PALB2 gene are rare but are thought to be associated with increased risks of developing breast cancer in various populations.The partner and localizer of breast cancer 2 (PALB2) is responsible for facilitating BRCA2-mediated DNA repair by serving as a bridging molecule, acting as the physical and functional link between the breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) proteins. Truncating mutations in the PALB2 germline mutations in 122 Asian women with breast cancer, all of whom had significant family history of breast and other cancers. Further screening for nine PALB2 mutations was conducted in 874 Malaysian and 532 Singaporean breast cancer patients, and in 1342 unaffected Malaysian and 541 unaffected Singaporean women.We evaluated the contribution of PALB2, we found two novel truncating mutations and ten missense mutations in families tested negative for BRCA1/2-mutations. One additional novel truncating PALB2 mutation was identified in one patient through genotyping analysis. Our results indicate a low prevalence of deleterious PALB2 mutations and a specific mutation profile within the Malaysian and Singaporean populations.By analyzing the entire coding region of  PALB2  is a protein that interacts with BRCA2, stabilizing the intranuclear accumulation of BRCA2 proteins at sites of DNA damage . BiallelPALB2 mutations are rare, but have been reportedly associated with increased predisposition to breast and other cancers. In multiple-breast cancer case families, germline PALB2 mutations have been reported in 1.1% (10 out of 920), 2.7% (3 out of 113), 2.0% (1 out of 50), and 0.6% (5 out of 779) of Western European families in the United Kingdom, Finland, French-Canada, and Australasian, respectively ,6,172,6,Multiplex genotyping of seven PALB2 mutations identified in exon 4 in this study and an additional two Asian recurrent mutations were performed using high-throughput Sequenom MassARRAY iPLEX platform . Mutations in other exons were identified after the completion of the genotyping study and were therefore excluded from analyses. All mutations identified were confirmed by direct sequencing in an independent DNA sample.In silico analysis of the effect of missense mutations on protein function was determined using Polyphen-2 (Polymorphism Phenotyping version 2)  and SIFTBRCA1 and 13 in BRCA2. The remaining 122 individuals tested negative for BRCA mutations were analyzed for PALB2 germline mutations by direct Sanger sequencing  and deleterious mutations that were reported to be recurrent in other Asian populations (Chinese PALB2 c.751 C>T and PALB2 c.1250_1251 delAAinsTCT)  require other methods, including in vitro functional characterization [Our study also identified 10 re novel ,26. Of trization  and co-sPALB2 c.1050 delAAinsTCT) [PALB2 c.1050 delAACA) at the same site. Sequence alteration caused by deletion of AACA resulted in mutant SNP that is similar to the interrogated SNP and was therefore regarded as a positive screen. This highlights the limitation of genotyping assay in distinguishing distinct mutations which share a common mutant SNP and therefore, confirmation by conventional sequencing is necessary.Our study uncovered an unusual situation where genotyping of a deleterious mutation (AinsTCT)  revealedPALB2 mutations, 2 have moderate family history with Manchester score of 12 and 14 respectively, whereas one developed breast cancer at late age in the absence of any family history of cancer (Manchester score of 2). Indeed, by contrast to BRCA1 and BRCA2 mutation status, we found that there was no association between PALB2 mutation status and family history of breast, ovarian, prostate or pancreatic cancer (data not shown). This is consistent with other studies that have also shown no familial clustering of breast cancer in PALB2 families compared to non-BRCA carrier families [PALB2 carrier families were not available for co-segregation analysis and therefore it was not possible to determine the increased risk caused by germline mutation of PALB2 in our population.Notably, of the 3 individuals with germline deleterious families . The famPALB2 deleterious mutation carriers in 122 high risk non-BRCA1 or BRCA2 breast cancer patients by DNA sequencing and 1 germline carrier in 1406 breast cancer patients by genotyping. Our data shows that PALB2 germline mutations are rare, and are associated with family history of breast cancer in some, but not all families.In summary, we found 2 germline Table S1Amplification primers used for the sequencing of PALB2 gene.(DOCX)Click here for additional data file.


App-Runx1 genetic interval, we showed that the Ts65Dn viability and ECG were improved by this reduction of gene copy number. Whole-genome expression studies confirmed gene dosage effect in Ts65Dn, Ms5Yah, and Ts65Dn/Ms5Yah hearts and showed an overall perturbation of pathways connected to post-natal lethality  and heart function . In addition cardiac connexins  and sodium channel sub-units  were found down-regulated in Ts65Dn atria with additional down-regulation of Cx40 in Ts65Dn ventricles and were likely contributing to conduction defects. All these data pinpoint new cardiac phenotypes in the Ts65Dn, mimicking aspects of human DS features and pathways altered in the mouse model. In addition they highlight the role of the App-Runx1 interval, including Sod1 and Tiam1, in the induction of post-natal lethality and of the cardiac conduction defects in Ts65Dn. These results might lead to new therapeutic strategies to improve the care of DS people.Down syndrome (DS) leads to complex phenotypes and is the main genetic cause of birth defects and heart diseases. The Ts65Dn DS mouse model is trisomic for the distal part of mouse chromosome 16 and displays similar features with post-natal lethality and cardiovascular defects. In order to better understand these defects, we defined electrocardiogram (ECG) with a precordial set-up, and we found conduction defects and modifications in wave shape, amplitudes, and durations in Ts65Dn mice. By using a genetic approach consisting of crossing Ts65Dn mice with Ms5Yah mice monosomic for the  App-Runx1 genetic interval. Further molecular characterization revealed an overall perturbation of transcription, a few candidates homologous to human chromosome 21 genes, and changes in the expression of several genes, including cardiac connexins  and sodium channel sub-units , likely contributing to ECG defects. This model provides a unique opportunity to study further the DS heart diseases and to propose therapeutic avenues for treatment of DS cardiac complications observed in adults.Down syndrome is a common cause of birth defects mainly due to heart diseases with an incidence of up to 50%. Nevertheless the pathophysiology of DS cardiac anomalies and complications are not well understood. Here we have been able to demonstrate using a series of DS mouse models that birth defects and changes in ECG, similar to those in humans, are linked to dosage sensitive genes located in the  Homo sapiens) is the most common chromosomal anomaly and cause of intellectual disabilities Down syndrome (DS), caused by trisomy of human chromosome 21 , 17  and 10 . The largest duplication created on the Mmu16, the Dp(16)1Yey, results in a mouse trisomic for the whole region from Lipi to Zfp295 that shows cardiac anomalies 16)65Dn (Ts65Dn) model, which carries a shorter trisomy ranging from Mrpl39 to Zfp295, shows specific cardiovascular malformations associated with post-natal lethality and reduced transmission rate of the Ts65Dn allele at weaning Tiam1-Kcnj6 region To further study the correlation between phenotype and genotype in DS, various mouse models have been created At present, while morphological and histological aspects of the cardiovascular phenotypes observed in DS mouse models during perinatal age have been described, the functional aspects remain unexplored in viable adult animals. Before the development of cardiac imaging, ASD, VSD and AVSD were scored by abnormal electrocardiographic recordings (ECG) in man. CHD could be predicted on this basis in 80% of the cases in DS people. Most characteristic features of CHD-induced changes in ECG are superior frontal QRS axis deviation, first degree block and partial bundle branch block App to Runx1 region and their implications in heart defects and lethality by a subtractive approach. For that we used a partial monosomic model, Ms5Yah, carrying a deletion for the previously mentioned region and we found that compound Ts65Dn/Ms5Yah mice were partially rescued for the early post-natal lethality and some aspect of ECG features. Furthermore re-establishing euploidy in the App-Runx1 region modified gene expression changes, highlighting several pathways involved in cardiac function that were deregulated in the Ts65Dn DS mouse model.The aim of the present study was to explore the cardiac function in the Ts65Dn mouse model. Moreover we wanted to assess the susceptibility to dosage effect of genes within the 2P = 1.74×10−8). Observation of litters showed partial loss of progeny with death in the first 48–72 hours and some complete loss of litters either abandoned or cannibalized by Ts65Dn mothers. Heart and great vessels microdissection and cardiac histology analyses showed one out of 18 Ts65Dn dead pups presenting both great vessel and cardiac malformations . Moreover, these ECG features were found to be highly predictive of a Ts65Dn genotype.Since DS patients can show electrocardiographic and functional changes even in the absence of overt CHD, an electrocardiographic investigation of Ts65Dn mice was performed to look at more subtle phenotypes than gross morphological anomalies. The six standard peripheral leads  ECG of wt and Ts65Dn mice are illustrated in −4). Noticeable changes in the wave amplitudes were also recorded in Ts65Dn as compared with wt . In the P = 9×10−4). PR was prolonged from 35.3±0.5 ms to 44.6±1.1 ms . QRS wave duration was also increased by 8%. Values were respectively 9.02±0.21 ms in wt and 9.89±0.2 ms in Ts65Dn . QT and QTc were also larger in Ts65Dn (73.0±2.0 ms and 73.0±1.3 ms respectively) than in wt . Since the above-described results point to conduction changes in Ts65Dn as compared to wt, a sub-group of these mice (N = 8) were treated with the Na channel blocker, flecainide. As reported in The results were at variance in the precordial leads . R wave Lipi to Zfp295Sod1 to Zfp295, nor in the Ts1Rhr carrying a trisomy from Cbr1 to Orf9Mrpl39-Sod1 interval. Thus we developed a partial monosomic model named Ms5Yah carrying a 7.7 Mb deletion on Mmu16 between the App and Runx1 genes  indicating that the region causes lethality due to haploinsufficiency. Interestingly the transmission rate was evaluated at 27.9% at post natal day 1  and 48.3% at embryonic day 18.5  indicating a lethality within this period. Thus we considered the App-Runx1 region as a haploinsufficient region inducing post-natal lethality. We checked whether the Ms5Yah pups were carrying cardiovascular defects as observed in the Ts65Dn newborns using microdissection and histological analyses. No cardiovascular malformations, either in the great vessels or in the intra-cardiac septation could be seen in 18 dead pups. 185 Ts65Dn/Ms5Yah offspring were obtained at weaning from the breeding of Ms5Yah males with Ts65Dn females. The expected ratios were calculated considering the previously described transmission rates obtained for Ts65Dn line and Ms5Yah line. Transmission ratios for Ts65Dn and Ms5Yah alone were not significantly different from the expected ratio . However, the transmission rate for combined Ts65Dn/Ms5Yah alleles was 24.9%, as expected for a Mendelian ratio showing that the Ts65Dn-allele-induced lethality was completely rescued by the monosomy of the App-Runx1 region (χ2P = 4.8×10−7). However the transmission rate for the Ms5Yah allele did not reach the expected value showing that Ms5Yah-induced lethality still occurs. Thus the deletion between App and Runx1 genes (Ms5Yah) induced a severe postnatal lethality that is partially rescued by combining Ms5Yah with the Ts65Dn model suggesting effect due to the new chromosomal configuration.The Ts65Dn allele induces early post-natal lethality which has also been described in Ts1Yu mice, a trisomic model for a larger region of Mmu16 extending from x1 genes . The traAs we found that the Ms5Yah monosomy rescued Ts65Dn post-natal lethality, we decided to look at ECG in Ts65Dn/Ms5Yah adult mice to determine whether the combination between these two models had an effect on electrocardiologic pattern. Ts65Dn/Ms5Yah mice showed wave shape anomalies similar to those described above for Ts65Dn mice. However, relative distribution of these features in Ts65Dn/Ms5Yah  showed aP = 0.014) but not from the Ts65Dn (P = 0.53).As Ts65Dn mice showed a variable orientation of the QRS axis, this item was also determined for Ts65Dn/MS5Yah mice. Repartition of this axis for wt, Ts65Dn and Ts65Dn/Ms5Yah by quadrants is reported in P = 0.23 and P = 0.052) or Ts65Dn (P = 0.57 and P = 0.48) mice. QTc was significantly reduced down to 65.5±2.8 ms  in Ts65Dn/Ms5Yah mice. This value was no longer different from the wt value (P = 0.075). Thus two copies of the App-Runx1 region in Ts65Dn/Ms5Yah mice led to a partial recovery of the Ts65Dn electrocardiographic phenotypes. Wave shape anomalies, wave amplitudes on precordial leads and QTc duration were rescued whereas wave amplitudes on peripheral leads were not and RR and PR were partially rescued as they showed an intermediate duration between Ts65Dn and wt values.In the frontal plane, wave amplitude of Ts65Dn/Ms5Yah mice showed the same trend as Ts65Dn mice when compared with wt, with significant decrease of the S wave amplitude in inferior leads, increase in superior leads and smaller changes in R waves. While the Ts65Dn/Ms5Yah model did not rescue the changes in wave amplitudes in the frontal plane, it completely rescued changes in the sagittal plane  as illustrated in App-Runx1 region to two copies in Ts65Dn mice points at the gene dosage effect related to this interval. Whole genome expression arrays were then performed on adult mice heart samples to determine genes that are dosage sensitive and to observe the deregulations on the whole genome. Wt, Ts65Dn, Ms5Yah and Ts65Dn/Ms5Yah heart samples were analysed using Affymetrix Gene Chip technology. RMA normalized data obtained from Affymetrix Expression Console software were filtered using an expression level threshold above 50 in raw data  and fold change (FC) between wt and transgenic mice >1.2 or <0.8. We measured the expression levels of 25,099 transcripts, representing 22,193 genes. Among those, 9,017 genes (40.6%) had a fluorescence signal above the threshold in wt samples and were considered as expressed genes in adult mouse heart. The ratio of gene expression across chromosomes between aneuploid and wt mice was 1.00±0.002 (ranging from 0.98±0.003 to 1.03±0.005) except for genes located on the monosomic and trisomic intervals located on Mmu16 and Mmu17 for the specific region found in the Ts65Dn minichromosome The rescue of Ts65Dn viability and electrocardiographic phenotypes by reestablishing the post hoc analysis. The clustering on arrays of most significantly deregulated genes  are represented on In order to further analyze gene expression modifications GeneSpring software was used to define the most significantly deregulated genes using one way ANOVA and a Tukey HSD App or downstream of Runx1, or on the Mmu17 centromeric regions of the Ts65Dn minichromosome. These genes were found over-expressed in both Ts65Dn and Ts65Dn/Ms5Yah arrays but not in Ms5Yah arrays. We also found a set of 12 genes located elsewhere whose expression follow the same rationale, 9 overexpressed and 3 down-regulated as shown on Msx2, and only 6 additional genes from different chromosomes that had similar expression variations correlated with changes both in the number of copy of the App-Runx1 region and in the Ts65Dn trisomy. The other groups were mainly composed of genes located outside the above-mentioned Mmu16–Mmu17 regions and were distinguished by their expression patterns. Genes from the third group were differentially expressed in Ts65Dn arrays, while comparable to wt in Ms5Yah and in Ts65Dn/Ms5Yah arrays. 3 genes from this group were found in the proximal region of Mmu17 that is trisomic in the Ts65Dn model and one, Tiam1, was located in the App-Runx1 region but whose expression is not sensitive to dosage in Ms5Yah mice. The expression of genes from the fourth group was modified only in the Ms5Yah model. Among those genes, one gene, Sft2d1, comes from the centromeric region of Mmu17 and 5 genes  are from the Ms5Yah interval. They were all down-regulated. In addition 13 down-regulated genes and 18 up-regulated genes are found outside the aneuploid regions. A fifth group contained 10 genes over-expressed in Ms5Yah and Ts65Dn/Ms5Yah arrays whereas 15 additional genes displayed a more complex expression pattern (groups 6 and 7).Gene expression profiles were categorized in seven distinct groups with respect to genotype-associated expression patterns that are listed in App-Runx1 region that were expressed at a ratio versus wt close to 1.0. As shown in Atp5j, Cldn14, Erg, Pcp4 and Prdm15) and four on Mmu17  were found expressed but not dosage sensitive in heart.Looking more closely at the 109 Mmu16 known protein coding genes present on the Ts65Dn minichromosome, we found that 4 were absent from the chip and 39 were expressed below background level. Among the 66 remaining genes expressed in adult mice heart, 78.8% were up-regulated in Ts65Dn arrays with a mean fold change of 1.36±0.01 (ranging from 1.20 to 1.61). Similarly, of the 43 Mmu17 known protein coding genes located on the Ts65Dn minichromosome, 8 were absent from the chip and 7 were not expressed. Of the 28 Mmu17 remaining trisomic genes expressed in adult mice heart, 78.6% were upregulated in Ts65Dn arrays with a mean fold change versus wt of 1.36±0.02 (ranging from 1.21 to 1.55). Thus the presence of the Ts65Dn minichromosome results in an overall upregulation of genes present in three copies as shown on Usp16, Cct8 and Bach1 (located between App and Runx1 genes) showed fold change versus wt above 1.2 in Ts65Dn, below 0.8 in Ms5Yah and close to 1.0 in Ts65Dn/Ms5Yah samples. Dyrk1a and Sh3bgr (located between Runx1 and Zfp295) showed over-expression (fold change above 1.2) in Ts65Dn and Ts65Dn/Ms5Yah and fold change close to 1.0 (Dyrk1a) and slightly under-expressed (Sh3bgr) in Ms5Yah samples. All these data confirm the results obtained from expression arrays. Thus variation of gene expression was scored and partly verified for several genes which belong to groups of genes misregulated as a consequence of Ms5Yah or of Ts65Dn aneuploidies. Some of them were located outside the aneuploid regions, indicating a genome-wide trans effect on gene expression in Ts65Dn, Ms5Yah andTs65Dn/Ms5Yah mice hearts.Five genes were selected to validate expression levels observed with arrays using quantitative PCR analysis , Table 6eGFP transgenic mouse model whose heart conduction system is labeled with Green Fluorescent Protein (GFP) eGFP/+;Ts65Dn mice affected by clearcut ECG phenotypes were compared to three Cx40eGFP/+ disomic mice showing no ECG anomaly. Left and right ventricles were dissected out as described by Miquerol et al.et al.To go further in the understanding of ECG anomalies, an additional experiment was designed by targeting and measuring expression levels of genes related to conduction anomalies in ventricles and atria separately. Action potential propagation in the cardiac cellular network mostly depends on three factors: geometry , cardiac connexins and ion channels availability, in particular sodium channels Cx40, Cx43, Cx45 and Cx30.2 are chamber- and tissue- specific in the heart Scn5a gene which codes for a pore forming protein and is mutated in human heart conduction diseases  and for which heterozygous mutant mice suffer from abnormal heartbeats and defects in the impulse conduction system and also at Scn10a recently implicated in heart conduction + channel Scn4b was found underexpressed in Ms5Yah mice using affymetrix array and we also investigated the other beta-subunit Scn1bCx40 and Cx43 by 34 and 39% respectively and sodium channels-coding genes Scn5a and Scn10a were also down-regulated by 42% and 31% respectively and Scn1b by 29% , frontal QRS axis dispersion, decrease in right precordial lead amplitude and P wave changes, replicating in part the defects observed in DS people. PR interval prolongation (first degree AV block) observed in Ts65Dn mice includes auricle, AV node and His bundle conduction time. Gross morphology of the His bundle, as deduced from CX40-GFP labeling, does not appear to be changed. The AV node is more likely to account for PR interval prolongation even though enlargement of the auricle, as suggested by P wave amplitude increase in V4 lead and P wave axis change, could also be involved. In DS patients, such AV block and P wave changes have been recorded Cx40, Cx43 and Na channels expression that we observed in the auricle of Ts65Dn mice could account for the first degree AV block supported by the prolongation of the PR interval that includes auricle, AV node and His conduction time. This could also account for the P wave amplitude changes. Enlargement of the auricle as suggested by P wave amplitude increase could also participate to PR prolongation. Both the effect of flecainide and the absence of Na channel expression changes in ventricles do not support a decrease in Na channel availability. In the absence of large patch of fibrosis and of clear-cut change in CX40 distribution in Ts65Dn, f-QRS could be related to local defect in CX40 associated to discrete local increase in collagen. This needs further investigation but could account for the observed loss of Cx40 expression.Most of the observed effects of the Ts65Dn trisomy on ECG are related to conduction known to depend on membrane excitability, intercellular coupling and tissue architecture App-Runx1 fragment in the Ts65Dn model (double transgenic Ts65Dn/Ms5Yah mice) resulted in the rescue of Ts65Dn postnatal lethality, indicating that one or more genes present on this region are responsible for the observed lethality and that Ts65Dn trisomic genes located on Mmu17 are not major players in this phenotype. Our finding, combined with the different observations of CHD and lethality present or absent from other DS models Sod1, Tiam1 and unknown predicted genes such as Gm10789 or Gm2771 that are trisomic in Dp16(2)Yey mice which display CHD but are only in two copies in the Ts1Cje, Dp(16)1Yey/Df(16)2Yey, and Ts65Dn/Ms5Yah models. Sod1 was found decreased in Ms5Yah but not overexpressed in Ts65Dn. On the contrary Tiam1 was found increased in Ts65Dn heart and not significantly downregulated in the Ms5Yah. Thus Tiam1 is a candidate for cardiac defect in Ts65Dn. Tiam1 encodes an ephrin related receptor that influences synapse functions and controls epithelial tight junctions Bach1 and Rcan1 which were found deregulated in Ts65Dn and Ms5Yah arrays but not in Ts65Dn/Ms5Yah samples. Nevertheless in Ts65Dn/Ms5Yah mice the monosomy-induced lethality of the Ms5Yah allele is in part rescued but not as complete as for the Ts65Dn allele. Somehow some genes that are not included in the Tiam1-Cbr overlap between Ms5Yah and the Df(16Tiam1-Kcnj6)Yey/+, but located at the boundaries of the considered region, i.e. in the Mrpl39-Tiam1 or Kcnj6-Zfp295 intervals, must have a major effect on survival of the Ms5Yah mice. However, the CHD observed in Ts65Dn dead pups is lower than that observed in DS patients, indicating that one or more gene outside of the Mrpl39-Zfp295 region are contributing to CHD.Restoring the disomy of the genes present in the App-Runx1 region in double transgenic Ts65Dn/Ms5Yah mice. Reduced heart rate and prolonged QRS and QT observed in Ts65Dn mice were all back to normal in Ts65Dn/Ms5Yah mice, whereas the first degree block (PR) was only partially restored. The essential role of the App-Runx1 region in the appearance of the Ts65Dn electrocardiographic pattern is hence highlighted by the reduced ECG phenotype in Ts65Dn/Ms5Yah mice. The obvious changes recorded in the waves registered by precordial recording between Ts65Dn and Ts65Dn/Ms5Yah and the rescue of the QT phenotype point again to a major contribution of the App-Runx1 region to Ts65Dn ECG phenotypes. However, ECG defects are not completely recovered in the Ts65Dn/Ms5Yah compound animals and are probably induced by complex interactions between genes located in distinct regions of the Mmu16.Many aspects of the ECG phenotypes observed in adult Ts65Dn mice were rescued by re-establishing euploidy of the Kcnj6Rcan1 is clearly involved in the formation of the annulus fibrosis between the auricles and ventricles as well as in the formation of the valvules App-Runx1 region that triggers conduction defects and contributes to the risk of CHD.Recent evidence suggests that the same genes that cause defects in heart development and CHD might be involved in cardiac dysfunction such as abnormal electrical conduction and diminished contractile function App-Runx1 interval whose expression was detected in the heart were down-regulated to 0.68±0.08 expression level compared to wt and 3 genes, Tiam1, Slc5a3 and Mrps6, were not affected by decrease in copy number. For most of those aneuploid genes, expression level returned to normal (1.10±0.1) in Ts65Dn/Ms5Yah double transgenic mice. The overall difference in gene expression levels between the different mouse models can be explained by gene dosage. Our data support the hypothesis that a triplicated Hsa21 causes a 50% increase in trisomic genes expression as a primary dosage effect Atp6j expression is not affected by gene copy number while Tiam1 is more sensitive to increase copy number. In Ts65Dn heart, trisomic genes Sod1, Atp5o, Hcls, Ripply3, Psmg1, and Sh3bgr were not affected by the trisomy while Slc5a3 and Mrps6, back to two copies in Ts65Dn/Ms5Yah mice, were still overexpressed.We compared transcriptional profiles of RNA from adult mouse heart of 2n, Ts65Dn, Ms5Yah and Ts65Dn/Ms5Yah mice in order to determine aneuploid genes that are sensitive to gene dosage and hence might be candidate for the heart phenotypes, and to attempt to correlate observed transcript level differences to pathways impacted by the different aneuploidies. Most of the expressed triplicated/monosomic genes were up-regulated in Ts65Dn, down-regulated in Ms5Yah and expressed at similar levels in wt and in Ts65Dn/Ms5Yah hearts. 73 genes located on the Ts65Dn chromosome were overexpressed in the heart with a ratio versus wt of 1.34±0.15; only 12 were not found deregulated. 19 of those genes were located on the Mmu17 region triplicated in the Ts65Dn App-Runx1 interval. 13 genes were found on the proximal part of the Mmu17 of the Ts65Dn chromosome, 14 trisomic from the Mmu16 and 12 located elsewhere in the genome. With the second group we identified 33 genes of which 26 from the App-Runx1 interval, that were deregulated in both Ts65Dn and Ms5Yah and compensated in Ts65Dn/Ms5Yah double mutant mice. Group 3 encompassed 16 genes specifically deregulated by in Ts65dn hearts while 40 genes specific of the Ms5Yah heart are found in group 4. We also identified 3 additional groups  containing 10, 10 and 7 genes respectively, with expression level affected by combination of aneuploidies. For example group 5 were specific for the Ms5Yah with similar level in the Ts65Dn/Ms5Yah mice. Interestingly genes from the seven groups contribute to pathways related to the observed phenotypes. 25 genes out of 151 are associated with embryonic lethality, growth defect, and premature death (Mouse Genome Database (MGD) November 2011, Dyrk1a, F5, Gabpa, Hmgn1, Pde10a, Morc3, Slc5a3, Tfb1m and Vwf) that could be involved in the Ts65Dn perinatal lethality and 9 from group 2 , for which mutation impaired embryonic viability and growth that could contribute to Ts65dn and to the Ms5Yah impaired viability. In addition Amot, Ccne2, Cerk, C1qa and Fzd3 from group 4, could contribute to growth retardation and premature death Grin2b or Psmg1 can still contribute to the perinatal lethality observed in the Ts65Dn/Ms5Yah Tfb1m (group 1) causes abnormal heart development and physiology that could affect the viability of the Ts65Dn/Ms5Yah mice Adam19, Slc8a1/Ncx1 and Rcan1 from group 2 are able to induce various types of heart defects from irregular heartbeat to ASD and VSD Ripply3 and Ccne2 (group 4) loss-of-functions potentially induce VSD App-Runx1 region play an important role in the heart defects and lethality observed in Ts65Dn and suggest some pathways altered in DS heart. Overall the analysis reveals the complexity of the phenotype with several trisomic genes along the Hsa21 working alone or in cooperation to contribute to the whole range of heart defects observed DS. Whole genome expression analysis pointed at some deregulated genes, whose contribution should be further analyzed. These data were obtained from adult trisomic mice, and thus do not give information about expression at the embryonic or postnatal states. We believe that further molecular and electrophysiological studies at postnatal states could thus give important information about genes involved in early postnatal lethality and should confirm or point to new candidate genes.Detailed expression analysis highlighted a list of 151 deregulated genes that were categorized in 7 groups . 82 of tMice were handled with the agreement of the local ethical committee and in accordance with the European Council Guidelines for the Care and Use of Laboratory animals (accreditation 7320). They were housed under a 12 h/12 h light-dark cycle in TAAM-CNRS husbandry at Orléans (France) (certificate C45-234-6) and fed on a standard rodent chow. YH, as the principal investigator in this study, was granted the accreditation 45-31 and 67–369 to perform the reported experiments.16)65Dn (Ts65Dn) mice were purchased from the Jackson Laboratory . They were mated with F1 B6C3B males, in which the B6 are C57BL/6J mice and C3B are sighted C3H/HeH, a congenic line for the BALB/c allele at the Pde6b gene et al.App and Runx1 genes were obtained by in vivo TAMERE App locus using MICER vector tm1YahApp mouse line was generated. tm1YgRunx1 mice were described previously Tg(Pgk1-Cre)1Lni, expressing the Cre recombinase under the control of the early acting phosphoglycerate kinase-1 promoter App-Runx1 region in females gametes and to an offspring carrying the Del(16App-Runx1)5Yah noted here Ms5Yah, monosomic for this region  and Mx1 (gene of interest). Apob forward (CACGTGGGCTCCAGCATT), Apob reverse (TCACCAGTCATTTCTGCCTTTG), Mx1 forward (TCTCCGATTAACCAGGCTAGCTAT) and Mx1 reverse (GACATAAGGTTAGCAGCTAAAGGATCA) primers were purchased from SIGMA-Aldrich, and Taqman MGB probes Mx1 FAM (6-FAM-CCTGGTCGCTGTGCA-MGB-NFQ) and Apob HEX (HEX-CCAATGGTCGGGCAC-MGB-NFQ) from Applied Biosystem. PCR conditions were as follows: (1) 50°C for 2 min, (2) 95°C for 10 min, (3) 95°C for 15 sec, (4) 60°C for 1 min (steps 3 and 4 were repeated 50 times). Alternatively we used the PCR–based protocol For identification of both Ms5Yah and Ts65Dn alleles, genomic DNA was isolated from tail biopsies using the NaCl precipitation technique. The Ts65Dn allele was identified using a Taqman qPCR protocol with differential analysis of quantity between 5′-ATCCGGGAATGGTCCCTA-3′) specific for the wt allele, one Fwd primer (5′-CAAGCACTGGCTATGCATGT-3′) specific for the Ms5Yah allele and a Ms5Yah/wt Rev (5′-GTTCGTTGCCTGAAGGAGAG-3′) primer common to both alleles. PCR reactions gave wt and Ms5Yah products of 482 bp and 328 bp long respectively.The Ms5Yah allele was identified by PCR using one Fwd primer  for 24 hours and rinsed in PBS. Micro-dissections were realized to assess cardiovascular malformations using a Leica MZFL-III dissecting microscope equipped with a Leica DC200 digital camera. Aortic arches were then removed and hearts embedded in paraffin using a Leica TP 1020 tissue processor. Serial sections between 5 µm and 8 µm were fixed on Stick-On coated slides (LABOnord). Slides were dewaxed in xylene, rehydrated and stained in hematoxylin and 0.2% eosin. Images were captured using a Leica M420 brightfield macroscope equipped with a Photometrics Cool Snap digital camera.3) with a 1–500 Hz bandwidth  and continuously monitored on a Gould oscilloscope . Acquisition was routinely performed at 2 kHz . R, S, and RS wave amplitudes and heart rate (HR) were averaged and monitored on line every 10 s. After 1 h of control recording, the 6 peripheral and 4 precordial leads were recorded at 5 kHz for one minute each.Electrocardiogram (ECG) was recorded under urethane anesthesia  as previously described et al. to label the second wave adjoining the QRS complex as a J wave 1/2. Electrical axis was determined from the algebraic sum of the QRS amplitude in aVF and DI. Flecainide  was prepared in distilled water and injected intraperitoneally.HR mode was obtained from HR averaging over 10 s. Off-line analysis of ECG was performed with ECG-auto software (v. 1.5.11 EMKA Technologies). The ECG wave analysis used ECG libraries made up from original tracings. Measurements were performed every 20 sec, and reported values are the average of five measurements made on individual wave complexes taken over the first seconds of each interval. As the mouse ECG displayed no real electrical zero, this was taken as the average of five consecutive points taken between the end of P wave and the beginning of Q (or R) wave (from time 15 ms before Q/R wave beginning) as in infants. We followed Liu P<0.05.Results are given as mean values ± standard error of the mean (sem). Statistical tests were performed with Sigma Stat software (Systat Software). We used the Student's t-test and ANOVA followed by the Student-Newman-Keuls (SNK) multiple-comparison tests. Proportions were analyzed with the Fisher exact test. Significance was set at For Affymetrix arrays and qPCR validation, hearts were isolated from Ts65Dn, Ms5Yah, Ts65Dn/Ms5Yah and wt control mice (N = 5 per group) at 5 months of age and flash frozen. For cardiac connexins and sodium channels expression, hearts were isolated from 7 Ts65Dn and 5 wt adult mice at 4 months of age, and ventricles were separated from atria and flash frozen. Total RNA was prepared using Trizol (Invitrogen) according to the manufacturer's instructions. Samples quality was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies).et al.P<0.1 were selected for clustering analysis. Hierarchical clustering was carried out with Cluster3.0 software using Euclidian distances to calculate the distances between the genes and between the samples. Calculated distances were then clustered by complete linkage clustering. Post-Hoc analysis using GeneSpring gave a list of statistically deregulated genes. Known mammalian phenotypes database  and functional annotation clustering using Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics were performed to estimate the potential impact of deregulated genes in transgenic mice. This latter tool mainly provides typical batch annotation and gene-GO term enrichment analysis to highlight the most relevant Gene Ontology (GO) terms associated with a given genes list Biotinylated cDNAs were prepared from total RNAs and hybridized onto GeneChip Mouse GENE 1.0ST arrays (Affymetrix). Chips were washed and scanned on the Affymetrix Complete GeneChip instrument system generating digitized image data files. Raw data was processed with the Robust Multiarray Average (RMA) algorithm developed by Irizarry Ppia, Gnas, Hprt1, and Pgk1 were selected as reference genes for normalization for both Affymetrix array validation and cardiac connexins and sodium channels gene expression experiments. Primers and Taqman probes were designed using Primer3 software (P<0.05.After DNase treatment with TurboDNA-free (Applied Biosystems), 1 µg of total RNA was converted to cDNA using Superscript III (Invitrogen) primed with poly d(T) and random hexamers. software . Taqman Figure S1App-Runx1 region (A) on Mmu16 was targeted for in vivo Cre/loxP recombination by inserting a loxP site on App locus in tm1YgRunx1 (B). Recombination using Tg(Pgk1-cre)1Lni led to deletion of the floxed fragment creating Ms5Yah mouse model. Chimeras and monosomic mice were distinguished using Southern Blot genotyping (C) and the deletion was confirmed by CGH arrays (D).Ms5Yah mouse model creation and validation. (TIF)Click here for additional data file.Table S1P<0.1. Genes in pink. orange and blue are genes located respectively within the centromere-Pde10a region on Mmu17. the Mrpl39-Runx1 and Runx1-Zfp295 region on Mmu16 trisomic in Ts65Dn mice. We calculated the mean, the standard error to the mean for the expression ratio and the Student-t-test comparing mutant versus wt and annotated with * if P<0.05. ** if P<0.01 and *** if P<0.001. Overexpressed genes with an expression ratio>1.2 are underlined in red whereas down-regulated gene with a ratio<0.8 are in green. Normal expression ration between 0.8 and 1.2 are underlined in grey.List of deregulated genes in Affymetrix hybridization arrays. This list was obtained by using ANOVA with Bonferroni correction and fixing a threshold of (PDF)Click here for additional data file.Table S2Sequences and Tm of primers and Taqman probes used for qPCR analyses. Genes in bold were used as housekeepers.(PDF)Click here for additional data file.


The enhanced autologous neutralization of pseudotyped viruses expressing the sensitive envelope (Env) was associated with increased sensitivity to reagents and monoclonal antibodies targeting distinct sites in Env. Chimeric viruses constructed by swapping fragments of sensitive Env into resistant Env backbone revealed that the presence of unique residues within C2V3 region of gp120 governed increased neutralization. The enhanced virus neutralization was also associated with low CD4 dependence as well as increased binding of Env trimers to IgG1b12 and CD4-IgG2 and was independent of gp120 shedding. Our data highlighted vulnerabilities in the Env obtained from cross neutralizing plasma associated with the exposure of discontinuous neutralizing epitopes and enhanced autologous neutralization. Such information may aid in Env-based vaccine immunogen design.Broadly neutralizing antibodies to HIV-1 usually develops in chronic infections. Here, we examined the basis of enhanced sensitivity of an  Individuals with chronic HIV-1 infection in absence of antiretroviral therapy (ART) tend to develop cross-neutralizing antibodies over a period of years env clones we mapped determinants in the C2V3 region which were found to modulate neutralization properties of autologous Envs amplified from this patient.In the present study, we examined the relationship between genetic and neutralization properties of autologous HIV-1 Envs that modulated neutralization properties of circulating envelopes obtained from a chronically infected Indian patient whose plasma showed BCN response env clones from plasma of this patient that showed presence of clade C and non-clade C mosaics; however only four functional Envs were obtained that gave detectable infectious titers as Env-pseudotyped viruses. The limited number of env clones obtained from the LT5 plasma showed genetic heterogeneity env clones were found to be functional and gave reasonable infectivity titers. Sequence analysis indicated that out of these four env clones, two were of pure clade C (LT5.J3b and LT5.J7b) and two were B/C recombinants (LT5.J4b and LT5.J12). First we examined the neutralization sensitivity of pseudotyped viruses expressing these four Envs to contemporaneous autologous plasma. As shown in We previously reported Next, we examined the degree of sensitivity of these four LT5 Envs to reagents that target distinct sites in Envs, such as CD4bs , V3 loop (MAbs 3074 and 3869), CD4-induced epitopes/coreceptor binding site (17b MAb) and MPER (MAbs 4E10 and 2F5) To dissect the sequence of the LT5.J4b Env conferring enhanced neutralization, first we swapped different segments between the LT5.J4b (sensitive) and LT5.J12 (resistant) Envs to construct chimeric Envs . PseudotAt this juncture, we wanted to fine map residue/s within C2V3 sequence in LT5.J4b Env to examine if there is/are any specific residue/s that possibly contributed to enhanced virus neutralization. When compared between LT5.J4b and LT5.JJ12 Envs, we found fifteen differences in amino acid residues in C2V3 region (twelve in the C2 and three in the V3 loop). Specific point substitutions were made in the LT5.J12 Env as described in Methods and pseudoviruses expressing mutant Envs were prepared. Interestingly, we did not find any specific substitution conferring sensitivity to LT5.J12 Env . The Q24We next assessed the relationship between presence of unique C2V3 sequence and relative binding of MAbs to Env trimers targeting different regions of Env, which showed enhanced neutralization of LT5.J4b Env. 293T cells were transfected with equal concentrations of Env plasmid DNA and the relative binding of MAbs or CD4-IgG2 to Env trimers expressed on 293T cells was assessed by measuring relative luminescence units (RLU) as described previously Since the unique sequence of the C2V3 region was found to modulate both the neutralization sensitivity and binding of Envs to IgG1b12 and CD4-IgG2 which are the prototypic ligands that bind to CD4bs on Env trimer, we subsequently examined the effect of this modulation on the infectivity and CD4 dependence of these Envs. For this, we tested the infectivity of the Envs expressing wild type Envs and C2V3 chimeric Envs in HeLa RC49 cells, expressing high amount of coreceptor CCR5 but limiting amount of CD4 Next we evaluated whether there was any association between neutralization sensitivity of both LT5.J4b (sensitive) and LT5.J12 (resistant) Envs with ligand-induced gp120 shedding. The MAbs and sCD4 (referred to as ligands here) were mixed with pseudotyped viruses expressing LT5.J4b and LT5.J12 Envs with equal infectious TZM-bl titers in a final concentration that showed 50% neutralization of these Envs (see Methods). These virus-ligand mixtures were incubated at 37°C for different time periods as indicated , and theSince we found that the LT5.J4b Env that was found to be very sensitive to contemporaneous autologous plasma also showed enhanced sensitivity to IgG1b12 and anti-V3 MAb, the LT5 patient’s plasma showing BCN activity In order to understand if presence of unique mutations in the C2V3 region  modulateIn the present paper, we examined the vulnerabilities in circulating HIV-1 Envs obtained from plasma of a chronically infected patient that showed cross neutralization of several heterologous HIV-1 Env-pseudotyped viruses. In essence, we examined the determinants in Env that conferred escape from neutralization by autologous antibodies. Although we obtained mixture of clade B and C and B/C recombinants from the plasma of this particular patient, the plasma antibodies exhibited bias towards clade C The presence of BCN antibodies in the plasma of the LT5 patient who was chronically infected for several years without any anti-retroviral therapy, possibly accounted for restricting clinical advancement to disease stage. The presence of BCN activity of plasma of the LT5 patient containing mixture of viral subtypes in vivo even in the pressure of autologous neutralizing activity in vivo as suggested by the low CD4 dependence of LT5.J4b Env. Compared to LT5.J4b other Envs exhibited high CD4 dependence for entry and might replicate with slower rate although they are neutralization resistant and possibly might contribute to the survival of sensitive species. The survival of the sensitive viral species could also be the decoy strategy to direct the immune response towards them and to retract the antibody pressure exerted on the resistant species. This phenomenon could possibly be the basis for slow disease progression as far as this particular patient is concerned. Irrespective of the basis of existence, both sensitive and resistant Envs provided an opportunity to dissect the region in the Env that conferred enhanced sensitivity autologous antibodies. The chimeric constructs made by using the LT5.J4b and LT5.J12 Envs revealed that presence of unique C2V3 sequence conferred LT5.J4b Env with enhanced neutralization susceptibility. Though the role of V1V2 in masking the epitopes has been well documented we did not observe any change in neutralization sensitivity to plasma antibodies when this region was transferred from LT5.J4b (sensitive) to LT5.J12 (resistant). The C3 and V4 regions in Env individually or combined was not found to be involved in forming neutralization epitopes. The C3 region contains highly variable α2 helix which was shown by previous studies to modulate the neutralization sensitivity to autologous plasma directly or indirectly by influencing CD4 binding loop in C3 and imparting more flexibility or structural adaptation to the protein in response to neutralizing antibodies et alAmong the four Envs from LT5 plasma LT5.J4b was found to be most sensitive to autologous plasma and MAbs targeting different regions on Env. Since, we were not able to analyze Env variants from plasma of earlier and later time points; the evolution dynamics of LT5 Envs could not be known. However, the sensitive viral species can emerge The sequence conservation of the C2V3 region and that it contains the CD4 contact residues signifies the functional role of this region The glycosylation on the Env trimer is another important factor that influences the neutralization sensitivity. Presence of a PNG at the 386 position shields the neutralizing epitopes in the CD4bs and its removal was previously shown to increase the sensitivity of HIV-1 Envs to IgG1b12 apparently by filling a cavity penetrated by W100 of IgG1b12 Our data provided important information on atypical determinants in the C2V3 region of Env that gave rise to greater exposure of neutralizing epitopes in CD4bs, V3 loop and coreceptor binding sites. Both CD4bs and V3 loop are important regions on gp120 which are highly antigenic and immunogenic in nature Although the specificity of LT5 plasma was partially mapped to CD4bs and V3 loop it is possible that other specificities were also present as the gp120 OD construct used to adsorb IgG1b12 like antibodies may also adsorb antibodies which bind further apart from CD4bs on gp120 outer domain. Multiple antibody specificities were reported in chronic infections E coli competent cells. Similar procedure was followed for site directed mutagenesis except the forward primer of insert and corresponding backbone primer which amplifies in opposite direction introduced single or multiple mutation.Chimeric Env cloning and site-directed mutagenesis was carried out by using tailor-made primers as shown in 4 TZM-bl cells were added into the mixture in presence of DEAE-Dextran  making final concentration 25ug/ml (Sigma Inc.). The plates were incubated in a CO2 incubator for 48 hours and the degree of virus neutralization was assessed by measuring relative luminescence units (RLU) by using BriteliteTM luciferase substrate (Perkin Elmer Inc.) in a luminometer .The neutralization assays were carried out as described earlier + 293T cells for 1 hour at 37°C and subsequently with goat-anti-human IgG conjugated with horse radish peroxidase . The cells were washed for four times with first blocking buffer  2, 1 mM MgCl2, 20 mM Tris, pH 7.5) The relative interaction of Env with MAbs or CD4-IgG2 was assessed by cell-based binding assay as described before et al.5 RLU/well); no MAbs) was incubated for the indicated time periods at 37°C in culture medium supplemented with a ligand in the concentration that gives approximately 50% neutralization at 1 hour of incubation. The virus incubated at different time points was then used to infect TZM-bl target cells, and virus infectivity was monitored by luciferase activity at 72 h after infection. Time of addition experiments were conducted to probe the kinetics of virus neutralization by different ligands and to assess the induction of gp120 shedding from the virions. Antibody concentrations were chosen to yield neutralization of approximately 50% after 1 h of pre-incubation to allow for the monitoring of increases in neutralization activity over time. The selection of precise ligand concentration was possible for LT5.J4b as it is sensitive to most ligands tested here but LT5.J12 was resistant at 10 ug/ml, highest concentration tested. For LT5.J12 therefore, the highest concentration was used to assess the gp120 shedding over the period of time.The gp120 shedding was assessed by measuring infectivity of virus incubated at 37°C as described by Ruprecht Escherichia coli-expressed protein based on the sequence of the HxBc2 strain which binds CD4 and the broadly neutralizing antibody IgG1b12 but not the non-neutralizing antibodies b6 and F105 5 RLU virus was then added in each well and incubated the plate for another 1 hour. After incubation 1×104 cells were added with 25 ug/ml dextran and incubated the plate for 48 hours. Infection of TZM-bl cells was measured by luciferase activity and the increase in infection in presence of probe was compared with the wells where probe was not added.Adsorption of LT5 plasma was performed by using an outer domain (OD) protein of gp120 The correlations between IgG1b12 and LT5 plasma sensitivities of a panel of 30 Envs Structural models of HIV-1 C2V3 regions were produced with SWISS MODEL homology modeling server in project mode The authors declare that they have no competing interests.Figure S1Neutralization sensitivity of LT5 Envs to VRC01 and 2F5 monoclonal antibodies. Note that all the LT5 Envs showed resistance to VRC01 MAb including the sensitive LT5.J4b Env.(TIF)Click here for additional data file.Figure S2Correlation between IgG1b12 and LT5 plasma sensitivities of a panel of 30 Env pseudotyped viruses .(TIF)Click here for additional data file.Figure S3Construction of chimeric and mutant Envs. (A) Chimeric Envs were constructed using a region exchange strategy and mutant Envs were made by introducing specific nucleotide change in primer. (a) The domain to be transferred for example C2V3 (light blue box) was PCR amplified from the env gene using primers (arrows) that annealed to conserved sequences flanking C2V3 and amplified inward. (b) The recipient Env (open box) plus plasmid vector was PCR amplified using primers (arrows) that annealed to sites adjacent but overlapping (at least 15 base pairs) to those of the C2V3 primers and amplified outward. (c) The two fragments were then mixed with PCR cloning cocktail containing pox virus DNA polymerase which forms cohesive ends in PCR fragments by its 3′–5′ exonuclease activity. The cohesive ends anneal and forms circular plasmids with a nick at each strand. The annealed product is directly transformed in competent cells where the nicks are sealed. (B) The PCR fragments after treatment with dry down cloning cocktail. The homologous sequence is annealed to yield circular plasmid. (a) chimeric and (b) mutant Env preparation is shown.(TIF)Click here for additional data file.


Based on this notion, different studies have evaluated the prognostic value of this maker in cancer but contradictory results have been found. Indeed, even within the same cancer population, the presence of CD4+FOXP3+T cells has been associated,with either a poor or a good prognosis, or no correlation has beenfound. Here, we demonstrate,in patients with oral squamous cell carcinoma (OSCC), that what really represents a prognostic parameter is not the overall expression of FOXP3 but its intracellular localization.While overallFOXP3 expression in tumor infiltrating CD4+T cells does not correlate with tumor recurrence, its intracellular localization within the CD4 cells does: nuclear FOXP3 (nFOXP3) is associated with tumor recurrence within 3 years, while cytoplasmicFOXP3 (cFOXP3) is associated with a lower likelihood of recurrence. Thus, we propose elevated levels of the cFOXP3/nFOXP3 ratio within tumor infiltrating CD4+ T cells as a predictor of OSCC recurrence.Forkhead box protein P3 (FOXP3) expression in tumor infiltrating CD4 While the infiltration of effector lymphocytesisgenerally associated with a good prognosis, the infiltration of other immune cell populations (i.e.Myeloid Derived Suppressor Cells (MDSC) and T regulatory cells (Treg)) isthought to promote tumor progression by restraining tumor immunity and promoting neoplastic cell invasion and metastasis +tumor infiltrating cells. The results indicate that the presence of CD4+ cells expressing FOXP3 in the cytoplasm is associated with a favorable prognosis whereas its nuclear localization correlates with an increasedrisk of recurrence. In light of these results,we propose the use of cFOXP3/nFOXP3 ratio as a prognostic factor in OSCC.In this retrospective case-control study, we examined the prognostic value of FOXP3 with respect to recurrence of OSCC taking into account the subcellular localization of FOXP3 within CD4This study was approved by the University of Miami IRB before initiation.Due to the retrospective nature of the study and the lack of personal identifier in the specimens evaluated the requirement for informed consent was waived by the IRB.We selected specimens from patients who underwent glossectomy (with or without neck dissection) and without prior treatment by either radiation or chemotherapy. Subjects were identified from among those treated at our tertiary referral academic medical center between 1/1/2001 and 12/31/2010 by search of a registry of CPT  codes for glossectomy . Only patients with a diagnosis of SCC of the oral tongue staged T1 or T2  whose surgery was limited to a partial glossectomy with or without neck dissection were included. We excluded patients with a diagnosis of in-situ carcinoma, T3 or T4 disease, involvement of other oral or oropharyngealsubsites, need for any form of mandibulectomy or other more extensive surgical procedure, a prior history of radiation to the head and neck or treatment with chemotherapy, or any history of immunosuppression or immune compromise.Additional criteria required availability of sufficient residual archival paraffin embedded tissue from the surgical resection for experimental analysis and documentation of disease status three years from date of surgery. A total of 392 clinical records were obtained, with 49 meeting inclusion criteria.Patients’ HPV status was determined by the head and neck pathology core at University of Miami using standard IHC protocols to detect p16 of Human papillomavirus.Four µm sections underwent deparaffinization, rehydratation, and incubation for 30 min at RT in a sodium borhydride solution  to reduce sample autofluorescence. Antigen retrievalwas performed by a 15 min incubation at 95°C in EDTA antigen retrieval solution pH = 9 . The slides were then incubated with Image-iT (Invitrogen) for 30 min at RT followed by incubation (1 h at RT) with PBS containing 1% BSA and 0.3% Triton-X100 to permeabilize the tissue and to block non-specific binding. Samples were incubated O/N at 4°C with the mouse monoclonal, anti-human FOXP3 antibody ab20034  and the goat polyclonal anti-human CD4 antibodyAF-379-NK,  in PBS with 1% BSA. After three washes with PBS, samples were labeled for 1 h at RT with the Alexa-555 conjugated anti-mouse antibody (for FOXP3) and with the Alexa-488 conjugated anti-goat antibody (for CD4). Both antibodies were used at a 1/400 dilution in PBS/BSA (1%). Finally, sections were counterstained in PBS containg 2 mMDAPI (Invitrogen),for 15 min at RT, rinsed with PBS and coverslips were mounted using Biomeda gel mounting media . Slides were analyzed with ZeissAxiovert Microscope and the Zeiss Axiovision LE software  at 20×magnification or with the SP5 spectral confocal inverted microscope (Leica) at the University of Miami imaging core facility.http://rsbweb.nih.gov/ij/www.cellprofiler.comAreas of interest corresponding to the neoplastic lesion of the tissue were identified by an experienced pathologist in serial section stained with H&E. Microphotographs of 5 random fields at 20X were taken in the corresponding area of the fluorescence labeled sections with a Zeiss Axiovert Microscope. Images for each patient were qualitatively evaluated with ImageJ . Consideration of multivariate models was limited to testing the addition of baseline patient characteristics to the single marker model with the best (highest) AUC. All statistical analysis was performed in SAS® v 9.3.+ CD4+ cells in HNSCC Different hypotheses have been proposed to explain the inconsistent results obtained by different studies on the predictive role of tumor infiltrating FOXP3To evaluate whether tumor infiltrating Treg can serve as a prognostic marker in this defined patient cohort, paraffin embedded sections of the tumor were stained with hematoxylin and eosin  and serial sections labeled with antibodies specific for human CD4 and human FOXP3 and counterstained with DAPI.The anti-human FOXP3 antibody clone 236A/E7 was selected and used because it targets an epitope between aminoacid 105 and 235 of human FOXP3 +FOXP3+ cells were quantified using a computer-aided method for image analysis  that could limit the power of theanalysis, CD4analysis . Brieflyanalysis  and the analysis , Empiricanalysis .+FOXP3+ cells was evaluated with this method for each patient as well as thedifferences between patients with or without recurrence.Despite the use of a homogenous population, validated antibodies, and a computer aided method for tumor infiltrating Treg enumeration, no association was found between the percentage of CD4+FOXP3+ within the and tumor recurrence ,further suggesting that two well defined subsets of FOXP3+CD4+T cells infiltrate the tumor. Furthermore, while the percentage of cytoplasmicFOXP3+T cells was associated with a favorable prognosis  within the CD4 significantly correlated with tumor recurrence , we evaluated whether the subcellular localization of FOXP3 within the CD4p<0.001, .In silico models of the interaction between regulatory and effector T cells +CD4+T cells , there was a significant positive linear correlationwhen in the 19 cases of diseaserecurrence . This analysis indicated a 1.37-fold increase in Treg cells per unit increase in %cFOXP3+/CD4+ cells  between cases and controls (The relationship between Treg and effector FOXP3controls .2(nFOXP3) and log2(ratio nFOXP3/cFOXP3) ) or 50% decrease (−log2(cFOXP3)) in the marker. Receiver operating curves (ROC) and corresponding area under the curve (AUC) are shown in + CD4+ cells gave the highest AUC, we estimated the effect of this marker after adjustment for each of the baseline characteristics previously considered. None of these covariates were significant although T-stage was marginally significant (p = 0.097)  .+ cells has been evaluated as a prognostic marker for different human malignancies. Nevertheless, while some studies indicate its association with a negative prognosis, others correlate its expression with a positive prognosis or show no association. Here we show that intracellular localization is an important factor to considerwhen assessing the prognostic importance of FOXP3.In oral squamous cell carcinoma,an elevated number of tumor infiltrating CD4+T cellsexpressingFOXP3 in the cytoplasm are indicative of a favorable prognosis (no recurrence within three years) whereas an high concentration of CD4+ T cells expressing nuclear FOXP3 isstrongly associated with recurrence.Furthermore, the ratio between nuclear and cytoplasmicFOXP3+ CD4+ T cells was significantly better than either localized measure as an indicator of the risk of recurrence in patients with OSCC. Thus, we suggest the exploration of this parameter in other malignancies.Since its discovery, FOXP3 expression within the tumor infiltrating CD4


Intracerebral inoculation of transgenic mice failed to demonstrate prion disease transmission. Sc). PrPSc propagate prion diseases within and between species and thus pose risks to public health. Prion infectivity or PrPSc presence has been demonstrated in urine of experimentally infected animals, but there are no recent studies of urine from patients with Creutzfeldt-Jakob disease (CJD). We performed bioassays in transgenic mice expressing human PrP to assess prion infectivity in urine from patients affected by a common subtype of sporadic CJD, sCJDMM1. We tested raw urine and 100-fold concentrated and dialyzed urine and assessed the sensitivity of the bioassay along with the effect of concentration and dialysis on prion infectivity. Intracerebral inoculation of transgenic mice with urine from 3 sCJDMM1 patients failed to demonstrate prion disease transmission, indicating that prion infectivity in urine from sCJDMM1 patients is either not present or is <0.38 infectious units/mL.Prion diseases are neurodegenerative conditions associated with a misfolded and infectious protein, scrapie prion protein (PrP C), which predominantly accumulates in the central nervous system  in 5 phenotypically distinct subtypes  accounts for ≈70% of all cases of sCJD and unquestionably is the most prevalent type of human prion disease , can be sporadic, inherited, or acquired by infection; the sporadic form alone accounts for the great majority of all the cases of CJD  affected by typical sCJDMM1 that was histologically and immunochemically confirmed  were provided by the National Prion Disease Pathology Surveillance Center. The patients were 54, 68, 60, and 69 years old with disease durations of ≈2, 2, 3, and 2 months, respectively  (4 L each time) at 4°C by using the Pierce Slide-A-Lyzer cassette  (10-kDa cutoff) with 2 additional changes over 2 days.Urine samples from sCJDMM1 patients 1–3 collected 2 weeks, 1 month, and 1 week before death, and from 3 healthy controls, were concentrated 100-fold by ultrafiltration with Millipore Centricon Plus 70  (10-kDa cutoff) by using a Beckman  centrifuge at 3,000 × g for 5 min. Microsomal fractions (MF) were prepared from the frontal cortex of the sCJDMM1 patients according to Reichl et al. , was centrifuged as above. This supernatant, pooled with the previous one, was recentrifuged at 10,000 × g for 7 min at 4°C. The pellet was discarded and the supernatant was centrifuged at 100,000 × g for 1 h at 4°C. The final pellet, representing the MF, was resuspended in PBS at 10% (wt/vol). Transgenic mice expressed full-length human PrP-129M at wild type level in mouse PrP null background, Tg(HuPrP-129M)0/0Prnp (Tg40) (Brain homogenates (BH) from inoculated mice  were prepared at 4°C in PBS cleared by centrifugation at 1, 000 × MF of sCJDMM1 patient 4 was spiked in 100× concentrated and dialyzed normal urine. The sample was 10-fold serially diluted and intracerebrally inoculated into Tg40 mice.MF (100 µL) of sCJDMM1 patient 3 was spiked into normal urine (100 mL) either before or after 100× concentration and dialysis. Subsequently, 30 µL of the differently processed urine samples, both containing an equivalent amount of 3 µL of MF, were intracerebrally injected into Tg40 mice. The infectivity titers of the 2 differently processed MF spiked urines were compared on the basis of their mean incubation times in Tg40 mice.Thirty microliters of MF from sCJDMM1 patients 1–4 suspended in PBS or in 100× concentrated and dialyzed normal urine were intracerebrally injected into Tg40 mice , Table 4Infectivity in MF from sCJDMM1 patients 1 and 4, the latter spiked and diluted in 100× concentrated and dialyzed normal urine, was estimated by endpoint dilution by using probit regression analysis . Incubation periods , x-axis 50]) by assuming a Poisson distribution in the response variable (The amount of prion infectivity that is below the threshold of detectability of our bioassay but that might be present in the urine of the sCJDMM1 patients was estimated in infectious unit (IU) per milliliter with 95% confidence interval (CI) . Titers from brain MF of sCJDMM1 patients 2–4 were estimated by interpolating the mean incubation times of the inoculated Tg40 mice to the dose-incubation period curve of 6 ID50 per gram of brain tissue equivalent . The histologic examination revealed no lesions (data not shown). These data show that 100× concentrated and dialyzed urine is not toxic following intracerebral inoculation into Tg40 mice.The effect on prion infectivity of the concentrated and dialyzed urine when used as carrier was assessed by comparing infectivity titers of MF from cerebral cortex of sCJDMM1 patient 4 diluted either in PBS or in 100× concentrated and dialyzed urine. The bioassay analyses showed that using concentrated and dialyzed urine as carrier the infectivity titer of MF was reduced ≈20-fold (1.3 log reduction) , indicat6 ID50/g and 6.9 × 106 ID50/g, respectively, were found in the 2 differently processed samples, showing that the concentration and dialysis procedure per se had no detectable effect on infectivity.The possible loss of prion infectivity during concentration and dialysis procedures was assessed by bioassay. We compared the infectivity titers of brain MF from sCJDMM1 patient 3 spiked in normal urine before the concentration and dialysis procedure with the infectivity of the same amount of MF spiked in normal urine already concentrated and dialyzed . SimilarSc was detected by Western blot, even after enrichment with NaPTA precipitation , no mice showed any evidence of clinical disease . No PK-rpitation , panel Apitation , panel BOn the basis of these negative results, we estimate that prion infectivity in urine of sCJDMM1 patients to be 0 IU/total volume of inoculated urine, which by Poisson distribution is less than 0.37 IU for patients 1 and 2 and 0–0.46 IU for patient 3 (upper limit of 95% CI). However, if we take into account the 100× concentration of urine and the finding that 100× concentrated and dialysized urine may reduce prion infectivity by ≈20-fold , the estSc was detected by Western blot in the brain even after PrPSc enrichment with NaPTA (data not shown). Thus, the estimated infectivity titer in raw urine of sCJDMM1 by Poisson distribution is 0–0.11 IU/mL (95% CI), not dissimilar to the value (0–0.24) obtained with the same urine after concentration and dialysis.To directly assess the prion infectivity in urine without concentration and dialysis, we inoculated 33 Tg40 mice with raw urine from sCJDMM1 patient 1. No inoculated mice showed clinical signs of prion disease over their normal lifespan (up to 857 dpi) . Similar5 or 104 dilutions of the brain tissue equivalent depending on whether the MF preparations were inoculated directly or after spiking into concentrated and dialyzed normal human urine.The present study demonstrates that the urine from patients affected by advanced sCJDMM1, the most common sCJD subtype that alone accounts for ≈60% of all human prion diseases, contains either no prion infectivity or an infectivity titer that is below the detection limit of our bioassays. The bioassays were done in Tg mice expressing human PrP-129M (Tg40) following inoculation with urine obtained from patients with sCJDMM1 and a variety of positive and negative controls. In limit dilution experiments, Tg40 mice inoculated with MF preparations obtained from the brains of 3 urine donors with sCJDMM1 had prion disease develop at up to 10,,Sc in urine from CJD patients is not known, this urine PrPSc species might show even higher loss of infectivity in the concentrated and dialyzed urine carrier than the brain PrPSc preparations used in the spiking experiments. To address this concern, we inoculated 33 Tg40 mice with raw urine from one of the 3 donors with sCJDMM1. No recipient mice showed evidence of prion disease suggesting an infectivity ranging from 0.0 and 0.11 IU/per mL (upper limit of 95% CI) as estimated by the Poisson distribution.To enhance the sensitivity of our system, urine samples were concentrated and dialyzed before inoculation. Similar procedures have been used in all the previous studies on prion infectivity of urine ,,, the infSc cannot be excluded, our inability to detect prion infectivity in human urine of patients with sCJDMM1 differs from several recent experimental studies on urine of prion-affected animals. Low prion infectivity has been demonstrated in urine from scrapie-infected hamsters  , only minute amounts of PrPSc have been detected in a few nonneural peripheral organs and tissues such as skeletal muscle and spleen (–,Sc to peripheral organs is much wider and typically involves lymph nodes, tonsil, spleen, portions of the intestinal tract, and the skeletal muscle (,,,The most likely explanation for the discrepancy between our negative results on human urine and the positive findings by bioassay in urine from animals resides in the different locale and mode of formation of the prion agents. In all the published animal experiments, including bioassays and PMCA, the prion disease was induced by intracerebral or oral administration of exogenous prions, whereas we examined urine infectivity in a naturally occurring sporadic human prion disease. In exogenously acquired prion diseases, PrPSc and prion infectivity (,Recent data have proved that feces from hamsters infected with scrapie by the oral route and to a lesser extent through intracerebral and intraperitoneal inoculation, contain a discrete amount of PrPSc are present in urine from patients with sCJD and to assess the presence of infectious prion in urine from patients with every other form and subtype of human prion diseases, our study shows that urine from patients with sCJDMM1, the most common subtype of sCJD, does not contain prion infectivity detectable by our bioassay and suggests that no significant prionuria occurs in this common subtype of human prion disease.Although additional studies are still needed to determine whether minute amounts of prion infectivity or PrP


Niemann-Pick C (NPC) disease is due to loss of NPC1 or NPC2 protein function that is required for unesterified cholesterol transport from the endosomal/lysosomal compartment. Though lung involvement is a recognized characteristic of Niemann-Pick type C disease, the pathological features are not well understood. We investigated components of the surfactant system in both NPC1 mutant mice and felines and in NPC2 mutant mice near the end of their expected life span. Histological analysis of the NPC mutant mice demonstrated thickened septae and foamy macrophages/leukocytes. At the level of electron microscopy, NPC1-mutant type II cells had uncharacteristically larger lamellar bodies , while NPC2-mutant cells had predominantly smaller lamellar bodies  than wild type. Bronchoalveolar lavage from NPC1 and NPC2 mutant mice had an approx. 4-fold and 2.5-fold enrichment in phospholipid, respectively, and an approx. 9-fold and 35-fold enrichment in cholesterol, consistent with alveolar lipidosis. Phospholipid and cholesterol also were elevated in type II cell LBs and lung tissue while phospholipid degradation was reduced. Enrichment of surfactant protein-A in the lung and surfactant of the mutant mice was found. Immunocytochemical results showed that cholesterol accumulated in the LBs of the type II cells isolated from the affected mice. Alveolar macrophages from the NPC1 and NPC2 mutant mice were enlarged compared to those from wild type mice and were enriched in phospholipid and cholesterol. Pulmonary features of NPC1 mutant felines reflected the disease described in NPC1 mutant mice. Thus, with the exception of lamellar body size, the lung phenotype seen in the NPC1 and NPC2 mutant mice were similar. The lack of NPC1 and NPC2 proteins resulted in a disruption of the type II cell surfactant system contributing to pulmonary abnormalities. Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disorder marked by excess intralysosomal cholesterol, progressive neurodegeneration and hepatosplenomegaly that has no effective approved treatments nih/nihNpc1 mice demonstrated vacuoles in type I and endothelial cells and foamy alveolar macrophages Npc1 mice has been documented but surfactant was not analyzed The most common mutations associated with NPC disease (95% of cases) are in the NPC1 gene with the affected patients demonstrating a broad clinical spectrum Pulmonary surfactant, produced by type II alveolar cells, performs the critical role of reducing surface tension in the alveoli. Cholesterol is the most abundant neutral lipid in surfactant, contributing 5–10% weight by volume, with the rest made up of phospholipids and protein Reports on the morphology of type II cells in lungs of patients or animal models of NPC deficiency describe the pneumocytes to be “unremarkable” The mice and felines were housed under the National Institutes of Health and USDA guidelines for the care and use of animals in research. The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approved all experimental protocols.Mice.nih/nihNpc1 and Npc2 hypomorph mice on a BALB/c background were obtained as a gift from Dr. Peter Lobel. Both types of mice are established models of NPC disease. The Npc1 mouse defect arose due to a spontaneous mutation in mouse chromosome 18 Npc2 mutant mice were produced by Sleat, et al. and contain an aberrant recombination event such that the targeted allele has an additional repeat of intron 1 and replacement of amino acid cysteine 42 with a stop codon Npc1 and Npc2 mice were determined by PCR of genomic DNA from tail tips. The primer pairs have been previously described The felines were housed in the School of Veterinary Medicine of the University of Pennsylvania. NPC1 mutant diseased felines and their normal counterparts were produced from the same line. The NPC1 felines have a spontaneous missense mutation (C955S) in the NPC1 protein which results in lysosomal accumulation of unesterified cholesterol The isolation of type II cells from mice lungs was performed as described previously Lamellar bodies were isolated as described previously Lung tissue from 9–14 week old mice was collected, homogenized, and separated using denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described (8). Primary antibody against rabbit anti-NPC1 , NPC2 , secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated , and ECL Plus Western Blotting Detection Reagent  were used.Lungs from wild type, NPC1 and NPC2 mutant mice were excised from 3 animals for each experimental group. From each mouse, the left lobe, upper right lobe, and lower right lobe were fixed overnight in a mixture of formaldehyde and glutaraldehyde, and embedded in paraffin. Tissue was cut into 5 µm sections and stained with hematoxylin and eosin. Random fields of distal airway were collected at 60× magnification. Alveolar septal thickness was determined using an overlaid test grid. The alveolar septal thickness was measured as the length of a grid line that extended from one alveolus to an adjacent alveolus. Regions containing large airways or capillaries were not included in this measurement. >200 probe “hits” of the alveolar septal thickness were gathered from more than 5 sections per mouse and 15 fields for each experimental condition.. Lungs were cut into 1 mm3 blocks, treated for 2 hours with 2% osmium, dehydrated, embedded in EPON, and polymerized for 48 hours at 60°C. Blocks were sliced into ultrathin (80 nm) sections in a Leica Ultracut UCT ultramicrotome. Samples were stained with 2% uranyl acetate and 0.5% lead citrate. All samples were imaged on a TEM .Lungs from mice were perfused through the pulmonary artery with 0.1 M sodium cacodylate buffer to remove blood cells. Lungs were excised and fixed by perfusion with 5% glutaraldehyde through the pulmonary artery and trachea. The trachea was removed, and whole lungs were fixed for at least 4 hours. A portion of the lung from a cat were perfused with cacodylate buffer through the bronchi and fixed by immersion in 5% glutaraldehyde4), heated for 20 minutes, and then cooled. Distilled water and ammonium molybdate were added to the sample and vortexed before the addition of reagent A . Samples were boiled for 10 minutes, cooled and colorimetric reading was done at 830 nm.To measure cholesterol, samples were prepared following instructions in the Amplex Red Cholesterol Assay Kit . Protein for each corresponding sample was determined using the Lowry assay and data are presented as µg lipid/µg protein 3H] dipalmitoyl phosphatidylcholine ([3H]-DPPC) by the isolated, perfused lung was performed as described previously 3H]-DPPC were prepared by freezing and thawing followed by extrusion under pressure through a 100-µm pore-size filter. The liposomes  were instilled into the lungs of anesthetized mice; the lungs were removed and placed in a perfusion apparatus under ventilation. After 5 min  or 2 hours, the lungs were lavaged and the lung tissue homogenized. The homogenate was extracted Uptake and degradation of liposomes labeled with [choline-methyl-Type II cells were fixed in 4% paraformaldehyde for 20 minutes. Cells were washed, fixative was quenched in 0.3% glycine, and cells were permeabilized in 0.2% triton for 30 minutes at room temperature. Cells were blocked in 10% BSA with 2% normal goat serum for 1 hour. Primary antibodies were incubated on cells overnight in dilute blocking buffer. The next day, cells were washed and incubated in secondary antibody for 1 hour. Cells were then washed and imaged.t-test or ANOVA. Results were considered statistically significance at P≤0.05.All data are expressed as mean ± SEM. Statistical comparisons were performed using SigmaStat . Data were analyzed using a Student’s NPC1 and NPC2 mutant mice have average life spans of 72 days Histological evaluation of the lungs of NPC1 and NPC2 mutant mice using hematoxylin/eosin stained sections was performed to determine pulmonary effects due to loss of cholesterol transport. The micrographs showed a thickening of the intra-alveolar septae with a slight enlargement of the airways in the NPC1 mice . QuantitUpon closer inspection of the alveolar architecture at the electron microscopic level, more pronounced pulmonary abnormalities were apparent. In Within mouse lung capillaries were enlarged polymorphonuclear leukocytes or circulating macrophages filled with vacuolar inclusions were found in both mutant mice, apparent in NPC1 mice 2 to 0.7 µm2. While some LBs in NPC1 mutant mice showed a distribution similar to wild type, a significant proportion of NPC1 type II cell LBs were enlarged  were similar in size as those of the smaller sized wild type LBs (<0.2 µm2) and were fairly uniform with none larger than 0.7 µm2. The mean average size of the LBs of the mice reflected these differences with NPC1 (0.81±0.02 µm2)>WT (0.43±0.02 µm2)>NPC2 (0.22±0.01 µm2), all statistically significantly different  from each other. Interestingly, compared to the number of LBs per cell in wild type alveolar type II cells , NPC2 alveolar type II cells exhibited 65% more LBs  while the LB numbers/cell between WT and NPC1 were approximately the same .It appeared that the lamellar bodies of type II cells from the affected mice differed in size. Thus, a morphometric analysis of lamellar body size of type II cells was performed for the NPC1 and NPC2 mutant lungs and their matching litter mates using electron microscopic images of the type II cells and ImageJ analysis. The size of the LBs of wild type mice were primarily distributed in the range from 0.2 µmenlarged . 42% of An enrichment in the cholesterol content of NPC1 mouse lungs has been documented previously As would be predicted from the lipidosis in the alveolar space observed in the electron microscopic data, there was a marked increase in surfactant phospholipid in both mutant mice. The phospholipid levels of the bronchoalveolar lavage (BAL) were elevated over control mice in the NPC1 mutant (4-fold) and in NPC2 mutant mice (3-fold) , mirroriAnalysis of the bronchoalveolar lavage showed that the NPC1 mutant mouse lung had a mild protein accumulation which was not the case for the NPC2 mutant mice . We furtLipid analysis was performed on lamellar bodies isolated from pooled samples of lung homogenates, either from NPC1 mutants, NPC2 mutants or the wild type counterparts. The data revealed an increase in phospholipid  and cholesterol  content compared to wild type animals , with a 3H-choline-labelled DPPC liposomes were instilled into the lungs of wild type or NPC1 mutant mice. After a two hour perfusion time, there was no significant difference in the endocytosis of the DPPC by the lungs . However, total degradation of 3H-DPPC was reduced by 46% in the NPC1 mice lungs as compared to wild type due to significant decreases in all lipid and aqueous fractions  versus 0.40±0.02 micron2  for wild type feline (P<0.05). Measurement of the lung lipid content  lamellar bodies and lipid-enriched lamellar bodies, surfactant and lung tissue. Thus, both mice and feline animal models are suitable for studies of disease progression and pulmonary complications related to loss of a functioning NPC pathway.The feline NPC1 disorder, which progresses more slowly, is felt to be an excellent model for the human pathogenesis We describe previously uncharacterized dysfunctions in type II cells of the NPC1 and NPC2 mutant mice that may help to clarify their contribution to the elevated surfactant cholesterol phenotype associated with Niemann-Pick type C. First, the surfactant storage organelles, the lamellar bodies, have significantly elevated phospholipid and cholesterol content, which correlate to elevated phospholipid and cholesterol in the surfactant. In support of this data, immunocytochemistry directly demonstrated excess cholesterol accumulated within lamellar bodies of type II cells isolated from the NPC mutant mice. Second, there were anomalies in the lamellar body size in the type II cells from both mutant mice: enlarged lamellar bodies in NPC1 mutant mice, mirrored in the NPC1 mutant felines; and small lamellar bodies in NPC2 mutant mice. The relationship between lamellar body size an pulmonary abnormalities is not yet clear although giant lamellar body formation was associated with impaired surfactant secretion in a mouse model of Hermansky-Pudlak syndrome (HPS) Lamellar body size is an important indication of type II cell health since lamellar body size is altered in a number of genetic diseases such as ABCA3 deficiency and HPS The absence of NPC1 protein in mice negatively affected pulmonary function, where inspiratory capacity, elastance and hysterisivity were increased in the diseased lungs In summary, we have identified defects in surfactant homeostasis in both NPC1 and NPC2-mutant mice including elevated surfactant phospholipid and cholesterol, elevated lamellar body phospholipid and cholesterol, and abnormal lamellar body sizes. Many of these pathologic changes were noted in the NPC1 mutant feline pulmonary system as well. The work underscores the importance of understanding the regulation of cholesterol trafficking to maintain proper pulmonary function. These studies elucidate novel characteristics of lung disease progression in Niemann-Pick C1 and C2, and demonstrate that epithelial cell dysfunctions can be a contributing source for lung disease.


In vitro, hCG stimulated the expression of Plk2 in granulosa cells, but not Plk3. This induction of Plk2 expression was mimicked by both forskolin and phorbol 12 myristate 13-acetate (PMA). Moreover, Plk2 expression was reduced by inhibitors of prostaglandin synthesis or the EGF pathway, but not by progesterone receptor antagonist (RU486) treatment. At the promoter level, mutation of the Sp1 binding sequence abolished the transcriptional activity of the Plk2 gene. ChIP assays also revealed the interaction of endogenous Sp1 protein in the Plk2 promoter region. Functionally, the over-expression of Plk2 and Plk3 arrested granulosa cells at the G0/G1 phase of the cell cycle. In contrast, the knockdown of Plk2 expression in granulosa cells decreased the number of cells in the G0/G1 stage of the cell cycle, but increased granulosa cell viability. In summary, hCG induced Plk2 and Plk3 expression in the rat ovary. Prostaglandins and the EGF signaling pathway are involved in regulating Plk2 expression. The transcription factor Sp1 is important for Plk2 transcriptional up-regulation. Our findings suggest that the increase in Plk2 and Plk3 expression contributes to the cell cycle arrest of granulosa cells which is important for the luteinization of granulosa cells during the periovulatory period.The highly conserved polo-like kinases (Plks) are potent regulators of multiple functions in the cell cycle before and during mitotic cell division. We investigated the expression pattern of Plk genes and their potential role(s) in the rat ovary during the periovulatory period. Plk2 and Plk3 were highly induced both in intact ovaries and granulosa cells  Drosophila melanogaster polo mutation In females, an acute rise of luteinizing hormone (LH) released from the pituitary  triggers ovulation and induces terminal differentiation of preovulatory granulosa cells to become luteal cells. The LH surge terminates granulosa cell proliferation and initiates a program of luteinization in which the cells stop their division and differentiate into luteal cells Among the five members in the mammalian Plks family, Plk1 has been most thoroughly studied. The major function of Plk1 is promoting mitotic entry by phosphorylation of Cdc25C in vivo expression of Plk2 and Plk3 was dramatically increased after treatment with hCG which was used to mimic the preovulatory LH surge. Moreover, hCG stimulated the expression of Plk2, but not Plk3, in granulosa cell cultures. Given the similar and dramatic induction of Plk2 by hCG both in vivo and in vitro, we further explored the regulatory mechanism of Plk2 expression and the potential role of Plk2 in periovulatory granulosa cells using an in vitro model.Considering the roles of Plk1-4 in regulating cytokinesis, we hypothesized that the LH surge induces these Plks and that their induction is involved in the transition of granulosa cells to luteal cells. In the present study, we investigated the periovulatory expression patterns of the Plk family members. We found that the in vivo and the action of LH in preovulatory granulosa cell cultures.The gonadotropin-primed immature female rat is a well accepted experimental model to examine ovulation as well as ovarian granulosa cell function in vivo. Both Plk2 and Plk3 protein levels increased at 4 h after hCG in the intact ovary  and protein kinase C (PKC) signaling pathways in preovulatory granulosa cells hCG also sets in motion a number of steps which are crucial for follicular rupture and oocyte release including activation of epidermal growth factor (EGF) signaling and induction of progesterone receptors (PGR) and prostaglandin-endoperoxide synthase 2 (PTGS2) To investigate which transcription factors are important for Plk2 transcription, three Plk2 promoter reporter constructs  were transfected into granulosa cells isolated from PMSG-primed immature rats. The transfected granulosa cells were treated with FSK+PMA which mimics the action of an ovulatory dose of LH/hCG in vivo, ChIP assays were performed on chromatin samples extracted from periovulatory granulosa cells. PCR analysis revealed that immunoprecipitation of endogenous Sp1 enriched chromatin fragments containing the Sp1 binding sequence in the promoter region compared to that of normal rabbit IgG at 8 h post-hCG  In response to the LH surge granulosa cells of preovulatory follicles stop proliferation and begin to differentiate into luteal cells. Many cell cycle regulators have been reported to play roles in the process of luteinization, such as p27 in vivo after hCG administration, but only Plk2 is induced by hCG in vitro. The induction of Plk2 is mediated by the PKA and PKC pathways, as well as the hCG-induced activation of EGF signaling pathway and prostaglandins. The transcription factor Sp1 is important for Plk2 transcription. Our novel findings suggest Plk2 and Plk3 may regulate the cell cycle arrest of granulosa cells after the LH surge which is critical for granulosa cell luteinization.In summary, the present study demonstrates that both Plk2 and Plk3 are highly expressed in granulosa cells The experimental protocol was approved by the University of Kentucky Institutional Animal Care and Use Committee and Institutional Animal Care and Use Committee at Zhejiang University and complied with the principles of laboratory animal care.Molecular biological enzymes and molecular size markers were purchased from Toyobo . Trizol™ and pCRII-TOPO Vector were purchased from Invitrogen™ Life Technologies, Inc. . Chemicals and reagents  were all purchased from Sigma Chemical Co.  unless mentioned.Immature female Sprague-Dawley rats were provided by Harlan, Inc. and Zhejiang University Laboratory Animal Center and maintained on a 12L:12D cycle as described previously 6/well and cultured at 37°C in a humidified atmosphere of 5% CO2. The cells were treated with various reagents  for time points outlined below for each experiment. Each experiment was performed at least three times.Ovaries were collected 48 h after PMSG administration and processed as described previously in vitro and in vivo. Oligonucleotide primers for rat L32 , rat Plk1 , rat Plk2 , rat Plk3  and rat Plk4  were designed using OMIGA 2.0 software  and synthesized by Shanghai Sangon Biological Engineering Technology & Services . The specificity for each primer set was confirmed by electrophoresis of the PCR products on a 2.0% agarose gel. The PCR products were sequenced before using. The melting (dissociation) curve was analyzed using a 7300 Real-Time PCR System  after each real-time PCR. The real-time PCRs were carried out as previously described −ΔΔCT method and normalized to the endogenous reference gene L32.Real-time PCR was used to measure expression of Plk1, Plk2, Plk3 and Plk4 mRNA Western blotting was performed as described previously CAC CCA GCA GGC GCG -3′).Genomic DNA was isolated from rat tail samples using an easy-DNA kit (Invitrogen). 921-bp (−884/+37), 163-bp (−126/+37) and 85-bp (−48/+37) fragment of the Plk2 gene were amplified using the primers attached with restriction enzyme sites (KpnI and NheI). Fragments of Plk2 promoter were cloned into the pGL3 basic vector . Site-directed point mutations of the Plk2 promoter were generated using a QuickChange II site-directed mutagenesis kit according to the manufacturer’s protocol (Stratagene). The sequences of the oligonucleotide primers used to generate Plk2 promoter containing mutation (shown in lowercase) are following: mutant  and Renilla luciferase vector (pRL-TK vector) using a Lipofectamine 2000 reagent (Invitrogen). The next day, cells were treated with forskolin  and phorbol 12-myristate 13-acetate  for 4 h. The cells were then harvested to measure firefly and Renilla luciferase activities using a dual-luciferase reporter assay system (Promega). Firefly luciferase activities were normalized by Renilla luciferase activities and each experiment was performed in triplicate at least three times.Granulosa cells isolated from immature rats (48 h after PMSG) were seeded into 96 well plates at 1×10ChIP assay was performed on Sp1 in the Plk2 promoter region using a ChIP kit  as described previously The AdEasy XL adenoviral vector system kit (Stratagene) was used to construct Plk2 and Plk3 recombinant adenovirus. The processes for generating and propagating recombinant adenoviruses were described previously Rat granulosa cells collected at 48 h after PMSG priming were cultured in HyQ MEM-RS medium for 4 h before addition of the adenoviruses. The granulosa cells were exposed to Ad-Plk2, Ad-Plk3 or Ad-GFP at a multiplicity of infection (MOI) of 50 pfu/cell for 2 h. Then the medium was replaced with fresh HyQ MEM-RS medium. Approximately a 70% infection efficiency of GFP-adenovirus in granulose cells was routinely observed. Granulosa cells were collected for total RNA isolation, protein extraction, or flow cytometric analysis after adenovirus exposure for 48 h.Granulosa cells were collected from immature rats 48 h after PMSG administration. Cells were transfected with the specific siRNA against Plk2  or scrambled siRNA (Invitrogen) using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Four hours later, transfection media were replaced with fresh culture media and the cells were treated with hCG (1 IU/ml) for further 48 h. The cells were collected for flow cytometric analysis or processed to prepare cell lysates for Western blot analyses.6) and incubated at 37°C for 30 min. Then, granulosa cells were resuspended in propidium iodide (50 µg/mL) and incubated for 15 min in the dark at 4°C. A FACS Calibur flow cytometer  at Zhejiang University was used to determine the cell cycle distribution at an excitation wavelength of 488 nm. Histograms of cell cycle were obtained from 3 determinations .To determine the impact of Plk2 and Plk3 on cell cycle kinetics, granulosa cells treated with Ad-Plk2 and Ad-Plk3 adenovirus or Plk2 siRNA as described above. Granulosa cells were suspended using 3% trypsin after culture and then stained for DNA content. Briefly, ribonuclease A was added to cells  for further 24 h. Cell viability was measured using CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) according to the manufacturer’s protocol (Promega) as described previously ρ <0.05 considered significant.All the results are presented as means ± SEM. Two-way ANOVA was used to test differences in Plk2 and Plk3 expression across time of culture and treatment. One-way and t-test analysis of variance (ANOVA) was used to test differences in Plks mRNA levels among treatments. If the effects of time of culture or treatment were revealed significant, the means were compared by Duncan’s test, with