histones are epigenetic modifications that determine active against repressive conformation of chromatin structure, thereby regulating gene expression and driving essential processes such as embryonic development, fetal organ development, cell differentiation and tissue-specific gene expression [21]. Genomic imprinting is a type of epigenetic gene regulation that uses epigenetic marks to silence specifically one of the parental alleles. There are approximately 100 known imprinted genes in humans [22]. Most imprinted genes are found in clusters across the genome and are regulated by parent-specific DNA methylation and histone modification marks at cis-acting imprinting centres, as well as non-coding RNAs. Most of the known imprinted genes have functions related to growth and behaviour; disruption of the normally programmed parental expression of imprinted genes can therefore result in disorders related to growth and neurodevelopment.Gametogenesis and embryogenesis are important stages of mammalian development that require genome-wide epigenetic reprogramming. During spermatogenesis, protamines replace most histone proteins to create a highly compacted DNA. Establishment of DNA methylation imprints at paternally methylated imprinting centres is complete in males at the time of birth. In females, the establishment of maternally methylated imprinting centres begins during puberty and is almost complete in ovulated oocytes. After fertilization, the paternal genome undergoes rapid active DNA demethylation in which protamines are replaced by histones, whilst the maternal genome is passively demethylated, so that DNA methylation patterns are lost through cell divisions. Although, the whole genome undergoes demethylation, parent-specific DNA methylation is maintained at imprinting centres. Subsequently, the genome is remethylated and cell-type-specific epigenetic patterns are established as embryonic development proceeds. The parent-specific DNA methylation at imprinting centres is maintained in somatic cells, but it is erased and re-established in the gametes starting a new cycle of imprinting (Fig 1; [23]). As the establishment and maintenance of imprinting marks coincides in timing with important stages of ART, such as oocyte maturation under supraphysiological hormone concentrations and embryo culture, it has been proposed that ART can lead to imprinting errors [24].Open in a separate windowFigure 1Life cycle of genomic imprinting and assisted reproductive technology. Erasure, re-establishment and maintenance of genomic imprinting occur during gametogenesis and preimplantation embryo development. Blue and red solid lines show paternal and maternal methylation at imprinting centres through gametogenesis and early stages of preimplantation development. Imprinting marks are erased at early stages of gametogenesis. Re-establishment of imprinting occurs throughout gametogenesis, but finishes much later in oocytes compared with sperm. During preimplantation development, both maternal and paternal imprinting marks are maintained whilst the rest of the genome is demethylated. The paternal genome is demethylated rapidly and actively (dashed blue line) whilst the maternal genome is demethylated at a slower rate passively through cell division (dashed red line). Various steps of assisted reproductive technology such as ovarian stimulation, ovulation induction, gamete and embryo manipulation and culturing create unusual environments for gametes and embryos and thus, can interfere with proper establishment of imprinting marks in oocytes or maintenance of imprinting marks in embryos. Subfertility can be associated with epigenetic errors in imprinting erasure and/or establishment in both oocytes and sperm. Adapted from [23].In 2001, the first evidence that genomic imprinting can be perturbed during ART procedures came from studying sheep fetuses derived from in vitro cultured embryos that presented with large offspring syndrome (LOS; [25]). LOS occurs sporadically in cattle and sheep conceived by IVF and is characterized by a 20–30% increase in birth weight frequently accompanied by congenital anomalies and placental dysfunction [24]. Owing to phenotypic similarities of LOS to the human overgrowth disorder Beckwith–Wiedemann syndrome (BWS), which is caused by the dysregulation of gene expression within an imprinted cluster on chromosome 11p15.5, the authors hypothesized that genes from the orthologous cluster in sheep or a closely related pathway could be dysregulated in LOS. They tested expression of the insulin-like growth factor 2 (IGF2) gene known to be overexpressed in BWS, and the IGF2R receptor gene, which is involved in clearance of IGF2 from the circulation. IGF2R is imprinted in sheep but not in humans. In sheep with LOS, no differences for IGF2 were found, but reduced expression of IGF2R was observed after loss of DNA methylation at the imprinting centre for this gene [25].In the following decade, several studies provided further evidence that children conceived by ART might be at increased risk of imprinting disorders. The strongest case has been made for BWS and Angelman syndrome. BWS is the most common human overgrowth syndrome characterized by prenatal and postnatal overgrowth, congenital anomalies and tumour predisposition [26]. Angelman syndrome is a neurodevelopmental disorder characterized by microcephaly, severe intellectual disability and a unique behavioural profile including frequent laughter, smiling and excitability [27]. Multiple case reports from various countries indicate an increased frequency of BWS and Angelman syndrome in ART children (3–10-fold) compared with the general population. However, two cohort studies failed to replicate this association [28]. The low incidence of both BWS (1 in 13,700) and Angelman syndrome (1 in 15,000) in the general population [28] makes epidemiological studies difficult—the two cohort studies reported 2,492 and 6,052 ART children, respectively, and are probably underpowered to detect an increased risk of BWS and Angelman syndrome. However, even if there might be increased relative risks for these syndromes in ART children, the absolute risks in this population remain low.The molecular causes of BWS and Angelman syndrome are heterogeneous. They include genomic (deletion, uniparental disomy and gene mutation) and epigenetic (loss of imprinting due to aberrant DNA methylation) alterations at imprinted gene clusters on chromosomes 11p5.5 and 15q11–q13, respectively. These alterations occur with specific frequencies for each of the two disorders [26,27]. Results of molecular testing in children with these syndromes and conceived using ART, reveal an excess of epigenetic compared with genetic molecular alterations. For example, loss of DNA methylation at imprinting centre 2 occurs in about 50% of BWS cases in the general population, whereas several studies found loss of DNA methylation at imprinting centre 2 in 96% (27/28) of BWS ART-conceived children. In Angelman syndrome, approximately 3% of cases in the general population have loss of methylation at 15q11–13, whereas 5 out of 19 (26%) Angelman syndrome children conceived by ART or naturally by parents with a history of subfertility had loss of DNA methylation at 15q11–13 (Fig 2).Open in a separate windowFigure 2Enrichment of epigenetic alterations in Beckwith–Wiedemann syndrome and Angelman syndrome after assisted reproductive technology. Loss of methylation (LOM) at imprinting centre 2 (IC2) on chromosome 11p15.5 contributes to 50% of Beckwith–Wiedemann syndrome (BWS) cases in the general population, whereas LOM at IC2 is found in 27 out of 28 cases (96%) in the BWS assisted reproductive technology (ART) population, which represents a 1.9-fold enrichment of this epigenetic defect. For Angelman syndrome (AS), methylation disruption at the 15q11–q13 imprinting centre contributes to 3% of AS cases, and in the AS ART and subfertility population it was found in 5 out of 19 cases (26%; eight fold enrichment). Data from the following publications were used for these calculations, BWS [31,32,33,34,35] AS [35,36].The data for loss of DNA methylation in Angelman syndrome cases conceived naturally by subfertile parents highlights the fact that epigenetic alterations could, at least in part, result from underlying parental subfertility. Indeed, several studies have shown that abnormalities of spermatogenesis, such as oligospermia (low sperm concentration), low sperm motility or abnormal sperm morphology are associated with altered DNA methylation at imprinted loci. These occur in both maternal and paternal alleles of imprinting centres in sperm and could be transmitted to offspring conceived by ART [26]. One study of chromosomally normal fetuses spontaneously aborted at six to nine weeks of gestation found that DNA methylation alterations at imprinted loci were sometimes inherited from sperm. Thus, it is possible that this dysregulation of imprinting in male gametes might be one cause of the association between imprinting disorders and ART.Studies of other known imprinted syndromes, such as Prader–Willi syndrome, Russell–Silver syndrome, maternal and paternal uniparental disomy of chromosome 14, pseudohypoparathyroidism type 1b and transient neonatal diabetes mellitus, have either not demonstrated an association with ART or have been inconclusive owing to their small size [29]. A link has also been suggested between ART and the newly defined ‘multiple maternal hypomethylation syndrome'', which clinically presents either as BWS or transient neonatal diabetes mellitus, and is associated with loss of DNA methylation at multiple maternally methylated imprinting centres; loss of methylation at paternal imprinting centres has not been reported so far. Thus, human imprinting disorders that have been observed with increased relative frequency in ART offspring are confined to loss of DNA methylation at maternally methylated imprinting centres, similar to epimutations of IGF2R in LOS. One could propose that ART has a greater impact on female than male gametes, as the eggs are subjected to more environmental exposures—supraphysiological doses of hormones—and more manipulation than the sperm. However, studies of mouse in vitro cultured embryos and ART-exposed human and mouse gametes suggest that ART can also be associated with either loss or gain of DNA methylation on both maternal and paternal alleles [23].Mouse models are a valuable method to investigate which stages of ART procedures can disrupt normal imprinting patterns. The advantage of using mouse models is the ability to investigate each of the parameters of ART—ovulation stimulation and embryo culturing—separately and at different stages of development. Furthermore, mouse models allow investigators to alter ART parameters, such as concentration of hormones or media for embryo culturing. Most importantly, studies in animal models have shown that ART procedures without the confounding factor of subfertility do have a negative impact on imprint regulation [23].The exposure of maturing oocytes from mice to abnormally high doses of gonadotropins has been suggested to alter imprint establishment. Yet, studies performed directly on superovulated oocytes are inconclusive, as not all of them have demonstrated increased rates of DNA methylation errors at imprint centres compared with spontaneously ovulated oocytes. Interestingly, studies of DNA methylation in mouse blastocysts harvested from superovulated mothers identified an increased rate of DNA methylation errors at imprint centres. This included loss of DNA methylation at the paternally methylated H19—the imprinting centre on human chromosome 11 and mouse chromosome 7 implicated in BWS and the related undergrowth Russell–Silver syndrome. It suggests that superovulation also impairs imprinting maintenance; probably by affecting the ability of the oocyte to synthesize and store sufficient maternal factors (RNA and proteins; [23]). In support of this hypothesis, four maternal effect proteins have been previously identified that are involved in imprinting maintenance in preimplantation embryos. It was also found that imprint errors arise in blastocysts in a dose-dependent manner—higher doses of hormones resulted in DNA methylation errors in a larger number of embryos [23].As the establishment and maintenance of imprinting marks coincides in timing with important stages of ART […] it has been proposed that ART can lead to imprinting errorsAnother factor that might contribute to imprinting errors is the micromanipulation of gametes during IVF and ICSI procedures. Evidence supporting this hypothesis includes the observation in mouse models that a higher number of IVF embryos—resulting from superovulation alone or superovulation and embryo culturing—have aberrant H19 DNA methylation compared with in vivo conceived embryos [23]. Media with varying compositions are used in ART clinics, and whilst all of the media are suboptimal for normal maintenance of all DNA imprints in mouse embryos, the number of embryos with aberrant DNA methylation at imprinting centres varies depending on the media [23]. Interestingly, it was also found that embryos with faster rates of development are more prone to loss of DNA methylation at imprinting centres [23].Though it is not yet clear how these findings relate to ART in humans, the mouse research is crucial for informing human studies about which variables should be addressed to optimize the safety and efficacy of ART procedures. Apart from ART itself, it has been shown that compromised fertility in mice results in loss or delay of DNA methylation acquisition in one of three tested imprinted genes. The compromised fertility is induced by genetic manipulation of a gene involved in communication between oocytes and surrounding follicular cells, which is crucial for proper oocyte maturation. The results suggest that the observed loss of DNA methylation could be caused by impaired transport of metabolites from follicular cells to oocytes, which is important for imprint establishment [23].Data linking dysregulation of imprinted loci and ART is limited to several imprinted gene clusters associated with clinically recognizable syndromes. However, there are more genes in the human genome that have been discovered to be, or are predicted to be, imprinted [22] but are not yet known to be associated with clinical phenotypes. Potentially, ART can lead to dysregulation of these imprinted genes, which might be another, as yet unrecognized factor contributing to neonatal and long-term health problems of ART-conceived children. At this point, it is also not clear whether epigenetic disruption during ART is limited to imprinted genes or has more global effects on the genome. The data for genome-wide DNA methylation analysis are limited in both human and mouse to individuals with no apparent disease phenotype. So far, these data have been inconclusive [23,28].One could propose that ART has a greater impact on female than male gametes, as the eggs are subjected to more environmental exposures […] and more manipulation than the spermDespite significant advances in the efficacy and success of ART procedures during the past few decades, the health risks, especially related to long-term outcomes in ART-conceived children, remain poorly understood. Moreover, the phenomena known as ‘fetal programming''—when maternal and in utero exposures can lead to various adult onset disease susceptibilities—have been suggested to be transmissible to the next generations, probably through epigenetic mechanisms [30]. In the case of ART procedures, the effect of ‘unusual'' environments during gametogenesis and early embryonic development on adult-onset disease and trans-generational inheritance is still not clear. Additional research is needed to elucidate the effects of ART on genome-wide epigenetic patterns and their link to human disease. As ART will continue to be an important medical intervention and the number of children born with the help of ART procedures will probably continue to rise in the future, it is crucial to understand the associated health risks and underlying molecular mechanisms of these technologies. This will increase the safety of this intervention and enable couples using ART to be fully informed regarding both present and future health-related risks.? Open in a separate windowDaria GrafodatskayaOpen in a separate windowCheryl CytrynbaumOpen in a separate windowRosanna Weksberg     

Effectiveness of interventions designed to reduce the use of imaging for low-back pain: a systematic review

Background:Rates of imaging for low-back pain are high and are associated with increased health care costs and radiation exposure as well as potentially poorer patient outcomes. We conducted a systematic review to investigate the effectiveness of interventions aimed at reducing the use of imaging for low-back pain.Methods:We searched MEDLINE, Embase, CINAHL and the Cochrane Central Register of Controlled Trials from the earliest records to June 23, 2014. We included randomized controlled trials, controlled clinical trials and interrupted time series studies that assessed interventions designed to reduce the use of imaging in any clinical setting, including primary, emergency and specialist care. Two independent reviewers extracted data and assessed risk of bias. We used raw data on imaging rates to calculate summary statistics. Study heterogeneity prevented meta-analysis.Results:A total of 8500 records were identified through the literature search. Of the 54 potentially eligible studies reviewed in full, 7 were included in our review. Clinical decision support involving a modified referral form in a hospital setting reduced imaging by 36.8% (95% confidence interval [CI] 33.2% to 40.5%). Targeted reminders to primary care physicians of appropriate indications for imaging reduced referrals for imaging by 22.5% (95% CI 8.4% to 36.8%). Interventions that used practitioner audits and feedback, practitioner education or guideline dissemination did not significantly reduce imaging rates. Lack of power within some of the included studies resulted in lack of statistical significance despite potentially clinically important effects.Interpretation:Clinical decision support in a hospital setting and targeted reminders to primary care doctors were effective interventions in reducing the use of imaging for low-back pain. These are potentially low-cost interventions that would substantially decrease medical expenditures associated with the management of low-back pain.Current evidence-based clinical practice guidelines recommend against the routine use of imaging in patients presenting with low-back pain.^1–3 Despite this, imaging rates remain high,^4,5 which indicates poor concordance with these guidelines.^6,7Unnecessary imaging for low-back pain has been associated with poorer patient outcomes, increased radiation exposure and higher health care costs.⁸ No short- or long-term clinical benefits have been shown with routine imaging of the low back, and the diagnostic value of incidental imaging findings remains uncertain.^9–12 A 2008 systematic review found that imaging accounted for 7% of direct costs associated with low-back pain, which in 1998 translated to more than US$6 billion in the United States and £114 million in the United Kingdom.¹³ Current costs are likely to be substantially higher, with an estimated 65% increase in spine-related expenditures between 1997 and 2005.¹⁴Various interventions have been tried for reducing imaging rates among people with low-back pain. These include strategies targeted at the practitioner such as guideline dissemination,^15–17 education workshops,^18,19 audit and feedback of imaging use,^7,20,21 ongoing reminders⁷ and clinical decision support.^22–24 It is unclear which, if any, of these strategies are effective.²⁵ We conducted a systematic review to investigate the effectiveness of interventions designed to reduce imaging rates for the management of low-back pain.     

Prevalence of and risk factors for persistent postoperative nonanginal pain after cardiac surgery: a 2-year prospective multicentre study

Background:

Persistent postoperative pain continues to be an underrecognized complication. We examined the prevalence of and risk factors for this type of pain after cardiac surgery.

Methods:

We enrolled patients scheduled for coronary artery bypass grafting or valve replacement, or both, from Feb. 8, 2005, to Sept. 1, 2009. Validated measures were used to assess (a) preoperative anxiety and depression, tendency to catastrophize in the face of pain, health-related quality of life and presence of persistent pain; (b) pain intensity and interference in the first postoperative week; and (c) presence and intensity of persistent postoperative pain at 3, 6, 12 and 24 months after surgery. The primary outcome was the presence of persistent postoperative pain during 24 months of follow-up.

Results:

A total of 1247 patients completed the preoperative assessment. Follow-up retention rates at 3 and 24 months were 84% and 78%, respectively. The prevalence of persistent postoperative pain decreased significantly over time, from 40.1% at 3 months to 22.1% at 6 months, 16.5% at 12 months and 9.5% at 24 months; the pain was rated as moderate to severe in 3.6% at 24 months. Acute postoperative pain predicted both the presence and severity of persistent postoperative pain. The more intense the pain during the first week after surgery and the more it interfered with functioning, the more likely the patients were to report persistent postoperative pain. Pre-existing persistent pain and increased preoperative anxiety also predicted the presence of persistent postoperative pain.

Interpretation:

Persistent postoperative pain of nonanginal origin after cardiac surgery affected a substantial proportion of the study population. Future research is needed to determine whether interventions to modify certain risk factors, such as preoperative anxiety and the severity of pain before and immediately after surgery, may help to minimize or prevent persistent postoperative pain.Postoperative pain that persists beyond the normal time for tissue healing (> 3 mo) is increasingly recognized as an important complication after various types of surgery and can have serious consequences on patients’ daily living.^1–3 Cardiac surgeries, such as coronary artery bypass grafting (CABG) and valve replacement, rank among the most frequently performed interventions worldwide.⁴ They aim to improve survival and quality of life by reducing symptoms, including anginal pain. However, persistent postoperative pain of nonanginal origin has been reported in 7% to 60% of patients following these surgeries.^5–23 Such variability is common in other types of major surgery and is due mainly to differences in the definition of persistent postoperative pain, study design, data collection methods and duration of follow-up.^1–3,24Few prospective cohort studies have examined the exact time course of persistent postoperative pain after cardiac surgery, and follow-up has always been limited to a year or less.^9,14,25 Factors that put patients at risk of this type of problem are poorly understood.²⁶ Studies have reported inconsistent results regarding the contribution of age, sex, body mass index, preoperative angina, surgical technique, grafting site, postoperative complications or level of opioid consumption after surgery.^{5–7,9,13,14,16–19,21–23,25,27} Only 1 study investigated the role of chronic nonanginal pain before surgery as a contributing factor;²¹ 5 others prospectively assessed the association between persistent postoperative pain and acute pain intensity in the first postoperative week but reported conflicting results.^{13,14,21,22,25} All of the above studies were carried out in a single hospital and included relatively small samples. None of the studies examined the contribution of psychological factors such as levels of anxiety and depression before cardiac surgery, although these factors have been shown to influence acute or persistent postoperative pain in other types of surgery.^1,24,28,29We conducted a prospective multicentre cohort study (the CARD-PAIN study) to determine the prevalence of persistent postoperative pain of nonanginal origin up to 24 months after cardiac surgery and to identify risk factors for the presence and severity of the condition.     

Erythropoietin Receptor (EpoR) Agonism Is Used to Treat a Wide Range of Disease

The erythropoietin receptor (EpoR) was discovered and described in red blood cells (RBCs), stimulating its proliferation and survival. The target in humans for EpoR agonists drugs appears clear—to treat anemia. However, there is evidence of the pleitropic actions of erythropoietin (Epo). For that reason, rhEpo therapy was suggested as a reliable approach for treating a broad range of pathologies, including heart and cardiovascular diseases, neurodegenerative disorders (Parkinson’s and Alzheimer’s disease), spinal cord injury, stroke, diabetic retinopathy and rare diseases (Friedreich ataxia). Unfortunately, the side effects of rhEpo are also evident. A new generation of nonhematopoietic EpoR agonists drugs (asialoEpo, Cepo and ARA 290) have been investigated and further developed. These EpoR agonists, without the erythropoietic activity of Epo, while preserving its tissue-protective properties, will provide better outcomes in ongoing clinical trials. Nonhematopoietic EpoR agonists represent safer and more effective surrogates for the treatment of several diseases such as brain and peripheral nerve injury, diabetic complications, renal ischemia, rare diseases, myocardial infarction, chronic heart disease and others.In principle, the erythropoietin receptor (EpoR) was discovered and described in red blood cell (RBC) progenitors, stimulating its proliferation and survival. Erythropoietin (Epo) is mainly synthesized in fetal liver and adult kidneys (1–3). Therefore, it was hypothesized that Epo act exclusively on erythroid progenitor cells. Accordingly, the target in humans for EpoR agonists drugs (such as recombinant erythropoietin [rhEpo], in general, called erythropoiesis-simulating agents) appears clear (that is, to treat anemia). However, evidence of a kaleidoscope of pleitropic actions of Epo has been provided (4,5). The Epo/EpoR axis research involved an initial journey from laboratory basic research to clinical therapeutics. However, as a consequence of clinical observations, basic research on Epo/EpoR comes back to expand its clinical therapeutic applicability.Although kidney and liver have long been considered the major sources of synthesis, Epo mRNA expression has also been detected in the brain (neurons and glial cells), lung, heart, bone marrow, spleen, hair follicles, reproductive tract and osteoblasts (6–17). Accordingly, EpoR was detected in other cells, such as neurons, astrocytes, microglia, immune cells, cancer cell lines, endothelial cells, bone marrow stromal cells and cells of heart, reproductive system, gastrointestinal tract, kidney, pancreas and skeletal muscle (18–27). Conversely, Sinclair et al.(28) reported data questioning the presence or function of EpoR on nonhematopoietic cells (endothelial, neuronal and cardiac cells), suggesting that further studies are needed to confirm the diversity of EpoR. Elliott et al.(29) also showed that EpoR is virtually undetectable in human renal cells and other tissues with no detectable EpoR on cell surfaces. These results have raised doubts about the preclinical basis for studies exploring pleiotropic actions of rhEpo (30).For the above-mentioned data, a return to basic research studies has become necessary, and many studies in animal models have been initiated or have already been performed. The effect of rhEpo administration on angiogenesis, myogenesis, shift in muscle fiber types and oxidative enzyme activities in skeletal muscle (4,31), cardiac muscle mitochondrial biogenesis (32), cognitive effects (31), antiapoptotic and antiinflammatory actions (33–37) and plasma glucose concentrations (38) has been extensively studied. Neuro- and cardioprotection properties have been mainly described. Accordingly, rhEpo therapy was suggested as a reliable approach for treating a broad range of pathologies, including heart and cardiovascular diseases, neurodegenerative disorders (Parkinson’s and Alzheimer’s disease), spinal cord injury, stroke, diabetic retinopathy and rare diseases (Friedreich ataxia).Unfortunately, the side effects of rhEpo are also evident. Epo is involved in regulating tumor angiogenesis (39) and probably in the survival and growth of tumor cells (25,40,41). rhEpo administration also induces serious side effects such as hypertension, polycythemia, myocardial infarction, stroke and seizures, platelet activation and increased thromboembolic risk, and immunogenicity (42–46), with the most common being hypertension (47,48). A new generation of nonhematopoietic EpoR agonists drugs have hence been investigated and further developed in animals models. These compounds, namely asialoerythropoietin (asialoEpo) and carbamylated Epo (Cepo), were developed for preserving tissue-protective properties but reducing the erythropoietic activity of native Epo (49,50). These drugs will provide better outcome in ongoing clinical trials. The advantage of using nonhematopoietic Epo analogs is to avoid the stimulation of hematopoiesis and thereby the prevention of an increased hematocrit with a subsequent procoagulant status or increased blood pressure. In this regard, a new study by van Rijt et al. has shed new light on this topic (51). A new nonhematopoietic EpoR agonist analog named ARA 290 has been developed, promising cytoprotective capacities to prevent renal ischemia/reperfusion injury (51). ARA 290 is a short peptide that has shown no safety concerns in preclinical and human studies. In addition, ARA 290 has proven efficacious in cardiac disorders (52,53), neuropathic pain (54) and sarcoidosis-induced chronic neuropathic pain (55). Thus, ARA 290 is a novel nonhematopoietic EpoR agonist with promising therapeutic options in treating a wide range of pathologies and without increased risks of cardiovascular events.Overall, this new generation of EpoR agonists without the erythropoietic activity of Epo while preserving tissue-protective properties of Epo will provide better outcomes in ongoing clinical trials (49,50). Nonhematopoietic EpoR agonists represent safer and more effective surrogates for the treatment of several diseases, such as brain and peripheral nerve injury, diabetic complications, renal ischemia, rare diseases, myocardial infarction, chronic heart disease and others.     

Ocular Infection of Mice with Influenza A (H7) Viruses: a Site of Primary Replication and Spread to the Respiratory Tract

Avian H7 influenza viruses have been responsible for poultry outbreaks worldwide and have resulted in numerous cases of human infection in recent years. The high rate of conjunctivitis associated with avian H7 subtype virus infections may represent a portal of entry for avian influenza viruses and highlights the need to better understand the apparent ocular tropism observed in humans. To study this, mice were inoculated by the ocular route with viruses of multiple subtypes and degrees of virulence. We found that in contrast to human (H3N2 and H1N1) viruses, H7N7 viruses isolated from The Netherlands in 2003 and H7N3 viruses isolated from British Columbia, Canada, in 2004, two subtypes that were highly virulent for poultry, replicated to a significant titer in the mouse eye. Remarkably, an H7N7 virus, as well as some avian H5N1 viruses, spread systemically following ocular inoculation, including to the brain, resulting in morbidity and mortality of mice. This correlated with efficient replication of highly pathogenic H7 and H5 subtypes in murine corneal epithelial sheets (ex vivo) and primary human corneal epithelial cells (in vitro). Influenza viruses were labeled to identify the virus attachment site in the mouse cornea. Although we found abundant H7 virus attachment to corneal epithelial tissue, this did not account for the differences in virus replication as multiple subtypes were able to attach to these cells. These findings demonstrate that avian influenza viruses within H7 and H5 subtypes are capable of using the eye as a portal of entry.Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have resulted in over 420 documented cases of human infection to date, have generally caused acute, often severe and fatal, respiratory illness (1, 50). While conjunctivitis following infection with H5N1 or human influenza viruses has been rare, most human infections associated with H7 subtype viruses have resulted in ocular and not respiratory disease (1, 9, 37, 38). Infrequent reports of human conjunctivitis infection following exposure to H7 influenza viruses date from 1977, predominantly resulting from laboratory or occupational exposure (21, 40, 48). However, in The Netherlands in 2003, more than 80 human infections with H7N7 influenza virus occurred among poultry farmers and cullers amid widespread outbreaks of HPAI in domestic poultry; the majority of these human infections resulted in conjunctivitis (14, 20). Additionally, conjunctivitis was documented in the two human infections resulting from an H7N3 outbreak in British Columbia, Canada, in 2004, as well as in H7N3- and H7N2-infected individuals in the United Kingdom in 2006 and 2007, respectively (13, 18, 29, 46, 51). The properties that contribute to an apparent ocular tropism of some influenza viruses are currently not well understood (30).Host cell glycoproteins bearing sialic acids (SAs) are the cellular receptors for influenza viruses and can be found on epithelial cells within both the human respiratory tract and ocular tissue (26, 31, 41). Both respiratory and ocular tissues additionally secrete sialylated mucins that function in pathogen defense and protection of the epithelial surface (5, 11, 22). Within the upper respiratory tract, α2-6-linked SAs (the preferred receptor for human influenza viruses) predominate on epithelial cells (26). While α2-3-linked SAs are also present to a lesser degree on respiratory epithelial cells, this linkage is more abundantly expressed on secreted mucins (2). In contrast, α2-3-linked SAs (the preferred receptor for avian influenza viruses) are found on corneal and conjunctival epithelial cells of the human eye (31, 41), while secreted ocular mucins are abundantly composed of α2-6 SAs (5). It has been suggested that avian influenza viruses are more suited to infect the ocular surface due to their general α2-3-linked SA binding preference, but this has not been demonstrated experimentally (30).The mouse model has been used previously to study the role of ocular exposure to respiratory viruses (6, 39). In mice, ocular inoculation with an H3N2 influenza virus resulted in virus replication in nasal turbinates and lung (39), whereas ocular infection with respiratory syncytial virus (RSV) resulted in detectable virus titers in the eye and lung (6). These studies have revealed that respiratory viruses are not limited to the ocular area following inoculation at this site. However, the ability of influenza viruses to replicate specifically within ocular tissue has not been examined.Despite repeated instances of conjunctivitis associated with H7 subtype infections in humans, the reasons for this apparent ocular tropism have not been studied extensively. Here, we present a murine model to study the ability of human and avian influenza viruses to cause disease by the ocular route. We found that highly pathogenic H7 and H5 influenza viruses were capable of causing a systemic and lethal infection in mice following ocular inoculation. These highly pathogenic viruses, unlike human H3N2 and H1N1 viruses, replicated to significant titers in the mouse corneal epithelium and primary human corneal epithelial cells (HCEpiCs). Identification of viruses well suited to infecting the ocular surface is the first step in better understanding the ability of influenza viruses of multiple subtypes to use this tissue as a portal of entry.     

Modelling mitigation strategies for pandemic (H1N1) 2009

Background

The 2009 influenza A (H1N1) pandemic has required decision-makers to act in the face of substantial uncertainties. Simulation models can be used to project the effectiveness of mitigation strategies, but the choice of the best scenario may change depending on model assumptions and uncertainties.

Methods

We developed a simulation model of a pandemic (H1N1) 2009 outbreak in a structured population using demographic data from a medium-sized city in Ontario and epidemiologic influenza pandemic data. We projected the attack rate under different combinations of vaccination, school closure and antiviral drug strategies (with corresponding “trigger” conditions). To assess the impact of epidemiologic and program uncertainty, we used “combinatorial uncertainty analysis.” This permitted us to identify the general features of public health response programs that resulted in the lowest attack rates.

Results

Delays in vaccination of 30 days or more reduced the effectiveness of vaccination in lowering the attack rate. However, pre-existing immunity in 15% or more of the population kept the attack rates low, even if the whole population was not vaccinated or vaccination was delayed. School closure was effective in reducing the attack rate, especially if applied early in the outbreak, but this is not necessary if vaccine is available early or if pre-existing immunity is strong.

Interpretation

Early action, especially rapid vaccine deployment, is disproportionately effective in reducing the attack rate. This finding is particularly important given the early appearance of pandemic (H1N1) 2009 in many schools in September 2009.Jurisdictions in the northern hemisphere are bracing for a “fall wave” of pandemic (H1N1) 2009.^1–3 Decision-makers face uncertainty, not just with respect to epidemiologic characteristics of the virus,⁴ but also program uncertainties related to feasibility, timeliness and effectiveness of mitigation strategies.⁵ Policy decisions must be made against this backdrop of uncertainty. However, the effectiveness of any mitigation strategy generally depends on the epidemiologic characteristics of the pathogen as well as the other mitigation strategies adopted. Mathematical models can project strategy effectiveness under hypothetical epidemiologic and program scenarios.^6–12 In the case of pandemic influenza, models have been used to assess the effectiveness of school closure⁷ and optimal use of antiviral drug^6,9,10 and vaccination strategies.⁸ However, model projections can be sensitive to input parameter values; thus, data uncertainty is an issue.¹³ Uncertainty analysis can help address the impact of uncertainties on model predictions but is often underutilized.¹³In this article, we present a simulation model of pandemic influenza transmission and mitigation in a population. This model projects the overall attack rate (percentage of people infected) during an outbreak. We introduce a formal method of uncertainty analysis that has not previously been applied to pandemic influenza, and we use this method to assess the impact of epidemiologic and program uncertainties. The model is intended to address the following policy questions that have been raised during the 2009 influenza pandemic: What is the impact of delayed vaccine delivery on attack rates? Can attack rates be substantially reduced without closing schools? What is the impact of pre-existing immunity from spring and summer 2009? We addressed these questions using a simulation model that projects the impact of vaccination, school closure and antiviral drug treatment strategies on attack rates.