Arabidopsis uncovers distinct temporal classes of gene expression and opens the door to detailed studies of the regulatory pathways involved.A transcriptome analysis of male gametophyte development in  The haploid male gametophyte generation of flowering plants consists of two- or three-celled pollen grains. This functional specialization is thought to be a key factor in the evolutionary success of flowering plants. Moreover, pollen ontogeny is also an attractive model in which to dissect cellular networks that control cell growth, asymmetric cell division and cellular differentiation. Our objective, and an essential step towards the detailed understanding of these processes, was to comprehensively define the male haploid transcriptome throughout development.Arabidopsis and used Affymetrix ATH1 genome arrays to identify a total of 13,977 male gametophyte-expressed mRNAs, 9.7% of which were male-gametophyte-specific. The transition from bicellular to tricellular pollen was accompanied by a decline in the number of diverse mRNA species and an increase in the proportion of male gametophyte-specific transcripts. Expression profiles of regulatory proteins and distinct clusters of coexpressed genes were identified that could correspond to components of gametophytic regulatory networks. Moreover, integration of transcriptome and experimental data revealed the early synthesis of translation factors and their requirement to support pollen tube growth.We have developed staged spore isolation procedures for The progression from proliferating microspores to terminally differentiated pollen is characterized by large-scale repression of early program genes and the activation of a unique late gene-expression program in maturing pollen. These data provide a quantum increase in knowledge concerning gametophytic transcription and lay the foundations for new genomic-led studies of the regulatory networks and cellular functions that operate to specify male gametophyte development. Development of eukaryotic cells towards particular cell fates is regulated by complex and dynamic changes in gene expression. These changes, when monitored on a genome-wide scale, provide a detailed framework for the analysis and modeling of cellular development. To monitor patterns of gene expression it is important to be able to isolate cells at precise stages along a developmental pathway. Well-developed procedures for cell culture and single-cell PCR techniques have allowed genome-wide changes in gene expression to be monitored during animal cell differentiation -4.Arabidopsis - which include the Affymetrix ATH1 gene arrays. Recently, a significant advance in transcriptome analysis of plant cell types has been achieved through fluorescence-activated cell sorting of cell-type marked and protoplasted root cells using Affymetrix ATH1 micorarrays [Transcriptomic studies of single cell types in plants have focused on diploid sporophytic cell types, including undifferentiated cell suspensions ,6, leaf orarrays . This haIn contrast to such enabling technologies and procedures developed for sporophytic cell types there have been no studies that provide a genome-wide perspective of cell fate determination and differentiation during haploid gametophyte development. The haploid male gametophyte generation of flowering plants has a simple and well-defined pathway of development and consists of two- or three-celled pollen grains that deliver two sperm cells via the pollen tube to the embryo sac at fertilization. The highly reduced cell lineage and functional specialization of the male gametophyte are thought to be key factors in the reproductive fitness and evolutionary success of flowering plants. Moreover, pollen ontogeny provides an attractive model of cellular development in which to dissect the regulation of cell growth and division, cellular differentiation and intercellular communication (for reviews see -17).Arabidopsis that affect all phases of pollen development [Plumbago zelanica [Arabidopsis and to analyze changes in gene expression from unicellular microspores to mature differentiated pollen grains. A particular advantage of the male gametophyte generation is that developing microspores and pollen grains are symplastically isolated. This facilitates access to viable cell populations at different stages of haploid development without contaminating sporophytic cells.Recent progress in understanding of molecular and cellular aspects of pollen development has emerged from genetic studies that have identified mutants in elopment -29. In pzelanica -33. DespArabidopsis using serial analysis of gene expression (SAGE) [Arabidopsis and analyzed mRNA populations only in mature differentiated pollen grains. These studies do not, therefore, provide a developmental perspective of gene expression during development and differentiation of the male gametophyte.Some initial progress has been made towards the definition of the male gametophytic transcriptome of n (SAGE)  and Affyn (SAGE) ,36. Thesn (SAGE) ). HoweveArabidopsis and the use of Affymetrix ATH1 Genome Arrays to analyze transcript expression profiles throughout four successive stages of male gametophyte development in Arabidopsis. Isolated spore populations were large enough to enable RNA extraction for direct microarray hybridization without any preceding amplification step that could lead to bias in expression signals between stages or between genes within individual stages. Progression from proliferating microspores to terminally differentiated pollen was characterized by large-scale repression and the activation of a unique collection of late-program genes during pollen maturation. Putative male gametophyte-specific genes and distinct clusters of coexpressed genes are identified, including key groups of regulatory factors including cell cycle, transcription and translation factors. Bioinformatic and experimental data are used to address the importance of transcription and translation during pollen germination and tube growthHere we describe spore isolation procedures for Arabidopsis required the introduction of a procedure for the isolation of homogeneous populations of viable spores at precisely defined stages of development. The method was based on centrifugation of isolated mixed spores in a Percoll step-gradient [Transcriptome profiling throughout microgametogenesis in gradient ,39. Larggradient .Microscopic examination of isolated spore populations revealed no contaminating sporophytic cells and little or no other cellular debris Figure . Vital sArabidopsis ATH1 Genome Arrays were used to explore the dynamics of gene expression throughout male gametophyte development in comparison with sporophytic tissues. Microarrays were hybridized with cRNA probes made from total RNA purified from isolated spores. Hybridization data from two biological replicates derived from independently grown populations of plants were compared. Only genes with a positive hybridization signal and a detection call value of 1 in both experiments were scored as expressed. Microarray data from each pair of replicates were highly correlated, with correlation coefficients of 0.986 (UNM), 0.972 (BCP), 0.991 (TCP) and 0.971 (MPG). Complete microarray data are publicly available at the European Arabidopsis Stock Centre (NASC) microarray database [database . Sporophdatabase . This prdatabase ), leavesWe have previously confirmed and validated the expression pattern of 15 putative pollen-specific genes identified using Affymetrix AG arrays by reverse transcription-PCR analysis . SimilarArabidopsis [The ATH1 Genome Array harbors oligonucleotide probes representing 22,591 genes based on the Arabidopsis Genome Initiative annotation. This represents 80.7% of the most recent estimate of 28,000 protein-coding genes in bidopsis . Of thesTo identify genes expressed preferentially or specifically in developing male gametophytes, hybridization data was compared with sporophytic ATH1 datasets . Transcripts with a consistent positive expression signal in at least one stage of male gametophyte development and a zero signal in any sporophytic dataset were considered male gametophyte-specific. In total, 1,355 specific transcripts were identified, representing 9.7% of the male gametophytic transcriptome. The number of male gametophyte-specific transcripts ranged from 857 (BCP) to 625 (MPG). Thus, in contrast to the decline in the total number of diverse transcripts expressed, the representation of male gametophyte-specific transcripts increased, from 6.9% and 7.2% at UNM and BCP-stages to 8.0% and 8.6% at TCP and MPG-stages respectively.Analysis of the distribution of transcripts among three abundance classes: high , medium (10- to 100- fold less) and low (more than 100-fold less) Figure , revealeR value) calculated  between the transcriptomes of UNM and BCP, the two earliest developmental stages , but with greater deviation than the early stages. The scatter plot of TCP and MPG revealed the shift between extreme mRNA abundance classes as described above. This was more evident when bicellular and tricellular stages were compared , provide evidence that the major quantitative shift in transcriptome size between BCP and TCP stages is not simply the result of repression, but also involves the activation of new groups of genes associated with pollen maturation. The lack of correlation (R = 0.194) between gene expression profiles in uninucleate microspores and mature pollen  than to mature pollen (R = 0.194). This is also in accord with the lack of correlation between transcriptome profiles of mature pollen and cell suspensions (R = 0.13).The relationship between cell proliferation activities and transcriptome profiles was examined by comparison of early UNM and late MPG stages with a publicly available suspension cell culture dataset. These comparisons demonstrated that the microspore transcriptome was significantly more similar to that of cell suspensions  were grouped into four clusters (25, 27, 29 and 35) comprising early expressed genes repressed after PMII. Several large gene clusters collectively containing 1,899 genes (13.6%) were associated with pollen maturation. These were activated or upregulated between BCP and TCP stages, forming clusters 5, 7, 11, 13, 18-24, 26, 28, 38 and 39. In contrast, a discrete set of 298 genes forming cluster 17 was upregulated only after TCP stage. In total, 3,342 late genes (24%), forming clusters 1-3, 6, 8 and in particular, cluster 17, encode proteins that are likely to function during post-pollination development.Arabidopsis transcription factor families were derived by compilation of data available at The Ohio State University Arabidopsis Gene Regulatory Information Server [We focused our further analysis on three key categories of genes with likely regulatory significance in male gametophyte development; core cell-cycle genes, transcription factors and core translation factors Figure . Core cen Server , data pun Server  and datan Server ,50, respn Server  were defn Server .Among 61 core cell-cycle genes, 55 genes were present on the ATH1 GeneChip and 45 (82%) of these were expressed in the male gametophyte . Representative(s) of all families and subfamilies were expressed. The majority of gametophytic core cell-cycle genes showed similar expression profiles Figure , with a We identified 1,594 genes encoding putative transcription factors that were divided into 34 gene families . Their representation on the ATH1 GeneChip was 1,350 (85%). Of these, 608 (45%) were expressed in the male gametophyte, including 54 (15.7%) that were male gametophyte-specific. There were distinct differences in the representation of large transcription factor families (with over 25 members) in the gametophyte. Among those over-represented were the p-coumarate 3-hydroxylase (C3H) family (67% of family members present on the ATH1 GeneChip), the CCAAT family (64%), C2H2 zinc finger proteins (57%), the WRKY family (53%), the bZIP family (51%), the TCP family (50%) and the GRAS family (50%). In contrast, the AUX/IAA (20%), HSF (33%), bHLH (34%), NAC (34%), AP2-EREBP (35%), HB (36%), R2R3-MYB (37%), MADS (37%) and C2C2 zinc finger (37%) gene families were all under-represented.The dominant expression pattern of transcription factor genes reflected the general repression of mRNA diversity between BCP and TCP stages Figure . BesidesAtPAB3 (At1g22760), AtPAB6 (At3g16380), AtPAB7 (At2g36660), AteIF2-B3 (At3g07920), AteIF4G-like (At4g30680) and AteIF6-2 (At2g39820). There was a striking over-representation of poly(A)-binding (PAB) proteins among the male gametophyte-specific genes; seven out of eight PAB genes were male gametophyte-expressed, three of which were specific. Moreover, two of these gametophyte-specific PAB genes were among the few late pollen genes encoding translation initiation factors  of these were expressed in the male gametophyte . The vast majority of translation factor genes belonged to the early group and these were strongly expressed Figure . Reflects Figure .de novo synthesis of a new set of late pollen transcription factors, suggested storage of translation factors and ongoing transcription after pollen germination. Therefore we investigated the dependence of Arabidopsis pollen germination and tube growth on transcription and translation. Pollen was cultured with increasing concentrations of actinomycin D and cyclohexmide to examine the importance of transcription and translation, respectively. Actinomycin D had only moderate effects on both pollen germination and tube growth even at high concentrations . Moreovview see -55. The Arabidopsis mature pollen transcriptome using Affymetrix 8K AG arrays led to the identification of 992 and 1,584 pollen-expressed mRNAs, respectively [Two recent studies of the ectively ,36. Resuectively . Howeverectively . Therefoectively  to arounectively  to arounDevelopmental analysis of transcriptome data revealed two striking features, a sharp reduction in transcript diversity after BCP stage and a major shift in mRNA populations between BCP and TCP stages. The decline in mRNA diversity after BCP stage is associated with terminal differentiation as well as the documented phenomenon of protein storage in pollen  documents the homogeneity in expression profiles of most early genes. In contrast, late gene clusters included a significant number of genes with similar profiles between BCP and TCP stages, followed by expression profiles that deviated between TCP and MPG stages. Cluster 1, and in particular cluster 17, contained genes strongly upregulated in TCP and MPG, with likely functions in post-pollination events. The differential fate of certain late gene clusters is likely to be a feature of their requirement during pollen maturation or post-pollination events.Arabidopsis PAB mRNAs were gametophytically expressed. Three PAB genes (PAB3, PAB6 and PAB7) appeared to be male gametophyte-specific and PAB5 was preferentially expressed in pollen. Moreover, PAB3 and PAB5 are the most abundant early and constitutive PAB mRNAs and PAB6 and PAB7 belong among the few late core translation-factor genes. Although these data suggest-specific expression, our data do not rule out expression in other sporophytic tissues, particularly in flowers. Indeed, previously published expression data confirmed the expression of these PABs in other reproductive tissues together with pollen [Our analysis revealed completely different expression profiles of transcription factors when compared to core translation factors. The majority of core translation factors belonged to the early-group genes with few that were male gametophyte-specific. This may be expected, given that many genes are involved in general cellular activities. However, genes encoding PAB proteins did not follow the general trend. Seven out of eight h pollen .Conversely, transcription factors showed more diverse spectra of expression profiles including early, constitutive and late. There was a considerable variation in the expression profiles of individual transcription factor families. The most over-represented was the C3H family, members of which are known to have roles in lignin and other phenylpropanoid pathways in plants . AlthougArabidopsis pollen germination and tube growth were relatively independent of transcription, and that active pollen-tube growth, and to a lesser extent pollen germination, were dependent upon protein synthesis. It is known for some plant species that mRNAs and rRNAs accumulate during pollen maturation and are stored for use during pollen germination [Arabidopsis pollen is charged with a diverse complement of stored mRNAs that could be used to support pollen germination and pollen tube growth. Moreover, the early synthesis of mRNAs encoding translation factors strongly suggests that these are preformed and stored in mature pollen grains to support rapid activation upon hydration and germination. We also suggest that some abundant late transcription factors could regulate maturation-associated genes or act as repressors of inappropriate transcription in growing pollen tubes.The majority of core translation factors belonged to the early gene clusters. In contrast, a significant number of transcription-factor genes were strongly expressed during pollen maturation. These data alone did not obviously support the fact that pollen germination and early tube growth in many species are largely independent of transcription, but vitally dependent on translation . Similarmination ,63. Our The key impact of this work is that it provides a genome-wide view of the complexity of gene expression during single cell development in plants. Analysis of the male gametophytic transcriptome provides comprehensive and unequivocal evidence for the unique state of differentiation that distinguishes the developing male gametophyte from the sporophyte. Male gametogenesis is accompanied by large-scale repression of gene expression that is associated with the termination of cell proliferation and the selective activation of new groups of genes involved in maturation and post-pollination events. Development is associated with major early and late transcriptional programs and the expression of about 600 putative transcription factors that are potential regulators of these developmental programs. This wealth of information lays the foundation for new genomic-led studies of cellular functions and the identification of regulatory networks that operate to specify male gametophyte development and functions.Arabidopsis thaliana ecotype Landsberg erecta plants were grown in controlled-environment cabinets at 21°C under illumination of 150 μmol/m2/sec with a 16-h photoperiod. Isolated spores from three stages of immature male gametophyte were obtained by modification of the protocol of Kyo and Harada [g, 3 min, 4°C). Concentrated spores were loaded onto the top of 25%/45%/80% Percoll step gradient in a 10-ml centrifuge tube and centrifuged (450 g, 5 min, 4°C). Three fractions were obtained containing: (1) microspores mixed with tetrads; (2) microspores mixed with bicellular pollen; and (3) tricellular pollen  and stored at -80°C. The purity of isolated fractions was determined by light microscopy and 4',6-diaminophenylindole (DAPI) staining according to [in vitro for 4 h according to [d Harada ,39. Afterding to . Mature rding to . Pollen Total RNA was extracted from 50 mg of isolated spores at each developmental stage using the RNeasy Plant Kit (Qiagen) according to the manufacturer's instructions. The yield and RNA purity was determined spectrophotometrically and using an Agilent 2100 Bioanalyzer at the NASC.24 primer fused to T7 RNA polymerase promoter. Biotin-labeled target cRNA was prepared by cDNA in vitro transcription using the BioArray High-Yield RNA Transcript Labeling Kit (Enzo Biochem) in the presence of biotinylated UTP and CTP.Biotinylated target RNA was prepared from 20 μg of total RNA as described in the Affymetrix GeneChip expression analysis technical manual. Double-stranded cDNA was synthesized using SuperScript Choice System (Life Technologies) with oligo(dT)Arabidopsis ATH1 Genome Arrays were hybridized with 15 μg labeled target cRNA for 16 h at 45°C. Microarrays were stained with streptavidin-phycoerythrin solution and scanned with an Agilent 2500A GeneArray Scanner.Sporophytic data from public baseline GeneChip experiments used for comparison with the pollen transcriptome were downloaded from the NASC website ,64. The All gametophytic and sporophytic datasets were normalized using freely available dChip 1.3 software . The relArabidopsis Genome Annotation Release 3.0 published by The Institute for Genomic Research [Arabidopsis thaliana Database [Microsoft Excel was used to manage and filter the microarray data. For annotation of genes present on the ATH1 Array, the Research  was usedDatabase , Kyoto EDatabase  and TAIRDatabase . HierarcDatabase , with coArabidopsis transcription factor families were derived by compilation of data available at the Ohio State University Arabidopsis Gene Regulatory Information Server [The following additional data is available with the online version of this article: Additional data file n Server , data pun Server  and datan Server  and 24]Arabidopsn Server . The chaThe complete transcriptomic datasets usedClick here for additional data fileThe complete set of 39 clusters of genes coexpressed during male gametophyte developmentClick here for additional data fileThe expression profiles of male gametophyte-expressed transcription factors sorted into individual gene familiesClick here for additional data file


FEZ1/LZTS1, FHIT, TP53, P27kip1, and BCL2).Collecting duct carcinoma (CDC) is a rare but very aggressive variant of kidney carcinoma that arises from the epithelium of Bellini's ducts, in the distal portion of the nephron. In order to gain an insight into the biology of this tumor we evaluated the expression of five genes involved in the development of renal cancer  cases. Fhit protein was absent in three cases (27%). The overexpression of p53 protein was predominantly nuclear and detected in 4 of 11 cases (36%). Immunostaining for p27 was absent in 5 of 11 cases (45.5%). Five of the six remaining cases (90%) showed exclusively cytoplasmic protein expression, where, in the last case, p27 protein was detected in both nucleus and cytoplasm. Bcl2 expression with 100% of the tumor cells positive was observed in 4 of 11 (36%) cases. Statistical analysis showed a statistical trend (P = 0.06) between loss and reduction of Fez1 and presence of lymph node metastases.These findings suggest that Fez1 may represent not only a molecular diagnostic marker but also a prognostic marker in CDC. Renal cancer accounts for 3% of adult malignancies. An estimated 35,710 new cases are expected to occur in the U.S. in the current year and 12,480 patients will die of this disease [Recent evidence suggests that solid renal parenchymal tumors arising in the distal portion of the nephron, such as oncocytomas, chromophobe renal cell carcinomas and collecting duct carcinomas represent an heterogeneous group of neoplasm from both clinical and biological perspectives . CollectHistochemically, CDC cells contain intracytoplasmic mucicarminophilic material, where RCCs do not . CDCs arLoss of heterozygosity (LOH) analysis in six CDCs revealed allelic loss at chromosomes 8p and 13q in 50% of the tumors, and rarely at the short arm of chromosome 3 ,13. ThisTo improve our understanding of the biology of CDC and to explore the possibility that different genes may be involved in the etiology and prognosis of this neoplasm, we analyzed by immunohistochemistry eleven cases of CDC for the expression of five genes (Fez1, Fhit, p53, p27, and bcl2) often involved in the development of many common cancers. These findings were correlated with conventional clinical-pathological features including clinical outcome.Eleven patients with primary CDC  underwent radical nephrectomy between 1983 and 2000 in the Department of Urology, University of Padua, Italy. Tissue specimens from these tumors were registered in the Department of Pathology at the same institution. All eleven patients had an adequate clinical follow up and were included in our study. Eight of the 11 patients had metastatic disease, five with lymph node metastasis and five with distant metastases at the time of surgery. None of these eleven patients received any systemic therapy before surgery. Samples were fixed in 10% buffered formalin for routine histological processing and were stained with hematoxylin and eosin.Ulex Europaeus Lectin, polyclonal antibody were performed as previously described [Immunohistochemical reactions using anticytokeratin (Keratins 5, 8, 10, an 18) monoclonal antibodies, and escribed . The tumescribed ,5-7 and escribed .Paraffin sections containing non-neoplastic kidney as well as neoplastic areas were deparaffinized according to standard procedures followed by rehydration through graded ethanol series, and mounted on positively charged slides. Immunostaining was performed as previously described . BrieflyWe evaluated the association between each marker's expression (positive or negative) and each clinical-pathological outcome (metastasis and stage) with Fisher's exact test. For survival, we used the Kaplan-Meier method and log-rank test, as well as Cox proportional hazards regression.All cases showed several common microscopic features. The main tumor consisted of a tubulo-papillary carcinoma showing pleomorphism and a high mitotic rate with several bizarre mitotic figures, and presence of small cystic spaces surrounded by a highly desmoplastic stroma.Two cases showed a predominant papillary pattern accompanied with dilated and atypical changes in the epithelium of the collecting ducts in the adjacent renal medulla, which was the most convincing feature supporting a collecting duct origin of these tumors. Metastases, when available for examination, were histologically similar to the infiltrating part of the primary tumors. Immunocytochemically, tumor cells from all cases examined did not express keratin 10, while strong positivity to anti-keratin 5, 8, and 18 antibodies, and anti-UEL antibody was observed. The positivity to high molecular weight keratin and UEL further substantiate the diagnosis of CDC. The clinical-pathological features and protein expression of the study's 11 cases are shown in Table Our results showed that Fez1 protein expression was undetectable in six of the eleven (55%) cases. One case showed a substantial reduction of expression with 60% of the tumor cells negative, while the remaining four showed diffuse positivity for Fez1 immunostaining Figures  and 1B. Fhit protein was absent in three of eleven cases (27%), and the remaining eight showed diffuse immunoreactivity Figure . Reductip53 protein was found to be overexpressed mainly in the nucleus in four of eleven cases (36%) Figure . One of Immunoreactivity for p27 was observed in the nuclei of most glomerular and tubular cells in the normal kidney. p27 immunostaining in CDC showed high variability. Protein expression was absent in five of eleven cases (45.5%). All the remaining six carcinomas showed a high percentage (> 40%) of p27 positive cells. The expression of p27 protein was detected exclusively in the cytoplasm in five of the six positive cases (90%) while a mixture of nuclear and cytoplasmic protein staining was observed in the last case Figure . OverallBcl2 protein expression in 100% of the CDC tumor cells was observed in four of eleven cases (36%) Figure , while tClinical-pathological features as well as immunohistochemistry results are listed in Table FEZ1, FHIT, P53, P27kip1, and BCL2) in a relatively large series of CDCs (eleven cases), considering the rarity of this tumor.The application of the most recent molecular cytogenetic techniques revealed that renal parenchymal tumors can be classified into distinct subtypes based on the combination of specific genetic alterations . The patFEZ1/LZTS1 (leucine zipper tumor suppressor 1) is a putative tumor suppressor gene located at 8p22 [ at 8p22 . Studies at 8p22 . LOH at  at 8p22 , suggestFEZ1 encodes a 67-kDa leucine-zipper protein with a region of similarity to cAMP-dependent activated protein [FEZ1 gene have been reported in several solid tumors, including prostate, breast, esophageal, and gastric carcinomas [FEZ1 into Fez1 null cancer cells reduced cell growth with the accumulation of cells at late S to G2/M phase of the cell cycle. Conversely, inhibition of Fez1 expression stimulated cells growth [FEZ1 gene lies (8p21-22) has been also associated with the invasive behavior of breast cancer [FEZ1 in several human cancers including CDC. protein . Mutatiorcinomas ,19. In arcinomas . Recent s growth . Furthert cancer  and witht cancer . These dFHIT maps to the short arm of chromosome 3 (3p14.2), encompasses the common FRA3B fragile region, and encodes for a protein of 16.8 kDa, with diadenosine triphosphate hydrolase activity [FHIT gene has been observed by immunohistochemistry in many types of cancers [FHIT gene in tumorigenesis of this rare tumor cannot be excluded.The tumor suppressor gene activity . Reducti cancers ,27. Alth cancers , Hadacze cancers . The sam cancers . While iTP53 tumor suppressor gene maps to chromosome 17p13.1 and plays a major role in DNA transcription, cell growth, proliferation and apoptosis process [The  process . In norm process  our dataP27 is a member of the universal cyclin-dependent kinase inhibitor (CDKI) family. The expression of this important protein is regulated by cell to cell contact inhibition as well as by specific growth factors, such as transforming growth factor (TGF-β). In addition to its role as a CDKI, p27 is considered a putative tumor suppressor gene, a major regulator of drug resistance in solid tumors, and a promoter of apoptosis. p27 acts also as a safeguard against inflammatory injury and has a role in cell differentiation . It has Nevertheless, we did not find statistical correlation to assess its involvement in CDC biology, possibly due to the limited number of tumors studied.BCL2, implicated in the regulation of cell death by inhibiting apoptosis, seems to be vital in normal kidney morphogenesis. In fact, Bcl2 deficient mice develop polycystic kidneys characterized by dilated proximal and distal tubules [The proto-oncogene  tubules . High le tubules . In the Our results suggest a potential association between FEZ1 expression and CDC pathology and prognosis. No similar patterns were seen for any of the other markers studied. Even so, the statistical power of the study was limited and negative findings should not be construed as evidence that these markers are not important. Rather, a larger study would need to be carried out to further investigate their role in CDC.FEZ1 gene alterations may be linked to the high frequency of LOH found at 8p22, where FEZ1 resides. The lack of similar association for the other four genes studied may be due to the low statistical power of the study.Our results suggest that Fez1 expression may be associated to both clinical-pathological features and survival in patients with CDC. None declared.CDC, Collecting Duct Carcinoma; RCC, renal cell carcinoma; LOH, loss of heterozygosityA.V. carried out the immunohistochemical analysis and reviewed the slides, he also contributed to the draft of the manuscript. T.P.G. was responsible for the clinical study. M.G. carried out the original histopathological diagnosis. H.I. participated in the immunohistochemical analysis and statistical analysis. E.G. participated in the immunohistochemical analysis and statistical analysis. F.P. was responsible for the clinical study. L.G.G. participated in designing the study and in drafting the manuscript. C.M.C. participated in designing the study. R.B. participated in the original design and coordination of the study, and in writing the manuscriptThe pre-publication history for this paper can be accessed here:


Pelvic lymph nodes metastasis is an important prognostic factor for patients with cervical carcinoma. However, the relationships between the number of positive nodes, site of metastases nodes, adjuvant therapy and the prognosis is controversial. The purpose of this study was to investigate the influence of positive lymph nodes on the prognosis of Chinese women with stage IB1-IIB cervical carcinoma.Between January 1992 and December 1997, 398 women with International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IIB cervical carcinoma underwent radical surgery in Cancer Hospital, Fudan University. Of these sixty-six patients (16.6%) who were histologically confirmed to have positive pelvic lymph nodes were analyzed retrospectively. The survival was estimated using Kaplan-Meier method. The differences in survival were compared with Log-rank test. Multivariate analyses were performed with the Cox proportional hazard model.P < 0.05). The 5-year survival of patients with one positive node was higher than that of those with two or more positive nodes (56.5% vs. 36.4%, P < 0.05). The distant metastasis rate in the former group (5.9%) was lower than the latter's (32.7%) (P = 0.05). However, there was no significant difference of pelvic recurrence between the two groups (P > 0.05). The number of positive nodes positively correlated with the level of positive nodes (P < 0.01). The 5-year survival of the patients who had no adjuvant therapy (12.6%) was much lower than that (53.7%) of those with adjuvant therapy (P < 0.05). However, there was no obvious difference between adjuvant radiotherapy, chemotherapy and chemo-radiotherapy (P > 0.05).The 5-year survival of the patients with pelvic lymph nodes metastases was 40.7%. Cox proportional hazard model analysis showed that cellular differentiation, the number of positive nodes and adjuvant therapy to be the independent prognostic factors (The prognosis of patients with stage IB1-IIB node-positive cervical carcinoma who underwent radical surgery alone was very poor. Adjuvant therapy increases the survival rate, decreases the pelvic recurrence and distant metastasis. Although radical radiotherapy (RT) and radical surgery can be the proper choices for patients with early stage cervical cancer, most of the patients in China prefer the radical surgery to RT. Hence, in China the radical surgery has been widely used as first-line therapy for this group of women. Some poor prognostic subgroups have been identified, among these the pelvic lymph node status has been considered as the most important prognostic factor. Radical hysterectomy with bilateral pelvic lymphadenectomy produces an expected 85–90% survival in women with stage IB and IIA cervical carcinoma without lymphatic spread. However, once tumors involve regional lymph nodes, 5-year survival has been reported to be only 30–60% . In mostBetween January 1992 and December 1997, 398 women with International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IIB cervical carcinoma underwent radical surgery at the Department of Gynecologic Oncology, Cancer Hospital of Fudan University. Of these 66 patients who had undergone Wertheims-Meigs' surgery  and were histologically confirmed to harbor positive pelvic lymph node were included in this study. The median age at diagnosis was 49 years (range 21 to71). Out of 66, 8 patients were in stage IB1 (12.1%), 37 patients (56.1%) in stage IIA and 21 patients (31.8%) in stage IIB. Histologically 41 women (62.1%) had squamous carcinoma, 20 (30.3%) had adenocarcinoma, 4 (6.1%) adenosquamous carcinoma and 1 patient (1.5%) had small cell carcinoma. The tumors in 4 patients (6.1%) were well differentiation, 46 cases (69.7%) moderately differentiated and 16 (24.2%) poorly differentiated. The average lymph nodes resected were 14.8 per patient while the average positive lymph nodes resected were 3.7 (1~28) per patient. The average diameter of the cervical tumors was 3.6 cm (1~7 cm). The details of the patients' clinical characteristics are listed in Table Sixty four of these women (97.0%) had brachytherapy in either three or four fractions with a total dose of 15~20 Gy at point A, two weeks prior to radical surgery because of bulky tumor or vaginal vault involvement. Intra-arterial chemotherapy was administered to 11 patients (16.7%) before surgery because of bulky tumor or parametrial extension. The regimen based on cisplatin (CDDP) + 5-Fluorouracil(5-FU) with 2~3 cycles at 3-weeks intervals was used. All patients underwent Wertheims-Meigs' procedure that included radical hysterectomy and bilateral pelvic lymphadenectomy. Three patients had para-aortic lymph node (PALN) sampling because there was suspicion of metastasis.Postoperative external beam irradiation was delivered to 18 patients (27.3%) with 6 MV X-ray routine anterior and posterior portal at a dose of 35~45 Gy at point B with 1.8 Gy daily fraction. Two patients with positive vaginal margin were given extra brachytherapy with 15 Gy 0.5 cm below the vaginal mucosa. 12 cases with positive common iliac node and 3 with PALN metastasis received an additional 40 Gy to the PALN chain area. 10 patients (15.2%) were given postoperative chemotherapy. The regimen consisted CDDP + 5-FU + Cyclophosphamide (CTX) for squamous carcinoma, and CDDP + 5-FU + Mitomycin (MMC) for adenocarcinoma with 2~6 cycles at 3~4-weeks intervals. A total of 19 patients (28.8%) received adjuvant radiotherapy and chemotherapy as mentioned above.Patients were evaluated every two months for the first two years and then six monthly for the additional years by clinical interview, or telephone, or letters. Disease-free survival (DFS) was defined as the time from the date of surgery to local or nodal recurrence or metastasis. Overall survival (OS) was calculated from the date of surgery to the date of death. Recurrences were defined as local if they were detected in the pelvis or vagina and distant metastases as detected in extra-pelvic locations. The median follow-up time was 32 months (range 2~108 months).P < 0.05 was considered significant.Data analysis was performed with Statistical package for social sciences (SPSS) version 10.0 statistical package. The survival was calculated by Kaplan-Meier method. The differences in survival were compared with Log-rank test. Multivariate analyses were performed with the Cox proportional hazard model. The correlation analysis was performed by Kendall's method. Pearson's chi-square or Fisher's exact test was used to compare the difference of proportions. A probability value of P < 0.05). The former's distant metastasis rate (5.9%) was lower than the latter's (32.7%) (P = 0.05). However, there was no significant difference of pelvic recurrence between them (P > 0.05)  was higher than that (36.4%) of those with two or more positive nodes (atter's 3.7% (P = 5) Table The 5-year survival 33.3%) of the patients with positive common iliac node and paraaortic lymph node (PALN) was less than that (43.1%) of   patients with lower than common iliac node involvement. However, the difference was not significant (P > 0.05). The former's pelvic recurrence rate (13.3%) was lower than the latter's (29.4%) (P > 0.05). However, the patients with common iliac or paraaortic nodes had significantly higher distant metastasis (53.3%) than that of the patients with lower nodes (17.6%) (P < 0.01). The number of the positive lymph nodes was closely correlated with the level of lymph node metastasis. The relative coefficient was 0.557 (P < 0.01)  of the   patients who had no adjuvant therapy was much lower than that (53.7%) of   those with adjuvant therapy (P<0.05). The 5-year survival rates of the adjuvant radiotherapy, adjuvant chemotherapy, and adjuvant radio-chemotherapy groups were 53.5%, 49.2% and 56.1% respectively (P > 0.05). The pelvic recurrence (42.1%) and distant metastasis (31.6%) of the surgery alone were higher than those with adjuvant therapeutic groups. However, the differences were not significant (P > 0.05) (Table .6% of thP < 0.05) . The 5-year survival of patients with one positive node (56.5%) was higher than that (36.4%) of those with two or more positive nodes (P < 0.05). There was no significant difference of pelvic recurrence between them. However, the former's distant metastases rate (5.9%) was lower than the latter's (32.7%) (P = 0.05). In the analysis of the site of lymph node metastases, the 5-year survival (33.3%) of the patients with positive common iliac node and paraaortic lymph node (PALN) was lower than that (43.1%) of patients with lower than common iliac node involvement. Unlike the series of Tsai the difference in our series was not statistically significant [P < 0.01). The result suggested that in patients with more positive pelvic nodes there is higher chance of nodal metastasis at higher level node basins, which supported the external irradiation of PALN chain area for the multiple pelvic nodes involvement. The distant metastasis rate was 32.7% in patients with multiple positive lymph nodes, which showed the limitation of adjuvant radiotherapy and theoretically supported the postoperative combined chemotherapy.Radical surgery has been found to be very effective in patients with early stage (IB-IIA) cervical carcinoma -13. Howenificant . This iset al [The prognosis of patients with positive lymph node was poor because of local recurrence and distant metastasis. How to improve the prognosis of these patients has been the focus of gynecological oncology. Stock et al  comparedPeters' study  demonstret al [Several randomized clinical trials -25 perfoet al  did a syet al ,27. Receet al .The therapy of patients with stage IIB cervical carcinoma is still a controversy . AlthougThe author(s) declare that they have no competing interests.XC collected the data, patients' follow-up and drafted the article.SC, ZL, MT, MX collectively designed the study, participated in collection of data and revising the article.RZ participated in collection and analysis of data, revising the articleNone


How trophic factors regulate the expression of FasL is presently unclear, but two regulators of FasL, FOXO3a (FKHRL1) and JNK have been described to play a role in other cell types. Thus, their potential function in motoneurons was investigated in this study.Programmed cell death of motoneurons in the developing spinal cord is thought to be regulated through the availability of target-derived neurotrophic factors. When deprived of trophic support, embryonic spinal motoneurons We show here that as a result of removal of neurotrophic factors and the consequent reduction in signalling through the PI3K/Akt pathway, Foxo3a translocates from the cytoplasm to the nucleus where it triggers cell death. Death is reduced in Fas and FasL mutant motoneurons and in the presence of JNK inhibitors indicating that a significant part of it requires activation of the Fas/FasL pathway through JNK.Therefore, in motoneurons as in other cell types, FOXO transcriptional regulators provide an important link between other signalling pathways and the cell death machinery. During development of higher vertebrates, motoneurons within the spinal cord are generated in excess, and about half the cells initially generated undergo programmed cell death (PCD) during the days following target muscle contact . The mosin vitro is activation of the Fas/CD95 death receptor by its cognate ligand, FasL [FasL expression is strongly upregulated in motoneurons cultured for 3 days without neurotrophic factors [cis between FasL and Fas on individual motoneurons [We have shown that a major driving force for the death of motoneurons deprived of neurotrophic factors nd, FasL . Fas andnd, FasL . While l factors , as in c factors . Moreoveoneurons . UnderstFasL promoter contains three FOXO DNA-binding sites, and Foxo3a-induced apoptosis of cerebellar neurons is decreased when Fas/FasL interaction is blocked by the decoy fusion protein Fas-Fc [FasL gene.The transcription factor Foxo3a  was a clear candidate. In conditions in which the PI3K/Akt survival and growth pathway is activated, Foxo3a is phosphorylated by Akt and exported to the cytoplasm where it is sequestrated by the 14-3-3 protein . Overexpn Fas-Fc . Thus, iFasL expression in some neuronal cells, through its effects on the transcriptional activity of the AP-1 complex. Although this pathway can play different roles, in neurons it is involved in apoptosis in response to several stresses, including withdrawal of survival factors [FasL upregulation in neurons induced to die results from JNK activation and phosphorylation of c-Jun [gld mice, which are defective for FasL, the killing effect induced by trophic deprivation is reduced compared to wt mice.The JNK pathway has also been shown to regulate  factors . In cereof c-Jun . MoreoveFasL activation both directly and through JNK is a major target of its actions.We therefore wished to study the function of Foxo3a in motoneurons and its relation to JNK signalling. We show that, in the absence of survival signalling through the Akt pathway, Foxo3a is translocated to the nucleus where it triggers motoneuron death. Using appropriate mouse mutants, we show that Fas signalling is required for roughly half of the cell death induced by Foxo3a, suggesting that Methods) to strongly activate the PI3K/Akt pathway . In these conditions, Foxo3a is predominantly detected in the cytoplasm  or the absence Fig.  of neuroResults in all conditions were quantified by counting 2 independent experiments Fig. . They clTo investigate the ability of Foxo3a to trigger death of cultured motoneurons, we took advantage of the electroporation technique we recently developed for high-efficacy transduction of primary neurons . We firsWe therefore used electroporation to analyse the effects of Foxo3a on motoneuron survival. Overexpression of HA-wt-Foxo3a with GFP had no significant effect as compared to GFP alone or GFP with the empty vector . To mimic Foxo3a activation and nuclear translocation, we then overexpressed HA-TM-Foxo3a together with GFP. The triple mutant triggered significant death of motoneurons cultured with or without NTFs Fig. . In the FasL, known to be regulated by Foxo3a and shown by us to be able to trigger motoneuron death. Motoneurons were therefore isolated from mice bearing mutations that lead to reduced signalling through the Fas pathway: gld mice (point mutation in FasL) and lpr mice (regulatory defect in Fas). As before, electroporation of HA-TM-Foxo3a in control motoneurons led to loss of 60 to 70 % of them compared to HA-wt-Foxo3a  show greatly exacerbated death responses to activation of this pathway [We overexpressed the constitutively active mutant TM-Foxo3a in the presence of neurotrophic factors to mimic the nuclear translocation of Foxo3a in their absence. We observed a 50% reduction in death induced by TM-Foxo3a when we used motoneurons that were mutant for Fas/FasL signalling. This could in theory result through several indirect mechanisms. However, the literature on direct regulation of y Foxo3a  and our  support  make it  complex ,18. The  pathway . In vitr pathway . It willlpr or gld mice. This may in part reflect the fact that these mutants are strong hypomorphs, rather than complete nulls. For example, in lpr mice, some Fas is still synthesized following splicing-out of the inhibitory transposon from primary transcripts [kip1 [The killing effect of Foxo3a was not totally abolished in motoneurons from nscripts . Howeverts [kip1 , the gluts [kip1 , transfots [kip1 , the DNAts [kip1  and the ts [kip1 . Of partS, BimL and BimEL [L and 89% by overexpression of BimS ([see During death of sympathetic neurons induced by NGF deprivation, Foxo3a directly activates Bim expression and thereby triggers cell death . Moreovend BimEL . We detend BimEL , part A)imS (see , part B)In conclusion, one of the upstream events now known to trigger death of neurons deprived of trophic support is the failure of Akt to phosphorylate Foxo3a and thereby prevent it from entering the nucleus. The fact that this mechanism is found to occur in motoneurons, a classical system for the study of neuronal cell death, opens the door to a better understanding of its role during development and in neurodegenerative pathology.lpr/lpr and gld/gld mice were purchased from the Jackson Laboratory (Bar Harbor, ME). lpr/lpr mutants present an insertion of an early transposon into the fas gene, resulting in fas transcriptional repression [Normal CD1 and C57BL/6 mice were obtained from Iffacredo (L'Arbresle, France). pression . gld/gldpression . All mutLY 294002 was purchased from Calbiochem and used at a final concentration of 100 μM. L-JNKl1 was purchased from Alexis Biochemicals and used at a final concentration of 1 μM. Rabbit polyclonal antibody to HA-tag was from Clontech. Antibodies against Foxo3A were as described previously  and wereWe are grateful to Anne Brunet for donating vectors used as the basis for our expression constructs. The vectors encoding HA-tagged Akt and Akt ca were as described previously . The vecMotoneuron cultures were prepared from E12.5 mouse spinal cords essentially as described , except Cells dissociated from E12.5 mouse ventral spinal cords were centrifuged over a 6.5% (v/v) Metrizamide cushion at 2000 rpm for 15 min. Cells at the interface were collected and washed on a BSA cushion at 1500 rpm for 5 min. Cells were resuspended in electroporation buffer at a density of 50,000 cells per 100 μL. 100 μL aliquots of the suspension were transferred to 4 mm gap cuvettes (Eppendorf) and five μg of the pCAGGS-GFP vector as well as the same molar amount of the vector of interest were added. After 15 min of incubation at room temperature, cells were electroporated using three pulses of 5 ms at 200 V with intervals of 1 s. Immediately after electroporation, 1.5 ml of culture medium was added to dilute the cells which were plated in four four-wells plates .For anti-HA staining, motoneurons electroporated with HA-tagged wt or TM Foxo3A were seeded on polyornithine-laminin-coated 12-mm diameter glass coverslips and cultured for 24 hr at 37°C in complete Neurobasal medium supplemented or not with NTFs. Motoneurons cultured without NTFs were treated with LY294002 for the last 90 min. They were then fixed in 3.6% (v/v) formaldehyde for 30 min, washed in PBS-50 mM L-Lysine, and blocked for 1 hr in 5% donkey serum, 4% BSA, 0.1% Triton X-100 in PBS-50 mM L-Lysine. Cells were incubated with the anti-HA antibody (dilution 1:500) in blocking buffer, followed by fluorochrome-conjugated anti-rabbit secondary antibody. The cells were then observed under a fluorescence microscope. Cells with typical motoneuron morphology that were both GFP- and HA-positive were analyzed. The fraction of motoneurons expressing HA-wt-Foxo3A or HA-TM-Foxo3A exclusively in the nucleus, exclusively in the cytoplasm or in both compartments was evaluated in a total of 20 cells per well.Staining for endogenous Foxo3A (dilution 1:100) was performed using the same method on non-electroporated motoneurons cultured for 24 hr with NTFs.Motoneurons were counted 48 hr after electroporation under a fluorescence microscope. Only green cells with healthy motoneuron morphology, i.e. with large cell bodies and long non-fragmented neurites, were taken into consideration. Two different wells for each condition were counted (between 50 and 350 green motoneurons) in all the experiments. Cross percentages of test versus control (being set at 100%) were calculated yielding 4 values, used to calculate the medium and the SD for each experiment.C.B. conducted most of the experiments, B.P. some. C.H and B.P. conceived the experiments and the experimental design. C.B., B.P. and C.H. wrote the paper.A – The 3 forms of Bim are expressed in motoneurons: RT-PCR was performed on extracts of 80,000 motoneurones cultured for 3 days in the presence of NTFs, in a 35 mm diameter dish. Primers used for Bim were the following: 5'-GTGACAGAGAAGGTGGACAAT-3' and 5'-ATACCAGACGGAAGATAAAGC-3'. The 3 products were:BimS 284 bp, BimSL 374 bp, BimL 542 bp; S or BimL kills a major proportion of motoneurons: B – Overexpression of Bimmotoneurons purified from E12.5 mice embryos have been electroporated with a vector coding GFP alone or coelectroporated with a vector coding GFP and a vector coding either BimL or BimS, obtained by direct cloning of the PCR products described above first cloned into pGEMTeasy, then subcloned into pcDNA3 into EcoRI sites. Conditions of electroporation were as described in Material and Methods. The surviving electroporated motoneurons were counted after 2 days in culture in the presence of NTFs.Click here for file


In quality of care research, limited information is found on the relationship between quality of care and disease outcomes. This case-control study was conducted with the aim to assess the effect of guideline adherence for stroke prevention on the occurrence of stroke in general practice. We report on the problems related to a variant of confounding by indication, that may be common in quality of care studies.Stroke patients (cases) and controls were recruited from the general practitioner's (GP) patient register, and an expert panel assessed the quality of care of cases and controls using guideline-based review criteria.A total of 86 patients was assessed. Compared to patients without shortcomings in preventive care, patients who received sub-optimal care appeared to have a lower risk of experiencing a stroke . This result was partly explained by the presence of risk factors (6.1 per cases, 4.4 per control), as reflected by the finding that the OR came much closer to 1.00 after adjustment for the number of risk factors . Patients with more risk factors for stroke had a lower risk of sub-optimal care . This finding represents a variant of 'confounding by indication', which could not be fully adjusted for due to incomplete information on risk factors for stroke.At present, inaccurate recording of patient and risk factor information by GPs seriously limits the potential use of a case-control method to assess the effect of guideline adherence on disease outcome in general practice. We conclude that studies on the effect of quality of care on disease outcomes, like other observational studies of intended treatment effect, should be designed and performed such that confounding by indication is minimized. There is a long tradition of studying at population level the quality of medical care provided to patients who died from conditions amenable to medical intervention. This type of study , aims to identify deficiencies in medical care that may have contributed to death. It was first systematically carried out on maternal death, and later on other causes of avoidable death -4. This An important limitation of this type of study, without control subjects, is its inability to fully establish a causal relationship between identified deficiencies in care and the adverse outcome, and to determine to what extent identified deficiencies are associated to the occurrence of such an event. Identified deficiencies in care are expected to indicate only to a certain extent an increase in risk of an adverse health outcome, while the probability of having an adverse outcome can be calculated only if we compare the care provided to patients who suffered an adverse outcome with that of patients who did not suffer such an event. For this reason it has been proposed to perform a case-control study with patients with an adverse event as 'cases' and a comparable group of patients without an adverse outcome as 'controls' .We performed a case-control study with the aim to assess the effect of guideline adherence for stroke prevention on the occurrence of stroke in general practice. Unfortunately, we encountered various obstacles in the design and conduct of this study, in particular related to the recruitment of cases and controls, in availability of information on the care delivery process in the GP's data registration system, and in controlling for differences other than differences in the quality of care. The aim of this paper is to highlight the problems related to a variant of confounding by indication, that may be common in quality of care studies.Observational studies of intended treatment effects are particularly prone to 'confounding by indication', and can produce misleading estimates on either, or both, the size and direction of treatment effects ,8. ConfoFrom the Dutch national GP register, a random sample was taken of 58 GPs working in Rotterdam and the surrounding region. The study was restricted to patients with a first-ever stroke meeting the following criteria for inclusion: (a) diagnosis of intracerebral haemorrhage or infarction according to the World Health Organization (WHO) definition of stroke , (b) ageIn a pilot study among 32 GPs, the quality of care measurement instruments (audit procedure and questionnaire) were tested. GPs participating in the pilot study did not participate in this study. Data on the process of care, two years prior to the occurrence of stroke (for controls from January 1995 to January 1997), were collected by means of structured face-to-face interviews with the GP, using separate questionnaires for each stroke patient. GPs were interviewed between March and October 1999. At the time of interview, GPs used either hand-written or electronic patient records to retrieve patient information. In case information was not available in the patient's record, information was drawn from the GP's memory. For each question, the type of data source was registered. The questionnaire comprised questions related to patient characteristics and family and medical history of CVD and risk factors, and the detection and treatment of cardiovascular risk factors such as hypertension, diabetes mellitus, transient ischemic attack (TIA) and cardiac failure. Similarly, data were collected on lifestyle-related risk factors such as smoking status, overweight, and excessive alcohol intake.The quality of preventive care and its potential to prevent stroke was assessed and valued by a six-member panel of experts. The panellists (three neurologists and three GPs) were selected on the basis of their clinical expertise with respect to stroke prevention, experience in quality of care evaluation, academic or non-academic background and professional discipline. Six practice guidelines relevant to stroke prevention  were selected by the panel . These gIn a two-round evaluation, with a final plenary round, cases were assessed by the panellists (panellists were divided in sub-panels). Each sub-panel assessed a specific number of cases. Based on identified elements of sub-optimal care and seriousness of shortcoming in terms of 'minor' and 'major', the panellists allocated grades on a scale of 0 to 3 Table .The two-round process was focussed on detecting consensus among the panellists (providing the same grade), and no attempt was made to force the panellists to consensus. The intersubpanel agreement was k = 0.63 . A detailed description of the assessment method is provided elsewhere .Analysis of the data was done by using simple cross-tabulations, and by using logistic regression analysis to model the chance of getting a stroke as a function of the presence of sub-optimal care (as ascertained by the panel), age and sex, and risk factors for stroke.The rate of participation was 62% (36 GPs). The main reason for GPs not to participate in the study was lack of time and interest (68%). Participating and non-participating GPs did not differ significantly in age, practice type, and date of qualification. Ninety-two percent of the GPs used electronic GP information systems. Among cases and controls there was a nonsignificant difference in mean age, however, cases were slightly older than controls (67 versus 65 years). Initially, before we excluded patients 'without' hypertension, GPs identified and selected 50 cases and 58 controls (1.4 case and 1.6 control per GP). Expected number of cases was 2.5 stroke patients per GP per year . After eOverall, data for verification of the initial diagnosis of stroke, assessment of GPs' guideline adherence, and judgement of the causality of the relationship between non-adherence and the occurrence of stroke could be collected from the patient records. However, information on risk factors such as family history of CVD, body weight (overweight), excessive alcohol intake, and smoking was less easily obtained. Depending on the type of risk factor, in 8–56% of all subjects, information on risk factors was unknown to the GP . In 41–58% information was taken from the GP's memory, instead of the patient register.In 43% of the cases and 31% of the controls, no sub-optimal care could be identified (grade 0), whereas in 57% and 69%, respectively, sub-optimal care was identified grade 1, 2 or 3). Thus the Odds Ratio for a case to receive sub-optimal care was 0.60 (95%CI 0.24 – 1.53) compared to a control (Table  or 3. ThThe mean number of risk factors among cases (6.1 per patient) was higher than among controls (4.4 per patient) Figure . If adjuPatients with a higher number of risk factors for stroke, indeed have a lower risk of sub-optimal care . As expected, higher numbers of risk factors per patient also increases the risk of stroke .This study demonstrated confounding by indication in a case-control study analysing the association between guideline adherence and the occurrence of stroke in general practice. It also provided insight into the possibilities for controlling for this confounding bias. We learned that, at present, difficulties in patient recruitment and data retrieval seriously limit the potential use of a case-control method to assess the relationship between guideline adherence for stroke prevention and stroke in general practice.We found that in specific domains data were incomplete and not readily available in the patient records. As a consequence, in many cases GPs were unable to identify stroke patients from their patient register, which most likely introduced under-reporting of stroke patients. As compared to national frequencies (2.5 stroke patients per GP per year) , GPs parStrong indications for the existence of confounding by indication were found, albeit different from how it is usually described in literature. Confounding by indication, which is conceived as a substantial problem in observational studies of treatment efficacy, usually refers to a situation in which patients who are more in need both receive more care have a higher risk of adverse health outcome . In our A more detailed analysis showed, similar to results found in previous studies, that this result partly emanates from a higher prevalence of risk factors for stroke among patients suffering stroke at a later stage in life, which not only increases the risk of stroke but also GPs' compliance to guidelines. We hypothesize that, on average, patients with more risk factors for stroke receive more attention or visit their GP more frequently, which in turn facilitates guideline adherence (e.g. compliance to quarterly follow-up of treated hypertensive patients) and at the same time results in better quality of care. Controlling for (recorded) risk factors reduced the counter-intuitive result by approximately one half, and we hypothesise that incomplete registration of risk factors for stroke explains why the risk of stroke in stroke-prone or high-risk patients associated with sub-optimal care remained below 1.00, even after controlling for risk factors. We hope that our paper draws the attention of quality of care researchers to this variant of confounding by indication, that may lead to biased associations between process measures of quality of care and care outcomes.This study shows that, at present, difficulties in patient recruitment and data retrieval seriously limit the potential use of a case-control method to assess the relationship between guideline adherence for stroke prevention and stroke in general practice. It demonstrates the role of confounding by indication, causing patients with an adverse health outcome to appear to receive better quality of care.The author(s) declare that they have no competing interestsJK, NK, PK, AP and JM conceived and designed the study. Analyses were performed by JK and GB. All authors contributed to this article and earlier drafts of the manuscript.The pre-publication history for this paper can be accessed here:


Tyger! Tyger! burning brightIn the forests of the nightWhat immortal hand or eyeCould frame thy fearful symmetry?Panthera tigris, three have gone extinct since the 1940s.When William Blake wrote these words in the late 1700s, the deforestation and habitat destruction that would decimate wild tiger populations had already begun. In 1900, an estimated 100,000 wild tigers lived throughout much of Asia, from India in the west to Sumatra and Indonesia in the south to Siberia in the east. Today, the ongoing stresses of habitat loss, hunting, and an illegal trade in tiger parts have spared fewer than 7,000 tigers. Of eight traditionally classified subspecies of Conservation strategies to combat this grinding attrition are tailored to each subspecies. But several lines of evidence suggest that subspecies designations—based on geographic range and morphological traits such as body size, skull traits, coat color, and striping patterns—may be flawed. An earlier molecular study of 28 tigers found little evidence of genetically distinct subspecies, while surveys of tiger habitat found few physical barriers sufficient for subspecies isolation.To get a clearer picture of the genetic structure of existing tiger populations, Shu-Jin Luo, Jae-Heup Kim, Stephen O'Brien, and nineteen colleagues performed a comprehensive genetic analysis of mitochondrial and nuclear genes from over 130 tigers. By identifying distinct patterns of variation within these gene families, the authors reconstructed the evolutionary distribution and ancestry of the tiger. Their results support many of the traditional subspecies designations and identify further subdivisions in others.DRB that's involved in pathogen recognition, and short repeating genetic elements called microsatellites—act as unique signposts that flag significant demographic and evolutionary events in the tiger populations.Luo et al. collected “voucher specimens”  of blood, skin, and hair from 134 tigers representing the entire tiger range, and examined them, along with samples of preserved pelts and hair, for three molecular markers. The markers—a stretch of mitochondrial DNA (mtDNA) sequence, a gene with highly variable DNA sequence called P. t. sumatrae) and (P. t. tigris) Bengal tigers—but others suggested that the Indochinese subspecies (P. t. corbetti) should be divided into two groups, representing a northern Indochinese and a peninsular Malaya population (which the authors designated respectively as P. t. corbetti and P. t. jacksoni, after the tiger conservationist Peter Jackson). Interestingly, clusters for the captive South China tigers also fell into two distinct lineages—P. t. amoyensis, the traditional grouping, and P. t. corbetti, though the designation is still tentative. These subdivisions were largely supported by the other genetic analyses.mtDNA sequences were extracted from tigers originating in the Russian far east (Siberian, or Amur, tigers), south China, northern Indochina, the Malaya Peninsula, Sumatra, and the Indian subcontinent. The mtDNA analysis identified 30 haplotypes—characteristic regions on a chromosome—that could be clustered. Some of the clusters supported traditional classifications—e.g., for the Sumatran (The distinct genetic patterns found in the tiger populations suggest six rather than five living subspecies. Reduced gene flow and genetic drift in isolated populations, as well as human activity, likely caused these partitions. The low genetic variability seen in the Siberian tigers, for example, might be explained by severe population declines: the animals were nearly exterminated in the early 1900s, and today only 500 remain. Sumatran tigers, on the other hand, show relatively high genetic variability and uniqueness, possibly reflecting a historically large breeding population that was later isolated.In what distant deeps or skiesBurnt the fire of thine eyes?Whether recent population and habitat declines, as opposed to earlier events, can fully explain these patterns is not clear. But these results offer valuable data for conservation strategies and captive breeding programs that rely on distinctions in subspecies taxonomy and geographic provenance. Evoking both the darker side of creation and humanity, Blake could not have imagined the modern fate of his “Tyger.” Scholars have long debated the multilayered meaning of his poem, including the second stanza, which starts,Will we reduce future generations to a literal reading?


The single-strand binding (Ssb) protein of phage T4 (T4 gp32, product of gene 32) is a mRNA-specific autogenous translational repressor, in addition to being a sequence-independent ssDNA-binding protein that participates in phage DNA replication, repair and recombination. It is not clear how this physiologically essential protein distinguishes between specific RNA and nonspecific nucleic acid targets. Here, we present phylogenetic evidence suggesting that ssDNA and specific RNA bind the same gp32 domain and that plasticity of this domain underlies its ability to configure certain RNA structures for specific binding. We have cloned and characterized gene 32 of phage RB69, a relative of T4 We observed that RB69 gp32 and T4 gp32 have nearly identical ssDNA binding domains, but diverge in their C-terminal domains. In T4 gp32, it is known that the C-terminal domain interacts with the ssDNA-binding domain and with other phage-induced proteins. In translation assays, we show that RB69 gp32 is, like T4 gp32, an autogenous translational repressor. We also show that the natural mRNA targets  for the 2 proteins are diverged in sequence from each other and yet can be repressed by either gp32. Results of chemical and RNase sensitivity assays indicate that the gp32 mRNA targets from the 2 related phages have similar structures, but differ in their patterns of contact with the 2 repressors. These and other observations suggest that a range of gp32-RNA binding specificities may evolve in nature due to plasticity of the protein-nucleic acid interaction and its response to modulation by the C-terminal domain of this translational repressor. T4 gp32, the single-strand binding (Ssb) protein of bacteriophage T4, is a well studied member of the Ssb protein family, and was the first such ssDNA-binding replication protein to be discovered . The proin vivo. The protein binds to a specific target  in the 5' leader segment of the mRNA from gene 32, and represses translation of this RNA [in vitro measurements of the protein's binding affinities to different nucleic acid ligands. It has been observed that ssDNA is favored over translational operator RNA as a ligand for T4 gp32 and that RNA of nonspecific sequence is the least preferred nucleic-acid ligand for this Ssb protein [In vivo, T4 encoded mRNA for gp32 is intrinsically more metabolically stable than the typical prokaryotic mRNA and is thought to have opportunities to undergo many cycles of gp32-mediated repression and depression during the replication and other processing of phage DNA. The potential for translation of this mRNA in the T4 infected E coli host is thought to be determined by availability of ssDNA in the metabolic pool [in vivo observations relating to levels of T4 gp32 biosynthesis under conditions of DNA damage or abnormal accumulation of ssDNA in the phage infected bacterial host [T4 gp32 has the interesting property of being able to control its own biosynthesis at the translational level this RNA . Anotherthis RNA ,9. It is protein -12. In vlic pool ,13,14. Dial host .In vitro studies suggest that the pseudoknot serves as the initial recognition (nucleation) site for the protein and that this gp32-RNA interaction leads to cooperative binding of additional gp32 monomers to the less structured downstream sequence containing the UUAAA/UAAA elements and ribosome-binding site (RBS) [It is not clear how T4 gp32 distinguishes between specific RNA and the non-specific nucleic acid sequence of ssDNA or ssRNA ligands. It appears that single-strandedness of the nucleic acid is not the most important criterion used by the protein to selectively bind its own message in the phage-induced mRNA pool. The translational operator for T4 gp32 has been mapped by RNA footprinting assays and determined to consist of two contiguous components, a 5' terminal ~28-nucleotide component that forms a folded structure (RNA pseudoknot) and an adjacent, less structured, >40-nucleotide component that lies 3' to the pseudoknot ,16. The The 3-dimensional structure of intact T4 gp32 has not been solved, although a number of biochemical and physiological observations have provided clues that the protein is modularly organized into 3 distinct domains . In partE coli K-12 strain K802  was used as host in cloning experiments and the E coli B strain NapIV (hsdRk+, hsdMk+, hsdSk+, thi, supo) was the host for plasmid-mediated gene expression studies that utilized lambda pL control. E coli B strain BL21(DE3), which harbors a T7 RNA polymerase gene under cellular lac promoter control [32 to bacterial cells.The AseI-digested RB69 genomic DNA to identify and retrieve an ~35-kb DNA fragment that hybridized to a T4 gene 32-specific riboprobe under stringent conditions. The riboprobe was prepared by methods described previously [32 clone pYS69 [AseI fragment in AseI-compatible Eco R1-generated ends of plasmid vectors. However, further digestion of the AseI fragment with ApoI (which generates Nde1-compatible ends) yielded a shorter, ~15-kb, fragment that could be cloned in the NdeI-EcoRI interval of vector pNEB193 (cat# N3051S, New England Biolabs, Beverly, MA). The cloned fragment was sequenced and found to be very similar to the T4 genetic segment extending from gene 59 through the 5' terminal ~2/3 of gene 32, except that the RB69-derived DNA appeared to lack a homologue of the T4 ORF 32.1 (see below). Comparisons between the T4 and RB69 gene 59-32 regions are diagrammed in Fig 32 from RB69 genomic DNA, through PCR amplification using Taq DNA polymerase. For this purpose, we utilized two primers, one perfectly matching a sequence in the cloned AseI-ApoI RB69 fragment (ie, upstream primer: 5'GCTGCTAAGAAATTGTTCATAG3') and the other (the downstream primer), an 18-mer bearing the sequence 5'CAGCAGCAGTGAAACCTTTA3', was chosen from a PCR screen of an RB69 primer library. DNA amplification was carried out under low-stringency conditions for primer annealing (30 sec at 25°C), which allowed activity from the imperfectly matched downstream primer. We obtained several products that we resolved by agarose gel electrophoresis Only one of these products, an ~35-kb DNA fragment, hybridized, although poorly, to the T4 gene 32-specific riboprobe initially used for the Southern blot analysis of Ase1-digested RB69 genomic DNA. This fragment was sequenced, using the PCR, and found to contain the 3' terminal segment of RB69 gene 32 as well as some of the region distal to RB69 gene 32 (relative to the T4 genetic map). Collectively, sequence analysis of the cloned and amplified RB69 genomic segments yielded sufficient information for designing new primers to amplify, from genomic DNA, the entire wild-type RB69 gene 32, as well as shorter segments of this gene and its putative control region in the untranslated RB69 IC59-32 region  as to maintain pL-mediated expression at undetectable levels. Minimizing plasmid-driven transcription from pL contributed to stable maintenance of the cloned wild-type RB69 gene 32, the product of which is highly toxic to bacterial cells. RB69 gene 32 mutants still emerged when such clones were grown at ≤30°C. Some of these mutants were archived for use as controls in certain studies , probably because of residual (constitutive) lac-promoter activity in this bacterial host. To circumvent potential toxicity, pSP72-based recombinants were propagated in hosts lacking a T7 RNA polymerase gene. The purified plasmid DNA from these hosts was used for in vitro transcription and translation assays. Methods for the radiolabeling of plasmid encoded proteins and their subsequent analysis by SDS-PAGE have been described elsewhere [We used the lambda r pLY965  to clonelsewhere ,25, and E coli NapIV clones of phage genes, was as described previously for T4 RegA protein [RB69 gp32 and T4 gp32 were purified from the overproducing clones pRBg32Δop (RB69 gp32) and pYS69 (T4 gp32), respectively. We used the gp32 purification protocol outlined by Bittner et al,  with min protein . Anionic protein . Under tin vitro studies originated from in vitro transcription of pSP72 clones of the desired gene 32 sequences Methods have been described elsewhere [32 transcripts and positions -96 to +161 in case of the T4 gene 32 transcripts. These products also included a 10-nt sequence from the plasmid's T7 promoter region RNA sequencing was carried out by using the RVT-catalyzed primer-extension (cDNA synthesis) method described elsewhere [in vitro translation assays, the RNA preparations included full length and truncated versions of the gene 32 open-reading frame from each of the 2 phage sources.RNA preparations used for footprinting and other lsewhere . Phage-slsewhere ,29. Sequ32 recombinant DNA (coupled transcription-translation assays) or purified RNA (DNA-free translation assays) to assess repressor activities of purified RB69 gp32 and T4 gp32. With plasmid-directed gene 32 expression, it was possible to use expression of the plasmid borne bla gene (β-lactamase) as an internal control. Each 50 μl in vitro assay reaction mixture  contained 1 μg of plasmid DNA template or 4 μg RNA, 5 μl of a mixture of all amino acids (1 mM each) except L-methionine, 1 μl of an S30-premix cocktail , 15 μl S30 extract and the balance of volume in nuclease-free water Reaction mixtures, including any added gp32, were constituted in an ice bath before transferring to 37°C for incubations (30 or 60 min). Reactions were stopped by rechilling in the ice bath. Proteins from 5 μl samples were precipitated with 20 μl acetone, collected by centrifugation, dried and suspended in SDS extraction buffer for analysis by SDS-PAGE and autoradiography. Analysis of plasmid encoded  gp32 fragments was carried out in SDS-PAGE (10% gels) using Tricine as the electrophoresis buffer. This buffer system allows for effective resolution of small polypeptides [We used E coli S30 cell-free extracts  with purified pSP72-based gene The RNA-modifying chemical reagents Dimethylsulfate  and Diethylpyrocarbonate  and the ribonucleases (RNases A1, T1 and V1 respectively) were used to probe RB69- and T4-derived operator RNAs for intrinsically structured regions. The RNases were also used for RNA footprinting (protection by gp32) studies.DMS was diluted in absolute ethanol at ratios of 1:2, 1:4, and 1:5 ratio v/v and its effects were analyzed at the three concentrations. The reaction buffer contained 30 mM HEPES pH 7.5, 10 mM MgCl2. Reactions were stopped in 0.5 M β-mercaptoethanol and 0.75 M sodium acetate. The protocol for DEPC treatment was identical to that for DMS, except that we used 1 μl of DEPC per 100 μl of reaction mix and incubated the reactions at room temperature for 10 min.4Cl, 10 mM Mg acetate, 10 mM Tris-HCl pH 7.4, and 6 mM β-Mercaptoethanol. The buffer for digestions with RNase V1 contained 25 mM Tris-HCl pH 7.2, 10 mM MgCl2, and 0.2 M NaCl Incubations were at 37°C in 30 μl buffer in all cases. RNase treatments were halted with an equal volume of buffer containing 0.4 M Na acetate pH 5.2, 20 mM EDTA, and 30 μg E coli tRNA. When used for RNA footprinting, RB69 gp32 or T4 gp32 was added at concentrations in the range between 1 μM and 5 μM.For the RNase-sensitivity assays, including gp32-mediated RNA footprinting, digestions with RNases A1 and T1 were carried out in 30 μl buffer containing 60 mM NHThe amino acid sequence of RB69 gp32 was deduced from the determined nucleotide sequence of the gene. An alignment between the predicted primary structures of this protein and its T4 homologue is shown in Fig IC59-32 region with its counterpart (the IC32.1-32 region) from T4 The T4 region (GenBank NC_00866) has been experimentally documented to harbor the translational operator for gene 32 expression [IC59-32 region that appears to have no T4 counterpart . However, such experiments, which require 10–30 μM purified protein to demonstrate repression  By these criteria, the T4 and RB69 proteins appeared to be similarly functional in each other's physiological systems. Yet, the natural targets for the 2 proteins are clearly different from each other in topography , the RB69 gp32 footprint on the RNA target from RB69 was 5 residues longer than the footprint of this protein on the T4-derived RNA target; however, the positions of the 2 footprints relative to the respective initiator AUG and 5' terminal boundary of the pseudoknot structure appeared to be identical , it has been reported that there are no indications that an RNA pseudoknot structure exists in the putative TIR for gene 32, although the UUAA/UUAAA sequence units are conserved in the RB49 IC59-32 region [In summary, we envisage that as a mediator of gp32's interactions with other phage induced proteins, the C-terminal domain of gp32 may co-diverge with its protein targets to maintain mutual recognition, and that structural plasticity of a conserved ssDNA-binding domain may allow an also diverging RNA target to establish rearranged contacts within a relatively conserved protein pocket. It is unclear if the 2 sets of divergence are interconnected, but together, they could facilitate the evolution of a high degree of diversity in how the synthesis and/or replication activity of this Ssb protein is regulated among phylogenetic relatives of T4. It will be important to find out if this diversity includes RNA ligands for gp32 that control the DNA-binding activity but not synthesis of gp32, or if autogenous translational repression has been replaced by other mechanisms for control of gene 32 in some T4 relatives. There is at least one reported example where evolution resulted in lack of RNA binding function in the Ssb protein of an M13-like phage . Also, a2 region . It remaORF32.1  homing endonuclease (now named SegG) that mediates its own transfer, along with T4 gene 32, to the ORF32.1-less genome of phage T2 in T4 × T2 genetic crosses. We note that the 5' terminal sequence of the RNA pseudoknot for T4 gp32 translational control overlaps the reading frame of the segG gene, in addition to being very similar (~83% identity) to the corresponding segment of the pseudoknot sequence of RB69, which lacks a segG gene  may be a chimera consisting of an extension of the parental segG reading frame into the recipient genome's pseudoknot sequence. Such lateral transfer events and subsequent mutation may have profound influences on evolution of the RNA binding functions of proteins that have relaxed sequence but stringent structural requirements for their RNA target.Finally, we should comment about None declared.Jamilah Borjac-Natour: Conducted most of the experimental work and initial data analysis and prepared summaries; wrote the first draft and participated in subsequent revisions of the manuscript. Vasiliy Petrov: Conducted independent analysis of data and generated summaries and composite figures for presentation in the manuscript. Participated in revision of the manuscript during later stages of preparation. Jim Karam: Directed the study, evaluated results on an ongoing basis, worked closely with the coauthors during preparation of Figures, played a major role during revision of manuscript drafts and communicated the manuscript to the journal.


In vitro studies showed that OVA-Mφ start to produce IL-10 upon interaction with allergen-specific T cells, which might mediate their immunosuppressive effects. Herein, we examined whether IL-10 is essential for the immunosuppressive effects of OVA-Mφ in vivo, and whether ex vivo stimulation of the IL-10 production by OVA-Mφ could enhance these effects.Previously, we demonstrated that OVA-loaded macrophages (OVA-Mφ) partially suppress OVA-induced airway manifestations of asthma in BALB/c mice. in vitro. The increase of IL-10 production was examined and, subsequently, ex vivo stimulated OVA-Mφ were used to treat (i.v.) OVA-sensitized mice. To further explore whether Mφ-derived IL-10 mediates the immunosuppressive effects, Mφ isolated from IL-10-/- mice were used for treatment.Peritoneal Mφ were loaded with OVA and stimulated with LPS or immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) -/-Mφ that were loaded with OVA and stimulated with ISS-ODN ex vivo, failed to suppress OVA-induced airway inflammation.We found that stimulation with LPS or ISS-ODN highly increased the IL-10 production by OVA-Mφ (2.5-fold and 4.5-fold increase, respectively). ISS-ODN stimulation of OVA-Mφ significantly potentiated the suppressive effects on allergic airway inflammation. Compared to sham-treatment, ISS-ODN-stimulated OVA-Mφ suppressed the airway eosinophilia by 85% (vs. 30% by unstimulated OVA-Mφ), IL-5 levels in bronchoalveolar lavage fluid by 80% (vs. 50%) and serum OVA-specific IgE levels by 60% (vs. 30%). Importantly, IL-10These results demonstrate that Mφ-derived IL-10 mediates anti-inflammatory responses in a mouse model of allergic asthma, which both can be potentiated by stimulation with ISS-ODN. The development of allergen-specific CD4c asthma ,2. The T+ Th3 cells and CD4+ T regulatory 1 (Tr1) cells [2, TGF-β and IL-10 [Dendritic cells (DC) are well defined as antigen presenting cells (APC) able to initiate and regulate T cell responses . Besides1) cells ,6. Macro1) cells ,8. To ex1) cells . Moreove1) cells -12. Alth1) cells ,14, it h1) cells ,16. Simind IL-10 ,9,17.in vitro [in vitro and in vivo [In the lung, alveolar Mφ participate in the maintenance of immunological homeostasis. By secreting pro-inflammatory cytokines and chemokines they direct the recruitment and activation of inflammatory cells, while they also play a key role in dampening immune responses against non-pathogenic antigens ,18. Alvein vitro ,20 and A in vivo . Additio in vivo -24. Inte in vivo . Moreove in vivo  and othe in vivo ,27 demonIn vitro studies demonstrated that allergen-specific T cells induced IL-10 production by OVA-loaded Mφ (OVA-Mφ), suggesting that the immunosuppressive effects of OVA-Mφ might be mediated by IL-10 [-/- mice, we examined whether Mφ-derived IL-10 is crucial in the suppression of allergen-induced allergic airway inflammation.by IL-10 . In thisby IL-10  and the by IL-10  increase-/- BALB/c mice were kindly provided by DNAX  and were housed in macrolon cages with filter top. All mice were provided with food and water ad libitum.Animal care and use were performed in accordance with the guidelines of the Dutch Committee of Animal Experiments. Specific pathogen-free  male BAEscherichia coli 0111:B4 were purchased from Sigma-Aldrich (St. Louis, MO). CpG-containing phosphorothioate ISS-ODN and control phosphorothioate mutated oligodeoxynucleotide were synthesized by Isogen Bioscience BV (Maarsen, The Netherlands). The ISS-ODN used had the sequence 5'-TGACTGTGAA-CGTTCGAGATGA-3' and the mutated-ODN had the sequence 5'-TGACTGTGAA-GGTTAGAGATGA-3' [OVA  and purified LPS from GATGA-3' .in vitro experiments, Mφ were plated in triplicate wells of a 96-well round-bottomed plate (Greiner Bio-One GmbH, Kremsmuenster, Austria) at 1 × 105 Mφ/well in RPMI 1640 enriched with 2% FCS, penicillin/streptomycin  and 50 μM β-mercaptoethanol (Sigma-Aldrich). Mφ were loaded with 2 mg/mL OVA and stimulated with different concentrations of LPS, ISS-ODN or mutated-ODN, for 20 h at 37°C and 5% CO2. Subsequently, supernatants were harvested and the amount of IL-10 was determined using an IL-10-specific sandwich ELISA. Stimulation with 10 μg/mL LPS or 3 μg/mL ISS-ODN triggered the highest IL-10 production by Mφ.Peritoneal Mφ were isolated from naïve BALB/c mice as described previously . For in in vivo studies, 1 × 107 Mφ/mL were loaded with 2 mg/mL OVA and were stimulated with 10 μg/mL LPS or 3 μg/mL ISS-ODN. After incubation for 3 h at 37°C and 5% CO2, the Mφ were extensively washed  to remove all residual soluble OVA, LPS, and ISS-ODN.For 5 Mφ in 50 μl saline. As an additional control group, mice were i.v. injected with 50 μL saline (sham treatment). One week after treatment, mice were exposed to OVA  aerosol challenges for 5 min on 8 consecutive days.Mice were sensitized to OVA by active sensitization with 7 i.p. injections of 10 μg OVA in 0.5 mL pyrogen-free saline on alternate days . TreatmeMice were sacrificed and were bled by cardiac puncture. Subsequently, serum was collected and stored at -70°C until analysis. OVA-specific IgE in serum was measured as described . A referBronchoalveolar lavage (BAL) was performed immediately after bleeding of the mice by lavage of the airways through a tracheal cannula with 1 mL saline containing 2 μg/mL aprotinine (Roche Diagnostics) and 5% BSA (Sigma-Aldrich). Cytokines in the supernatant of this first mL of the BAL fluid (BALF) were determined by ELISA. Subsequently, mice were lavaged 4 times with 1 mL saline. The cells in the BALF were pooled in cold PBS (including those from the first mL) and subsequently differentiated into mononuclear cells (monocytes, Mφ and lymphocytes), eosinophils, and neutrophils as described previously .IL-5, IL-10, IL-12p70 ELISAs  were performed according to the manufacturer's instructions. The detection limit of the IL-5 ELISA was 10 pg/mL, of the IL-10 ELISA 15 pg/mL, and of the IL-12p70 ELISA 62.5 pg/mL.U test (2-tailed). For ELISA, results were statistical analyzed using a Student's t test (2-tailed, homoscedastic). Results were considered statistically significant at the P < .05 level.All data are expressed as mean ± standard error of mean (SEM). Statistical analysis on BALF cell counts was performed using the non-parametric Mann-Whitney In vitro studies suggest that the immunosuppressive effects of OVA-Mφ could be mediated by Mφ-derived IL-10 [in vitro, while control mutated oligodeoxynucleotide did not. The IL-10 levels produced by OVA-Mφ increased 2.5-fold upon stimulation with LPS and 4.5-fold upon stimulation with ISS-ODN. IL-12p70 was not detectable in any of these cultures (data not shown).ed IL-10 . To furted IL-10 ,29, we tin vivo effect of the increased production of IL-10 by OVA-Mφ, peritoneal Mφ were isolated and subsequently loaded with OVA and stimulated with LPS (10 μg/mL) or ISS-ODN (3 μg/mL) for 3 h. The stimulated and OVA-loaded Mφ were administered (i.v.) to OVA-sensitized mice.To examine the P < .05) the up-regulation of serum OVA-specific IgE , compared with sham-treated mice  suppressed by 85% compared to sham-treated mice and by 79% compared to mice treated with OVA-Mφ  the IL-5 levels in the BALF by 80%, compared to sham-treated mice. IL-10 was not detectable in the BALF of any of the mice (data not shown).The BALF of sham-treated mice contained high levels of the Th2 cytokine IL-5 Figure , that coSince ISS-ODN-stimulated OVA-Mφ produced the highest levels of IL-10 and most strongly suppressed OVA-induced airway inflammation, we used these Mφ to further analyze the underlying mechanism of immunosuppression by allergen-loaded Mφ.-/- BALB/c mice. After loading with OVA and stimulation with ISS-ODN ex vivo, the IL-10-/- Mφ were administered (i.v.) to OVA-sensitized BALB/c mice.To prove that IL-10 produced by OVA-Mφ indeed mediates the observed immunosuppressive effects, we isolated peritoneal Mφ from IL-10-/- Mφ were as high as that of sham-treated mice  than in mice treated with ISS-ODN-stimulated OVA-Mφ.Serum OVA-specific IgE levels of allergic mice that were treated with ISS-ODN-stimulated IL-10e Figure . Howeverφ Figure . The ser-/- OVA-Mφ did not suppress airway eosinophilia , compared to sham-treated mice  upon treatment with ISS-ODN-stimulated OVA-Mφ  and others  found thex vivo, promote a switch from Th2 cells to Th1 cells [+ T lymphocytes, ex vivo transduced to express IL-10, it was shown that allergen-specific lymphocytes can suppress allergen-induced asthma manifestations via production of IL-10 [Mycobacterium vaccae induced allergen-specific Treg cells that produced IL-10 and TGF-β [In this study and in our previous study , we obseh1 cells ,27. Thish1 cells , while oh1 cells . Likely,h1 cells ,11, wherh1 cells . This T-h1 cells . By usinof IL-10 . Recentlof IL-10 ,48. Furtnd TGF-β . In agreHere, we demonstrated, in a mouse model of allergic airway inflammation, that treatment with allergen-loaded Mφ suppress asthma manifestations in an IL-10-dependent manner. Importantly, the IL-10 production and anti-inflammatory effects of allergen-loaded Mφ can be potentiated by stimulation with ISS-ODN. Further detailed analysis of the mechanisms underlying this Mφ-based immunotherapy may lead to the development of new strategies to induce tolerance against allergen-specific Th2 responses in allergic diseases, including asthma.JLMV carried out the allergic model, subsequent analysis, writing and preparation of the manuscript. BCAMvE, PVJ and GAH assisted with the allergic model. AJMvO participated in the direction of the study as well as writing and preparing the manuscript. All authors read and approved the final manuscript.