Traditionally, lysozyme c has been considered to be part of a small family that includes genes for two other proteins, lactalbumin, which is found only in mammals, and calcium-binding lysozyme, which is found in only a few species of birds and mammals. More recently, additional testes-expressed members of this family have been identified in human and mouse, suggesting that the mammalian lysozyme gene family is larger than previously known.Lysozyme Here we characterize the extent and diversity of the lysozyme gene family in the genomes of phylogenetically diverse mammals, and show that this family contains at least eight different genes that likely duplicated prior to the diversification of extant mammals. These duplicated genes have largely been maintained, both in intron-exon structure and in genomic context, throughout mammalian evolution.c and lactalbumin proteins have acquired very different functions during evolution, it is likely that many of the other members of the lysozyme-like family will also have diverse and unexpected biological properties.The mammalian lysozyme gene family is much larger than previously appreciated and consists of at least eight distinct genes scattered around the genome. Since the lysozyme  Calcium-binding lysozymes appear to be relatively rare; they have been found in the milk of only a few mammalian species , as well as in the eggs  and stomachs  of some bird species [The vertebrate lysozyme gene family has traditionally been considered to be composed of three genes: lysozyme lysozyme -4. Lysoz species ,2,5. Lys species ,5,6. Lac species ,2,4,7. L species . Lactalb species ,9. Indeec, lactalbumin, and calcium-binding lysozyme sequences had suggested that the earliest divergences within this gene family occurred between lysozyme c and the ancestor of the genes for lactalbumin and calcium-binding lysozyme, and that this initial gene duplication may have preceded the divergence of the lineages leading to fish and mammals [Previous phylogenetic analyses of lysozyme  mammals ,11. The  mammals ,12 sugge mammals ,11. ThusEnsembl [LYZL (lysozyme-like): LYZL2, LYZL4, LYZL6 and LYZL3  are expressed predominantly in the testes, it has been suggested that they might have a role in reproduction [Lyzl4 and Spaca3 in mice [Recently, cDNAs for several additional lysozyme-like sequences have been identified from human testis cDNA libraries -15. ThesEnsembl  as LYZL as SPRSA  and SLLPA [SLLP1 ). The prA [SLLP1 ,15. Sincoduction -15,17. S in mice ,19.LYZL genes in the human genome suggests that the mammalian lysozyme-like gene family is larger than previously appreciated, and raises the possibility that the lysozyme-like proteins encoded by these genes may have novel biological functions. Here we have used extensive similarity searches of the human and other vertebrate genomes. We thereby identified three additional intact lysozyme-like genes in the human genome; these have been annotated in the databases, but not reported in the literature. We have also identified multiple lysozyme-like genes in the genomes of diverse vertebrates. Using a combination of phylogenetic and genomic neighborhood (or synteny) analyses, wherein the relationships of the genes that flank the lysozyme-like genes in diverse species were examined, we demonstrate that orthologs of the human lysozyme-like genes are found in the genomes of diverse mammalian species. Our analyses suggest that there were at least six, and perhaps as many as nine, diverse types (or subfamilies) of lysozyme-like genes in the genome of the common ancestor of all extant mammals, and that these diverse genes have been maintained on most mammalian lineages. This suggests that their protein products probably have essential biological functions that are yet to be identified.The identification of these BLAST [c, and thereby identified a total of nine annotated genes (Table c (LYZ) [LALBA, Synonym: LYZL7) [LYZL2, LYZL4, LYZL6 [SPACA3  [BLAST searches -- the LYZL1, SPACA5 (Synonym: LYZL5), and SPACA5B genes -- had been annotated as lysozyme-like in Ensembl [ψLYSC1). These ten lysozyme-like sequences are distributed over five chromosomes in humans, with two genes each on chromosomes 10, 17, and X, three genes on chromosome 12, and one gene on chromosome 3  , lactalb: LYZL7) , LYZL2, 4, LYZL6 , and SPA, SLLP1) ,14. The  Ensembl , but havLYZL1 and LYZL2 genes are both located on human chromosome 10, but are separated by about 1 Mb; moreover, these genes are embedded within 60 kb long repeated sequences that are greater than 95% identical (not shown). This suggests that the LYZL1 and LYZL2 gene duplicates were generated as part of a recent segmental duplication on chromosome 10. Likewise, the SPACA5 and SPACA5B genes are both on the X chromosome, separated by about 120 kb, and are within long (~100 kb) repeated DNA sequences that have high sequence identity (not shown). Thus, the LYZL1/LYZL2 and SPACA5/SPACA5B gene pairs both appear to have originated from relatively recent and large genomic segmental duplications, a common form of gene duplication in mammals [Most of the predicted human proteins Figure  show bet mammals . The hig mammals , as discEnsembl and Pre!Ensembl databases  [Xenopus tropicalis, the lone representatives of reptiles and amphibians with sequenced genomes, had 8 and 16 lysozyme-like genes, respectively.To determine whether an expanded lysozyme-like gene family is a general feature of mammalian (and other vertebrate) genomes, we conducted intensive homology searches for lysozyme-like genes in all vertebrate genomes available in the ex.html) ,25. ThesBLAST searches identified potential orthologs in most mammalian genomes . Initial assignments of orthology of these mammalian genes were based upon sequence similarity, but were subsequently confirmed by performing genomic neighborhood and phylogenetic analyses (see below). Our combined findings about gene number and orthologous relationships of the mammalian lysozyme-like gene family members are outlined in Figure c gene. Thus, none of the non-mammalian vertebrate lysozyme-like genes could be definitively classified as orthologs of any of the lysozyme-like mammalian genes, other than Lyz itself.Importantly, the s Figure  of all oLyzl1/2-like gene found in the treeshrew (from Genescaffold_6044), which had a nearly full-length coding sequence that contained stop codons and frameshifts, but no introns; thus, this gene appears to be a processed pseudogene. Taken together, these observations suggest that essentially all of the vertebrate lysozyme-like genes have been generated by duplications of genomic DNA, rather than by reverse-transcription and insertion into genome.Many of the genes that were identified in our searches were only partial sequences, most likely due to the incomplete nature of the genomes in question. However, all but one of these genes were consistent with a structure similar to that of the mammalian lysozyme and lactalbumin genes -- that is, their coding regions appeared to be composed of four exons having similar intron-exon structures ,6. The lThe presence of multiple lysozyme-like genes in all mammalian genomes, as well as in the genomes of several other vertebrate species, raises the possibility that the lysozyme-like gene family may have amplified early in vertebrate evolution. To examine this issue, and to further establish the orthology-paralogy relationships of lysozyme-like genes, we conducted a series of phylogenetic analyses . However, the genes from the other vertebrates did not consistently group with any of these mammalian orthologs, with the exception of some of the lysozyme c sequences.Importantly, as illustrated by the Bayesian analysis shown in Figure In addition, it is clear that most, if not all, of the eight mammalian lysozyme-like genes duplicated and diverged from each other prior to the divergence of the earliest mammalian lineages. This is clear for at least two reasons. First, both the platypus and the eutherian genomes contain copies of most of these gene duplicates; therefore, these genes must have diverged earlier than did the species lineages. Second, some of the non-mammalian vertebrate genes appear to have phylogenetic affinity for some of the mammalian gene lineages, although few have much statistical support. This is particularly evident for many of the lizard genes which, as illustrated in Figure Lalba clade was the earliest diverging lineage and that most of the Lyzl and Spaca genes  were most closely related to each other, these relationships were not consistently found , which would agree with the hypothesis generated by the phylogenetic analyses , elephant (8 genes), armadillo (5 genes), sloth (6 genes), opossum (4 genes), and wallaby (8 genes)  and marsupials .The number of and rats -29, rabband rats ,31, and and rats -36. In armadillo  genes, slineages ,29,32-37rmadillo  genes, sLyzl1 and Lyzl2 genes in the human genome appear to have been generated recently via a genomic segmental duplication. Indeed, with the exception of those primate species that are close relatives of human, most other mammals have only a single gene sequence similar to Lyzl1 or Lyzl2  showed little similarity to any of the other lysozyme-like genes (or proteins) in our BLAST searches. When the platypus Lyzl8 gene was used as a query to search mammalian genomes, only one genomic sequence -- from the sloth  is located adjacent to the Lalba gene in the dog  and avian calcium-binding lysozyme genes are not closely related in our phylogenies, a finding in agreement with some earlier analyses [Lysc1-like genomic sequences all were found on short genomic contigs . In the little brown bat, the Lalba gene is embedded in a large genomic fragment that was not annotated to include c12orf41 . A more careful examination of the little brown bat genomic contig revealed that most of the genomic region is composed of unsequenced gaps.An intriguing observation from our genomic neighborhood analysis of was that the mammalian calcium-binding lysozyme gene  to ~250 kb (cow and opossum). For all of these genomes, except the opossum (see below), the only genes (or pseudogenes) annotated as existing between Lalba and c12orf41 were olfactory receptor-like genes, which are not very useful for identifying orthologous and conserved genomic neighborhoods due to their abundance. In the opossum, in addition to the olfactory receptor-like genes, three additional genes were annotated between Lalba and c12orf41: the genes Mip, Spryd4, and Gls2. Unfortunately, the wallaby genome is poorly assembled near the Lalba and c12orf41 genes, and thus the neighboring genes could not be identified. Although the Mip, Spryd4, and Gls2 genes reside on the same chromosome as Lalba and c12orf41 in many mammals , they are found greater than 8 Mb away; furthermore, in some species  they are on different chromosomes. These observations suggest that the organization of the Lalba, c12orf41, Mip, Spryd4, and Gls2 genes, and potentially a Lysc1 gene, has changed between the opossum  and placental mammals.For the 17 genomes that did have linked Lalba and c12orf41 genes for the 17 genomes where these two genes were linked was aligned with MultiPipMaker [Lysc1 gene were not observed in 9 of the genomic sequences -- those from marmoset, mouse, rat, guinea pig, rabbit, treeshrew, cow, little brown bat, and opossum . Thus, there are only 6 species with nearly complete genomic sequences spanning the Lalba and c12orf41 genes for which we have good evidence for the actual absence of a Lysc1 gene or pseudogene -- mouse, rat, guinea pig, rabbit, treeshrew, and opossum. Pairwise sequence alignments between the mouse, rat, or guinea pig genomic sequences with those from dog or horse (or primates) using PipMaker [Lysc1 gene, is missing from these rodent genomes (results not shown). This suggests that this genomic region, including the Lysc1 gene, was deleted either early on the rodent lineage or in the common ancestor of rodents and close relatives , but after the divergence of the rodent lineage from the primate lineage  of the horse and dog Lysc1 genes  and the elephant -- do possess sequences between the Lysc1 genes that predict potentially functional calcium-binding lysozymes were found in only a few species , whereas pseudogenes were found on several lineages . Phylogenetic and genomic analyses suggested that the pair of Lysc1 genes found in the shrew resulted from a tandem gene duplication event on the lineage leading to this species  and treeshrews. Taken together, the above observations suggest that the Lysc1 gene likely arose from a duplication of the lactalbumin gene early in mammalian evolution, and was inactivated several times independently, as summarized in Figure Intact Lyz, Lalba, Lysc1, Lyzl1/2, Lyzl4, Lyzl6, Spaca3, and Spaca5) in most mammalian species. These observations suggest that this family experienced several duplication events prior to the origin of mammals. Several other gene families also experienced such amplifications near the origin of mammals, such as those generating the gene families for keratin-associated proteins [e.g., hair (keratin-associated proteins), skin , and diet (bitter taste receptors) [Spaca3 and Lyzl4 genes have recently been shown to be involved in fertilization in mice [Here we have shown that the mammalian lysozyme gene family is much larger than previously anticipated, and is composed of at least eight distantly-related members  -47. Intr in mice ,19. MuchLYZL1/LYZL2 and SPACA5/SPACA5B in the human genome). The lysozyme-like gene family also shares with the keratin associated protein [Lyz and Lyzl1/2 subfamilies. The Lyz subfamily showed the greatest tendency to tandemly duplicate and evolve in concert, whereas the other lysozyme-like genes typically showed conservation in copy number. Tandem duplication or amplification of the Lyz gene has previously been observed in certain mammals, including the ruminants and rodents, where lysozyme appears to function as a digestive enzyme in the gut [Lyz genes -- e.g., elephant and wallaby -- are also herbivorous species, and thus may use lysozyme as a digestive enzyme upon gut bacteria. The need for higher levels of digestive lysozymes in the guts of fermenting herbivores could have driven the fixation of the tandem duplications in these lineages. Gene conversion between the tandem duplicates might then provide a mechanism whereby favorable mutations in one gene copy could spread to the other copies in the cluster [Similar to the keratin-associated protein  and bitt protein , kallikr protein , and bit protein  gene fam protein ,23, whic the gut -34. It i cluster ,36, as w cluster  in well-Ensembl [Pre!Ensembl [tBLASTn algorithm [c and lactalbumin sequences. Subsequent tBLASTn searches used all of the identified putative lysozyme-like protein sequences. Similar searches were conducted using additional databases  available at the NCBI website [Ensembl database were searched using these sequences using tBLASTn for similar sequences. All sequences that had E-scores below 0.01 were examined. Sequences identified by BLAST searches were used in reciprocal BLASTx searches of the human, mouse and dog proteomes to ensure that their best matches were lysozyme-like sequences. Sequences that were unannotated to encode lysozyme-like sequences  algorithm. MEGA4 [PAUP [Phylogenetic trees of the sequences were generated by a variety of methods including es 3.1.2 ,61, Phylyes 3.2f , and Phyf [PhyML , MEGA4.0EGA4.0.2 , and PAU 4beta10 . Bayesiat server -68. MrBay Tracer  to verifby PhyML  on the Pebserver  using pam. MEGA4  was usedA4 [PAUP , with 10MAFFT [Clustal [Guidance scores [i.e., trimmed alignments or protein sequences) or simpler models of sequence evolution  tended to yield weaker support for the earlier diverging lineages, but none of our analyses were in significant conflict with the key inferences of the phylogeny presented in Figure With respect to orthology-paralogy issues, the choice of outgroup, the alignment method , and the scores ,59) aligLalba sequences were arbitrarily used to root the trees. When only mammalian lysozyme-like gene sequences were used for the phylogenetic analyses, then stronger support for each of the orthologous groups was found with all of the phylogenetic methods used including Bayesian inference, maximum likelihood, distance, and parsimony (see Additional file Lysc1 and Spaca5 where the platypus does not have these sequences. For Lysc1, the sloth sequence was used to root the tree, whereas for Spaca5 the elephant and tenrec sequences provided the root.For phylogenies that contained just mammalian lysozyme-like sequences, DMI and CBS together designed the research and outlined the manuscript. DMI, JMB, and CBS obtained and analyzed the data. DMI drafted the manuscript. All of the authors have read, edited, and approved the final manuscript.Supplementary Table 1. This file is in PDF format. Location of lysozyme genes in vertebrate genomes.Click here for fileSupplementary Figure 1. This file is in PDF format. Phylogeny of vertebrate lysozyme-like sequences generated by PhyloBayes.Click here for fileSupplementary Figure 2. This file is in PDF format. Phylogeny of vertebrate lysozyme-like sequences generated by PhyML.Click here for fileSupplementary Figure 3. This file is in PDF format. Phylogeny of only mammalian lysozyme-like sequences generated by MrBayes with support for the orthologous genes by different phylogenetic methods.Click here for fileSupplementary Figure 4. This file is in PDF format. Conservation of genomic organization near Lyzl1/2 genes.Click here for fileSupplementary Figure 5. This file is in PDF format. Conservation of genomic organization near Lyzl4 genes.Click here for fileSupplementary Figure 6. This file is in PDF format. Phylogeny of Lyzl4 genes.Click here for fileSupplementary Figure 7. This file is in PDF format. Conservation of genomic organization near Lyzl6 genes.Click here for fileSupplementary Figure 8. This file is in PDF format. Phylogeny of Lyzl6 genes.Click here for fileSupplementary Figure 9. This file is in PDF format. Conservation of genomic organization near Spaca3 genes.Click here for fileSupplementary Figure 10. This file is in PDF format. Phylogeny of Spaca3 genes.Click here for fileSupplementary Figure 11. This file is in PDF format. Conservation of genomic organization near Spaca5 genes.Click here for fileSupplementary Figure 12. This file is in PDF format. Phylogeny of Spaca5 genes.Click here for fileSupplementary Figure 13. This file is in PDF format. Phylogeny of Lalba genes.Click here for fileSupplementary Figure 14. This file is in PDF format. Conservation of genomic sequences between Lalba and c12orf41 genes.Click here for fileSupplementary Figure 15. This file is in PDF format. DNA sequences of Lysc1 genes.Click here for fileSupplementary Figure 16. This file is in PDF format. Phylogeny of Lysc1 genes.Click here for fileSupplementary Table 2. This file is in PDF format. Outgroup lysozyme sequences used for phylogenetic analysis.Click here for fileSupplementary Figure 17. This file is in Word format. FASTA formatted MAFFT alignment of lysozyme DNA sequencesClick here for file


Recently, workers' mental health has become important focus in the field of occupational health management. Depression is a psychiatric illness with a high prevalence. The association between job stress and depressive symptoms has been demonstrated in many studies. Recently, studies about the association between sleep quality and depressive symptoms have been reported, but there has been no large-scaled study in Korean female workers. Therefore, this study was designed to investigate the relationship between job stress and sleep quality, and depressive symptoms in female workers.From Mar 2011 to Aug 2011, 4,833 female workers in the manufacturing, finance, and service fields at 16 workplaces in Yeungnam province participated in this study, conducted in combination with a worksite-based health checkup initiated by the National Health Insurance Service (NHIS). In this study, a questionnaire survey was carried out using the Korean Occupational Stress Scale-Short Form(KOSS-SF), Pittsburgh Sleep Quality Index(PSQI) and Center for Epidemiological Studies-Depression Scale(CES-D). The collected data was entered in the system and analyzed using the PASW (version 18.0) program. A correlation analysis, cross analysis, multivariate logistic regression analysis, and hierarchical multiple regression analysis were conducted.Among the 4,883 subjects, 978 subjects (20.0%) were in the depression group. Job stress and sleep quality were strongly associated with depressive symptoms. Hierarchical multiple regression analysis revealed that job stress displayed explanatory powers of 15.6% on depression while sleep quality displayed explanatory powers of 16.2%, showing that job stress and sleep quality had a closer relationship with depressive symptoms, compared to the other factors. The multivariate logistic regression analysis yielded odds ratios between the 7 subscales of job stress and depressive symptoms in the range of 1.30-2.72 and the odds ratio for the lack of reward was the highest. In the partial correlation analysis between each of the 7 subscales of sleep quality (PSQI) and depressive symptoms, the correlation coefficient of subjective sleep quality and daytime dysfunction were 0.352 and 0.362, respectively.This study showed that the depressive symptoms of female workers are closely related to their job stress and sleep quality. In particular, the lack of reward and subjective sleep factors are the greatest contributors to depression. In the future, a large-scale study should be performed to augment the current study and to reflect all age groups in a balanced manner. The findings on job stress, sleep, and depression can be utilized as source data to establish standards for mental health management of the ever increasing numbers of female members of the workplace. Since the currency crisis in 1997, Korea has experienced dramatic social and economic changes. Large and small businesses went bankrupt or underwent layoffs, staff reduction and restructuring. With the gradual deterioration of the work environment, many workers have been exposed to various kinds of job stress, including job insecurity . Under tAccording to 2010 data of the Korean National Statistical Office , there wThe relationship between job stress, among many factors that may impact depression, and depressive symptoms has already been demonstrated in many studies -15. It hAccording to previous Korean and international studies ,20, womeAlthough female workers are relatively more susceptible to depression and job stress factors, previous Korean studies related to depression have been conducted mostly in male workers with limited consideration of females, focusing on major companies and small and medium-sized companies ,13,21 orFrom Mar 2011 to Aug 2011, self-administered questionnaires were collected from 4,966 female workers in manufacturing, finance, and service fields at 16 0077orkplaces in Yeungnam province while worksite-based health checkups were conducted by the National Health Insurance Service (NHIS). Among them, 4,833 female workers were included in the final analysis, excluding 83 with incomplete responses. This study was reviewed by the institutional review board (IRB) before implementation and all questionnaires were obtained under consent of the participants(IRB No. 2012-SCMC-059-00).2 indicating low weight; BMI ≥18.5 and ≤ 24.9 kg/m2 indicating normal weight; and BMI ≥25 kg/m2 indicating obesity [Socio-demographic characteristics and health behaviors such as age, educational level, marital status, body mass index (BMI), smoking habit, alcohol drinking, regular exercise, and job tenure (years) were reviewed. The subjects' age ranged from 17 to 59 years. They were categorized by age , educational level  and marital status . For the obesity level, WHO criteria pertaining to obesity for Asia-Pacific region was employed: BMI < 18.5 kg/mThe Korean Occupational Stress Scale-Short Form(KOSS-SF)  was usedSleep quality was assessed using the Korean language version of the Pittsburgh Sleep Quality Index(PSQI) , originaThe depression level was assessed using a validated Korean translation by Cho et al.  of the sSocio-demographic characteristics, smoking habit, lifestyle, job characteristics, job stress and sleep quality with subscales were summarized using descriptive statistics with frequencies and percentages. The t-test and ANOVA test were employed to analyze the level of depressive symptoms using each factor as an independent variable. To determine the prevalence, the chi-squared test was used based on the total CES-D score of 21. A multivariate logistic regression analysis was conducted to determine the impact of job stress, the 7 subscales, and sleep quality on depressive symptoms and the significance.To investigate the stepwise influence of independent variables on depressive symptoms, hierarchical multiple regression was performed using a variable with significant results in univariate analysis as an independent variable and the depression level score as a dependent variable. All Variation Inflation Factor(VIF) values to confirm multi-collinearity between variables were below 2 points, not showing any specific issues regarding multi-collinearity. When a significant independent variable was not a continuous variable, it was converted to dummy variable for use. Logistic regression analysis was conducted among the 7 subscales of job stress and depressive symptoms, while multiple regression analysis was conducted among the 7 subscales of sleep quality and depressive symptoms. The collected data was entered in a system and statistically analyzed using the PASW (version 18.0) program at a statistical significance of 0.05.Based on the 21-point threshold of the total CES-D score, 978 subjects  obtained 21 or more points   were adjusted, the odds ratio of the high stress group compared to the low stress group in each job stress subscale for depression was 2.72(95% CI=2.32-3.19) for lack of reward, 2.38(95% CI=2.03-2.78) for occupational climate, 2.32(95% CI=2.00-2.70) for interpersonal conflict, 2.24(95% CI=1.93-2.60) for job insecurity, 2.10(95% CI=1.81-2.45) for job demand, 1.97(95% CI=1.70-2.29) for organizational system and 1.30(95% CI=1.12-1.51) for insufficient job control. Lack of reward showed the highest odds ratio, while insufficient job control had the lowest odds ratio among the subscales.In multiple regression analysis among the 7 sleep quality (PSQI) subscales and depressive symptoms (CES-D), regression coefficients of daytime dysfunction and subjective sleep quality were 3.05 and 2.32, respectively, showing higher values than the other 5 sleep quality subscales  was significantly higher in the high stress group than the low stress group. As a result of multivariate logistic regression analysis, the high stress group had a higher odds ratio for depression. This result is consistent with previous studies reporting that job stress is a cause of increased risk of mental health including depression ,37,38.The depression rate according to sleep quality (PSQI) was significantly higher in the poor sleep quality group than the good sleep quality group. As a result of multivariate logistic regression analysis conducted to determine the association between sleep quality and depressive symptoms, the poor sleep quality group showed a higher odds ratio than the good sleep quality group. This result was consistent with a study involving employees of small-scale manufacturing companies  that hasAs in studies suggesting that people with depression have a higher chance of insomnia ,40, sleeIn the hierarchical multiple regression analysis using 4 different models, the overall explanatory power of the variables associated with depressive symptoms was 32.9%. Among the factors that may affect depressive symptoms, the general characteristics employed in Model I showed a limited explanatory power of only 6.9%. Model II and Model III showed that both job stress and sleep quality have a substantial influence on depressive symptoms, while Model IV demonstrated a dramatic increase in the explanatory power of job stress and sleep quality together to 26.1%. This suggested that these two factors have a considerable synergic effect on depressive symptoms.This study in female workers showed that job stress is a significant contributor to depressive symptoms. With the finding, a logistic regression analysis was conducted to confirm which job stress items of the 7 subscales can affect the depression level. It was found that all 7 subscales were strongly associated with depressive symptoms, among which the lack of reward showed the closest relationship, while insufficient job control showed the least. This was not consistent with a previous study of Kim et al . that reIt has been found that among employees of small and medium-sized companies and office workers, the occupational climate was a significant factor associated with depressive symptoms ,48. SimiMultiple regression analysis was conducted to confirm the association between 7 subscales of sleep quality (PSQI) and depressive symptoms (CES-D) showed higher regression coefficients in subjective sleep quality and daytime dysfunction, compared to the other 5 subscales. This result can be interpreted to mean that rather than objective factors like sleep duration and use of sleeping medication, subjective factors assessed by the subjects themselves according to their own interpretation such as subjective sleep quality and daytime dysfunction indicating limited social activities and difficulty in work concentration due to sleepiness are closely related with depressive symptoms . TherefoThe study had some limitations. First, given its cross-sectional study design, this study can determine the association among job stress, sleep quality, and depressive symptoms but may not be able to fully explain the causal relationship among them. Second, regarding sleep, the shift rotation factor was not reflected although it is closely related to sleep quality or sleep duration. In addition, because the proportion of young female workers in their 20s was remarkably higher than that of older female workers in their 30s and above, the results cannot be generalized as an assessment on the mental health of female workers across all age groups.In spite of these limitations, this study is meaningful in that it has shown that the depressive symptoms of female workers are closely related to job stress and sleep quality, given the fact that females are relatively vulnerable to depression compared to males and the current situation in which more and more women are entering the workforce. A study with a large-scale sample should be performed in the future to supplement the study limitations and to identify occupational factors of the development of depression, reflecting all age groups. Based on this study, depression management standards for female workers should be established and a suicide prevention program should be developed and introduced. In addition, this study can be used as source data for mental health management in increasing female workforce across all fields.The authors declare that they have no competing interests.YWK conceived and designed this study. HSC and YWK and BGJ were involved in writing the manuscript. HSC and KHL performed the data collection. HOP and KSP performed the statistical analysis, the interpretation of data. YSK had critically revised the manuscript. All authors read and approved the final manuscript.


Human papillomavirus (HPV) is one of the most common sexually-transmitted pathogens. A number of studies in the literature have estimated the burden of HPV-related diseases by collecting data at primary care level, while a comprehensive assessment of the global burden of HPV-related diseases on hospital resources is still lacking.This was a retrospective cohort study based on hospital discharge data collected from 2000 to 2010 in the Veneto Region . All hospitalizations for diseases potentially associated with HPV were identified by searching the hospital discharge records, then the proportion of these hospitalizations relating to diseases attributable to the HPV infection was calculated.Overall, the annual hospitalization rate for HPV-related diseases was 21.3 per 100,000 individuals in the general population, 15.8 per 100,000 males, and 27.6 per 100,000 females. Hospitalizations were due mainly to genital warts, and peak among 15- to 44-year-olds in both genders. Taking both sexes together, the hospitalizations attributable to HPV dropped from 24.5/100,000 in 2000 to 17.5/100,000 in 2011, showing a significant decline during this period, with an average annual percent change (AAPC) of −1.9% . On the other hand, the hospitalization rate for genital warts tended to increase significantly , whilst there was a significantly declining trend in the hospitalization rate for anal cancer , genital cancer  and oropharyngeal cancer .Data derived from the hospital records indicate that HPV-related diseases are an important public health issue. Human papillomavirus (HPV) is the most common sexually-transmitted pathogen . In the Most infections are asymptomatic and transient, but certain HPV types are oncogenic. Numerous studies have identified many biochemical features that contribute to HPV-induced carcinogenesis, such as the ability of the viral proteins E6 and E7 to bind cellular factors, and biological properties like the capacity of HPV proteins to induce cell immortalization .Genital infection with oncogenic or high-risk types of HPV is a causal factor in cervical cancer; the HPV types mainly involved are 16 and 18, which are responsible for approximately 70% of cases of this cancer. HPV also has a well-known role in premalignant lesions of the cervix, vulva and vagina, and the International Agency for Research on Cancer (IARC) has recently supported (with “sufficient” or “limited” evidence) the carcinogenicity of HPV in the penis, anus, oral cavity, oropharynx, tonsils and larynx .HPV is responsible for several benign diseases as well, e.g. genital warts, that have a significant economic and psychological impact ,11. GeniA number of studies in the literature have estimated the burden of HPV-related diseases by collecting data at primary care level, while only one European study (conducted in Spain) used the hospitalization records to ascertain the burden of HPV related to anal and penile neoplasms . No compThe aim of the present study was to estimate the burden of hospitalization for HPV-related diseases, stratified by sex, in a population of almost five million citizens.To estimate the annual HPV-associated hospitalization rates, we analyzed the data in the hospital records from 2000 to 2011 in the Veneto Region  concerning all discharges from all public and accredited private hospitals, including day hospital services. HPV-associated hospitalization were identified by selecting all the hospital discharge records containing one of the following codes in the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM): genital warts; 078.11 “Condyloma acuminatum”; anal cancer; 154.2-154.8 “Malignant neoplasm of anus”; oropharyngeal cancer; 146.0-146.9 “Malignant neoplasm of oropharynx”; 171.0 “Malignant neoplasm of head, face, and neck”; genital cancer; 187.1-187.9 “Malignant neoplasm of penis”; 180.0-180.9 “Malignant neoplasm of cervix uteri”.Only admissions for which HPV-related diseases were indicated as the main diagnosis at the time of discharge, were drawn from the database. The proportion of hospitalizations for diseases potentially associated with HPV infection was calculated on the assumption that all discharges for cervical cancer and genital warts were HPV-related, while this was true of 50% and 88%, respectively, of cancers of the male genitalia (penis) and anus, and applied to 26% of oropharyngeal cancers –9]. The. The9]. Significant trends over the years considered were assessed as average annual percent changes (AAPC), a summary measure of the trend over a given fixed interval. It is computed as a weighted average of the annual percent change (APC) emerging from the join-point model, using weights equating to the length of the APC interval. If an AAPC lies entirely within a single join-point segment, the AAPC is the same as the APC for that segment .Data were obtained from the Regional Statistics Office and analyzed using EPI-Info 2000 software  and the Joinpoint Regression Program, rel. 4.0.4. May 2013 .The study was conducted on data routinely collected by the health services linked to anonymized records that make it impossible to identify the individuals concerned. The data analysis was performed on the anonymized aggregated data. Data in the Local Health Authority registries are recorded with the patient’s consent and can be used as aggregated data for scientific studies without further authorization . This stWe identified 16,659 hospitalizations for diseases potentially associated with HPV between January 1, 2000 and December 31, 2011. The proportion of these hospital admissions attributable to HPV was calculated to be 12,410 , comprising 4,384 males (35.3%) and 8,026 females (64.7%). Genital warts were the most common HPV-related condition prompting admission to hospital (44.9%).Genital warts were usually associated with age under 45 years 4,613; 82.8%), while the HPV-related cancers were more common in patients aged ≥ 45 years . Table 3; 82.8%,Overall, the average hospitalization rate for HPV-related diseases in the years 2000–2011 was 21.8 per 100,000 . Table Considering both sexes together, the hospitalization rate attributable to HPV dropped from 24.5/100,000 in 2000 to 17.5/100,000 in 2011; this decline was statistically significant, with an AAPC of −1.9%  from 2000 to 2011. On the other hand, the trend of the hospitalization rate for genital warts increased significantly , whilst there was a significantly declining trend in the hospitalization rate for anal cancer , genital cancer , and oropharyngeal cancer .Over the whole study period, the HPV-attributable hospitalization rate for males remained stable, going from 16.1/100,000 in 2000 to 14.6/100,000 in 2011 , while for females it decreased from 32.5/100,000 in 2000 to 20.2/100,000 in 2011 .Looking at the admissions for HPV-related cancer in males, the hospitalization rate decreased significantly for anal cancer  and oropharyngeal cancers , while for genital (penis) cancer it remained stable  , but not in females  , Figure The present study examined the trends in the hospitalization rates for different types of HPV-related disease in a cohort of 5 million citizens and the results show that the burden of HPV-related diseases on hospital resources is an important public health issue.It is not easy to estimate the morbidity of HPV-related diseases in the general population, partly because the currently-available epidemiological picture is hazy due to differences in study designs and population profiles, access to health care and registration methods . Hospital discharge data can, however, be useful for assessing the cases severe enough to warrant hospitalization .Using discharge diagnoses to identify cases of HPV-related diseases is more useful for cancer than for benign diseases. Patients are only hospitalized for genital warts in the more complex cases, and particularly for those requiring surgical removal .The overall hospitalization rate identified was 21.8/100,000 population during the calendar years 2000–2011, and it was 1.8 times higher for females than for males. This might suggest a gender-related difference in the natural history of HPV, with lower infection and higher disease rates in women, and vice versa in men ,18. The Our data show that a drop in the hospitalization rates for women from 2000 to 2011. This concerns the hospitalization for cancer, and cancer of the uterine cervix in particular, and relates to the introduction of organized screening programs for detecting and treating this disease in our region. In accordance with Italian national guidelines, cervical cancer screening programs have been implemented in the Veneto Region since 1997. They are based largely on European guidelines and women from 25 to 64 years of age are personally invited for a Pap test every three years .There was also a smaller drop in the hospitalization rate for anal cancer, that was statistically significant in males. A possible explanation for this could lie in changes in the management of chemotherapy and radiotherapy for cancer: since 2007 they have been administered not in hospital but at ambulatory outpatient services .The overall trend of our HPV-related hospitalizations nonetheless revealed an increase among males during the study period. The hospitalization rates rose for genital warts in males during the period considered (pointing to the need for prevention programs), but not in females. A higher prevalence among males of severe HPV-related warts warranting hospitalization could be expected to induce a corresponding increase in women too, given the transmissible nature of HPV , but thiThis study shows that hospitalization for genital warts has a considerable impact on the health services in the Veneto Region. Vaccination strategies can be used for the primary prevention of HPV infection, and could reduce the burden of HPV-related disease. Two vaccines against HPV have been developed and are currently used in population vaccination programs in many countries. Both have proved highly effective in preventing HPV 16 and 18, which are jointly responsible for approximately 70% of cervical cancers . A quadrPresently there has been ongoing debate over the introduction of HPV vaccination. Before introducing the vaccination in males should be considered the vaccination coverage rate in females, the burden of HPV-related diseases and whether it is programmatically feasible and economically sustainable.In Italy, since 2007, free anti-HPV vaccination has been offered to all girls in their 12th year of life. Two different vaccines have been available in Italy for the past three years: a bivalent vaccine directed against HPV types 16 and 18 (involved in cancer of the uterine cervix and other cancers) and a quadrivalent vaccine against HPV 16 and 18, and 6 and 11  . As HPV Our study has some limitations. Among them, we should consider the dubious accuracy of the total number of HPV-related hospital admissions identified due to coding errors in the hospital records. Another limit lies in that the responsibility of HPV could not be tested because no virology data were available. Judging from our hospitalization records, it would seem important to conduct prospective epidemiological studies with the cooperation of specialists in this field, with a view to obtaining a more precise picture of the epidemiological situation regarding HPV-related diseases.The authors declare that they have no competing interest.VB: data analysis, supervision, drafting of the manuscript; FP statistical expertise, drafting of the manuscript CS: study conception and design, drafting of the manuscript; MS: study conception and design, data collection; TB: study conception, supervision; CB: intellectual content, drafting of the manuscript; AB: statistical expertise, data interpretation and supervision. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/462/prepub


Canine Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are uncommonly reported in the ulnar, since they are underestimated relative to the more common spindle cell tumours of soft tissue. In dogs, MPNST accounts for 27% of nervous system tumours. In man, MPNST represents 5-10% of all soft tissue sarcomas and is often associated with neurofibromatosis type 1 (NF-1).An 8-year-old, 9 kg, female mixed-breed dog with a subcutaneous mass on the upper right side of the ulnar region was presented to the small animal research and teaching hospital of Tehran University. The dog was anorexic with general weakness. The mass (7 × 4 cm) was removed surgically and processed routinely. Microscopically, the mass was composed of highly cellular areas with a homogeneous population of round or spindle cells, high cellular pleomorphism, high mitotic index and various morphologic patterns. Furthermore, spindle cells arranged in densely or loosely sweeping fascicles, interlacing whorls, or storiform patterns together with wavy cytoplasm, nuclear palisades, and round cells were arranged in sheets or cords with a meshwork of intratumoral nerve fibers. In addition, in this case the presence of neoplastic cells within the blood vessels was observed. Immunohistochemically, tumor was positive for vimentin and S-100 protein. The histopathologic features coupled with the S-100 and vimentin immunoreactivity led to a diagnosis of malignant neurofibroma.To the best of our knowledge, primary ulnar MPNST has not been reported in animals. This is the first documentation of an ulnar malignant peripheral nerve sheath tumour in a dog.http://www.diagnosticpathology.diagnomx.eu/vs/1310907815984587The virtual slide(s) for this article can be found here:  Canine peripheral nerve sheath tumors (PNSTs) are uncommonly reported, and their clinical behavior has not been well documented ,2. TheseIn human MPNSTs, variable histologic patterns and heterogenous differentiation have been reported -7, incluHistologically, in human MPNSTs, the malignant nature of these tumours were evident upon their microscopic inspection, as they were comprised of an infiltrative and cellular proliferation of atypical, mitotically active spindle cells. Most MPNSTs are high-grade tumours with a high mitotic rate and commonly induce necrosis. The most common histological patterns include a high-grade fibrosarcomatous mass composed of densely packed sheets of plump but relatively uniform spindle or oval cells ,22-26.Immunohistochemical studies have contributed to the definition of clear diagnostic criteria for PNSTs. In human medicine, the expression of S-100 protein is used to differentiate between spindle cell tumours of neural and non-neural origins . In veteThis report describes the histologic and immunohistochemical diagnosis of a neurofibrosarcoma in the ulnar of a dog.An 8-year-old, 9 kg, female, mixed-breed dog referred to the Small Animal Clinic of Tehran University. Clinical examination revealed a rapidly growing, nodular, subcutaneous mass, 7×4 cm in diameter, invading and strongly adhering to the underlying tissue. The mass was located on the upper right side of the ulnar region. The dog had anorexia, general weakness and inability to stand. Ultrasonography revealed a subcutaneous mass with a central depth of 5.4 cm. ventrally, the margins of the lesion appeared to be well defined and the abdominal organs were not visibly affected.The blood cell count was undertaken manually and calcium and phosphorus concentration were measured by commercial kits  using a semi-automatic analyzer . Haematological and biochemical profiles  were within normal ranges.For partial excisional biopsy, an intravenous combination of diazepam (0.27 mg/kg)  and ketamine hydrochloride (5.50 mg/kg)  were used for induction and maintenance of anesthesia and the dog received normal saline solution 0.9% intravenously, to a total of 0.5 L (12 mL/kg/h) . Atropine sulphate (0.03 mg/kg)  was given subcutaneously as pre-medication. The mass was removed surgically and processed routinely. The sample was fixed in formalin and embedded in paraffin for sectioning. The sections were stained with haematoxylin and eosin. For further study, paraffin sections were stained immunohistochemically with S-100, vimentine markers .2O2 in Tris-buffered saline  for 15 minutes at 20°C before the antibodies used included those for vimentin , S-100 protein .For immunohistochemistry, sections from tumor were mounted on adhesive-coated slides , processed through xylene, and rehydrated in ethanol. Antigen retrieval was by boiling in a microwave oven (700 W) twice for 5 minutes in Tris–EDTA buffer—1.21 g Tris base  and 0.372 g EDTA —in 1 liter of distilled water and pH 9. Endogenous peroxidase was blocked with 0.6% (v/v) HHistopathologically, neurofibrosarcoma tumour cells were not circumscribed by connective tissue and neoplastic cells often exhibited an aggressive behavior, high cellularity, cellular pleomorphism, and various morphologic patterns. Atypical mononuclear or multinucleated cells were consistently observed. More than three mitotic figures per high-power field (400×) were found in this case Figure B and 1C.In some areas, neurofibrosarcoma was composed mainly of a homogeneous population of round cells Figure C. These However, in some regions of the tumor tissue, fusiform or spindle cells arranged in densely or loosely sweeping fascicles, interlacing whorls, or storiform patterns together with wavy cytoplasm, nuclear palisades were predominant Figure F. In addImmunohistochemical features of the spindle-shaped neoplastic cells were predominantly positive for S-100 and vimentin Figure B and 3C PNSTs have also been reported in cats, dogs, cattle, rats, and horses ,29,30. IMany of the first group are consistent with MPNSTs and have metastatic potential . Those oThe histological differentiation between malignant and benign PNST can be difficult because both may show undefined edges and some degree of cellular pleomorphism . It has , neurofibrosarcoma, and neurogenic sarcoma) and may be used as a single diagnostic tool [In dogs and humans, divergent differentiation is usually associated with a poor prognosis ,37. The tic tool ,39 or intic tool ,41. The In this study, based on their morphologic features diagnostic for human neurofibrosarcoma, i.e., growth pattern and microscopic features , together with the presence of intratumoral nerve fibers and the restriction of the S100 and vimentin immunostaining to a subpopulation of the neoplastic cells, the tumour was diagnosed as neurofibrosarcoma. But the cause of MPNST in domestic animals has not yet been determined. Due to its anatomical location and difficulties encountered in complete surgical removal. Canine MPNST often recurs after surgery and the prognosis is generally poor . In addiNeurofibrosarcoma in this report had marked morphologic variation. The round cell type found in this case was morphologically similar to the primitive neuroectodermal tumour described as the small round-cell type in human MPNST  or one oImmunohistochemical expression of S100 protein and vimentin by the neoplastic cells prompted consideration of peripheral nerve sheath tumour in the differential diagnosis. Various immunohistochemical markers have been used to define MPNST. S-100 is commonly expressed in normal nervous tissue and in most MPNST , but alsOn the basis of gross morphology, histopathological and immunohistochemical features, the final tumour in this study, a diagnosis of neurofibrosarcoma was made. For understanding of these complex neoplasms and the development of the effective differential diagnosis, further investigation will be needed into the clinical features and the basic science.This study described histopathology and immunohistochemical features of canine subcutaneous neurofibrosarcoma of the ulnar region. The histological features of these tumours would suggest that most should be classified as high-grade MPNST. A subcutaneous MPNST may be diagnosed on the basis of observing the histopathological pattern described in present study. In addition, S-100 and vimentin immunohistochemical expression may be used to help confirm the diagnosis of neurofibrosarcoma. Finally, the use of immunohistochemistry may be helpful in distinguishing this type of neoplasm from other malignancies with similar morphology. The incidence of neurofibrosarcoma in animals is unknown; we hope this will become clearer. To our knowledge, this is the first report of ulnar MPNST in a dog, suggesting that this tumour should be included as a differential diagnosis for ulnar spindle cell tumours.The authors declare that they have no competing interests.AT and FK participated in the histopathological evaluation, performed the literature review, acquired photomicrographs and drafted the manuscript and gave the final histopathological diagnosis. JJperformed sequencing alignment and manuscript writing. EH and AB carried out the immunohistochemical stains evaluation. MAH and MMM edited the manuscript and made required changes. All authors have read and approved the final manuscript.


Previous studies have suggested that there may be an association between some immune-mediated diseases and risk of tuberculosis (TB).We analyzed a database of linked statistical records of hospital admissions and death certificates for the whole of England (1999 to 2011), and a similar database (the Oxford Record Linkage Study (ORLS)) for a region of southern England in an earlier period. Rate ratios for TB were determined, comparing immune-mediated disease cohorts with comparison cohorts.In the all-England dataset, there were significantly elevated risks of TB after hospital admission for the following individual immune-mediated diseases: Addison's disease, ankylosing spondylitis, autoimmune hemolytic anemia, chronic active hepatitis, coeliac disease, Crohn's disease, dermatomyositis, Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic thrombocytopenia purpura (ITP), myasthenia gravis, myxedema, pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus (SLE), thyrotoxicosis and ulcerative colitis. Particularly high levels of risk were found for Addison’s disease (rate ratio (RR) = 11.9 (95% CI 9.5 to 14.7)), Goodpasture’s syndrome (RR = 10.8 (95% CI 4.0 to 23.5)), SLE (RR = 9.4 (95% CI 7.9 to 11.1)), polymyositis (RR = 8.0 (95% CI 4.9 to 12.2)), polyarteritis nodosa (RR = 6.7 (95% CI 3.2 to 12.4)), dermatomyositis (RR = 6.6 (95% CI 3.0 to 12.5)), scleroderma (RR = 6.1 (95% CI 4.4 to 8.2)) and autoimmune hemolytic anemia (RR = 5.1 (95% CI 3.4 to 7.4)).These two databases show that patients with some immune-mediated diseases have an increased risk of TB, although we cannot explicitly state the direction of risk or exclude confounding. Further study of these associations is warranted, and these findings may aid TB screening, control and treatment policies. Mycobacterium tuberculosis, but active tuberculosis (TB) occurs in only 5% to 10% of exposed individuals [About one-third of the world’s population is infected with Links between other diseases and TB have also been uncovered. The risk of people infected with HIV developing TB is more than 20 times greater than that of people not infected with HIV . DiabeteIn 1855, Thomas Addison described autopsy findings of six patients with adrenal tuberculosis, which continues to be one of the most common causes of adrenal insufficiency in the developing world . FurtherTable To investigate the association between TB and immune-mediated disease further, we undertook record linkage studies to determine the risk of TB specifically in individuals with selected immune-mediated diseases using an English national linked Hospital Episode Statistics (HES) dataset and a similar regional dataset.For our principal analyses, we used a dataset of English national HES (1999 to 2011), covering the population of England of about 50 million people, combined with statistical records of mortality. The HES data are statistical records of hospital care that are compiled for every episode of day case care or hospital admission in all National Health Service (NHS) hospitals in England. The death data derive from death certificates. The dataset used in this study, in which successive records for each individual were linked together (version 13yr-V06), was constructed by the Oxford record linkage group.International Classification of Diseases, tenth edition (ICD-10) codes used for each immune-mediated disease are shown in Table The basic methods were the same for the analysis of each disease and are described for rheumatoid arthritis followed by TB. A cohort of people with a record of day case care or inpatient admission for rheumatoid arthritis was constructed for those with a diagnosis of rheumatoid arthritis in any diagnostic position on the record for hospital care, by identifying the first episode of day case care, or admission, for rheumatoid arthritis during the study period. The tion ICD-0 codes uPeople were included in the rheumatoid arthritis or reference cohort if they did not have an admission for TB either before or at the same time as the admission for rheumatoid arthritis or the reference condition. We then searched the database for any subsequent NHS hospital care for, or death from, TB in these cohorts. We included cases of TB regardless of whether TB was recorded as the main or secondary diagnosis on the hospital record, except where stated otherwise. We considered that rates of TB in the reference cohort would approximate those in the general population of the region while allowing for migration in and out of it .We calculated rates of TB based on person-years. We took ‘date of entry’ into each cohort as the date of first admission for rheumatoid arthritis, or reference condition, and ‘date of exit’ as the date of first record of TB, death, or the end of data collection (28 February 2011), whichever was the earliest. We first calculated rates for TB, stratified and then standardized by age (in 5-year age groups), sex, calendar year of first recorded admission, region of residence, and quintile of patients’ Index of Deprivation score. Since the 1970s the UK government has calculated local measures of deprivation in England . Seven dRA/ERA)/(Oref/Eref). The confidence interval for the rate ratio of TB and χ2 statistics for its significance were calculated as described elsewhere [We used the indirect method of standardization, using the combined rheumatoid arthritis and reference cohorts as the standard population. The stratum-specific rates in the combined rheumatoid arthritis and reference cohorts were then applied to the number of people in each stratum in the rheumatoid arthritis cohort, separately, and then to those in the reference cohort, to give an observed (O) and expected (E) number of people with TB in each of the two cohorts. The ratio of the standardized rate of occurrence of TB in the rheumatoid arthritis cohort was calculated relative to that in the reference cohort using the formula  after TB. TB was taken as the exposure cohort, rheumatoid arthritis as the outcome, and rates of rheumatoid arthritis were calculated in the TB exposure cohort and compared with those in the reference cohort, as described above. In this analysis, we excluded people from the TB or reference cohort if they had rheumatoid arthritis before their first admission for TB or the reference condition. This, together with similar exclusions described in the section above on rheumatoid arthritis as the exposure, ensured that no individual was included in the analysis of both TB as the exposure and as the outcome .This is a similar dataset, unique in England in its time, which commenced in 1963. We used the dataset from 1963 to 1998 and the same methods, originally developed to study disease associations in the ORLS  to invesWe looked at the effects on the outcomes of each condition, separately, in the reference cohort. Where a reference condition appeared to be associated with the outcome, we removed this condition from the reference cohort to see if this made a difference to the results. In the great majority of the analyses, doing this made no material difference; in the two instances where it did make a difference, albeit small, we specify in Additional file The construction and analysis of the datasets were undertaken with the approval of the Central and South Bristol Research Ethics Committee .Table There were significantly elevated risks of TB after hospital admission for the following individual immune-mediated diseases: Addison's disease, ankylosing spondylitis, autoimmune hemolytic anemia, chronic active hepatitis, coeliac disease, Crohn's disease, dermatomyositis, Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic thrombocytopenia purpura, myasthenia gravis, myxedema, pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, SLE, thyrotoxicosis and ulcerative colitis Table .Particularly high levels of risk were found for Addison’s disease (rate ratio (RR) = 11.9 ), Goodpasture’s syndrome (RR = 10.8 (95% CI 4.0 to 23.5)), SLE (RR = 9.4 (95% CI 7.9 to 11.1)), polymyositis (RR = 8.0 (95% CI 4.9 to 12.2)), polyarteritis nodosa (RR = 6.7 (95% CI 3.2 to 12.4)), dermatomyositis (RR = 6.6 (95% CI 3.0 to 12.5)), scleroderma (RR = 6.1 (95% CI 4.4 to 8.2)) and autoimmune hemolytic anemia (RR = 5.1 (95% CI 3.4 to 7.4)).We studied the occurrence of a first admission for TB at least 1 year and at least 5 years after the first admission for each immune-related disease to help establish whether any elevated risk of TB was confined to the short term or was more prolonged, although this reduced the statistical power within each time period Table . This anWe also investigated the risk of respiratory and non-respiratory TB separately Table . As expeWhen stratifying data by sex, there were no significant differences between males and females.When restricting analyses to those where an admission for TB was recorded as the main diagnostic reason for admission after admission for an immune-mediated disease, there were significantly elevated risks for: Addison's disease, ankylosing spondylitis, autoimmune hemolytic anemia, chronic active hepatitis, coeliac disease, Crohn's disease, Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic thrombocytopenia purpura, polyarteritis nodosa, polymyositis, psoriasis, rheumatoid arthritis, scleroderma, SLE and ulcerative colitis  and 9,021,678 individuals in the reference cohort . There were significantly elevated risks in people with TB for the following immune-mediated diseases: Addison’s disease, autoimmune hemolytic anemia, chronic active hepatitis, Crohn’s disease, Goodpasture's syndrome, idiopathic thrombocytopenia purpura, polyarteritis nodosa, polymyositis, Sjögren's syndrome, SLE and thyrotoxicosis (Table P <0.001) for myxedema and 0.81  for rheumatoid arthritis. For males the rate ratios were 1.04  and 1.25  for myxedema and rheumatoid arthritis, respectively.When stratifying the data by sex, there were significant differences between males and females for myxedema and rheumatoid arthritis. Females with TB had rate ratios of 0.63  in order to minimize the risk of detection bias or any misdiagnosis between immune-mediated diseases and subsequent recognition that they were, instead, manifestations of TB. We also did this to help interpretation of the causal direction of the associations. Of interest in this respect are Addison’s, Sjögren’s and SLE showing associations in both directions in the 5-year analysis. It is likely that the great majority of cases of Addison’s are caused by TB. There is also evidence to suggest that TB may be an infective trigger for immune-mediated disease, mechanistically by molecular mimicry, bystander activation or acting as an adjuvant ,23.The associations between other immune-mediated diseases, and their mechanisms, warrant further attention. One possible mechanism could be immune dysfunction predisposing to both immune-mediated disease and TB. Whether this dysfunction leads to an increased susceptibility to infection with TB or a greater risk of TB activation will be important to tease out. Another possible mechanism is the effect of treatment of autoimmune diseases on TB risk. Immunosuppressive drugs such as corticosteroids have long been associated with the risk of TB, and more recently, TNF antagonists have also been shown to increase risk . In addiBased on our data, screening for latent TB at immune-mediated disease diagnosis and regular timely screening thereafter may be beneficial. This will be especially true where we have identified particularly high levels of risk and/or where pre-existing data corroborate our findings. Although we have grouped these disorders together as immune mediated, these diseases are looked after by a diverse range of specialists . Raising awareness of the importance of screening for TB in this context is more difficult but needs to be put in place.P values, as well as confidence intervals, so that the reader can judge the degree of significance for each immune-mediated disease and subsequent TB. It is possible that some of the associations that are significant at a level of P <0.05 may result from making multiple comparisons and the play of chance. This may particularly be so where there is no prior hypothesis to support the finding. However, many of the associations are very strong, and levels of significance high, and it is unlikely that most of these are attributable to chance alone. The ORLS data is also helpful in this regard: associations between several diseases and increased risks of TB were seen consistently in this smaller dataset suggesting that, taking the findings from both datasets together, these are not chance findings. The fact that we see the classic association between Addison’s and TB, and confirm previous findings of association between coeliac disease and rheumatoid arthritis and TB [A key strength of the dataset is its size with large numbers of fairly uncommon diseases. The risk of TB was studied within a single population, using the same methodology, which means that direct comparisons of risk between immune-mediated diseases can be made. The dataset has limitations. The data is based on prevalent cases of immune-mediated diseases and of TB  rather than being a cohort with follow-up from the date of first diagnosis. Data are not recorded on patients who move out of the area covered by data collection or who are treated in hospitals outside the area. The dataset is limited to people who were admitted to hospital, or who received day case specialist care and thus there exists the potential for selection bias. This would not capture all people with each immune-mediated disease, although it should identify the great majority with subsequent diagnosed TB. We lack treatment data for immune-mediated diseases. There is very limited information on potential confounding factors such as detailed socioeconomic characteristics, other co-infections , ethnicity and smoking. The majority of individuals in the ORLS were born in Britain, and thus associations seen in the ORLS are in a reasonably homogeneous population in respect of ethnicity. The effect of making multiple comparisons needs to be considered. For this reason, we have given exact s and TB ,5 adds fOur results should be regarded as speculative rather than definitive. They need further work, in different study designs, to confirm or refute the findings. Further studies should employ prospective longitudinal cohorts of individuals with TB and, separately, cohorts of individuals with immune-mediated disease to truly understand the mechanism behind the associations described. In countries where TB is uncommon the relative risks present translate into a small absolute risk increase. Nevertheless, these data may help to increase awareness among clinicians of a possible increased risk of TB in patients with autoimmune diseases. This could, in turn, lead to better screening, and treatment, for latent TB in this patient population.E: Expected; HES: Hospital episode statistics; ITP: Idiopathic thrombocytopenia purpura; ICD: International classification of disease; NHS: National health service; O: Observed; ORLS: Oxford record linkage study; RR: Rate ratio; TB: Tuberculosis; TNF: Tumor necrosis factor; SLE: Systemic lupus erythematosus.The authors declare no competing interests.MJG is the guarantor and designer of the study. RG led the analysis. SVR, AS and MJG contributed to the analysis and interpretation of the data. SVR wrote the first draft and all authors contributed to subsequent drafts and the final paper. All authors have read and approve the manuscript for publication.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/97/prepubTable S2. Shows the data analysis results for the rate ratios of immune-mediated disease following a principal diagnosis of tuberculosis in England, 1999 to 2011.Shows data analysis results for the rate ratios of primary diagnosis of tuberculosis following selected immune-mediated diseases (IMD) in England, 1999 to 2011. Click here for file


TERT gene, was identified and reported to have an effect on TERT expression and telomerase activity. We examined whether the functional MNS16A was related to the risk of occurrence or progression of NPC in the Chinese population.Increases in human telomerase reverse transcriptase (TERT) expression and telomerase activity are frequently seen in nasopharyngeal carcinoma (NPC). Recently, a variable tandem-repeats polymorphism, MNS16A, located in the downstream region of the S) allele and 302 or 333 bp as the long (L) allele. The genetic associations with the risk of NPC were analyzed by logistic regression.We genotyped the MNS16A polymorphism in a case-control study of 855 patients with NPC and 1036 cancer-free controls using PCR, and determined genotype by classifying the DNA band of 243 or 272 base pairs (bp) as the short  had a significantly reduced risk of NPC occurrence compared with those carrying the LL genotype (odds ratio (OR) = 0. 71, 95% confidence interval (CI) = 0. 52 to 0. 96, P = 0. 025). Using a immunohistochemical assay on the NPC tissues, the SL genotype carriers were found to have lower TERT expression than the LL genotype carriers (P = 0. 035).The MNS16A genotype was not associated with the progression of NPC. However, individuals carrying the TERT MNS16A polymorphism may contribute to the risk of NPC onset in Chinese population.Our study indicates that the  Nasopharyngeal carcinoma (NPC) is an epithelial malignancy with strikingly variable racial and geographic distribution. It is particularly prevalent in populations from southeast Asia, with an incidence rate ranging from 15 to 50 per 100,000, which is ~100 times higher than that in Caucasian populations . In addiTERT) is the core component responsible for its enzymatic activity [Telomeres are structures that cap the distal ends of chromosomes, and function to prevent chromosome degradation, end-to-end fusions, rearrangements and chromosome attrition . Telomeractivity . In humaactivity . The exiactivity -12.TERT play an important role in the occurrence and progression of NPC [With regard to NPC, increased telomerase activity and TERT expression is also common in tumor tissues, and can be detected at high frequencies compared with normal nasopharyngeal epithelium and tissues ,13. It hn of NPC .TERT might be an excellent biological candidate susceptibility gene for NPC, and that functional polymorphisms within TERT might result in genotype-dependent differences in susceptibility to NPC.Based on the functional relevance of TERT in the pathogenesis of NPC described above, we hypothesized that the TERT gene on chromosome 5p15. 33 [TERT mRNA level and the activity of telomerase [TERT gene might contribute to the tumor susceptibility. Indeed, several genetic epidemiological studies have shown MNS16A to be a potential risk factor and/or a prognostic marker of lung [TERT, MNS16A, has any bearing on the occurrence or progression of NPC in the Chinese population.In a previous study, a functional variable number of tandem repeats (VNTR) polymorphism, MNS16A, was identified in the downstream region of the 5p15. 33 . The reglomerase . Therefo of lung -23, brea of lung , brain [ of lung -27 and c of lung  cancers.The study was approved by the ethics committee of Beijing Institute of Radiation Medicine , and written informed consent was obtained from all the participants involved.This case-control study enrolled 855 patients with incident NPC and 1036 control subjects  classification of the 1997 American Joint Committee on Cancer (AJCC) system. All TNM classifications were determined by senior pathologists of the hospital, on the basis of the postoperative histopathologic examination. All the controls were recruited in the same regions during the same time that the NPC cases were collected. The selection criteria for the controls included no individual history of cancer, and frequency matching to the cases on gender and age (± 5 years). The response rate for the controls was 96%.From the group of 855 patients with incident NPC, 41 patients who had undergone resection before receiving any further treatment at Guangxi Cancer Hospital were selected, and primary NPC biopsies were collected from them . Briefly, PCR was performed in a total volume of 15 μl with an initial denaturing step for 5 minutes at 95°C, followed by 43 cycles of 30 seconds at 95°C, 45 seconds at 61°C and 1 minutes at 72°C, and a final extension step with 10 minutes at 72°C. The PCR products were separated in a 2. 5% agarose gel, visualized with UV light, and photographed. Four lengths of repeat polymorphisms were seen: 243, 272, 302 and 333 bp. As reported previously [The MNS16A polymorphism was genotyped by PCR with the primers described previously  (forwardAF128894. 1). PCR was performed in a total volume of 15 μl with an initial denaturing step for 5 minutes at 95°C, followed by 35 cycles of 30 seconds at 95°C, 45 seconds at 52°C and 1 minute at 72°C, and a final extension step of 10 minutes at 72°C. Using the new primers, four lengths of repeats polymorphism were also seen: 306, 335, 365 and 396 bp, corresponding to the previously seen bands of 243, 272, 302 and 333 bp, respectively. One extra band for every heterozygous genotype was still visible in a 2. 5% agarose gel and disappeared in an 8% denatured polyacrylamide gel . After washing, the slides were treated with a commercial kit . All slides were then incubated with 3,3-diaminobenzidine tetrahydrochloride (DAB), and the cells were counter-stained with hematoxylin. Negative and positive controls were performed at the same time. The slides were mounted with neutral balsam for examination. Photographs were taken  for study comparison.NPC tissues (n = 41) and non-tumor nasopharyngeal tissues (n = 13), which had been fixed with paraformaldehyde and embedded in paraffin wax, were analyzed for protein expression of TERT. Two slides from each biopsy were stained with hematoxylin and eosin for routine histological evaluation. The slides were washed in xylene to remove the paraffin wax, and then rehydrated through serial dilutions of alcohol, followed by washing in phosphate-buffered saline (PBS) pH 7. 2. All subsequent washes were buffered via the same protocol. The slides were then incubated with 3% HThe IHC signals were scored as previously described . Briefly2 test was used to compare gender, ethnicity, tobacco and alcohol use, and family history of first-degree relatives between patients and controls. The unpaired t-test was used to analyze differences in mean age and mean smoking level between patients and controls.The χet al [Genotype and allele frequencies for the MNS16A polymorphism were determined by gene counting, and departures from Hardy-Weinberg equilibrium were tested using the random-permutation procedure implemented in the Arlequin package . The asset al . FPRP ofL and S allele carriers were assessed by a Wilcoxon signed-ranks test and a trend χ2 test, respectively. Logistic regression analysis was also performed for MNS16A genotypes, with the IHC staining scores as the outcome variable.Differences in the protein levels detected by IHC between the tumor and non-tumor tissue, and between the MNS16A P < 0. 05 was considered significant, and all statistical tests were two-sided. These analyses were performed using SPSS software .P = 0. 0011, t-test), positive history of NPC in their first-degree biological relatives  and non-Han ethnicity . An absolute majority of cases (97. 0%) was classified as having poorly differentiated SCC. According to the TNM system, 4. 8%, 46. 2%, 30. 3% and 18. 7% of patients had stage I, II, III and IV disease, respectively.The selected demographic and clinical characteristics of patients with NPC and control subjects were described in detail previously  and controls (P = 0. 17). When the commonest genotype  was designated as reference group, all the other genotypes were still not associated with the risk of NPC  = 2), and this difference was mainly caused by the lower frequencies of the SL and SS genotype in the case group compared with the control group. Based on logistic regression analysis with adjustment for age, gender, tobacco and alcohol use, ethnicity, and family history of NPC, individuals carrying the S alleles (SL + SS genotype) had a significantly reduced risk of NPC occurrence compared with those carrying the LL genotype . This association remained robust given prior probabilities of actual association of 25% (FPRP = 0. 13) and 10% (FPRP = 0. 30), but not with a prior probability of 1% (FPRP = 0. 83) [P = 0. 0068). The association between the MNS16A and risk of NPC occurrence were further examined with stratification by age, gender, tobacco and alcohol use, smoking level, ethnicity, and family history (Table SL + SS genotypes was more pronounced in subjects who were older (≥ 45 years), male, smokers, drinkers, Han Chinese and with a negative family history, these differences could be attributed to chance , indicating that these potential confounding factors had no modification effect on the risk of NPC related to MNS16A genotypes.In accordance with a previous report , the 243= 0. 83) . When caSL + SS genotypes on severity of NPC (as measured by the TNM staging system). However, the distributions of the MNS16A genotypes were not significantly different between subgroups with different clinical stages or different TNM classifications .In 798 patients with NPC in the Guangxi population, the mean ± SD age at diagnosis was 46. 75 ± 11. 82 years for subjects with the SL genotype carriers having lower TERT expression than the LL genotype carriers .We further investigated the protein expression of TERT in NPC tissues and non-cancerous nasopharyngeal epithelium tissues by IHC assay  was over-represented in controls compared with cases. Furthermore, carriers of the S alleles tended to have an older age of diagnosis than those of the LL genotype, suggesting a protective role of the MNS16A S allele against the onset of NPC. It was reported that the region containing MNS16A had some promoter activity that was influenced by the length of the MNS16A tandem-repeats polymorphism [S allele, compared with the L allele, was associated with higher expression of the TERT antisense RNA transcripts. In our study, we further investigated the protein expression of TERT in NPC and non-cancerous nasopharyngeal epithelium tissues by IHC. We found that the SL genotype was significantly associated with lower TERT expression than the LL genotype  over a lifetime, would have a lower susceptibility to developing this disorder. However, the exact functional molecular mechanisms of MNS16A remain unknown. Further studies will be needed to clarify how MNS16A affects antisense RNA transcription and how antisense RNA affects TERT expression.In our study population, the morphism . The MNSpe Table . Given tTERT MNS16A has been studied in many cancers, but the results are conflicting. Consistent with this study in NPC, one study reported an association of the MNS16A S allele with a decreased risk of non-small-cell lung cancer (NSCLC) [S allele conferring an increased risk of breast cancer, glioblastoma and anaplastic gliomas [S and L combining system, and assessed directly the association of the raw alleles with the diseases. For example, Jin et al. found that the 243 bp allele, but not the 272 bp allele, was associated with the risk of lung cancer in a Korean population [et al. reported that only the 274 bp allele was associated with the risk of colorectal cancer [Recently, the  (NSCLC) . By cont (NSCLC) , and som gliomas ,26. Recepulation . Hofer el cancer . In thisSS, SL and LL genotype in our controls were 0. 2%, 8. 9% and 90. 9% . It remains to be determined whether these differences between ethnic groups influence the risk of developing NPC.We compared the genotype frequencies of the MNS16A polymorphism in populations from other studies based on available published data to identify differences from our own study. We found that the frequencies of the ectively ,26-28. OTERT MNS16A polymorphism was associated with clinical progression in some types of cancer, including clinical stage, tumor stage, lymph-node metastasis and survival time, although the results were contradictory. Wang et al. observed a significantly prolonged survival time for patients with glioblastoma with the SS genotype compared with the SL or LL genotype [LL genotype was significantly associated with longer survival time in patients with glioblastoma [et al., which did not find any survival differences for patients with glioblastoma or anaplastic glioma with different MNS16A genotypes [SL + SS genotypes relative to the carriers of the LL genotype. Therefore, we urge that the role of the MNS16A in the progression of NPC be investigated in additional studies with larger sample sizes.Previous studies have also reported that the genotype . Howeverblastoma  and earlblastoma , and wasblastoma . It is aenotypes . In patienotypes ,19. HoweTERT-CLPTM1L locus on 5p15. 33 to be significantly associated with many different types of cancer, including basal cell carcinoma, glioma, lung, prostate, bladder, testicular germ cell, pancreatic, and cervical cancers [TERT locus contains risk factors for cancer is compelling. Further studies are needed to systematically verify the SNPs at the TERT locus, investigate the interactions between these SNPs and MNS16A, and and assess their contributions to the endemics of NPC in the Chinese population.Recently, numerous genome-wide association studies (GWAS) have been carried out to identify associations between cancer risk and single-nucleotide polymorphisms (SNPs). Multiple independent GWAS have all shown the  cancers . Thus, tIn reviewing the results of this study, several potential limitations should be kept in mind. First, as this was a hospital-based study, our NPC cases were enroled from the hospitals, and the controls were selected from the community population, thus inherent selection bias cannot be completely excluded. However, through further adjustment and stratification in data analyses, we aimed to minimize the potential confounding effect. Second, several association studies have reported identification of the genes that may relate to the susceptibility to NPC -39; howeS allele, which is associated with higher expression of the TERT antisense RNA and lower expression of the TERT protein, seems to be a genetic protection factor for NPC risk in a Chinese population. If confirmed by other studies, knowledge of genetic factors contributing to the pathogenesis of the NPC as presented here may have implications for the cancer screening and treatment of this disorder in the future.Our results reveal an association for the first time between the functional tandem repeat polymorphism MNS16A downstream of TERT and susceptibility to NPC. Furthermore, the The authors declare that they have no competing interests.YZ conceived and designed the experiments, performed the experiments, analyzed the data, and drafted the manuscript. HZ contributed to the experimental design and data analysis. YZ, ZW, PL and LY contributed to the sample preparation and genotyping. FM, HW, YC contributed to sample collection. YZ contributed to the denatured polyacrylamide gel electrophoresis. GZ and FH conceived and designed the experiments, and drafted and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/9/106/prepubSupplemental Table S1, S2 and S3. Supplemental Figure S1, S2 and S3.Click here for file


The concept of immunization was started in Japan in 1849 when Jenner’s cowpox vaccine seed was introduced, and the current immunization law was stipulated in 1948. There have been two turning points for amendments to the immunization law: the compensation remedy for vaccine-associated adverse events in 1976, and the concept of private vaccination in 1994. In 1992, the regional Court of Tokyo, not the Supreme Court, decided the governmental responsibility on vaccine-associated adverse events, which caused the stagnation of vaccine development. In 2010, many universal vaccines became available as the recommended vaccines, but several vaccines, including mumps, zoster, hepatitis B, and rota vaccines, are still voluntary vaccines, not universal routine applications. In this report, immunization strategies and vaccine development are reviewed for each vaccine item and future vaccine concerns are discussed. The dawn of vaccinology was the first scientific systematic investigation of the cowpox vaccination by Edward Jenner in 1796, although several variations in approach were performed using dried pus from smallpox skin lesions in Central Asia, China, and Turkey . Jenner’Haemophilus influenzae type b conjugated with tetanus toxoid (Hib) was introduced in 2008, and 7-valent Streptococcus pneumoniae conjugated vaccine with recombinant diphtheria toxoid (PCV7) and human papilloma virus vaccines (HPV) became available in 2010. Rotavirus vaccines were introduced in 2012. Several issues concerning vaccines in Japan are discussed in this article.The Japanese government in the Meiji era decided that all Japanese people should be immunized with the vaccine for smallpox, which was stipulated in 1876, and a vaccination law against smallpox started in 1910. The present immunization law was implemented in 1948 under occupation by the United States (US). Issues related to vaccine development and immunization policies are summarized in Table The Japanese immunization law is complicated with double-standard categories: routine recommended and voluntary vaccination. Routine recommended vaccines consist of BCG, acellular pertussis vaccine (DTaP), measles and rubella combined vaccine (MR), inactivated polio (IPV), Hib, PCV7, HPV, and Japanese encephalitis vaccine (JEV). Voluntary vaccines are hepatitis B (HBV), mumps, varicella, and rotavirus vaccines. The difference between the two is the cost of immunization; routine recommended vaccines are principally covered by the regional government , 11. UntIn 2010, Hib, PCV7, and HPV began to be used as temporarily recommended vaccines, and the cost was partially supported by the regional governments . VaccinaIn Japan, live attenuated measles vaccines were developed in the 1970s, and four strains were licensed (three strains are used at present) with the implementation of recommended immunization in 1978 . Five stRegarding the reporting system for measles in Japan, through 3,000 sentinel clinics or hospitals for pediatric infectious diseases and 450 clinics for adult measles surveillance, patients with clinically suspected measles were reported to Regional Health Care Centers. In the late 1990s to early 2000s, 20,000–30,000 cases of measles, including several dozen deaths, were reported yearly. A total of 2,034 cases of measles, including 8 deaths, were reported in a severe measles outbreak in Okinawa in 1998–1999 . Many ofTo reduce the number of patients with measles, an additional MR catch-up campaign was started for teenagers at the age of 13 and 18 years (MR III and IV) from 2008 for a 5-year schedule. After 2008, all cases with measles had to be registered, and the number of patients with measles was reduced to 457 cases in 2010 (3.58 cases per million), with some imported genotypes . In 2011Global measles vaccination coverage increased from 72 % in 2000 to 82 % for the first dose in 2007, and the two-dose immunization strategy was recommended for countries with high coverage of the first-dose measles vaccine, at more than 95 %. Most countries (88 %) now implement the two-dose strategy . HoweverThe rubella vaccine strategy was markedly changed in 1994. Before 1989, the rubella vaccine was administered to 14-year-old girls, but the vaccine target has changed to all children aged 12–90 months. Boys more than 90 months of age and girls from 90 months to 14 years of age were not enrolled as immunization targets in the transition period. Even though a temporal catch-up campaign was conducted to cover the immunization gap, vaccine coverage was extremely low , 28. AccSurveillance data of reported cases of polio paralysis are shown in Fig. Pertussis is still a serious illness in young infants, and causes whooping cough, apnea, cyanosis, choking, and encephalopathy . In JapaBordetella or detection of the pertussis genome is not always successful because of the short duration of excretion of Bordetella influenced by the empirical administration of antibiotics or vaccination history [In 1981, a new type of acellular pertussis was developed, and a combined vaccine (DTaP) was introduced into recommended immunization practice. Principally, two types of DTaP vaccine  were developed: the B-type consisted of two major antigens, pertussis toxin (PT) and filamentous hemagglutinin (FHA), and the T-type contained pertactin and fimbriae in addition to PT and FHA , 39. Nat history , 42. A s history .DTaP was adopted by global vaccine makers because of the lower incidence of adverse events than that with DTwP, and multivalent combined vaccines, such as DTaP/Hib/IPV/HBV hexavalent vaccines, were developed based on DTaP. Pertussis is principally an infectious children’s illness causing whooping and prolonged cough, and the Advisory Committee on Immunization Practices (ACIP) recommended a five-dose DTaP schedule, at ages 2, 4, 6, 15–18 months, and 4–6 years, instead of the previous DTwP in the US in 1997 . In the Two types of influenza virus vaccines are now globally available, inactivated and cold-adapted live attenuated vaccines. There are three types of inactivated vaccines: whole virion, split, and subunit inactivated vaccines. The whole virion inactivated vaccine induced febrile reactions after the vaccination, and thereafter the split vaccine was licensed in 1972 in Japan, which has been used for more than 40 years with a lower incidence of febrile reactions. The split vaccine is made by destroying the structure of virus using detergents and ether to remove their lipid components from the formalin-inactivated whole virion. The HA subunit vaccine is purified from the HA fragments zone . ChangesThree pandemics of influenza occurred in the 20th century. The most devastating pandemic dated back to 1918 and was known as Spanish flu. It was caused by a highly pathogenic H1N1 influenza virus transmitted through some animals from an avian pathogenic virus and is estimated to have killed 40–50 million people . In 1957Several types of H5 vaccines have been developed, basically based upon the HA split, subunit vaccine, or whole virion inactivated with adjuvant. In Japan, alum-adjuvanted H5N1 whole virion inactivated vaccine (WIV)  was developed using a genetically engineered reassortant, the NIBRG-14 strain, originating from H5N1/A/Vietnam/1194/2004. In a clinical phase II/III trial in healthy adults, alum-adjuvanted WIV  led to favorable immunogenicity, >70 % sero-conversion rate in neutralization tests (NT) antibodies, without causing any serious systemic illnesses . HoweverStreptococcus pneumoniae 6B and 19A serotypes uncovered by PCV7 are increasing, with a higher number of penicillin-resistant strains [S. pneumoniae has approximately 100 serotypes, using serotype replacement after the introduction of PCV7 and PCV13 to be licensed.Hib and PCV are the major pathogens of bacterial meningitis and invasive systemic bacteremia, and they cause serious pneumonia. In the past, bacterial infection was believed to be treatable with antibiotics through early diagnosis and was not a target for vaccine development before 2000 in Japan. However, a shift led to the development of vaccines in the late 1980s in the US. The appearance of resistant strains provided an impetus for the introduction of vaccines. In Japan, the surveillance study of the incidence of Hib meningitis was conducted, which showed the incidence was 8.3 per 100,000 children less than 5 years of age , 63. The strains , 66. HibFive live mumps vaccine strains were developed in the 1970s from domestic wild strains isolated in the 1960s and 1970s , 68. MMRThe varicella zoster virus vaccine OKA strain was developed in Japan in 1974, and is the only strain available in the world . InitialMumps and zoster vaccines were universal vaccines in the US and EU but were voluntary in Japan , 12, 75.The disease surveillance system in Japan is based on 3,000 sentinel clinics or hospitals for pediatric infectious diseases and reflects the tendency of infectious diseases, not population-based incidences. The immunization strategy is decided based upon disease surveillance, and monitoring of vaccine-associated adverse events is important to assess the safety. It is now based on postmarketing surveillance in Japan and should be developed in a systematic administrative form, together with laboratory investigations. It is difficult to identify the relationship of vaccination to serious adverse events occurring within a few weeks after immunization, and, in most cases, a direct relationship could not be identified. In 2005, a serious case of acute disseminated encephalomyelitis (ADEM) was reported after vaccination with the Japanese encephalitis vaccine (JEV). At that time, JEV was produced from purified virus particles from mouse brains infected with Japanese encephalitis virus. Therefore, JEV has the potential to cause allergic encephalomyelitis. JEV was suddenly discontinued in a shortsighted political decision, without considering the effects of blank periods without JEV. At that time, tissue-culture JEV was ready to be marketed. Comprehensive decisions are required and should be made after scientific discussion.No organization for decision making on immunization is currently systematized in Japan, such as the Advisory Committee on Immunization Practices (ACIP) of the US . An inve