Intraduct papillary mucinous neoplasm (IPMN) is a pancreatic tumour that is often associated with chronic pancreatitis (CP) in the surrounding pancreas. Type 1 autoimmune pancreatitis (AIP) is a fibro-inflammatory condition with characteristic histological features and prominent IgG4+ plasma cells and is part of the spectrum of IgG4-related disease (IgG4-RD). The aim of this study was to determine whether CP associated with pancreatic IPMN commonly represents AIP.We identified two consecutive ‘index’ cases of pancreatic IPMN during routine reporting in which the adjacent pancreas showed morphological features suggestive of AIP. These cases were investigated using the Boston criteria for the histopathological diagnosis of IgG4-RD and the HISORt criteria for the clinical and histopathological diagnosis of AIP. Using the same criteria, we proceeded to a clinical review of 12 extra cases of IPMN in which the tumour or the surrounding pancreas showed significant lymphoplasmacytic inflammation and/or fibrosis.Both of the ‘index’ cases fulfilled the HISORt criteria for AIP and both had morphological features characteristic of IgG4-RD using the Boston criteria, although only one possessed features ‘highly suggestive of IgG4-RD’ after immunohistochemistry. Additionally, both ‘index’ cases had radiological features that could represent extrapancreatic manifestations. Review of the 12 additional cases of IPMN revealed no further examples showing co-existent AIP.While pancreatic IPMN and AIP may co-exist, most CP associated with IPMN does not represent AIP. Intraduct papillary mucinous neoplasm (IPMN) of the pancreas is characterised by mucin production, cystic dilation of the pancreatic ducts and neoplastic intraductal papillary epithelial growth. Co-existent invasive carcinoma may be present. Chronic pancreatitis (CP) of the surrounding pancreas is a recognised feature. The CP associated with two consecutive cases of non-invasive IPMN identified during routine reporting was more florid than would usually be associated with a non-invasive pancreatic neoplasm and was suggestive of type 1 autoimmune pancreatitis (AIP). Type 1 AIP is the pancreatic manifestation of IgG4-related disease (IgG4-RD); a chronic and often multisystem fibroinflammatory condition associated with characteristic clinical, serological, radiological and histological features . Both underwent appropriate work-up in order to confirm or exclude AIP/IgG4-RD. In association with collaborative research into IgG4-RD between Southampton and Oxford, 12 further cases (2008–11) of pancreatic IPMN from the Oxford Radcliffe Hospitals NHS Trust  in which the tumour or surrounding pancreas showed features raising the possibility of AIP/IgG4-RD, that is, lymphoplasmacytic inflammation and/or fibrosis, were reviewed with the specific aim of ensuring that a diagnosis of AIP/IgG4-RD had not been missed. These 12 cases were identified within the 23 consecutive IPMN resections performed in Oxford during this period.All 14 cases underwent detailed histological examination and review of clinical records. Each case was assessed for the presence of morphological features that could indicate the presence of AIP/IgG4-RD, that is, lymphoplasmacytic inflammation, storiform fibrosis and obliterative phlebitis. An IgG4+ plasma cell count was performed within areas of lymphoplasmacytic inflammation, calculated as the mean IgG4+ plasma cell count per high power field  within the three HPF containing the greatest number of these cells. In cases in which the IgG4+ plasma cell count was 10 or more cells/HPF, IgG immunohistochemistry was performed in order to calculate an IgG4+/IgG+ ratio. The case records were examined for clinical, serological and/or radiological features that would support a diagnosis of AIP according to the HISORt criteria.The histological and clinical features of each case are given in The two ‘index’ cases (cases 1 and 2) showed CP immediately surrounding the IPMN as well as within the distant pancreas. The characteristic morphological features of lymphoplasmacytic inflammation, storiform fibrosis and obliterative phlebitis were present in both cases. Both cases also showed very prominent IgG4+ plasma cell numbers within the infiltrate  while the IgG4+/IgG ratio was significantly elevated in only case 1 . Eleven The 14 patients with IPMN were predominantly man in the sixth to seventh decades of life and presented with abdominal pain and weight loss rather than jaundice. All had a focal mass on imaging, most involved the head of the pancreas and were associated with a dilated pancreatic duct. Only one patient had undergone serum IgG4 measurement as AIP was in the differential diagnosis. No patient received a trial of corticosteroids. Case 1 had indeterminate pulmonary nodules and prominent retroperitoneal lymph nodes and case 2 had a solitary indeterminate pulmonary nodule and peri-pancreatic nodes . These mOn the basis of histological and clinical review, case 1 had features ‘histologically highly suggestive of IgG4-RD’ according to the Boston criteria and fulfilled the HISORt criteria for AIP. Using the same guidelines, case 2 possessed histological features suggestive of IgG4-RD but without an IgG4+/IgG+ ratio over 40% that is ‘mandatory’ for a diagnosis of IgG4-RD according to the Boston criteria, although the morphological characteristics and IgG4+ plasma cells met the HISORt criteria for AIP. None of the remaining cases possessed histological or clinical features that fulfilled either set of diagnostic criteria.Pancreatic neoplasms are commonly associated with CP in the immediately surrounding parenchyma. Distant pancreatic tissue may also show CP if there has been obstruction of the main pancreatic duct, and this feature can be particularly marked in invasive tumours such as ductal adenocarcinoma. The severity and extent of CP associated with non-invasive IPMN is usually less than that seen classically with ductal adenocarcinoma. Therefore, the presence of widespread severe CP within the two ‘index’ cases in this study was striking and unexpected.Histological examination of the two ‘index’ cases revealed morphological features  characteristic of IgG4-RD according to the Boston criteria.IPMN of the pancreas may be associated with morphological features in the surrounding pancreas that raise the possibility of type 1 AIP/IgG4-RD.IPMN of the pancreas may be associated with AIP/IgG4-RD but the majority of cases showing histological evidence of CP in the adjacent tissue do not represent examples of AIP/IgG4-RD.A histological diagnosis of IgG4-RD requires the presence of key morphological characteristics—lymphoplasmacytic inflammation, storiform fibrosis and obliterative phlebitis—together with prominent IgG4+ plasma cell numbers and an IgG4+/IgG+ plasma cell ratio of over 40%.Despite the sometimes very characteristic histological features of IgG4-RD, it is always essential to interpret histological findings in the overall clinical context of the case, for example, through the use of guidelines such as the HISORt criteria.Our review has confirmed one case ‘histologically highly suggestive of AIP/IgG4-RD’ and one further case of possible AIP/IgG4-RD, associated with pancreatic IPMN. These were identified during routine histopathology reporting due to morphological features within the adjacent and distant pancreas. However, our clinical and histological review of 12 additional cases has suggested that an association between pancreatic IPMN and the presence of AIP/IgG4-RD is not common. It does, however, highlight the importance of careful study of the ‘background’ pancreas within surgical specimens removed for pancreatic neoplasia as well as purely for CP. IgG4 immunohistochemistry is an important component of this assessment, but the tissue IgG4+ count (and IgG4+/IgG+ ratio) must always be interpreted in the context of the morphological features of a case and the clinical scenario. Recognition of IgG4-RD is essential as the condition is potentially multisystem in nature and may be underdiagnosed, especially if the clinical presentation is atypical. Furthermore, the disease usually responds well to corticosteroid therapy, but relapse is common and therefore follow-up in a specialist centre is recommended.


To use multilevel functional principal component analysis to exploit the information inherent in the shape of longitudinally sampled glucose curves during pregnancy, and to analyse the impact of glucose curve characteristics on neonatal birth weight, percentage fat and cord blood C-peptide.A cohort study of healthy, pregnant women (n = 884). They underwent two oral glucose tolerance tests , which gave two glucose curves per woman.Glucose values were higher, and peaked later in third trimester than in early pregnancy. The curve characteristic “general glucose level” accounted for 91% of the variation across visits, and 72% within visits. The curve characteristics “timing of postprandial peak”, and “oscillating glucose levels” accounted for a larger part of the variation within visits (15% and 8%), than across visits (7% and <2%). A late postprandial peak during pregnancy, and high general glucose levels in third trimester had significant, positive effects on birth weight (p<0.05). Generally high glucose levels during pregnancy had a significant, positive impact on neonatal percentage fat (p = 0.04). High general glucose level in third trimester had a significant, positive impact on cord blood C-peptide (p = 0.004).Shape information in entire OGTT curves provides significant physiological information of importance for several outcomes, and may contribute to the understanding of the metabolic changes during pregnancy. High maternal glucose levels in pregnancy have adverse short-term and long-term health effects for both the mother and the child Changes in glucose metabolism during pregnancy include increasing insulin resistance and increasing gluconeogenesis in the liver Functional data analysis (FDA) is a collection of statistical methods developed to analyse curve data In a previous article The STORK study, a Norwegian prospective cohort study of 1031 healthy, pregnant women, provided OGTT data from gestational weeks 14–16 and 30–32 The study was approved by the Regional Committee for Medical Research Ethics, Southern Norway, Oslo, Norway (reference number S-01191), and performed according to the Declaration of Helsinki. All participating women provided written informed consent.The STORK study is a prospective cohort of 1031 healthy, Norwegian women of Scandinavian heritage who registered for obstetric care at Oslo University Hospital Rikshospitalet from 2001 to 2008. The overall aim of the study was to extend insights into maternal metabolic syndrome and determinants of foetal macrosomia Blood samples were drawn in the morning, between 0730 and 0830 after an overnight fast, and were obtained from veni-puncture in tubes containing Ethylenediaminetetraacetic acid (EDTA). Plasma glucose was measured immediately in a drop of fresh, whole EDTA blood. During the OGTT, blood samples were taken every 30 minute for 2 hours. Glucose measurements were done by the Accu-Chek Sensor (ACS) glucometer . Due to an unexpected increasing trend in the fasting glucose measurements over the 7 years of recruitment, all glucose measurements were de-trended prior to the analyses Women with premature births or non-complete OGTT data were excluded, giving a study sample of 884 women and their neonates .Birth weight was recorded within two hours after the birth. In a subsample of the cohort, the percentage of neonatal body fat was measured by Dual-energy X-ray absorptiometry (DXA) scanning, and C-peptide in cord blood from the time of birth was measured  [30], [3Descriptive statistics of registered data are presented as mean, standard deviation (SD) and range, or frequency and percentage (%).The OGTT measurements from the two visits were converted into 884 continuous, smooth OGTT glucose curves , from gestational weeks 14–16, and 884 continuous, smooth glucose curves from gestational weeks 30–32. The curve fitting procedure was based on B-splines basis functions and is described in When functional data like glucose curves are observed on two or more occasions for each individual, we apply a multilevel model for functional data to extract information Assume that the individual, true blood glucose curve s curves , and expe curve  , and thecurve    are founIn order to quantify each individual glucose curve's characteristics, we estimate individual scores for each FPC curve. A woman's FPC scores reflect how her individual curve trajectories at the two visits correspond to the common characteristics expressed by the FPC curves. Then we can study how glucose curve characteristics vary from woman to woman, and from visit to visit.across visits  were used to compare AUC with FPC scores.The traditional, simple summary measure AUC was calculated directly from the de-trended glucose measurements by the trapezoid method. Pearson's correlation coefficients , early pregnancy BMI, age and parity as explanatory variables. The multivariable analyses involved stepwise variable selection procedures based on Akaike's information criterion (AIC), analyses of several models considered to be of importance, and considerations of physiological importance of the findings. Model diagnostics were thoroughly checked during the analysis. The final multiple models presented in the In supplementary analyses, we repeated the analyses of birth weight for the reduced samples where percentage fat and C-peptide were available.All analyses were performed in R 3.0.0. The estimation of FPC scores was done by the R2WinBUGS package that runs WinBUGS from R p≤1.00). There was a small increase in fasting glucose and a large increase in fasting insulin from inclusion to weeks 30–32. The 2-h glucose levels were elevated in third trimester, and the prevalence of GDM increased from 0.3% at inclusion to 6% in third trimester.Characteristics of the study sample at inclusion at gestational weeks 14–16, at gestational weeks 30–32 and at birth are shown in The smoothed glucose curves at gestational weeks 14–16  and 30–3across visits, and the first three subject- and visit-specific FPCs explained 92% of the residual variation within visits. Further analyses were restricted to these FPC curves and the corresponding FPC scores . The FPC1 and FPC2 curves had very similar temporal appearances across and within visits.In the multilevel FPCA, the first two subject-specific FPCs explained 98% of the variation subj, FPC14–16 and FPC30–32 scores are high or low. The dominating curve characteristic for the variation across visits, FPC1subj, accounting for 91% of this variation, was given the interpretation “general glucose level”. Women with high FPC1subj scores had glucose curves above the overall mean, and a somewhat later postprandial peak, whereas women with low FPC1subj scores had glucose curves below the overall mean  was “timing of postprandial peak”. Women with low FPC2subj scores had a clear early peak and low glucose values at the end of the OGTTs .The second most important curve characteristic across visits (FPC2he OGTTs . Women wwithin visits (accounting for 72% of this variation), was “general glucose level within visits”, i.e. the general glucose level not accounted for by the general glucose level across visits. A woman with a high subject- and visit-specific FPC114–16 (FPC130–32) score had a glucose curve above the subject-specific mean at weeks 14–16 (30–32) (14–16 (FPC130–32) score had a glucose curve below the subject-specific mean at weeks 14–16 (30–32). An example of this is the lower curve in The dominating curve characteristic for the residual variation  (30–32) . An exam14–16 (FPC230–32) score had a clear early peak, and low glucose values at the end of this OGTT (14–16 (FPC230–32) score at weeks 14–16 (30–32) had a later postprandial peak, with high glucose values at the end of this OGTT .The second most important curve characteristic for the variation within visits was “timing of postprandial peak within visits”. A woman with a low FPC2his OGTT . A woman14–16 or FPC330–32 scores had two postprandial peaks during the corresponding OGTT, whereas women with low FPC314–16 or FPC330–32 scores had only one glucose peak during the OGTT (across visits (7% and less than 2%), than within visits (15% and 8%).The third most important curve characteristic for the variation within visits was “oscillating glucose within visits”. The glucose curves of women with high FPC3the OGTT . The chasubj scores were high. The women with the green, red and purple curves had low FPC1subj scores. The woman with cyan curves and generally high glucose levels on both OGTTs, had a curve that was high above the mean at gestational weeks 14–16, but less so at gestational weeks 30–32. Thus, her FPC114–16 score was high, and her FPC130–32 score low.subj scores and AUC at weeks 14–16 and weeks 30–32 , between FPC114–16 and AUC14–16 (r = 0.73), and between FPC130–32 and AUC30–32 (r = 0.87). All correlations are given in ≤r≤0.35).There were strong correlations between FPC1subj), and of the subject- and visit-specific general glucose level at weeks 30–32 (FPC130–32) (subj) on birth weight.Crude analyses showed significant positive effects on the three neonatal outcomes of the general glucose level across visits (FPC1C130–32) . There wsuband FPC130–32 scores remained significant (subj scores) would be expected to have babies with higher birth weight than women with early postprandial peaks (high FPC2subj scores), and women with the highest residual glucose levels in third trimester (high FPC130–32 scores) would be expected to have babies with higher birth weight than women with low residual glucose levels in third trimester (low FPC130–32 scores).In multivariable analyses of birth weight, only FPC2nificant : Women wsubj scores remained significant (subj scores) would be expected to have babies with a higher percentage of fat than women with generally low glucose levels during their OGTTs (low FPC1subj scores). According to AIC, FPC2subj scores also held information important for this outcome, although not statistically significant in the final model: Low FPC2subj scores, implying late glucose peaks, corresponded with high values of neonatal percentage fat.In multivariable analyses of neonatal percentage of fat, the effect of the FPC1nificant : Women w30–32 scores remained significant  had somewhat lower C-peptide levels than those with one glucose peak during the OGTT (low FPC314–16 scores).In multivariable analyses of C-peptide in cord blood, the effect of FPC1nificant : Women wsubj in both subsamples, of FPC214–16 in the percentage fat subsample, and a non-significant contribution of FPC230–32 in the C-peptide subsample.Supplementary analyses showed that alternative models chosen due to physiological theories, and to explore the effects of colinearity, gave the same results for all three outcomes. Analyses of birth weight in reduced samples where percentage fat or C-peptide was available showed significant effects of FPC1The present study successfully used multilevel FDA to analyse changes in longitudinally observed glucose curves during pregnancy. The general glucose levels, in particular postprandial glucose, increased from early pregnancy to gestational weeks 30–32, and postprandial glucose peaked later in gestational weeks 30–32. The glucose characteristics extracted by FPCA had significant impact of glucose curve characteristics on birth weight, neonatal percentage of fat, and C-peptide in cord blood, demonstrating physiological relevance.The physiological interpretation of FPC curves is essential for the usefulness of FPCA. The identification of the general glucose levels as the most dominant characteristics of individual glucose curves was supported by the strong associations between FPC1 scores and the AUCs. The elevated postprandial levels in third trimester, the small increase in fasting glucose, and the large increase in fasting insulin and prevalence of GDM from inclusion to weeks 30–32 , are in The regression results for birth weight in The sample size in the present study was substantial, but the women were healthy and relatively homogenous. This may have caused less variation in the individual glucose curves and made it more difficult to extract important discriminating curve characteristics. Also, the scarce sampling of glucose during the OGTT is likely to obscure the extraction and interpretation of the curve characteristics. More physiologically interesting temporal details and better discriminating abilities of the FPCs may be expected in a more heterogeneous population, and from OGTT curves over more than 2 hours or with a more frequent OGTT sampling. With more measurements per OGTT, it is also possible to apply alternative smoothing strategies Compared to studies presenting intravenous glucose tolerance tests The finding of the general glucose level as the most important glucose curve characteristic is in accordance with the numerous studies focusing on elevated glucose of various types, e.g. fasting, 1-h, 2-h or HbA1c values, in diabetes research th percentiles), for high fasting, 1-hour and 2-hour glucose levels The HAPO study is a reference study for the impact of maternal blood glucose levels on pregnancy outcomes, and has found statistically significant higher odds ratios for high birth weight, cord-blood serum C-peptide level and percentage body fat Click here for additional data file.Appendix S1Multilevel functional data analysis (FDA), technical details.(PDF)Click here for additional data file.Appendix S2Program code.(PDF)Click here for additional data file.


Therefore, the confounding effects of their pro-inflammatory profile at baseline need careful consideration. Moreover, despite the prevailing view that AM have an anti-inflammatory bias, our data clearly show that they can adopt a striking pro-inflammatory phenotype, and may have greater capacity for presentation of exogenous antigens than MDM.Alveolar macrophages (AM) are thought to have a key role in the immunopathogenesis of respiratory diseases. We sought to test the hypothesis that human AM exhibit an anti-inflammatory bias by making genome-wide comparisons with monocyte derived macrophages (MDM). Adherent AM obtained by bronchoalveolar lavage of patients under investigation for haemoptysis, but found to have no respiratory pathology, were compared to MDM from healthy volunteers by whole genome transcriptional profiling before and after innate immune stimulation. We found that freshly isolated AM exhibited a marked pro-inflammatory transcriptional signature. High levels of basal pro-inflammatory gene expression gave the impression of attenuated responses to lipopolysaccharide (LPS) and the RNA analogue, poly IC, but in rested cells pro-inflammatory gene expression declined and transcriptional responsiveness to these stimuli was restored. In comparison to MDM, both freshly isolated and rested AM showed upregulation of MHC class II molecules. In most experimental paradigms  Streptococcus pneumoniae infection Tissue resident macrophages play a key role in host defense by generating potent inflammatory responses to innate immune stimulation, but also regulate anti-inflammatory mechanisms which are crucial for resolution of inflammatory responses and restoration of tissue homeostasis. Alveolar macrophages (AM) are of specific interest as the principal cellular sentinels of the distal respiratory tract, because they are continuously exposed to potentially inflammatory stimuli, within a delicate tissue microenvironment that is particularly susceptible to damage. Innate immune responses by AM are crucial for immunosurveillance and host protection against inhaled pathogens, but excessive inflammatory responses by these cells also underpins the pathogenesis of numerous diseases including sarcoidosis, pulmonary fibrosis, tuberculosis and Inflammatory responses by macrophages are subject to extensive research, but findings are commonly extrapolated from experiments with peripheral blood monocyte derived macrophages (MDM). In humans, AM can readily be obtained by bronchoalveolar lavage (BAL), providing a population of genuine primary tissue macrophages which are amenable to study. Experimental observations are often interchangeably extrapolated between these two common cellular models, but in view of the inherent phenotypic heterogeneity between different macrophage populations the extent to which this is appropriate is uncertain. By comparison with other macrophages, AM are reported to exhibit a highly specialised phenotype characterised by enhanced pattern recognition receptor (PRR) and scavenger receptor expression combined with poor antigen presentation capacity in vivo is thought to be driven principally by endocrine function of colony stimulating factor (CSF)1, also known as macrophage .Bronchoscopy and bronchoalveolar lavage are commonly used for investigation of haemoptysis with normal radiographic imaging, in order to exclude respiratory tract pathology Next we sought to make a comprehensive comparison of AM and MDM using genome-wide expression data. This analysis showed extensive gene expression differences . In ordeMacrophages play a key role in innate immune inflammatory responses, which are predominantly reflected by changes in gene expression. Therefore we tested transcriptional responses to innate immune stimulation of AM and compared these to MDM. Four hour stimulation of MDM with LPS induced upregulated expression of a well validated set of cytokines and chemokines, but in freshly isolated AM all of these genes showed high level expression at baseline and no further upregulation after LPS stimulation . In ordeIn our gene array analysis, we noted that the expression of inflammatory genes was lower in unstimulated AM cultured for 24 hours, when compared to cells cultured for 4 hours . InteresWe extended these studies to test the expression levels of a panel of six inflammatory genes in AM from healthy participants, which were either used immediately or rested in culture for 48 hours before LPS stimulation for 4–24 hours . In all In view of the reduction of pro-inflammatory gene expression in rested AM to comparable levels seen in MDM, we assessed whether rested AM approximated more closely to MDM at genome-wide level also. Hierarchical clustering of whole genome expression data, showed that rested AM grouped more closely to MDM than freshly isolated AM but still formed a distinct cluster . We therAM are widely considered to be important cellular constituents of the lower respiratory tract and by analogy to macrophage biology in general, are thought to play a key role in innate immune recognition of microbial pathogens or other danger signals, generation of inflammatory responses, control of intracellular pathogens and homeostatic regulation of immune responses. They also serve as a bridge between innate and adaptive immunity through antigen presentation and provision of co-stimulatory signals. MDM are commonly used to model AM, but few studies have directly compared human AM with MDM, hence we have very little genuine insight into the specific phenotype and function of AM. In the present study we used genome-wide transcriptional profiling to compare AM with MDM and focussed specifically on transcriptional responses to innate immune stimulation with LPS. We obtained AM from patients undergoing bronchoscopy for reported haemoptysis, but found to have normal radiology and no other evidence of respiratory tract pathology after follow up. Nonetheless it is possible that these selection criteria may have affected our findings. In addition, cigarette smoking has been reported to affect gene expression profiles in AM from different subjects ex vivo, was reduced by depletion of AM with clodronate in vivo, such as prostaglandin E2 (PGE2), TGFβ or surfactant, which have all been shown to attenuate AM inflammatory mediator production, may account for our ex vivo observations in vivo tissue microenvironment. In order to test this hypothesis further studies on tissue samples from the lung are necessary to establish whether AM show any features of a pro-inflammatory phenotype in situ.We demonstrate that freshly isolated AM bear the major transcriptional features of MDM, but AM showed greatly increased expression of genes strongly associated with pro-inflammatory responses. Although these data conflict with a widely held view that AM have a predominant anti-inflammatory or regulatory phenotype Importantly, freshly isolated AM did not further upregulate pro-inflammatory gene expression following innate immune stimulation. However, pro-inflammatory mediator expression progressively declined with time in culture, such that by 24 hours, LPS and poly I:C stimulated upregulation of these genes was discernible. These findings are consistent with previous reports of inflammatory cytokine induction by AM after LPS stimulation for 18–24 hours Transcriptional profiles from rested AM were clearly more similar to MDM than freshly isolated AM, but still represented by a distinct cluster. Interestingly the most highly enriched group of genes that were persistently upregulated in AM compared to MDM, were MHC II antigen molecules. This finding is consistent with previous reports of high MHC II expression in AM that may also be attributed to the role of GM-CSF in AM biology The study was approved by the joint University College London/University College Hospitals National Health Service Trust Human Research Ethics Committee, and written informed consent was obtained from all participants.5 cells per well in 12 well plates (TPP), removing non-adherent cells after one hour. Ultrapure LPS (100 ng/ml) from Escherichia coli O111:B4 and poly IC (10 µg/ml), both from Invivogen, were used to stimulate inflammatory responses. All cell lines were obtained from the American Type Culture Collection (ATCC). THP1 and SUPT1 cell lines were cultured as suspension cells in RPMI and HeLa cells as adherent cells in DMEM with 10% foetal calf serum and antibiotics (as above). THP1 cells were differentiated by addition of 5 ng/mL phorbol myristate acetate (Sigma Aldrich) for 72 hours Blood samples were obtained from healthy volunteers for isolation of PBMC and production of MDM by differentiation with M-CSF as previously described Total RNA was purified from cell lysates collected in RLT buffer (Qiagen) using the RNEasy Mini kit (Qiagen). Human universal standard reference RNA representing an equal mixture of ten human cell lines was obtained from Stratagene. Samples were processed for Agilent microarrays and data were normalised as previously described CGGTCCTGGCGCTCAG, reverse primer: CCGGGTACAATCGCACTTATACTG, and probe: CCATACAGCAAATCCTT. Expression levels of target genes were normalised to that of HPRT1 (Hs99999909_m1) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as previously described cDNA was synthesised using the qScript cDNA Supermix kit (Quanta BioSciences) and quantitative (q)PCR of selected genes was performed using the following inventoried TaqMan assays (Applied Biosystems); HPRT1 (Hs99999909_m1), CCL20 (Hs01011368_m1), IL23 (Hs00372324_m1), IL1β (Hs01555410_m1), IL6 (Hs00985639_m1) and TNFα (Hs00174128_m1). PTGS2 expression was quantified using: forward primer: MDM and AM culture supernatant cytokine concentrations were quantified by ELISA (eBioscience) according to the manufacturer’s instructions.Figure S1Gene ontology associations of expression differences between AM and MDM. The top three most significantly enriched gene ontology associations are presented for the 50 most highly (A) upregulated and (B) downregulated genes in AM compared with MDM.(TIF)Click here for additional data file.Figure S2Poly I:C induced pro-inflammatory gene expression in rested AM. Freshly isolated alveolar macrophages (AM) show no pro-inflammatory transcriptional response to stimulation with poly IC (10 µg/ml), but basal levels of pro-inflammatory gene expression are reduced in AM which have been rested for 48 hours and these show significant upregulation following 4 hour stimulation with poly IC (10 µg/ml). Bars represent mean ±SEM for 3 separate experiments .(TIF)Click here for additional data file.Table S1Clinical information for AM donors. Patient details for samples used for freshly isolated AM arrays* and ELISAs‡, time course study of PTGS2 expression† and 48 hour rested AM§. DIP =  desquamative interstitial pneumonia.(DOC)Click here for additional data file.Table S2Supporting literature for macrophage transcriptional signature. In genome-wide expression data, genes that showed significantly higher expression (>8 fold) in monocyte derived macrophages compared to standard reference RNA were subjected to PubMed searches to identify those that are known to be expressed by macrophages, and thereby generate a transcriptional signature for macrophages. The first author, publication year, journal, direct object identifier (DOI) and PubMed identifier (PMID) is provided for representative citations associated with each of these genes.(DOC)Click here for additional data file.Table S3Summary of AM enrichment and culture methods. Recently published studies involving alveolar macrophages were identified from PubMed using the following search criteria: “(((((macrophages) AND human) AND bronchoalveolar lavage) NOT mouse) NOT murine) NOT mice” with Limits: Humans, Journal Article, English, published in the last 5 years. The method of AM enrichment and the time in culture before AM were assessed is indicated for each study, reflecting the common contemporary practice of enrichment by plastic adherence before use of freshly isolated AM. The first author, publication year, journal, direct object identifier (DOI) and PubMed identifier (PMID) is provided for each study.(DOC)Click here for additional data file.


Ndufs4 gene ablation. Heart-specific Ndufs4-null mice had a decrease of ∼50% in complex I activity within the heart, and developed severe hypertrophic cardiomyopathy as assessed by magnetic resonance imaging. The decrease in complex I activity, and associated cardiac dysfunction, occurred absent an increase in mitochondrial hydrogen peroxide levels in vivo, accumulation of markers of oxidative damage, induction of apoptosis, or tissue fibrosis. Taken together, these results indicate that diminished complex I activity in the heart alone is sufficient to drive hypertrophic cardiomyopathy independently of alterations in levels of mitochondrial hydrogen peroxide or oxidative damage.Mitochondrial complex I, the primary entry point for electrons into the mitochondrial respiratory chain, is both critical for aerobic respiration and a major source of reactive oxygen species. In the heart, chronic dysfunction driving cardiomyopathy is frequently associated with decreased complex I activity, from both genetic and environmental causes. To examine the functional relationship between complex I disruption and cardiac dysfunction we used an established mouse model of mild and chronic complex I inhibition through heart-specific  The heart is particularly susceptible to mitochondrial dysfunction due to its strict dependence on aerobic metabolism As persistent disruption of complex I activity from a range of causes is frequently observed in cardiomyopathies Ndufs4 gene, which encodes a 18 kDa subunit of complex I that is not directly involved in electron transport but which plays a role in assembly or stability of the entire complex Ndufs4-null mice have been established as a model for Leigh syndrome; a devastating early onset neurological disorder often associated with mutations to respiratory complexes Ndufs4-null mouse strain has no reported pathological phenotype up to 1 year of age despite significant inhibition of complex I activity Ndufs4-null mouse to explore how persistent inhibition of mitochondrial complex I activity affected cardiac function specifically.To assess the potential primary role of complex I disruption in driving cardiomyopathy through these processes, we chose to disrupt the Ndufs4 gene in the heart leads to a ∼50% decrease in complex I activity, that in turn drives severe hypertrophic cardiomyopathy, as determined by magnetic resonance imaging (MRI). However, Ndufs4 gene ablation had no effect on mitochondrial hydrogen peroxide levels, oxidative damage, apoptosis, or tissue fibrosis in the heart. This work suggests that disruption to complex I alone is sufficient to drive myocardial dysfunction. Furthermore, this complex I driven cardiomyopathy occurs through disruption of bioenergetic function and is independent of ROS-dependent pathways.Here we demonstrate that selective disruption of the All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and the University of Cambridge Animal Welfare Policy, and were approved by the UK Home Office under project license 80/2374.Ndufs4-null mouse model was a kind gift from the lab of Prof Nils Göran-Larsson, Max Planck Institute for Ageing, Cologne, Germany The Ndufs4Loxp/+ mice with mice carrying the heart specific cre recombinase, CKM-NLS-cre and established on a C57Bl/6 background LoxP/LoxP mice LoxP/LoxP) and control mice, the male CKM-NLS-cre knockouts  were crossed to control females (Ndufs4LoxP/LoxP). Mice with genotype Ndufs4+/LoxP or Ndufs4LoxP/LoxP were used as controls LoxP/LoxP males and females between the ages of 8–24 weeks were used for all experiments. As described previously, within this age range there was no discernable difference in health, appearance or behaviour between heart-specific Ndufs4-null mice and controls.Previously, these mice were generated by breeding 2) and maintenance (1.25–2% in 1 l/min O2). A pressure sensor for respiration rate was used to monitor anaesthesia depth, breathing rate was maintained in the range 30–60 breaths per minute. Prospective gating of the MRI sequences was achieved with ECG monitoring. Body temperature was monitored using a rectal thermometer and kept constant at 37°C using a water-heated blanket.Animals (8–24 weeks of age) were anaesthetized with gaseous isoflurane both for induction After the acquisition of the standard protocol, MRI was performed using a multi-slice inversion recovery sequence as described previously.For image analysis the papillary muscles and trabeculations were excluded from the delineation of the LV at each phase of the cardiac cycle. The regions from each slice were combined using Simpson's rule to provide LV mass, end diastolic volume, end systolic volume, stroke volume and ejection fraction using Segment v1.9 Mitochondrial complex I activity was assessed as described previously in vivo using the MitoB mass spectrometric probe as described previously Mitochondrial hydrogen peroxide was measured Protein carbonylation levels in the heart were determined by rapid homogenization of entire hearts and protein quantification of heart homogenate, followed by quantification of protein carbonyls using the BioCell Protein Carbonyl Assay Kit according to the manufacturers instructions.The expression of caspase 3 and cleaved caspase 3  was analyzed by standard Western blotting techniques. The band intensity of three independent experiments was measured using SigmaGel software and normalized to Actin .The expression of manganese superoxide dismutase (MnSOD)  was analyzed by standard Western blotting techniques. The band intensity of three independent experiments was measured using SigmaGel software and normalized to Actin .Hearts were excised, stored overnight in 10% formalin and embedded in paraffin before 9 mircons thick section were taken throughout the heart. Masson's Trichrome staining was performed according to the manufacturers specifications . Briefly, slides were deparaffinized in deionized water and fixed in Bouin's solution. After washing and incubation in Weigert's Irono Heamatoxylin solution, the slides were incubated in Biebrich Scarlet Acid Fuchsin and Anilin Blue solution for staining. Finally the slides were dehydrated in ethanol and mounted. The nuclei were stained black, the collagen fibers blue and muscles redData are presented as mean ± standard error from the mean. Statistical analysis was performed using one-way ANOVA. The differences were considered significant at P<0.05.Ndufs4-null mouse model Ndufs4-null mice exhibited ∼50% lower myocardial complex I activity compared to controls  was significantly higher in modeling .Ndufs4-null mediated hypertrophic cardiomyopathy correlated with changes in mitochondrial ROS levels. To do so we used the recently developed MitoB probe for in vivo quantification of mitochondrial hydrogen peroxide production Ndufs4-null mouse hearts exhibited mitochondrial hydrogen peroxide levels indistinguishable from controls  to form mitochondrial hydrogen peroxide Ndufs4-null mouse hearts to compare the endogenous superoxide dismutation capacity, finding levels to be comparable Click here for additional data file.Video S2Long-axis MRI of heart function of Ndufs4-null mouse hearts.(WMV)Click here for additional data file.


Culicoides midges that become infected after biting a viraemic host. The use of live attenuated vaccines has been vital for the control of this disease in endemic regions. However, there are safety concerns over their use in non-endemic countries. Research efforts over the last two decades have therefore focused on developing alternative vaccines based on recombinant baculovirus or live viral vectors expressing structural components of the AHS virion. However, ethical and financial considerations, relating to the use of infected horses in high biosecurity installations, have made progress very slow. We have therefore assessed the potential of an experimental mouse-model for AHSV infection for vaccine and immunology research. We initially characterised AHSV infection in this model, then tested the protective efficacy of a recombinant vaccine based on modified vaccinia Ankara expressing AHS-4 VP2 (MVA-VP2).African horse sickness (AHS) is a lethal viral disease of equids, which is transmitted by  Culicoides, which become infected after taking a blood meal from an infected and viraemic host. African horse sickness (AHS) is a lethal viral disease of equids, caused by an orbivirus that is closely related to bluetongue virus (BTV). The African horsesickness virus (AHSV) is transmitted by biting midges of the genus The AHSV genome is composed of ten linear segments of dsRNA, encoding seven structural proteins VP1 to VP7 and four non-structural proteins NS1, NS2, NS3 and NS3a One of the most effective intervention strategies to combat AHS is vaccination, allowing horses to survive in endemic regions. Live attenuated strains of AHSV that were developed as vaccines have been available for more than 60 years and are still routinely used in South Africa and other endemic countries For these reasons a number of recombinant vaccination strategies have been pursued over the years, as an alternative to attenuated vaccines. Baculovirus expressed AHSV capsid proteins VP2, VP5, VP7 and VP3, either individually or combined to form virus-like particles (VLP), have shown promising results Although more work is needed to improve vaccines against this disease, progress is hampered by the need to perform vaccine efficacy studies in horses. Work with AHSV infected horses in high biosecurity installations represents a logistical and financial burden. For these reasons we have initiated a programme of research based on developing an experimental mouse model for AHS, based on recent successes with a similar model for bluetongue We present the results of a pilot study using a small animal model for AHS, based on interferon-α receptor knock-out (IFNAR −/−) mice, and an exploration of the suitability of this system to test AHS vaccine efficacy. We have used a recombinant modified vaccinia Ankara virus, expressing AHS-4 VP2 (MVA-VP2), that was previously shown to induce a virus neutralising antibody response in ponies 5.8 (Group A) or 104 pfu (Group B) per mouse via the subcutaneous route. Two uninfected mice were kept as controls (Group C). Clinical signs were monitored and virus titres in blood samples determined.An experimental infection with AHSV-4 was conducted in IFNAR −/− mice using two different virus dose levels. Thus, two groups of five mice were infected respectively with 10All mice infected in group A presented clinical signs, which were evident by day 4 post-infection. These included presentation of rough hair coat, a hunched posture and reduction of mobility, which in some cases led to lethargy. Lacrimation and swelling of the eyelids was also observed. Neurological signs were also seen including ataxia, with the presentation of ‘circling gait’, and paresis of one, two or all four limbs often leading to paralysis. One animal from this group died of the disease, whilst two others had to be euthanized on humane grounds due to paralysis and ataxia. In contrast, mice in group B presented a milder clinical syndrome characterised by reduction of mobility, which was common to all animals, and eye swelling in just one individual. There were no neurological signs present and all animals infected in this group survived. Controls in group C remained healthy until the end of the study.Mice in group A developed a significantly higher viraemia (mean virus titres) than those in group B . FurtherMicroscopically, the spleen showed a loss of its normal structure and it was not possible to observe the separation between red and white pulp due to a marked lymphoid depletion . In addi6 pfu/mouse induced clinical signs, viraemia and significant levels of lethality, we evaluated the protective efficacy of a recombinant MVA expressing VP2 of AHSV-4 (MVA-VP2). In earlier studies this recombinant MVA virus was shown to induce virus neutralising (VN) antibodies against AHSV-4 in ponies 7 pfu of MVA-VP2 per mouse. These animals and six unvaccinated IFNAR −/− control mice (C1–C6) were subsequently challenged on day 35 with 106 pfu of AHSV-4. Blood samples were collected at regular intervals for virus titration and assessment of antibody responses by VN assays and VP7- ELISA. The clinical signs, percentage survival, virus titres and Ct values determined by real-time RT-PCR in blood, were compared between the two groups of animals, to evaluate the level of protective immunity afforded by MVA-VP2 vaccination.Once the challenge infection of IFNAR −/− mice with AHSV-4 had been characterised and that doses in the order of 10The percentage survival and viraemia showed that all of the vaccinates survived the challenge and were negative for viraemia .In contrPost-mortem examination of control mice revealed the same type of lesions as observed in the previous challenge experiment performed with AHS-4. In addition to hematoxylin-eosin staining, immuno-fluorescence experiments were used to demonstrate the presence of AHSV antigen in tissues. These were performed on paraformaldehyde fixed cryosections of AHSV-4 challenged mice and uninfected control mice. A rabbit polyclonal antiserum raised against AHSV- VP7 was used as primary antibody. Distribution of antigen in spleen was widespread and uniform, whereas in the liver it was observed as scattered foci  and 6.Data from a semi-quantitative analysis of the virus content in blood samples by real-time RT-PCR were compared with virus isolation data . Only twAnalysis of antibody responses was performed by VN test and VP7-ELISA. All of the mice vaccinated with MVA-VP2 developed VN antibodies after the second dose of vaccine was given . FollowiMost of the recent African horse sickness research has focused on development of vaccine technologies that are safer, effective and compatible with their application in non-endemic countries. However, any new vaccine need to be evaluated in protective efficacy studies. Such experiments are difficult to perform in horses (the target species), due to high costs and logistic difficulties relating to the use of statistically significant number of animals in high bio-containment. For these reasons we have tested a mouse model for AHS, similar to that recently used for bluetongue Culicoides transmitted AHSV in the natural host.Laboratory animals, notably mice, have been used for many years to study infections with AHSV. Intracerebral inoculation of suckling mice is one of the primary techniques used to isolate AHSV from clinical specimens. But apart from this diagnostic application, mouse experimental mouse models have also been used in studies of AHSV pathogenicity and vaccination Recently, Calvo-Pinilla and co-workers successfully used IFNAR −/− mice as a model for bluetongue The Madrid 87 strain used in our studies was chosen because the VP2 gene employed to construct the MVA-VP2 vaccine virus was derived from it. This virus was initially isolated in mouse brain, further passaged once in mouse brain and three times in BHK before used in our mouse experiments. This virus strain, although obviously not exactly the same stock, was used in challenge experiments in horses inducing typical severe signs of AHS including death (10).We used a subcutaneous route of infection because it most closely resembles the natural route of entry/infection during the natural AHSV infection cycle. BTV infection of IFNAR −/− mice via this route induces similar levels of lethality, viraemia and pathology to those generated via an intravenous route . However, it is uncertain whether the route of challenge would influence the outcome of AHS virus infection in IFNAR −/− mice. Vaccination was performed by the intraperitoneal route since this is a well established route and it had been used successfully in IFNAR −/− mice vaccinated with recombinant MVA (20).Interferon alpha (IFN-α) can play an important role in the innate immune response against viruses. IFN-α is produced by plasmacytoid dendritic cells in response to viral dsRNA, which binds to cellular receptors of neighbouring cells, stimulating of a series of antiviral mechanisms that help to clear infection. It is not known yet whether AHSV specifically induces IFN-α, or if IFN-α plays an important role in controlling AHSV infection, but evidence from studies with BTV suggest this possibility One concern over the use of IFNAR −/− mice in vaccination studies is the potentially detrimental effect that the absence of IFN-α signalling could have on the adaptive immune response of the host. However, vaccination studies in IFNAR −/− mice with non-infectious agents, such as recombinant vesicular stomatitis virus glycoprotein, have shown that IFNAR −/− mice can develop normal levels of neutralising antibodies and cytotoxic T-lymphocyte responses In our studies we have used the highly attenuated vaccinia MVA strain as a vaccine vector and showed that IFNAR −/− mice vaccinated with MVA-VP2 developed VN antibodies and protective immunity against AHSV. The levels of immunity are very high since none of vaccinates presented viraemia and only two animals produced blood samples that were positive by highly sensitive real-time RT-PCR techniques. Furthermore, only one vaccinate developed VP7 specific antibodies after challenge suggesting that the vaccinates were exposed to only very low levels of virus antigen. Our data confirms that a lack of IFN-α in mice did not affect the immune responses elicited by vaccination with an MVA based vaccine. These findings are consistent with similar studies performed with MVA based vaccines against BTV The protective effect against AHSV infection that was provided by MVA-VP2 vaccination was not surprising. We have previously demonstrated induction of VN antibodies in ponies vaccinated with the same recombinant virus Our study confirms the potential MVA-VP2 as a vaccine against AHSV. This vector vaccine stimulated VN antibodies in ponies, shows protective capacity in the IFNAR −/− model and because it is based on a restricted antigenic repertoire of the virus, is suited for differential diagnostics with an appropriate differential ELISA test (DIVA strategy). A VP7-ELISA that has already been developed as a diagnostic assay for AHSV specific antibodies could serve this purpose, since the control animals that survived the infection all presented VP7 specific antibodies, in contrast to those that had only been vaccinated with MVA-VP2, which showed no response to VP7. Most of the vaccinated animals that were subsequently also challenged with AHSV, still failed to develop VP7 specific antibodies, because they did not become infected. However, in one case M2 in  a positiIn conclusion, we have shown that IFNAR −/− mice can be infected by a subcutaneous route, inducing clinical signs, lethality, viraemia and pathology that are appropriate for immunology and pathogenesis studies of AHSV. To gain further evidence of the potential of this system, we conducted a vaccination and challenge experiment in this model, demonstrating the protective efficacy of the recombinant MVA-VP2 prototype vaccine. More work is needed to refine and improve this model and to determine whether our findings can be extended to other strains and serotypes of AHSV. This experimental system will facilitate work on individual and even cross-reactive vaccines as well as other aspects of AHS research.Baby hamster kidney cells (BHK-21), and Vero cells, obtained from the Institute for Animal Health Central Service Unit, were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM glutamine, antibiotics and 10% fetal calf serum (FCS). Standard virus titrations were performed in Vero cells. The AHSV-4 virus stock used for challenge and for virus neutralisation tests was derived from a Spanish isolate of AHSV (Madrid-87) passaged twice in mouse brain and three times in BHK-21 cells. Virus stocks were generated by infection of confluent BHK-21 cells with this virus using a multiplicity of infection (MOI) of 0.1. When total cytopathic effect (CPE) was visible, the cells and supernatants were harvested and centrifuged. The supernatant was kept at −80C.The MVA-VP2 virus used in this study was derived by single passage in the chicken embryo fibroblast cell line DF-1 of the construct described elsewhere All experiments with live animals were performed under the guidelines of the European Community (86/609) and were approved by the Centro de Investigación en Sanidad Animal, INIA, Madrid (CISA), reference number: 2008/007. Mice were maintained under pathogen-free conditions and allowed to acclimatize to the biosafety level 3 (BSL3) animal facilities at CISA for 1 week before use in our experiments.For challenge, mice were innoculated subcutaneously with the challenge strain. Mice were examined for clinical symptoms daily. Animals showing severe symptoms were humanely euthanized. These clinical signs included: severe ataxia, paralysis, circling gait, lethargy and any other condition that affected the animals capacity to eat or drink. Whole blood was collected in EDTA from all animals at regular intervals after inoculation for virus isolation. 200 µl of blood was washed in PBS and resuspended in 900 µl of water to lyse the cells. 100 µl of 10x PBS was added to the sample and 10-fold dilutions of this material were used for quantification of viraemia using a standard plaque assay on Vero cells as described 7 pfu/mouse. Mice were bled for serum collection on days 0, 28, 35  and 23 days post-challenge.IFNAR −/− mice were immunised by two intraperitoneal injections of MVA-VP2 administered on days 0 and 28 using 1050 of AHSV. A virus titration was included to check the virus dose of the assay was correct and a titration of horse antisera positive for AHSV-4 VN antibodies were included in the assay as positive controls. Vero cells were added at 2×104 cells/well/100 µl and plate was incubated at 37°C for 3 days. Wells were observed for development of cytopathic effect.Sera were tested for neutralising antibodies using standard procedures using AHSV-4.Samples were preliminary screened for neutralising activity at a single dilution of 1/5. Positive samples were further titrated to determine the VN titre of the sera. Briefly, in a microtitre flat bottomed plate, 50 µl of serum samples dilutions in DMEM without bovine serum were incubated for 1 hour at 37°C with 50 µl of 100 TCIDA commercial blocking ELISA test  was used to detect the presence of VP-7 specific antibodies in serum. Manufacturer recommendations were used.Samples from different tissues and organs were taken and fixed in 10% buffered formalin (pH 7.2) for histopathological studies. After fixation, samples were dehydrated through a graded series of alcohol to xylol and embedded in paraffin wax. Sections of 4-micron-thick were cut and stained with hematoxylin and eosin for histopathological analyses.In addition, tissues were also cryopreserved in OCT, cut in 7 micron-thick sections, fixed in 4% paraformaldehyde and processed for immuno-fluorescence. Briefly, a rabbit antiserum raised against AHS-9 -VP7 crystals was diluted 1/1000 in PBS containing 0.5% bovine serum albumin (PBS-A) and applied to the sections. After 1 hour incubation at room temperature (RT), the slides were washed in PBS and incubated with 488-Alexafluor conjugated anti-rabbit IgG (Invitrogen) diluted 1/200 in PBS-A. A final wash in PBS preceded a 5 minute incubation step with rhodamin-phalloidin and a 5 minute incubation step with DAPI. Sections were then mounted and observed by confocal microscopy.et al. Total RNA was extracted from blood samples processed as indicated above using BioRobot Universal (Qiagen). After heat denaturation the RNA was tested by real time RT-PCR on an Mx3005p machine  essentially as described by Shaw


We have recently described an increased lymphocytic infiltration rate in breast carcinoma tissue is a significant response predictor for anthracycline/taxane-based neoadjuvant chemotherapy (NACT). The aim of this study was to prospectively validate the tumor-associated lymphocyte infiltrate as predictive marker for response to anthracycline/taxane-based NACT.The immunological infiltrate was prospectively evaluated in a total of 313 core biopsies from HER2 negative patients of the multicenter PREDICT study, a substudy of the neoadjuvant GeparQuinto study. Intratumoral lymphocytes (iTuLy), stromal lymphocytes (strLy) as well as lymphocyte-predominant breast cancer (LPBC) were evaluated by histopathological assessment. Pathological complete response (pCR) rates were analyzed and compared between the defined subgroups using the exact test of Fisher.Patients with lymphocyte-predominant breast cancer (LPBC) had a significantly increased pCR rate of 36.6%, compared to non-LPBC patients . LPBC and stromal lymphocytes were significantly independent predictors for pCR in multivariate analysis . The amount of intratumoral lymphocytes was significantly predictive for pCR in univariate  but not in multivariate logistic regression analysis .Confirming previous investigations of our group, we have prospectively validated in an independent cohort that an increased immunological infiltrate in breast tumor tissue is predictive for response to anthracycline/taxane-based NACT. Patients with LPBC and increased stromal lymphocyte infiltration have significantly increased pCR rates. The lymphocytic infiltrate is a promising additional parameter for histopathological evaluation of breast cancer core biopsies. Primary systemic therapy is the treatment of choice in locally advanced breast cancer. Besides the well-established adjuvant therapy regimens neoadjuvant chemotherapy (NACT) is increasingly used in patients with operable cancers The adaptive immune system is thought to play an important role in suppressing the progression of malignant cancers Using core biopsies of untreated breast carcinomas for the analysis of predictive markers, NACT regimen can be used as in vivo chemotherapy-sensitivity test with pCR as indicator of beneficial outcome from chemotherapy A total of 313 FFPE primary tumor core biopsies were evaluated in the prospective PREDICT study, a substudy of the GeparQuinto trial. The GeparQuinto trial (NCT 00567554) was a prospective, randomized, open label, multicentre phase III trial program exploring the integration of Bevacizumab, Everolimus (RAD001) and Lapatinib into current neoadjuvant chemotherapy regimes for primary breast cancer. Chemotherapy consisted of 4 cycles of epirubicine, cyclophosphamide followed by taxane. The PREDICT study was designed as a substudy of GeparQuinto for prospective validation of molecular biomarkers in HER2 negative tumors in the neoadjuvant setting. Only HER2-negative patients in setting 1 that did not receive Bevacizumab were included in the PREDICT study. 93 centers  have participated in the Predict substudy and have provided tumor samples in parallel to the randomization. Everolimus was administered to the non-responders in a second randomization, at that time the lymphocyte analysis had already been performed. 37 patients investigated for lymphocyte parameters were randomized to the Everolimus arm of GeparQuinto. Written informed consent for use of biomaterials was obtained from all patients, ethic committee approval was obtained for all centres participating in the clinical study and from the institutional review board of the Charité hospital.All clinical data, including the immunohistochemical data on estrogen receptor, progesterone receptor and HER2 status were extracted from the clinical study databases and represent the local assessment. This was predefined in the prospective statistical analysis plan for the PREDICT study.All samples were formalin-fixed, paraffin-embedded pretherapeutic core biopsies collected before randomization, with written informed consent. Samples were stored in the GBG tumor bank at the Institute of Pathology, Charité Hospital, Berlin, Germany.The following inclusion criteria were used: 1) HER2 negative patients that were randomized to setting 1 of GeparQuinto and did not receive Bevacizumab, 2) available primary tumor sample for biomarker analysis, 3) available data on pathological complete response (pCR). pCR was defined as the absence of residual invasive tumor cells in breast and lymph nodes .Tissue samples were fixed in neutrally buffered formalin. From each paraffin block, a 2 µm section was prepared and stained with haematoxylin and eosin (H&E). Only samples with a proportion of tumor tissue of at least 30% of the whole tissue area were included in the analysis. Stromal Lymphocytes, intratumoral lymphocytes as well as LPBC were defined as previously described in Appendix Table A1 (online only) of Intratumoral lymphocytes (iTu-Ly) were defined as intraepithelial mononuclear cells within tumor cell nests or in direct contact with tumor cells. They were reported as the percentage of the tumor epithelial nests that contain infiltrating lymphocytes. Stromal lymphocytes (str-Ly) were defined as the percentage of the tumor stroma area that contains a lymphocytic infiltrate without direct contact to tumor cells. The lymphocyte-predominant breast cancer (LPBC) was defined as tumors with either intratumoral lymphocytes in ≥60% of tumor cell nests or lymphocytes in ≥60% of the stromal area.In this study the TILs were evaluated by only one pathologist. The interobserver variability for this evaluation has been tested by our group in the previous study, with a kappa score of 0.61 Statistical analysis was performed using SAS 9.2 under SAS Enterprise Guide 4.3 . pCR rates were reported in subgroups defined by binary parameters  and compared between subgroups using exact test of Fisher. The probability of pCR as a function of immunological parameters was determined by univariate logistic regression analysis. The multivariate logistic regression was used to adjust analysis for the known clinical parameters having influence on pCR.An independent prospective validation was performed in the PREDICT study, a substudy of the GeparQuinto trial. The CONSORT statement and workflow of the tumor samples are described in High-grade tumors had a significantly increased pCR rate of 30.9% compared to the group of G1–G2 tumors with a pCR rate of 11.9% . Hormone receptor negative tumors showed a significant increase of pCR rate . The pCR rate of invasive ductal carcinomas was 21.9%, compared to 2.9% for invasive lobular tumors (p = 0.01). The pCR rate of nodal negative patients was significantly increased compared to patients with tumor infiltrated lymph nodes . The evaluation of tumor stage (cT1–2 vs. cT3–4) and age of the patients (<50 vs. ≥50 years) showed no significant influence on the pCR rates after NACT .The group of lymphocyte-positive breast cancer (LPBC) had a significantly increased pCR rate of 36.6%, whereas non-LPBC tumors had a pCR rate of 14.3% .In logistic regression analysis , all thrThe multivariate analysis revealed LPBC and increased stromal lymphocytes (strLy) as significant independent predictors for pCR with an OR of 2.7  and OR of 1.2 , respectively. The presence of intratumoral lymphocytes (iTuLy) was no significant independent parameter for pCR in multivariate analysis . Another significant independent parameter in multivariate testing was a negative hormone receptor status p<0.05, .In an exploratory analysis, we have analyzed the pCR rate according to hormone receptor status: In our cohort, HR positive tumors had a pCR rate of 12%, while HR negative tumors had a pCR rate of 36.5%. In HR positive tumors (n = 209), the pCR rate for LPBC was increased to 28.2% (11 pCRs of 39 tumors), while it was only 8.2% (14 pCRs of 170 tumors) for non-LPBC tumors . For the HR negative tumors (n = 104) the pCR rate was increased to 44.2.% for LPBC, compared to 31.1% for non-LPBC .Previous data from our group demonstrated that the degree of lymphocyte infiltration can be used as a continuous predictive factor for response to NACT. Tumors with a particular strong lymphocytic infiltrate, designated LPBC, had a significantly increased pCR rate compared to tumors with low lymphocyte infiltration + T cells mediated by DNA-damaging agents such as cyclophosphamide + T cell infiltration remained unchanged in complete responders There are several distinct mechanisms of the tumor-immune interaction in response to chemotherapy that may be considered for our results. Chemotherapy can increase the susceptibility of tumor cells to lysis by cyctotoxic CD8+ T cells and T helper CD4+ T cells infiltrated in the tumor tissue lead to a reduction of the tumor growth +CD25highFoxP3+ regulatory T cells (Treg cells) can stimulate tumor growth + T cells on the clinical outcome in 1334 breast cancer patients. The authors demonstrated that the rate of tumor-infiltrating cytotoxic CD8+ T cells significantly correlates with improved clinical outcome + T cells through direct cell-cell contact and/or secretion of transforming growth factor β (TGFβ). High infiltration rates of Treg cells were found in the peripheral blood as well as in the tumor tissue in a variety of tumors including breast cancer The large group of infiltrating lymphocytes can encompass T cells as well as B cells with their respective different subgroups. It is known that the composition of the immune infiltrate can influence the prognostic and/or predictive impact in different tumor types including breast carcinoma Increased levels of mature B cells can be located in secondary lymphoid tissues as well as in the tumor tissue of different tumor entities including breast cancer In our current data we evaluated the lymphocytic tumor-infiltrate without further identification of the subgroups of the different T and B cell subpopulations. Nevertheless, our previous investigation analyzed immunohistochemically the composition of the lymphocytic infiltrate consisting of CD20 positive B cells and CD3 positive T cells In conclusion, we show in a prospectively evaluation that tumor-associated lymphocyte infiltration in breast carcinoma tissue is a predictor for response to NACT. Confirming our previous data, we demonstrated that an increased lymphocytic infiltration rate is predictive for response to anthracycline/taxane-based NACT. Our findings suggest that the comparably simple evaluation of tumor-associated lymphocytes in H&E sections could be used as an additional parameter to define a group of patients that might benefit from NACT.


LDLR) gene in an effort to provide an improved large animal model of familial hypercholesterolemia and atherosclerosis. Yucatan miniature pigs are well established as translational research models because of similarities to humans in physiology, anatomy, genetics, and size. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, male and female LDLR+/− pigs were generated. Subsequent breeding of heterozygotes produced LDLR−/− pigs. When fed a standard swine diet , LDLR+/− pigs exhibited a moderate, but consistent increase in total and LDL cholesterol, while LDLR−/− pigs had considerably elevated levels. This severe hypercholesterolemia in homozygote animals resulted in atherosclerotic lesions in the coronary arteries and abdominal aorta that resemble human atherosclerosis. These phenotypes were more severe and developed over a shorter time when fed a diet containing natural sources of fat and cholesterol. LDLR-targeted Yucatan miniature pigs offer several advantages over existing large animal models including size, consistency, availability, and versatility. This new model of cardiovascular disease could be an important resource for developing and testing novel detection and treatment strategies for coronary and aortic atherosclerosis and its complications.Recent progress in engineering the genomes of large animals has spurred increased interest in developing better animal models for diseases where current options are inadequate. Here, we report the creation of Yucatan miniature pigs with targeted disruptions of the low-density lipoprotein receptor ( CFTR) gene closely recapitulate human CF, including lung disease The application of gene targeting technology in livestock animals, particularly pigs, has generated significant interest in the development of new models of human diseases. In cases where existing rodent models fail to recreate key clinical phenotypes, pigs are an attractive alternative. Perhaps the best example of this is the recent development of porcine models of cystic fibrosis (CF) Atherosclerosis is characterized by the accumulation of proliferative smooth muscle cells, macrophages, lipids, cholesterol, calcium deposits, and cellular debris in vessel walls LDLR) gene lead to familial hypercholesterolemia (FH) LDLR gene. These heterozygotes typically have twice the normal plasma LDL levels and a significantly increased risk for atherosclerosis. Individuals with two defective LDLR alleles face early-onset atherosclerosis and often die of the disease at a young age.In humans, loss-of-function mutations in the LDL receptor  gain-of-function mutation (D374Y), which is associated with human autosomal dominant FH, was reported Recently, the development of transgenic Yucatan miniature pigs expressing a proprotein convertase subtilisin/kexin type 9  to create Yucatan miniature pigs with targeted disruptions in one or both This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Institutional Animal Care and Use Committee of Exemplar Genetics approved all animal experiments. All animals were housed in Exemplar Genetics' AAALAC-accredited facilities. Standard procedures for animal husbandry were used throughout.Sry gene Fetal fibroblasts were previously isolated from approximately day 35 Yucatan miniature pig fetuses as previously described Genomic DNA was isolated from Yucatan fetal fibroblasts (Qiagen). A 8.6 kb PCR product which included a region from LDLR exon 2 to exon 6 was amplified using a high fidelity polymerase  and LDLR primers 2F2 and 6R2 (see R cassette. The primers for the 5′ arm were LDLR5′armF (XhoI) and LDLR5′armR (EcoRV). The primers for the 3′ arm were LDLR3′armF (HindIII) and LDLR3′armR (HindIII). The engineered termination codon is at amino acid position C128 (C128X). This targeting construct (pLDLR-Neo) was used as a template to create the amplicon for the generation of the LDLR-targeting proviral vector.The 5′ and 3′ homology arms were amplified by PCR using plasmid pLDLR2/6 and subcloned sequentially into a plasmid containing a PGK-NeoNotI) and AAV-LDLR-R (NotI). This product was subcloned into the rAAV proviral plasmid, pFBAAV2-CMVP.NpA (obtained from University of Iowa Gene Transfer Vector Core) and grown in Sure2 cells (Stratagene) to ensure ITR integrity. This proviral plasmid is referred to as pAAV-LDLR-Neo. The rAAV targeting virus was produced by the University of Iowa Gene Transfer Vector Core.PCR amplification of a 4.5 kb amplicon from plasmid pLDLR-Neo was achieved by using the following primers: AAV-LDLR-F  were performed as previously described LDLR and RNeo were produced by PCR amplification using the following primers: LDLR XmnI probe1F/LDLR XmnI probe 1R and PGK-NeoF/NeoR-R, respectively.Genomic DNA isolation, whole genome amplification, and Southern blotting procedures were performed as previously described Nuclear transfer was performed by Viagen, Inc.  as previously described LDLR+/+, LDLR+/−, and LDLR−/− pig liver. Poly A+ RNA was obtained from liver total RNA by using FastTrack MAG mRNA Isolation Kits (Invitrogen). Northern blot analysis was performed using the NorthernMax formaldehyde-based system (Ambion). LDLR and GAPDH probe templates were amplified using the primer sets LDLR North8F/9R and pGAPDH North1F/2R, respectively.Total RNA was isolated  from LDLR exons 1–5.Total RNA was isolated as described above. First strand cDNA was synthesized with 2 μg of total RNA using SuperScript III (Invitrogen) and following manufacturer's instructions. PCR was performed using primers LDLR North4F and LDLR Exon 5R1, which spans Pig liver was homogenized on ice in RIPA buffer  supplemented with protease inhibitor cocktail (Sigma), 1 mM PMSF (Sigma), and 1 mM DTT. The homogenate was centrifuged at 4°C, 10,000×g for 30 minutes, and the supernatant was combined with SDS-PAGE sample buffer . The liver lysate samples were heated in boiling water for 5 minutes, cooled on ice, and sonicated with a Sonic Dismembrator (Fisher Scientific). The samples were electrophoresed in a 7.5% SDS-PAGE gel with Tris-Glycine running buffer. Protein was transferred onto a PVDF Immobilon-P Transfer Membrane (Millipore) using XCell II Blot Module (Invitrogen) according to manufacturer's instructions. Immunoblot analysis was performed using primary antibodies against LDLR , β–tubulin , and secondary antibodies conjugated with horseradish peroxidase . Signal was detected using Chemiluminescent Detection Kit (Pierce).Whole blood was collected in EDTA-containing tubes, and plasma was prepared and frozen at −80°C. Lipid analysis of most samples was performed by the Lipoprotein Analysis Laboratory in the Department of Pathology at Wake Forest University School of Medicine . Total cholesterol was measured by enzymatic assay, lipoproteins were measured by gel filtration chromatography. Total cholesterol of samples from 15–18 month old heterozygote animals was determined by LipoScience .LDLR+/+ and LDLR−/− littermates were flushed with normal saline and perfusion-fixed under systemic pressure with 10% buffered formalin. Abdominal aortas were immersion-fixed in 10% buffered formalin. Sections of each coronary artery were taken perpendicular to the direction of blood flow at one cm intervals from their origin and embedded in paraffin. The number of sections reflects the distance covered. In general there are an average of 20 total coronary artery sections per pig heart: 7 left anterior descending coronary artery, 10 right coronary artery, 3 to 4 circumflex. The coronary artery sections were stained with Verhoeff-van Gieson to highlight the internal and external elastic lamina. Adjacent sections were stained with hematoxylin and eosin to confirm lesion details. The abdominal aorta between the renal and iliac arteries was photographed for measuring the percent surface area with raised lesions when macroscopically present as follows: % surface area with raised lesions  =   ×100. Measurements were confirmed by Sudan IV staining. The abdominal aorta was then divided into 1 cm sized opened segments taken perpendicular to the direction of blood flow from the level of the renal arteries to the origin of the iliac arteries, producing on average 6 sections per pig and paraffin embedded. Light microscopic sections were prepared from the proximal half of each segment and stained with Verhoeff-van Gieson and hematoxylin and eosin.The heart and coronary arteries from female Analysis for significance was performed with GraphPad Prism software (GraphPad Software) using one-way ANOVA with post hoc comparison using the Tukey test or unpaired Student's t test, as specified.LDLR gene. Specifically, a neomycin-resistance cassette (RNeo) was inserted into exon 4 of porcine LDLR  was used to deliver the targeting vector to male and female fibroblasts harvested from Yucatan miniature pig fetuses. Following rAAV infection and antibiotic selection, sing PCR . The gensing PCR  [1], [40egration .LDLR+/− cells were used as nuclear donors for SCNT LDLR+/− piglets   . Souther female) .LDLR-targeted Yucatan miniature pigs, total cholesterol was measured in a group of 15 to 18 month old male LDLR+/− cloned pigs (n = 16) and wild-type controls (n = 7) fed a standard diet (∼2% fat and no cholesterol). LDLR+/− pigs total cholesterol was elevated relative to control pigs   .LDLR+/− males and females were retained for breeding purposes. For this study, sixteen litters were produced and yielded the expected Mendelian inheritance of 20 LDLR+/+, 40 LDLR+/−, and 21 LDLR−/− pigs. Since the sires and dams were each derived via SCNT from single, LDLR-targeting events, each litter produced from these matings is essentially from the same cross.Cloned RNeo cassette and inserted a premature termination codon. The most likely consequence of this mutation is the induction of nonsense-mediated mRNA decay LDLR transcripts , while LDLR−/− pigs had dramatically elevated levels . At three to four weeks of age, pigs were weaned and placed on a standard diet consisting of no cholesterol and ∼2% fat. When measured again at 26 weeks of age, the cholesterol levels in these pigs were similar to what was seen pre-suckle. These cholesterol data are summarized in LDLR−/− pigs would show the presence of atherosclerosis having been fed a standard pig diet with no added cholesterol or fat. We necropsied three LDLR−/− pigs and two LDLR+/+ controls at 7 months of age and assessed the presence of atherosclerosis in the coronary arteries and the abdominal aorta. The LDLR+/+ pigs had no visible atherosclerosis in the abdominal aorta, while the LDLR−/− pigs had 45% of the abdominal aorta covered with raised atherosclerotic lesions that were distributed fairly equally over proximal and distal portions. Atherosclerotic lesions had areas of probable foam cells and small areas of necrosis, hallmarks of human atherosclerosis  and Verhoeff-van Gieson (VVG) and evaluated for the presence of atherosclerosis. The LDLR+/+ pigs had no visible atherosclerotic lesions in the right coronary artery (RCA), left anterior descending artery (LAD), or circumflex artery (CIRC). Small areas of intimal thickening were seen in the proximal portion of each. The LDLR−/− pigs however, had small atherosclerotic lesions, as well as intimal thickening in sections from the RCA, LAD, and CIRC. These were predominantly in the proximal portion of the arteries but there was some extension into the distal portions as well. A representative lesion is shown in Coronary arteries from the LDLR-targeted pigs by feeding a high fat, high cholesterol diet. For this study, we included only females and barrows  since they are commonly used for atherosclerosis studies in industry settings. All pigs were raised on a standard diet until the age of 5 months, at which time they were transitioned to a diet containing ∼40% saturated fat and ∼1% cholesterol from natural sources including eggs and butter.We next sought to increase the severity and accelerate the onset of atherosclerosis in the Cholesterol levels were measured following 90 and 180 days on the diet . There wLDLR−/− pigs had an appreciable amount of atherosclerosis in the abdominal aorta. While the total abdominal aortic surface area was comparable in all three groups, LDLR−/− pigs had significantly higher surface area with raised lesions and percent aortic surface area covered with lesions  than either the LDLR+/− or LDLR+/+ pigs  suggested that FH is a recessive disease in pigs, because cholesterol levels in heterozygotes did not differ from wild-type controls. However, this is likely due to the high genetic variability seen in these animals, including the presence of various ApoB alleles and other genetic modifiers LDLR-targeted Yucatan miniature pigs allows the difference between LDLR+/+ and LDLR+/− animals to be more readily observed.Our results are consistent with human FH, a dominant disorder exhibiting a gene dosage effect in which LDLR-targeted Yucatan miniature pigs offer several advantages over existing large animal models of hypercholesterolemia and atherosclerosis. First, at 60–75 kg, mature Yucatan miniature pigs are one-third to one-half the size of domestic pigs (including the Rapacz FH pigs) making them more representative of average human size and easier for most researchers to conduct studies. This is important for studies directed at the development of new human devices and diagnostic equipment. Second, due to the “knock-out” nature of the gene disruption, a more severe, and thus more rapidly developing, atherosclerosis is possible in the homozygotes compared to the Rapacz or PCSK9-D374Y transgenic pigs. This will decrease the duration and costs of studies. Furthermore, since the heterozygote pigs have only one functional LDLR allele, they are susceptible to accelerated, diet-induced hypercholesterolemia and atherosclerosis . This makes them uniquely suited for the development of therapeutic approaches that directly, or indirectly, induce LDL receptor expression and the evaluation of a treatment's ability to impede or even reverse the progression of atherosclerosis. This may not be possible with the constitutive transgene expression in the PCSK9-D374Y pig. Finally, because of the homogeneous genetic background of the miniature pig line, the variability in phenotype and response to interventions seen in current models should also be reduced.This model also has some limitations, with perhaps the greatest being its size and the challenges that come with it. A large animal model requires specialized housing, trained staff, unique study facilities, and in some cases, specially formulated diets. Most current animal models of hypercholesterolemia and atherosclerosis require a highly modified diet often consisting of 1–4% pure cholesterol and sodium cholate, which are not typical of human diets and with the latter being possibly toxic and counterproductive LDLR-targeted Yucatan miniature pigs remain to be addressed. We describe the presence of advanced atherosclerotic lesions in eleven-month-old LDLR−/− pigs fed a high fat, high cholesterol diet, but we did not observe plaque rupture or thrombosis. A longer study is underway to better establish the full spectrum and timeline of phenotypic development, though the results presented here are encouraging. The present study also does not address the impact of gender and puberty on phenotype. Our standard diet study only looked at pigs up to 6–7 months, which is the point at which pigs undergo puberty. We saw no discernable differences between males and females up to that point. The high fat, high cholesterol diet study only looked at castrated males and females, and again, no differences in atherosclerosis severity were seen. Finally, it is obvious that an animal model with improved predictive efficacy is needed. While that will take more time to demonstrate, we are currently seeking to validate that drugs that have been successful in humans are equally effective in the porcine model. Perhaps more importantly, we would also like to know whether LDLR-targeted pigs would have predicted the failure in humans of drugs that were initially shown to be efficacious in mice.Several issues regarding the LDLR-targeted Yucatan miniature pigs provide a versatile new model system for pursuing research into new devices, diagnostic tools, and therapeutics for human cardiovascular disease. This study further validates genetically engineered pigs as useful biomedical models of human disease.The Figure S1LDLR-targeted pig fetal fibroblasts.Genotyping results from  A. Example of PCR results. Primers amplified a 1.5 kb product from the wild-type allele and 3.2 kb product from the LDLR-targeted allele. Lanes 1–5 are examples of PCR-positive cell clones. Lane 6 is a wild-type cell clone. B. Southern blot of whole genome amplified DNA. (Left) XmnI digested genomic DNA was hybridized with a probe that detects porcine LDLR downstream of the targeting vector boundary. The LDLR-targeted allele produced an approximately 7.8 kb band, and the wild-type band is approximately 6.0 kb. (Right) The same DNA was hybridized with a probe that detects the RNeo cassette, yielding only the targeted 7.8 kb band. Lane 3 is an example of a properly targeted cell line. Lane 5 is a wild-type control.(PDF)Click here for additional data file.Table S1List of PCR and Sequencing Primers.(PDF)Click here for additional data file.


The diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns.We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium  was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture.Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced.The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia. Primary ciliary dyskinesia (PCD) is a rare inherited multi-genic disorder of cilia, impairing mucociliary clearance. It has a prevalence of approximately 1∶10,000 to 40,000 in Europe Subjects with an indicative clinical history should be referred to a specialist PCD service for investigations that may include nasal nitric oxide (nNO) concentration, assessment of ciliary function and ultrastructural axonemal defects Using their ability to regrow healthy ciliated epithelium from an initial sample with a high degree of secondary damage Jorrisen et al The PCD diagnostic centres in the UK Local and national R&D and ethical approvals were obtained . All subjects gave written consent for sampling and for their data to be used.Between 2009 and 2012, 158 subjects referred to centres in Leicester and Southampton had analysis of cilia before and after culture at ALI as part of routine PCD diagnostics. An additional 7 subjects had insufficient cilia for analysis prior to culture, but sufficient following culture at ALI. Leicester and Southampton PCD centres are two of the three national diagnostic centres in England working to share protocols and standards. The HSV and TEM data is audited between the centres to assess accuracy of reporting and to maintain consistency of reporting across the service.A cytology brush  was inserted approximately 5 cm into the patients naris and the nasal epithelium was brushed. This method obtained strips of nasal ciliated epithelium, and each brushing contained up to 10,000 cells. Each sample was divided in to three to allow, ciliary functional analysis, fixation for TEM and cell culture. Brushings were only performed if patients had been free from an upper respiratory tract infection for at least four weeks. HSV analysis was conducted on all samples pre- and post- ALI culture. TEM was conducted on all samples with any abnormality of CBP or CBF pre-culture, but not necessarily if function was entirely normal. TEM was generally only conducted following culture if it was expected to aid diagnostic decision making e.g. if the pre-culture TEM sample was insufficient for full analysis, had severe secondary damage or provided an equivocal result.Protocols were similar across the centres, but equipment differed, hence baCiliated epithelium for TEM was fixed in 3–4% glutaraldehyde and prepared, imaged and analysed in a blinded fashion as previously described 16. In brief, nasal brush biopsies were grown on collagen  coated tissue culture trays (12 well) in bronchial epithelial growth medium (BEGM)  for approximately a week. Confluent basal cells were then expanded into collagen coated flasks (25 or 75 cm2) and the BEGM replaced three times per week. Suspensions of the non-ciliated basal cells were then seeded  onto collagen-coated transwell inserts (1.2 cm2)  in a 12 well culture plate under ALI medium (1:1 BEBM:DMEM 4.5 g/L D-glucose including SingleQuots) until confluent. The monolayer of primary human basal epithelial cells was then exposed to an air-liquid interface, feeding them basolaterally only with ALI-medium supplemented with an additional 100 nM all-trans retinoic acid . The medium was exchanged three times per week and any apical surface liquid or mucus removed. Once cilia were observed (3–6 weeks post transition to ALI), the ciliated epithelium was recovered (by gentle scraping using a spatula) from the transwell for analysis of ciliary function and TEM analysis of the axonemal structure, as described above (Leicester), or a quarter segment of the ALI culture was mounted in the chamber slide, prior to CBF determination (x100 objective lens) by fast fourier transformation analysis (Southampton).The cell culture methodology was as previously describedThe degree of ciliation per well varied between 5–50% in each subject and the time to ciliogenesis was also variable between 3–8 weeks post ALI culture. There was no difference in the ciliogenesis time between PCD and non-PCD cultures.CBF was determined from at least 6 separate strips of epithelium sideways on to the observer and the mean and confidence intervals reported for each subject. Data was normally distributed. Paired t-tests were used to compare mean CBF before and after culture, statistical analysis was performed in SPSS version 20 for Windows  and figures produced in GraphPad prism 5 . A p-value less than 0.05 was considered significant.4  and Leicester (n = 73) for diagnostic testing. We included consecutive subjects who had paired analysis of ciliary function before and following ALI culture as part of their PCD diagnostic assessment. Seven subjects whose samples were insufficient for analysis but whose post-culture sample was used for diagnostic analysis are not included in the study. Forty-one participants from Leicester and 13 from Southampton were diagnosed with PCD based on a combination of clinical history, ciliary function and ultrastructure4 . The cen4 . All theThe ciliary beat pattern of samples from subjects who were finally diagnosed with PCD was uniformly abnormal prior to culture. Dyskinetic patterns included static cilia, static in combination with twitching cilia, cilia with rigid stroke lacking full sweep and a rotating pattern. Beat frequency was outside the normal range for the majority of PCD cases . All samEight of 13 Southampton PCD samples and 13 of 41 Leicester PCD samples demonstrated a combination of static and abnormal motile cilia pre-culture, however post- culture at ALI went on to exhibit completely static or near static (<1 Hz) cilia (supplementary data: video 1 a and b). An additional 9 samples with slow ciliary beat frequency had further reduction of the frequency following culture. All samples continued to demonstrate universal dyskinesia after culture. The mean* ciliary beat frequency decreased following culture  . , outer dynein arm defects (n = 5), inner dynein arm defect (n = 12) and microtubular disorganization (n = 3). The outer and inner and the outer dynein arm defects had the least ciliary movement compared with the inner arm and microtubular disorganisation group before culture. The degree of motion seen in these EM phenotypes was decreased following ALI culture and there was no individual EM defect that was more prone to becoming static after ALI culture.Samples from subjects who finally had the diagnosis of PCD excluded, demonstrated a range of functional ciliary abnormalities when analysed immediately after nasal brushing. These included areas of poor coordination, lack of full sweep and mucus impedance (example of poor coordination: supplementary data file: video 1). The mean ciliary beat frequency of most, but not all samples was within the normal range before culture . TEM anaFollowing culture, all subjects were considered not to have PCD on the basis of normal post-ALI culture ciliary function (supplementary data file: video 2), combined with other diagnostic criteria including TEM. The mean ciliary beat frequency tended to decrease following culture in the Southampton cohort only,  but the size of this drop in frequency was small and not considered physiologically relevant  . SamplesUltrastructure was re-analysed by TEM post-culture in 41 non-PCD samples. These continued to show no ultrastructural defects suggestive of PCD.CBP and CBF was determined in all samples including the fresh biopsy and post cultured cells. In non-PCD subjects the dyskinesia was significantly reduced after cell culture Click here for additional data file.Video S2The same subjects cilia as in  The image was recorded at 500fps and played back at 30fps.(MPG)Click here for additional data file.Video S3High speed video images of ciliary function from a subject with PCD showing abnormal movement. Recorded at 500fps and played at 30fps.(MPG)Click here for additional data file.Video S4The same PCD subject as in  Recorded at 500fps and played at 30fps.(MPG)Click here for additional data file.