Besides their role in diagnosis of acute myocardial infarction (MI), troponins may be powerful biomarkers for risk stratification in the general population. The objective of our study was to compare the performance of three troponin assays in cardiovascular disease (CVD) risk prediction in a population-based cohort without a history of CVD events.Troponin I concentrations were measured using a contemporary-sensitivity, high-sensitivity, and super-sensitivity assay in 7,899 participants of the general-population based FINRISK 1997 cohort. We used Cox proportional hazards regression to determine relative risks, followed by measures of discrimination and reclassification using 10-fold cross-validation to control for over-optimism.As outcome measures we used CVD, MI, ischemic stroke, heart failure (HF), and major adverse cardiac events (MACE). During the follow-up of 14 years 1,074 incident MACE were observed.Values above the lower limit of detection were observed in 26.4%, 81.5% and 93.9% for the contemporary-sensitivity, high-sensitivity and super-sensitivity assay, respectively. We observed significant associations of troponin concentrations with the risk of future CVD events and the results tended to become stronger with increasing assay sensitivity. For the super-sensitivity assay the multivariate adjusted hazard ratios (per one standard deviation increase) for different outcomes were: MI 1.24 [95% CI 1.11–1.39], stroke 1.14 [1.01–1.28], CVD 1.15 [1.07–1.24], HF 1.28 [1.18–1.39], and MACE 1.18 [1.11–1.25]. In subjects with intermediate risk, we found an improvement of net reclassification for HF , and MACE .Using a super-sensitivity assay, cardiac troponin was detectable in almost all healthy individuals. Its concentration improved risk prediction and reclassification for cardiovascular endpoints. The cardiac-specific protein complex troponin is released in conditions of myocardial damage, and therefore its use as a necrosis marker for the diagnosis of acute myocardial infarction (MI) is well established Identifying individuals with increased risk for incident cardiovascular diseases (CVD) is a major aim in primary prevention The use of more sensitive troponin assays has revealed a high prognostic potential of low troponin concentrations, but their clinical value in risk prediction has not been established and may depend on the sensitivity of the assay in question. Therefore, the objective of our study was to examine the hypothesis that a more sensitive assay detects more individuals at risk of future cardiovascular disease in a population-based cohort without a history of prior MACE. To address this issue, we measured troponin concentrations using a contemporary-sensitivity, high-sensitivity, and super-sensitivity troponin assay in 7,899 participants of the FINRISK 1997 cohort followed up for 14 years.The present study included 8,444 individuals from the FINRISK study enrolled in 1997. This prospective population-based study was carried out in five districts of Finland, including North Karelia, Northern Savo (former Kuopio), Southwestern Finland, Oulu province, and the region of Helsinki and Vantaa. A stratified, random sample was drawn from the national population register, the age-range was 25–74 years. All individuals enrolled in the study received a physical examination, a self-administered questionnaire, and a blood sample was drawn. Altogether, 11,500 individuals were invited and 8,444 (73%) participated in the clinical examination. Individuals with a prevalent history of MACE (n = 470) and pregnant women (n = 76) were excluded from the present analysis. During a follow-up of up to 14 years, the National Hospital Discharge Register, the National Causes of Death Register and the National Drug Reimbursement Register were used to identify the endpoints The blood pressure measurement was performed on the right arm in a sitting position after a 5-minute phase of rest. The cuff length was 40 cm. The mean of two measurements was used in the analyses. Data on the use of antihypertensive medications were collected with the questionnaire. Arterial hypertension was defined according to the American Heart Association (AHA) definition, meaning a blood pressure ≥140 mmHg systolic or ≥90 mmHg diastolic, or the use of antihypertensive medication. Smokers were classified by questionnaire as active smokers (smoking regularly ≥1 year and during the past 6 months), former smokers (smoked regularly ≥1 year and quit smoking ≥6 months before the survey), and non-smokers (never smoked regularly). Diabetes was defined either as previously diagnosed diabetes or impaired glucose tolerance by a physician, or by taking any hypoglycemic drugs.st, 2010) were lost to follow-up. The study endpoints were defined as follows. CVD included MI, coronary death, hospitalized unstable angina pectoris, any coronary revascularization, and ischemic stroke. Further endpoints were incident ischemic stroke (hemorrhagic strokes were excluded), incident HF, and MACE (CVD or HF). In a sub-analysis we further distinguished fatal MI from non-fatal MI. The use of Finnish national health care registries for identifying these cardiovascular outcomes has been validated The follow-up rate was 100% for the participants who continued living in Finland. Those who had permanently moved abroad , and the different troponin assays were performed at the MORGAM Biomarker Laboratory, University Heart Center Hamburg, Germany, which was formerly located at the University Medical Center Mainz, Germany. The contemporary-sensitivity troponin I assay  was considered valid for values above the limit of detection (LOD) of 10 pg/mL, but observed values below this limit were also used in the analysis . The 10% coefficient of variation was at 32 pg/mL. Troponin was also assessed using a prototype high-sensitivity cardiac troponin assay . The established LOD for the assay ranges from 0.8–1.9 with a median of 1.5 pg/mL. For analyses, a LOD of 1.9 pg/mL was considered. Observed values below this limit were also included in the analysis . The 10 percent coefficient of variation was at 5.2 pg/mL. The concentration representing the 99Baseline characteristics are presented as counts and percentages for dichotomous variables, and as median and IQR for continuous variables. Age and sex adjusted Kaplan-Meier-curves for MACE and HF were produced using categorized troponin concentrations. For the contemporary-sensitivity assay, we used 1 pg/mL  and 10 pg/mL (assay threshold) as cut-points. For the high-sensitivity assay, we used 1.9 pg/mL (assay threshold), and 5.1 pg/mL (same percentile as 10 pg/mL for the contemporary assay) as cut-points. We proceeded the same way for the super-sensitivity assay, using cut-points of 1.0 pg/mL (median limit of detection) and 5.1 pg/mL (same percentile as 10 pg/mL for the contemporary assay). To describe the association of troponin with clinical endpoints, Cox regression models adjusting for the variables of the Framingham risk score  and region of Finland  were computed. We used the Framingham equation meant for estimating the 10-year risk of cardiovascular event in primary care The FINRISK 1997 study enrolled 7,899 individuals (50.3% women and 49.7% men) after exclusion of those with prevalent MACE and pregnant women Table 1. and Net reclassification for the different events did not improve in the overall analysis Table 4.The use of more sensitive troponin assays has revealed a high potential for diagnosis of acute cardiac events, but also for identifying individuals at risk of a future cardiovascular event. This study compared the predictive power of baseline troponin concentrations in a population-based setting using three troponin assays of different sensitivity, ranging from a contemporary-sensitivity and high-sensitivity to a super-sensitivity assay, in individuals without a history of MACE. We observed a generally stronger association of troponin concentrations with MACE, CVD, MI, HF, and stroke, when the super-sensitivity assay was used compared to the high-sensitivity and contemporary-sensitivity assays. The associations remained significant after adjustment for the classical risk factors included in the Framingham equation, and additional adjustment for CRP and NT-proBNP for the endpoints MACE, CVD, MI, and HF. Net reclassification improvement was not significant among all subjects. In subjects with intermediate risk, however, reclassification was significantly improved for various endpoints. The strongest clinical reclassification improvement was found with the super-sensitivity troponin assay for MACE and HF.th percentile according to gender with mostly higher concentrations in males. In our study median troponin concentrations measured by either the high-sensitivity or the super-sensitivity assay were higher in males. The measured HRs however did not differ significantly. Nevertheless gender-specific analysis should be considered for the interpretation of absolute troponin concentrations. Furthermore, recent results from the Scottish Heart Health Extended Cohort study suggested that the optimal (in terms of sensitivity and specificity) cutpoints of high-sensitivity troponin I differed between men and women Previous studies have shown that troponin predicts cardiovascular events in the general population A particular strength of the present study is the comparison of three troponin assays with different sensitivities in risk prediction. However, various limitations merit consideration. First, measurements have been performed in frozen samples that were stored for 14 years at −70°C and had been thawed twice. Second – and most important – we do not have sufficient data at the moment to fully understand the clinical consequences of reclassification in those individuals reclassified from the intermediate risk group to the high-risk group. A recent report suggested medical intervention with statin therapy after such reclassification obtained using measurements of fibrinogen or CRP Using a super-sensitivity assay, troponin I was detectable in almost all healthy individuals in this population-based setting. In terms of relative risk, troponin I was a significant predictor for future CVD events over and above the standard Framingham equation and remained significant even after further adjustment for CRP and NT-proBNP. In subjects at intermediate risk of CVD, the super-sensitivity troponin assay significantly improved reclassification for HF and MACE outcomes. Although both, the super-sensitivity and the high-sensitivity assays are predictors for incident CVD, application of the super-sensitivity assay might hold a slightly higher potential to reclassify the outcome.File S1File containing Tables S1–S3. Table S1: Hazard Ratios from Cox Regression Models for Baseline Troponin Assessed by Contemporary-Sensitivity, High-Sensitivity and Super-Sensitivity Assays for Various Endpoints by Gender after Adjustment for the Framingham Risk Score. Table S2: Hazard Ratios from Cox Regression Models for Baseline Troponin Assessed by Contemporary-Sensitivity, High-Sensitivity and Super-Sensitivity Assays for Various Endpoints, after Adjustment for Framingham Risk Score, CRP and NT-proBNP. Table S3: C-Statistics and Integrated Discrimination Improvement for Baseline Troponin Assessed by Contemporary-Sensitivity, High-Sensitivity and Super-Sensitivity Assays for Various Endpoints after Adjustment for the Framingham Risk Score.(DOC)Click here for additional data file.


Neoplastic dissemination to the leptomeninges is an increasingly common occurrence in patients with both haematological and solid tumors arising outside the central nervous system. Both refinement of diagnostic techniques (Magnetic resonance imaging) and increased survival in patients treated with targeted therapies for systemic tumors account for this increased frequency. Cerebrospinal fluid cytological analysis and MRI confirm clinical diagnosis based on multifocal central nervous system signs/symptoms in a patient with known malignancy. Overall survival in patients with leptomeningeal neoplastic dissemination from solid tumors is short, rarely exceeding 3-4 months. However, selected patients may benefit from aggressive therapies, Apart from symptomatic treatment, intrathecal chemotherapy is used, with both free  and liposomal antitumor agents . Palliative radiotherapy is indicated only in cases of symptomatic bulky disease, surgery is limited to positioning of Ommaya recervoirs or C5F shunting. We report clinical data on a cohort of 26 prospectively followed patients with neoplastic leptomeningitis followed in Lombardia, Italy, in 2011. Prognostic factors and pattern of care are reported. Neoplastic meningitis is due to dissemination of malignant cells to the leptomeninges and the subarachnoid space. It occurs in 10–15% of haemolymphoproliferative malignancies and in 5–10% of solid cancers [It more frequently represents late complication of long-standing neoplastic disease, but in 10–15% of patients may be the first-ever manifestation of otherwise occult cancer [The pathways for tumor dissemination to the leptomeninges and subarachnoid space include haematogenous route, perineural blood/lymphatic vessels, and direct infiltration from contiguous sites .Not only extra-CNS tumors, but also tumors arising within the CNS  display relapses and/or multifocal presentations with distant foci and a supposedly intra-CSF pathway of dissemination of neoplastic cells.Guidelines for effective treatment of neoplastic meningitis are lacking, due to the low levels of evidence, which is mostly present for haemolymphoproliferative disease.In meningeal dissemination from solid extra-CNS tumors, and more so in distant spread of primitive CNS tumors, there is a lack of uniform approach due to a number of factors: among these, the belief of oncologists that neoplastic meningitis invariably implies a dismal prognosis in the short-term has limited patient recruitment in clinical trials.Although this assumption holds true in a high number of cases, it does not apply to the totality of patients, however.This consideration, together with the more widespread availability of MRI facilities in neurooncological diagnosis and with the progress in survival in extra-CNS cancers achieved by chemotherapy and molecularly targeted therapies , increasThe clinical signs and symptoms of neoplastic meningitis are classically subdivided in those pointing to cerebral, cranial nerve, or spinal cord/roots involvement. Typically, in a high proportion of patients symptoms are present suggesting simultaneous involvement of both cerebral and spinal levels, but some patients present with isolated deficits .Cerebral signs and symptoms may either be localized  or suggestive of a widespread brain dysfunction , or be even more unspecific, such as headache.The literature reports that the presence of signs at the neurological examination is more frequent as compared to the reporting of symptoms by the patients during history collection.Neoplastic meningitis not infrequently coexists with intraparenchymal or dural metastases, especially in the case of breast cancer and leukemia/lymphoma.The diagnosis of neoplastic meningitis is straightforward in the majority of cases, but a number of cases may pose diagnostic challenges.This happens more frequently when the gold standard for diagnosis  does not yield unequivocally positive results. This may be the case—according to the literature—in a proportion of patients ranging from 20 to 50–60%; reasons for this include too little volume of CSF analyzed, distance of the CSF sampling site from the bulk of leptomeningeal disease, and delay in CSF processing and analysis , 4. The In such cases, CSF analysis may yield negative results for malignant cells, yet display other abnormal features , such as increase in total proteins and reduced glucose levels, as well as moderate reactive pleocytosis.Such CSF pattern may pose serious difficulties in differential diagnosis with CNS infections, which may mimic the neuroradiological picture of NM and are not unexpected in heavily treated cancer patients .Some reports have stressed that the closer the CSF sampling to the site of disease, the higher the percentage of positivity for CSf malignant cells; ventricular CSF or lumbar CSF may thus provide different information as far as cytology is concerned.In exceptional cases, leptomeningeal biopsy is deemed necessary.In neoplastic meningitis from heamatological malignancies, CSF cytofluorimeter analysis has been reported to be more often diagnostic as compared to standard cytomorphological analysis , 7.As far as the role of MRI is concerned, the features of leptomeningeal dissemination include both indirect and direct evidence of neoplastic cell CSF seeding. Among the former, hydrocephalus is not rare, due mostly to alterations in the CSF flow and particularly in CSF reabsorption at the skull vault. Direct evidence of neoplastic dissemination includes linear or nodular enhancement at leptomeningeal/ependymal level.More subtle signs of alterations in the CSF dynamics include exclusion of part of cerebral sulci, with limited volumes with increased protein content.The role of surgery is limited to resection of symptomatic, bulky disease, and/or biopsy in order to achieve diagnosis in selected cases; in some patients, positioning of an Ommaya recervoir may allow intraventricular chemotherapy without the need for repeated lumbar punctures, but the dynamics of CSF flow need to be carefully assessed in order to possibly achieve tumoricidal drug concentrations in the sites of disease. Ventriculoperitoneal shunting procedures to relieve symptomatic hydrocephalus carry a risk for the development of neoplastic dissemination to the peritoneum and are often complicated by shunt dysfunction/occlusion.Intrathecal chemotherapy should preferably be delivered in patients with good PS (see below), with limited extra-CNS disease and with linear contrast enhancement at MRI (the penetration of drugs within bulky disease areas is limited to 2-3 mm).The NCCN 2012 Guidelines for diagnosis and management of CNS tumors include brain and spine MRI as well as CSF examination in the workup of patients with suspected leptomeningeal tumor dissemination. According to these guidelines, either positivity of CSF cytology alone or positive radiologic findings with supportive clinical findings or else signs and symptoms with suggestive CSF in a patient known to have a malignancy may be sufficient for diagnosis.After diagnosis, patients are stratified in either poor risk , or else good risk .In the former group, only fractionated external beam RT is considered to symptomatic sites, and palliative care is the standard. An exception is possible in patients with highly chemosensitive tumors such as lymphoma and SCLC.On the other hand, in good risk patients both radiotherapy to bulky disease or symptomatic sites may be delivered and intrathecal chemotherapy is a worthwhile option.Of note, assessment of CSF flow is strongly recommended before initiating intrathecal chemotherapy. This assessment is more frequently performed in northern America, while it is less a frequent practice in Europe.With normal CSF flow, either craniospinal irradiation—in the case of breast cancer or lymphoma—or high dose methotrexate i.v in the case of breast cancer or lymphoma or intrathecal chemotherapy with methotrexate or AraC or liposomal AraC are the treatment of choice.Unless an Ommaya recervoir is positioned by the neurosurgeon, repeated intrathecal administration of antineoplastic drugs is usually performed via lumbar punctures. With methotrexate, twice weekly administrations are performed during the induction phase, due to the short half life of the drug in the CSF.μG/mL persisting at 14 days.Analogous schedules are needed with nonliposomal cytarabine, whereas a pegylated formulation of cytarabine allows sustained tumoricidal concentrations in the CSF which make once every 2 weeks treatment possible. The development of cytarabine encapsulated in multivesicular liposomes has led to detection of CSF concentrations of more than 0.1 In this technology, microscopic particles made of aqueous chambers separated from each other by bilayer lipid membranes , deliver gradually the incorporated drug, with subsequent metabolization of the membrane remnants via normal pathways. Cytarabine, a highly hydrophyilic compound, is an ideal molecule for this approach .The achievement of tumoricidal concentrations of cytarabine in the CSF is of crucial importance since cytarabine is a phase-specific drug affecting only cells in the S phase. In the CSF, very little activity of the inactivating enzyme cytidine deaminase enables cytarabine to persist in its biologically active form for longer time as compared to systemic delivery .Only few randomized trials have been conducted on the effectiveness and toxicity of intrathecal chemotherapy in neoplastic meningitis (reviewed in ).In the 1999 published trial by Glantz et al. on neoplastic meningitis from solid tumors , intrathAlso the 2006 trial by Shapiro and colleagues provides data pointing to a nonsignificantly different effect of liposomal AraC versus methotrexate in 103 patients with neoplastic meningitis froms solid tumors .In the other 1999 paper by Glantz et al. , liposomOther nonrandomized studies have been performed , 15 inveThe main reason for continuing use of liposomal AraC in these patients—apart from the lack of a consolidated and effective standard of care—is the need for less frequent lumbar punctures in often severely ill patients. However, the levels of evidence in favour of this approach are weak. A recent determination of EMA has temporarily suggested to consider alternative therapies to liposomal AraC after an inspection to the production site of the drug in California; treating physicians are waiting for a solution of this possibily temporary problem. Other widely adopted intrathecal treatments apart from liposomal AraC include methotrexate and thiotepa.Preliminary experiences show the feasibility of associating rituximab with liposomal cytarabine in patients with recurrent neoplastic meningitis . Also sySome effect has been reported for systemic treatment with systemic gefitinib or erlotinib in NSCLC with neoplastic meningitis, and with sorafenib in renal cancer, whereas the role of trastuzumab in breast cancer with neoplastic meningitis is still debatable (reviewed in ).In 2011 a prospective collection of patients diagnosed with neoplastic meningitis from solid tumors was started in a number of Centers in Lombardia. The aim of this study is to assess the pattern of care in this often underdiagnosed and undertreated condition. Previous work from an analogous initiative in Piedmont  supportsIn 12 months, 26 patients with neoplastic meningitis from solid extra-CNS tumors have been diagnosed. Their clinical features are reported in Tables Cerebrospinal fluid analysis was performed in 22 out of 26 patients, yielding the following results: in 18/22 patients, CSF analysis revealed malignant cells. Mean values of CSF total protein were 152 mg% , whereas mean CSF glucose was 51.5 mg/dL . Lower than normal glucose levels were only seen in 3 patients out of 22.As reported in In this cohort, no patient was treated by radiotherapy after diagnosis of neoplastic meningitis.Assessment of possible prognostic factors showed that at univariate analysis, higher performance status, primary histology (breast versus others), less elevated CSF protein, and linear contrast enhancement at MRI versus nodular disease, as well as intrathecl chemotherapy versus no intrathecal chemotherapy were associated with more prolonged survival.However, probably due to the low number of patients, no statistically significant differences were detected in subgroups at multivariate analysis.In This case underscores the heterogeneity of clinical course in neoplastic meningitis, since it conflicts with 2 other cases (both from a primary breast cancer) who are still alive at the present followup. Differences in the molecular biology profile of tumors within the same histotype are well known and may indeed play a role also in the more aggressive or indolent course of neoplastic meningitis. Note that in this case series the majority of patients did not present meningeal irritation signs/symptoms at disease onset.When considering the toxicity profile, only one grade 4 toxicity occurred. In a melanoma patient, an inflammatory encephalopathy picture with seizures, stupor, signs of meningeal irritation, nausea, moderate increase in temperature took place starting 24 hours after intraventricular administration of 50 mg of liposomal AraC; concomitantly, a slight intraventricular CSF lymphocytosis was detected. The encephalopathy improved progressively leading to recovery of the premorbid status within 72 hours. CSF culture was negative for infectious complications.4 more patients displayed moderate postinjection headache and slight fever, usually starting within 24 hours from intrathecal delivery of liposomal AraC and receding in 1 to 2 days.2 patients—both affected by metastatic breast cancer—are alive at a followup ranging from 11 to 23 months.Intrathecal chemotherapy for neoplastic meningitis may be a worthwhile option for a number of patients with this very serious disease. Technological developments allowing slow-release delivery of potentially active drugs may in the future be combined with targeted treatments  focused on multistep inhibition of neoplastic cell survival, growth, and spreading within the neuraxis.However, a better basic knowledge of the biological mechanisms underlying selective homing of neoplastic cells to the leptomeninges, together with strict monitoring of the risk/benefit ratio , 21, wilThis is very important, since increased survival times are  the consequence of more aggressive systemic treatments, which may significantly enhance the neurotoxicity of intrathecal therapies –24.


G12D, broadly in epithelial tissues. We used a mouse model in which the G12D mutation is placed in the endogenous Kras locus controlled by inducible, Cre-mediated recombination in tissues expressing cytokeratin 19 including the oral cavity, GI tract, lungs, and ducts of the liver, kidney, and the pancreas. Introduction of the KrasG12D mutation in adult mouse tissues led to neoplastic changes in some but not all of these tissues. Notably, many hyperplasias, metaplasias and adenomas were observed in the oral cavity, stomach, colon and lungs, suggesting that exposure to products of the outside environment promotes KrasG12D-initiated tumorigenesis. However, environmental exposure did not consistently correlate with tumor formation, such as in the small intestine, suggesting that there are also intrinsic differences in susceptibility to Kras activation. The pancreas developed small numbers of mucinous metaplasias with characteristics of early stage pancreatic intraepithelial neoplasms (PanINs), supporting the hypothesis that pancreatic ducts have the potential to give rise pancreatic cancer.Activating mutations in the Kras gene are commonly found in some but not all epithelial cancers. In order to understand the susceptibility of different epithelial tissues to Kras-induced tumorigenesis, we introduced one of the most common Kras mutations, Kras Cancer is thought to arise from a series of genetic and epigenetic changes that confer oncogenic properties on a cell and its descendents  which has Kras mutations in approx. 95% of cases G12D-initiated tumorigenesis in adult mice. Because epithelial tumors of some tissues are frequently associated with Kras mutation and those of other tissues are rarely associated, we tested the hypothesis that some tissues are refractory to the effects of Kras mutation, as opposed to being susceptible to its effects but rarely undergoing its mutation, by introducing the mutation into multiple tissues. We found that KrasG12D could induce neoplastic changes in multiple, although not all, assayed tissues. Susceptible tissues included those which in humans rarely have Kras mutations as well as those that frequently do. There was some correlation of susceptibility with exposure to the outside environment, suggesting that environmental factors may play a major role in promotion of KrasG12D-initiated tumors. However, this correlation was not uniform indicating that there are also intrinsic differences in how cells respond to Kras activation. Additionally, we demonstrate that pancreatic ducts are capable of giving rise to mucinous metaplasias with characteristic of early PanIN lesions.We have recently developed a genetic tool that allows us to investigate the susceptibility of different epithelial tissues to KrasG12D mutation was targeted to multiple epithelial tissues via the CK19CreERTG12DG12D allele cannot be expressed until Cre recombinase removes a transcriptional stop cassette, positioning the mutated coding sequence in line with the endogenous Kras promoter. CK19CreERT encodes the Cre recombinase fused to a region of the estrogen receptor that has been mutated to bind tamoxifen. This ERT domain prevents Cre from entering the nucleus and inducing recombination until it binds tamoxifen. For these studies, tamoxifen was injected into adult mice, abrogating embryonic effects. The CreERT coding region was inserted in exon 1 of the Cytokeratin 19 gene, a structural protein found in many epithelial tissues. We have shown previously that CK19CreERT can activate a loxP-flanked reporter in cells throughout the gastrointestinal tract and in ducts of the pancreas, liver, and kidney CreERT can activate KrasG12D in cells of all these tissues.KrasCreERT was active in lung and oral cavity. In the lung, CK19 protein is widely expressed in all epithelium CreERT recombined the R26REYFP reporter largely in bronchi and bronchioles (28+/−10% of cells labeled) and rarely in alveoli (0.22+/−0.08% labeled)  were positive for the EYFP reporter. Other tissues including the stomach, small intestine, colon, pancreas and liver were labeled as previously described In the course of these studies, we also found that CK19CreERT; LSL-KrasG12D mice and littermate controls were given tamoxifen at 6–8 weeks of age and then followed for up to 6 months. Mice were monitored weekly for signs of distress including weight loss. Over the six month time period, 9 of 15 CK19CreERT; LSL-KrasG12D mice were euthanized due to loss of at least 20% body weight . All CK19CreERT; LSL-KrasG12D mice at 6 months post-tamoxifen had visible tumors up to 3 mm in diameter. Upon histological analysis, all CK19CreERT; LSL-KrasG12D mice had multiple benign lung tumors consisting primarily of papillary adenomas that became densely packed in the larger lesions . Thus, KrasG12D mutation alone is not sufficient to alter homeostasis in this tissue.We have reported previously that the CK19G12D susceptibility could be a difference in the extent of recombination of the LSL-KrasG12D allele. While we had shown that CreERT produced from the CK19 allele could recombine the R26REYFP allele in all these tissues, different alleles may have different rates of recombination due to loxP flanking sequences or higher level chromatin structure. Therefore, we performed PCR analysis of DNA extracted from each of the tissues that had little or no phenotype in CK19CreERT; LSL-KrasG12D mice, as well as from stomach which had a strong phenotype, and from tail DNA as a negative control. As expected, the KrasG12D allele was recombined in stomach but was also recombined to some extent in small intestine, colon, liver, pancreas, and kidney while no recombination was detected in tail DNA . As expected, the amount of recombination  in humans One of these tissues is the oral mucosa. Caulin et al. G12D can induce frequent metaplasia in gastric fundus. These metaplastic glands displayed characteristics of foveolar hyperplasia with an extended zone of PAS-positive surface mucous cells, oxyntric atrophy, and basal expression of TFF2. Foveolar hyperplasia is associated with conditions such as Menetrier's disease rather than gastric cancer In stomach cancer, there have been reports that Kras may also be regulated by microRNAs and/or by gene amplification CreERT allele produced recombination mostly in bronchi and bronchioles leading to the papillary adenomas observed in the CK19CreERT; LSL-KrasG12D mice. These tumors were quite different than those observed when the LSL-KrasG12D allele was activated by nasal installation of adenovirus encoding Cre recombinase CreERT is active primarily in bronchi and bronchioles, it is likely that inhaled viral delivery targets mainly other cell types such as alveolar cells.In lungs, the CK19G12D results in a low frequency of mPanIN1A lesions by 2 months post-tamoxifen treatment and progression to later stage PanINs as mice age G12V in acinar cells also led to mPanIN and PDAC but only in conjunction with chemically induced pancreatitis The cell of origin of pancreatic ductal adenocarcinoma has been widely debated. While these lesions in humans consist entirely of ductal cells, recent studies in mice have pointed to an amazing plasticity in the adult pancreas, showing that acinar cells readily give rise to ductal epithelium, notably to mucinous, hyperplastic epithelium in the case of transgenic overexpression of the growth factor TGFα G12D mutation to pancreatic ducts have been lacking and testing the ductal compartment as the cell of origin for PanIN or PDAC was not possible. We now show, using the CK19CreERT allele to activate the KrasG12D allele, that pancreatic ducts can give rise to lesions with the morphological and molecular characteristics of early PanINs. While these cells have the characteristics of early PanIN lesions, we cannot conclude at this point that they are true “PanINs,” because they did not give rise to PDAC in the course of our analysis. Future studies will determine the ability of these lesions to progress to invasive or metastatic disease when combined with loss of tumor suppressor genes or with induction of pancreatitis.Until now, the tools to direct KrasCreERT is expressed in a small percentage of acinar cells G12D model had 50% of acinar cells recombined and yet had a similar number of PanIN lesions arise as we did in our study CreERT labels <1% of acinar cells th the number) of lesions than those seen from elastase-driven activation of KrasG12D if acinar cells were the cell of origin in our study. Two, the PanIN-like lesions resulting from CK19CreERT activation were continuous with normal ductal epithelium. Comparing our results and those described above, we conclude that both duct and acinar cells are capable of giving rise to mucinous lesions with PanIN-like characteristics and therefore potentially to PDAC.Because CK19CreERT is widely expressed in this mucosa G12D allele was seen  promotes oncogenic events that do not necessarily become apparent until after birth.We did not observe hyperplasia in the intestinal tract although CK19was seen . Jacks aG12D-initiated tumorigenesis. This differential susceptibility is most evident in the small intestine which had a high rate of recombination of the LSL-KrasG12D allele with no apparent resulting phenotype. This could be due to multiple mechanisms. It could be that Kras regulates different signaling pathways in different cells and that some pathways are more oncogenic than others. Supporting differences in Kras function, high level of KrasG12D may induce tumorigenesis in some cells but will induce senescence in other cells G12D to induce liver tumors but does not explain the inability of KrasG12D to induce intestinal tumors. The activity of oncogenes is frequently abrogated by the expression of tumor suppressor genes. Tissues may vary in which tumor suppressors are expressed, how many are expressed, and how readily they are silenced. It is likely that future studies will uncover multiple mechanisms that vary from tissue to tissue, allowing some cells to maintain homeostasis in the face of Kras activation while others become transformed.The studies presented here clearly show that different tissues have different susceptibilities to KrasCreERTG12DEYFPCreERT; LSL-KrasG12D mice were analyzed as well as 15 littermate controls that were wildtype or had either the CK19CreERT or the LSL-KrasG12D allele but not both.All experiments were performed with approval from the Vanderbilt Institutional Animal Care and Use Committee, Animal Welfare Assurance number A3227-01, protocol numbers M/06/078 and M/09/394. CK19Tissues were fixed in 4% paraformaldehyde for 4–5 h at 4°C, except tissues for EYFP detection which were fixed overnight, washed in 70% ethanol and embedded in paraffin after dehydration through a series of increasing ethanol concentrations and Histoclear . Paraffin sections were cut at a depth of 5 µm. Antibodies used were rabbit anti-GFP , mouse anti-TFF2 (a kind gift of Nick Wright), rabbit anti-phosphohistone H3 , rabbit anti-claudin-18  and mouse anti-Ki67 . Mucin was detected by staining with periodic acid-Schiff reagent  as per manufacturer's instructions or with Alcian blue, pH 2.5 (Sigma).5′CAGTGCAGTTTTGACACCAGCT3′, and KRasLox-R1: 5′GCATAGTACGCTATACCCTGTGGA3′ and 30–35 cycles of amplification as indicated.Tissue was scraped from slides and digested with proteinase K in DirectPCR lysis buffer . Lysates were heat-inactivated, extracted with phenol and chloroform, precipitated with isopropanol, resuspended and 100 ng used in polymerase chain reaction (PCR) as described


Over the last years there has been an increased focus on the importance of knowing the effect of pre-analytical influence on the proteomes under study, particularly in the field of biomarker discovery. We present three proteomics studies examining the effect of blood contamination and the rostro-caudal gradient (RCG) on the cerebrospinal fluid (CSF) proteome, in addition to plasma/CSF protein ratios. The studies showed that the central nervous system (CNS) derived proteins appeared to be unaffected by the RCG, while the plasma-derived proteins showed an increase in concentration towards the lumbar area. This implies that the concentration of the plasma-derived proteins in CSF will vary depending on the volume of CSF that is collected. In the CSF samples spiked with blood, 262 of 814 quantified proteins showed an abundance increase of more than 1.5 fold, while 403 proteins had a fold change of less than 1.2 and appeared to be unaffected by blood contamination. Proteins with a high plasma/CSF ratio appeared to give the largest effect on the CSF proteome upon blood contamination. The results give important background information on how factors like blood contamination, RCG and blood-CNS-barrier influences the CSF proteome. This information is particularly important in the field of biomarker discovery, but also for routine clinical measurements. The data from the blood contamination and RCG discovery studies have been deposited to the ProteomeXchange with identifier PXD000401. Protein quantification in human body fluids is of high interest in clinical proteomics, and pre-analytical factors may influence the quantitative analysis of the proteome. Knowledge about which and how proteins are affected is important for sample selection and enables exclusion of samples with inadequate quality. Cerebrospinal fluid (CSF), an ultra-filtrate of plasma produced in the choroid plexi, is the most proximal fluid to the central nervous system (CNS) and an important material in the search for biomarkers in neurological diseases. The effect of the pre-analytical influence on the human CSF proteome has previously been studied using proteomics et al.Blood contamination is one of the most important pre-analytical factors in CSF analysis. CSF becomes blood contaminated in up to 20% of all lumbar punctures (LP) et al.As the CSF flows uni-directionally along the spinal cord down to the lumbar subarachnoid space nd to the 45th mL of CSF from one patient with progressive supranuclear palsy (PSP) undergoing LP. In the last experiment, the plasma/CSF ratio was calculated for 152 proteins in five individuals using dimethyl shotgun proteomics. The presented work will be particularly important for quantitative protein measurements in CSF and provides important results for the understanding of the RCG and blood/CSF protein relation.In the present study, we have examined the effect of blood contamination on the CSF proteome by monitoring the abundance of more than 800 CSF proteins at two different blood spike-in levels through a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) LC-MS approach. We also monitored the effect of centrifugation on blood-spiked CSF samples in a related experiment. In a separate experiment, we examined the RCG effect on the CSF proteome using iTRAQ (isobaric tags for relative and absolute quantitation) shotgun proteomics. Multiple reaction monitoring (MRM) MS in combination with stable isotope dilution (SID) was used to monitor 48 proteins derived from blood or CNS (leptomeninges and brain). For this experiment we analyzed CSF from the 22 EDTA tubes and centrifuged at 4°C at 1100×g for 10 min. Plasma and CSF was collected at the same time. All CSF and plasma samples went through one freeze/thaw cycle. In the blood contamination study we used fresh blood donated by a healthy volunteer.CSF was collected from patients undergoing diagnostic LP at the Department of Neurology, Haukeland University Hospital, Bergen, Norway. CSF used in analysis of the CSF/plasma protein ratio was collected from patients undergoing spinal anesthesia at the Department of Orthopedic surgery, Haukeland University Hospital, Norway. The CSF was collected and treated after the guidelines presented in the consensus protocol if not stated otherwise The study was approved by The Regional Committee for Medical and Health Research Ethics (REC), Western Norway, and written informed consent was obtained from all patients and controls.The pre-analytical factors we have studied are: Blood contamination of CSF, the RCG of CSF proteins, and plasma/CSF protein ratio .st mL sample, which was used for routine analysis and thus not included in our dataset. Among the proteins unaffected by the RCG were mainly proteins expected to predominantly arise from the CNS, like cystatin-C and contactin-1, whereas in the uncertain category we found a mixture of both blood- and CNS-derived proteins.In the iTRAQ shotgun experiment , 577 pro13C and 15N. Formaldehyde-d2 (CD2O) , 13CD2O , and sodium cyanoborodeuteride (96% D) were from Isotec .Urea, dithiothreitol (DTT), iodoacetamide (IAA), N-octyl-β-D-glucopyranoside (NOG) and calcium chloride were purchased from Sigma-Aldrich . Trypsin Porcine was purchased from Promega. Tris (hydroximethyl) amonimethane was from Merck KGaA . Water, acetonitrile (ACN) and formic acid (FA) were MS grade, purchased from Fluka Analytical, Sigma-Aldrich. The iTRAQ kit was purchased from AB SCIEX . 4-12% Bis-Tris gel (NuPAGE®), MES SDS Running Buffer, lithium dodecyl sulfate (LDS) sample buffer, and SeeBlue Plus2 Pre-Stained Standard were from Invitrogen, Life Technologies . Stable Isotope Standards (SISs) were in crude quality unless stated otherwise, and were purchased from Thermo Fisher Scientific  and JPT Technologies . Their C-terminal lysine or arginine was labeled with Total protein concentrations for all samples were measured using Qubit™ Fluorometer (Life Technologies).g for 10 min. The samples were analyzed in experiment A (SDS-PAGE label-free) and B (in-solution label-free) with experimental setup as shown in CSF samples with <500 erythrocyte/μL were chosen for the blood contamination experiments. Hemoglobin beta was not detected in any of the CSF samples using SID-MRM (data not shown). One mL CSF from four patients were pooled and divided into four aliquots. One aliquot (1 mL) was kept as reference CSF without added blood, one was spiked with 20 µL blood/mL CSF (2%) and two were spiked with 5 µL blood/mL CSF (0.5%). The sample spiked with 2% blood and one of the samples spiked with 0.5% blood were centrifuged at 4°C at 400×CSF was protein depleted according to the manufacturer's protocol using the Multiple Affinity Removal System (MARS-HU-14) 4.6×100 mm LC column  coupled to Dionex Ultimate 3000 LC system . The total protein amount ranged from approximately 500-1400 µg prior to depletion due to different amount of added blood. When depleting five identical aliquots from the same sample, the %CV of the measured protein concentration over all samples was 6.8%. The protein depleted CSF was concentrated using 3 kDa ultra-centrifugation filters  which were pre-rinsed with 0.1% NOG for increased protein recovery, and freeze dried. The pellet was dissolved in 2X LDS sample buffer , reduced with 10 mM DTT and alkylated with a final concentration of 20 mM IAA for 20 min in darkness before the samples were loaded on 4-12% gradient Bis-Tris gels . Each lane was cut into ten fractions which were washed, trypsin digested, and peptide eluted essentially as described elsewhere m/z) were acquired for 80 min in the Orbitrap with a resolution R = 60 000 at 400 m/z . The seven most intense eluting peptides above an ion threshold value of 1000 counts, and charge states ≥ +2, were sequentially isolated to a target value of 1e4 (maximum accumulation time was 200 ms) and fragmented in the high-pressure linear ion trap by low-energy CID (collision-induced-dissociation) with normalized collision energy of 40% and wideband-activation enabled. The isolation width was maintained at 2 Da, activation q = 0.25, and activation time of 10 ms. The resulting fragment ions were scanned out in the low-pressure ion trap at normal scan rate, and recorded with the secondary electron multipliers. One MS/MS spectrum of a precursor mass was allowed before dynamic exclusion for 20 s. Lock-mass internal calibration was not enabled. The raw files were analyzed and pre-processed using the Progenesis LC-MS software 2.7 . The MS/MS spectra were analyzed using the database homo sapiens SwissProt  using the open-source graphical user interface SearchGUI (version 1.7.3) http://peptide-shaker.googlecode.com), and filtered at the stringent threshold of 1% false discovery rate (FDR). The FDR was estimated using the reversed version of the database Survey full scan MS spectra . The 0-1st mL was blood contaminated (erythrocytes 37 000/μL); this one mL was used for routine analysis and was not included in this study. The workflow for the RCG experiment is shown in To detect a possible RCG of proteins we sampled the seven following points of the RCG from a PSP patient: 1-2In the iTRAQ discovery experiment equal volumes representing the seven RCG points were processed as shown in the experimental setup in The combined and labeled tryptic peptides were dried and fractionated by mixed mode reversed phase-anion exchange (MM (RP-AX)) chromatography on a 1260 Infinity LC system  using a Promix MP column  . The duration of the gradient was 70 min, with 50 µL/min flow, using the following buffers: Buffer A  and Buffer B . The dry peptide mixture was re-suspended in Buffer A and fractionated into 21 fractions with the following gradient: Hold at 15% B from 0-10 min. From 10-45 min 15-60% B, from 45-55 min 60-100% B, hold at 100% B from 55-60 min, and ramp from 100-15% B from 60-65 min, then hold at 15% B from 65-70 min. The fractions were freeze-dried and re-suspended in 0.1% FA before injection on the Orbitrap system. MM (RP-AX) fractions 1-4 were excluded, as they were found to contain excess iTRAQ reagents, and the two latest fractions were combined before injection.5 at a maximum allowed accumulation time of 1000 ms and isolation width at 3 Da. Fragmentation in the HCD cell was performed with a normalized collision energy of 40%, and activation time of 0.1 ms. Fragments were detected in the Orbitrap at a resolution of 7500 with first mass fixed at m/z 160. Two MS/MS spectra of a precursor mass were allowed before dynamic exclusion for 10 s, and lock-mass internal calibration was not enabled.Approximately 0.5 µg of each MM (RP-AX) fraction was loaded on an Ultimate 3000 RSLC nano system online connected to an Orbitrap Velos Pro using the same settings and columns as described for the blood contamination study. The seven most intense eluting peptides above an ion threshold of 1000 counts and charge states ≥ +2, were sequentially isolated in a back-to-back analysis of the same precursors using two different fragmentation techniques, CID and HCD (higher-energy collision dissociation). The CID fragmentation was performed as described for the blood contamination study except collision energy of 35% compared to 40%. For the HCD fragmentation, the ions were isolated in the high-pressure linear ion trap to a target value of 5e12C with maximum charge 7. The resulting data were searched against the homo sapiens SwissProt database  with following search parameters: Precursor mass tolerance 10 ppm and fragment mass tolerance 0.7 Da, maximum two missed cleavages allowed, and MMTS (Cys) and iTRAQ  as fixed modifications. Reverse database scores were calculated. The default fixed auto validation settings were applied for both proteins and peptides, including calculate FDR using reverse hits. In protein mode, the threshold score was >20, proteins were accepted at precursor charge +2 if score >6, Scored Percent Intensity >60%, delta rank1-rank2 >2, and at charge +3 if >8, >70%, >2. The FDR was <1%. The first and seventh RCG points were included twice in the experimental design and the average protein abundances were reported. In Spectrum Mill, a ratio was calculated for every protein by dividing the protein abundance in an individual RCG sample by the abundance in the reference sample. Using these ratios, we calculated the R-squared value and the fold change values for every protein between the different RCG points.The generated raw files were extracted, searched and validated using the Spectrum Mill software package v4.0 beta . The data extraction settings were as follows: Fixed modifications were methyl methanethiosulfonate (MMTS) of Cys and iTRAQ , variable modifications were oxidation of Met, fragment mass tolerance MH+ interval 300-4000, scan time range 0-300 min, and sequence tag length >0. Merge CID and HCD MS/MS, and find precursor µElution 96 well plate  as described in To verify the findings from the RCG iTRAQ discovery, MRM assays were built. Equal volumes of CSF from the seven RCG points were reduced, alkylated and trypsin digested according to the protocol available as The samples were re-suspended in 3% ACN/5% FA, and equal volumes of each sample (ranging from 0.6-1 µg) were analyzed on a triple quadrupole tandem mass spectrometer ) online coupled to a Dionex Ultimate 3000 RSLC nano system. The spiked CSF digest was loaded onto the pre-column and analyzed using the same MS settings and LC gradient as described elsewhere homo sapiens . The following settings were used: Carbamidomethylation of Cys as fixed modification, oxidation of Met as variable. 10 ppm for precursor and 0.7 Da for fragment tolerance, two missed cleavages were allowed, and FDR was set to 1%. Only unique peptides were used for quantification and only proteins with two or more peptides in two or more patients were included. The abundance of every protein was calculated in every sample by Proteome Discoverer, and a ratio was calculated by dividing the abundance in the plasma sample by the abundance in the corresponding CSF sample. This ratio was used to calculate an average ratio and a %CV for every protein across the five individuals.To investigate the plasma/CSF ratio for a larger number of proteins we collected corresponding plasma and CSF from five patients undergoing surgery using spinal anesthesia. These patients had no neurological history and did not have any inflammatory diseases. None of the CSF samples had visible amounts of blood, and hemoglobin beta was not detected by SID-MRM. The abundance of proteins present in both plasma and CSF were quantified using dimethyl labeling. A reference mix was prepared of total 10 µg protein from both plasma and CSF from each of the five patients. The individual samples were digested and labeled as shown in To investigate how blood contamination affects the CSF proteome, a reference CSF sample was compared to CSF spiked to achieve 0.5% and 2% blood  reference versus 0.5% blood and (2) reference versus 2% blood, are given in To examine how blood contaminated CSF is affected by centrifugation, we compared two CSF samples spiked to achieve 0.5% blood where only one was centrifuged  reflects the protein concentration change along the RCG as previously suggested In this study we used shotgun and targeted proteomics to quantify peptides and proteins in seven RCG points ranging from the 2nd mL) and 7 (the 44-45th mL) was 1.63, indicating an approximate 60% total protein concentration decrease from the lumbar towards the ventricular area. These results show a good linearity with an R-squared value of 0.89 (st and 20th mL of the RCG was measured in two new patients (with free CSF circulation). The fold change of the protein measurements were 1.3 and 1.45 (data not shown), verifying the trend observed from the first patient.The fold change of the overall protein concentration between RCG point 1 (the 1-2 of 0.89  and corrA SID-MRM study of 48 proteins (70 peptides) was designed to further confirm the trend observed during the iTRAQ quantification . Based oCorresponding plasma and CSF samples from five individuals without neurological symptoms were analyzed by dimethyl-based shotgun proteomics using an Orbitrap Velos Pro. Our goal was to obtain estimates on the plasma/CSF ratios for a portion of the proteins present in these proteomes. This information could be used to further indicate which CSF proteins that appears to be affected by or origins from plasma/blood. It will also give information about these proteins ability to cross the blood-brain barrier (BBB), and indicate if it would be useful to quantify a given biomarker candidate protein in both CSF and plasma in a biomarker verification experiment. Equal total amount of protein (10 µg) from CSF and plasma was par-wise compared for the five patients, and the plasma/CSF ratio for each protein was averaged across the five individuals. From 434 proteins identified across all experiments (data not shown), 152 proteins were quantified with two or more unique peptides in two or more individuals . AlbuminIt is known that several neurological disorders lead to impaired BBB which may cause increased influx of plasma proteins into CSF, and in addition, CSF can be blood contaminated during LP. Comparing the list of 201 proteins previously reported to be differentially abundant in CSF of multiple sclerosis patients  from plasma, and which proteins that appear to be unaffected by blood contamination and are likely to be CNS-derived. The correct endogenous amounts of the latter category of proteins could likely be measured in CSF even if the samples are blood contaminated. Some of the proteins observed in the unaffected group in For the plasma/CSF comparison experiment, a wide range of ratios were observed as described in the results section. The reasons for the different plasma/CSF ratios between plasma-derived proteins remain to be determined, but it has been suggested that factors like protein size, hydrogen bonds and hydrodynamic volume are important for a proteins ability to cross the BBB In theory, the higher the plasma/CSF ratio of a protein, the more influence will the blood contamination have on the CSF concentration. Exceptions would be if there is high intrathecal synthesis of the protein, particularly in combination with low plasma concentrations. As can be observed from et al.et al.et al, using prostaglandin-H2 D-isomerase. Compared to their study, we found the RCG effect on prostaglandin-H2 D-isomerase to be uncertain with a fold change of 1.3 from the first to seventh RCG point. In addition, we verified our iTRAQ results using SID-MRM and included several other brain-derived proteins such as chitinase 3-like protein, secretogranin-3 and S100B and found that none of them decreased down the RCG. Hence, from our results it seems like the concentration of the brain-derived proteins is not affected by the RCG. It should be pointed out that we studied this effect in one patient only, a PSP patient, and examined CSF up to the 44th collected mL which is a limitation of this work. However, our results are supported by the findings by Brander et al. st to the 20th mL CSF collected. It therefore seems apparent that the collected volume of CSF affects the protein concentration, and most importantly, that the brain-derived proteins does not appear to be affected. Whether this is true for all conditions and diseases needs to be further tested.For the RCG study, the plasma proteins show an increase in concentration towards the lumbar area and steady influx due to diffusion laws are believed to be the cause of this gradient. In contrast to Reiber nd mL CSF) and 7 (the 44-45th mL CSF), it will be approximately 30% difference in concentration for the plasma proteins if only 1 mL CSF is collected compared to if all 45 milliliters are collected as one sample. In clinical practice between 5 and 20 milliliters of CSF are normally collected, which would give a range of approximately 10-15% variation in the plasma protein concentrations based on the results obtained from our examined patient. This number will, as we have shown, vary from patient to patient, and the effect should be examined in a larger number of patients to get a clear picture of the variation.Given the linearity for the plasma proteins in the RCG and that there is a 1.6 fold change (60%) between RCG point 1  from plasma, which is useful information when determining if the concentration of a given CSF biomarker candidate also should be assessed in plasma. In sum, our study gives important information about the influence of factors like blood contamination, RCG and BBB on protein concentration measurements in CSF, particularly important in the field of biomarker discovery, but also for routine clinical measurements. A limitation of our work is the low number of samples included, and future studies including larger number of samples should be conducted to verify our findings.http://proteomecentral.proteomexchange.org) via the PRIDE partner repository The mass spectrometry proteomics data from the blood contamination and RCG discovery studies have been deposited to the ProteomeXchange Consortium (Document S1In-solution digestion protocol.(PDF)Click here for additional data file.Table S1Peptides used for quantification in the RCG verification study. The table contains protein ID, Uniprot accession number, peptide sequence, transition used for quantification, precursor m/z (Q1) and fragment m/z (Q3) of the endogenous and SIS peptides, and collision energy used in the MRM assay. a indicate that the peptides were of AQUA quality.(PDF)Click here for additional data file.Table S2Results from the blood contamination study, experiment A. Proteins from the blood contamination study A  quantified with two or more peptides. CS is the confidence score of the proteins, obtained in the Progenesis LC software. The ratios for 0.5% blood/reference CSF, 2% blood/reference CSF and 0.5% blood/2% blood are calculated. Proteins with ratio >1.5 in either of the two spike-in levels were classified as affected (n = 262). Ratio <1.2 was classified as not affected (n = 403) and ratios in between were uncertain (n = 149). The protein and peptide reports from Progenesis are available in the table.(XLSX)Click here for additional data file.Table S3Results from the blood contamination study, experiment B. Comparison of the effect of centrifugation of 0.5% blood contaminated CSF with label free quantification. Ratio >1.5 for 0.5% blood not centrifuged/0.5% blood centrifuged were considered affected by blood contamination, while ratio <1.2 was considered as unaffected. CS is the confidence score from the Progenesis output. The data are identified using the database homo sapiens, SwissProt. The protein and peptide reports from Progenesis are available in the table.(XLSX)Click here for additional data file.Table S4Results from the rostro caudal gradient discovery study. Results from the iTRAQ discovery experiment applied on the seven RCG points of CSF. Proteins were quantified using Spectrum Mill and identified using homo sapiens, SwissProt. The table lists identification and quantification of 279 proteins with two unique peptides or more with R square value calculated of the seven RCG points, and the fold change (FC) of the 1-2nd and 44-45th mL, corresponding to RCG point 1 and 7. Included in the table is also R squared value of the six RCG points from the 10-11th to the 44-45th mL and the FC between the 10-11th and 44-45th mL (RCG point 2 and 7). The proteins were considered affected or not according to the criteria in (XLSX)Click here for additional data file.Table S5Comparison of the R-squared values and fold changes for the peptides quantified with SID-MRM and the iTRAQ experiment for the RCG study. Proteins are categorized into groups based on the major expected contributing source of origin, Uniprot is the primary reference unless otherwise stated.(XLSX)Click here for additional data file.Table S6Results from the plasma/CSF study. Results from the plasma/CSF study . The proteins are quantified using dimethyl labeling in Proteome Discoverer (PD), identified using Mascot and the homo sapiens database from SwissProt. The average protein ratio, in addition to standard deviation and %CV are included for each protein. The protein and peptide reports from PD are available in the table.(XLSX)Click here for additional data file.Table S7Summary of  This combined table gives an overview over which proteins that are affected by blood spike-in, centrifugation, RCG and their plasma/CSF ratio, and also how the relation is between the experiments for each protein. The proteins are combined based on accession numbers, except for the iTRAQ discovery study where the leading protein was used.(XLSX)Click here for additional data file.


Sir,Polyorchidism is a rare congenital anomaly with more than 100 cases reported in the literature. We report a case of a 47-year-old man presented to our clinic  complaining of a painless mass in left hemiscrotum for 1 year. On admission, the patient denied any history of trauma, chronic illnesses, or major operations. The patient had no fertility problems and was a father of 3 children. On physical examination, a soft mass that is approximately the same size as left testis, and 2 vasa deferentia were noted in the left scrotum. The right testis and systemic examination were normal. The ultrasonography study of the left scrotum demonstrated a normal left testis measuring 28 × 34 × 36 mm and supernumerary testis measuring18 × 21 × 23 mm. The size and echogenicty of the right testis was normal. There were no suspicious malignant features on ultrasonography of the bilateral scrotum. The patient had normal serum levels of alfa fetoprotein, beta subunit of human chorionic gonadotrophin (hCG), lactate dehydrogenase, and testesterone. Further evaluation by magnetic resonance imaging (MRI) showed that the mass had intermediate signal intensity on T1-weighted image and high signal intensity on T2-weighted image. MRI confirmed the presence of supernumerary testis, which had the same signal characteristics as the normal testicles on T1- and T2-weighted images . No otheet al., based on anatomy is a useful and more appropriate classification to use. A testis being drained by an outflow path (vas deferens) was coded as type A, whereas undrained testes without connection to a draining vas deferens were coded as type B. Type A testes were further divided into 4 subgroups. A1: Supernumerary testis has its own epididymis and vas deferens, A2: Supernumerary testis has its own epididymis but common vas deferens with neighboring testis, A3: Supernumerary testis has its common epididymis and vas deferens with neighboring testis, and A4: Supernumerary testis has its own vas deferens but common epididymis with neighboring testis. Type B testes were further divided into 2 subgroups. B1: Supernumerary testis has its own epididymis and B2: Supernumerary testis lacks an epididymis, thus consisting of testicular tissue only.[The most common presentation of polyorchidism is triorchidism, or tritestes, where 3 testes are present. The supeThere is no consensus in the literature regarding the management of polyorchidism, especially in cases where the supernumerary testes found incidentally are posing a surgical dilemma. Some authors have suggested orchiectomy of the supernumerary testes in adults because of 4%-7% risk of malignacy.6 The low


Maintaining the fitness of captive amphibian populations is essential to the success of ex situ conservation projects. Here we examined the impact of plant cover on measures of fitness and behaviour in captive red-eyed tree frogs . We found significant effects of plant provision on body size, growth rates and cutaneous bacterial communities that together demonstrate a compelling fitness benefit from cover provision. We also demonstrate a strong behavioural preference for planted rather than non-planted areas. We also assessed the impact of plant provision on the abiotic environment in the enclosure as a potential driver of these behavioural and fitness effects. Together this data provides valuable information regarding enclosure design for a non-model amphibian species and has implications for amphibian populations maintained in captivity for conservation breeding programmes and research.Despite the importance of  Ex situ conservation has been identified as a major component of the conservation response, and the only possibility for some species that are declining rapidly in the wild ex situ conservation and reintroduction programmes comprise population ‘rescue’ from the wild, breeding of healthy offspring, and (re)establishment of self-sustaining wild populations once threats have been alleviated Amphibian species are globally threatened as a result of synergistic threats including disease, over-collection, habitat loss and degradation, and climate change Environmental complexity, and specifically shelter or cover provision, may be an important component of enclosure design and captive husbandry for amphibians. Wild amphibians use cover for a variety of reasons, including behavioural homeostasis Xenopus laevisXenopus, shelter seems to confer behavioural benefits, but has no impact on growth or body condition Existing research on effects of captive environmental conditions, although limited, is biased towards laboratory populations of Agalychnis callidryas), and therefore other facets of captive husbandry may do so too Current research in amphibian husbandry tends to use measures of growth rates, body condition and behaviour to assess fitness effects. Captive husbandry protocols may also have more subtle effects on amphibian health and fitness. Bacterial communities living on the skin of amphibians are emerging as an important component of amphibian immunity . Enclosures were housed in a climate-controlled room at the University of Manchester at a diurnal ambient temperature of 24°C and nocturnal ambient temperature of 22°C, and an ambient relative humidity of 60–80%. Circulation fans prevented air stagnation. Reptisun 10.0 UVB fluorescent tubes (ZooMed Inc.) and plant growth tubes  with reflectors (Arcadia) were provided on top of terraria on a 12∶12 hr light cycle. All UVB bulbs were subject to a 100 hour burning-in period before use to reduce emission fluctuations associated with new tubes, and replaced within one year of use .Group 1 consisted of frogs (N = 19) reared from metamorphosis in planted or non-planted enclosures. These frogs were used to examine environmental variables, growth and body condition and bacterial communities. Frogs were raised from a single clutch of eggs produced by a pair of captive adult frogs. Tadpoles were reared in a single large aquarium (100×50×30 cm) at 23–25°C. Tadpoles were provided with plastic plants for shelter and water quality was maintained using an air-driven sponge filter and partial water changes. Tadpoles were fed a diet of crushed fish flakes (Tetramin) and dried powdered Agalychnis frogs Gryllus bimaculatus), following Ogilvy et al. All enclosures contained a small water dish and a substrate of moistened paper towels, frequently used as a substrate for Sciandapsus sp. – ‘Devil’s ivy’), with no other cover provided in either enclosure type. On day 180 , Dieffenbachia plants were introduced into all tanks, and Sciandapsus plants removed  as the larger leaves provided a better resting site for sub-adult frogs.Enclosures were either planted (n  =  9 frogs) or non-planted (n  =  10 frogs), with three enclosures per treatment group. Sample sizes were not equal as one froglet died at metamorphosis. Froglets were allocated alternately to treatment enclosures on metamorphosis and enclosure types were positioned alternately on the same shelf. Planted enclosures contained living plants (Sciandapsus sp.) and non-planted enclosures (ExoTerra ‘Faunarium Medium’).A FLIR 5 Infrared camera (FLIR Systems) was used to thermally image planted (A FLIR 5 Infrared Camera (FLIR Systems) was used to measure the skin temperature of frogs (N = 19) under planted and non-planted conditions. Measurements were taken on three consecutive days at 14:00, when terraria had reached their peak daytime temperature. Mean values were calculated for each frog over the three days and t-tests were used to compare mean skin temperatures. At the same time, tanks were visually split into thirds  and the height of the frogs’ resting positions was recorded as top, middle or bottom .Four extra of each planted and non-planted enclosures (ExoTerra ‘Faunarium Medium’) were set up identically to those used in this experiment with the exception that frogs were not housed in them. The drying rate of the enclosure was assessed by saturating paper towel substrate in enclosures maintained at 25°C with 100 ml of water and measuring weight loss of the entire paper towel to the nearest gram after 2, 4, 6, 8 and 24 hours. Rate of water loss was calculated for each enclosure and the data was analysed using a Mann-Whitney U-test.Planted and non-planted vivaria  housing Group 1 experimental frogs were used for the measurement of UV index (UVi). UVi is a unitless measure of UVB radiation, weighted for its biological significance, and is becoming widely used in the study of UVB exposure in herptiles e.g. . The Rephttp://imagej.nih.gov/ij/) was used to calculate the snout-to-vent length (SVL) of frogs, and the mean taken from the three measurements of each individual. The mass of each frog was recorded on days 90 and 180, subsequent to a four-day fasting period (the routine maximum period between feedings in the husbandry schedule) to avoid variation in mass due to foraging success 0/L]R where M is the mass, L is the snout-vent length for a given individual, L0 is the arithmetic mean of the SVL's for the whole population, and R  is equivalent to the regression value  of M on L for the whole population Frogs were photographed three times each against a scale after 6 (once tail had fully disappeared), 90 and 180 days post metamorphosis. Image J (freeware available at Scindapsus sp.) or non-planted conditions; henceforth ‘Phase 1’ swabs. The second set of swabs were taken two months after Dieffenbachia plants were added to all enclosures, after the end of the planted/non-planted study; henceforth ‘Phase 2’ swabs.Frogs were swabbed twice for cutaneous bacterial communities. The first swabs were taken while frogs were being maintained under planted  bacteria from their skin 2 solution up to a concentration of 10-2 for each sample. 100 µl of each dilution (10−1 and 10−2) were plated out on R2A agar media  and incubated at 25°C (the same temperature at which frogs were maintained). New morphologically distinct bacteria colonies (‘morphotypes’) were counted up until 12 days post swabbing, after which negligible new colony growth was observed.Eppendorf tubes containing swabs were vortexed to dissociate bacteria from the swab. The swab was removed and samples were serially diluted by pipetting 100 µl into 900 µl of 0.8% w/v NaClBacterial counts were multiplied by their respective dilution factors and averaged for each morphotype. Data for the dorsum and ventrum of each frog were combined, and differences in the overall bacterial community composition during each phase were analysed using the Adonis function in the Vegan package in RStudio. Adonis is a permutational multivariate analysis that uses distance matrices to analyse the variation in the overall bacterial community structure associated with frogs according to treatment group. The effect of treatment group on species richness  and total abundance  was analysed using t tests in JMP 10.5′-GTGCTGCAGAGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CACGGATCCTACGGGTACCTTGTTACGACT-3′) http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify each morphotype to genus level. Morphotypes with 99% sequence similarity or greater were considered the same species Representative colonies of each morphotype were streaked out on R2A agar until a pure culture was obtained. Bacterial species were identified using 16S rDNA sequencing with universal primers 27F  as their ‘prior treatment’. Frogs were then transferred individually to anexperimental arena for four days, starting on day 185 after metamorphosis. The arena consisted of a glass terrarium (90×45×45 cm) with one end furnished with artificial terrarium plants and the other no plants. A line was drawn at the centre of the arena and the water bowl positioned to exactly straddle the line on the floor. Substrate was damp paper towels. Frogs were allowed to acclimatise for one night and then for the following three days the position of the frog in the tank was scored twice a day (10:00 and 16:00 hrs). Frogs were scored ‘1’ if they were positioned at the end of the arena with live plants, and ‘0’ if they were at the end with artificial plants. A frog exactly straddling the centre of the tank would have been scored 0.5, although this never occurred. Mean scores over the three days were calculated and analysed using a non-parametric Wilcoxon signed rank test.Adult frogs previously reared under the same conditions as one another were maintained in ‘home’ enclosures for 2 weeks with (n = 15) or without plants , with UVi falling in a steep gradient with increasing distance from the lamp. UVi was also predicted by treatment , with planted tanks having a steeper UVi gradient. Mean UVi was not significantly different between treatments immediately under the lamp , but became significantly lower in planted setups at the middle and bottom of the tank .UVi was significantly influenced by position  in the tank , day 90  or day 180  of the experiment. Similarly, there was no significant effect on body condition on day 90  or day 180 . We therefore ran proceeding models without the ‘enclosure’ term. There was no significant difference in SVL of frogs in planted and non-planted treatments at the start of the experiment . However, the SVL of frogs in planted tanks was significantly larger than that of frogs in non-planted tanks on day 90  and day 180  and accession numbers are listed in Comamonadaceae, Enterobactereacae, and Flavobacteriaceae.Overall, 29 bacterial morphotypes were isolated , but oneA. callidryas maintained with and without Sciandapsus plants . Frogs in the planted environment supported a significantly greater bacterial load , bacterial abundance , or the Adonis analyses .A total of 14 bacteria morphotypes were cultured, with a range of 7 to 13 morphotypes per individual. There were significant differences in the overall composition of the bacterial community associated with Dieffenbachia plants to all tanks there were no significant differences in the overall composition of the bacterial community associated with A. callidryas previously kept with and without Sciandapsus sp. . There was no significant difference in species richness  did not have a statistically significant effect on species richness , bacterial abundance , or the Adonis analyses .A total of 15 bacteria morphotypes were cultured, with a range of 6 to 12 morphotypes per individual. After the addition of Sciandapsus plants and then changed to Dieffenbachia plants experienced a decrease in species richness (Sciandapsus to Dieffenbachia) and half maintained on Sciandapsus for the duration. These frogs were swabbed for bacterial communities (using exactly the same methods as before) both prior to the plant change and then again one month later. A two-way ANOVA showed there was no significant effect of sampling point (pre- or post-plant change), treatment group (plant species changed and plant species not changed) or their interaction on species richness . However, both before and after the plant change, the average species richness for frogs where the plant species remained the same (7.5±0.5 and 7.8±0.2 respectively) and for those where the plant species was changed (7.3±0.7 and 7.4±0.3 respectively) was closer to the values obtained from the group of frogs that had the plant species changed (8.7±0.7) during Phase 2, compared to those that had plants introduced to their enclosure during Phase 2 (9.8±0.5). In addition, a two-way ANOVA model for sampling point, treatment group and their interaction was statistically significant , with a Tukey's post-hoc analysis showing frogs in tanks that changed plant species had significantly increased bacterial abundance one month after the plant change. This is consistent with the results from the frogs in Group 1 in Phase 2 that also had their plant changed.Although there were no statistically significant differences in species richness between the two groups during Phase 2 of the study, the group previously kept with richness . In orde29  =  186.500, p < 0.001).Adults given a choice between planted and unplanted ends of an enclosure showed a significant preference for the planted end, with the mean position score for the population (0.872) being significantly greater than a score expected for random selection (0.5) . The variance was also significantly smaller for this group  indicating these frogs show a more consistent response than those that came from planted home enclosures.When adult frogs from a previous treatment of non-planted conditions in their home enclosure where given a choice between planted and unplanted ends of an enclosure  they showed a significantly higher mean position score (0.989) than frogs with plants present in their home enclosure (0.756) . The authors attributed these differences to more favourable microclimates provided by the cover and to the effects of being able to hide from perceived predation risk X. laevisX. laevis is well known for its adaptability, which may allow it to cope more easily with a lack of cover.Our data show that the provision of plant cover in the tank leads to significantly increased body size and BCI of A. callidryas growth shown in the paper presented here is likely to represent an important real-world fitness cost, influencing survivorship both as adults and juveniles, as well as impacting on fecundity A. callidryas, body size is linked to fitness traits in both males  of bacteria associated with their skin than A. callidryas maintained without plants. A richer bacterial community is likely to be advantageous as this may increase the stability and productivity of the community, making it less susceptible to pathogenic infection, and it may also increase the potential for the presence of a species that can protect the host from pathogens such as the chytrid fungus The presence of Plethodon cinereus) maintained with either soil collected from the wild or sterile Provasoli medium supported different bacterial communities after 28 days, indicating the environment and its associated bacteria (or lack of) affects the community associated with the host amphibian. However, given that the same species of bacterial morphotypes were isolated from frogs maintained in both planted and non-planted environments  than the plant change itself, which may have been buffered in frogs previously without plants when these were introduced in Phase 2. However, changing the plant species does cause a subsequent increase in the bacterial abundance for frogs, which lasts for months after the event. Whether this is of benefit or detriment to frogs remains to be tested.The results from Phase 2 of the bacterial study show that when ants see , whereasAcinectobacter sp.), although there were some similarities between the two phases at the family and genus level  spent more time under shelters when available than in the open.We found that In our study, frogs that had been previously housed without plants clearly showed a stronger preference for planted enclosures than those previously housed with plants. This coupled with the overall preference for cover suggests that frogs seek out plants for shelter if given the option, and exhibit a stronger behavioural response once access to plants is restored after a period of plant absence. This is potentially an effort to alleviate some level of physiological stress experienced from lack of cover, although analysis of corticosteroid ‘stress’ hormones see  would beX. laevis as a model for designing enclosures for other amphibians. We used a single sibship of frogs in this study so, given the prevalence of clutch effects on a variety of characters in amphibians A. callidryas, with a similar ecology, until more species-specific data is available. It is also important to expand this sort of research to include other non-model amphibians, particularly for those taxa and ecotypes that are not yet represented, including salamanders and caecilians.In this study we found a large and positive effect of providing shelter to frogs on growth, body condition, bacterial communities and behaviour in captive amphibians, which are at least partly linked to an increased heterogeneity as a result of plants in enclosures. This suggests that captive enclosure design may influence the long-term fitness of captive populations, and the chances of success for reintroduction attempts. This is important for institutions of all types that maintain amphibians in captivity for both conservation and research. Our data also extends work on non-model amphibians to include arboreal anurans, which have thus far not been investigated, and calls to further attention the problems associated with using laboratory


Aquaporin-4 (AQP4) is a water channel mainly located in the ventricular ependymal cells (brain-CSF barrier), the sub-ependymal glia, glia limitans and in end-feet of astrocytes in at the blood–brain barrier (BBB).In the present work, the expression of AQP4 in the cerebrospinal fluid (CSF) in control and congenital human hydrocephalus infants (obstructive and communicating), was analysed by Western-blot and enzyme immunoassay (ELISA).p < 0.05) although the increase was not significant in obstructive hydrocephalus (obstructive: 14.7%). The AQP4 quantification by ELISA also showed that, the mean concentration of AQP4 in CSF was significantly higher in communicating hydrocephalus . However, there was no increase over control in obstructive hydrocephalus (obstructive: 8.65 ± 0.80 ng/ml).AQP4 was found to be high compared to the control in the CSF in congenital hydrocephalus patients. Western-blot showed higher values for AQP4 than controls in communicating hydrocephalus (communicating: 38.3%, control: 6.9% AQP4 has a modulatory effect on ependyma stability and acts in CSF production and reabsorption. Therefore, the increase of AQP4 in the CSF in congenital hydrocephalus could be due to the fact that AQP4 passes from the parenchyma to the CSF and this AQP4 movement may be a consequence of ependyma denudation. Aquaporin-4 (AQP4) is the principal water channel of the central nervous system ,2 which in vitro conditions but also in in vivo[Many studies report that there is an up-regulation of AQP4 in animal hydrocephalus models which suggest that this up-regulation may be due to a compensatory effect of hydrocephalus -9. Howevn in vivo. Moreoven in vivo,16.Therefore, AQP4 could participate in the physiopathology of the water channels in CSF production, in the adequate formation of the gap-junctions and consequently in the ependyma denudation during ventricular dilation. The aim of the present work is to examine the expression of AQP4 in the CSF in patients with congenital obstructive and communicating hydrocephalus.. One of the samples was excluded due to blood contamination. Parents had given verbal informed consent. The medical ethical committee of the University of La Laguna and the University Hospital of the Canary Islands approved the study. The CSF was always extracted for diagnostic and / or therapeutic motives. The diagnosis of the hydrocephalus was determined by ultrasound methods during pregnancy and not controlled by pelvicephalometry just before birth. The type of the hydrocephalus was established by ultrasound methods: tetraventricular hydrocephalus of unknown origin was considered to be communicating hydrocephalus; Dandy-Walker syndrome and Sylvian aqueduct obstruction were considered to be obstructive hydrocephalus  and reduced using β-mercaptoethanol. Samples were heated to 95°C for 5 min and run on a SDS-PAGE gel (10% polyacrylamide) for 2 h at 80 V and the proteins were then blotted to a PVDF membrane  at 400 mA for 2 h in blotting buffer . After 1 h blocking , samples were incubated with target antibody at a dilution of 1:1000 for AQP4  in blocking buffer at 4°C overnight. Detection was performed by enhanced chemiluminescence  after an hour’s incubation with horseradish peroxidase-conjugated anti-rabbit 1:20,000 . The primary antibody was omitted to control for the method specificity.. A non-parametric Kruskal-Wallis test was used for data comparison between the control and hydrocephalus groups, which was conducted using the IBM SPSS statistic 19 software where data were considered as statistically significant at p < 0.05.Densitometric analysis was completed in Image J . The 'Mean Gray Value' was measured on all Western-blot samples. This value gives the average stain intensity in greyscale units for all threshold pixels. The data for triplicate samples were averagedp < 0.05 which was conducted using the IBM SPSS statistics 19 software.In order to give a more accurate quantification of the AQP4 in the CSF of hydrocephalus and control patients, seven additional samples were added to those used for Western blot Table ; four coThe Western blots showed only one AQP4 band of 34 KDa in all samples. The mean grey value in CSF was higher in both types of hydrocephalus; obstructive  and communicating hydrocephalus  when compared to the control CSF   and when compared to obstructive hydrocephalus (χ 22 = 4.32 p < 0.05). The average concentration of the controls was 8.61 ± 0.31 ng/ml, of obstructive hydrocephalus was 8.65 ± 0.80 ng/ml and of communicating hydrocephalus was 11.32 ± 0.69 ng/ml.ELISA also, showed Figure  that AQPAquaporins (AQPs) are membrane proteins that facilitate water and small solute movement in tissues. Hydrocephalus is the main central nervous system disorder associated with defective CSF turnover ,6. AquapA study  detectinin vitro studies [Thus, despite their inherent relationship in the processes of water transport in the previously mentioned conditions , AQP4, by means of connexin-43 (Cx43) regulation, is also a modulator of the formation and development of the gap junctions in the ependymal basolateral membrane and astrocyte feet. It has been reported that knockdown and knockout AQP4 mice downregulate the expression of Cx43 in different  studies ,21,22. F studies ,16.The CSF protein concentration is compartmental related, thus in the lumbar compartment the protein concentration is higher than in the ventricular compartment . DespiteWe have shown that the AQP4 concentration is higher in the CSF of communicating hydrocephalus infants than in the CSF of non-communicating hydrocephalus patients or controls. It is possible that AQP4 may freely leak from the parenchyma to the CSF during the early stages, when ependymal denudation is occurring and the hydrocephalus remains communicating. The leaking of AQP4 to the CSF may decrease as a glial scar-like layer covers the ventricular surface and subsequent stenosis and obstruction occurs.There are no conflicts of interest.LCR and IGM participated in conception and design of the study and in the collection of data. JMGT, ACR, HPC participated in performing the methods and contributed to analysis and interpretation of data. LCR, IGM, ACP and ECC participated in conception, design manuscript, analysis and interpretation of data and preparation of the final version. All authors have read and approved the final version of the manuscript.


Cystic dysplasia of the testis (CDT) is a benign, congenital malformation of the testis anda rare cause of painless scrotal swelling in children, mimicking testicular cancer. It is commonly unilateral, often associated withipsilateral wolffian duct and ureteral abnormalities. Cystic dysplasia of the rete testis (CDT) represents a diagnostic challenge made easierif age, precise localisation, typical ultrasonographic features, the presence or absence of associated genitourinary malformations,as well as tumor markers are considered. The definite treatment of such a benign lesion is testis-sparing surgery, however in mostcases watch and wait strategy can be recommended. We present a case of cystic dysplasia of the testis in a 17-month-old boy withright multicystic dysplastic kidney, epididymal cyst, history of vesicoureteral reflux (VUR), as well as of solitary umbilical artery. We performedepididymidal cyst enucleation and right testicular biopsy. Cystic dysplasia is an unusual benign congenital lesion of the rete testis often associated with ipsilateral renal and ureteral abnormalities. Compression of the surrounding parenchyma generally occurs and may result in testicular atrophy. We present a case of cystic dysplasia of the testis in a 17-month-old boy.A 17-month-old white boy was admitted to the hospital with painless right hemiscrotal enlargement. The patient had a prior medical history of right multicystic dysplastic, ectopic, non functional on DMSA, kidney (MCDK) followed since gestation, a left VUR, and solitary umbilical artery at birth. On physical examination a paratesticular, possibly cystic mass, was palpated and the testis was found to be enlarged and nontender. The left testicle felt normal. Preoperative ultrasound showed right testicular enlargement (1.95 × 1.01 cm) compared to the left (1.50 × 0.62 cm). The multicystic lesion comprised approximately 90% of the testicular volume and was compressing the normal parenchyma to the periphery . Cystic Surgical operation was carried out through an inguinal incision to remove the paratesticular mass. Given the opportunity the testis was inspected and was noted to be enlarged, whitish and rubbery on palpation. The decision was made to perform a wedge biopsy. The paratesticular mass was found to be a simple epididymal cyst that was enucleated intact .Histological examination showed multiple, anastomosing, irregular cystic spaces of varying sizes and shapes predominantly located in the mediastinum of the testis, consistent with cystic dysplasia of the rete testis Figures .First described in 1973, CDT is a rare congenital monolateral lesion resulting from abnormal development of the rete testis . Other aCDT likely has an embryologic pathogenesis thought to arise during the fifth week of gestation. Normal testis development requires the interaction of the epigenitalis of the mesonephros, a Wolffian duct structure, and the rete tubules . CDT is This extremely rare cause of scrotal swelling may appear on physical examination as a painless transilluminating testicular mass . Pain isSonography is recognized as the imaging method of choice for the evaluation of the nature of testicular abnormalities as well as for the followup of untreated forms , 10, 11.Because of the frequent association of CDT with other ipsilateral genitourinary abnormalities, sonographic evaluation of the urinary tract should also be included in the diagnostic work up. Kogan proposes diagnostic criteria for CDT. They include the identification of cystic lesions on ultrasound, normal tumor markers, and evidence of associated ipsilateral mesonephric anomalies. If these criteria are met, he states that open biopsy is not necessary for diagnosis , 12. Testicular tumor markers are an important tool in the evaluation of testicular tumors in children and adults . High leThe differential diagnosis of cystic testicular lesions in children include epidermoid, dermoid, inflammatory, posttraumatic, or congenital testicular cysts . Cystic On histologic examination of cystic dysplasia of the rete testis, the lesion consists of dilatation of the ducts and compression of the surrounding testicular tissue. The cystic spaces are lined with cuboidal cells and separated by fibrous septa. The cysts can be microscopic or measure up to several millimeters in size .Historically, orchiectomy was recommended . With im