accession_id (string)	pmid (string)	introduction (sequence)	methods (sequence)	results (sequence)	discussion (sequence)	conclusion (sequence)	front (sequence)	body (sequence)	back (sequence)	figure (sequence)	table (sequence)	formula (sequence)	box (sequence)	code (sequence)	quote (sequence)	chemical (sequence)	supplementary (sequence)	footnote (sequence)	graphic (sequence)	media (sequence)	unknown_pub (string)	glossary (sequence)	n_references (int32)	license (string)	retracted (string)	last_updated (string)	citation (string)	package_file (string)
"PMC7114196"	"25175676"	[ "<title>Introduction</title>", "<p id=\"par0005\">Gastrointestinal and hepatic diseases in dogs are the third most frequent problem reported by owners in United States and Australia (##REF##16910851##Freeman et al., 2006##). Diarrhea represents one of the most frequent disorders in dogs examined at private veterinary practice, with a prevalence of 2.2% (##REF##10319174##Lund et al., 1999##), young dogs under 6 months of age being at a higher risk of diarrhea than adult dogs (##REF##22812470##Tupler et al., 2012##). In puppies, degradation of feces quality is associated with a reduced daily weight gain and an increased risk of death (##REF##22520179##Grellet et al., 2012##).</p>", "<p id=\"par0010\">A great variety of parasites and viruses are described to be enteropathogens during the weaning period in puppies. <italic>Giardia duodenalis</italic>, <italic>Cryptosporidium parvum</italic>, <italic>Toxocara canis</italic>, <italic>Cystoisospora ohioensis</italic> complex, <italic>Cystoisospora canis</italic>, canine parvovirus type 2 (CPV2) and canine coronavirus (CCV) are the most prevalent (##REF##12549614##Hackett and Lappin, 2003##). However, as in other species, diarrhea is multifactorial, involving factors intrinsic to the dog (breed size and age), nutritional factors (diet change without transition, food type and quality), together with lifestyle and environmental stressors (##REF##12013468##Weber et al., 2002##, ##REF##14511146##Weber et al., 2003##, ##REF##16008226##Sokolow et al., 2005##, ##REF##16454643##Hernot et al., 2006##, ##REF##21420191##Stavisky et al., 2011##). Most studies on risk factors of diarrhea in young dogs focused on one single pathogen or a group of pathogens without taking into account environmental stressors (##REF##8545969##Finlaison, 1995##, ##REF##17123427##Buehl et al., 2006##, ##REF##22520179##Grellet et al., 2012##, ##REF##22812470##Tupler et al., 2012##). Moreover most of the studies considering multiple enteropathogens infections were performed in shelters, in a context far different from that in breeding kennels (##REF##16008226##Sokolow et al., 2005##, ##REF##22812470##Tupler et al., 2012##). The purpose of this epidemiological study was to determine prevalence of enteropathogens in puppies in breeding kennels and to perform a risk factors analysis for diarrhea during the weaning period including enteropathogens, environment and management procedures.</p>" ]	[ "<title>Materials and methods</title>", "<title>Animals and breeding kennels</title>", "<p id=\"par0015\">A total of 266 puppies (60 litters) from 29 French breeding kennels were included in this study between May and September 2009 (mean of 9 puppies included per kennel; range: 2–18). Puppies were between 5 and 14 weeks of age (mean: 7.8 weeks of age) (##FIG##0##Fig. 1##\n). These breeding kennels were randomly selected from a data base of breeders registered at Alfort Veterinary School for training programs. Only puppies with a normal clinical examination were included (puppies with clinical signs of prostration, dehydration and/or anorexia were excluded of this study). For each kennel, data concerning environmental factors (number of puppies sold per year, and litter size for each puppies included), management of the kennel and puppies (number of meals distributed per day, access to outdoor, vaccination) and puppies’ characteristics (age, breed, sex), were collected. Puppies vaccinated within the preceding 10 days before the visit were not included.</p>", "<p id=\"par0020\">Depending on the mean adult body weight of their respective breed, puppies were divided in two groups (small if mean adult body weight < 25 kg; large otherwise). Small breed dogs represented 25.6% (68/266) of the total number of dogs included. Based on the mean number of puppies sold per year (calculated over the last two years and considered as the size of the kennel), kennels were also separated into “small” (i.e. less than 30 puppies sold per year) and large kennels (i.e. more than 30 puppies sold per year). Puppies housed in breeding kennels producing 30 puppies or more per year represented 51.1% (136/266) of the total number dogs included. Puppies were divided into two groups according to the number of meal per day: puppies receiving less than 4 meals per day and puppies receiving 4 meals per days or more.</p>", "<title>Evaluation of feces consistency</title>", "<p id=\"par0025\">For each puppy, fecal consistency was evaluated by a single operator using a 13-point scale, based on the texture and shape of the feces (from liquid to hard and dry) (##REF##22520179##Grellet et al., 2012##). Based on growth rate, thresholds for abnormal feces were previously validated and appeared to vary with breed stature and age (##REF##22520179##Grellet et al., 2012##). Briefly, feces with a score ≤ 5 was classified as abnormal for large breed puppies whatever the age, for small breed puppies, fecal scores ≤6 and ≤7 were classified as abnormal for 4–5 weeks old puppies and for older puppies between 6 and 8 weeks old, respectively.</p>", "<p id=\"par0030\">After collection, stools were separated in three samples, one being stored at +4 °C for coproscopy and other frozen (−20 °C) for <italic>Giardia intestinalis</italic> and <italic>Cryptosporidium parvum</italic> copro-antigens quantification.</p>", "<p id=\"par0035\">A rectal swab was performed for each puppy immediately after stool collection for detection of canine parvovirus type 2 (CPV2) and canine coronavirus (CCV). The swabs were stored at −20 °C until DNA extraction.</p>", "<title>Intestinal parasites</title>", "<p id=\"par0040\">By the standard McMaster flotation technique using saturated magnesium sulphate solution (density: 1.28 g/ml) (##REF##20502918##Bauer et al., 2010##), all eggs and oocysts were identified according to their morphological characteristics under light microscopy by a single operator (##REF##5857291##Levine and Ivens, 1965##, ##REF##8241078##Baek et al., 1993##).</p>", "<p id=\"par0045\">Copro-antigens of <italic>G. intestinalis</italic> and <italic>C. parvum</italic> were quantified on 100 mg of feces using respectively the ProSpecT-Giardia and the ProSpecT-Cryptosporidium Microplate Assay kit (Remel, France) (##REF##12669352##Decock et al., 2003##, ##REF##17939549##Mekaru et al., 2007##, ##REF##17320291##Rimhanen-Finne et al., 2007##). An optical density value > 0.05 was considered positive according to the manufacturer's instructions.</p>", "<title>Coronavirus and parvovirus fecal excretions</title>", "<p id=\"par0050\">CPV2 and CCV detection were performed by qPCR and qRT-PCR respectively as already described (##REF##22520179##Grellet et al., 2012##). Results from duplicate analyses (mean of two results) were expressed semi-quantitatively as viral load levels. Puppies were defined as excreting CPV2 and CCV for high viral loads over 10<sup>10.3</sup> copies and 10<sup>9.3</sup> copies respectively (##REF##22520179##Grellet et al., 2012##).</p>", "<title>Data management and statistical analysis</title>", "<p id=\"par0055\">Statistical analyses were performed with the SAS version 9.3 software (SAS Institute Inc., Cary, NC, USA).</p>", "<title>Statistical analysis for prevalence of enteropathogens</title>", "<p id=\"par0060\">Number of puppies with fecal positive and negative test results for each enteropathogen was tabled by different factors under study like age of puppies, size of the kennel, breed size, and litter size. Univariate analyses of the putative risk factors for each enteropathogen infection were performed. The significance of the univariate associations was determined using the <italic>χ</italic>\n<sup>2</sup>-tests. A <italic>P</italic> value < 0.05 was considered statistically significant.</p>", "<title>Statistical analysis for risk factors of abnormal feces</title>", "<p id=\"par0065\">Correlation matrix of quantitative and dichotomous variables (excretion of CPV2, CCV, <italic>G. intestinalis</italic>, <italic>C. parvum</italic>, <italic>T. canis</italic>, <italic>C. ohioensis</italic> complex, and <italic>C. canis</italic> and number of meal per day, litter size, breeding kennel size) was determined with Kendall's Tau-b measure of correlation coefficient (Proc CORR). These highly correlated variables were defined as predictors in a partial least squares regression (Proc PLS) with fecal consistency as the response variable. The Variable Importance for Projection (VIP) statistic of ##UREF##4##Wold (1994)## was used to assess the contribution of each predictor to the model (##UREF##4##Wold, 1994##). Only predictors with a VIP value over 0.8 were selected to be included in a new partial least squares regression (##UREF##5##Wold, 1995##). Variables of the final partial least squares regression with a VIP value over 0.8 were not collinear (<italic>r</italic>\n<sup>2</sup>\n < 0.10). These variables were subsequently integrated as independent variables and assessed as a fixed effect in a generalized linear mixed model (proc GLIMMIX) with fecal consistency as a binary outcome (logit transformation). As data on puppies were nested within naturally occurring hierarchies (puppies within litter, litters within breeding kennel), litter variable nested within breeding kennel, written as litter (breeding kennel), was defined as a random term. The respective influence of litter and breeding kennel as random effects was also determined.</p>" ]	[ "<title>Results</title>", "<title>Prevalence of enteropathogens</title>", "<p id=\"par0070\">77.1% (205/266) of the puppies were infected by at least one enteropathogen with 29.3% of them excreting 3 pathogens or more (##TAB##0##Table 1##, ##TAB##1##Table 2##\n). Seven different viruses and parasites were identified. 14.7% of puppies (39/266) were infected by CPV2, 20.3% (54/266) by CCV, 41% (109/266) by <italic>Giardia</italic> sp., 25.9% (69/266) by <italic>C. parvum</italic>, 25.6% (68/266) by <italic>C. ohioensis</italic> complex, 22.2% (59/266) by <italic>T. canis</italic>, and 13.2% (35/266) by <italic>C. canis</italic>. All enteropathogens except <italic>T. canis</italic> presented a significantly higher prevalence in large breeding kennels. Puppies between 5 and 8 weeks of age presented a significantly higher prevalence of CPV2 and <italic>C. ohioensis</italic> complex and a lower prevalence of CCV and <italic>G. duodenalis</italic> than puppies between 9 and 14 weeks of age (##TAB##2##Table 3##\n).</p>", "<title>Risk factors of abnormal feces</title>", "<p id=\"par0075\">Sixty six out of 266 feces evaluated (24.8%) were classified as abnormal (##FIG##1##Fig. 2##\n). In the initial partial least squares regression CPV2, <italic>C. canis</italic>, <italic>G. intestinalis</italic> and the number of meal per day presented a VIP over 0.8. These four factors were included in a new partial least squares regression. CPV2 and number of meal per day were two factors keeping a significant impact on the incidence of abnormal feces with a VIP over 0.8 (VIP = 1.7 and VIP = 1.0 respectively). In the final model only fecal excretion of CPV2 increased risk of weaning diarrhea (<italic>P</italic>\n = 0.003, odds ratio = 5; confidence interval 95%: 1.7–14.7). 61.5% (24/39) of puppies infected by CPV2 presented abnormal feces compared to 15.2% (42/277) of puppies not infected by CPV2. A global significant effect of litter and breeding kennel was observed, with a significant effect of the litter level (<italic>P</italic>\n < 0.001), and no significant effect of the breeding kennel level (<italic>P</italic>\n = 0.101).</p>" ]	[ "<title>Discussion</title>", "<p id=\"par0080\">The present study represents the first investigation of the prevalence of weaning diarrhea in puppies living in a breeding kennel. Prevalence of this clinical sign, affecting both growth and survival was high with 24.8% of puppies between 5 and 14 weeks concerned. Among the seven different enteropathogens (2 viruses and 5 parasites) tested in this study, 77.1% of puppies were infected by at least one virus or parasite, and 55.3% carried multiple organisms. Prevalence of parasites was higher than the prevalence of viruses (74.4% vs 34.6%). This high prevalence of multiple infections is in accordance with a previous study on dogs entering animal shelters in which 55% of them presented multiple digestive infections (##REF##22812470##Tupler et al., 2012##). In our study, 14.7% of puppies were excreting CPV2, 20.3% by CCV. However, only 0.4% of puppies presented a mixed infection by these two viruses. Prevalence of these viruses depends on age, lifestyle and health status of dogs. Serological and virological investigations demonstrated that CCV and CPV2 are highly prevalent in kennels and animal shelters compared to single owned dogs (##REF##1850889##Rimmelzwaan et al., 1991##, ##REF##8382389##Tennant et al., 1993##, ##REF##10458093##Bandai et al., 1999##, ##REF##11256282##Naylor et al., 2001b##, ##REF##18086157##Schulz et al., 2008##). Moreover a higher prevalence of CCV and CPV2 was described in young animals under 6 months of age compared to adult dogs (##UREF##3##Sakulwira et al., 2003##, ##REF##19709815##Gates and Nolan, 2009a##, ##REF##19972636##Gates and Nolan, 2009b##, ##REF##20637546##Epe et al., 2010##). In addition to the wide distribution and contagiosity of these viruses, the methods used for detection of these viruses can also contribute to this high prevalence. PCR assays have been proven to be up to 4 × 10<sup>4</sup> times more sensitive than electronic microscopy and virus isolation for detection of CCV (##REF##11230424##Naylor et al., 2001a##). This method is able to detect virus in the feces of low-grade shedding animals below 10<sup>6</sup> particles per gram of unprocessed feces, which is considered the detection limit for electronic microscopy. In our study, a higher prevalence of CPV2, CCV was observed in breeding kennels producing 30 puppies per year or more. The higher prevalence in this sub-population could be linked to the contagiousness of these pathogens and their stability in the environment (##REF##17601636##Terpstra et al., 2007##, ##UREF##1##Eterpi et al., 2010##). The close contact between animals and the density of puppies could promote the environmental contamination and subsequently the spread of the infection.</p>", "<p id=\"par0085\">17.9% of puppies in our study were found excreting a high load of CPV2. This virus is well described as inducing hemorrhagic diarrhea associated with vomiting, anorexia, dehydration and depression (##REF##2983478##Meunier et al., 1985##, ##UREF##2##Prittie, 2004##). However, in our study, CPV2 also increased risk of weaning diarrhea but without systemic signs, as already described, and 12.5% of dogs without gastrointestinal disease excreted this virus (##REF##22520179##Grellet et al., 2012##). Thus our study demonstrated that puppies can excrete high viral loads of CPV2 without any systemic sign. This observation is in accordance with one previous study in which fecal excretion was also quantified by PCR (##REF##19407086##Schmitz et al., 2009##). However studies using less sensitive methods (fecal antibody-based antigen tests, immune-electron microscopy) did not observed this healthy carrier status (##REF##12549614##Hackett and Lappin, 2003##, ##REF##15847935##Desario et al., 2005##, ##REF##16008226##Sokolow et al., 2005##, ##REF##18086157##Schulz et al., 2008##, ##REF##19407086##Schmitz et al., 2009##). The lack of systemic clinical signs on these puppies could be linked either to an efficient systemic immunity or to local intestinal immunity (##REF##7152659##Rice et al., 1982##, ##REF##2842925##Macartney et al., 1988##). Fecal IgA, endogenous or provided by milk, protect intestinal mucosa by inhibiting the adherence of pathogens, thereby preventing adhesion of these pathogens. Canine milk was found rich in IgA (##REF##806517##Heddle and Rowley, 1975##) with high levels of CPV2 antibodies (##REF##15646052##Decaro et al., 2004##). These antibodies, repeatedly ingested in large quantities by the puppies during the first weeks of life, could provide some protection to the intestinal mucosa against CPV2 deleterious effects decreasing the systemic clinical signs associated to CPV2 infection (septicaemia, dehydration). Nevertheless these clinically healthy animals probably represent major sources of virus for other animals and for the environmental contamination. Interestingly, in our study, young puppies (between 5 and 8 weeks of age) presented a higher infection rate by CPV2 than older ones. This result highlights the interest of vaccination before 8 weeks of age in breeding kennels to limit CPV2 spreading. The interference with maternally derived antibodies considered as one of the most important causes of immunization failure in puppies (##REF##2842925##Macartney et al., 1988##), can be overcome by the use of high titer CPV2 vaccines (##REF##21111542##De Cramer et al., 2010##).</p>", "<p id=\"par0090\">Other infectious agents tested were not associated with weaning diarrhea. In our study, 20.3% of puppies were infected by CCV, but this virus was not identified as a risk factor of abnormal feces. Implications of CCV in acute dog diarrhea are controversial. No relation between coronavirus and diarrhea was observed in different studies, with more healthy dogs infected by this virus than dogs with diarrhea in some of these studies (##REF##16008226##Sokolow et al., 2005##, ##REF##18086157##Schulz et al., 2008##, ##REF##22812470##Tupler et al., 2012##). However, CCV was also described as a virus inducing severe gastroenteritis, lethal in some cases (##REF##16475526##Evermann et al., 2005##, ##REF##16704791##Buonavoglia et al., 2006##, ##REF##18031959##Decaro et al., 2008##, ##REF##19887130##Decaro et al., 2009##). These variations in clinical signs could be linked to variations in pathogenicity between strains (##REF##17533554##Escutenaire et al., 2007##), to the age of infected dogs (##REF##19887130##Decaro et al., 2009##), to the number of genotypes infecting puppies simultaneously (##REF##16024100##Decaro et al., 2005##) or to the association of the coronavirus with other enteropathogens (##UREF##0##Appel, 1988##). Neither <italic>C. Ohioensis</italic> complex nor <italic>C. canis</italic> were associated with weaning diarrhea in our study. Impact of these parasites on weaning diarrhea is still controversial (Buehl, 2006). This difference of clinical signs observed between studies may be explained by differences in the age of infected dogs, the environmental conditions and the virulence of species.</p>" ]	[ "<title>Conclusion</title>", "<p id=\"par0095\">Based on this study, CPV2 infection was the major risk factors of weaning diarrhea. Some central strategies can be suggested like a targeted sanitary and medical prophylaxis against CPV2, particularly in large breeding kennels.</p>" ]	[ "<p>Diarrhea represents one of the most frequent disorders in dogs. In puppies, degradation of feces quality is associated with a reduced daily weight gain and an increased risk of death. Prevention of diarrhea in puppies requires a global approach encompassing enteropathogens, environment and management practices especially when housed in groups. The purpose of this study was to determine prevalence of enteropathogens in puppies in breeding kennels and to identify risk factors of diarrhea. Two hundred and sixty six puppies (between 5 and 14 weeks of age) from 29 French breeding kennels were included. For each kennel, data about environment, management of the kennel and puppies’ characteristics (age, sex and breed) were collected. For each puppy, fecal consistency and fecal excretion of enteropathogens (viruses and parasites) was evaluated. At least one enteropathogen was identified in 77.1% of puppies and 24.8% of puppies presented abnormal feces. The main risk factor of weaning diarrhea was fecal excretion of canine parvovirus type 2 (odds ratio = 5; confidence interval 95%: 1.7–14.7). A targeted sanitary and medical prophylaxis against canine parvovirus type 2 should be implemented to decrease risk of weaning diarrhea.</p>", "<title>Keywords</title>" ]	[]	[]	[ "<fig id=\"fig0005\"><label>Fig. 1</label><caption><p>Distribution of puppies with age (<italic>n</italic> = 226).</p></caption></fig>", "<fig id=\"fig0010\"><label>Fig. 2</label><caption><p>Incidence of abnormal feces depending on breed size (<italic>n</italic> = 266 puppies).</p></caption></fig>" ]	[ "<table-wrap position=\"float\" id=\"tbl0005\"><label>Table 1</label><caption><p>Frequency of identification of enteropathogens in fecal samples.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"3\" align=\"left\">No. of viruses identified per puppy<hr/></th><th colspan=\"5\" align=\"left\">No. of parasites identified per puppy<hr/></th><th colspan=\"6\" align=\"left\">Total number of enteropathogens identified per puppy<hr/></th></tr><tr><th align=\"left\">0</th><th align=\"left\">1</th><th align=\"left\">2</th><th align=\"left\">0</th><th align=\"left\">1</th><th align=\"left\">2</th><th align=\"left\">3</th><th align=\"left\">4</th><th align=\"left\">0</th><th align=\"left\">1</th><th align=\"left\">2</th><th align=\"left\">3</th><th align=\"left\">4</th><th align=\"left\">5</th></tr></thead><tbody><tr><td align=\"char\">65.4 (174)</td><td align=\"char\">34.2 (91)</td><td align=\"char\">0.4 (1)</td><td align=\"char\">25.6 (68)</td><td align=\"char\">34.6 (92)</td><td align=\"char\">28.6 (76)</td><td align=\"char\">9.0 (24)</td><td align=\"char\">2.3 (6)</td><td align=\"char\">22.9 (61)</td><td align=\"char\">21.8 (58)</td><td align=\"char\">32 (85)</td><td align=\"char\">17.7 (47)</td><td align=\"char\">4.1 (11)</td><td align=\"char\">1.5 (4)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tbl0010\"><label>Table 2</label><caption><p>Frequency of coinfection between enteropathogens in puppies.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">CPV2</th><th align=\"left\">CCV</th><th align=\"left\"><italic>T. canis</italic></th><th align=\"left\"><italic>C. ohioensis</italic> complex</th><th align=\"left\"><italic>C. canis</italic></th><th align=\"left\"><italic>Giardia</italic> sp.</th><th align=\"left\"><italic>C. parvum</italic></th></tr></thead><tbody><tr><td align=\"left\"><bold>CPV2</bold></td><td align=\"left\">0.4 (1)</td><td align=\"left\">2.3 (6)</td><td align=\"left\">1.5 (4)</td><td align=\"left\">7.5 (20)</td><td align=\"left\">11.3 (30)</td><td align=\"left\">5.6 (15)</td></tr><tr><td/><td align=\"left\"><bold>CCV</bold></td><td align=\"left\">1.9 (5)</td><td align=\"left\">4.1 (11)</td><td align=\"left\">3.8 (1)</td><td align=\"left\">15.8 (42)</td><td align=\"left\">4.9 (13)</td></tr><tr><td/><td/><td align=\"left\"><italic><bold>T. canis</bold></italic></td><td align=\"left\">11.7 (31)</td><td align=\"left\">4.1 (11)</td><td align=\"left\">3.8 (10)</td><td align=\"left\">9 (24)</td></tr><tr><td/><td/><td/><td align=\"left\"><italic><bold>C. ohioensis</bold></italic><bold>complex</bold></td><td align=\"left\">11.3 (3)</td><td align=\"left\">3.8 (10)</td><td align=\"left\">9.4 (25)</td></tr><tr><td/><td/><td/><td/><td align=\"left\"><italic><bold>C. canis</bold></italic></td><td align=\"left\">10.2 (27)</td><td align=\"left\">4.5 (12)</td></tr><tr><td/><td/><td/><td/><td/><td align=\"left\"><italic><bold>Giardia</bold></italic><bold>sp.</bold></td><td align=\"left\">11.7 (31)</td></tr><tr><td/><td/><td/><td/><td/><td/><td align=\"left\"><italic><bold>C. parvum</bold></italic></td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"tbl0015\"><label>Table 3</label><caption><p>Prevalence of enteropathogens depending of puppies’ characteristics and environmental factors.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Pathogens</th><th align=\"left\">Total prevalence</th><th colspan=\"4\" align=\"left\">Age of puppies<hr/></th><th colspan=\"3\" align=\"left\">Size of the kennel<hr/></th><th colspan=\"3\" align=\"left\">Breed size<hr/></th><th colspan=\"4\" align=\"left\">Litter size<hr/></th></tr><tr><th/><th/><th align=\"left\">5–6 weeks %<break/><italic>n</italic><sub><italic>i</italic></sub>/<italic>n</italic></th><th align=\"left\">7–8 weeks %<break/><italic>n</italic><sub><italic>i</italic></sub>/<italic>n</italic></th><th align=\"left\">9–14 weeks %<break/><italic>n</italic><sub><italic>i</italic></sub>/<italic>n</italic></th><th align=\"left\">Global <italic>P</italic> value</th><th align=\"left\"><30%<break/><italic>n</italic><sub><italic>i</italic></sub>/<italic>n</italic></th><th align=\"left\">≥30%<break/><italic>n</italic><sub><italic>i</italic></sub>/<italic>n</italic></th><th align=\"left\"><italic>P</italic> value</th><th align=\"left\">Large %<break/><italic>n</italic><sub><italic>i</italic></sub>/<italic>n</italic></th><th align=\"left\">Small %<break/><italic>n</italic><sub><italic>i</italic></sub>/<italic>n</italic></th><th align=\"left\"><italic>P</italic> value</th><th align=\"left\">≤4%<break/><italic>n</italic><sub><italic>i</italic></sub>/<italic>n</italic></th><th align=\"left\">5–7%<break/><italic>n</italic><sub><italic>i</italic></sub>/<italic>n</italic></th><th align=\"left\">≥8%<break/><italic>n</italic><sub><italic>i</italic></sub>/<italic>n</italic></th><th align=\"left\">Global <italic>P</italic> value</th></tr></thead><tbody><tr><td align=\"left\">CPV2</td><td align=\"left\">14.7<break/>39/266</td><td align=\"left\">23.3<sup>a</sup><break/>10/43</td><td align=\"left\">16.7<sup>a</sup><break/>23/138</td><td align=\"left\">7.1<sup>b</sup><break/>6/85</td><td align=\"left\">0.032</td><td align=\"left\">0<break/>0/130</td><td align=\"left\">28.7<break/>39/136</td><td align=\"left\"><0.001</td><td align=\"left\">14.1<break/>28/198</td><td align=\"left\">16.2<break/>11/68</td><td align=\"left\">0.682</td><td align=\"left\">22.6a<break/>14/62</td><td align=\"left\">20a<break/>18/90</td><td align=\"left\">6.1<sup>b</sup><break/>7/114</td><td align=\"left\">0.003</td></tr><tr><td align=\"left\">CCV</td><td align=\"left\">20.3<break/>54/266</td><td align=\"left\">7<sup>b</sup><break/>3/43</td><td align=\"left\">13.8<sup>b</sup><break/>19/138</td><td align=\"left\">37.6<sup>a</sup><break/>32/85</td><td align=\"left\"><0.001</td><td align=\"left\">0<break/>0/130</td><td align=\"left\">39.7<break/>54/136</td><td align=\"left\"><0.001</td><td align=\"left\">23.2<break/>46/198</td><td align=\"left\">11.8<break/>8/168</td><td align=\"left\">0.043</td><td align=\"left\">8.1<sup>b</sup><break/>5/62</td><td align=\"left\">32.2<sup>a</sup><break/>29/90</td><td align=\"left\">17.5<sup>b</sup><break/>20/114</td><td align=\"left\">0.001</td></tr><tr><td align=\"left\"><italic>T. canis</italic></td><td align=\"left\">22.2<break/>59/266</td><td align=\"left\">44.2<sup>a</sup><break/>19/43</td><td align=\"left\">22.5<sup>b</sup><break/>31/138</td><td align=\"left\">10.6<sup>c</sup><break/>9/85</td><td align=\"left\"><0.001</td><td align=\"left\">29.2<break/>38/130</td><td align=\"left\">15.4<break/>21/136</td><td align=\"left\">0.007</td><td align=\"left\">22.7<break/>45/198</td><td align=\"left\">20.6<break/>14/68</td><td align=\"left\">0.714</td><td align=\"left\">16.1<break/>10/62</td><td align=\"left\">17.8<break/>16/90</td><td align=\"left\">28.9<break/>33/114</td><td align=\"left\">0.069</td></tr><tr><td align=\"left\"><italic>C. ohioensis</italic> complex</td><td align=\"left\">25.6<break/>68/266</td><td align=\"left\">30.2<sup>a</sup><break/>13/43</td><td align=\"left\">31.9<sup>a</sup><break/>44/138</td><td align=\"left\">12.9<sup>b</sup><break/>11/85</td><td align=\"left\">0.005</td><td align=\"left\">23.8<break/>31/130</td><td align=\"left\">27.2<break/>37/136</td><td align=\"left\">0.002</td><td align=\"left\">29.3<break/>58/198</td><td align=\"left\">14.7<break/>10/68</td><td align=\"left\">0.017</td><td align=\"left\">19.4<sup>b</sup><break/>12/62</td><td align=\"left\">13.3<sup>b</sup><break/>12/90</td><td align=\"left\">38.5<sup>a</sup><break/>44/114</td><td align=\"left\"><0.001</td></tr><tr><td align=\"left\"><italic>C. canis</italic></td><td align=\"left\">13.2<break/>35/266</td><td align=\"left\">41.9<sup>a</sup><break/>18/43</td><td align=\"left\">6.8<sup>b</sup><break/>8/138</td><td align=\"left\">10.6<sup>b</sup><break/>9/85</td><td align=\"left\"><0.001</td><td align=\"left\">1.5<break/>2/130</td><td align=\"left\">24.3<break/>33/136</td><td align=\"left\"><0.001</td><td align=\"left\">17.2<break/>34/198</td><td align=\"left\">1.5<break/>1/68</td><td align=\"left\">0.001</td><td align=\"left\">4.8<break/>3/62</td><td align=\"left\">16.7<break/>15/90</td><td align=\"left\">14.9<break/>17/114</td><td align=\"left\">0.081</td></tr><tr><td align=\"left\"><italic>G. duodenalis</italic></td><td align=\"left\">41<break/>109/266</td><td align=\"left\">32.6<sup>b</sup><break/>14/43</td><td align=\"left\">30.4<sup>b</sup><break/>42/138</td><td align=\"left\">62.4<sup>a</sup><break/>53/85</td><td align=\"left\"><0.001</td><td align=\"left\">17.7<break/>23/130</td><td align=\"left\">63.2<break/>86/136</td><td align=\"left\"><0.001</td><td align=\"left\">41.4<break/>82/198</td><td align=\"left\">39.7<break/>27/68</td><td align=\"left\">0.805</td><td align=\"left\">51.6<sup>a</sup><break/>32/62</td><td align=\"left\">46.7<sup>a</sup><break/>42/90</td><td align=\"left\">30.7<sup>b</sup><break/>35/114</td><td align=\"left\">0.011</td></tr><tr><td align=\"left\"><italic>C. parvum</italic></td><td align=\"left\">25.9<break/>69/266</td><td align=\"left\">37.2<break/>16/43</td><td align=\"left\">22.5<break/>31/138</td><td align=\"left\">25.9<break/>22/85</td><td align=\"left\">0.156</td><td align=\"left\">20.8<break/>27/130</td><td align=\"left\">30.9<break/>42/136</td><td align=\"left\">0.06</td><td align=\"left\">26.3<break/>52/198</td><td align=\"left\">25<break/>17/68</td><td align=\"left\">0.838</td><td align=\"left\">27.4<sup>a</sup><break/>17/62</td><td align=\"left\">12.2<sup>b</sup><break/>11/90</td><td align=\"left\">36<sup>a</sup><break/>41/114</td><td align=\"left\">0.001</td></tr></tbody></table></table-wrap>" ]	[]	[]	[]	[]	[]	[]	[ "<table-wrap-foot><fn><p>Data are given as % (number) of puppies.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn><p>Data are given as % (number) of puppies.</p></fn></table-wrap-foot>", "<table-wrap-foot><fn id=\"spar0045\"><p>For each line, categories with different letters (a,b,c)were significantly different (<italic>P</italic> < 0.05).</p></fn><fn id=\"spar0050\"><p><italic>n</italic><sub><italic>i</italic></sub>/<italic>n</italic> = number of puppies infected for the category considered/total number of puppies in the category considered.</p></fn></table-wrap-foot>" ]	[ "<graphic xlink:href=\"gr1_lrg\"/>", "<graphic xlink:href=\"gr2_lrg\"/>" ]	[]	"[{"surname": ["Appel"], "given-names": ["M.J.G."], "article-title": ["Does canine coronavirus augment the effects of subsequent parvovirus infection?"], "source": ["Vet. Med."], "year": ["1988"], "fpage": ["360"], "lpage": ["366"]}, {"surname": ["Eterpi", "McDonnell", "Thomas"], "given-names": ["M.", "G.", "V."], "article-title": ["Virucidal activity of disinfectants against parvoviruses and reference viruses"], "source": ["Appl. Biosaf."], "volume": ["15"], "year": ["2010"], "fpage": ["165"], "lpage": ["171"]}, {"surname": ["Prittie"], "given-names": ["J."], "article-title": ["Canine parvoviral enteritis: a review of diagnosis, management, and prevention"], "source": ["J. Vet. Emerg. Crit. Care"], "volume": ["14"], "year": ["2004"], "fpage": ["167"], "lpage": ["176"]}, {"surname": ["Sakulwira", "Vanapongtipagorn", "Theamboonlers", "Oraveerakul", "Poovorawan"], "given-names": ["K.", "P.", "A.", "K.", "Y."], "article-title": ["Prevalence of canine coronavirus and parvovirus infections in dogs with gastroenteritis in Thailand"], "source": ["Vet. Med. \u2013 Czech"], "volume": ["48"], "year": ["2003"], "fpage": ["163"], "lpage": ["167"]}, {"surname": ["Wold"], "given-names": ["S."], "part-title": ["PLS for multivariate linear modeling"], "source": ["Chernornetric Methods in Molecular Design"], "year": ["1994"], "publisher-name": ["VCH"], "fpage": ["359"]}, {"surname": ["Wold"], "given-names": ["S."], "part-title": ["Multivariate analysis (3-day course)"], "year": ["1995"], "publisher-name": ["Umetrics Inc."], "publisher-loc": ["Winchester, MA"]}]"	{ "acronym": [], "definition": [] }	48	"NO-CC CODE"	"no"	"2023-06-06 23:35:26"	"Prev Vet Med. 2014 Nov 1; 117(1):260-265"	"oa_package/fe/03/PMC7114196.tar.gz"
"PMC7128992"	"31400641"	[ "<title>Introduction</title>", "<p id=\"p0005\">Lung transplantation is an established treatment option for selected patients with end-stage lung disease. Chronic lung allograft dysfunction (CLAD) and infections are the main factors limiting long-term survival in lung transplant recipients (LTRs) ##UREF##0##[1]##. Acute cellular rejection (ACR) is a potential risk factor for the development of CLAD ##UREF##1##[2]##, ##REF##15996255##[3]##, ##REF##16340783##[4]##, ##REF##11834356##[5]##. During the first postoperative year, ACR affects 28% of LTRs at least once, necessitating treatment with steroid augmentation ##UREF##0##[1]##, ##REF##21471083##[6]##. Symptoms of ACR are nonspecific, including dyspnea, cough, sputum production, fever and/or hypoxia ##REF##15597291##[7]##, ##REF##29312755##[8]##. Non-invasive tests like pulmonary function testing and chest imaging are useful indicators for potential complications, but have no discriminatory value between ACR and infection ##REF##20354931##[9]##. For these reasons, transbronchial biopsies (TBB) remain the gold standard for the diagnosis of ACR ##REF##27819763##[10]##. However, TBB are invasive and bear potential risks such as pneumothorax or bleeding ##REF##9041985##[11]##, ##REF##10027438##[12]##. Moreover, TBB are prone to sampling error and inter-observer variability ##REF##23370547##[13]##, ##REF##21219569##[14]##, ##REF##15949723##[15]##. The clinical and prognostic role of grade A1 ACR remains unclear. Depending on clinical management guidelines and practice standards at different transplant centers, a finding of grade A1 ACR might be ignored, prompt repeat biopsy or might result in augmented immunosuppression ##REF##9041985##[11]##, ##UREF##2##[16]##, ##REF##28637080##[17]##.</p>", "<p id=\"p0010\">Immense work in multiple laboratories worldwide is currently under way to determine the potential role of cytokines in diagnosis, treatment and monitoring disease progression in various fields such as heart failure, neuro-degeneration and gastrointestinal diseases. Cytokine production by BAL T lymphocytes and mast cells has been shown to be part of pro- and anti-inflammatory processes leading to airflow limitation and exacerbations of obstructive lung disease ##REF##16751017##[18]##, ##REF##7582279##[19]##. Cytokines are furthermore implicated in rejection after organ transplantation and induction of fibrotic pathways ##REF##30510964##[20]##.</p>", "<p id=\"p0015\">ACR is driven by T cell recognition of foreign major histocompatibility complexes ##REF##29312755##[8]##, ##REF##17072801##[21]##. Cytokines play a key role in this process by stimulating proliferation, chemotaxis and activation of cytotoxic T lymphocytes, neutrophils and alveolar macrophages AM ##UREF##3##[22]##, ##REF##9723771##[23]##, ##REF##22138818##[24]##. We have recently reviewed the potential role of surrogate markers such as cytology and cytokines in bronchoalveolar lavage (BAL) and plasma samples ##REF##27323950##[25]##, ##REF##28784117##[26]##. Analyzing cytokines in BAL may provide information on allograft status, a potentially useful diagnostic tool. Advances in detection of biomarkers are urgently needed to identify ACR and reliably predict increased risk for the development of CLAD ##UREF##4##[27]##.</p>", "<p id=\"p0020\">In this retrospective single-center study, we analyzed a panel of pro-inflammatory cytokines, including interleukin (IL-)6, IL-8, interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in a large cohort of LTRs. The aim of this study was to correlate cytokine levels in BAL fluid of surveillance bronchoscopies with the development of complications including ACR and infection during the first year following lung transplantation. Such correlation might provide a feasible and specific diagnostic potentially allowing early recognition and subsequent targeted treatment of complications in LTRs.</p>" ]	[ "<title>Patients and methods</title>", "<title>Study population</title>", "<p id=\"p0025\">Starting in 1998, the four cytokines IL-6, IL-8, IFN-γ and TNF-α were analyzed in BAL fluid of LTRs during surveillance bronchoscopies for future research purposes. We enrolled all patients whose medical records included BAL cytokine analyses and concomitantly obtained TBB as well as microbiologic studies. This study was approved by the Cantonal Ethics Committee of Zurich (KEK-ZH number 2016-02148).</p>", "<title>Immunosuppression protocol and prophylaxis regimen</title>", "<p id=\"p0030\">In general, all primary LTRs at University Hospital Zurich receive induction therapy (antithymocyte globulin or basiliximab) and life-long triple immunosuppressive therapy ##REF##23986418##[28]##. At our center, cyclosporine A, tapered dose prednisone, and azathioprine or mycophenolate mofetil (since 1999) are used ##REF##22832220##[29]##. Anti-infective prophylaxis is used as previously described ##REF##14745663##[30]##, ##REF##23572438##[31]##. All patients classified as cytomegalovirus (CMV) intermediate-risk or high-risk received prophylaxis with valganciclovir ##REF##26222822##[32]##, ##REF##30069386##[33]##. In case of CLAD, macrolides are given for immunomodulation ##REF##22832220##[29]##.</p>", "<title>Predictors</title>", "<p id=\"p0035\">At regular intervals BAL fluid and TBB were obtained during routine surveillance bronchoscopies during the first year after transplantation (at one, two, three, four, six and 12 months following lung transplantation) ##REF##14745663##[30]##. Levels of C-reactive protein (CRP) were routinely measured for all patients before surveillance bronchoscopies. Patients did not undergo surveillance bronchoscopy if clinically unstable, showing clinical signs of infection or infection-associated lung allograft dysfunction or if elevated inflammatory markers in the laboratory. Bronchoscopies performed for specific clinical indications or events were excluded from the study. Lavage was performed in a sub-segmental bronchus of either the lingula or the middle lobe using three to four 50 ml aliquots of 0.9% saline solution ##REF##8726927##[34]##. Five to eight TBB were taken from the lower lobe. After fixation in 4% formaldehyde solution and serial section biopsy specimens were stained with hematoxylin and eosin, elastic Van Gieson and Grocott.</p>", "<p id=\"p0040\">BAL fluid cytokine concentrations of IL-6, IL-8, IFN-γ and TNF-α were determined by University Hospital Zurich Immunology Laboratory using commercially available, validated quantitative sandwich enzyme immunoassays. The immunoassays use microplates pre-coated with polyclonal antibodies specific for the cytokine and enzyme-linked polyclonal antibodies for their detection in a blinded fashion (R&D Systems; Minneapolis, MN, USA). ELISA kits included Quantikine® ELISA Human IL-6 Immunoassay (catalog # D6050), Quantikine® ELISA Human CXCL8/IL-8 Immunoassay (catalog # D8000C), Quantikine® ELISA Human IFN-γ Immunoassay (catalog # DIF50) and Quantikine® HS ELISA Human TNF-α Immunoassay (catalog # HSTA00D). All cytokines were measured according to the manufacturer’s instruction. The absorbance was measured in an enzyme-linked immunosorbent assay (ELISA) reader (Dynex Opsys MR™ Microplate Reader) at 450/630 nm. The respective cytokine concentration was determined by interpolation from standard curves and expressed as pg/ml. Sensitivity of the assays was 0.7 pg/ml for IL-6, 3.5 pg/ml for IL-8, 8 pg/ml for IFN-γ and 0.1 pg/ml for TNF-α. BAL fluid cytokine concentrations were measured once a week. BAL fluid cytokine samples were stored at 4 °C until processed, for a maximum of one week. Starting in 2013, samples were centrifuged for 10 min at a speed of 2370 g at 4 °C immediately prior to the measurements. The supernatant was removed from the pellet after centrifugation. In summary, 50 μl/well (TNF-α) or 100 μl/well (IFN-γ, IL-6, IL-8) of Assay Diluent were added to the well of the cytokine microplates, then 50 μl/well (IL-8), 100 μl/well (IFN-γ, IL-6) or 200 μl/well (TNF-α) of the respective BAL sample, standards and controls were added as suggested by the manufacturer and incubated for 2 h (IFN-γ, IL-6, IL-8) or 3 h (TNF-α). Plates were washed four times (IL-6), five times (IFN-γ, IL-8) or six times (TNF-α). Removing excess liquid and washing thoroughly is essential; hence, plates with IFN- γ and IL-8 were washed once more than suggested by the provider. Then, 100 μl/well (IL-8) or 200 μl/well (IFN-γ, IL-6, TNF-α) of the respective Conjugate was added and incubated at room temperature for one hour (IL-8) or two hours (IFN-γ, IL-6, TNF-α). Plates were washed four times (IL-6), five times (IFN-γ, IL-8) or six times (TNF-α). Only in the case of TNF- α 50 μl/well of Amplifying Solution had to be added before the addition of the Substrate Solution and incubated at room temperature for 30 min. Then, 50 μl/well (TNF-α) or 200 μl/well (IFN-γ, IL-6, IL-8) of the respective Substrate Solutions were added and incubated at room temperature for 30 min (IFN-γ, IL-6, IL-8) or 60 min (TNF-α). 50 μl/well of Stop Solution was added (IFN-γ, IL-6, IL-8, TNF-α). Plates were read at OD 450/630 nm within 30 min.</p>", "<title>ACR and microbial detection</title>", "<p id=\"p0045\">ACR was assessed and graded in the TBB specimens by experienced pathologists using standard International Society for Heart and Lung Transplantation (ISHLT) nomenclature ##REF##18096473##[35]##, ##REF##8820078##[36]##. Both Grade A ACR and grade B ACR were considered. Episodes of clinically suspected ACR, antibody-mediated rejection (AMR) and CLAD were not included in the analysis.</p>", "<p id=\"p0050\">Specimens were classified as infected if BAL fluid microbiologic studies identified bacteria, viral pathogens, fungi or mycobacteria. Bacterial cultures were considered positive if cultures showed growth greater than 100000 viable organisms per ml, excluding oral flora. PCR was used for detection of respiratory viruses (adenovirus, bocavirus, coronavirus, enterovirus, influenza A, influenza B, metapneumovirus, parainfluenzavirus, parechovirus, rhinovirus and RSV). CMV culture results were not included in the analysis. For this study we did not include the clinical presentation, radiologic findings, macroscopic appearance of BAL fluid and concomitant antibiotic, antiviral or antifungal treatment. Bronchoscopy samples were classified as “no pathologic process” if both TBB and BAL fluid microbiologic studies did not show a pathologic process, irrespective of clinical or radiologic presentations.</p>", "<title>Statistical analysis</title>", "<p id=\"p0055\">Statistical analysis was performed in R (version 3.4.1; R Foundation for Statistical Computing, Vienna, Austria) using “pROC”, “broom”, “lmerTest” and “survival” libraries. Baseline recipient characteristics were expressed as mean (standard deviation) or as median (interquartile range) for continuous variables and as frequency (percentage) for categorical variables. Cytokine concentrations were log transformed prior to analysis to ensure normal distribution. Concentrations too low to detect by ELISA assay were assigned a value of 0.01 pg/ml. Boxplots were generated using default settings in the R graphics library, with whiskers at the default of 1.5 times the interquartile range. Continuous variables were compared using the Mann-Whitney-<italic>U</italic> test or Kruskal-Wallis test. Categorical variables were compared using Fisher’s exact test. In a multivariable analysis, we controlled for patient characteristics frequently associated with rejection. These included age, underlying lung disease and type of infection (bacterial, viral or fungal). ROC curves for all four cytokines were calculated using the “pROC” package ##UREF##5##[37]##. Overall survival of LTRs was assessed using the Kaplan-Meier method and compared using the log-rank test, with censoring at 31 December 2016. Because the ACR status was assessed after transplantation, we considered it to be a time-varying co-variate in this part of the analysis and had to reshape the data accordingly.</p>" ]	[ "<title>Results</title>", "<title>Study population</title>", "<p id=\"p0060\">During the study period from February 1998 to November 2016, 425 subjects underwent lung transplantation; 106 subjects were excluded because no cytokine data was recorded in the patient health record system and/or no bronchoscopies were performed. In the remaining 319 subjects, 747 BAL fluid samples were analyzed and compared with TBB specimens obtained during the same bronchoscopy. Median number of TBB and BAL samples per patient was 3 (IQR 1–4). Median time to first TBB sample was 43 (IQR 29–83) days. Patient characteristics are provided in ##TAB##0##Table 1##\n. Compared with ISHLT Thoracic Transplant Registry data, our study population included a greater proportion of patients receiving transplantation for CF (33.5% vs. 22.9%) and pulmonary fibrosis (29.1% vs. 24.7%) ##UREF##0##[1]##. Conversely, the patients included in the study received slightly fewer transplants for chronic obstructive pulmonary disease (COPD) (31.3% vs. 31.8%) and pulmonary hypertension (5.3% vs. 6%) ##UREF##0##[1]##. Overall 31.4% of the total 319 patients experienced at least one episode of ACR during the first year post transplantation (vs. 28% ISHLT) ##UREF##0##[1]##.</p>", "<title>Discrimination between specific groups</title>", "<p id=\"p0065\">Bronchoscopy results were grouped based on microbiological and pathological analyses. Of the 747 bronchoscopy specimens, 214 (28.65%) showed “no pathologic process”. Of the remaining samples, 69 (9.24%) showed ACR, 358 (47.93%) infection and 106 (14.19%) “combined ACR and infection”. ##TAB##1##Table 2##\nprovides an overview of the cytokine concentrations in the different groups.</p>", "<p id=\"p0070\">##FIG##0##Fig. 1## shows levels of log(IL-6) among the four subgroups; no significant difference was observed. IFN-γ, IL-8 and TNF-α also did not show significant differences by specific group. As shown in ##FIG##1##Fig. 2##\n, none of these cytokines had an optimal cut-off to diagnose “combined ACR and infection” or isolated episodes of ACR.</p>", "<p id=\"p0075\">Depending on the guidelines of different transplant centers, ACR may only be treated in >A1 ACR episodes. Levels of log(IL-6) during ACR (median 0.37 vs. 0.21) did not differ from “combined ACR and infection” (median 0.67 vs. 0.51), when ACR was defined ≥A2 as opposed to ≥A1 ACR (p = 0.22). No differences were observed in the levels of log(IL-8), log(IFN-γ) and log(TNF-α) (p = 0.21, p = 0.52, p = 0.21, respectively).</p>", "<title>Factors influencing cytokine pattern</title>", "<p id=\"p0080\">Using a linear mixed effects model, we found no significant differences in the cytokine levels by specific group for any of the cytokines. After adjusting for age, underlying disease leading to lung transplantation and type of infection at the time of surveillance bronchoscopy, IL-8 showed significantly lower log(IL-8) levels in patients with infection (2.14) than in patients with “no pathologic process” (2.41, p = 0.02). Also, log(IFN-γ) was lower in patients with ACR only (-1.07), than with “no pathologic process” (−0.77, p = 0.05). log(IL-6) (ACR only 0.39, p = 0.76; infection only 0.19, p = 0.27; “combined ACR and infection” 0.37, p = 0.91, “no pathologic process” 0.35) and log(TNF-α) (ACR only −0.98, p = 0.86; infection only −0.88, p = 0.40; “combined ACR and infection” −0.93, p = 0.65, “no pathologic process” −1.00) did not vary in a significant manner by specific group.</p>", "<title>Number of events</title>", "<p id=\"p0085\">ACR was detected in 175 (23.43%) of all TBB samples (91 showing A1 ACR). 59 patients (18.5%) experienced one event of ACR during the first year after transplantation, 22 patients (7%) experienced two events, and 19 patients (6%) experienced more than two events according to surveillance bronchoscopies. Using a generalized linear mixed model ACR decreased slightly in the first year, with odds ratio of 0.915 per month (p = 0.02). No differences in the rates of ACR event were observed by type of pathogen detected during surveillance bronchoscopies (bacterial infection, p = 0.95; viral infection, p = 0.20; fungal infection, p = 0.79).</p>", "<title>Survival</title>", "<p id=\"p0090\">Among patients undergoing surveillance bronchoscopies with TBB, eight died within the first year after transplantation. Median 1-year conditional survival was 8.6 years for patients without ACR and 7.9 years for patients with a minimum one biopsy-proven ACR in the first year after transplantation, respectively. As shown in ##FIG##2##Fig. 3##\n, the 5-year survival rate was similar between patients without (70%) or with episodes of ACR (69%) and higher compared to ISHLT Registry data (57% in era 2009 – June 2015) ##UREF##0##[1]##. There were no significant differences in overall survival between patients with and without ACR (hazard ratio 1.18, 95%-confidence interval 0.69–2.02, p = 0.54), and among patients with ACR, between grade A1 ACR and grade > A1 ACR (hazard ratio 1.00, 95%-confidence interval 0.58–1.71, p = 1.00). There was no correlation with ACR events per patient and survival (p = 0.96).</p>" ]	[ "<title>Discussion</title>", "<p id=\"p0095\">In this observational study we could not detect a relevant role of IL-6, IL-8, INF-γ and TNF-α in BAL fluid samples of LTRs to identify complications including ACR, infection or both.</p>", "<p id=\"p0100\">After adjustment for age, underlying disease leading to lung transplantation and type of infection at time of surveillance bronchoscopy minor differences in the cytokine levels were observed. These data show that the pattern of BAL cytokines is of minor value as a diagnostic marker in LTRs.</p>", "<p id=\"p0105\">As reported before ##REF##28784117##[26]## data on the role of IL-6 in ACR is conflicting. Whereas some experimental and clinical data showed a significant increase in IL-6 in ACR ##UREF##3##[22]##, ##REF##7692639##[38]##, other studies found no significant association at all ##REF##24025324##[39]##, ##REF##15135367##[40]##. Despite the high number of samples no correlation was found in our analyses. IL-8 was significantly lower during infection than in LTRs with “no pathologic process” after adjustment for age, underlying disease and type of infection. No correlation was found between IL-8 and ACR, which is along the line with most previous studies ##UREF##3##[22]##, ##REF##9272913##[41]##, ##REF##16585086##[42]##. While IL-8 has been linked with the development of CLAD, its role in detecting ACR seems negligible according to our data ##REF##25394537##[43]##, ##REF##23821509##[44]##. Levels of IFN-γ were significantly lower during ACR only after correction for age, underlying disease and type of infection. However, in the light of the results from previous studies, these findings should be interpreted with caution ##REF##24025324##[39]##, ##REF##10202618##[45]##, ##REF##10452338##[46]##, ##REF##7544620##[47]##. Levels of TNF-α did not correlate with the four prespecified groups. Accordingly, previous studies have suggested no correlation between TNF-α and ACR ##UREF##3##[22]##, ##REF##24025324##[39]##, ##REF##7544620##[47]##. Of technical note, ELISA is not the best method to detect TNF-α since it is limited in the detection of cytokines that are active in the membrane bound form ##REF##10811142##[48]##, ##REF##3349526##[49]##.</p>", "<p id=\"p0110\">Survival did not differ between patients with ACR and “no ACR” and no difference was seen in patients with multiple ACR events. However, we only included data obtained from surveillance bronchoscopies without clinical signs of allograft dysfunction. Patients are followed up every 1–2 weeks within the first six months following transplantation and 2–4 weeks thereafter just at our center due to the limited size of Switzerland. Any clinical signs of infection or allograft rejection prompt immediate treatment. In addition, patients with multiple ACR events and early signs of CLAD are treated with extracorporeal photophoresis, potentially explaining the higher survival rate compared to the data from the ISHLT registry. In summary, no correlation was found between the cytokines studied here and survival of patients suggesting that these cytokines are not suitable markers for monitoring.</p>", "<p id=\"p0115\">This study has several limitations: First, this is a retrospective study. Thus, even though we planned surveillance bronchoscopies at regular intervals, a selection bias can’t be excluded and patients with clinically relevant allograft dysfunction or infection did not undergo surveillance bronchoscopies. This might explain why we derived data from 319 patients, missing the potential of 425 subjects. Also, while we obtained total cell count in BAL samples we did not include this information in our analysis. An increase in total cell count is non-specific after lung transplantation and has also been found in periods with no infection or rejection ##REF##10202611##[50]##. Second, we restricted cytokine analysis to a small number of cytokines. When this study was initiated in 1998, these were the only commercially available and validated kits used at our center. All four cytokines had been associated with rejection in previous studies ##REF##28784117##[26]##. Also, we did not perform immunologic analyses reflecting the innate immune system such as alpha defensins or matrix metalloproteinases. Further, combining BAL cytokine and cytology levels might contribute to a composite score, increasing diagnostic accuracy for BAL to diagnose ACR. However, evaluating such a score was not part of the study. Third, due to the fact, that AMR was an ill-defined condition before the publication of the 2016 ISHLT Consensus Report ##REF##27044531##[51]## we did not include patients with AMR in our study. Fourth, we did not perform analyses to associate cytokines with CLAD development ##UREF##6##[52]##, ##REF##25050473##[53]##. The diagnosis of CLAD is an indication to treat patients with an immunomodulatory macrolide. As such, even if the absolute number of patients receiving macrolides at our institution during the first year after lung transplantation is low, a bias of such treatment for cytokine distribution in BAL cannot be excluded ##REF##29593707##[54]##. Fifth, statistical methods were not used to adjust for storage time of the BAL samples, number of analyses per patient and date of collection after transplantation. Sixth, infection was defined based on microbiological findings in BAL fluid only and did not take into account clinical symptoms, imaging studies, macroscopic appearance of BAL fluid and concomitant antibiotic, antiviral or antifungal medication. Detecting infection and differentiating infection from ACR based on clinical symptoms may be difficult in LTRs ##REF##30069386##[33]##, ##REF##10194239##[55]##. Thus, in patients on multiple immunosuppressive drugs the mere presence of pathogens in BAL fluid in the absence of clinical deterioration may fulfill criteria of infection and prompt anti-infective treatment. Indeed, at our center, all pathogenic findings in BAL fluid except for oral flora and candida species prompted anti-infective treatment ##REF##14745663##[30]##, ##REF##23572438##[31]##.</p>", "<p id=\"p0120\">Some aspects of our immunological analysis warrant further discussion. We did not centrifuge BAL specimens before 2013. In a recent internal review conducted by our Immunology Lab comparing cytokine concentrations in BAL samples with and without prior centrifugation, no difference was seen in the majority of samples (unpublished data). Further, we measured BAL cytokine levels once a week and therefore not necessarily on the day they were obtained. Finally, BAL fluid samples were stored at 4 °C until processed, at a maximum of one week. An internal analysis performed in our Immunology Lab in 2012 showed a difference in cytokine concentration <20% between BAL samples stored at 4 °C and samples stored at −20 °C immediately after arrival until processing one week later.</p>", "<p id=\"p0125\">There is a need of standardization in BAL technique in LTRs. The ISHLT has recently established a Working Group to address this issue as far as the BAL procedure is concerned, the quotient of instilled volume and aspirated volume should be calculated and provided. This facilitates comparability of cellular and protein concentrations between studies. This would be particularly important for cytokines, as their concentrations are typically low. To further correct for dilution factors of BAL fluid, the urea method has been described, whereby the measured BAL fluid cytokine concentration is adjusted to a urea plasma/BAL coefficient ##REF##28787761##[56]##. We did not apply this method in our study to normalize the data. Levy and colleagues recently showed that sequential BAL samples reflect distinct pulmonary compartments ##REF##29801490##[57]##. This might have implications for future research. Along this line, centers should agree on a standardized cytokine detection method as comparability between methods is low ##REF##10811142##[48]##. ELISA has been a reliable method at our center. However, it is costly and requires strict adherence to time protocols. FACS or Luminex® assays may be alternatives for selected cytokines, yet these methods have yielded less reliable results in our laboratory. Also, numerous potential confounders related to medication (dosage of immunosuppression, anti-infective prophylaxis and treatment, additional individual medication) and immunological analysis make interpretation of cytokine data in LTRs challenging. In the field of rheumatology attempts have been made to harmonize autoantibody nomenclature, thereby optimizing antinuclear antibody usage ##REF##27252255##[58]##.</p>", "<p id=\"p0130\">Based on the data shown here, the identification of a single biomarker or a profile of different biomarkers is unlikely to provide conclusions on lung allograft function. Whether computed algorithms and precision medicine might help to translate this complex data into the clinical setting is unclear at the moment. In our opinion, this can only be achieved in a collaborative approach by using standardized and validated methods in large prospective multi-center cohort studies. This effort is crucial, however, to optimize survival and quality of life for LTRs ##UREF##4##[27]##.</p>", "<p id=\"p0135\">In summary, this is one of the largest retrospective studies to analyze BAL fluid cytokine profiles in LTRs. The cytokines investigated here have no role to diagnose complications after lung transplantation. It is unlikely that further attempts to study these BAL fluid cytokines in the context of ACR, infection or both in LTRs will add novel valuable insights.</p>" ]	[]	[ "<p>Early diagnosis and treatment of acute cellular rejection (ACR) may improve long-term outcome for lung transplant recipients (LTRs). Cytokines have become valuable diagnostic tools in many medical fields. The role of bronchoalveolar lavage (BAL) cytokines is of unknown value to diagnose ACR and distinguish rejection from infection. We hypothesized that distinct cytokine patterns obtained by surveillance bronchoscopies during the first year after transplantation are associated with ACR and microbiologic findings.</p>", "<p>We retrospectively analyzed data from 319 patients undergoing lung transplantation at University Hospital Zurich from 1998 to 2016. We compared levels of IL-6, IL-8, IFN-γ and TNF-α in 747 BAL samples with transbronchial biopsies (TBB) and microbiologic results from surveillance bronchoscopies. We aimed to define reference values that would allow distinction between four specific groups “ACR”, “infection”, “combined ACR and infection” and “no pathologic process”. No definitive pattern was identified. Given the overlap between groups, these four cytokines are not suitable diagnostic markers for ACR or infection after lung transplantation.</p>", "<title>Keywords</title>" ]	[ "<title>Authorship</title>", "<p id=\"p0140\">NES conducted data collection and statistical analysis, provided draft versions and revised the manuscripts. EP supervised the laboratory analyses, reviewed all versions of the manuscript and assisted to write the final version. SH performed statistical analysis and assisted to write the final version. CB and MK reviewed all versions of the manuscript and assisted to write the final version. LCH designed the project, revised all versions of the manuscript and assisted to write the final version. CAR designed and supervised the project, revised all versions of the manuscript and provided the final version. All authors read and approved the final manuscript.</p>", "<title>Funding sources</title>", "<p id=\"p0145\">This study was partially funded by <funding-source id=\"gp005\">Lunge Zürich</funding-source>.</p>", "<title>Disclosure statement</title>", "<p id=\"p0150\">The other authors have no conflicts of interest to disclose. This work was supported by a grant from Lunge Zürich (grant number 2017-01).</p>", "<title>Credit author statement</title>", "<p id=\"p0155\">NES conducted data collection and statistical analysis, provided draft versions and revised the manuscripts. EP supervised the laboratory analyses, reviewed all versions of the manuscript and assisted to write the final version. SH performed statistical analysis and assisted to write the final version. CB and MK reviewed all versions of the manuscript and assisted to write the final version. LCH designed the project, revised all versions of the manuscript and assisted to write the final version. CAR designed and supervised the project, revised all versions of the manuscript and provided the final version. All authors read and approved the final manuscript.</p>", "<title>Declaration of Competing Interest</title>", "<p id=\"p0160\">The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.</p>" ]	[]	[ "<fig id=\"f0005\"><label>Fig. 1</label><caption><p>Concentration of IL-6 in BAL fluid for different specific groups. The log transformed concentrations (log([pg/ml]) of IL-6 at the time of ≥A1 rejection; viral, bacterial or fungal infection; “no pathologic process” or “combined ACR and infection” is shown as box-and-whiskers plots. Significance for differences between specific groups was determined using Kruskal-Wallis test. Significance for differences between two categories was determined using Wilcoxon Rank Sum test. Log levels of IL-6 were lowest during ACR and highest during “combined ACR and infection” compared with “no pathologic process” (p = 0.87 and p = 0.11, respectively).</p></caption></fig>", "<fig id=\"f0010\"><label>Fig. 2</label><caption><p>Left: ROC curves for IFN-γ, IL-6, IL-8 and TNF-α to diagnose isolated ACR in surveillance bronchoscopies with TBB during the first year after transplantation. Area under the curve was 0.48 (IFN-γ), 0.55 (IL-6), 0.51 (IL-8) and 0.52 (TNF-α). No optimal cut-off can be derived from these data. Right: ROC curves for IL-6, IL-8, IFN-γ and TNF-α to diagnose “combined ACR and infection” in surveillance bronchoscopies with TBB during the first year after transplantation. Area under the curve for IFN-γ was 0.517 and for IL-8 was 0.531. Area under the curve for TNF-α was 0.509. No optimal cutoff can be derived from these data, particularly where higher scores of TNF-α do not appear to correspond with higher probability of “combined ACR and infection”.</p></caption></fig>", "<fig id=\"f0015\"><label>Fig. 3</label><caption><p>Kaplan-Meier analysis of survival after lung transplantation. No significant differences in overall survival were observed between patients with and without at least one episode ACR diagnosed in surveillance bronchoscopies during the first year after transplantation (p = 0.54).</p></caption></fig>" ]	[ "<table-wrap position=\"float\" id=\"t0005\"><label>Table 1</label><caption><p>Patient characteristics.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Demographics</th><th/></tr></thead><tbody><tr><td>Patients, n</td><td>319</td></tr><tr><td>Female, n (%)</td><td>142 (44.5%)</td></tr><tr><td>Male, n (%)</td><td>177 (55.5%)</td></tr><tr><td>Age at transplantation, mean ± standard deviation (range), year</td><td>51.8 ± 15.5 (18–77)</td></tr><tr><td>CMV high-risk, n (%)</td><td>134 (42.1%)</td></tr><tr><td colspan=\"2\">  </td></tr><tr><td>Transplant indication</td><td/></tr><tr><td>COPD, n (%)</td><td>100 (31.3%)</td></tr><tr><td>Cystic fibrosis, n (%)</td><td>107 (33.5%)</td></tr><tr><td>Pulmonary fibrosis, n (%)</td><td>93 (29.1%)</td></tr><tr><td>Pulmonary hypertension, n (%)</td><td>17 (5.3%)</td></tr><tr><td>Re-Transplantation, n (%)</td><td>2 (0.6%)</td></tr></tbody></table></table-wrap>", "<table-wrap position=\"float\" id=\"t0010\"><label>Table 2</label><caption><p>BAL cytokine concentrations for different specific groups. The concentrations [pg/ml] in the given group at the time of grade ≥A1 ACR (R); viral, bacterial or fungal infection (I); “no pathologic process” (None) or “combined ACR and infection” (R+I) is shown as median and interquartile range (IQR). Significance for differences between categories was determined using Kruskal-Wallis test.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>Group</th><th>Median [pg/ml]</th><th>IQR [pg/ml]</th><th>p-value</th></tr></thead><tbody><tr><td rowspan=\"4\"><bold>IL-6</bold></td><td>None</td><td>2.20</td><td>1.00–5.50</td><td rowspan=\"4\">p = 0.045</td></tr><tr><td>R</td><td>1.60</td><td>1.17–5.93</td></tr><tr><td>I</td><td>2.50</td><td>0.90–5.45</td></tr><tr><td>R+I</td><td>4.10</td><td>1.40–7.10</td></tr><tr><td colspan=\"5\">  </td></tr><tr><td rowspan=\"4\"><bold>IL-8</bold></td><td>None</td><td>475.00</td><td>212.00–891.00</td><td rowspan=\"4\">p = 0.7</td></tr><tr><td>R</td><td>453.00</td><td>225.00–829.00</td></tr><tr><td>I</td><td>476.00</td><td>220.00–1151.50</td></tr><tr><td>R+I</td><td>585.00</td><td>255.00–1115.00</td></tr><tr><td colspan=\"5\">  </td></tr><tr><td rowspan=\"4\"><bold>TNF-α</bold></td><td>None</td><td>0.11</td><td>0.01–0.30</td><td rowspan=\"4\">p = 0.2</td></tr><tr><td>R</td><td>0.10</td><td>0.00–0.45</td></tr><tr><td>I</td><td>0.20</td><td>0.03–0.50</td></tr><tr><td>R+I</td><td>0.10</td><td>0.00–0.68</td></tr><tr><td colspan=\"5\">  </td></tr><tr><td rowspan=\"4\"><bold>IFN-γ</bold></td><td>None</td><td>0.10</td><td>0.00–1.20</td><td rowspan=\"4\">p = 0.1</td></tr><tr><td>R</td><td>0.10</td><td>0.00–0.20</td></tr><tr><td>I</td><td>0.10</td><td>0.00–1.02</td></tr><tr><td>R+I</td><td>0.10</td><td>0.010–1.00</td></tr></tbody></table></table-wrap>" ]	[]	[]	[]	[]	[]	[]	[]	[ "<graphic xlink:href=\"gr1_lrg\"/>", "<graphic xlink:href=\"gr2_lrg\"/>", "<graphic xlink:href=\"gr3_lrg\"/>" ]	[]	"[{"label": ["1"], "surname": ["Chambers", "Yusen", "Cherikh"], "given-names": ["D.C.", "R.D.", "W.S."], "article-title": ["The registry of the international society for heart and lung transplantation: thirty-fourth adult lung and heart-lung transplantation report-2017; focus theme: allograft ischemic time"], "source": ["J. Heart Lung Transplant."], "year": ["2017"]}, {"label": ["2"], "surname": ["Swanson", "Mentzer", "Reilly"], "given-names": ["S.J.", "S.J.", "J.J."], "article-title": ["Surveillance transbronchial lung biopsies: implication for survival after lung transplantation"], "source": ["J. Thorac. Cardiovasc. Surg."], "volume": ["119"], "year": ["2000"], "fpage": ["27"], "lpage": ["37"]}, {"label": ["16"], "surname": ["Stephenson", "Flint", "English"], "given-names": ["A.", "J.", "J."], "article-title": ["Interpretation of transbronchial lung biopsies from lung transplant recipients: inter- and intraobserver agreement"], "source": ["Can. Respir. J. J. Can. Thorac. Soc."], "volume": ["12"], "year": ["2005"], "fpage": ["75"], "lpage": ["77"]}, {"label": ["22"], "surname": ["Patella", "Anile", "Del Porto"], "given-names": ["M.", "M.", "P."], "article-title": ["Role of cytokine profile in the differential diagnosis between acute lung rejection and pulmonary infections after lung transplantation"], "source": ["Eur. J. Cardiothorac. Surg."], "year": ["2014"]}, {"label": ["27"], "surname": ["Meyer"], "given-names": ["K.C."], "article-title": ["Recent advances in lung transplantation"], "source": ["F1000Res."], "volume": ["7"], "year": ["2018"]}, {"label": ["37"], "surname": ["Robin", "Turck", "Hainard"], "given-names": ["X.", "N.", "A."], "article-title": ["pROC: an open-source package for R and S+ to analyze and compare ROC curves"], "source": ["BMC Bioinf."], "volume": ["12"], "year": ["2011"], "fpage": ["77"]}, {"label": ["52"], "surname": ["Berastegui", "Gomez-Olles", "Sanchez-Vidaurre"], "given-names": ["C.", "S.", "S."], "article-title": ["BALF cytokines in different phenotypes of chronic lung allograft dysfunction in lung transplant patients"], "source": ["Clin. Transplant."], "volume": ["31"], "year": ["2017"]}]"	{ "acronym": [ "ACR", "AMR", "BAL", "CLAD", "CMV", "COPD", "CRP", "ELISA", "IFN-γ", "IL", "IQR", "ISHLT", "LTR", "TBB", "TNF-α" ], "definition": [ "acute cellular rejection", "antibody-mediated rejection", "bronchoalveolar lavage", "chronic lung allograft dysfunction", "cytomegalovirus", "chronic obstructive pulmonary disease", "C-reactive protein", "enzyme-linked immunosorbent assay", "interferon gamma", "interleukin", "interquartile range", "International Society for Heart and Lung Transplantation", "lung transplant recipient", "transbronchial biopsy", "tumor necrosis factor alpha" ] }	58	"NO-CC CODE"	"no"	"2023-06-06 23:35:01"	"Cytokine. 2020 Jan 7; 125:154794"	"oa_package/73/b5/PMC7128992.tar.gz"
"PMC7129780"	"31479875"	[ "<title>Introduction</title>", "<p id=\"p0020\">Interferons (IFNs) are a group of cytokines that serve as the first line of defense against viruses. In addition to their protective role against viral infection, the interferon (IFN) family - consisting of types I, II, and III IFNs, have numerous additional functions that influence cellular growth and immune surveillance against tumor cells ##REF##15864272##[1]##, ##REF##25614319##[2]##, ##REF##26194286##[3]##. All three IFN members activate the JAK/STAT pathway and induce interferon-stimulated gene (ISG) expression by binding to their respective receptors: IFNαR1 and IFNαR2 for type I interferon (IFNα/β), IFNγR1 and IFNγR2 for type II interferon (IFNγ), and IFNλR1 and IL10Rβ for type III interferon (IFNλ1~4) ##REF##15864272##[1]##, ##REF##28289095##[4]##. In contrast to types I and II, type III IFN was only recently identified and plays not only antiviral functions but also novel immunomodulatory functions in oncology and autoimmune diseases ##REF##26552337##[5]##, ##REF##30399529##[6]##. IFNλ1~3 were identified through computational based prediction from genome sequencing ##REF##12483210##[7]##, ##REF##12469119##[8]## and IFNλ4 was discovered in genome-wide association studies (GWAS) on hepatitis C virus (HCV)-infected patients ##REF##23291588##[9]##. The ΔG allele of a dinucleotide genetic variant (rs368234815) that is upstream of the IFNL3 locus on chromosome 19 creates the functional IFNλ4, while the TT allele leads to a frameshift, thereby rendering it a pseudogene ##REF##23291588##[9]##. Interestingly, HCV patients with the ΔG allele and hence expressing IFNλ4, responded poorly to PEGylated-IFNα-ribavirin treatment as compared to patients with the TT allele ##REF##25534433##[10]##. However, IFNλ4 still induces the major hepatic ISG expression during chronic HCV infection and is able to drive the anti-viral response against other viruses such as the MERS-CoV <italic>in vitro</italic>\n##REF##24169568##[11]##. Similar to IFNα (Roferon-A for hairy cell leukemia) and IFNβ (Avonex for multiple sclerosis), successful phase 2 clinical trials of PEGylated IFNλ1 against hepatitis D virus (HDV) infection highlight the pharmaceutical potential of the IFNλ family.</p>", "<p id=\"p0025\">Previously, the transient expression of wild-type IFNλ4 in mammalian cells failed to produce significant amounts of recombinant IFNλ4. It was suggested that a weak signal peptide in IFNλ4 may be responsible for its impaired secretion and that proper glycosylation of IFNλ4 may be required for its secretion ##REF##23291588##[9]##. Although recombinant IFNλ4 can be purified from a bacterial expression system through refolding the inclusion body ##REF##24169568##[11]##, a lack of glycosylation may affect the efficacy of IFNλ4. Recently, glyco-engineering, which introduces new glycosylation sites or alters the glycan composition of CHO cells, has been widely used to produce improved therapeutic proteins, because glycan moieties can affect various protein properties, such as solubility, stability, <italic>in vivo</italic> activity, and serum half-life. For example, improved half-life and productivity are obtained from glyco-engineered hIFNβ-1a and hIFNα ##UREF##0##[12]##, ##REF##18039474##[13]##. Moreover, increased secretion of lipase, cutinase, llama V<sub>HH</sub> antibody, and macrophage inhibitory cytokine 1 results from the addition of a single N-glycosylation site ##REF##11055947##[14]##, ##REF##19637381##[15]##. Therefore, we propose that glyco-engineering IFNλ4 is a viable option for improving its expression level and possibly altering other properties.</p>", "<p id=\"p0030\">In this study, we used mutagenesis to introduce new potential N-glycosylation sites based on the model structures of the IL10Rβ-IFNλ4-IFNλR1 complex. Our results indicate that, among several IFNλ4 variants, three - L28N, P73N, and L28N + P73N - exhibited enhanced productivity, although only P73N was glycosylated <italic>de novo</italic>. Moreover, these HEK293-expressed IFNλ4 variants retained their binding affinity to the specific IL10Rβ and IFNλR1 receptors, and showed a more potent IFNλ4-mediated signaling and antiviral activity than did <italic>E. coli</italic>-derived IFNλ4 (eIFNλ4).</p>" ]	[ "<title>Materials and methods</title>", "<title>Modeling process</title>", "<p id=\"p0035\">The human IFNλ4 amino acid sequence (22~179, NCBI Accession Number: AFQ38559.1) was used in SWISS-MODEL homology modeling with three templates (PDB code: 5T5W.1.C, 3OG6.1.A, 3OG4.1.A). The model with the highest QMEAN-Z (Qualitative Model Energy ANalysis-Z) score (−2.56) was aligned to the IL10Rβ-IFNλ3-IFNλR1 structure (PDB code: 5T5W) to create the IL10Rβ-IFNλ4-IFNλR1 model.</p>", "<title>Cell lines, cell culture, and reagents</title>", "<p id=\"p0040\">Expi293F (#A14527, Gibco®) cells were cultured according to ATCC guidelines and used within 6 months of receipt. They were maintained in suspension in Expi293F expression medium (#14351, Gibco®) at 37 °C and 8% CO<sub>2</sub> with 125 rpm agitation. Huh-7.5 cells (Apath) were maintained at 37 °C with 5% CO<sub>2</sub> in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (WelGENE), 4.5 g/l glucose, L-glutamine, and 1% penicillin/streptomycin (WelGENE). Small-interfering RNAs (siRNAs) against IFNλR1 and scrambled sequences were obtained from Santa Cruz Biotechnology. Transfection of IFNλR1 siRNA was performed using lipofectamine RNAi MAX (Invitrogen). Recombinant IFN-α-2a was obtained from PBL Assay Science, recombinant IFN-β was obtained from PeproTech, and recombinant human IFNλ1 (1598-IL), λ2 (8417-IL), λ3 (5259-IL), and eIFNλ4 (9165-IL) were obtained from R&D Systems.</p>", "<title>Expression and purification of recombinant proteins</title>", "<p id=\"p0045\">Gene encoding human IFNλ4 (1~179) was cloned into a modified pcDNA3.1 (#V79020, Invitrogen™) containing a C-terminal 6x-His tag. IFNλ4 variants were generated by site-directed mutagenesis (QuikChange site-Directed Mutagenesis Kit, #200519, Agilent) using the IFNλ4 wild-type construct as the PCR template. The primers for site-directed mutagenesis are listed in <xref rid=\"s0105\" ref-type=\"sec\">Supplementary Table 1</xref>. For IFNλ4-Protein A expression, the C-terminal 6x-His in the IFNλ4 constructs were replaced with a Protein A gene derived from PEZZ18 (#VPT4033, GE Healthcare life Sciences). A thrombin cleavage sequence (LVPRGS) was introduced between the IFNλ4 genes and the Protein A gene using the PCR primer, in order to remove Protein A. IFNλ4 wild-type and variants containing 6x-His or Protein A were transfected into Expi293F cells using ExpiFectamine 293 Transfection Kits (#A14524, Invitrogen<sup>M</sup>), following the manufacturer’s protocol. For the purification of IFNλ4 variants, the supernatant containing secreted IFNλ4-Protein A was loaded onto IgG Sepharose resin (#17096902, GE Healthcare Life Sciences). After three washes with 1x PBS, the protein-bound resins were incubated overnight with thrombin (1% (v/v) in 1x PBS) at 4 °C to remove the C-terminal Protein A tag. Eluted IFNλ4 variants were subsequently purified by gel-filtration chromatography in a Superdex 200 Increase 10/300 GL column (#28990944, GE Healthcare Life Sciences) equilibrated with 1x PBS.</p>", "<title>Immunoblotting</title>", "<p id=\"p0050\">The cells were lysed with RIPA buffer (Thermo Fisher Scientific) to prepare total cell lysates. Ten micrograms of each cell lysate were loaded on SDS-PAGE gels prior to immunoblotting. The antibodies used for immunoblotting were: IFNλ4 (1:200, mouse, Millipore MABF227), IFNλ4 (1:200, rabbit, Abcam ab196984), STAT1 (1:1000, rabbit, BD Biosciences 610120), PY-STAT1 (1:1000, mouse, BD Biosciences 612233), STAT2 (1:1000, rabbit, Santa Cruz Biotechnology sc-476), IRF9 (1:1000, rabbit, Santa Cruz sc-496), SOCS1 (Abcam #62584), USP18 (Cell Signaling Technology #4813), horseradish peroxidase (HRP)-conjugated rabbit IgG (1:5000, Abcam ab97051), and HRP-conjugated mouse IgG (1:5000, Abcam ab97023).</p>", "<title>PNGase F treatment</title>", "<p id=\"p0055\">N-glycans of IFNλ4 were removed using a PNGase F kit (#P0704S, New England Biolabs) according to the manufacturer’s instructions. Briefly, IFNλ4 variants were boiled with Glycoprotein Denaturing Buffer (10×) and chilled on ice. GlycoBuffer(10×), NP-40(10×), and 1 μl of PNGase F were added onto denatured proteins and the mixture was incubated at 37 °C for 1 h before the Western blot analysis.</p>", "<title>Glycosylation site analysis</title>", "<p id=\"p0060\">The glycopeptides resulting from non-specific digestion were prepared as previously described ##REF##24016182##[16]##. Briefly, 50 µg/µL IFNλ4 variants were incubated with 50 µg/µL pronase E for 1 h at 37 °C. The digested glycopeptides were enriched by graphitized carbon solid-phase extraction (PGC-SPE) and analyzed by nanoLC-Chip Q-TOF MS (Agilent Technologies). The LC-MS and MS/MS data were processed and interpreted as previously described, using MassHunter Qualitative Analysis software (version B.07.00, Agilent Technologies) and GP Finder software ##REF##17022651##[17]##.</p>", "<title>Determination of binding kinetics</title>", "<p id=\"p0065\">The IFNλ4 variant binding kinetics to IFNλR1 and IL10Rβ were measured by biolayer light interferometry on a BLItz system (ForteBio, Pall Life Sciences). The mixtures were agitated at 2200 rpm in washing buffer (200 mM NaCl, 20 mM Tris-HCl pH 8, 5% glycerol, 0.01% Tween-20). Assays were performed at room temperature. Biotinylated IFNλ4, at concentrations of 0.25 mg/ml, were loaded onto the surfaces of streptavidin biosensors (ForteBio) for 1 min, followed by washing of the loaded biosensors for 2 min with washing buffer (200 mM NaCl, 20 mM Tris-HCl pH 8, 5% glycerol, 0.01% Tween-20) to remove any unbound protein. The biosensor tips were immersed in drops containing indicated concentration of IFNλR1 and IL10Rβ (500, 1000 and 2000 nM). Associations (on rate, k<sub>on</sub>) were measured over a 2 min interval. The sensors were subsequently immersed in washing buffer for 2 min to measure dissociation (off-rate, k<sub>off</sub>). K<sub>D</sub>, measured in nanomoles, was calculated as the ratio of off-rate to on-rate. The resulting data were analyzed by fitting to a 1:1 ligand model with the global fitting function.</p>", "<title>Production and infection of cell culture-derived HCV (HCVcc)</title>", "<p id=\"p0070\">The Japanese fulminant hepatits-1 (JFH-1) strain (genotype 2a) of HCVcc was produced as described previously ##REF##26216956##[18]##. DMEM containing 5% human serum was used to culture the Huh-7.5 cells, in order to produce highly infectious JFH1 HCVcc. HCVcc infectivity was quantified by a colorimetric focus-forming assay, as described previously ##UREF##1##[19]##. Huh-7.5 cells were infected with JFH-1 HCVcc at 0.5 multiplicity of infection (MOI).</p>", "<title>RNA extraction and real-time quantitative PCR</title>", "<p id=\"p0075\">Total RNA isolation and TaqMan real-time quantitative PCR were performed as described previously ##REF##24899196##[20]##. In brief, total RNA was isolated with GeneAll Ribospin™ (GeneAll), after which TaqMan Gene Expression Assays (Applied Biosystems) were used to determine the mRNA levels of the target genes. Quantification of intracellular HCV RNA copies was performed as described previously ##REF##24899196##[20]##. The results were standardized to the mRNA levels of GAPDH and the data are presented as means ± standard error of the mean. TaqMan Assay (Applied Biosystems) used in this study are: IFNLR1 (Hs00417120_m1), ISG15 (Hs01921425_s1), MX1 (Hs00895608_m1), SOCS1 (Hs00705164_s1), USP18 (Hs00276441_m1), GAPDH (Hs02758991_g1). IFNL proteins (R&D Systems) used in this study are: IFNL1 (1598-IL), IFNL2 (8417-IL), IFNL3 (5259-IL), eIFNL4 (9165-IL).</p>", "<title>Statistical analysis</title>", "<p id=\"p0080\">Data from experiments with cell lines are presented as means ± standard error of the mean. Unpaired <italic>t</italic>-tests or two-tailed Mann-Whitney U-tests were performed for statistical analysis. All of the analyses for real-time quantitative PCR were performed with GraphPad Prism version 7.01. P values less than 0.05 were considered to be statistically significant.</p>" ]	[ "<title>Results</title>", "<title>Design and expression of IFNλ4 variants</title>", "<p id=\"p0085\">The low affinity of wild-type IFNλ to its receptor, IL10Rβ, hampers the production of the stable ternary complex - IL10Rβ-IFNλ-IFNλR1. Therefore, only the structures of IFNλ3 alone ##REF##19457860##[21]## or IFNλ1 in complex with IFNλR1 ##REF##20934432##[22]## have been determined. Recently, Mendoza, <italic>et al</italic>., introduced affinity-enhancing mutations on IFNλ3 which stabilized its interaction with IL10Rβ, and elucidated the crystal structure of the type III interferon signaling complex, IL10Rβ-IFNλ3-IFNλR1 (PDB code: 5T5W) (##FIG##1##Fig. 1##\nA) ##REF##28329704##[23]##. Although IFNλ4 shares only ~30% sequence identity with IFNλ1~3, the sequence alignment of IFNλ1~4 suggests that IFNλ4 interacts with IFNλR1 and IL10Rβ in a similar manner as the IL10Rβ-IFNλ3-IFNλR1 ternary complex ##REF##28329704##[23]## for two reasons. First, the amino acids of the IFNλ family that are critical for IFNλR1 binding are well-conserved in IFNλ4 (P37, L40, K44, R47, D48, I108, F159, and R163) (##FIG##1##Fig. 1##B). Second, hydroxyl groups of several aromatic residues of IL10Rβ (Y59, Y82, Y140, and W143) form a hydrogen bonding network with IFNλ3 (S44, L45, Q48R, and E106D); these are also well conserved in IFNλ4 (S34, L35, R48, and Q100). Therefore, we modeled the IFNλ4 structure using the crystal structure of IFNλ3 and IFNλ1 (##FIG##1##Fig. 1##A) and structurally aligned it to the IL10Rβ-IFNλ3-IFNλR1 structure to build the IL10Rβ-IFNλ4-IFNλR1 model (##FIG##1##Fig. 1##B). Interestingly, the model structure of IL10Rβ-IFNλ4-IFNλR1 indicates that critical hydrophobic pockets for harboring the hydrophobic residues of IL10Rβ (Y82 and W143) are well maintained on the surface of IFNλ4 (##FIG##1##Fig. 1##C).</p>", "<p id=\"p0090\">Using the IL10Rβ-IFNλ4-IFNλR1 model structure, we searched the new N-glycosylation candidate sites of IFNλ4 based on three criteria. First, the sites had to be outside the receptor binding region to minimize the change in the receptor-ligand binding and signal activation. Second, they had to be exposed to the solvent to allow access to oligosaccharyltransferase (OST), which catalyzes the initial transfer of glycan from the lipid-linked oligosaccharide onto the substrate asparagine ##REF##16356726##[24]##, ##REF##3896128##[25]##. Third, the consensus sequence (NXS/T, X = any amino acid except proline) had to be achieved by single point mutation to minimize the structural distortion caused by the mutation. Only six candidate sites were available that met all three criteria: L28N, A54N, P73N, H97N, K154N, and A173N (##FIG##1##Fig. 1##A and B). We named them M1 ~ M6, respectively.</p>", "<p id=\"p0095\">Next, we examined the expression levels of each of the IFNλ4 variants (M1 ~ M6) by western blot and found that two IFNλ4 variants, M1 (L28N mutation) and M3 (P73N mutation), resulted in enhanced protein expression (##FIG##2##Fig. 2##\nA). Interestingly, only M3 showed the prominent up-shift in SDS-PAGE that indicates successful hyperglycosylation. We also checked the expression level of the double mutants (L28N and P73N, M7), showing further enhanced protein expression compared to M1 and M3 variants (##FIG##2##Fig. 2##A). The constructs used in the western blot for hit discovery carried a C-terminus 6x Histidine tag, which may interfere with proper secretion of the protein, given the extensive distribution of positively-charged amino acids in IFNλ4. Therefore, we substituted the 6x histidine tag with a protein A tag and purified three IFNλ4 variants (M1, M3, and M7) using affinity chromatography followed by thrombin digestion, in order to remove the protein A tag and subsequent size exclusion chromatography. Final IFNλ4 variants (M1, M3, and M7) were analyzed by SDS-PAGE and Coomassie blue staining under reducing and non-reducing condition. The resulting bands indicate that three IFNλ4 variants (M1, M3, and M7) are monomer (##FIG##2##Fig. 2##B). The elution profile of standard proteins indicates that each monodispersed peak corresponds to the IFNλ4 variants (~44 kDa) (##FIG##2##Fig. 2##C). Most likely, this oversized elution is due to the presence of N-glycosylation on IFNλ4 variants, which was confirmed by the results shown in the following section.</p>", "<title>Identification of N-glycans on IFNλ4 variants</title>", "<p id=\"p0100\">To identify the presence of N-glycans on the three IFNλ4 variants, we treated them with PNGase F and compared their sizes with SDS-PAGE. The M3 (P73N) and M7 (L28N + P73N) IFNλ4 variants were located at higher molecular weight positions compared to the M1 (L28N) IFNλ4 variant. After de-glycosylation with PNGase F, however, the molecular weight of the three IFNλ4 variants decreased to the same level, indicating the presence of N-glycans in all IFNλ4 variants, but the status or position of M1 N-glycosylation may be slightly different from those of M3 and M7 IFNλ4 variants (##FIG##3##Fig. 3##\nA).</p>", "<p id=\"p0105\">We used mass spectrometry to determine the exact position of N-glycans on IFNλ4 variants ##REF##26964748##[26]##. Briefly, purified IFNλ4 variants were treated with pronase E to produce glycopeptides, and ultimately to determine the glycosylation site. The glycopeptides were then separated and analyzed by nanoLC-Chip Q-TOF MS. The LC/MS data indicate that the mutated L28N in M1 and M7 IFNλ4 variants were not glycosylated, whereas an original N-glycosylation site, Asn61, and mutated P73N were fully occupied by N-glycans (##FIG##3##Fig. 3##B–D). These are in accordance with the PNGase F treatment results, where M1 (L28N) migrated more quickly than either M3 (P73N) or M7 (L28N + P73N).</p>", "<title>Receptor binding affinity and biological activity of IFNλ4 variants</title>", "<p id=\"p0110\">To investigate whether the mutation and the additional glycan on IFNλ4 variants affect their binding to their receptors, IL10Rβ and IFNλR1, we examined the <italic>in vitro</italic> binding affinity of IFNλ4 variants to IL10Rβ and IFNλR1 by Biolayer Light Interferometry (BLI) and then compared them with that of IFNλ4 WT purified from <italic>E. coli</italic> (eIFNλ4). Similar to eIFNλ4, the three IFNλ4 variants properly bound to their receptors and their binding affinities to IFNλR1 were higher than to IL10Rβ (##FIG##4##Fig. 4##\n). Moreover, our variants had a slightly higher affinity towards IL10Rβ than the eIFNλ4 does (For IL10Rβ, K<sub>D M1</sub> = 49 nM, K<sub>D M3</sub> = 51 nM, K<sub>D M7</sub> = 49 nM, K<sub>D eIFNλ4</sub> = 71 nM), while their binding affinity to IFNλR1 was similar to each other (For IFNλR1, K<sub>D M1</sub> = 14 nM, K<sub>D M3</sub> = 22 nM, K<sub>D M7</sub> = 17 nM, K<sub>D eIFNλ4</sub> = 19 nM). The modifications elicited by mutation and glycosylation does not inhibit their interaction with their specific receptors, even further stabilize the interaction between IFNλ4 and IL10Rβ.</p>", "<p id=\"p0115\">In order to determine whether the mutation and additional glycan on the IFNλ4 variants affected their functional activity, we investigated their IFNλR1-dependent phospho-STAT1 signaling upon treatment with the IFNλ4 variants. Our results indicate that treatment with the M1, M3, and M7 IFNλ4 variants also induced phosphorylation of STAT1, just as in other type III interferons, IFNλ1~3, and that the suppression of IFNλR1 expression by small interference RNA specific to the IFNλR1 gene (siIFNλR1) abolished the phosphorylation of STAT1, even after treatment with the IFNλ4 variants (##FIG##5##Fig. 5##\nA). IFNλ4 stimulation reportedly leads to the assembly of the ISGF3 transcription factor complex, which consists of phospho-STAT1, phospho-STAT2, and IRF9 and induces the expression of ISG15 ##REF##28630501##[27]##, which is critical for anti-viral activity ##REF##22328912##[28]##. We showed that the M1, M3, and M7 IFNλ4 variants also induced the expression of ISG15 and inhibited HCV replication in HCV-infected Huh-7.5 cells (##FIG##5##Fig. 5##B and 5C). Interestingly, the M1, M3, and M7 IFNλ4 variants showed a significantly more potent ISG induction and anti-viral activity than eIFNλ4.</p>", "<p id=\"p0120\">Prolonged exposure to IFNλ proteins induces the production of unphosphorylated ISGF3 (U-ISGF3) consisting of STAT1, STAT2 and IRF9 without tyrosine phosphorylation while the expression of phosphorylated ISGF3 are diminished ##REF##26216956##[18]##. As a result, the upregulation of the U-ISGF3-specific set of genes, such as Mx1, is maintained long-term. In order to assess whether the M1, M3, and M7 variants display a similar functionality during prolonged treatment, we evaluated the protein levels of the U-ISGF3 components. The protein levels of STAT1, STAT2, and IRF9 were equally upregulated by all IFNλ4s (##FIG##5##Fig. 5##D). Nevertheless, our IFNλ4 variants maintained the upregulation of Mx1 more robustly than did eIFNλ4, but IFNλ1, 2 and 3 maintained more strongly the upregulation of Mx1 expression compared to our IFNλ4 variants (##FIG##5##Fig. 5##E).</p>", "<p id=\"p0125\">Previously, eIFNλ4 is shown to induce the expression of negative regulators of IFN signaling ##REF##28630501##[27]##, ##REF##29070670##[29]## such as SOCS1 and USP18. To assess the effect of glycosylation on this functionality, we examined the expression level of SOCS1 and USP18 upon the treatment of M1, M3 and M7 variants on Huh7 cell line. USP18 was significantly increased upon treatment of IFNλ1, 2 and 3. While eIFNλ4 resulted in slight increase of USP18, our IFNλ4 variants (M1, M3 and M7) showed a comparable activity to IFNλ1, 2 and 3 (##FIG##5##Fig. 5##F). The protein expression of SOCS1 was not significantly increased by the treatment of any form of IFNλs (##FIG##5##Fig. 5##F). However, the mRNA expression of SOCS1 was slightly upregulated by IFNλs, although there was no significant difference among IFNλs (##FIG##5##Fig. 5##G). These results suggest that our structure-based approach on selecting <italic>de novo</italic> glycosylation maintained the biological activity of IFNλ4 and our IFNλ4 variants expressed from HEK293 have superior activity compared to eIFNλ4.</p>" ]	[ "<title>Discussion</title>", "<p id=\"p0130\">Until recently, research on IFNλ4 and its use as a clinical therapeutic was hindered by the inability to obtain appreciable amounts of this protein. Purification of bacterial-derived recombinant IFNλ4 through refolding ##REF##24169568##[11]## still poses a number of problems, including the complexity of purification steps, lack of glycosylation, and endotoxin contamination. Our study is an example of how glycoengineering aided by structural information can be used to overcome such limitations; it is the first to report the successful production of IFNλ4 protein from a mammalian cell line with enhanced properties compared to eIFNλ4 and requiring a simpler purification protocol. There are several possibilities as to why our IFNλ4 variants displayed enhanced expression and potency. First, the presence of acidic N-glycans may stabilize the protein through a charge-balance, since IFNλ4 is unusually abundant with positively charged amino acids (~23%). Second, considering how the immune response is more actively triggered by our variants without critically affecting the receptor binding activity, compared to eIFNλ4, extra N-glycans may have extended the half-life, as in other reports ##UREF##0##[12]##, ##REF##15959882##[30]##, thereby increasing the fraction of functional protein during treatment. Finally, since N-glycosylation in eukaryotes is co-translational ##UREF##2##[31]##, the protein folding may have also been affected. Nevertheless, the mechanism behind the enhanced expression induced by unmodified L28N remains unanswered. We speculate that L28 may serve as the hydrophobic aggregation nuclei interacting with nearby hydrophobic residues, such as L29 or Y32 ##REF##19571001##[32]##, ##REF##24908382##[33]##; perhaps L28N alleviates this effect.</p>", "<p id=\"p0135\">This study is unique, in that we were able to successfully identify a viable <italic>de novo</italic> N-glycosylation site by structural elucidation of glyco-peptides. We used endogenous serine, threonine, or asparagine to introduce the glycosylation modification, which minimized the mutation-induced structural distortion. Other IFNλ4 candidates containing N-glycosylation sites at random locations were also tested (data not shown) but no additional modification or expression changes were observed, indicating the efficiency of our structure-oriented approach. However, glyco-peptides containing L28N were not detected by mass spectrometry, suggesting that the site may have remained unmodified. According to the recent cryo-EM structure of oligosaccharyltransferase (OST) ##REF##29466327##[34]##, ##REF##29519914##[35]##, ##REF##29301962##[36]##, which catalyzes the initial transfer of glycan from the lipid-linked oligosaccharide onto the substrate asparagine, substrate binding to the catalytic subunit, STT3, requires structural flexibility near the glycosylation sequence of the substrate. Our IFNλ4 model suggests that P73 is located on the flexible loop, while other eliminated candidate sites (A173 and K154) are part of the α-helix. This may partly explain the successful glycosylation of P73N. On the contrary, L28N may not be physically accessible by OST. Elucidating the structure of IFNλ4 may provide insights to this hypothesis.</p>", "<p id=\"p0140\">A number of interferons are already targets of drug development, due to their ability to generate strong antiviral and antitumor responses or to modulate immune responses. IFNα and its PEGylated variants are used against cancers such as hairy cell leukemia (Roferon A), melanoma (Multiferon), and AIDS-related Kaposi's sarcoma (Intron A) ##REF##3730612##[37]##, ##REF##16760174##[38]##, ##REF##3492260##[39]##. IFNβ is a well-known treatment for multiple sclerosis, with a number of different versions available (Rebif – IFNβ 1a, liquid form, Avonex – IFNβ 1a, lyophilized, Cinnovex – IFNβ 1a, biogeneric, Betaseron – IFNβ 1b, Plegridy – PEGylated IFNβ 1a) ##REF##21132998##[40]##, ##REF##21942977##[41]##, ##UREF##3##[42]##, ##UREF##4##[43]##. Similar to this, IFNλs also have a therapeutic potential, because it has been shown that IFNλs can protect hosts from various viruses, including influenza virus, West Nile virus, norovirus and rotavirus ##REF##16760174##[38]##, ##REF##3492260##[39]##, ##REF##21132998##[40]##, ##REF##21942977##[41]##. It will be very interesting to test if glycosylated IFNλs exert anti-viral activity in hosts infected by such viruses. In this regard, a recent study demonstrated that IFNλs can suppress influenza virus without the inflammatory side effects of IFNα ##REF##27520969##[44]##. However, it was previously shown that the expression of functional IFNλ4 is associated with unresponsiveness to IFNα treatment among HCV-infected patients ##REF##23291588##[9]##, and a subsequent study showed that long-term exposure to IFNλ4 leads to cellular unresponsiveness to IFNα treatment by upregulation of USP18 or SOCS1 ##REF##28630501##[27]##, ##REF##29070670##[29]##, indicating that IFNλ4 treatment may be detrimental to virus-infected patients. On the other hand, our result shown in ##FIG##5##Fig. 5##C indicates that IFNλ4 treatment can directly suppress HCV replication. Whether IFNλ4 treatment will be beneficial or detrimental to virus-infected patients might be determined by duration of the treatment and use of IFNα following IFNλ4 treatment. Although much further research and insight into the mode of action of IFNλ4 is required to understand if its effects are beneficial or detrimental to human health, our engineered IFNλ4 variants can be utilized as an alternative platform of IFNλ4 wild-type.</p>" ]	[]	[ "<p id=\"np005\">These authors contributed equally to this work.</p>", "<title>Graphical abstract</title>", "<p>Interferon lambda 4 (IFNλ4) has been recently known and studied for its role in hepatitis C virus (HCV) infection, but its clinical potential is significantly hampered due to its poor expression <italic>in vitro</italic>. Our study reports the successful production of IFNλ4 from a mammalian cell line through a glycoengineering and structure-based approach. We introduced <italic>de novo</italic> N-glycosylation of IFNλ4, guided by structural analysis, and produced IFNλ4 variants in Expi293F that displayed improved expression and potency. To preserve the structure and functionality of IFNλ4, the model structure of the IFNλ4 signaling complex was analyzed and the N-glycosylation candidate sites were selected. The receptor binding activity of engineered IFNλ4 variants and their receptor-mediated signaling pathway were similar to the E. coli version of IFNλ4 (eIFNλ4), while the antiviral activity and induction levels of interferon-stimulated gene (ISG) were all more robust in our variants. Our engineered IFNλ4 variants may be further developed for clinical applications and utilized in basic research to decipher the immunological roles of IFNλ4.</p>", "<title>Keywords</title>" ]	[ "<title>Declaration of Competing Interest</title>", "<p id=\"p0145\">The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.</p>" ]	[ "<title>Supplementary material</title>", "<p id=\"p0165\">The following are the Supplementary data to this article:\n</p>", "<title>Acknowledgments</title>", "<p id=\"p0150\">This work was supported by the <funding-source id=\"gp005\"><institution-wrap><institution-id institution-id-type=\"doi\">10.13039/501100003725</institution-id><institution>National Research Foundation of Korea (NRF)</institution></institution-wrap></funding-source> grant (2015M3A9B5053960 to H.M.K. and H.J.A) and the <funding-source id=\"gp010\">Korean Health Technology R&D Project, Ministry of Health and Welfare</funding-source> (HI18C0135 to H.M.K.).</p>", "<title>Author contributions</title>", "<p id=\"p0155\">J.H.C., S.H.H., H.J.A, E.C.S., and H.M.K. designed the experiments; J.H.C., S.H.H., and N.S. contributed to the experimental work; J.H.C., P.L., E.C.S., and H.M.K. wrote and edited the manuscript. E.C.S. and H.M.K. supervised the project. All authors reviewed the manuscript.</p>" ]	[ "<fig id=\"f0030\" position=\"anchor\"></fig>", "<fig id=\"f0005\"><label>Fig. 1</label><caption><p>Design of IFNλ4 variants. (A) Model structure of IL10Rβ-IFNλ4-IFNλR1 (right) built from the IL10Rβ-IFNλ3-IFNλR1 complex structure (PDB code: 5T5W, Left). Mutation sites for potential N-glycosylation are displayed as the orange stick model (L28, A54, P73, H97, K154, A173) and seen in two different views (bottom). The endogenous N-glycosylation site – N61 – is shown as the blue stick. (B) Sequence alignment of IFNλ proteins. Amino acids important in binding with IFNλR1 and IL10Rβ are highlighted in green and cyan, respectively. Mutation sites for potential N-glycosylation are labeled from M1 to M6 and are boxed in orange. The endogenous N-glycosylation site for IFNλ4 (N61, M0) is boxed in blue. Sequence conservation from the highest to lowest order is colored in red, blue, and black. The *IFNλ3 sequence was obtained from the crystal structure of the IL10Rβ-IFNλ3-IFNλR1 complex ##REF##28329704##[23]##, which contains affinity-enhancing mutations on IFNλ3 for stabilizing its interaction with IL10Rβ. (C) Binding mode of IFNλ3 and IFNλ4 towards IL10Rβ. The hydrogen bond network between IFNλ3 and IL10Rβ is shown and similar interactions between IFNλ4 and IL10Rβ are mapped based on the model structure of IL10Rβ-IFNλ4-IFNλR1 (left). Surface representation of binding sites for IL10Rβ Y82 and W143 within IFNλ3 ##REF##28329704##[23]## and IFNλ4 (right). Hydrophobic residues are colored in orange and grey. Hydrophobic pockets for harboring the hydrophobic residues of IL10Rβ (Y82, W143) were found within both IFNλ3 and IFNλ4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</p></caption></fig>", "<fig id=\"f0010\"><label>Fig. 2</label><caption><p>Production of IFNλ4 variants. (A) Expression test of IFNλ4 variants. Expression level of IFNλ4 wild-type and variants containing C-terminal 6x-histidine tags were monitored by Western blot with anti-his antibody. M1 (L28N), M3 (P73N), and M7 (L28N + P73N) showed enhanced expression and were selected for larger scale expression. (B) Coomassie blue staining of purified M1, M3, and M7 under reducing and non-reducing condition. The proteins were purified by affinity chromatography with IgG sepharose followed by thrombin digestion and gel filtration chromatography. (C) Gel filtration chromatogram of M1, M3, M7, and standard proteins. Each gel filtration peak corresponds to standard proteins – thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa). A bar connecting the maximum and the x-axis is drawn to help read the elution volume. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</p></caption></fig>", "<fig id=\"f0015\"><label>Fig. 3</label><caption><p>Identification of N-glycosylation on IFNλ4 variants. (A) SDS-PAGE analysis with and without PNGase-F treatment of IFNλ4 variants, M1, M3, and M7. (B) Schematic diagram of IFNλ4 variants (M1, M3, M7) marked with the confirmed position of N-glycosylation by mass spectrometry. (C) Collision-induced dissociation (CID) tandem MS spectra of precursor ion at <italic>m</italic>/<italic>z</italic> 813.95 [M + 3H]<sup>3+</sup> corresponding to Hex5HexNAc4Fuc1NeuAc1 with peptide back bone (NCS) on N61 from IFNλ4 variants (M1, M3, M7). (D) CID tandem MS spectra of precursor ion at <italic>m</italic>/<italic>z</italic> 745.93 [M + 3H]<sup>3+</sup> corresponding to Hex5HexNAc4Fuc1 with peptide back bone (NSSC) on P73N from IFNλ4 variants (M3 and M7). Mutated L28Ns in M1 and M7 were not glycosylated.</p></caption></fig>", "<fig id=\"f0020\"><label>Fig. 4</label><caption><p>Binding kinetics of IFNL4 variants with IFNλR1 or IL10Rβ. (A) Binding curves of IFNλR1 and IL10Rβ toward IFNλ4 variants (M1, M3, M7) and eIFNλ4 at the indicated concentrations of IFNλR1 and IL10Rβ (500, 1000, 2000 nM). Sensorgrams were obtained from BLItz instrument. Data points are shown in grey and the corresponding fits are shown in red (IFNλR1) and blue (IL10Rβ). K<sub>D</sub> values were calculated from 1:1 global fitting. (B) Binding kinetics of IFNλR1 and IL10Rβ to immobilized IFNλ4 variants (M1, M3, M7) and eIFNλ4. Goodness of fit was assessed by evaluating the χ<sup>2</sup> and R<sup>2</sup> values generated for all fitting analyses. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</p></caption></fig>", "<fig id=\"f0025\"><label>Fig. 5</label><caption><p>Biological activity of IFNλ4 variants. (A) Activation of the JAK-STAT pathway by IFNλ4 variants via the IFNλ receptor. Huh-7.5 cells were treated with IFNλs or IFNλ4 variants (10 nM) for 30 min. The expression of IFNλR1 was suppressed by IFNλR1-specific siRNA (siIFNλR1) to show changes in the phosphorylation level of STAT1 (pSTAT1) triggered by IFNλ4 variants (eIFNλ4, M1, M3, and M7 IFNλ4 variants). (B) Induction of interferon-stimulated gene 15 (ISG15) expression by IFNλ4 variants in Huh-7.5 cells. Huh-7.5 cells were treated with 10 nM IFNλs for 10 h. (C) Inhibition of HCV replication by IFNλ4 variants. HCVcc-infected Huh-7.5 cells were treated with the indicated concentration of IFNλs for 48 h. (D) Production of U-ISGF3 by the prolonged treatment of IFNλ4 variants. Huh-7.5 cells were treated with 10 nM IFNα, IFNβ, IFNγ, or IFNλs for 72 h. Extended exposure to type III interferons induced the expression of U-ISGF3, which was composed of IRF9, unphosphorylated STAT1, and unphosphorylated STAT2. Similar responses by IFNλ4 variants were monitored. (E) Upregulation of Mx1 after prolonged treatment with IFNλs (10 nM). Mx1 is preferentially expressed by U-ISGF3 after prolonged treatment with type III interferons. (F) Immunoblotting of USP18 and SOCS1 at 48 h after treating IFNλs with final concentration of 10 nM on Huh7.5 cells. Band intensity of USP18 versus β-actin is shown below the blot image. (G) Induction of SOCS1 expression by 10 nM IFNλs treatment in Huh-7.5 cells. Relative expression was determined at 8- and 24-hour post-treatment by real-time quantitative PCR. In (A), (D) and (F), immunoblots are representative of three independent experiments with similar results. In (B), (C), (E), and (G), all analyses were done with triplicates and the graphs are representative of three independent experiments with similar results.</p></caption></fig>" ]	[]	[]	[]	[]	[]	[]	[ "<supplementary-material content-type=\"local-data\" id=\"m0005\"><caption><title>Supplementary data 1</title></caption></supplementary-material>" ]	[ "<fn-group><fn id=\"s0100\" fn-type=\"supplementary-material\"><label>Appendix A</label><p id=\"p0160\">Supplementary data to this article can be found online at <ext-link ext-link-type=\"uri\" xlink:href=\"https://doi.org/10.1016/j.cyto.2019.154833\" id=\"ir005\">https://doi.org/10.1016/j.cyto.2019.154833</ext-link>.</p></fn></fn-group>" ]	[ "<graphic xlink:href=\"ga1_lrg\"/>", "<graphic xlink:href=\"gr1_lrg\"/>", "<graphic xlink:href=\"gr2_lrg\"/>", "<graphic xlink:href=\"gr3_lrg\"/>", "<graphic xlink:href=\"gr4_lrg\"/>", "<graphic xlink:href=\"gr5_lrg\"/>" ]	[ "<media xlink:href=\"mmc1.docx\"/>" ]	"[{"label": ["12"], "surname": ["Song", "Yoon", "Kim", "Kim", "Lee", "Lee"], "given-names": ["K.", "I.S.", "N.A.", "D.H.", "J.", "H.J."], "article-title": ["Glycoengineering of interferon-beta 1a improves its biophysical and pharmacokinetic properties"], "source": ["PLoS ONE"], "volume": ["9"], "year": ["2014"], "object-id": ["e96967"]}, {"label": ["19"], "surname": ["Kang", "Shin"], "given-names": ["W.", "E.C."], "article-title": ["Colorimetric focus-forming assay with automated focus counting by image analysis for quantification of infectious hepatitis C virions"], "source": ["PLoS ONE"], "volume": ["7"], "year": ["2012"], "object-id": ["e43960"]}, {"label": ["31"], "surname": ["Kelleher", "Gilmore"], "given-names": ["D.J.", "R."], "article-title": ["An evolving view of the eukaryotic oligosaccharyltransferase"], "source": ["Glycobiology"], "volume": ["16"], "year": ["2006"], "fpage": ["47R"], "lpage": ["62R"]}, {"label": ["42"], "surname": ["Pakdaman", "Abbasi", "Gharagozli", "Ashrafi", "Delavar Kasmaei", "Amini"], "given-names": ["H.", "M.", "K.", "F.", "H.", "Harandi A."], "article-title": ["A randomized double-blind trial of comparative efficacy and safety of Avonex and CinnoVex for treatment of relapsing-remitting multiple sclerosis"], "source": ["Neurol. Sci.: Official J. Italian Neurol. Soc. Italian Soc. Clin. Neurophysiol."], "volume": ["39"], "year": ["2018"], "fpage": ["2107"], "lpage": ["2113"]}, {"label": ["43"], "mixed-citation": ["Peginterferon beta-1a (Plegridy) for multiple sclerosis. The Medical letter on drugs and therapeutics 2015; 57: pp. 67\u20139."]}]"	{ "acronym": [], "definition": [] }	44	"NO-CC CODE"	"no"	"2023-06-06 23:35:02"	"Cytokine. 2020 Jan 31; 125:154833"	"oa_package/c6/ea/PMC7129780.tar.gz"
"PMC7132398"	"25175674"	["<title>Introduction</title>","<p id=\"par0030\">Three quarters of all emerging infectious diseases(...TRUNCATED)	["<title>Methods</title>","<title>Study design</title>","<p id=\"par0045\">This study was conducted (...TRUNCATED)	["<title>Results</title>","<p id=\"par0070\">The socio-demographic, professional and HeV experience (...TRUNCATED)	["<title>Discussion</title>","<p id=\"par0105\">Overall, the results of this study show that by 2011(...TRUNCATED)	["<title>Conclusion</title>","<p id=\"par0165\">Despite a marked increase in the number of HeV cases(...TRUNCATED)	["<p>Following the emergence of Hendra virus (HeV), private veterinarians have had to adopt addition(...TRUNCATED)	[]	["<title>Acknowledgements</title>","<p id=\"par0175\">The authors would like to thank all veterinari(...TRUNCATED)	[]	["<table-wrap position=\"float\" id=\"tbl0005\"><label>Table 1</label><caption><p>Demographic and ed(...TRUNCATED)	[]	[]	[]	[]	[]	[]	["<table-wrap-foot><fn id=\"tblfn0005\"><label>a</label><p id=\"npar0005\"><italic>n</italic> = 20(...TRUNCATED)	[]	[]	"[{\"surname\": [\"Animal Health Australia\"], \"part-title\": [\"Response Policy Brief: Hendra Viru(...TRUNCATED)	{ "acronym": [], "definition": [] }	37	"NO-CC CODE"	"no"	"2023-06-06 23:35:25"	"Prev Vet Med. 2014 Nov 1; 117(1):40-51"	"oa_package/7c/5d/PMC7132398.tar.gz"
"PMC7529095"	"33024412"	["<title>Introduction</title>","<p id=\"Par2\">The situation of the world for the people is very ris(...TRUNCATED)	["<title>Proposed methodology</title>","<p id=\"Par43\">In this segment, we proposed the methodology(...TRUNCATED)	[]	[]	["<title>Conclusion</title>","<p id=\"Par85\">The novel 2019 Coronavirus, SARS-CoV-2 (COVID-19), ori(...TRUNCATED)	["<p>Communicated by Valentina E. Balas.</p>","<p id=\"Par1\">The control of spreading of COVID-19 i(...TRUNCATED)	["<title>Preliminaries</title>","<p id=\"Par15\">In this section, for better understanding of the sp(...TRUNCATED)	["<title>Acknowledgements</title>","<p>This work was supported by the Deanship of Scientific Researc(...TRUNCATED)	["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>COVID-19 confirmed cases distribution</p></capti(...TRUNCATED)	["<table-wrap id=\"Taba\"><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Not(...TRUNCATED)	["<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t(...TRUNCATED)	[]	[]	[]	[]	[]	["<table-wrap-foot><p>Bold value indicates the best alternative in critical path strategy</p></table(...TRUNCATED)	["<graphic xlink:href=\"500_2020_5287_Fig1_HTML\" id=\"MO39\"/>","<inline-graphic xlink:href=\"500_2(...TRUNCATED)	[]	"[{\"surname\": [\"Amiri\", \"Golozari\"], \"given-names\": [\"M\", \"F\"], \"article-title\": [\"Ap(...TRUNCATED)	{"acronym":["Step-1","Step-2","Step-2(a)","Step-2(b)","Step-2(c)","Step-2(d)","Step-3","Step-4","Ste(...TRUNCATED)	61	"NO-CC CODE"	"no"	"2023-06-06 23:35:14"	"Soft comput. 2023 Oct 1; 27(3):1809-1825"	"oa_package/3b/f9/PMC7529095.tar.gz"
"PMC7825904"	"33608162"	[]	[]	[]	[]	[]	[]	["<p content-type=\"salutation\"><italic>Sr. Director:</italic></p>","<p id=\"par0005\">La aspergilo(...TRUNCATED)	[]	[]	[]	[]	[]	[]	[]	[]	[]	[]	[]	[]	"[{\"label\": [\"1\"], \"source\": [\"REGISTRO S.E.N. COVID-19. INFORME 16 (18 marzo \\u2013 3 octub(...TRUNCATED)	{ "acronym": [], "definition": [] }	10	"NO-CC CODE"	"no"	"2023-06-06 23:35:03"	"Nefrologia. 2022 Jan 23 May-June; 42(3):359-360"	"oa_package/3d/7b/PMC7825904.tar.gz"
"PMC7856850"	"33551675"	["<title>Introduction</title>","<p id=\"Par42\">The coronavirus (COVID-19) has unfolded very swiftly(...TRUNCATED)	["<title>Methodology</title>","<p id=\"Par53\">A three-stage methodology has been used in the curren(...TRUNCATED)	[]	[]	["<title>Conclusion, limitations and future scope</title>","<title>Conclusion</title>","<p id=\"Par8(...TRUNCATED)	["<p>Communicated by V. E. Balas.</p>","<p id=\"Par1\">In work-from-home (WFH) situation due to coro(...TRUNCATED)	["<title>Relevant literature</title>","<p id=\"Par45\">This section is classified into two parts: (1(...TRUNCATED)	["<title>Appendix: The ratings for seven alternatives by four decision-makers</title>","<p id=\"Par9(...TRUNCATED)	["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Different categories of MSD risk factors (primar(...TRUNCATED)	["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Rating scale used for BWM and VIKOR tech(...TRUNCATED)	["<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t(...TRUNCATED)	[]	[]	[]	[]	[]	["<fn-group><fn><p>This article has been retracted. Please see the retraction notice for more detail(...TRUNCATED)	["<graphic xlink:href=\"500_2021_5592_Fig1_HTML\" id=\"MO1\"/>","<graphic xlink:href=\"500_2021_5592(...TRUNCATED)	[]	"[{\"surname\": [\"Ahmadi\", \"Zakerian\", \"Salmanzadeh\"], \"given-names\": [\"M\", \"SA\", \"H\"](...TRUNCATED)	{"acronym":["Age","B","BWM","BFO","COVID-19","DW","FE","GE","HHD","IF","JSA","JS","MCDM","MSDs","OB"(...TRUNCATED)	47	"NO-CC CODE"	"no"	"2023-06-06 23:35:14"	"Soft comput. 2023 Feb 3; 27(6):3283-3293"	"oa_package/74/7d/PMC7856850.tar.gz"
"PMC7906623"	"33636145"	["<title>Introduction</title>","<p id=\"para10\">Patients admitted to hospital with COVID-19 show va(...TRUNCATED)	["<title>Methods</title>","<title>Databases</title>","<p id=\"para110\">In this study, we used data (...TRUNCATED)	["<title>Results</title>","<p id=\"para220\">The features of the patients in the cohorts used for th(...TRUNCATED)	["<title>Discussion</title>","<p id=\"para310\">We identified three phenotypes based on demographics(...TRUNCATED)	[]	["<p id=\"cenpara10\">Members of the REIPI-SEIMC COVID-19 group and COVID@HULP group are listed in a(...TRUNCATED)	["<title>Data sharing</title>","<p>Data collected for the study, including deidentified participant (...TRUNCATED)	["<title>Supplementary Material</title>","<p id=\"para460\">\n\n</p>","<title>Acknowledgments</title(...TRUNCATED)	["<fig id=\"fig1\"><label>Figure 1</label><caption><p>Chord diagram of the distribution of groups of(...TRUNCATED)	["<table-wrap position=\"float\" id=\"tbl1\"><label>Table 1</label><caption><p>Bivariate analysis of(...TRUNCATED)	[]	["<boxed-text id=\"cetextbox10\"><caption><title>Research in context</title></caption><p id=\"para40(...TRUNCATED)	[]	[]	[]	["<supplementary-material content-type=\"local-data\" id=\"ecomp10\"><caption><title>Supplementary a(...TRUNCATED)	["<table-wrap-foot><fn><p>OR=odds ratio.</p></fn></table-wrap-foot>","<table-wrap-foot><fn><p>The va(...TRUNCATED)	["<graphic xlink:href=\"gr1_lrg\"/>","<graphic xlink:href=\"gr2_lrg\"/>","<graphic xlink:href=\"gr3_(...TRUNCATED)	[ "<media xlink:href=\"mmc1.pdf\"/>" ]	"[{\"label\": [\"3\"], \"surname\": [\"Docherty\", \"Harrison\", \"Green\"], \"given-names\": [\"AB\(...TRUNCATED)	{ "acronym": [], "definition": [] }	15	"NO-CC CODE"	"no"	"2023-06-06 23:35:22"	"Lancet Infect Dis. 2021 Jun 23; 21(6):783-792"	"oa_package/8e/1b/PMC7906623.tar.gz"
"PMC7929434"	"33567449"	[]	[]	[]	[]	[]	[]	["<p id=\"p0005\">See corresponding article on <ext-link ext-link-type=\"uri\" xlink:href=\"https://(...TRUNCATED)	[ "<p id=\"p005a\">The author reports no conflicts of interest.</p>" ]	[]	[]	[]	[]	[]	[]	[]	[]	["<fn-group><fn id=\"d36e223\"><p id=\"np0005\">The author is supported by the Wellcome Trust, Our P(...TRUNCATED)	[]	[]	"[{\"label\": [\"1\"], \"mixed-citation\": [\"World Health Organization. Impact of COVID-19 on peopl(...TRUNCATED)	{ "acronym": [], "definition": [] }	9	"NO-CC CODE"	"no"	"2023-06-06 23:35:02"	"Am J Clin Nutr. 2021 Apr 31; 113(4):763-764"	"oa_package/87/2e/PMC7929434.tar.gz"
"PMC8079233"	"33935377"	["<title>Introduction</title>","<p id=\"Par2\">With the advent of the COVID-19 pandemic, a massive a(...TRUNCATED)	["<title>Methodology</title>","<p id=\"Par10\">The process of medical image-based COVID-19 detection(...TRUNCATED)	["<title>Results and discussion</title>","<p id=\"Par24\">In this section, we present the multi-clas(...TRUNCATED)	["<title>Results and discussion</title>","<p id=\"Par24\">In this section, we present the multi-clas(...TRUNCATED)	["<title>Conclusion and future scope</title>","<p id=\"Par31\">The COVID-19 pandemic has clearly put(...TRUNCATED)	["<p id=\"Par1\">The demand for automatic detection of Novel Coronavirus or COVID-19 is increasing a(...TRUNCATED)	["<title>Literature of review</title>","<p id=\"Par7\">Recent developments in deep learning have bee(...TRUNCATED)	["<title>Acknowledgements</title>","<p>No Applicable.</p>","<title>Funding</title>","<p>This work wa(...TRUNCATED)	["<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Process of computer vision-enabled classificatio(...TRUNCATED)	["<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Comparative analysis of the study</p></c(...TRUNCATED)	["<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t(...TRUNCATED)	[]	[]	[]	[]	[]	["<fn-group><fn><p><bold>Publisher's Note</bold></p><p>Springer Nature remains neutral with regard t(...TRUNCATED)	["<graphic xlink:href=\"530_2021_794_Fig1_HTML\" id=\"MO1\"/>","<inline-graphic xlink:href=\"530_202(...TRUNCATED)	[]	"[{\"label\": [\"1.\"], \"mixed-citation\": [\"Cengil, E., \\u00c7inar, A.: A deep learning based ap(...TRUNCATED)	{ "acronym": [], "definition": [] }	56	"NO-CC CODE"	"no"	"2023-06-06 23:35:29"	"Multimed Syst. 2023 Apr 28; 29(3):1729-1738"	"oa_package/63/8a/PMC8079233.tar.gz"

Dataset Card for PMC Open Access XML

Dataset Summary

The XML Open Access includes more than 3.4 million journal articles and preprints that are made available under license terms that allow reuse. Not all articles in PMC are available for text mining and other reuse, many have copyright protection, however articles in the PMC Open Access Subset are made available under Creative Commons or similar licenses that generally allow more liberal redistribution and reuse than a traditional copyrighted work. The PMC Open Access Subset is one part of the PMC Article Datasets

This version takes XML version as source, benefiting from the structured text to split the articles in parts, naming the introduction, methods, results, discussion and conclusion, and reference with keywords in the text to external or internal resources (articles, figures, tables, formulas, boxed-text, quotes, code, footnotes, chemicals, graphics, medias).

The dataset was initially created with relation-extraction tasks in mind, between the references in text and the content of the references (e.g. for PMID, by joining the refered article abstract from the pubmed dataset), but aims in a larger extent to provide a corpus of pre-annotated text for other tasks (e.g. figure caption to graphic, glossary definition detection, summarization).

Supported Tasks and Leaderboards

[Needs More Information]

Languages

[Needs More Information]

Dataset Structure

Data Fields

"accession_id": The PMC ID of the article
"pmid": The PubMed ID of the article
"introduction": List of <title> and <p> elements in <body>, sharing their root with a <title> containing "introduction" or "background".
"methods": Same as introduction with "method" keyword.
"results": Same as introduction with "result" keyword.
"discussion": Same as introduction with "discussion" keyword.
"conclusion": Same as introduction with "conclusion" keyword.
"front": List of <title> and <p> elements in <front> after everything else has been searched.
"body": List of <title> and <p> elements in <body> after everything else has been searched.
"back": List of <title> and <p> elements in <back> after everything else has been searched.
"figure": List of <fig> elements of the article.
"table": List of <table-wrap> and <array> elements of the article.
"formula": List of <disp-formula> and <inline-formula> elements of the article.
"box": List of <boxed-text> elements of the article.
"code": List of <code> elements of the article.
"quote": List of <disp-quote> and <speech> elements of the article.
"chemical": List of <chem-struct-wrap> elements of the article.
"supplementary": List of <supplementary-material> and <inline-supplementary-material> elements of the article.
"footnote": List of <fn-group> and <table-wrap-foot> elements of the article.
"graphic": List of <graphic> and <inline-graphic> elements of the article.
"media": List of <media> and <inline-media> elements of the article.
"glossary": Glossary if found in the XML
"unknown_references": JSON of a dictionnary of each "tag":"text" for the reference that did not indicate a PMID
"n_references": Total number of references and unknown references
"license": The licence of the article
"retracted": If the article was retracted or not
"last_updated": Last update of the article
"citation": Citation of the article
"package_file": path to the folder containing the graphics and media files of the article (to append to the base URL: ftp.ncbi.nlm.nih.gov/pub/pmc/)

In text, the references are in the form ##KEYWORD##IDX_REF##OLD_TEXT##, with keywords (REF, UREF, FIG, TAB, FORMU, BOX, CODE, QUOTE, CHEM, SUPPL, FOOTN, GRAPH, MEDIA) referencing respectively to "pubmed articles" (external), "unknown_references", "figure", "table", "formula", "box", "code", "quote", "chem", "supplementary", "footnote", "graphic" and "media".

Data Splits

[Needs More Information]

Dataset Creation

Curation Rationale

Internal references (figures, tables, ...) were found using specific tags. Deciding on those tags was done by testing and by looking in the documentation for the different kind of possible usage. Then, to split the article into introduction, methods, results, discussion and conclusion, specific keywords in titles were used. Because there are no rules in this xml to tag those sections, finding the keyword seemed like the most reliable approach to do so. A drawback is that many section do not have those keywords in the titles but could be assimilated to those. However, the huge diversity in the titles makes it harder to label such sections. This could be the work of further versions of this dataset.

Source Data

Initial Data Collection and Normalization

Data was obtained from:

ftp.ncbi.nlm.nih.gov/pub/pmc/oa_bulk/oa_noncomm/xml/
ftp.ncbi.nlm.nih.gov/pub/pmc/oa_bulk/oa_comm/xml/
ftp.ncbi.nlm.nih.gov/pub/pmc/oa_bulk/oa_other/xml/

Additional content for individual articles (graphics, media) can be obtained from:

ftp.ncbi.nlm.nih.gov/pub/pmc + "package_file"

Who are the source language producers?

[Needs More Information]

Annotations

Annotation process

[Needs More Information]

Who are the annotators?

[Needs More Information]

Personal and Sensitive Information

[Needs More Information]

Considerations for Using the Data

Social Impact of Dataset

[Needs More Information]

Discussion of Biases

The articles XML are similar accross collections. This means that if a certain collection handles the structure in unusual ways, the whole collection might not be as well annotated than others. This concerns all the sections (intro, methods, ...), the external references (pmids) and the internal references (tables, figures, ...). To illustrate that, references are sometime given as a range (e.g. 10-15). In that case, only reference 10 and 15 are linked. This could potentially be handled in a future version.

Other Known Limitations

[Needs More Information]

Preprocessing recommendations

Filter out empty contents.
Remove unwanted references from the text, and replace either by the "references_text" or by the reference content itself.
Unescape HTML special characters: import html; html.unescape(my_text)
Remove superfluous line break in text.
Remove XML tags (<italic>, <sup>, <sub>, ...), replace by special tokens?
Join the items of the contents' lists.

Additional Information

Dataset Curators

[Needs More Information]

Licensing Information

https://www.ncbi.nlm.nih.gov/pmc/about/copyright/

Within the PMC Open Access Subset, there are three groupings:

Commercial Use Allowed - CC0, CC BY, CC BY-SA, CC BY-ND licenses Non-Commercial Use Only - CC BY-NC, CC BY-NC-SA, CC BY-NC-ND licenses; and Other - no machine-readable Creative Commons license, no license, or a custom license.

Citation Information

[Needs More Information]

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Homepage:

ncbi.nlm.nih.gov