Uterine decidualization supports embryo implantation and placentation as well as subsequent events, which together ensure a successful pregnancy. Accordingly, impaired decidualization results not only in implantation failure or early fetal miscarriage, but also may lead to potential adverse outcomes in all three pregnancy trimesters. Transcriptional reprogramming on a genome-wide scale underlies progesterone dependent decidualization of the human endometrial stromal cell (hESC). However, identification of the functionally essential signals encoded by these global transcriptional changes remains incomplete. Importantly, this knowledge-gap undercuts future efforts to improve diagnosis and treatment of implantation failure based on a dysfunctional endometrium. By integrating genome-wide datasets derived from decidualization of hESCs in culture, we reveal that the promyelocytic leukemia zinc finger (PLZF) transcription factor is rapidly induced by progesterone and that this induction is indispensable for progesterone-dependent decidualization. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) identified at least ten progesterone response elements within the PLZF gene, indicating that PLZF may act as a direct target of PGR signaling. The spatiotemporal expression profile for PLZF in both the human and mouse endometrium offers further support for stromal PLZF as a mediator of the progesterone decidual signal. To identify functional targets of PLZF, integration of PLZF ChIP-Seq and RNA Pol II RNA-Seq datasets revealed that the early growth response 1 (EGR1) transcription factor is a PLZF target for which its level of expression must be reduced to enable progesterone dependent hESC decidualization. Apart from furnishing essential insights into the molecular mechanisms by which progesterone drives hESC decidualization, our findings provide a new conceptual framework that could lead to new avenues for diagnosis and/or treatment of adverse reproductive outcomes associated with a dysfunctional uterus.Progesterone,  Following embryo attachment to the uterine epithelium, the underlying decidualized stroma is critical for further invasion by the conceptus into the maternal compartment. Because endometrial decidualization is required early in the continuum of events that lead to a successful pregnancy, abnormal decidualization can contribute not only to implantation failure or early miscarriage, but may initiate adverse reproductive outcomes that manifest in subsequent pregnancy trimesters. Genome-wide transcriptional changes by progesterone are known to underlie decidualization; however, the pivotal signals that functionally enable progesterone-driven decidualization are not fully known. Using an integrative analysis of genome-scale data along with studies on primary human endometrial stromal cells (hESCs), we reveal that the promyelocytic leukemia zinc finger (PLZF) transcription factor is rapidly induced by progesterone, and its induction is essential for progesterone-dependent hESC decidualization. Although PLZF in turn governs a remarkable array of target genes in the hESC, we demonstrate that PLZF tightly regulates the expression level of the early growth response 1 (EGR1) transcription factor, the perturbation of which compromises progesterone dependent decidualization. Together, our findings provide a new mechanistic perspective on progesterone action in the uterus which may furnish new opportunities for the formulation of more effective fertility solutions in the future. For healthy couples, the chance for conception per menstrual cycle is only thirty-to-forty percent , underscSuccessful advancement of embryo implantation into a receptive endometrium requires decidualization of the endometrial stromal cell layer, a critical cellular transformation process in which quiescent stromal fibroblasts proliferate and differentiate into transient epithelioid decidual cells . In addipromyelocytic leukemia zinc finger (PLZF) transcription factor is rapidly induced by P4 in human endometrial stromal cells (hESCs) and that this induction is indispensable for P4-driven hESC decidualization. Additionally, we show that PLZF tightly regulates the expression levels of the early growth response 1 (EGR1) transcription factor, the perturbation of which impairs hESC decidualization.Along with exposure to preovulatory and nidatory estradiol-17β (E2), the endometrial stroma requires progesterone (P4) “the hormone of pregnancy” and its nuclear receptor (the progesterone receptor (PGR)) for decidualization . While mTogether, our studies have uncovered a new mediator pathway of the P4 signal that enables quiescent hESCs to differentiate into decidual cells, a cellular transformation process that is essential for early pregnancy establishment.PLZF  is listed in the top 20 genes which are significantly induced in hESCs during decidualization [S1A Fig). Additionally, we previously showed by microarray analysis that Plzf is present in the top 100 genes induced in total uterine tissue of the mouse in response to acute P4 administration [PLZF . In response to a hormone decidual stimulus (17β-estradiol (E2), medroxyprogesterone acetate (MPA), and cAMP (EPC)), rapid induction of high transcript levels for PLZF was observed by quantitative real time PCR . The induction of transcripts encoding insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL) . Similar to findings in Fig 1A, PLZF protein expression was not detected in pre-decidual hESCs or with vehicle treatment . Treatment of hESCs with E2, cAMP, or MPA revealed that only MPA significantly induced PLZF transcription in hESCs , providing strong support for the progestin in the decidual hormone cocktail as the key driver of PLZF induction in decidualizing hESCs. Moreover, PLZF transcript levels are induced with acute progestin treatment (as early as 2 hours post MPA administration), which is not dependent on new protein synthesis . These results support PLZF as an early progestin responsive gene in hESCs. As further support for this conclusion, immunohistochemical analysis of human endometrial biopsy samples show that PLZF protein is highly expressed in the nucleus of the endometrial stromal cell during the P4-dominant secretory phase of the menstrual cycle but is expressed at low levels in endometrial tissue biopsied during the E2-dominant proliferative phase of the cycle . In the uterus of the ovariectomized mouse, Plzf transcript and protein levels are also significantly and rapidly induced by P4 , validating our previous microarray analysis of this tissue which showed Plzf transcription is induced by short-term P4 exposure [S2A and S2B Fig). Furthermore, expression levels of Plzf protein are low in the endometrium of the adult virgin mouse but are significantly increased in the uterine stromal compartment of the early pregnant mouse . It should be noted that global knockout of Plzf in the mouse results in a number of embryonic and developmental phenotypes [Comparing undecidualized hESCs with decidualized hESCs in culture, we recently revealed by RNA-seq analysis that lization   rather than by rapid non-genomic progestin effects [PGR were administered to hESCs . Following three days of culture with MPA or MPA plus RU486, PLZF transcript levels were examined in hESCs by quantitative real time PCR . Compared to vehicle treatment, MPA significantly increased the transcript levels of PLZF; however, this level of induction was markedly attenuated with the co-administration of RU468 . Further confirming the requirement of the PGR in this transcriptional induction, progestin induction of PLZF transcription was significantly attenuated with PGR knockdown . In addition, acute hormone treatment of the ovariectomized Pgr knockout (PRKO) mouse revealed that rapid P4-induced Plzf transcription in the murine uterus requires endometrial Pgr . Together, the above results strongly support an indispensable role for PGR in the rapid induction of PLZF transcription by progestin or P4 in human and murine uterine cells.To confirm that PLZF is rapidly induced by progestin  effects , RU486 ) in the PLZF gene were revealed from an analysis of a recently published chromatin immunoprecipitation-sequencing (ChIP-Seq) dataset derived from PGR cistrome studies on decidualized hESCs [PLZF gene as compared to a negative control region . Collectively, these results suggest that progestin transcriptional induction of PLZF involves binding of PGR to multiple sites across the PLZF gene.Ten PGR binding sites . Consistent with this block in stromal cell decidualization, a significant attenuation in the induction of decidual biomarkers (PRL and IGFBP-1) was observed in hESCs with PLZF knockdown . Although these results underscore a critical functional role for PLZF in advancing progestin dependent hESC decidualization, surprisingly the expression level of a number of previously published PGR target genes in hESCs  is not changed with PLZF knockdown . These results indicate that the PLZF transcription factor is either positioned below these targets in the hierarchy of target genes for PGR action or PLZF operates in parallel to control its own gene target ensemble, which in turn underpins PGR dependent hESC decidualization.To determine whether PLZF is functionally required for d HOXA10 –15) is nFig 4). Having passed Illumina’s purity filter, the PLZF cistrome consists of 19,092,785 unique alignments (without duplicate reads) from more than 36 Million reads and aligns with no more than 2 mismatches to the genome. A total of 1,120 sequence intervals are enriched for PLZF binding across all chromosomes (supporting information). Peak distribution analyses revealed significant enrichment for PLZF binding near transcription start sites (TSS) with 48% of PLZF intervals located within 500 base pairs (b.p.) of TSS, indicating preferential binding of the PLZF transcription factor to gene promoter regions (S1 Table). DNA sequence motif enrichment analysis revealed the zf-C2H2 DNA binding domain (MC00418) in the top enrichment cluster of motifs in the PLZF cistrome, a common DNA binding motif for many ZBTB transcription factors including PLZF [Fig 4B). This cluster also contained DNA binding motifs for breast cancer 1 early onset (BRCA1) (MC00299) and v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1) (MC00018) (S2 Table). Following these enrichment groups, are motifs that are commonly found in cistromes of TSS-proximal binding transcription factors, such as motifs recognized by CCAAT-enhancer binding proteins (or C/EBPs), interferon-regulatory factors, SP1, and by leucine zipper family of transcription factors (i.e. JUN and FOS) (S2 Table).To identify possible PLZF target genes, ChIP-Seq was performed to identify the genome-wide binding events for PLZF in decidualizing hESCs bona fide PLZF gene targets, we next compared 689 genes that have at least one PLZF ChIP binding region within 1kb of TSS with genes in hESCs for which expression changed with EPC treatment [S3 Table). By DAVID and GSEA analysis, the majority of these genes are associated with mitosis and cell cycle progression (S4 Table).To identify S3 Table); (b) belongs to a small family of transcription factors that enable early cellular growth responses [Fig 5A and 5B).Because PLZF was previously shown to act as a transcriptional repressor of cellular proliferative programs in a number of physiological systems; reviewed in , we firsesponses ; and (c)EGR1 transcript levels are significantly reduced as hESCs differentiate into decidual cells . Interestingly, analysis of our recent RNA-seq data from pre-decidual and decidualized hESCs shows that transcript levels of EGR1 along with EGR2 and EGR3 are significantly reduced with decidualization ; however, only EGR1 shows PLZF binding (S3 Table). Importantly, knockdown of PLZF expression results in a significant attenuation in the normal reduction of EGR1 transcript levels following three days of EPC treatment , suggesting PLZF represses EGR1 transcription as hESCs decidualize. Moreover, lentiviral-mediated overexpression of EGR1 significantly attenuated hESC decidualization in response to EPC treatment as evidenced by a marked reduction in the levels of the decidual biomarkers: IGFBP1 and PRL at the molecular and cellular level . Interestingly, immunohistochemistry disclosed an inverse correlation between stromal PLZF and EGR1 protein expression in the human endometrium during the E2 dominant proliferative and P4 dominant secretory phase of the menstrual cycle . It should be noted that the human endometrial stroma can decidualize during the secretory phase of the cycle even in the absence of a conceptus [). During the secretory phase of the cycle, significant PLZF expression is found in endometrial stromal cells; however, EGR1 expression is negligible during this time period . Together, these immunohistochemical findings further support the proposal that PLZF acts as a novel transcriptional repressor of EGR1 in endometrial stromal cells to enable decidualization.In keeping with its role as a driver of cell growth and proliferation, onceptus . In the Egr1 is markedly induced with E2 treatment alone (E2) whereas this induction is significantly blunted with the inclusion of P4 (P4E2) . Further confirming inhibition of E2 induction of Egr1 by P4, inclusion of RU486 in the P4E2 hormone treatment returns Egr1 levels to those observed with E2 alone . Interestingly, P4 induction of PLZF expression during this period parallels P4 suppression of Egr1 expression , providing correlative support for the proposal that P4 suppression of E2 induction of uterine Egr1 is mediated by PLZF. Together, our human and mouse studies support the proposal that E2-induced EGR-1 expression is suppressed by P4-induced PLZF in the endometrium.In the uterus of the ovariectomized mouse, In sum, recent genome-wide studies at the cistrome and transcriptome level underscore the regulatory complexity of P4-dependent hESC decidualization. Within this complexity, we have uncovered a new signaling pathway (PGR-PLZF-EGR1) that is crucial for P4-driven hESC decidualization.poxvirus and zinc finger (POZ)/ broad-complex, tramtrack, and bric-`a-brac (BTB) and Kruppel zinc finger (or POK) family of transcription factors; reviewed in [Extensive transcriptional reprogramming underpins hESC decidualization , we repoiewed in . Previouiewed in  to spermiewed in , 19. Dueiewed in , 21, PLZiewed in –26. At tiewed in ; howeveriewed in –31. Recriewed in –34.in vitro [In different physiological contexts, PLZF is induced by the steroid hormones: cortisone , testostin vitro , a functin-vitro and in-vivo, endometrial stromal fibroblasts undergo proliferation and differentiation before their transformation into polygonal epithelioid decidual cells [Observed both al cells . Using aal cells , 41, a cal cells .The rapid P4-induction of PLZF transcription in uterine stromal cells suggests that this transcription factor acts as a direct functional mediator of the P4 signal. This proposal is supported by our ChIP-Seq studies which identified at least ten potential PGR binding sites that span introns 1–3 of the human PLZF gene. This DNA binding pattern concurs with previous studies which show that nuclear receptor binding sites are commonly found in introns located far beyond the confines of the promoter-proximal region of target genes , 43. Loo2-His2 zinc finger transcription factor, which belongs to an immediate early response gene family that includes EGR 2–4 [, suggesting that Egr1 may mediate the known mitogenic effects of E2 in this tissue. Interestingly, results from these studies are similar to our findings in the mouse in which Egr1 is rapidly induced to high levels by E2 in the uterus; however, we further showed that this induction is also markedly reduced by P4 with simultaneous induction of PLZF. Intriguingly, recent investigations show that unscheduled elevation of EGR1 levels compromises hESC viability in vitro [in vivo and in vitro artificial decidualization [decidual/trophoblast prolactin-related protein (Dtprp) in murine uterine stromal cells, while inhibition of Egr1 upregulated the expression of Dtprp under in vitro decidualization conditions [Recognizing the GC-rich consensus sequence GCG(T/G)GGGCG in promoter regions of target genes, EGR1  is a Cys EGR 2–4 . A wide  EGR 2–4 . For exa EGR 2–4 , suggest EGR 2–4 ; however EGR 2–4 , 50, sugin vitro , indicatin vitro , Egr1 exlization . Moreovenditions . CollectInterestingly, we demonstrate that PLZF knockdown does not change the expression levels of FOXO1A, HOXA10 or HAND2 during hESC decidualization; these three genes have been shown to be important for the hESC decidual response , 53–57. In conclusion, pregnancy success relies on the execution of a programmed sequence of events, with uterine decidualization following embryo implantation representing a crucial early event which ensures not only the establishment but also the long-term maintenance of the fetomaternal interface. Advancement in our mechanistic understanding of P4’s involvement in decidualization is predicated upon identifying the key effectors that mediate the P4 signal into a decidual response. An integrative analysis of genome-scale data, together with primary hESC studies, we disclose PLZF as a potent mediator of P4 responsiveness which is essential for hESC decidualization. We also reveal that PLZF controls EGR-1 expression levels, the perturbation of which can compromise decidual progression. Given that further understanding of the molecular mechanisms that underlie P4-driven decidualization is essential if we are to formulate new clinical modalities in the diagnosis, prognosis, and treatment of infertility based on a defective endometrium, we believe our findings offer a new mechanistic perspective on P4 responsiveness in the uterus which may contribute to novel fertility solutions in the future.in vitro decidualization studies, endometrial biopsies were obtained using a pipelle catheter or curette under sterile conditions from the uterine fundus of healthy women of reproductive age during the proliferative phase of their menstrual cycle. Subjects ranged in age between 27–38 years and had a normal uterus as evaluated by transvaginal ultrasound. Volunteers provided written informed consent prior to endometrial tissue biopsy, which was conducted in accordance with a protocol approved by the Institutional Review Board (IRB) at Baylor College of Medicine and the guidelines of the Declaration of Helsinki [For Helsinki . A portiHelsinki , 62. IsoPLZF (L-005196-00-0005), or PGR (L-006763-00-0005) or FOXO1A (L-003006-00-0005) or HAND2 (L-008698-02-0005) or HOXA10 (L-006336-00-0005)  in six-well culture plates. In each well, siRNAs (60 pmol) were transfected into hESCs using Lipofectamine 2000 reagent in 1× Opti-MEM I reduced-serum media (Invitrogen Corporation). Forty-eight hours after transfection, in vitro decidualization of hESCs was initiated by treating cells with 1× Opti-MEM I reduced-serum media containing 2% FBS, 10nM E2, 1μM MPA (Sigma-Aldrich), and 50 μM cAMP (Sigma-Aldrich) (termed EPC media). The EPC medium was changed every two days, and hESCs harvested at defined time points as indicated [PRL and IGFBP-1 transcript levels served as a positive molecular readout for hESC decidualization.For RNA interference based studies, hESCs were transfected in triplicate with non-targeting (or control) small interfering (si)RNAs (D-001810-10-05), siRNAs targeting ndicated . Quantithttp://david.abcc.ncifcrf.gov/)) and Gene Set Enrichment Analysis (GSEA) on Molecular Signatures Databases (MSigDB) collection (http://software.broadinstitute.org/gsea/index.jsp) analyses were performed on protein coding genes to identify enriched biological themes. Integration of PLZF ChIP-Seq data with RNA-Seq data sets was performed using a relational database system [Human ESCs were cultured in EPC media for 72 hours before PLZF and Input ChIP-Seq were performed by Active Motif, Inc. . Human ESCs derived from six subjects were pooled before being fixed in 4% formaldehyde, snap-frozen, and shipped on dry ice to Active Motif. A goat polyclonal anti-human PLZF antibody  was used for the PLZF ChIP assay. Enriched DNA from the ChIP assay was amplified and DNA libraries were sequenced on an Illumina platform, followed by alignment of sequenced reads (24 million reads) to the human genome . Peak calling was performed by applying a threshold of 18  before the resultant-called peaks were stored as Browser Extendable Data (BED) files. Due to the pronounced enrichment for TSS-proximal binding of PLZF by Cis-regulatory Element Annotation System (CEAS) analysis, genes associated with PLZF binding were called if the ChIP binding intervals were located within 950 bp from the TSS. Galaxy/Cistrome’s implementation of the Cis-regulatory Element Annotation System (CEAS) and Genomic Regions Enrichment of Annotations Tool (GREAT) tools were used for enrichment analyses on chromosomes and genomic annotations. Database for Annotation, Visualization, and Integrated Discovery Bioinformatics Resources (DAVID) v6.7 . Complementary DNA (cDNA) was synthesized using the Bio-Rad Reverse Transcription kit . Quantitative real time PCR analyses were performed using validated primers  and TaqMan 2× master mix; ribosomal RNA (18S) was used as an internal control for gene specific primers; the primer list is shown in For western analyses, total protein was extracted from uterine tissue using 1× protein lysis buffer , pH 7.4). An equal amount of protein extract (20 μg) was run on 4–15% gradient SDS-PAGE gels  and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 1× TBS containing 0.5% Tween-20 and 5% non-fat dry milk powder. Subsequently, membranes were incubated with rabbit polyclonal anti-PLZF  or rabbit anti-human PGR (H-190) antibody  or monoclonal anti-β-actin (Sigma-Aldrich) antibodies to detect PLZF, PGR and β-actin proteins respectively. Horseradish peroxidase-conjugated antibodies  were used as secondary antibodies and the SuperSignal West Pico Chemiluminescent Substrate kit  was used to detect the chemiluminescent signal.When hESCs reached 60–70% confluence on eight-well chamber slides in triplicate wells, media was changed to EPC media. The EPC media was changed every two days for a total of six days before hESCs were fixed in 2% paraformaldehyde and permeabilized in 0.2% Triton-X detergent. Cells were washed and blocked in 1× PBS with 0.5% normal goat serum before incubation overnight with the rabbit polyclonal anti-human PLZF primary antibody ((H-300) Santa Cruz Biotechnology, Inc.). To detect immunoreactivity, hESCs were incubated with the Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody  to visualize immunopositive cells (green fluorescence). Cell nuclei were visualized using the 4, 6-diamidino-2-phenylindole stain . Finally, slides were separated from the chambers and mounted using Slowfade mounting media before imaging.A lentiviral vector expressing EGR1 (EX-OL00487-LX304) and a control vector expressing EGFP (EX-EGFP-LX304-B) were purchased from GeneCopoeia . Lentivirus was produced in the Gene Vector Core at Baylor College of Medicine. Twenty four hours following transduction of hESCs with control virus harboring empty vector or lentivirus expressing EGR1, media was replaced with EPC media to induce decidualization; lentiviral-mediated EGR1 overexpression was confirmed by real-time PCR.Briefly, paraformaldehyde-fixed tissues were processed and embedded in paraffin as previously described . Tissue vivarium maintained by the Center for Comparative Medicine at the Baylor College of Medicine. A diet of irradiated Tekland global soy protein-free extruded rodent food pellets  and fresh water were provided ad libitum to mice. Animal studies were undertaken following the guidelines described in the Guide for the Care and Use of Laboratory Animals ). Animal protocols  used in this research received prior approval from the Institutional Animal Care and Use Committee (IACUC) at Baylor College of Medicine. The PRKO mouse has been previously described [2 euthanasia was achieved with automated CO2 euthanasia chambers (EUTHANEX).In a recurrent photocycle of 12h lights on-off, mice were housed in temperature controlled (22°C ± 2°C) rooms in an AAALAC accredited escribed . The folTo evaluate uterine responsiveness to steroid hormone, mice were ovariectomized at six weeks-of-age and then rested for two weeks to ensure removal of serum ovarian steroid hormones before administration of exogenous hormone. Subcutaneous (s.c.) injection within the interscapular region of 100 μl sesame oil (vehicle control), 1 mg P4 , 100 ng E2 (Sigma), or 1mg RU486 (Sigma) in 100 μl vehicle was used to administer hormones and the antiprogestin. Following hormone treatment, whole uterine tissue was processed from euthanized mice for RNA, protein, and/or histological analysis.post-hoc test were performed using the Instat tool package version 3.0 . Data with p values less than 0.05 were considered statistically significant.For statistical analyses, a two-tailed student’s t test and one-way ANOVA with Tukey’s S1 FigA) Table shows the change in ZBTB16 (PLZF), IGFBP1, and PRL expression in hESCs during decidualization from a recently published RNA-seq dataset [B) Relative transcript levels of PLZF in hESCs treated with MPA alone at indicated time points. C) Relative transcript levels of PLZF in hESCs pre-treated with cyclohexamide (10ug/ML) for an hour and treated with MPA for four hours as indicated. D) Comparative protein levels of PGR in hESCs cultured for zero days (lanes 1 and 2) or four days (lanes 3 and 4) or six days (lanes 5 and 6) with the decidual stimulus  or MPA and cAMP  compared to vehicle treatment cells (lanes 1 and 2). Note: The PGR isoforms (A and B) are elevated in EPC and PC treated cells; β-actin was used as loading control. dataset . The rel(TIF)Click here for additional data file.S2 FigA) Relative Plzf transcript levels in uteri from ovariectomized wild-type mice treated with sesame oil (vehicle control) or progesterone for the indicated times. B) Western blot analyses for Plzf and β-actin protein levels in uteri from ovariectomized wild-type mice treated with sesame oil (vehicle control) or progesterone for the indicated times. Note: uteri from ovariectomized PRKO mice treated with progesterone for 6 hours were used as Pgr negative controls. C) Immunohistochemical detection of Plzf in uteri from adult virgin and early pregnant (5 days post coitum (d.p.c)) mice. Note the low levels of Plzf expression in the uterus of the virgin mouse compared to striking expression levels of Plzf in the stromal compartment of uterus of the early pregnant mouse (white arrow). At this stage of pregnancy, however, expression of Plzf was not detected in the luminal or glandular epithelium (LE and GE respectively); scale bar denotes 100μm and applies to all panels. D) Relative Plzf transcript levels in uteri from ovariectomized wild type control and PRKO mice injected with sesame oil (vehicle control) or progesterone and euthanized 6 hours post-injection. Results represent the mean ± SE; n = 3 mice/group. *P<0.05, **P<0.01, ns = not significant.(TIF)Click here for additional data file.S3 FigA) Table depicting EGR1, EGR2, and EGR3 transcript expression changes during EPC-driven decidualization [B) Relative transcript levels of EGR1, EGR2, EGR3, and EGR4 in hESCs cultured for 3 days in vehicle control (red bar) or EPC cocktail (black bar). lization ; gene ex(TIF)Click here for additional data file.S4 FigImmunohistochemical detection of PLZF (A to F) and EGR1 (G to L) expression in human endometrial tissue biopsied during proliferative phase (A-C and G-I) and secretory phase (D-F and J-I) of the menstrual cycle. Black and white arrowheads indicate glandular epithelium and stroma respectively; scale bar in panel A indicates 100 μm and applies to all panels.(TIF)Click here for additional data file.S5 FigPlzf and Egr1 transcript levels in uteri from ovariectomized wild-type mice treated with sesame oil (vehicle control) or E2 (3 h) or P4E2 in presence or absence of RU486. Note: The RU486 antagonist was added 30 min prior to P4 treatment (P4 was administered 3 hours prior to E2 treatment). Results represent the mean ± SE; n = 3 mice/group. *P<0.05, **P<0.01, ns = not significant.Relative (TIF)Click here for additional data file.S6 FigA) Transcript levels of PLZF in hESCs transfected with control siRNA or FOXO1A siRNA, HAND2 siRNA, or HOXA10 siRNA and cultured in EPC media for the indicated time period. B) Efficient knockdown of FOXOA1, HAND2, and HOXA10 was confirmed with the measurement of transcript levels of FOXOA1, HAND2, and HOXA10 in hESCs transfected with siRNA against respective protein as indicated. Results are reported as the mean ± SE from triplicates. *P<0.05, **P<0.01and ns>0.05.(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.


Ventilator-associated pneumonia (VAP) is the most common infectious complication in the intensive care unit. It can increase duration of mechanical ventilation, length of stay, costs, and mortality. Improvements in the administration of empirical antibiotic therapy have potential to reduce the complications of VAP. This review will discuss the current data addressing empirical antibiotic therapy and the effect on mortality in patients with VAP. It will also address factors that could improve the administration of empirical antibiotics and directions for future research. Ventilator-associated pneumonia (VAP) is nosocomial infection that develops following at least 48 h of mechanical ventilation. It can be divided into the following two categories based on duration of mechanical ventilation: early onset VAP (occurring on days 2–4) and late onset VAP (occurring on days ≥5). A third category is based on the risk of VAP being caused by multidrug resistant pathogens, but occurring on days 2–4 . Ventilain vitro susceptibilities of the identified pathogens. Empirical regimens are deemed appropriate if the isolated pathogen(s) is sensitive (S) in vitro to at least one antibiotic. Regimens are deemed inappropriate if the pathogen(s) is intermediate (I) or resistant (R) in vitro to all of the empirical antibiotics. As inappropriate empirical antibiotic therapy (IEAT) requires a change in therapy, patients with IEAT essentially receive delayed appropriate antibiotic therapy. The duration of the delay is dependent on laboratory procedures and clinician awareness of the results. While this may seem to be an overly simplistic approach to evaluating an antibiotic regimen, appropriate empirical antibiotic therapy (AEAT) has been associated with decreased mortality in patients with many different types of infection [Empirical antibiotic therapy entails the initial selection of an antibiotic regimen that aims to be effective against any pathogen suspected of causing the infection. Empirical therapy has been classified as appropriate/adequate or inappropriate/inadequate based on the nfection ,12. A de5 or ≥106 colony forming units/milliliter (cfu/mL). The study population sizes ranged from 38 to 183 patients with a total of 1,106 patients included in the analyses. Mortality rates were reported as either crude or attributable in-hospital mortality and ranged from 8.3% to 81.6%. The considerable variability in mortality should be emphasized. Of note, the study by Clec’h et al was only able to find a benefit in AEAT in the less sick patients (Logistic Organ Dysfunction score of ≤4), suggesting that there are patient characteristics that may play an important role in the response to empirical antibiotic therapy and infection [et al [I2 = 28.5%) between studies included in the unadjusted meta-analysis and a high degree of heterogeneity (I2 = 89.2%) in the adjusted analysis. They concluded that differences in the studies require the reader to interpret the results cautiously. The VAP studies demonstrating an overall mortality benefit with administration of AEAT are listed in n [et al . They inn [et al ,24,25 ann [et al ,16,26. T5 cfu/mL. Some clinicians may argue that using this higher threshold would lead to antibiotic therapy being inappropriately withheld for patients who may have had a clinically significant infection. Together these studies included 801 [p = 0.0001) [The VAP studies that did not demonstrate an overall mortality benefit with appropriate empirical antibiotics are included in uded 801 ,29 patieuded 801  evaluatiuded 801 ,29. Thes 0.0001) . It is aEscherichia coli, Proteus mirabilis, or Klebsiella pneumoniae and evaluated the effect of aminoglycoside (either gentamicin or kanamycin) administration at varying times following inoculation. When the aminoglycoside was administered at the time of inoculation (0 h), mortality was 0%, but increased to nearly 100% as the time to antibiotic administration increased to 4–8 h following inoculation. Unfortunately, current diagnostic techniques do not immediately identify infected patients. Two studies in humans evaluated the administration of delayed appropriate empirical antibiotic therapy (DAEAT) in patients with clinically and microbiologically confirmed VAP. Unfortunately, their definitions were significantly different. Luna and colleagues defined DAEAT as appropriate therapy given within 24 h of the clinical diagnosis of VAP in patients that had a clinical pulmonary infection score (CPIS) of ≥5 on the day before the clinical diagnosis was made [i.e., CPIS ≥ 7). In other words, patients were classified as receiving DAEAT because they could have been identified as having VAP at least one day sooner using the CPIS. These patients had a mean CPIS on the day before clinical diagnosis of 6.1 ± 0.2, which is just below the proposed CPIS diagnostic threshold for VAP. Patients who received DAEAT had a higher mortality rate than those with adequate therapy that was not delayed . Based on the definitions used in this study, it is difficult to determine the exact duration of delay in antibiotic therapy. If the diagnosis of VAP should have been made on the day prior to the actual diagnosis, it could be concluded that the delay in therapy was ≥24 h. Iregui et al. defined DAEAT as antibiotic therapy that was active in vitro to the identified VAP pathogen(s), and was administered ≥24 h after the diagnostic criteria for VAP were first documented [p < 0.01), as was VAP-attributable mortality . Together, these two studies confirm the importance of administering AEAT within the first 24 h following VAP diagnosis. However, there are no data in humans defining the optimal time of empirical antibiotic administration. Logic would suggest that sooner is better.It has long been the belief of clinicians that beginning appropriate empirical antibiotic therapy as soon as possible is essential. In fact, the Surviving Sepsis Campaign recommends administering antibiotics within one hour of suspected severe sepsis or septic shock . Intereswas made . Becausecumented . PatientThe diagnosis of VAP is a complex subject and a minority (~40%) ,35 of paet al investigated gene expression in eight patients developing VAT or VAP [et al validated preliminary data from a mouse model of VAP [et al was unable to identify biomarkers that were associated with the development of infection. They noted the transcriptional signatures of 165 trauma patients contained a generalized pattern that was more related to their trauma [Methods to improve the speed at which the diagnosis is confirmed or ruled out could significantly affect the use of empirical antibiotics. The use of gram stain to confirm the diagnosis and bacterial pathogens has been evaluated with mixed results. The use of preliminary culture results has shown promise, but studies utilizing this method are from a single institution and have not been validated externally. A more detailed discussion of these two methods can be found in another review by the authors . Gene exT or VAP . They foT or VAP . Patientl of VAP  by showil of VAP . These sr trauma . A meta-analysis conducted in 2008 compared specific antibiotic regimens for the empirical treatment of VAP . The autet al used MultiExperiment Viewer software and hierarchical clustering to analyze organisms included in an antibiogram [Incorporation of local antibiotic susceptibilities is essential in monitoring the effectiveness of empirical antibiotic regimens. Guidelines from the Clinical and Laboratory Standards Institute recommend establishing a unit-specific antibiogram . This isibiogram . The sofet al studied the use of a direct E-test on BAL fluid to determine VAP pathogen susceptibilities [3 cfu/mL for PSB and TPC, 104 or 105 cfu/mL for BAL, or 106 cfu/mL for EA) [The best method to improve the timing of AEAT is to reduce the lag time between the diagnosis of VAP and the reporting of pathogen susceptibilities. Boyer bilities . They cobilities ,51,52,53bilities . IdentifThe data evaluating the effect of appropriate empirical antibiotic therapy on mortality for patients with VAP are mixed, but the majority of trials demonstrate some benefit. No specific empirical antibiotic regimen has been found to be superior, so selection should be based on local pathogen susceptibility patterns. Novel VAP diagnostic methods and pathogen susceptibility testing may enhance the clinician’s ability to improve the likelihood of administering AEAT, but further research is needed.


Daclatasvir (DCV) is a potent hepatitis C virus (HCV) NS5A replication complex inhibitor with pangenotypic (1–6) activity in vitro. Methadone (MET) and buprenorphine (BUP) are opioid medications used to treat opioid addiction; patients on HCV therapy may require MET or BUP treatment. The effect of DCV on the pharmacokinetics (PK) of MET or BUP/naloxone (NLX) was assessed in subjects on stable MET or BUP.max and AUCTAU were derived from linear mixed effects models.An open-label, two-part study assessed the effect of steady-state oral administration of DCV on the PK of MET  or BUP/NAL . Safety/tolerability and pharmacodynamics  were also assessed. Subjects  received daily single-dose oral MET (40–120mg) or BUP/NLX (8/2–24/6mg) based on their prescribed stable dose throughout, in addition to DCV (60mg QD) on Days 2–9. Serial PK sampling occurred predose and postdose till 24 hours on Day 1 (MET/BUP) and Day 10 (MET/BUP/DCV). Noncompartmental PK were derived. Geometric mean ratios (GMR) and 90% confidence intervals (90% CI) for MET/BUP/norBUP CSubjects were aged 19–39 years, mostly white  and male . All subjects completed the study. No clinically meaningful effect was demonstrated as the GMR and 90% CIs fell within the prespecified interval (P1, 0.7–1.4; P2, 0.5–2.0: see Steady-state administration of DCV 60mg QD had no clinically meaningful effect on the PK of MET or BUP/NLX and was generally well-tolerated, suggesting that no dose adjustments will be required.


Heroin addiction is a complex psychiatric disorder with a chronic course and a high relapse rate, which results from the interaction between genetic and environmental factors. Heroin addiction has a substantial heritability in its etiology; hence, identification of individuals with a high genetic propensity to heroin addiction may help prevent the occurrence and relapse of heroin addiction and its complications. The study aimed to identify a small set of genetic signatures that may reliably predict the individuals with a high genetic propensity to heroin addiction. We first measured the transcript level of 13 genes  in lymphoblastoid cell lines in a sample of 124 male heroin addicts and 124 male control subjects using real-time quantitative PCR. Seven genes  showed significant differential expression between the 2 groups. Further analysis using 3 statistical methods including logistic regression analysis, support vector machine learning analysis, and a computer software BIASLESS revealed that a set of 4 genes  could predict the diagnosis of heroin addiction with the accuracy rate around 85% in our dataset. Our findings support the idea that it is possible to identify genetic signatures of heroin addiction using a small set of expressed genes. However, the study can only be considered as a proof-of-concept study. As the establishment of lymphoblastoid cell line is a laborious and lengthy process, it would be more practical in clinical settings to identify genetic signatures for heroin addiction directly from peripheral blood cells in the future study. Hence, heroin addicts have a very high relapse rate.,4 Heroin addiction became a serious problem in southeastern and southwestern Asia recently. Like that in other countries, heroin users not only had a high recidivism rate but also had the highest mortality rate among the addictive substance users in Taiwan.,7Heroin was synthesized as a legal drug in 1895. It was found to be a highly addictive drug in the early twentieth century, and became popularly abused after the mid-twentieth century. Previous studies have demonstrated that the estimated heritability of drug addiction ranges from 0.39 to 0.72 in different drugs.,10 In a twin resemblance study for illicit psychoactive substance use, heavy use, abuse, and dependence in a US population, the heritability of illicit psychoactive substance use ranges from 60% to 80%. These findings suggest that genetic factor plays an important role in the pathogenesis of heroin addiction.Heroin addiction is a complex disorder resulting from the interactions between genetic susceptibility and environmental factors.,9 However, there is still a gap of applying the genetic knowledge of heroin addiction to help manage the patients with heroin addiction in clinical settings. The study aimed to find a small set of genes that can reliably predict the individuals with a high propensity to heroin addiction. The tests of these genes shall be conveniently implemented in the clinical laboratory, and hopefully, the assay of these genes can be used in combination with other social, familial programs to help prevent the occurrence and the relapse of heroin addiction in clinical settings.Many genes associated with heroin addiction have been reported using different approaches, supporting the high genetic heterogeneity of heroin addiction.22.1Male adult patients (≥20 years old) met the diagnostic criteria of heroin use disorder according to the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5) was recruited into this study. The diagnosis was made based on medical records and interview by senior psychiatrists with consensus. Patients co-morbid with other major psychiatric diagnoses of DSM-5 such as neurodevelopmental disorders, schizophrenia spectrum and other psychotic disorders, bipolar related disorders, depressive disorders, and neurocognitive disorders were excluded. Male adult subjects (≥20 years old) who received regular medical checkups at a local medical center were recruited into this study as controls. A senior psychiatrist evaluated their mental statuses and histories of mental illness. Those who had a history or currently had a diagnosis of major psychiatric disorders according to DSM-5 such as substance-related and addictive disorders, neurodevelopmental disorders, schizophrenia spectrum and other psychotic disorders, bipolar-related disorders, depressive disorders, and neurocognitive disorders were excluded. A total of 8 mL venous blood was collected from each subject for the establishment of lymphoblastoid cell lines (LCLs). Total RNA was extracted from the LCL for the measurement of cDNA of selected genes. The study protocol was approved by the Ethical Committee of Bali Psychiatric Center , and written informed consent was obtained after full explanation of the protocol.2.2 After the establishment of LCL, total RNA from the LCL was extracted using TRIZOL Total RNA Isolation Reagent according to manufacturer's instruction . The cDNA was prepared using Superscript II RNase H− Reverse Transcriptase following the instructions provided by the manufacturer (Invitrogen Life Technologies). The details were also described in our previous report.LCL from each subject was established by transforming the lymphocytes with Epstein–Barr virus following the procedures described in our previous paper.2.3 The relative standard curve method was used for the measurement of the gene transcript level according to the procedure described in User Bulletin #2 ABI PRISM 7700 sequence detection system (Applied Biosystems). In this method, serial dilutions of known amount of RNA from a reference sample  were used to generate the external standard curve. For each unknown sample, the relative amount was calculated using linear regression analysis from their respective standard curves. The transcript level of measured gene in each subject was normalized by his/her 18S rRNA level. The reference 18S rRNA level was measured using predeveloped TaqMan assay reagents 18S rRNA MGB according to the manufacturer's protocol (Applied Biosystems). All experiments were performed in duplicate. The sequences of all the primer sets and optimal annealing temperature used for RT-qPCR are listed in Table A total of 13 genes  were measured in this study. The genetic transcript level was measured using real-time quantitative PCR (RT-qPCR) that was performed by SYBR Green method and implemented in StepOnePlus Real-Time PCR System according to the manufacturer's protocol . The detailed procedures were described in our previous report.2.4 The BIASLESS is a software developed for integrative analysis of single nucleotide polymorphisms (SNP) and gene expression, which is an efficient tool for selecting key SNP and gene expression markers and then building models for sample subdivision.To cross-validate the findings of genetic signatures for heroin addiction, we used 3 statistical methods to search for the genes that can reliably predict the diagnosis of heroin addiction in this dataset, including multiple logistic regression (LR) analysis, support vector machine (SVM) method, and the online computer program BIASLESS (Biomarkers Identification and Samples Subdivision). LR analysis was implemented using the Statistical Package for the Social Science V18.0 . The SVM model analysis was implemented using the LIBSVM package by Chang and Lin.33.1All the subjects were Han Chinese from Taiwan. A total of 124 male adults (37.5 ± 9.4 years) fulfilling the diagnostic criteria for heroin use disorders as defined by the DSM-5 were recruited into this study. Also, a total of 124 male adults (42.9 ± 14.6 years) without history or current diagnosis of substance use disorders and other major psychiatric diagnoses were recruited as the control groups. Their LCLs were established, cDNAs were prepared for the genetic assessment.3.2A total of 13 genetic transcript levels were measured for each subject and normalized by the total amount of their 18S rRNA. The mean transcript levels of each of the 13 genes in the patient and control groups are listed in Table 3.3In LR analysis, subjects with and without the diagnosis of heroin addiction were regressed with a linear combination of the gene expression levels. The accuracy rates of different models containing 13, 7, 5, and 4 genes are listed in Table 3.4In SVM analysis, we conducted the kernel function as radial basis function as it is the most accepted in a similar analysis. We used leave-one-out cross-validation on the patient gene expression dataset, and all subjects were built-in for both training and testing using the same formula. Individuals in the patient and control groups were randomly partitioned into 10 subsets for a cross-validation. Nine subsets formed a training set and the remaining 1 subset function a testing set. The training set was used to classify individuals in a testing dataset and calculated a testing accuracy. The accuracy rates of different gene combinations are summarized in Table 3.5In the analysis using BIALESS software, the data were run with the default parameter values. For each gene, gene expression level was normalized by subtracting the sample mean and dividing by the sample standard deviation of all 248 individuals (from 6 batches). Individuals in the patient and control groups were randomly partitioned into 10 subsets for a cross-validation. Nine subsets formed a training set, and the remaining 1 subset was a testing set. A flexible discrimination analysis was applied to the training set first. The genes with the highest increment of training accuracy were included into the classification model sequentially until the training accuracy reached 1.0 or its increment was less than a threshold of 0.001. Next, the classification model in the training set was used to classify individuals in a testing dataset and calculated a testing accuracy. The previous procedures were repeated until each of the 10 subsets had been analyzed as a testing dataset. Finally, among the 10 candidate classification models, the 1 with the highest cross-validation consistency was selected as the best classification model in this study. A leave-one-out testing accuracy of the best model was calculated.Our classification analysis identified JUN, CEBPG, ENO2, and PRKCB as the key gene expression signatures for a subdivision of heroin dependence and controls. The suggested classification model with 4 gene signatures had a cross-validation consistency of 4/10 in our 10-fold cross-validation analysis. The average training accuracy rate was 86.7%, and the leave-one-out testing accuracy was 85.5%. Distributions of gene expression levels in the heroin dependence and control groups are displayed in box-whisker plots  signaling were found among in these 3 groups of alcohol drinkers  using expression array technology. Our group also conducted a comparative gene expression profiling analysis of LCLs in a small sample of male heroin addicts and male control subjects previously. We detected 924 differentially expressed gene transcripts between these 2 groups, including 279 upregulated and 645 downregulated gene transcripts in individuals with heroin dependence compared with control subjects. These findings support the complex polygenic nature of heroin addiction.Heroin addiction, like the other substance use disorders, is a complex disorder resulting from the interplays between environment and genetic predisposition. An 8-gene expression signature could help to predict the survival and time to the treatment of chronic lymphocytic leukemia, and a 6-gene signature was found to be able to distinguish 4 subgroups of neuroblastoma. In drug abuse, no such study was reported yet in the literature to our knowledge. Hence, the study aimed to test the hypothesis whether a small set of signature genes can reliably differentiate heroin addicts from control subjects.Results from microarray-based gene expression studies usually contain dozens of genes, which are difficult to be applied in clinical settings. Hence, several groups have attempted to use a small set of genes as signatures to represent the findings from microarray-based studies. For example, a 21-gene assay was found helpful to assist in adjuvant treatment decision in hormone receptor-positive breast cancer patients. We originally intended to validate the differentially expressed genes found in expression microarray using RT-qPCR. Then, we came up with the idea that if we could find a small set of genes that could reliably predict the individuals with a high genetic propensity to heroin addiction, which would be useful in preventing the occurrence and relapse of heroin addiction. To test this hypothesis, we randomly selected 13 genes based on our preliminary study and measured the transcript levels of 13 genes in 124 male heroin addicts and 124 male control subjects using RT-qPCR. Among these 13 genes, we found 7 genes were differentially expressed that reached statistical significance after correcting for multiple testing. These 7 genes were further analyzed for building genetic signatures using 3 different statistical methods. We finally obtained 4-gene signatures that can predict the diagnosis of heroin addiction with the accuracy rate of around 85% in our dataset.The study is an extension of our previous study of comparative gene expression profiling analysis of LCLs in a small sample of 20 male heroin addicts and 20 male control subjects.These 4 genes include JUN, CEBPB, ENO2, and PRKCB in LR modeling and SVM analysis while in the analysis using BIALESS computer program, the CEBPB gene was replaced by the CEBPG. The discrepancy between these methods can be attributed to the different algorithms of these methods. Nevertheless, the identification of these genes supports the idea that a small set of gene transcripts can function as genetic signatures of heroin addiction. However, no other studies reported elevated intrinsic JUN in heroin addicts to our knowledge. Both CEPBP and CEPBG are members of CEPB family that contain a leucine zipper domain to form homodimer and heterodimer with each other. These 2 transcription factors are involved in many physiological and pathological conditions in humans.–25 PRKCB is a member of the protein kinase C (PRKC) family that are threonine-specific protein kinases that play an important role in signal transduction, regulation of gene expression, and control of cell division and differentiation. Studies reported that PRCKB played an essential role in dendritic cell differentiation and was involved in the pathogenesis of autism.,28 However, the relation between PRKCB and substance use disorders remains to be studied. Enolase is involved in the glycolysis and gluconeogenesis pathways. Human enolase is a homodimeric protein complex consisted of 2 subunits from 3 isoforms of enolase that are encoded by ENO1, ENO3, and ENO2, respectively. Tannu et al reported increased gamma enolase that was encoded by the ENO2 in the nucleus accumbens of cocaine overdose victims compared with control subjects, suggesting the involvement of the ENO2 in the pathophysiology of cocaine dependence. Our previous study also indicated that ENO2 was associated with heroin dependence. These signature genes may not directly link to heroin addiction at first look; they may represent the complex genetic pathways underlying the genetic disposition of heroin addiction. Hence, a combination of the expression of these signature genes can reliably distinct heroin addiction from control subjects with high accuracy rate as shown in this study.The interpretation of the biological significance of these signature genes of heroin addiction can be intriguing. JUN is proto-oncogene and a transcription factor that regulates the expression of its target genes. A recent paper reported that increased c-Jun expression in the cerebellum of rats under long-term heroin administration, which was attributed to be one of the mechanisms underlying the heroin-induced cerebellum neuronal damage. SVM is a supervised machine learning algorithm that is used for both linear and nonlinear classification. Interestingly, both LR and SVM analyses obtain the same 4 genes that had the accuracy rate of approximately 85.5% and 83.9%, respectively, supporting the robustness of the 4-gene signatures of heroin addiction. In the analysis using BIASLESS, the CEBPB was replaced by the CEBPG. Still, the 4-gene model had the accuracy rate of 85.5% in predicting the diagnosis of heroin addiction.LR is a powerful modeling tool for predicting the outcome; it assumes a linear relationship between predicting variables and outcome. Hence, the specificity of our findings in this study should be tested in another kind of substance use disorders. Also, the present study only measured the gene expression at mRNA level, which may not represent the gene expression at the protein level. It would be interesting to examine the changes of these signature genes at protein level in the postmortem brains of heroin addicts or the brains of rodents under long-term heroin administration in the future study.The study has several limitations. Due to limited resources, we were not able to measure more gene transcripts to find better genetic signatures with higher accuracy rate than the current findings in the prediction of heroin addiction. Also, the relatively small sample size can also affect the predicting power of our model in this study. Furthermore, the gene signatures found in this study may not be specific for heroin addiction, as substance use disorders share some common genetic susceptibility.Heroin addiction is a complicated result from interactions between genetic susceptibility and environmental factors. To reduce the confounding environment factors, we used LCLs as our experimental materials instead of peripheral blood cells, because the LCL was cultured in the medium for a long period, the influence of environmental factors was controlled to the minimum. Hence, the genes identified in this study can be considered as trait markers than as state markers. Also, the establishment of LCLs is a laborious and lengthy process, which also limits its clinical use. Hence, this study can only be considered as a proof-of-concept study. We suggest that it would be more useful in clinical settings if the future study can identify a small set of signature genes of heroin addiction from peripheral blood cells rather than from LCL.In conclusion, the present study suggests the feasibility of detecting a small set of genetic signatures from peripheral tissues that can classify the diagnosis of heroin addiction with reasonable accuracy rate. The approach in this study shall facilitate the search for trait genetic markers and biomarkers of heroin addiction, and promote the translational research of heroin addiction in clinical settings.


To investigate the time-dependent changes in plasma levels of interleukin-6, C-reactive protein, and tumor necrosis factor-α in patients with type A aortic dissection (TAAD) who received unoptimal medical management since the onset of dissections.Plasma levels of interleukin-6, C-reactive protein, and tumor necrosis factor-α were detected by ELISA and immuno-turbidimetric assay in 92 TAAD patients at hospital admission. Blood samples from 78 patients with uncontrolled hypertension and 82 healthy volunteers were also analyzed as controls. The occurrence of TAAD-related complication and its relationship with the plasma levels of these inflammatory biomarkers was also investigated.2/FiO2 ratio  and creatinine . The time to the peak level of CRP was shorter and the duration of persistently high CRP level was longer in the complication group than those in the complication-free group.The concentrations of inflammatory mediators were significant higher in TAAD than those in the uncontrolled hypertension and the healthy group. The time to peak plasma level of IL-6.and TNF-α was shorter than that of CRP in TAAD group. In the TAAD group, 51 patients suffered TAAD-related complications, and their plasma level of CRP was significantly higher than that in patients without TAAD-related complications . Also, CRP levels strongly correlated with the value of PaOElevated and persistently high levels of plasma CRP, IL-6 and TNF-α were associated with progressively development of the TAAD. The changing pattern of CRP might be a marker for diagnosis and prophylactic treatment of complications. Our findings suggested a critical role of the inflammation in the progression of dissection and TAAD-related complications. The Stanford type A aortic dissection (TAAD) is a catastrophic cardiovascular condition associated with severe morbidity and mortality ,2. PatieAortic dissection is characterized by medial degeneration with intimal tear and crossing of blood into the artery wall, leading to the formation of a false lumen and subsequent systemic inflammatory responses . InterleThe present study was a prospective, single-center, observational study. From August 2013 to February 2014, 156 patients admitted with TAAD at West China hospital were screened. Of these patients, 138 patients (88.5%) were transferred from rural and community hospitals and patients there received unoptimal medical management. After admission, all patients were managed according to current guidelines and 118 patients (75.6%) were treated surgically. Patients received stringent anti-hypertensive management, or treatment with statins, angiotensin-converting enzyme inhibitors or angiotensin receptor blocker within last 3 months at the time of enrollment were excluded. Moreover, patients with history of autoimmune or inflammatory systemic disease were also excluded. Of the 156 patients screened for participation, 21 patients refused participation in the study, 43 patients met at least one exclusion criteria, leaving 92 patients in the study (TAAD group). Meanwhile, 78 patients with uncontrolled hypertension alone (uHT group) and 82 healthy volunteers (Control group) selected among those attending our outpatient department were also enrolled in the present study. Our investigation was approved by the Institutional Review Board of West China Hospital in compliance with the Declaration of Helsinki (reference number: 2012150). Written informed consent was obtained from all patients before inclusion in this study.2/FiO2 ratio ≤ 300 was considered to be lung injury [The diagnosis of TAAD was confirmed in all patients according to the Stanford classification. Patients were divided according to the time of admission since the onset of symptoms   according to the manufacturer’s instructions. The plasma level of CRP was determined by immuno-turbidimetric assay . The detection limits were 0.5 pg/ml, 0.7 pg/ml and 0.17 mg/L for TNF -α, IL-6 and CRP, respectively.t test for normally distributed data, and Mann–Whitney U-test was used for the comparison of parameters that did not exhibit a normal distribution. Categorical variables were compared using Chi-square test or Fisher’s exact test or other nonparametric tests as appropriate. The relationship between the values of inflammatory biomarkers and the incidence of TAAD-related complications was assessed by Spearman’s correlation. Two-tailed p value less than 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 16.0 .Continuous variables are expressed as mean ± standard deviation, and categorical data as percentages or absolute numbers unless otherwise specified. Independent continuous variables were compared by unpaired Student’s Apart from the plasma levels of inflammatory cytokines at admission, no significant differences were observed regarding age, sex and other clinical variables between the TAAD group and the uHT group. In addition, patients in the TAAD and uHT groups tended to be older than healthy volunteers. Baseline data of the TAAD group and other 2 control groups are summarized in Table The concentration of IL-6 was significantly higher in patients with TAAD than that in the uHT  and Control group . The level of CRP was significantly elevated in TAAD patients (68.02 ± 35.75 mg/L) compared with those in the uHT (4.68 ± 3.89 mg/L) and Control groups (3.08 ± 2.46 mg/L). Similarly, a significant increase of the plasma level of TNF-α was detected in TAAD patients (Table Of the 92 TAAD patients recruited in our study Table .Table 22/FiO2 ratio and serum creatinine. The time-dependent changes of CRP level in the complicationgroup (51 cases) and complication-free group (41 cases) were shown in Figure In TAAD patients, a significant negative correlation was found between CRP expression and PaO2/FiO2 ratio  Figure A, whereaee group 1 cases wIn the present study, we showed that the plasma levels of CRP, IL-6 and TNF-α were significantly increased in acute TAAD patients (before T6) compared to those in uncontrolled hypertension and healthy volunteers. The expression levels of these inflammatory cytokines peaked at the acute phase of TAAD and gradually declined at different stages of TAAD progression. More importantly, the time-dependent changes of CRP level, particularly the CRP pattern provided more information in terms of prediction of TAAD-related inflammatory complications. Our data strongly suggested a critical role of inflammatory responses in the development of TAAD and resultant systemic complications.th day (T5) after symptoms onset and declined thereafter. Secondly, because CRP is a non-specific inflammatory marker, it reflects not only the TAAD itself but also systemic inflammatory responses, leadig to pulmonary or renal injury [CRP, a circulating and nonspecific inflammatory biomarker, has been shown to be elevated and varied with different stages of aortic dissection ,11-13. Pl injury ,15. MoreSignificant elevation of IL-6 and TNF-α levels were also detected in patients admitted at the acute phase of TAAD (before T7) when compared with other 2 control groups. However, in patients with chronic TAAD (T7-T9), no significant difference between the chronic TAAD subgroup and the uHT group was observed. In addition, the changing patterns of these two inflammatory cytokines were slightly different. Nevertheless, the plasma levels of IL-6 and TNF-α both peaked during the 12 hrs to 24 hrs period, which was much earlier than CRP. Current evidence demonstrated a significant infiltration of macrophages and neutrophils in the dissected aortic wall . These rIn the current study, the average values of CRP, IL-6 and TNF-α in patients with chronic TAAD and hypertension tended to be higher than those in the healthy controls . As the mechanism of TAAD remained large unknown , our finThere are several limitations in the present study. Firstly, because of the absence of serial blood samples spanning the first few hours period of TAAD progression, it is difficult for us to fully elucidate the changing patterns of CRP, IL-6.and TNF-α and their clinical values for diagnosis and prophylactic treatment of complications. Secondly, in order to avoid significant influences of several known drugs on the expression levels of inflammatory biomarkers, we recruited TAAD patients without optimal medical management. However, the rationality of the inclusion criteria remains to be fully discussed. Nevertheless, to the best of our knowledge, the present study firstly provided information on the changes of the inflammatory biomarkers, which were less affected by anti-hypertensive and anti-inflammatory medications. In addition, we observed such a surprisingly high percentage of inappropriately treated patients from rural areas , indicating the urgent necessity and importance of the primary prevention for hypertension-related disease in the rural areas of China. Although this study was strengthened by its prospective design, the patient population is relatively small, and we cannot exclude the possibility that other confounders such as genetic factors (Marfan syndrome etc.), smoking, diabetes and use of β-blockers and missed medication history may have influenced our results.In this small sample size, prospective study, we demonstrated that increased plasma CRP, IL-6 and TNF-α were significantly associated with TAAD. More importantly, the changing pattern of CRP might be a marker for diagnosis and prophylactic treatment of complications. Taken together, our data clearly indicated the critical role of inflammation in the progression of dissection and TAAD-related complications.Written informed consent was obtained from the patients for publication of this study. Copies of the written consent are available for review by the Editor-in-Chief of this journal.


Policy, interventions and research concerning southern African children remain dominated by a focus on AIDS-related orphanhood, although the association between orphanhood and disadvantage is highly questionable. I argue that the trope of the AIDS orphan serves a range of agendas, including for academic research. In particular, orphans represent the quintessential child-agent, celebrated in fairytales and fiction. Finally, I examine how this has led to a policy response – education bursaries – that cannot adequately address childhood poverty in the region. Over the past decade, public policy and non-governmental organisation (NGO) interventions relating to children in southern Africa have coalesced around a population of ‘orphans and vulnerable children’, self-evidently produced by the region’s AIDS pandemic. This policy focus is paralleled in burgeoning academic research on the impacts of AIDS and various dimensions and outcomes of orphanhood . Such a perspective is recognised to be problematic because it neglects the structural poverty that affects both orphans and many other children . Yet, I I begin by presenting evidence that contests the significance of orphanhood in determining disadvantage, and then move on to explore why AIDS orphans have been afforded such attention in both policy and research. I explore how the focus on orphans prefigures the adoption of educational bursaries as a solution – a solution that fails both to mitigate any individual disadvantage experienced by AIDS orphans and to address the structural poverty that shapes the lives of a much greater number of southern African youth. I conclude by advocating that childhood studies might usefully adopt a social justice lens, although this could require an alternative methodological approach.1 a research project in Malawi and Lesotho that sought to explore the processes through which AIDS impacts on food insecurity. In 2007 and 2008, I undertook, with colleagues,2 (we categorised these as ‘AIDS-affected’) and half had not (these were categorised as ‘unaffected’). The young people participated in nine research activities, most of which involved them in self/group-directed production of a diagram, visual or dramatic output. The majority of older youth (aged 18–24) from both villages, irrespective of whether they had participated in the initial research activities, subsequently took part in life history interviews.Our research set out to examine these mechanisms among young people in one rural village in each country. We surveyed all households, collecting individual and household level data. Based on this data, in each village we selected a broadly representative sample of approximately 40 young participants, more than half of all those aged 10–24. Half of the participants had experienced the long-term chronic illness and/or premature death of a close adult family memberThe research very quickly confounded our expectations that there would be observable differences between the young people we defined as ‘AIDS-affected’ and those we defined as ‘unaffected’. We had anticipated that those affected by AIDS would more likely have left school prematurely to care for relatives, work for their households or to earn an income. Equally, we expected AIDS-affected young people to lack the skills or resources required for more lucrative occupations. None of these expectations held true. While our research was certainly not statistically representative, we were struck by the absence of any clear relationship between being ‘AIDS-affected’ and individual disadvantage in accessing livelihood opportunities.Agricultural land in both communities was scarce. While a minority of young people had their own fields, few had sufficient land to support a livelihood. Although orphanhood had an impact, it was one among many factors shaping access to land and did not operate in a predictable way to deprive young people of their inheritance. In neither country is land generally transferred only at the point at which the land-holder dies. Practices differ between the two contexts and gender is a key determinant. In Lesotho, fields are traditionally allocated to a man when he marries, but today household land is usually passed entirely to the eldest son. Despite recent legal changes, very few women held land in their own right. In Malawi, by contrast, girls are often allocated a small field before marriage, and men generally need to marry to access land through their wives. The effects of AIDS were not clear-cut. While some young people complained they had been denied access to their parents’ land when they were orphaned, ostensibly because they were too young to make use of it, others had acquired land prematurely, and found themselves with access to a resource that most of their contemporaries lacked.Similar stories were told about accessing other resources. Where parents had engaged in artisanal activities, some children told of their struggles to maintain ownership of equipment – struggles that sometimes failed. In general, however, very few young people had access to productive resources, irrespective of their family status.In terms of accessing livelihood skills and knowledge, most young people said that they learned skills not from parents but from peers. Moreover, most children who could not reside with parents stayed with grandparents, aunts, uncles or stepparents and were certainly not spared the opportunity to engage in agricultural and other forms of work to support their households. They doubtless picked up skills they might otherwise have acquired by working with and for their parents.Not only was it impossible to identify generalised disadvantages among the AIDS-affected young people in terms of their access to livelihood resources; livelihood outcomes too were indistinguishable from those of unaffected youth. In Malawi, roughly equal numbers of affected and unaffected young people were engaged in small businesses, in irrigated (and therefore more lucrative) agriculture and in casual work. In Lesotho, affected and unaffected boys were equally likely to be employed herding.The only striking distinction between the two groups, particularly in Lesotho, was that AIDS-affected young people were more likely to attend school . AlthougWhile novel in examining impacts on livelihoods, ours is not the only empirical research to question the association between AIDS orphanhood and individual disadvantage. Relatedly, research has confounded common misconceptions about AIDS and orphanhood. OVC [orphans and vulnerable children] is not simply an innocuous bureaucratic label created to measure parental death and child vulnerability across diverse contexts; it also shapes thinking about who the world’s vulnerable children are, and to funnel global funds to support them. (p. 546)Some scholars have observed that by obscuring the effects of poverty, focusing on AIDS orphanhood is positively harmful. The discourse around orphanhood has not remained entirely static. Rather than AIDS orphans, most organisations now refer to ‘orphans and (other) vulnerable children’ (OVC). The last ‘Children on the Brink’ report  clearly The attention given to orphans in the international child protection discourse suggests that orphanhood is a major, if not the major factor affecting child vulnerability in sub-Saharan Africa. : 537A growing body of research recognises that this is not the case, but little attention has been paid to the reasons for the singular focus on AIDS orphanhood. I suggest below a number of possible explanations, and some of the international and national agendas that are served by the trope of the AIDS orphan.From a Western perspective, alarm at the scale and possible consequences of orphanhood is unsurprising. In the early 2000s, a third or more of adults in some African countries were believed to have contracted a disease that would kill them within a decade. Many children would be left with neither biological parent. Although orphanhood had been relatively common in poorer countries, the spectre of the AIDS pandemic constructed it as an issue of global concern.Orphanhood is particularly concerning to Western society for several reasons. First, in the West, nuclear families have long predominated and are viewed as the ‘natural’ milieu for child rearing. Where almost all children live with at least one parent, parents are viewed as indispensable to the successful raising of children. Second, attachment theory – the idea that every child requires a near-exclusive relationship with a mother or ‘permanent mother-substitute’ for its first few years – has been influential since the mid-20th century. Third, both folklore and research  suggest 3 it also serves a functional role. The AIDS orphan has become the ‘quintessential vulnerable child’ , explaining that children may react to stress through aggressive and anti-social behaviour, and arguing that the crisis may compromise countries’ development prospects. The motivation for focusing on AIDS orphans relates not only to their appeal as a humanitarian cause but also reflects fear of the potential social consequences. 4 Many other multi-lateral and bilateral donors, UN agencies, NGOs and philanthropic organisations also devote substantial funds to AIDS. Work with orphans holds a particular appeal to these organisations. While HIV prevention interventions can be highly controversial, and many aspects of AIDS remain sensitive or stigmatised, donors, NGOs and governments are generally happy to be associated with child-saving. Detailed data on funding allocations are difficult to obtain, but PEPFAR sets aside 10% of its programme funding to address the needs of orphans and vulnerable children and claims to have supported more than 5 million of them thus far  spent over $5 billion on bilateral HIV/AIDS programmes in 2012 . The Glothus far . Similar5Not only are children appealing to funders of AIDS projects, but those working with children see the funding attached to the theme of HIV and AIDS as a valuable resource. Much work with children in Africa is thus refracted through a lens of ‘OVC’ in order to secure funding see . UNICEF,Funding of interventions for AIDS orphans relates not only to their popular appeal, but also serves the political interests of donors committed to neoliberal agendas. Both childhood and the aid industry are depoliticised in popular discourse although beneath the surface both are highly political . HighligResponses to AIDS orphanhood, therefore, do not engage with systemic causes of poverty. In line with neoliberal thinking, responsibility for poverty and disadvantage is located in individuals and families rather than social, economic or political processes, and Western governments or society are certainly not implicated. Moreover, rather than transforming the structures that make people vulnerable, solutions are seen to lie in the appropriate targeting of individuals. Before undertaking our field research in Lesotho and Malawi, we consulted potential ‘stakeholders’ – representatives of government, UN agencies and NGOs. These stakeholder groups were particularly concerned that our research findings should help them with targeting.6 The task is simply to bring orphaned children to the same living standards as non-orphans – which is clearly a less demanding task than resolving poverty.As Interwoven with policy agendas is research, which often finds funding and seeks to make ‘impact’ in and through the same organisations that fund interventions. Below, I outline first how academic research is at times distorted by those seeking support for their own agendas, and then, more significantly, the problems that arise from the nature of the research that has been undertaken in relation to children in southern Africa.The reasons researchers focus on the impacts of AIDS on children reflect many of the contextual factors noted above. Orphanhood, from a Western perspective, is an alarming aberration from expectations of childhood. There is a large amount of funding available associated with AIDS; it is a particularly useful source of funding for children-focused research. It is also likely that the emphasis on ‘impact’ in funding decisions and in the assessment of research quality has encouraged researchers to work in areas that are of interest to policy makers and service providers.The point I want to argue in this article, however, is that researchers are drawn to AIDS-affected children, and orphans in particular, because they are viewed as particularly illustrative of children’s social agency. I will mention a few recent examples. The celebration of children’s agency through such research is not only a reflection of the realities of the lives of the children concerned, but is also, ironically, intended to contest the representation of AIDS-affected children as vulnerable and passive. Seeking to elaborate a key tenet of the ‘new social studies of childhood’  – that c7 Orphans also feature in classic fiction  and almost all comic book superheroes are orphans , succeeding against all odds, in face of a wicked adult society. orphans . Such ch8 While challenging the stereotype is doubtless valuable, the attention afforded to AIDS-affected children is problematic for the reasons outlined above: it deflects attention from the poverty that is experienced by many non-orphans and risks commodifying those who are orphaned. It also, I will argue, serves the same agendas as the policy focus on orphans that are set out above.Childhood studies research with AIDS-affected children echoes these fictional depictions in focusing on their social agency. In so doing, it has simultaneously undermined the stereotype of the vulnerable orphan and contributed to a body of examples of children’s agency.Returning to the fairy tale orphan, s/he has three further characteristics that recur in research accounts: s/he is both separate and different from the rest of society, and s/he acts alone . First, Second, fairytales represent orphans as fundamentally different from their peers – an ‘eternal other’ . This inFinally (and relatedly), fairytale orphans usually act alone. They are profoundly independent and isolated individuals. This emphasis on the individual hero fits tidily with the neoliberal ideal of autonomy, and for this reason, research that celebrates agency can serve a neoliberal agenda as much as the policy approaches outlined above. Focusing on the ways in which the freedoms children exercise are discursively constructed in policy and research, a number of authors have suggested that the focus on children as social actors is just another historical construction of childhood, related to the international economy and the free market’s need for autonomous entrepreneurial individuals or rational unitary subjects. This neoliberal focus on autonomy is problematic because, like the focus on orphanhood as a technical question, it shifts attention away from the structural causes of hardship. To draw together the previous two sections, I return to the data from our research to explore the outcomes of a policy focus on orphanhood. Once orphanhood is defined as a significant problem, interventions targeted towards orphans are required. Targeting is a means of addressing poverty as an individualised issue that affects some within a community, but not others, and which can be addressed on an individual basis.9 The decision to focus funding on education reflects a number of assumptions: that orphans are less likely to enrol (or remain enrolled) in secondary school than other children; that the economic cost of education is an obstacle for them; and most significantly, that failure to receive an education will disadvantage them, and perhaps wider society, in the future.In both Malawi and Lesotho, a substantial share of the finance devoted to AIDS orphans has been invested in the provision of bursaries for secondary education. Such bursaries are provided by philanthropic organisations  or by the World Bank and bilateral donors via the Ministry of Education and Training (in Lesotho).The assumption is that orphans are different – less ‘advantaged’ than other children. School attendance will build their human capital and enhance their autonomy. It is, however, a highly individualised solution that relates only to those targeted and which casts responsibility for the future onto the individual. Perhaps more than other forms of neoliberal social policy, education systems are geared to the production of autonomous, individual neoliberal subjects . VandenbAlthough, assisted in many cases by bursaries, the AIDS-affected young people in both case study villages had on average pursued their education further than those we deemed unaffected, none of those still resident in the villages had been able to employ that education to their own advantage. While we do not have information concerning young people from the Malawian village who were resident elsewhere, evidence from Lesotho suggested that those with secondary education were no more likely than their peers to find lucrative employment outside their own communities. Thus, although orphans are increasingly accessing schooling, schooling itself does not appear to have mitigated their disadvantage. Rather than addressing social injustice, bursary schemes incorporate orphans into a mechanism that functions to filter off a very small occupational elite but neglects the interests of the majority of poor children . While qThe purpose of this article is not to downplay the trauma and hardship that many children face as a consequence of orphanhood, but rather to highlight how the trauma and hardship many children face for other reasons are currently obscured by a narrow focus of both research and policy on orphanhood. I have argued that the reason for this narrow focus relates in part to the tendency through the duration of the AIDS pandemic for those engaged in childhood studies to frame their research in relation to the concept of agency or the idea that children are social actors. While this lens has highlighted important aspects of the lives of such young people, it has failed to shed so much light on children who less obviously embody agency. It has also supported interventions that fail both to address the structural causes of widespread child poverty and to remediate any disadvantage experienced by individuals as a consequence of their orphan status. Thus not only does such research draw further (arguably undue) attention to AIDS orphanhood, but it also, inadvertently, advances a neoliberal agenda, locating poverty in the individual and proposing ‘solutions’ that separate out categories of youth and focus on expanding individual autonomy.What type of research is required if we are to more adequately understand the pervasive poverty experienced by young people across the southern African region? I would argue that rather than honing in on young people’s agency, research should adopt a social justice lens to examine the contextually situated processes through which poor southern African children are systematically oppressed see . Young (


Histological examination is essential for definitive diagnosis, as other pathological conditions can also present with serum IgG4 elevation. However, IgG4-RD frequently involves vital or internal organs that are difficult to perform biopsies. We herein report a unique case of IgG4-RD involving vital organs that could be successfully diagnosed by alternative lip biopsy, an accessible, little invasive procedure, despite no apparent manifestation demonstrating the involvement in labial salivary gland.Immunoglobulin G4-related disease (IgG4-RD) is a recently recognized new disease entity characterized by elevated serum IgG4 and infiltration of IgG4+ plasma cells leading to the diagnosis with IgG4-RD. Treatment with prednisolone resulted in the remarkable improvement of organ involvements and the normalization of serum IgG4 level after 3 months. Prednisolone was gradually tapered without the relapse of disease.A 60-year-old man with swelling of both submandibular glands and elevated serum creatinine level visited our hospital. His labial salivary glands appeared normal. His blood test showed high serum IgG4, and positron-emission computed tomography revealed abnormal uptake in submandibular glands, periaorta, and left kidney with hydronephrosis. We suspected him of IgG4-RD; however, the involved organs were difficult to approach for histological examination. Alternatively, we performed lip biopsy and proved massive infiltration of IgG4The early recognition and diagnosis of IgG4-RD is clinically important because delay in the treatment initiation leads to fibrosis with irreversible organ damage. Our case highlights the possibility that lip biopsy is a promising option for histological examination in patients with IgG4-RD in whom affected organs are difficult to access, leading to early diagnosis with appropriate treatment. More recently, international consensus guidance statement on the management and treatment of IgG4-RD has also strongly recommended the diagnostic confirmation by biopsy for the exclusion of malignancies and other IgG4-RD mimics. However, vital organ involvement sometimes causes difficulty in obtaining specimen necessary for the pathological diagnosis. Delay in the diagnosis and the treatment initiation leads to fibrosis with irreversible organ damage in this disease. In this article, we report a unique case of IgG4-RD involving vital, inaccessible organs that could be successfully diagnosed with alternative lip biopsy, leading to the early diagnosis with appropriate treatment.Immunoglobulin G4-related disease (IgG4-RD) is a new disease entity characterized by elevated serum IgG4 and infiltration of IgG42A 60-year-old man with past history of allergic rhinitis noticed the swelling of bilateral submandibular glands (SMGs) in June 2014. Serum creatinine elevation (1.37 mg/dL) was pointed out at routine health checkup around the time and was admitted to our hospital for work-up.Physical examination revealed normal blood pressure 127/83 mm Hg and body temperature 36.6°C. The findings of ocular, lung, cardiovascular, abdominal, neurological, and skin examination were normal. Although his bilateral SMGs were swelling with no tenderness, his labial salivary gland (LSG) appeared normal.Laboratory tests revealed elevated serum creatinine , IgG , IgG4 , and slightly elevated C-reactive protein . Other blood tests, including blood count, serum electrolyte levels, liver enzyme levels, and blood glucose were within normal ranges. Antinuclear antibody, anti-SS-A, and SS-B antibodies were all negative. Urinalysis showed neither proteinuria, occult blood, white blood cells nor casts.Computed tomography (CT) revealed enlarged bilateral SMGs and the left kidney, the left hydronephrosis, and high-density area at the periaorta soft tissue Fig. A. Positr Thus, we performed less invasive lip biopsy in an attempt to obtain pathological specimen with patient agreement. Histological examination of his LSG revealed heavy infiltration of lymphocytes and hyperplastic germinal center formation  of prednisolone was initiated. Three months later, although the reevaluation of LSG tissue after glucocorticoid treatment was not conducted, the swelling of SMGs and left kidney and periaortitis in CT remarkably improved (Fig. We diagnosed him as IgG4-RD based on the 2011 comprehensive diagnostic criteria,3The present case suggested that LSG tissues obtained by lip biopsy can be one of the options for the pathological diagnosis of IgG4-RD when performing biopsies of underlying organs is difficult, even though physical and image examinations did not suggest the LSG involvement.–7 However, tissues from deep organs such as pancreas, retroperitoneum, kidney, ocular cavity, and periaorta are difficult to perform biopsy. In addition, IgG4-RD mainly affects middle-aged to elderly men who often have comorbidities, which places them at risk for complications. As was the present case, when the difficulty, the contraindication, or the risk in access to affected organs is tangible, lip biopsy could be tried for the definitive diagnosis of IgG4-RD because lip biopsy is less invasive and easy to perform.The importance of pathological examination for diagnosis of IgG4-RD has been increasingly recognized because other diseases mimicking IgG4-RD sometimes present with elevated serum IgG4. Abe et al also reported the sensitivity of lip biopsy as 63.0%, and more recently, Moriyama et al reported the sensitivity of lip biopsy as 55.6% for IgG4-RD.,15 Likewise, a case with only retroperitoneal fibrosis without Mikulicz's disease was successfully diagnosed by a lip biopsy. Another case report also indicated the usefulness of lip biopsy to support the diagnosis of IgG4-RD. In the previous study, 40.0% of IgG4-RD patients without lesions of salivary glands were positive for lip biopsy, and the mean number of affected organs and serum IgG4 levels in the positive cases were significantly higher than in the negative cases. Our present case also had multiple organ involvements, suggesting the association between the positivity of lip biopsy and the number of affected organs. Taken together, a certain amount of patients with IgG4-RD could be pathologically diagnosed by lip biopsy.Although the positivity of lip biopsy in our case might have been accidental, some reports have revealed that it is worthwhile to pay attention on the usefulness of lip biopsy. We previously reported that the sensitivity of lip biopsy for diagnosis of IgG4-RD was 83.3%. The initiation of treatment was recommended for all patients with active IgG4-RD. Importantly, the organ involvement could be subclinical through the late stages of IgG4-RD, by which time chronic inflammation with fibrotic change might have led to the irreversible organ injury. Thus, it is recognized that the early intervention is important for the prevention of irreversible organ injury associated with progressive fibrosis in the salivary glands and pancreas lesions.,19 In the present case, we successfully diagnosed him with IgG4-RD by lip biopsy, and treatment with glucocorticoid led to the improvement of his renal function, swelling of SMGs, and periaortitis. As patients with IgG4-RD usually respond well to treatment with glucocorticoid,,20 early recognition and diagnosis is clinically important to prevent the irreversible organ injury.The international statement regarding the management and treatment of IgG4-RD has been recently published.4In summary, we report a case of IgG4-RD affecting SMGs, periaorta, and kidney that could be pathologically diagnosed by lip biopsy. With lip biopsy being minimally invasive, convenient, and useful for pathological confirmation for IgG4-RD, we emphasize this procedure as one of the options when affected organs are difficult to approach. Early recognition and diagnosis of this disease is clinically important because most patients respond well to glucocorticoid, which prevents irreversible organ damage.


Each hospital, private clinic (PC), or rehabilitation facility (RF) in France has to produce a computer-based annual report on the organization and actions developed for its antibiotic policy to the French Ministry for Health. This report consists of a list of nationally pre-defined items that the physician responsible for the infection control unit has to fill online. A scoring computer program operates at the end of the process.The aim of the study was to analyze the results of the annual report provided by every hospital, PC, or RF in Paris city in 2014.Assessment of the reports was performed by the Regional Agency for Health Ile-de-France, a dependency of the French Ministry for Health.Evaluation data included the general grade (decreasing from A to E) and the review of all the items structuring every annual report.The reports of 69 healthcare organizations  were examined, including 17 university hospitals (UH), 16 public general hospitals (GH), 25 PC, and 11 RF.Distribution of the classes A and B taken together was 65% in UH, 87% in GH, 92% in PC, and 82% in RF.Results appeared to be perfectible in the items dealing with the means dedicated to prevention and control activities: only 47% of UH and 36% of PC had a full computerized physician drug-order entry system, and more than one third of PC or RF had no antibiotic therapy specialist.In contrast with better results from the other healthcare organizations, respectively, 35% of UH and 59% of UH had no defined collaboration between the antibiotic specialist, the pharmacist, and the microbiologist, and had no list of selected antibiotics under increased control.Less than two thirds of GH or RF complied with the items dealing with antibiotic utilization evaluation.Analysis of the annual reports from all the hospitals, clinics, and rehabilitation facilities in Paris city on the nationally-required activities in antibiotic policy highlighted relatively good global results since over 80% of all these organizations taken together was graded A or B. However, the results in those items dealing with the means allocated to prevention of antibiotic misuse or with evaluation of antibiotic prescribing practices need to be improved.None declared.