MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNA translation/decay, and may serve as biomarkers. We characterised the expression of miRNAs in clinically sampled oral and pharyngeal squamous cell carcinoma (OSCC and PSCC) and described the influence of human papilloma virus (HPV).Biopsies obtained from 51 patients with OSCC/PSCC and 40 control patients were used for microarray analysis. The results were correlated to clinical data and HPV status. Supervised learning by support vector machines was employed to generate a diagnostic miRNA signature.One hundred and fourteen miRNAs were differentially expressed between OSCC and normal oral epithelium, with the downregulation of miR-375 and upregulation of miR-31 as the most significant aberrations. Pharyngeal squamous cell carcinoma exhibited 38 differentially expressed miRNAs compared with normal pharyngeal epithelium. Differences in the miRNA expression pattern of both normal epithelium and SCC were observed between the oral cavity compared with the pharynx. Human papilloma virus infection revealed perturbations of 21 miRNAs, most significantly in miR-127-3p and miR363. A molecular classifier including 61 miRNAs was generated for OSCC with an accuracy of 93%.MicroRNAs may serve as useful biomarkers in OSCC and PSCC. The influence of HPV on miRNA may provide a mechanism for the distinct clinical behaviour of HPV-infected tumours. Head and neck cancer is the fourth most common cancer among men in The European Union , treatment comprises excision of the primary tumour followed by sentinel node biopsy or selective neck dissection  are small 21–22In a translational perspective, we examined the global miRNA expression in a series of consecutive tumours or biopsies obtained from patients with OSCC, PSCC and controls, respectively. We report remarkable tissue specificity in both normal and malignant tissue and that HPV status affects the miRNA expression pattern. On the basis of these observations, we generated an miRNA-based classifier that could differentiate between normal and malignant tissue in the oral cavity. We conclude that progression from normal epithelium to SCC is followed by changes in miRNA expression and that miRNA-based signatures may in the future support diagnosis in head and neck cancer.The Regional Scientific Ethical Committee (H-C-2008-080) approved the study. All patients gave informed consent for the use of tissue harvested at surgery.INK immunohistochemistry and in situ hybridisation (ISH).In total, 100 patients had biopsies taken for RNA extraction. Twelve patients were excluded because the tumour cell content was low or the subsequent histological examination revealed another diagnosis than SCC. Eighty-eight cancer and control patients were eligible for analysis. Thirty patients with primary OSCC (stages I–IV) and 19 patients with primary SCC of oro- and hypopharynx (stages I–IV) were included. All cancer patients had histological documented SCC and the tumour grade of differentiation was evaluated according to the WHO criteria by an experienced pathologist . Tumour Biopsies were harvested during primary surgery and placed directly in ‘RNA later’. The control group consisted of patients who obtained surgery for non-neoplastic diseases of the head and neck in the Department of Otolaryngology, Head and Neck Surgery, Rigshospitalet, and they were chosen to match the cancer patient group with regard to age, smoking status and biopsy site.Control tissue from the tonsils was collected after surgery by carefully scraping the epithelium from the lymphoid tissue to avoid prominent infiltration of lymphoid tissue from the samples. Biopsies were frozen to −80°C within 10 min.Total RNA was extracted and isolated by homogenising the frozen samples in Trizol  using a homogeniser . Briefly, total RNA was extracted from tissue lysate by phase separation with BCP (1-bromo-3-chloropropane) and RNA precipitation with isopropanol. After washing with alcohol, the RNA was eluted in RNAse free water. Total RNA concentration of samples was measured using a Nanodrop ND-1000 Spectrophotometer . For microarray analysis, 1000 ng of total RNA was labelled with FlashTag Biotin RNA Labelling Kits from Genisphere and hybridised to Affymetrix GeneChip miRNA array according to the manufacturer's instructions.Affymetrix miRNA array chips identify miRNAs in 71 organisms, including small nucleolar RNAs and small Cajal body-specific RNAs in human beings. Content is derived from Sanger miRbase miRNA database V11. It is a one-colour array and includes 847 human miRNAs. Four copies of each miRNA probe are distributed on the array. To minimise batch variation, each batch was run with equal numbers of OSCC, oral controls, PSCC and pharyngeal controls. Twenty samples were labelled and hybridised in each batch. Raw data files were imported into Affymetrix miRNA QCTool and normalised using the quantiles normalisation and median Polish summarisation following a background correction that corrects for the GC content of the each particular probe.RNA was amplified and labelled using the Ambion WT Expression Kit  according to the manufacturer's instructions. The labelled samples were hybridised to Human Gene 1.0 ST GeneChip array . The arrays were washed and stained with phycoerythrin-conjugated streptavidin using the Affymetrix Fluidics Station 450, and the arrays were scanned in the Affymetrix GeneArray 2500 scanner to generate fluorescent images, as described in the Affymetrix GeneChip protocol. Cell intensity files were generated in the GeneChip Command Console Software (AGCC) (Affymetrix).DNA was extracted from the interphase of the Trizol and precipitated with ethanol. It was dissolved in lysate buffer and proteinase K, and after another precipitation with ethanol, the solutions were transferred to spin columns (NucleoSpin Tissue). After washing, DNA was eluted with water of 70°C.−ΔCt method (Quantitative polymerase chain reaction (qPCR) validation was carried out on miRNAs selected from the miRNA microarray analysis in 20 samples. cDNA was prepared from 25 ng total RNA using TaqMan MicroRNA Reverse Transcription Kit and TaqMan MicroRNA Assays containing pre-designed primers for miR-125b, miR-187, miR-31, miR-181b and miR-375. hsa-miR-191 was used for endogenous control. Quantitative reverse transcription–PCR (QRT–PCR) reaction was performed using TaqMan Universal PCR Master Mix No AmpEras UNG, according to the manufacturer's instructions, all from Applied Biosystems. Each amplification reaction was performed in triplicate, and the median value of the three-cycle threshold was used for further analysis. For calculations of fold changes, we used the 2t method .GAPDH.Polymerase chain reaction for HPV was performed using general primers MY09 and MY11 and GP5+/GP6+  antibody, Santa Cruz , and visualised using DAKO Envision Flex+ system. Slides were evaluated by a pathologist as positive, if there was a strong diffuse staining of more than 50% of the tumour cells.Immunohistochemistry for p16In situ hybridisation was performed on formalin-fixed paraffin-embedded, 4-μm tissue sections using PathoGene HPV Screening Probe  in accordance with the manufacturer's instructions. The probe used was a ready to use cocktail of biotin-labelled HPV6, HPV11, HPV16, HPV18, HPV31, HPV33 and HPV51.Positive staining was identified as red nuclear dots. Any definitive nuclear staining in the tumour cells was considered positive.t-test. Oral cavity and pharynx samples were analysed separately. For each tissue type, a comparison of cancers vs controls was performed, and an miRNA was defined as being differentially expressed between the groups if the P-value was below 0.01 and the fold change above 1.5. In the PSCC subgroup, we compared HPV-positive vs -negative cases, and in the OSCC group, we compared patient samples with and without lymph node metastases. In these analyses, an miRNA was defined as being differentially expressed between the groups if the P-value was below 0.02 and the fold change above 1.5.Log 2 intensities of the 847 human miRNAs were imported into the Data Analysis software package Qlucore Omnics Explorer 2.1. Class comparison analysis was performed using Student's The built-in principal component analysis (PCA) tool in Qlucore Omnics Explorer was used only for visualisation of the grouping of samples using the genes selected in the class comparison. Hierarchical clustering was used for the visualisation of selected differentially expressed miRNAs using average linkage and Euclidean distance. Hierarchical clustering visualisation of both samples and variables is carried out using the Euclidean metric and data, where each variable has been normalised to mean 0 and variance 1.P-value cutoffs  and the cutoff resulting in a gene signature of optimal performance was used in the final model. The gene signature in the classifier was selected with Student's t-test with a P-value below 5E-04. Model fitting was carried out by training an SVM with a Gaussian kernel  loop consists of two steps: a univariate probe ranking and selection step, followed by fitting a support vector machine (SVM) on the sample division using the selected probes as covariates. All models were optimised by a grid search of n kernel .To validate that the classifier can distinguish between OSCC and normal oral mucosa outside the training setting, a collection of biopsies from seven patients with OSCC and seven control patients were harvested independently of the original 48 oral patients in the study. All control biopsies were taken from the gingival mucosa from patients operated with tooth extractions at the Department of Odontology, Copenhagen University. RNA was extracted, labelled and hybridised to Affymetrix GeneChip miRNA Array with the same procedure as used for the original samples. Raw data files from the test set were imported together with the original training data set into Affymetrix miRNA QCTool and normalised. The 14 samples were used as an independent test set; the classifier was applied using the signature obtained from the original data set of 48 samples with the criterions described in the classifier.To predict mRNA changes, based on the observed miRNA profile, we implemented the method developed by The effect of the up- and downregulated miRNAs was calculated separately as described by P-value of less than 0.05 was regarded as statistically significant.To assign function to the genes with the highest and lowest miRNA inhibitor score, biological function analysis was performed using the Ingenuity system software. This analysis estimates whether a biological function or pathway is enriched in the gene list provided compared with a Reference Set of molecules (the Human Gene 1.0 ST GeneChip array platform (Affymetrix) using a right-tailed Fisher's exact test. It gives a significance value as a measure of the likelihood that the association between a set of genes in our experiment and a given process or pathway was due to random chance. A Benjamin–Hochberg (FDR) corrected vs non-smokers was similar among the groups. Patients’ tumour and controls’ biopsy characteristics are listed in INK4A immunostaining revealed 15 were HPV positive and by ISH seven were HPV positive. For ensuring to find the ‘true’ HPV caused and clinically relevant HPV-positive patient samples, all three tests  for HPV were carried out. Only patients who were tested positive for at least two of the three parameters were considered HPV positive. Only eight PSCC and one OSCC fulfilled this criterion and could be used to evaluate the significance of HPV.Patient characteristics are summarised in To obtain an overview of the differences and similarities of the miRNA expression among the different cancers and normal tissues groups, we first visualised the miRNA expression of all samples in a PCA including all variables. In the uncensored  PCA plot, the oral control samples and the OSCC samples clustered into two distinct groups . FurtherP<0.01 and q<0.06, 114 miRNAs were found to be differentially expressed between OSCC and oral cavity control samples  of q<0.6 . Twenty-one miRNAs were upregulated and 25 miRNAs were downregulated. None of the miRNAs were exclusive to the groups and the fold changes were moderate. Two samples did not cluster correctly according to their anatomical subsite, of which one was in the soft palate just on the border of oral cavity and pharynx and one sample was an oral HPV-positive SCC. Comparing PSCC with normal pharyngeal epithelium revealed only 38 differentially expressed miRNAs  .As shown in the PCA plot, OSCC and PSCC samples were more similar to each other compared with the normal epithelia . In a di, q<0.2) . Twenty , q<0.2) . Within ound see , and a nound see . The mosvs 38) differed among pharyngeal cancers and the normal pharyngeal epithelium, indicating that miRNAs were different in the two epithelial linings. Consequently, we compared the miRNA composition in the two anatomical locations . Fifty-six were upregulated and 71 were downregulated, respectively, in the normal oral vs pharyngeal epithelium. There was almost a 10-fold less miR-150 and a fivefold more miR-1224 and miR-375 expression in the oral epithelium. This suggests that miRNAs may be important for maintaining distinct phenotypes of oral epithelium in the oral cavity and pharynx. The trend towards different expression profiles in patients with and without lymph node metastases was not observed in PSCC samples (as opposed to the OSCC samples). In addition, no differences in the miRNA expression were observed when comparing histological differentiation, stage or smoking status.Although OSCC and PSCC were partially similar , we obseocations . The sign=5) with oral cavity control samples (n=5). MicroRNA-375, miR-187 and miR-31 were used for qPCR in comparing all four groups (OSCC (n=5), oral cavity controls (n=5), PSCC (n=5) and pharyngeal controls (n=5)). The results of the qPCR validation are shown in Finally, five miRNAs and 20 samples were selected for validation by qPCR. MicroRNA-125b and miR-181b were used only for oral cavity samples comparing oral cavity cancer samples . The most significant aberrations were an 87-fold upregulation of POSTN , 83-fold upregulation of MMP1 ) and a 56-fold downregulation of TMPRSS11B , as well as a 27-fold downregulation of KRT4 (keratin4). The 20 most up- and downregulated genes are listed in P<0.007, q<0.05, fold change >2/−2) were imported into the Ingenuity Systems Pathway Analysis and a gene set enrichment analysis (Core IPA) was made to define pathways or biological functions, which have a likelihood of being regulated in the oral cavity cancers. Summary of core pathway analysis revealed that the following molecular and cellular functions were most significantly enriched in the gene list: cellular movement, cell morphology, cellular growth and proliferation, cell-to-cell signalling and interaction together with cellular development. One of the top canonical pathways was, as in the miRNA predicted pathways, the TGFβ signalling. On the basis of the biological functions that were enriched in the regulated gene list, the software proposes the diseases where these biological functions may be affected. The proposed diseases were as follows: connective tissue disorder, genetic disorder and cancer. One thousand eight hundred eighty-three genes were differentially expressed between oral cancer and oral controls with an adjusted β and IGF-1 signalling. From these biological functions, a suggestion of disease was made and satisfactorily the number one predicted disease was cancer. From the miRNA that changed more than fivefold up or down between OSCC and oral controls, we found 452 predicted target genes, which had a combined weighted inhibitor score of more than 2/−2. Fifty genes with the highest and lowest combined score as well as their matching fold changes identified in the gene expressions analysis are shown in Even though advances in diagnostic tools and treatments have been achieved over the past three decades, the mortality of head and neck cancer patients is still high and the offered treatment modalities are still associated with major adverse effects with considerable impact in the quality of life . ClinicaThere is a considerable variation in the miRNA profiles across the various miRNA profile studies of head and neck cancer. Differences may reflect both technically issues and differences in stage, grade and sampling from multiple anatomical sites as well as the use of cell lines.We performed a prospective translational and multidisciplinary study comprising a large and clinically well-defined patient material, which allowed us to generate site-specific miRNA expression profiles from oral cavity and pharyngeal cancers, as well as determine the influence of HPV on miRNA expression profile in head and neck cancers.The oral cavity samples showed very consistent expression profiles and benign and malignant samples separated clearly even in the uncensored PCA plots. Homogeneity was particularly striking among the OSCC and oral cavity control samples, whereas the PSCC and pharyngeal control samples were more diverse. One of the reasons for this may be a higher contamination of non-tumour cells in the biopsies from the PSCC patients, which are much more difficult to examine and perform biopsies from, compared with oral cavity cancers, which normally present themselves as a clear visible tumour easy to distinguish from surrounding structures. The difference between cancer samples and controls was also less pronounced in the pharynx. Many of the miRNAs lost in oral cancers and highly expressed in oral controls were more highly expressed in the pharyngeal cancers and only slightly higher expressed in the pharyngeal controls, indicating a higher resemblance in miRNA profile of pharyngeal epithelium with cancer. We also noted a marked difference in the miRNA expression between the controls from the oral cavity and pharynx. This may be related to the higher cell turnover of pharyngeal mucosa than oral mucosa . In addition, nodal metastasis occurs in 20–50% of patients with clinically N0 head and neck cancer. Treating the neck with either surgical neck dissection or radiotherapy is accompanied with significant side effects, and the treatment strategy of cervical lymph nodes is still under debate (Even though there was no correlation with miRNA profile and stage or grade, a distinct miRNA expression pattern was associated with lymph node metastasis in the oral cavity cancer group. MicroRNA-181b was upregulated and miR-942 and miR-181cHuman papilloma virus-positive HNSCC appears to be different from HPV-negative HNSCC in both molecular and clinical features . In geneA number of miRNAs were perturbed in the HPV-positive pharyngeal SCC samples compared with the HPV-negative pharyngeal SCC. The observed fold changes were as described modest and the FDR relatively high. This is at least partly due to a prominent infiltration of normal tissue in the HPV-negative PSCC and low number of HPV-positive samples. MicroRNA-145, miR-125a and miR-126, which are reduced in the HPV-positive PSCC, are also downregulated in cervical cancer tissue (vs non-smokers in patients with malignant mesothelioma and this may reflect the limited number of smokers in the HPV-positive PSCC group. The present material comprised only had five tonsillar SCC. To make a clear HPV miRNA classifier, it probably needs to include micro-dissected formalin-fixed paraffin-embedded tonsillar specimens to ensure a high proportion of HPV-positive samples and even tissue specificity.MiR-363 was elevated in the HPV-positive PSCC samples. This is in concordance with a most recent study, in which HPV-positive head and neck cancer cell lines showed a considerable upregulation of miR-363 and an siRNA knockdown of HPV16 E6 was accompanied by a reduction of miR-363 levels (We finally examined whether or not the distinct miRNA signatures could provide a solid basis for diagnostic classification between normal epithelium and malignant lesions in the oral cavity. A robust classifier was generated for oral cavity cancer, with an accuracy of 93% in the test samples. The test sample that failed was obtained from a patient who had clear inflammation in the area where the biopsy was taken. This sample clearly clustered with cancer samples and it may indicate that there are some similarities in the miRNA profiles between cancer and inflammation. Even though the test set was small, the classifier seemed robust, but needs to be confirmed in a larger validation study. A validation study should preferably also include inflamed tissue, which seems to have a degree of resemblance with malignant tissue as indicated above.In conclusion, we found significant differences in miRNA expression profiles of malignant and benign tissue samples as well as remarkable site-specific differences. In addition, the presence of lymph node metastasis at the time of diagnosis in oral SCC seems to influence the miRNA expression. Human papilloma virus infection in pharyngeal SCC shows a distinct miRNA expression profile, but needs to be further elucidated in a specific tonsillar SCC material.


Punctate inner choroidopathy (PIC) is an ocular inflammatory disease. Spectral domain optical coherence tomography (SD-OCT) allows detailed visualization of retinal and choroidal structures. We aimed to describe the retinal changes on SD-OCT associated with PIC lesions localized in the macula.Retrospective case series: PIC lesions not associated with choroidal neovascularization (CNV) and captured by macular SD-OCT scans were identified and characterized.Twenty-seven PIC lesions from seven patients (eight eyes) were identified and classified into four categories according to disease activity and temporal changes. Among clinically inactive patients, two main patterns were noted on OCT: (1) retinal pigment epithelium (RPE) elevation with sub-RPE hyper-reflective signals and (2) localized disruption of outer retinal layers with choroid and Bruch's membrane (BM) generally spared. Clinically active patients demonstrated lesions with intact BM with RPE elevation that fluctuated with disease activity and sub-RPE hyper-reflective signals. Photoreceptor-associated bands on SD-OCT (PRs) were not visible during active disease, but returned to normal visibility when lesions were clinically stable. Seven lesions in patients without clinically detected activity demonstrated alteration of RPE elevation.SD-OCT can provide detailed structural characteristics of PIC lesions. RPE elevation is noted in many lesions while BM and choroid are spared. Photoreceptor-associated bands on SD-OCT appear compressed during clinically active stages and are visible during stabilization. OCT may provide information on activity not detected clinically.The online version of this article (doi:10.1007/s12348-011-0054-6) contains supplementary material, which is available to authorized users. Punctate inner choroidopathy (PIC) is an ocular inflammatory disease mostly affecting young myopic women. It was described by Watzke et al. in 1984; diagnosis was based on the visualization of yellowish white punctate lesions on funduscopy apparently in the absence of ocular inflammation , 3. AutoIn recent years, optical coherence tomography (OCT) has become a common non-invasive imaging modality for visualizing retinal structures in detail. The advances in OCT technology have given ophthalmologists a detailed morphological view of the retina, previously only possible from microscopic analysis of cadaveric eyes. Hassentein and Meyer, in a recent review, reported a high correlation between histological structure of the retina and the image acquired by spectral domain (SD)-OCT . Other sWe sought to utilize the enhanced imaging capability of SD-OCT to describe the retinal changes of PIC lesions.Medical records of patients with a diagnosis of PIC, who were being followed in the outpatient clinic at the Wilmer Eye Institute between March 2008 and March 2010, by the uveitis specialist (QDN), were reviewed. The diagnosis of PIC was made clinically by one of the authors (QDN), based on funduscopic appearance of multiple punctate chorioretinal lesions localized mainly in the posterior pole and in the absence of iridocyclitis and vitreal inflammation. Institutional Review Board (IRB)/Ethics Committee approval was obtained, and HIPAA guidelines were followed for the study.clinically active or clinically stable. The criteria for development of clinical activity were eye-specific as a patient may have active disease in one eye and not in the other. Clinical activity was identified if a patient met one or more of the following criteria at any visit:Visual disturbances/changes noticed by the patient that are consistent with symptoms of previous attacksIndistinct borders of lesions with surrounding edema on contact lens biomicroscopyHyperfluorescence of the lesion with blurred borders on FA Fig. .PIC patients enrolled in the study were on immunomodulatory therapy (IMT) and were evaluated in the clinics approximately every 2 months. Clinic visits during which the patients had SD-OCT measurements were included in the index study. All ophthalmic examination, fundus photographs, and FAs of each patient for those particular visits were reviewed to identify all PIC lesions for possible inclusion into the study. The patients were then classified according to clinical activity of the disease as Patients who did not meet any of the above criteria were considered to have clinically stable disease. SD-OCT scans from selected patient were then reviewed. Scans of lesions within the area of retina covered by the macular raster  were included in the study. Lesions that were immediately adjacent to choroidal neovascularization (CNV) scars, with distorted surrounding retinal layers, were excluded.The selected lesions were reviewed systematically in the Retinal Imaging Research and Reading Center (RIRRC) at Wilmer to identify and characterize the abnormalities, which included the presence of abnormal signals, distortion of the normal retinal architecture, and loss or disruption of individual retinal layers. The characterized abnormalities were then classified according to the disease activity.SD-OCT findings of all patients were studied on the first visit available in our records; this visit was designated BL. SD-OCT scans were then reviewed for all available visits after the BL visit. These visits were labeled according to their temporal relationship to BL. For example, a visit that occurred 1 month after BL was labeled “M1.” After all observations have been analyzed, we describe the SD-OCT characteristics of the lesions in clinically active and clinically stable patients including the temporal changes observed on SD-OCT in both categories.The OCT scans from our study patients were captured using Spectralis HRA + OCT™ , which provides an axial resolution of 3.9 μm and a transverse resolution of 14 μm in tissue. It can acquire up to 100 B-scans at a particular location, allowing reduction of noise artifact .Twelve eyes of nine patients met the clinical diagnosis of PIC. Two eyes were excluded because of extensive scarring secondary to CNV. Another two eyes were excluded because none of the PIC lesions fell within the area covered by the macular raster of the SD-OCT. Twenty-seven PIC lesions were identified in eight eyes of seven patients . Six patients had scans available for multiple visits, with length of follow-up ranging from 8 to 23 months; one patient was scanned only once. Ages ranged between 23 and 57 years with a median of 30 years Table .Table 1DAt their first visit to our clinic, all seven patients had a history of CNV in at least one eye, had experienced recurrent disease activity in the past, and were on maintenance IMT during the entire period in which the index study was conducted.Two patients were identified as having clinically active disease and five had clinically stable disease. Twenty-four of the 27 lesions included in the study showed involvement of the RPE layer on SD-OCT. Based on clinical activity of the disease and temporal changes of lesions on SD-OCT, we classified the 27 PIC lesions into four categories: 1) clinically active patients with lesions that showed temporal SD-OCT changes (five lesions from two patients), (2) clinically active patients with lesions that showed no temporal changes on SD-OCT (three lesions from one patient), (3) clinically stable patients with temporal changes on SD-OCT (seven lesions from two patients), and (4) clinically stable patients with no temporal changes on SD-OCT (seven lesions from three patients). Additional five lesions were identified in a patient with clinically stable disease but were not categorized as above because there were no follow-up SD-OCT scans available. Figure  clinicalAmong a total of five lesions that were included in this category, RPE elevation with hyper-reflective sub-RPE signals was seen in four lesions. Bruch's membrane (BM) appeared to be intact in three lesions, disrupted in one, and not visible in one lesion. PRs were not visible in three lesions and partly visible in two lesions.Lesion 13: Lesion 13 was observed in the right eye of Patient 3 and demonstrated on the SD-OCT scan at BL as a small irregularity in RPE. It was observed to increase progressively in size over the course of 18 months prior to the detection of clinical activity.Lesion 14: Review of SD-OCT scans of the left eye, acquired at the BL visit , revealed that the patient had a lesion with RPE elevation and displaced PRs. At the time of clinical activity, RPE elevation had increased, and PRs were not visible. Over the course of the next 19 months, RPE elevation decreased, and PRs became visible again  and outer plexiform layer (OPL).Among the clinically stable patients, two main patterns were noted on SD-OCT: (1) RPE elevation with sub-RPE hyper-reflective signals and (2) no RPE elevation, but there was localized disruption of outer retinal layers including the ONL, OPL, PR, and RPE layers, with sparing of choroid and BM.visibility of the PRs was related to the extent of RPE elevation; non-visibility of PRs was associated with progression of the RPE elevation, and the visibility was associated with the regression and resolution of the RPE elevation. BM was intact in all six lesions  outwards with back shadowing, and there was an associated focal choroidal thinning  comparisons of lesions over time. The majority of lesions  in our study, irrespective of clinical activity, showed involvement of the RPE. RPE changes included RPE elevation with sub-RPE signals and RPE discontinuity. In the first description of PIC lesions, Watzke et al. described serous retinal detachments occurring over choroidal lesions . In our Temporal SD-OCT changes were observed in all clinically active patients and also in seven lesions of two other patients who were not identified to be clinically active. Temporal SD-OCT changes in four of the five lesions in clinically active patients were very similar to the changes observed in seven lesions in the two clinically stable patients. There could be several explanations for such observation. One possible explanation is that the high-resolution property of SD-OCT makes it a sensitive imaging modality which may identify changes before they become clinically detectable. Such hypothesis is further supported by the fact that SD-OCT changes in three lesions of Patient 3 were visible up to 21 months before identification of clinical activity.PIC is usually a benign disease but has the potential to cause severe visual loss if complicated by CNV and sub-retinal fibrosis: rates of up to 69% and 56%, respectively, were reported in our study of 77 PIC patients, and 75% of patients in a series of 12 patients had CNV . Essex eAn interesting phenomenon was noted while we were analyzing the SD-OCT scans. Patients 2, 3, and 7 had several lesions with RPE elevation and non-visibility of the overlying PRs. Review of latter scans of the same lesions revealed that the RPE elevation gradually resolved, and the PRs became visible. A possible explanation for this phenomenon may be that in the initial stages, there is elevation of RPE and compression of PRs. Resolution of the RPE elevation restored the PRs to their original position. The non-visibility of PRs persisted during follow-up in three lesions of Patient 5. Spaide et al. demonstrated loss of PRs in AZOOR complex of diseases . They deIn our scans, we were unable to identify changes in the choroid in all except one lesion. It was surprising because the first report from Watzke mentioned choroidal involvement of lesions, and eyes with PIC have been shown to have involvement of choriocapillaris on ICG –3. The lThe Japanese literature has previously reported TD-OCT findings of four patients with PIC disease , 17. TheThe definitive pathophysiology of white dot syndromes has been much debated throughout the years . SD-OCT One of the hypotheses for the pathogenesis of PIC is that the disease is secondary to the development of myopic cracks in BM. However, PIC is mostly seen in patients with moderate myopia. The assessment of integrity of BM was limited in some lesions due to the abnormal reflectivity of light by adjacent structures. However, when clearly visible, it appeared to be intact in the majority of the lesions in our study. Review of SD-OCT scans of Patient 3 demonstrated destruction of the BM on later scans when compared to earlier scans.SD-OCT may be able to provide detailed structural characteristics of PIC lesions and may serve as a useful, non-invasive means of identifying disease activity in patients with PIC. RPE elevation is noted in many lesions, while BM and choroid are spared. PRs appear to be compressed during RPE elevation and resume clear visibility upon resolution of the RPE elevation. Therefore, SD-OCT may provide information on disease activity that is not detected clinically or on FA. Further systematic, prospective SD-OCT studies of PIC lesions will most likely provide additional insights about the clinical course of PIC, and may provide clinicians with a non-invasive imaging tool in the management of patients with PIC.


Although test-retest reliability of mean values of spatio-temporal gait parameters has been assessed for reliability while walking alone , little is known about the test-retest reliability of stride time variability (STV) while performing an attention demanding-task . The objective of this study was to examine immediate test-retest reliability of STV while single and dual tasking in cognitively healthy older individuals (CHI) and in demented patients with frontotemporal degeneration (FTD).® and SMTEC® footswitch systems.Based on a cross-sectional design, 69 community-dwelling CHI  and 14 demented patients with FTD  walked alone  and while counting backward (CB) aloud starting from 50 . Each subject completed two trials for all the testing conditions. The mean value and the coefficient of variation (CoV) of stride time while walking alone and while CB at self-selected walking speed were measured using GAITRiteICC of mean value in CHI under both walking conditions were higher than ICC of demented patients with FTD and indicated perfect reliability (ICC > 0.80). Reliability of mean value was better while single tasking than dual tasking in CHI , whereas it was the opposite in demented patients . ICC of CoV was slight to poor whatever the group of participants and the walking condition (ICC < 0.20), except while dual tasking in demented patients where it was fair (ICC = 0.34).The immediate test-retest reliability of the mean value of stride time in single and dual tasking was good in older CHI as well as in demented patients with FTD. In contrast, the variability of stride time was low in both groups of participants. Stride time variability (STV), as measured by the coefficient of variation (CoV), is considered as a marker for the control of limb-coordinated movements ,2. In teDemented older adults demonstrate greater gait impairments than those expected with normal aging process ,8. In paCounting backward (CB) is an attention-demanding task frequently used in dual task paradigm involving gait . CompareSTV may be interpreted as surrogate marker for gait control only after it has been determined if dual-task-related changes of STV are due to a pathological process affecting older adults, to a normal biological variability following consecutive gait measures from trial to trial or to a measurement error. Although test-retest reliability of mean values of spatio-temporal gait parameters has been assessed for reliability while walking alone, little is known on the test-retest reliability of gait variability while dual tasking -12. ReceOut of 80 community-dwelling CHI participating in a large fall prevention program in Erlangen, Germany, 69 (90.0%) CHI  with at least 3 consecutive measured gait cycles were included in this study, with respect to the European guidelines for clinical applications of spatio-temporal gait analysis in older adults . In addi® and SMTEC® footswitches systems which are two validated devices providing similar measures of stride time  × 100) of stride time while walking alone  and while counting backward (CB) aloud starting from 50  were collected. Gait measurements were made according to the guidelines for clinical applications of spatio-temporal gait analysis in older adults . The meaide time . The GAI® and the SMTEC® footswitches systems used the same definition of stride time which was the time elapsed between the first contacts of two consecutive footfalls of the same foot expressed in ms. Walking trials were recorded on a 3.5-meter walkway for GAITRite® and 10-meters walkway for SMTEC®. To assure measuring of steady-state walking among CHI, participants started walking 2 meters before the active measuring electronic surface area and stop 2 meters after. In the group of demented patients with FTD, stride time parameters were collected on a 14-meter long walkway but were analyzed only over a distance of 10 meters. The first and last 2 meters corresponding to the acceleration and deceleration phase of each pass were excluded from analysis. All participants were asked to perform the walking tasks without prioritizing walking or cognitive task. Before testing, a trained evaluator gave standardized verbal instructions regarding the test procedure with a visual demonstration of the walking test. Each subject completed two trials for all the testing conditions. The walking trial was performed in a well-lit environment. The participants walked at their self-selected speed and wore their own footwear.The GAITRiteImmediate test-retest reliability of STV was evaluated comparing the first and the second trial performed while walking alone and while CB using intraclass correlation coefficient (ICC). Separated analyses were used for single and dual task condition. Using Landis and Koch interpretation of agreement, an ICC > 0.80 indicated almost perfect reliability, 0.61-0.80 substantial, 0.41-0.60 moderate, 0.21-0.40 fair, 0.00-0.20 slight, below 0.0 poor [All participants were able to complete single and dual-task walking, without falling. As shown in Table Our results showed that the immediate test-retest reliability of the mean value of stride time was perfect , higher than the reliability of the CoV and better in CHI than in demented patients with FTD. In contrast, the reliability of the CoV was slight to poor in both groups of participants, except while dual tasking in the group of demented patients with FTD by whom it was fair. In addition, the reliability of both stride parameters was better while dual tasking compared to single tasking in demented patients with FTD but not in CHI.The very good immediate test-retest reliability of mean value of stride time while single tasking showed in the studied sample of CHI and demented patients with FTD is consistent with the literature. Indeed, previous studies showed a high ICC for most temporo-spatial gait parameters including stride time while walking alone at usual walking speed -23. ImmeIn contrast to the mean value, little is known about the test-retest reliability of stride-to-stride variability. Our results show that the immediate reliability of the CoV of stride time while single and dual tasking is poor, which is in concordance with the few previous published data. For instance, Hollman et al. recently reported among healthy older adults that immediate test-retest reliability for variability in stride velocity was poor while dual tasking . This loWhilst the reliability of CoV was slight to poor in most case, our results highlight that the reliability while dual tasking in demented patients with FTD was higher compared to single tasking  and compared to CHI (ICC = 0.11). One explanation could be a practice effect related to the repetition of trials. We showed that demented patients had a higher mean value of CoV while dual tasking compared to CHI and compared to walking alone, which is in concordance with previously published data. We have earlier shown that CB in demented participants with impaired EF provoked severe perturbation in gait control resulting in an increased CoV of stride time compared to the mean value while dual tasking ,6,9. ThuThe main limitation of our study was the short length of the walkway used to assess STV. The European GAITRite network group recommends the highest number of gait cycles possible from a practical standpoint, with a minimum of three consecutive gait cycles . AnotherMeasurements of stride time variability had low immediate test-retest reliability in older CHI as well as in demented patients with FTD either in single or dual task condition. In contrast, the reliability of the mean value was good in both groups. This result suggests a normal biological variability for stride time variability between two immediate consecutive gait measurements.All authors have no conflicts of interest. There were not any financial and personal relationships with other people or organization that could influence this study.No authors have relevant financial interest in this manuscript.OB has full access to the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analyses; study concept and design: OB and GA; acquisition of data: GA and EF; analysis and interpretation of data: OB, GA, CA and FH; drafting of the manuscript: OB, GA, CA and EF; critical revision of the manuscript for important intellectual content: FH and RK; obtained funding: not applicable; statistical expertise: FH; administrative, technical, or material support: OB; study supervision: OB and GA.All the authors  have participated in the research reported, have seen and approved the final version of the manuscript, and have agreed to be an author of the paper.Gilles Allali was supported by a grant from the Swiss National Science Foundation (No 33CM30-124115).


The ventricularis and fibrosa of porcine aortic valve leaflets were exposed simultaneously to different combinations of sub-physiologic/physiologic/supra-physiologic levels of FSS magnitude and frequency for 24, 48 and 72 hours in a double cone-and-plate device. Endothelial activation and paracrine signaling were investigated by measuring cell-adhesion molecule  and cytokine  expressions, respectively. Extracellular matrix (ECM) degradation was characterized by measuring the expression and activity of the proteases MMP-2, MMP-9, cathepsin L and cathepsin S. The effect of the FSS treatment yielding the most significant pathological response was examined over a 72-hour period to characterize the time-dependence of FSS mechano-transduction. While cytokine expression was stimulated under elevated FSS magnitude at normal frequency, ECM degradation was stimulated under both elevated FSS magnitude at normal frequency and physiologic FSS magnitude at abnormal frequency. In contrast, combined FSS magnitude and frequency abnormalities essentially maintained valvular homeostasis. The pathological response under supra-physiologic FSS magnitude peaked at 48 hours but was then maintained until the 72-hour time point. This study confirms the sensitivity of valve leaflets to both FSS magnitude and frequency and suggests the ability of supra-physiologic FSS levels or abnormal FSS frequencies to initiate CAVD mechanisms.Calcific aortic valve disease (CAVD) is an active process presumably triggered by interplays between cardiovascular risk factors, molecular signaling networks and hemodynamic cues. While earlier studies demonstrated that alterations in fluid shear stress (FSS) on the fibrosa could trigger inflammation, the mechanisms of CAVD pathogenesis secondary to side-specific FSS abnormalities are poorly understood. This knowledge could be critical to the elucidation of key CAVD risk factors such as congenital valve defects, aging and hypertension, which are known to generate FSS disturbances. The objective of this study was to characterize  As the underlying cause of 30,000 deaths annually and 55,000 hospital discharges, aortic valve disorders have tremendous economic and societal impact While CAVD has been described historically as a passive degenerative process, it has now emerged as a highly regulated pathology presumably triggered by a combination of conventional cardiovascular risk factors ex vivo exposure of porcine aortic valve leaflets to side-specific alterations in FSS magnitude and/or frequency and the biological assessment of the acute pathological response in terms of inflammation and remodeling via immunostaining, immunoblotting and zymography. The results reveal the key role played by FSS abnormalities in the active regulation of early CAVD pathogenic events.Therefore, the goal of this study was to determine for the first time the role of FSS magnitude and/or frequency abnormalities in early CAVD pathogenesis. The methodology involved the in vitro2 on the ventricularis and a bidirectional oscillatory shear stress varying between −8 and +10 dyn/cm2 on the fibrosa. In addition to the physiologic FSS signal (group 1), eight abnormal FSS conditions were generated by decreasing/increasing the magnitude and/or frequency of the physiologic signal , 0.2% Trixon-100 (Sigma-Aldrich) and 1% dimethyl sulfoxide  in 1x PBS for 1 hour at room temperature. Following the blocking step, the slides were then incubated overnight at 4°C in primary antibody in 2–10% blocker at the following dilutions: VCAM-1 , ICAM-1 , TGF-β1 , BMP-4 , cathepsin L , cathepsin S , MMP-2  and MMP-9 . Following primary antibody incubation, sections were washed 3 times in 1x PBS and incubated with anti-rabbit or anti-mouse  secondary antibodies at 1∶100 dilution for 2 hours at room temperature. The tissue sections were then washed 3 times in 1x PBS, counterstained with 1 4′,6-Diamidino-2-phenylindole , mounted with fluorescence mounting medium (Dako), coverslipped and stored at 4°C. Slides were subsequently imaged under the mercury lamp of a Nikon E600 imaging microscope using a TR/FITC/DAPI filter.Following each mechanical treatment, the specimens were pulverized using a mortar and pestle in liquid nitrogen, homogenized in ice-cold RIPA buffer (Santa-Cruz) and centrifuged at 7,000 g to pellet extracellular matrix debris for 8 minutes at 4°C. The supernatant was assayed for protein concentration using a bicinchoninic acid (BCA) protein assay (Pierce). Equal amounts of tissue lysates were resolved by reducing SDS-PAGE. After transfer to a polyvinylidene difluoride (PVDF) membrane (EMD Millipore) using a mini trans-blot cell (Bio-Rad), the blots were blocked with 5% non-fat drymilk and probed with a primary antibody against BMP-4 , TGF-β1 . Depending on the primary antibody, appropriate anti-rabbit and anti-mouse HRP secondary antibody  was then used. The membranes were finally incubated in horseradish peroxidase-conjugated streptavidin. Immunopositive bands were then detected using a luminol-based chemiluminescence reagent (Pierce) against standard radiography film in a darkroom. The films were then analyzed by densitometry using ImageJ (NIH).Zymography was performed to quantify the activity of the proteolytic enzymes MMP-2 and MMP-9. Equal amounts of tissue lysates assayed by BCA were resolved by sample buffer (Bio-Rad) and loaded in the gel (Bio-Rad). After running, the gels were washed in renaturation buffer (Bio-Rad) and developing buffer (Bio-Rad). The gels were stained by adding stain solution (Sigma-Aldrich), de-stained in deionized water and scanned. The images were then analyzed by densitometry using ImageJ.The semi-quantitative assessment of immunostained images was carried out using ImageJ. Briefly, the intensities of positively stained regions were estimated and normalized by the number of cells visible in the images to yield a quantity consistent to a biomarker expression per cell. Distinction was not made between endothelial and interstitial cells during the cell counting.The dedicated macro of ImageJ was used to plot the histogram of individual lanes/bands in blots/gels. These histograms were then integrated to obtain the mean intensity of each immune-positive band. For the blots, these intensities for a particular protein were then normalized by the intensity values for β-actin (Santa Cruz), which was used as a housekeeping protein.Each experimental group consisted of six leaflet samples. All quantitative data were expressed as means ± SE and then normalized with respect to the values measured in fresh tissue (control). Data from all experiments were first tested for normality by the Anderson-Darling method, then analyzed using ANOVA to determine if there was significant contribution by a particular mechanical treatment on the measured parameters, followed by the Bonferroni post-hoc test. A p-value of less than 0.05 was used as a measure of statistical significance. All statistical analyses were performed using SPSS (IBM).The net effect of a particular FSS environment on pro-inflammatory processes after 48 hours of conditioning was estimated by calculating a pro-inflammatory state index Similarly, the contributions of FSS abnormalities to valvular remodeling were quantified via a tissue remodeling index An index smaller than one indicates a net decrease in pro-inflammatory/remodeling response, while an index larger than one reflected a net increase in tissue pro-inflammatory/remodeling processes. The nine pro-inflammatory and remodeling state indices resulting from the nine FSS magnitude-frequency treatments were interpolated using a biharmonic spline in order to predict the dependence of the tissue pathologic state on FSS magnitude and frequency.Following tissue exposure to physiologic and non-physiologic levels of FSS magnitude and frequency, immunostaining was performed to examine endothelial activation. Positive staining for ICAM-1  and VCAMBMP-4 and TGF-β1 immunostaining and immunoblotting were performed to investigate the effects of FSS abnormalities on paracrine signaling. Similar to the cell-adhesion molecules, BMP-4 and TGF-β1 expression  which suThe effects of FSS abnormalities on valvular remodeling were investigated by comparing the expression of the proteolytic enzymes MMP-2 and MMP-9 and the elastolytic proteases cathepsin L and cathepsin S. MMP-2 and MMP-9 immunostaining  support With most pro-inflammatory state index variations confined in the supra-physiologic FSS magnitude range, the results suggest the particular vulnerability of leaflet inflammation to elevated FSS levels . The symThe time-dependence of the leaflet pathological response following conditioning to supra-physiologic FSS under normal frequency  was studied by assessing the biological response of tissue exposed to this environment for 24 and 72 hours and by comparing it to those obtained in the fresh controls and tissue conditioned for 48 hours . The semThe objective of this study was to elucidate the respective and combined effects of abnormalities in FSS magnitude and frequency on the early mechanisms of CAVD in porcine aortic valve leaflets. The key results of the study can be summarized as follows: 1) leaflet tissue is selectively more sensitive to alterations in FSS magnitude than frequency; 2) FSS abnormalities promote paracrine signaling via BMP-4 and TGF-β1-dependent pathways and ECM degradation via MMP- and cathepsin-dependent pathways; and 3) the FSS mechanisms of early CAVD development are time-dependent. Therefore, the results confirm the key contribution of FSS alterations to valvular pathogenesis and suggest the capability of FSS abnormalities to trigger early CAVD events in the absence of any other risk factor.in vivo. Tachycardia conditions, which may result in acute FSS frequency alterations, are not chronic and therefore unlikely to contribute to a long-term pathological response. However, the rationale for varying the frequency of the FSS signal was to investigate the effects of changes in the harmonics of the FSS waveform experienced locally by the leaflets. Such phenomenon has been evidenced on bicuspid aortic valve leaflets The focus of this study on the characterization of the acute effects of FSS abnormalities on endothelial activation, paracrine signaling and ECM degradation is motivated by the established involvement of those biological processes in CAVD pathogenesis in vivo and in vitro models of CAVD proposed to date have relied on two leading hypotheses. The cholesterol hypothesis focuses on the traditional risk factors to study the biological events leading to CAVD Experimental ex vivo in the contexts of elevated pressure, stretch and FSS. Exposure of porcine aortic valve leaflets to hypertensive levels in a pressure chamber was shown to upregulate pro-inflammatory gene networks in interstitial cells ex vivo exposure of porcine valve leaflets to elevated stretch with an osteogenic culture medium demonstrated stretch magnitude-dependent tissue mineralization mediated by BMP-4 and TGF-β1 pathways in vivo evidence of the role played by BMP-4 in CAVD pathogenesis Our results demonstrated the particular sensitivity of valvular pro-inflammatory pathways to elevated FSS levels as illustrated by the increased expressions of the cytokines BMP-4 and TGF-β1. The ability of supra-physiologic mechanical stress environments to trigger pro-inflammatory processes has been observed ex vivo to promote collagen and sulfated glycosaminoglycan (sGAG) production but to decrease cathepsin L, MMP-2 and MMP-9 expression and activity in porcine leaflets ex vivo to combined pathological levels of pressure and stretch exhibit increased ECM synthesis and a progressive switch of the valve interstitial cells from a contractile to a more synthetic phenotype Our study also indicated increases in elastolytic enzyme expression and proteolytic enzyme expression and activity under elevated FSS magnitude  and abnormal FSS frequency , suggesting the progressive loss of valvular homeostasis and the alteration of valvular biosynthetic activity. The ability of supra-physiologic mechanical stress environments to break the delicate balance between ECM synthesis and degradation has been observed previously in the contexts of elevated pressure, stretch or a combination of these. Hypertensive levels have been shown It is important to note that the immunofluorescence images included in the paper focused on the fibrosa side due to the complete absence of positive stain on the ventricularis. Depending on the mechanical treatment, protein and enzyme expressions were distributed either in the subendothelial layer of the fibrosa or deeper in the tissue. Those results may correlate with the previously established phenotypic difference between the endothelial cells lining the leaflet fibrosa and ventricularis An interesting result is the apparent synergistic effects of FSS magnitude and frequency abnormalities on ECM degradation and paracrine signaling. In fact, leaflets exposed to physiologic FSS or combined abnormalities in FSS magnitude and frequency , cytokine and protease expressions peaked after 48 hours of exposure to FSS and then remained relatively constant until the 72-hour mark. Similar temporal responses have been observed previously in the contexts of valvular mechano-response to cyclic FSS The comparison of the results obtained in the fresh control group and in the group exposed to physiologic FSS for 48 hours (group 1) revealed no statistical difference in the expression of the different biomarkers considered in the study. Only MMP-9 activity was significantly downregulated under physiologic FSS as compared to the control group (78% decrease; see Finally, it is important to note that our experiments were conducted over a 48-hour period, which is much shorter than the timescale over which CAVD typically develops. However, the acute response observed and described in this study is relevant as it may shape the long-term processes involved in the formation of calcific lesions. In addition, the experiments were performed with porcine valve leaflets due to well-characterized antibody specificities and the extensive use of this tissue source in previous valvular mechanobiology studies. However, given the intricate differences between porcine and human valve tissues, the translation of the present findings to human valves should be done with caution.In conclusion, this study demonstrates the sensitivity of valve leaflets to FSS abnormalities and the ability of supra-physiologic FSS magnitude or sub-/supra-physiologic FSS frequency to upregulate key CAVD mechanisms such as paracrine signaling and ECM degradation . The res


Receptor phosphorylation is thought to be tightly regulated because phosphorylated receptors initiate signaling cascades leading to cellular activation. The T cell antigen receptor (TCR) on the surface of T cells is phosphorylated by the kinase Lck and dephosphorylated by the phosphatase CD45 on multiple immunoreceptor tyrosine-based activation motifs (ITAMs). Intriguingly, Lck sequentially phosphorylates ITAMs and ZAP-70, a cytosolic kinase, binds to phosphorylated ITAMs with differential affinities. The purpose of multiple ITAMs, their sequential phosphorylation, and the differential ZAP-70 affinities are unknown. Here, we use a systems model to show that this signaling architecture produces emergent ultrasensitivity resulting in switch-like responses at the scale of individual TCRs. Importantly, this switch-like response is an emergent property, so that removal of multiple ITAMs, sequential phosphorylation, or differential affinities abolishes the switch. We propose that highly regulated TCR phosphorylation is achieved by an emergent switch-like response and use the systems model to design novel chimeric antigen receptors for therapy. Recognition of antigen by the T cell antigen receptor (TCR) is a central event in the initiation of adaptive immune responses and for this reason the TCR has been extensively studied. Multiple studies performed over the past 20 years have revealed intriguing findings that include the observation that the TCR has multiple phosphorylation sites that are sequentially phosphorylated by the kinase Lck and that ZAP-70, a cytosolic kinase, binds to these sites with different affinities. The purpose of multiple sites, their sequential phosphorylation by Lck, and the differential binding affinities of ZAP-70 are unknown. Using a novel mechanistic model that incorporates a high level of molecular detail, we find, unexpectedly, that all factors are critical for producing ultrasensitivity (switch-like response) and therefore this signaling architecture exhibits systems-level emergent ultrasensitivity. We use the model to study existing therapeutic chimeric antigen receptors and in the design of novel ones. The work also has direct implications to the study of many other immune receptors. T cells patrol the body in search of infection and cancer derived antigens The importance of the TCR in initiating and regulating adaptive immune responses has meant that TCR proximal proteins have been extensively studied. The TCR itself is a multi-subunit receptor that contains subunits for ligand binding  . As expeMs (XX3) , reflectMs (XX3)  and sensMs (XX3) . These eWe next investigated the contribution of ZAP-70 binding to potency and sensitivity. To do this, we calculated the total concentration of phosphorylated f ZAP-70 .The increase in potency  not the more commonly performed assay that involves titrating the number of target cells (with a fixed antigen concentration). Optimal CARs should exhibit low potency ,The profiles of ITAM-bound ZAP-70 and total TCR Figure S1Alignment of the TCR (EPS)Click here for additional data file.Figure S2Ultrasensitivity in the absence of ZAP-70 can be mediated by differential affinities of Lck, CD45, or both. A) The total phosphorylation of TCR (EPS)Click here for additional data file.Figure S3An alternate measure of sensitivity for the heat map in (EPS)Click here for additional data file.Text S1BioNetGen source code used to generate the wild-type systems model of T cell receptor proximal signaling.(PDF)Click here for additional data file.


Smooth-pursuit eye movements allow primates to track moving objects. Efficient pursuit requires appropriate target selection and predictive compensation for inherent processing delays. Prediction depends on expectation of future object motion, storage of motion information and use of extra-retinal mechanisms in addition to visual feedback. We present behavioral evidence of how cognitive processes are involved in predictive pursuit in normal humans and then describe neuronal responses in monkeys and behavioral responses in patients using a new technique to test these cognitive controls. The new technique examines the neural substrate of working memory and movement preparation for predictive pursuit by using a memory-based task in macaque monkeys trained to pursue (go) or not pursue (no-go) according to a go/no-go cue, in a direction based on memory of a previously presented visual motion display. Single-unit task-related neuronal activity was examined in medial superior temporal cortex (MST), supplementary eye fields (SEF), caudal frontal eye fields (FEF), cerebellar dorsal vermis lobules VI–VII, caudal fastigial nuclei (cFN), and floccular region. Neuronal activity reflecting working memory of visual motion direction and go/no-go selection was found predominantly in SEF, cerebellar dorsal vermis and cFN, whereas movement preparation related signals were found predominantly in caudal FEF and the same cerebellar areas. Chemical inactivation produced effects consistent with differences in signals represented in each area. When applied to patients with Parkinson's disease (PD), the task revealed deficits in movement preparation but not working memory. In contrast, patients with frontal cortical or cerebellar dysfunction had high error rates, suggesting impaired working memory. We show how neuronal activity may be explained by models of retinal and extra-retinal interaction in target selection and predictive control and thus aid understanding of underlying pathophysiology. The simplest way to assess pursuit performance is to examine the response to the sudden onset of an unexpected, constant velocity target motion (a ramp stimulus). Figure Following initial response onset eye velocity frequently overshoots target velocity and may oscillate at a frequency of 3–4 Hz in humans Figure  Robinso. OscillaOcular pursuit is an example of a negative feedback control system and if it were linear, the response evoked by termination of a ramp stimulus should be the inverse of that at initiation; eye velocity should thus oscillate when reaching zero velocity . However, when target motion ceases unexpectedly, following a latency of ~100 ms, eye velocity generally decays to zero with a time constant of ~100 ms  at the beginning of the ramp and was then extinguished for a period (ED) up to 600 ms. Crucially, the direction, speed, initial presentation duration and time of initiation of the ramp were randomized with the objective of determining whether subjects were able to extract motion information within the brief presentation so as to scale their smooth eye velocity to target velocity during occlusion. Various sources of behavioral  they must select which particular moving object to pursue. One way to accomplish this would be to enhance the visual feedback of the selected object in relation to other stimuli by increasing the open-loop gain  can have a passive influence on smooth pursuit . This predictive pathway enables motion information to be retained during fixation and thereby allows appropriately scaled anticipatory movements to be released in advance of future eye movement, given appropriate expectation of target appearance. The results of numerous experiments . This includes a mechanism for detecting mismatch between the predictive velocity and available visual feedback. This reflects the fact that, in anticipatory mode, the system has changed from being one that relies on visual feedback to one that generates a predictive estimate of the required motor drive and uses feedback to check that this is correct. Importantly, it would not be possible for the reactive and predictive pathways to operate simultaneously since this would overestimate target velocity, so it must be assumed that activation of the predictive pathway automatically leads to inhibition of the reactive pathway. This model has been used to provide a very effective simulation of responses in the Mid-ramp, Short ramp, Initial Extinction and Control conditions  or interstitial nucleus of Cajal (INC), to extraocular motoneurons Figure . These iSmooth-pursuit is required even when our head and/or whole body moves  or not to pursue . Based on the memory of visual motion-direction presented at cue 1 , and these saccades were followed by smooth-pursuit. The lack of an initial smooth-pursuit component before saccades  was consistent with the finding that vector averaging was used to combine visual inputs arising from two moving spots , saccade latency to spot motion shortened usually to about 220 ms, and preceding the saccades, initial smooth-pursuit appeared with latencies typically of 130–150 ms Figure , arrow. Using the memory-based smooth-pursuit task, signals for directional visual motion-memory and movement-preparation have been identified in the SEF and caudal FEF. Three groups of neurons were found; two of them carried these signals separately  and the third carried both signals . Although the two regions carried qualitatively similar signals, consistent with the anatomical studies that show reciprocal connections between the SEF and FEF . The delay 1 discharge was not significantly influenced by the monkey's preparation of pursuit (B1 vs. C1). This was also seen when the monkey erred . Moreover, it did not exhibit directional responses during delay 2 of go  or no-go trials . These results suggest that the delay 1 activity of visual memory neurons reflected memory of the visual motion-direction presented by cue 1. Although it exhibited a build-up activity during go trials . Since this neuron was unrelated to pursuit  must have reflected visual response to rightward spot motion. Notice selective burst discharge to the identical visual motion stimuli during the action period, i.e., the clear burst during the action period appeared only in Figure B2, C1,2), indicating that the spot motion responses clearly depended on the visual motion-direction memory and go/no-go instructions. This interpretation is confirmed in Figure Possible neural correlates for the putative priming effects by cues during the action period . In addition, when the cue 2 instructed go to prepare to pursue in the congruent direction (A1), it exhibited robust discharge during the late period of delay 2. Figure go trials in their preferred directions. While the initial response to cue 1 for visual memory neurons  was larger, the two groups of neurons displayed similar discharge during the delay 1 and cue 2. During delay 2, the discharge of the two groups of neurons diverged. Figures  showed cgo trials .Visual memory + movement-preparation neurons exhibited congruent directionality during delay 1 and delay 2 of  Figures ,B, blue. Figures . This inC1), rightward pursuit (C2) and no-go (C3) of the same neuron (A). When the monkey made leftward pursuit , indicating that the delay 2 activity indeed reflected preparation for pursuit. In addition, the stronger discharge during the delay 1 in the same trials (C1 vs. C2) suggests that this discharge during delay 1 was also related to the monkey's choice and preparation for the subsequent pursuit direction independent of the cue 1 stimulus itself, which was non-directional.Figure Second, to evaluate these results, we calculated choice probability  and MST and sometimes even as early as some neurons in V1  was significantly higher in the SEF than caudal FEF . When the cue 2 instruction was no-go , this no-go neuron nearly stopped discharging at cue 2 and during delay 2, suggesting that the discharge during these periods reflected the monkey's decision not to pursue during go trials. This interpretation was supported by the analysis of choice probability  or if they pursued a moving spot, regardless of its directions , despite that the monkeys fixated a stationary spot during these periods  exhibited significant modulation during delay 1 or 2 of go or no-go trials, suggesting that the floccular pathway is specifically involved in executing smooth-pursuit eye movements per se as reported earlier  in the oculomotor vermis showed In our task, some task-related Purkinje cells (10/76) in the oculomotor vermis were pursuit-related during memory-based pursuit. Discharge characteristics of these neurons during pursuit using a single spot were similar to those reported previously , similar to no-go neurons in the SEF and caudal FEF, despite that the monkeys fixated a stationary spot during these periods , the major response type (46/77 = 60%) was also s Figure .no-go neurons in the oculomotor vermis/cFN signal? We believe that no-go neurons in these regions are non-motor neurons that receive inputs from SEF/caudal FEF no-go neurons  memory during delay 2 for the following reasons. (1) Discharge characteristics of no-go neurons in all these areas were basically similar  than those of SEF/caudal FEF no-go neurons , but choice probability quickly decreased during the action period. (4) During the action period of no-go trials, the monkeys occasionally made small saccades with amplitudes 1–6°  and part of cerebellar involvement in memory  are consistent with the differences in the effects of chemical inactivation  are difficulties in initiating volitional movements and, when initiated, slow and hypo-metric movement  followed by corrective saccades , and postsaccadic enhancement of smooth-pursuit was rarely observed. Moreover, consistent with many previous reports, peak pursuit eye velocities after saccades were significantly lower  in PD patients than those of controls during pursuit maintenance , and since postsaccadic enhancement of smooth-pursuit was also seen at least for the first saccades after spot motion .The lack of initial pursuit and deficient postsaccadic enhancement in most PD patients are unlikely to be due to impairments of smooth-pursuit eye movements  Figures  vs. B2*go instruction enhance visual motion responses of SEF and caudal FEF neurons in the cued direction  which required the patients to prepare and execute smooth-pursuit to a selected spot using the cue information , suggesting that both the cerebellar floccular region and oculomotor vermis were dysfunctional. In addition, during memory-based pursuit, most cerebellar patients tested exhibited direction errors during the action period  in the indirect pathway and concomitantly initiating extra-retinal activity in the efference copy loop . Raising gain in the indirect pathway (wT1) is the primary factor responsible for the initial high acceleration pursuit response, the extra-retinal component giving a lower level of eye acceleration and developing later than the visually driven component  in the appropriate channel but does not allow open-loop gain (wT1) to be immediately increased, thus leading to a low initial acceleration. Prior to initiation of the extra-retinal component weightings wD1 and wD2 are assumed to be equal and thus to cancel each other as a result of vector averaging  signals are summated (junction B in Figure FEF is probably the site at which retinal error and internal drive (either The control of open-loop gain is another function frequently associated with FEF. Tanaka and Lisberger  showed tObservation of reduced pursuit performance is common in patients with various neurological conditions, such as cerebral cortical lesions, cerebellar degeneration, PD, and schizophrenia (Leigh and Zee, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NAs) are used to treat numerous viral infections and cancer. They compete with endogenous nucleotides (dNTP/NTP) for incorporation into nascent DNA/RNA and inhibit replication by preventing subsequent primer extension. To date, an integrated mathematical model that could allow the analysis of their mechanism of action, of the various resistance mechanisms, and their effect on viral fitness is still lacking. We present the first mechanistic mathematical model of polymerase inhibition by NAs that takes into account the reversibility of polymerase inhibition. Analytical solutions for the model point out the cellular- and kinetic aspects of inhibition. Our model correctly predicts for HIV-1 that resistance against nucleoside analog reverse transcriptase inhibitors (NRTIs) can be conferred by decreasing their incorporation rate, increasing their excision rate, or decreasing their affinity for the polymerase enzyme. For all analyzed NRTIs and their combinations, model-predicted macroscopic parameters  were consistent with observations. NRTI efficacy was found to greatly vary between distinct target cells. Surprisingly, target cells with low dNTP/NTP levels may not confer hyper-susceptibility to inhibition, whereas cells with high dNTP/NTP contents are likely to confer natural resistance. Our model also allows quantification of the selective advantage of mutations by integrating their effects on viral fitness and drug susceptibility. For zidovudine triphosphate (AZT-TP), we predict that this selective advantage, as well as the minimal concentration required to select thymidine-associated mutations (TAMs) are highly cell-dependent. The developed model allows studying various resistance mechanisms, inherent fitness effects, selection forces and epistasis based on microscopic kinetic data. It can readily be embedded in extended models of the complete HIV-1 reverse transcription process, or analogous processes in other viruses and help to guide drug development and improve our understanding of the mechanisms of resistance development during treatment.Nucleoside analogs ( NAs) represent an important drug class for the treatment of viral infections and cancer. They inhibit DNA/RNA polymerization after being incorporated into nascent DNA/RNA, which prevents primer extension. Viruses are particularly versatile and frequently develop mutations enabling them to avert the effects of NAs. The mechanisms of resistance development are, however, still poorly understood. Through mathematical modeling, we assess the mechanisms by which HIV-1 can develop resistance against nucleoside analog reverse transcriptase inhibitors (NRTI). We quantify the effects of treatment and estimate the fitness of drug resistant mutants. We correctly predict that HIV-1 can develop resistance by decreasing NRTI incorporation rate, increasing its excision rate, or decreasing its affinity for the viral polymerase enzyme. Our model also allows quantification of the cell specific factors affecting NRTI efficacy. Resistance development also changes drug susceptibility distinctly and we show, for the first time, that selection of drug resistance can occur in particular target cells. This finding could provide an explanation of how clinically observed resistant viral mutants may arise. It also pin-points important parameters that may impact clinical efficacy of NAs used to treat other viruses.Nucleoside analogs ( We propose that NRTIs delay the process of DNA polymerization, rather than permanently terminating it, simultaneously keeping in mind that any delay of the process decreases the number of viral progeny and the likelihood of target cell infection by the virus. The developed mathematical formulation allows us to study viral polymerase inhibition by NRTIs as well as fitness effects related to drug resistance development. By integrating fitness effects and drug susceptibility, it is further possible to quantify the selective pressure exerted by NRTIs and to study epistasis. The derived analytical expressions can be used to study the effects of single- and multiple NRTIs on DNA polymerization in the absence and presence of resistance mutations and can be useful for drug design. Chain termination by A schematic view of the process of viral DNA polymerization in the presence of   states , where eAt each state Taking into account the mode of action of chain terminating nucleoside analogs, we conclude that polymerization will be decelerated in the presence of these inhibitors, because the overall time required to go from state ‘0’ (initiation of polymerization) to state Analogously, we can define the effect of chain terminating nucleoside analogs on some viral mutant, Based on the definitions above, we can also assess the combined effects of selection and drug pressure for any viral strain, i.e. Finally, we can assess epistatic interactions for combinations of mutations with regard to viral replication. Briefly, in a two-locus-two-allele model, epistasis is positive if some double mutant m12 replicates better than expected from the single mutants m1 and m2, normalized by the replication of the wild type wt (background). Epistasis is negative if the replication of the double mutant is less than expected from the single mutants. Along the same lines, epistasis has been used to study interactions of mutations in the absence of drugs mean first passage timeThe process of DNA polymerization  defines see e.g. . Eq. (8)In the case where no chain-terminating inhibitor is applied, we have Eq. (8)–(10) can subsequently be used to estimate the residual polymerase activity in the presence of Using eq. (10), it is possible to compute the average polymerization time –(11). In a homo-polymeric sequence, we have mean first passage timeresidual polymerase activity during homo-polymeric sequence, While in a hetero-polymeric sequence context, polymerase inhibition by Eq. (19) highlights the two distinct mechanisms by which inhibition can be conferred, namely a) inhibitor incorporation (and subsequent quasi-termination of the polymerization reaction) and b) competition for binding with natural nucleoside substrates. The efficacy of quasi-termination of the nascent DNA chain depends on the efficacy of inhibitor incorporation After substituting the enzymatic rate expressions eqs. (12)–(14) into equation (19), we can solve for the fifty percent inhibitory concentration Viruses can infect numerous activated- and resting cells. HIV-1, for example, has been shown to infect activated- and resting In the context of nucleoside analog efficacy, the major cell-specific factors (apart from pharmacokinetics), are cell type-, or cell stage specific dNTP pools see  and possUnder the parameters used, a 100 fold increase in dNTP concentrations would result in a 19 fold increase in the Resting cells on the other hand might insufficiently phosphorylate NRTIs and subsequently contain lower levels of activated compound. However, these cells do not simultaneously require smaller NRTI concentrations for inhibition (Excision of nucleoside reverse transcriptase inhibitors (NRTIs) of HIV-1 from terminated primers has been shown to be mediated by pyrophosphate (PPi) and ATP dependent mechanisms The enzymatic properties of a viral polymerase can be adapted in an evolutionary process to counteract inhibition by selective attrition of inhibitor incorporation , and is only marginally affected by lower dNTP levels in resting cells, as decreasing dNTP levels may not induce hyper-susceptibility as shown in in vivo.In in vivo mechanism of AZT resistance Excision of AZT-MP from the terminated primer is the major mechanism by which AZT resistance is thought to be mediated Selection of drug resistance depends on the competitive advantage of some resistant mutant over its competitors  in a particular environment. In order to quantify whether drug resistant mutants become selected in an environment that is modified by In Two major findings can be derived from Secondly, and quite surprisingly, in vivo concentrations ranges of TFV-DP (light grey area) the selective pressure towards the Q151M and the Q151Mc strains is relatively weak The selective advantages of intermediate viral strains of the Q151M-complex (multi-drug) resistance pathway  and Q151Mc/K70Q ) with respect to increasing tenofovir diphosphate (TFV-DP) concentrations are shown in Pol gene). It is therefore likely, that the combination of mutations produce a phenotype that has unexpected/novel properties. The intention of this analysis is to elucidate how epistasis depends on the environment in which the virus replicates  markedly (8 fold) and had only marginal impact on tenofovir diphosphate (TFV-DP), whereas lamivudine triphosphate (3TC-TP) and emtricitabine triphosphate (FTC-TP) were unaffected .We subsequently defined a therapeutic index as the ratio of the mean inhibition of We presented a novel mechanistic mathematical model of HIV-1 polymerase inhibition by in vitro assays. These parameters can usually be precisely determined with standard errors The developed model can be parameterized in terms of physiological parameters (such as dNTP concentrations) and microscopic kinetic rates (e.g. Based on the developed model of polymerization and its inhibition by Eq. (20) revealed that the rate of Various reports indicate cell-specific differences in V-1  see . For AZTV-1  see . In summWe analyzed the specific mechanisms of AZT resistance through TAMs. It is well known, that TAMs induce resistance through increasing the excision of incorporated ment see , becauseWe quantified the inhibitory effects of AZT during RNA- and DNA dependent polymerization and we analyzed how TAMs (‘D67N/K70R/T215Y/K219Q’) induce susceptibility changes. We found that AZT inhibition during HIV-1 reverse transcription is more efficient during RNA-dependent polymerization than during DNA-dependent polymerization, see in vivo. The developed model can be used to facilitate such an analysis: In While the emergence of a particular viral strains depends on a) the probability that the mutant is generated , it also critically depends on the likelihood that the generated mutant becomes selected subsequently. However, if inhibition- and selection forces are different in distinct target cells see , then thages see . We founages see  at clini“Selection of Resistance”.Epistasis has been suggested as a method to study evolutionary dynamics of virus populations Cell type specific susceptibility to AZT and impact of resistance and in in vivo maximally achievable AZT-TP concentration is close to the clinically achieved AZT-TP concentration in peripheral blood mononuclear cells (PBMCs), when 300 mg AZT is given twice daily, see Based on the developed model, we predicted that the ‘D67N/K70R/T215Y/K219Q’ mutation induces a 4.1 to 22.6 fold increase in the ells see  might noqualitatively in good agreement with results from cell culture assays  in comparison to RNA template may be present in vivo. The cooperativity of multiple RT enzymes can also explain the distinct shape of the dose-response curve observed in primary human cells with inhibitors that directly target the enzyme, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs), in contrast to inhibitors that target the RNA/DNA template (NRTIs) Recent mean first passage time (MFPT) to go from state ‘0’ (initiation of polymerization) to the state In this section we will derive the analytical solution for the polymerization time given in eq. (10), which is based on ideas given in Starting point for the derivation are the MFPT-equations (Equation (27) is satisfied byIn case where the sequence to be polymerized is homo-polymeric, e.g. ‘Poly-A’, all rates are uniform, i.e., Starting point for calculating the fifty percent inhibitory concentration (for polymerization of uniform sequences) is equation (19). We setTable S1Pre-steady state kinetic constants for nucleoside incorporation by wild type HIV-1 reverse transcriptase. Indicated parameters are average values from the respective literature sources.(PDF)Click here for additional data file.Table S2Fold change of kinetic parameters for DNA-dependent polymerization in various HIV-1 reverse transcriptase mutants, relative to wildtype RT.(PDF)Click here for additional data file.Table S3Pre-steady state kinetic constants for AZT excision by HIV-1 reverse transcriptase wildtype and ‘D67N/K70R/T215Y/K219Q’ mutant.(PDF)Click here for additional data file.Table S4Pre-steady state kinetic constants for nucleoside incorporation by human mitochondrial polymerase-. (PDF)Click here for additional data file.Text S1The supplementary text contains the modelling required to compute the probability to successfully complete reverse transcription (RT) in HIV-1, based on the parameters presented in the main manuscript.(PDF)Click here for additional data file.