Pulmonary Alveolar Proteinosis (PAP) is a rare syndrome characterized by pulmonary surfactant accumulation within the alveolar spaces. It occurs with a reported prevalence of 0.1 per 100,000 individuals and in distinct clinical forms: autoimmune  auto-antibodies; GMAbs), secondary (is a consequence of underlying disorders), congenital (caused by mutations in the genes encoding for the GM-CSF receptor), and PAP-like syndromes (disorders associated with surfactant gene mutations). The clinical course of PAP is variable, ranging from spontaneous remission to respiratory failure. Whole lung lavage (WLL) is the current standard treatment for PAP patients and although it is effective in the majority of cases, disease persistence is not an unusual outcome, even if disease is well controlled by WLL.In this paper we review the therapeutic strategies which have been proposed for the treatment of PAP patients and the progress which has been made in the understanding of the disease pathogenesis. Pulmonary alveolar proteinosis (PAP) is a diffuse pulmonary disease characterized by the accumulation of periodic acid-Schiff (PAS)-positive lipoproteinaceous material, primarily phospholipid surfactant and surfactant apoproteins, in the distal air spaces, which results in impaired gas transfer.PAP is an extremely rare disorder, occurring worldwide with an estimated prevalence of 0.1 per 100,000 individuals. The clinical course of PAP is variable, ranging from spontaneous remission to respiratory failure. The onset of clinical disease is insidious, with a subacute indolent course that often delays the diagnosis by months to years. This delay is secondary to the time required for sufficient surfactant accumulation in the alveoli to impair gas exchange. In fact, decreased clearance of surfactant from the alveoli has been postulated as the basis of PAP pathogenesis.Major advancements have been achieved in the understanding of the pathogenesis in the last two decades. The first milestone was the discovery in GM-CSF knockout mice that surfactant lipids and proteins progressively accumulate in the alveolar space, as in human PAP ,2. TheseThe vast majority of PAP  are classified as the autoimmune disease type, associated with the presence of GM-CSF autoantibodies. Less commonly is the congenital type  or the secondary type .When reviewing the therapeutic strategies for PAP, it is important to note that the various forms of PAP may require different treatments, even though whole lung lavage (WLL) is the current therapeutic standard.Autoimmune PAP has been treated successfully since the early 1960s by WLL, with significant improvements in symptoms and radiographic results. In the pre-whole lung lavage era, death occurred mostly because of progressive respiratory failure and, to a lesser extent, superimposed respiratory opportunistic infections. The high frequency of respiratory infections, including those from opportunistic agents, supports the hypothesis of an impairment in lung immunity. However the presence of infections occurring also outside the lung, would suggest a systemic impairment of host defences.Current therapy for the congenital form of the disorder depends on the patient’s age at presentation, severity of symptoms and anticipated disease course. In these cases the therapeutic approach is supportive, although successful lung transplantation has been reported.Therapy for secondary PAP generally involves treatment of the underlying condition. For example, when the disorder is associated with a hematologic cancer, successful chemotherapy or bone marrow transplantation corrects the associated pulmonary disorder.The first advance in the treatment of PAP came in 1960, when Dr. José Ramirez-Rivera applied repeated “segmental flooding” as a means of physically removing the accumulated alveolar material. This “segmental flooding” provided proof that physical removal of adequate amounts of the material provided functional improvement, but as initially described, this was clearly an impractical therapy for broad application. Over the next fifty years, this procedure was sequentially refined through the routine use of general anesthesia ,8, increThe principal criticism of this procedure is that WLL has not been standardized and no perspective clinical trials have been performed. As a result, WLL has been modified by each center  and a nudecubitus position; the lung is lavaged in the uppermost position, while the non-lavaged lung is mechanically ventilated. 500–600 mL of warmed (37°C) saline is injected in the lung. Fluid is then collected by gravity after opening the outflow tube. Manual chest percussion may be performed to improve drainage. When the outflow, initially milky, becomes clear, chest wall percussion, which restarts and greatly enhances the removal of proteinaceous material, is added. Lavage and percussion are continued until the outflow fluid became definitively clear, which may take 3 hrs and a total of 15–20 L saline for a single lung [The WLL technique at the Pavia Center is performed under general anaesthesia in an operating room or an intensive care unit. The patient is intubated with a double-lumen endotracheal tube and fibre-optic bronchoscopy is performed to confirm the appropriate tube placement. The patient is placed in the lateral Although there are no clearly established criteria for when to perform WLL, the recommendations indicated in available reports include: presence of persistent or progressive respiratory failure; absence of respiratory difficulty at rest, but presence of exercise desaturation (≫5% points); in selected cases, WLL may be discussed if a PAP patient, in particular a young adult, reports a significant limitation in daily or sport activities .WLL is generally well tolerated. The major adverse effect of WLL is hypoxemia, especially during the emptying phase, which decreases airway pressure and increases the perfusion of the lavaged lung. Among potential complications, intraoperative resistance tends to be more common when the first lung is being lavaged. Other more common and less dangerous complications include pneumothorax, pleural effusion, and hydropneumothorax, which can be avoided by meticulous charting of the infused saline solution versus the output, and by taking care not to allow instilled fluid to exceed the fluid drained by more than a few hundred milliliters in consecutive lavages .There are few contraindications, the most common is uncorrectable low oxygen saturation, convulsions and fever, which may indicate infection. Cardiopulmonary instability is a relative contraindication since WLL may lead to a fairly rapid improvement in oxygenation once the proteinaceous material is washed out of the alveoli . HoweverWLL improves survival, with the proportion of subjects free from recurrent PAP reaching ≫70% at 7 yrs . In a loWhen WLL with generalized anesthesia may be hazardous and in paediatric patients, where the use of WLL is less well established mainly because of the technical difficulties associated with the use of large endotracheal tube, the treatment with multiple segmental or lobar lavage by fiberoptic bronchoscopy is considered safer -24. The Although WLL is still considered the gold standard for treatment, the discovery of alveolar macrophage involvement and anti GM-CSF neutralizing antibodies led to the use of GM-CSF as a potential therapeutic approach for PAP. Since it is conceivable that the alveolar space is the site of GM-CSF signal disruption, with the impairment of surfactant catabolism in autoimmune PAP, then it is reasonable to propose that local GM-CSF supplementation would result in better treatment outcome.Evaluation of GM-CSF augmentation as a potential therapy in autoimmune PAP was initially prompted by the demonstration of restorative activities of this cytokine on impaired surfactant metabolism and innate immunity in GM-CSF deficient mice . So far,Later, Seymour and colleagues treated 14 patients over a three month period. Patients not responding to the initial dose of 5 μg/kg/d GM-CSF underwent stepwise dose escalation until a therapeutic response (represented by improvement in oxygenation at the lung level) was obtained.The overall response rate was 43% and lasted a median duration of 39 weeks. Exogenous GM-CSF was well tolerated, and no late side effects were reported .Subsequently, in an open-label study on 25 patients, using escalating doses of GM-CSF from 5 to 18 Mg/kg/day, GM-CSF treatment was associated with amelioration of clinical and quality-of-life-related parameters only in 48%, with relapse rates of 25% among responders . HoweverThe results on the role of GM-CSF antibody titer as a predictor of response to GM-CSF therapy are conflicting. Two papers by Bonfield and coworkers ,30 suggeIn a retrospective series, patients with idiopathic PAP were treated with aerosolized GM-CSF . In thisSubsequently, Tazawa et al. reported on the role of inhaled GM-CSF in 39 patients with autoimmune PAP. The study group received high-dose GM-CSF (125 μg twice daily on days 1–8 for six 2-week cycles) followed by low-dose (125 μg once daily on days 1–4 for six 2-week cycles) over a six month study period. The authors showed a response rate of 62% that was maintained in 83% patients at a one year follow up . OverallIt has also been demonstrated that GM-CSF inhalation therapy decreases markers of surfactant accumulation, including total protein and SP-A (Surfactant Preotein-A) in the BAL fluid of high responders . In partVery recently, the results of a meta-analysis of observational studies  suggest The authors of this meta-analysis speculate on the theoretical possibility of inadequate deposition of GM-CSF with the inhaled route, due to the presence of surfactant in the alveolar spaces, which would not be a problem with the subcutaneous route. However, we have to keep in mind that administration of GM-CSF via the inhalation route shows the best efficacy and prevents hematopoietic toxicity, especially on the long-term basis, encountered with subcutaneous administration .There is usually a lag period of 6–12 weeks in the subcutaneous treatment group and 4–12 weeks in the inhaled group prior to clinical response. This is probably due to the time required for GM-CSF to mobilize hematopoietic progenitor cells into peripheral blood and stimulate them to differentiate into functional alveolar macrophages in the lung.The overall response rate of GM-CSF therapy is lower compared with WLL, in fact it seems only to limit disease progression. For this reason, it has been proposed as stand-alone therapy in PAP patients with less severe disease and as a supplementary therapy to WLL in patients with more advanced autoimmune PAP .In this context, a decrease in GM-CSF requirements was shown by performing WLL, followed by nebulized GM-CSF . It alsoAt the Pavia center, a study protocol has been recently started with WLL followed by inhaled GM-CSF (Sargramostin) in the treatment of Autoimmune PAP (AIFA FARM7MCPK4).The presence of systemic anti GM-CSF antibodies in idiopathic PAP led to the hypothesis that PAP could be an autoimmune disease and hence the rationale for plasmapheresis as a therapeutic option. A 41-year old nonsmoker woman with a 5-year history of non-resolving pulmonary infiltrates was the first case of PAP treated with plasmapheresis. She was refractory to repeated WLL sessions and subcutaneous GM-CSF. On a compassionate basis, she was treated with plasmapheresis, 1.5 plasma volume exchanges on ten separate sessions over a 2-month period. One of these episodes was complicated by gram-negative sepsis and respiratory failure. She subsequently recovered from this, and her anti–GM-CSF antibody titer was reduced, with improvement in symptoms, oxygenation, and radiographs . No longSubsequently, a patient with autoimmune PAP, presenting with persistent disease despite three WLL treatments over 10 months, was treated with plasmapheresis with ten 1.5-L plasma exchanges. Plasmapheresis lowered the serum autoantibody level, but did not improve respiratory impairment. Further WLL therapy was required, but it was transiently effective, with increased length of symptom-free periods between subsequent WLLs .CO and oxygen saturation at rest and during exercise dramatically improved.Reducing the autoantibody level by depleting B cells is a novel approach for PAP. Rituximab is a monoclonal antibody directed against the CD20 antigen of B-lymphocytes. It has been demonstrated to be effective in various diseases mediated by autoantibodies, such as rheumatoid arthritis, idiopathic thrombocytopaenicpurpura and systemic lupus erythematosus. Rituximab was observed to be useful in the treatment of patients with unresponsive PAP. Two single-cases have been described: Borie et al.  describeThe first prospective, open-label, proof-of-concept rituximab trial was conducted in 10 PAP patients . InterveOver the last two decades we have greatly increased our understanding of the pathogenesis of PAP. Studies in mice, in primates and lastly in humans, represent the successful pathway of translational research, by which the results obtained in animal models were quickly transferred to patients. The discovery of the critical role played by neutralizing autoantibody against GM-CSF, which causes a defect in the function of alveolar macrophages linked to the disruption of surfactant homeostasis, has led to the development of a diagnostic test with a very high sensitivity and specificity. Following this research line we will soon be able to attain a better comprehension of the biological and immunological mechanisms regulating not only PAP etiology, but also lung homeostasis.In this context, even if a new alternative therapy to WLL has not yet emerged, modulation of the GM-CSF signaling pathway or novel biological approaches seem to be promising.The authors declare that they have no competing interests.IC wrote the first draft of the manuscript; ZK, FM and EP participated in the design of the review and helped to draft the manuscript; GR, FM and AB revised the description of WLL treatment; ML coordinated the writing of the manuscript and revised substantially all the drafts. All the authors read and approved the final manuscript.


Axonal damage is considered a major cause of disability in multiple sclerosis (MS) and may start early in the disease. Specific biomarkers for this process are of great interest.To study if cerebrospinal fluid (CSF) biomarkers for axonal damage reflect and predict disease progression already in the earliest stages of the disease, that is, in clinically isolated syndrome (CIS).We assessed CSF levels of neurofilament heavy (NFH), neurofilament light (NFL) and N-acetylaspartate (NAA) in 67 patients with CIS and 18 controls with neuropsychiatric diseases of non-inflammatory aetiology (NC). Patients with CIS underwent baseline magnetic resonance imaging (MRI) at 3T, and a follow-up MRI after 1 year was obtained in 28 of them.p=0.05) and NFL (p<0.001) levels. No significant group differences were found for NAA. Patients’ NFH levels correlated with physical disability  and with change in brain volume over 1 year of follow-up  but not with change in T2 lesion load.Compared with NC, patients with CIS had higher NFH (Our results confirm increased neurofilament levels already in CIS being related to the level of physical disability. The association of NFH levels with brain volume but not lesion volume changes supports the association of these markers with axonal damage. Moreover, it is also unclear if these markers are predictive of the evolution of subsequent brain tissue damage. With this study we therefore aimed to confirm previous findings on CSF levels of NF light and heavy chain proteins and NAA in CIS and to explore if they could also serve as predictive markers for disease progression in CIS, that is, to analyse if their concentrations might indeed predict accumulating tissue damage as indicated by MRI.12 from our MS outpatient clinic at the Medical University of Graz  score,with CIS . None ofWe included 18 patients with other neuropsychiatric diseases of non-inflammatory aetiology and a normal CSF as controls . This grThe study was approved by the ethics committee of the Medical University of Graz. All participants gave written informed consent.14 All CSF analyses were performed by trained technicians or biochemists blinded to clinical information. In 46 patients (68.7%) lumbar puncture was performed during their first clinical attack, in 21 patients (31.3%) CSF was obtained during remission after their first attack. Remission was defined as a time lag of at least 14 days between the clinical attack and CSF withdrawal in conjunction with clinical improvement as documented in our patient files.A total volume of 6–10 ml of CSF was obtained by lumbar puncture. After diagnostic work-up, excess of CSF was stored at -80°C until use in agreement with international consensus guidelines.15 All analyses were performed in duplicate and normalized. Inter-assay coefficient of variance (CV) was 17.9 per cent and intra-assay CV was 3.9 per cent. The hook effect16 caused by NF aggregates was avoided by further dilution of the samples to ensure accurate quantification of the immunoassay.Neurofilament heavy (NFH) concentrations were assessed in 67 patients with CIS and 18 controls by an in-house developed multiplex assay as described previously.www.umandiagnostics.com). This ELISA kit has recently been validated in a multicentre study, which identified critical factors such as accurate standard preparation.17 Due to depletion of some CSF samples, NFL levels were obtained from 47 patients with CIS and 15 controls.Neurofilament light (NFL) concentrations were measured using a commercial available ELISA kit from Uman-Diagnostics AB  months between the lumbar puncture and brain MRI at 3T. Follow-up MRI at the same magnet using identical protocols after 1 year was obtained from 28 patients with CIS  using a phased-array head coil with 12 receiver elements and a consistent imaging protocol as described previously.with CIS .All image analyses were performed by trained and experienced technicians and MR readers, blinded to clinical information.19 In addition, separate estimates of volumes of grey matter, white matter, cortical grey matter and ventricular CSF, normalized for subject head size, were estimated using SIENAX, which is part of FSL.20 For assessing T2 lesion load, masks defining the lesions were created and the total lesion load was calculated by multiplying the area of all masks by the slice thickness.Brain tissue volume and hyperintense T2-lesion load were measured as described previously.U test. Student’s t test was applied to compare mean age values between groups. Spearman correlations served to calculate the correlation coefficients between CSF, imaging and clinical data. Pearson partial correlations were performed on ranked variables to correct for age.Statistical analyses were performed using SPSS 18.0 . The Kolmogorov–Smirnov test assessed normality of data distribution. Groups were compared by Mann–Whitney Median levels of CSF of NFH and NFL were 1.5 fold and 2.3 fold higher, respectively, in patients with CIS compared with controls. NAA levels were not significantly different –C. CSF Nr=0.713, p<0.005) and NFL  levels correlated with age. In patients with CIS, this correlation was less strong for NFH  and absent for NFL levels . NAA levels did not correlate with age at time of lumbar puncture.In controls, both NFH , NFL  and NAA levels . After adjusting for age, this correlation remained significant for NFH  and NAA .EDSS score at initial assessment at clinical stability after the first attack positively correlated with NFH .r=-0.518, p<0.01; data adjusted for age) . Higher CSF NFH levels correlated with accelerated global brain volume decrease over a median follow-up of 1.0 years (for age) . There wr=0.551, p<0.001; data adjusted for age). NAA levels did not correlate with NFH and NFL levels.NFH levels correlated with NFL levels in the total group of patients  and NFL  levels significantly correlated with CSF cell count. r=0.252, p<0.05, data adjusted for age) and NFL  levels correlated with the IgG index and NFL levels correlated with albumin quotient . No significant correlations between NAA and IgG index or albumin quotient were present.Routine CSF parameters are listed in 11 This preliminary observation suggests that CSF NFH protein levels may predict progressive brain damage specifically caused by axonal damage.The association of NFH levels with subsequent brain tissue damage over a period of 1 year as defined by MRI is a main and new finding. Our study confirms that CSF NFH and NFL levels are already increased in patients with a CIS suggestive of MS.21 None of the CSF markers of axonal damage analysed in our study correlated with T2 lesion volume at baseline or its change over time. This partly contrasts a previous finding where a correlation of NFL levels with the number of T2 lesions has been described in a combined analysis of patients with CIS or MS.6 Another study found an association of CSF NFH levels with the volume of enhancing lesions in patients with MS.22 However, in our CIS cohort, the prevalence of enhancing lesions was too low to allow such an analysis.The relationship between CSF NF and 3T MRI measures of tissue damage in CIS has not been investigated so far. One study reported a correlation between the CSF/serum index of antibodies directed against NFL and brain atrophy, but this study analysed only patients with relapsing remitting or progressive forms of MS, and no relation with NF proteins was observed.11 and argues for axonal damage to occur already in the earliest stage of the disease, which is also corroborated by recent neuropathological findings.1 NFs are the major axonal cytoskeletal proteins.7 After axonal injury they are released into the extracellular compartment, and subsequently into the CSF and peripheral blood.23 Thus analysis of CSF/serum NF levels may provide a valuable tool to estimate the extent of axonal damage in patients with MS.24 Apart from measuring NF levels in CSF a few reports have also suggested NFH in peripheral blood as a potential marker of neurodegeneration in MS26 but no reports are yet available regarding NFL. Thus further studies on NF in peripheral blood are needed before recommending these tests for assessing MS prognosis. Potential clinical applications of assessment of neuroaxonal damage markers have been illustrated by two recent studies showing that NF levels may indicate treatment efficacy with the monoclonal antibody natalizumab,27 and further capture neurotoxic side effects of aggressive treatment regimens.28Our findings of increased CSF NFH and NFL levels in CIS support earlier suggestions7 Quantification of NAA in the extracellular fluid of the CNS was first described in patients with traumatic brain injury using cerebral microdialysis.29 Evidence for a clinical relevance of NAA comes from several studies, showing that both higher serum and CSF NAA levels correlated with physical disability in relapsing–remitting patients with MS.30 Compared with controls, CSF NAA levels were not altered in CIS in the current study, supporting previous findings of decreased NAA levels rather in later stages of the diseases such as in secondary progressive MS.6Another neuroaxonal marker is NAA.11 these findings suggest that age-related changes in the levels of CSF NF may at least partly be caused by ongoing neurodegeneration, which increases with age. In contrast, this age effect may be covered by pathological conditions, where increased brain tissue damage leads to higher CSF NF levels. Due to the correlation of NFs with age, statistical analyses require correction for this variable to ensure adequate clinical interpretation.11In the control group, we observed a close correlation of NFH and NFL levels with age. This correlation was less strong in CIS for NFH and absent for NFL levels. In line with a recent report on NFH,8 In order to compare both assays direct comparison studies need to be performed, exchanging protein standards, using the same quality controls (low and high) and measuring the same low- concentration CSF samples in every assay.In this respect one should also mention that compared with our luminex-based NFH assay, another electrochemiluminescence immunoassay for NFH assessment with higher sensitivity has recently been developed.31 our results still need to be regarded with caution since the range of the EDSS score in our CIS cohort was relatively small. Previous results on correlations of NFL with EDSS32 could not be confirmed by our study when correcting for age.After adjusting for age, NFH and NAA levels were correlated with EDSS in remission after the first attack, but the correlation was not very strong. Although NFH levels have been shown to correlate with EDSS also by several other groups,6 Whereas in early disease phases CSF NAA levels increase upon axonal damage, decreased levels are observed in advanced stages of the disease.10Although we did not observe any group differences for NAA, higher NAA levels correlated with the EDSS score. This argues for a temporal dynamic of NAA levels as suggested previously.33 In our CIS cohort, we could also not find any differences for NFH, NFL and NAA comparing patients whose CSF was obtained in remission compared with acute patients, as reported previously.32When comparing levels of NF and NAA in patients who converted to MS during follow-up compared with non- converters, no significant differences in biomarker levels emerged. Thus, we could not confirm previous reports on altered NFH, NFL or NAA levels in patients with CIS who converted to definite MS compared with non-converters.32 but not for NFH11 and no reports are yet available regarding NAA.10 Our findings suggest that NFH and NFL may thus be rather related to inflammatory disease activity.34 The correlation of NFH and NFL with the IgG index further supports this notion, but this correlation has not been found in a recently published study.11 We found a correlation of the albumin quotient only with NFL levels, which has not been described in other studies on CSF NFL chain protein.32 In line with previous reports no such relation was found for NFH levels11 and the NFH and NFL levels were correlated.6Another interesting finding of our study is that both NFH and NFL but not NAA levels correlated with CSF white cell count. Such association has been reported previously for NFL6 our results indicate that it is of minor importance in patients with CIS.Our results indicate that analysis of both NF subunits in the CSF markers is relevant in CIS since their relation to clinical and paraclinical findings are different. Whereas NFL more than NFH is closer related to inflammatory signs such as CSF white cell count and IgG index, NFH rather indicates permanent axonal damage and neuronal degeneration which is documented by its relation to physical disability and brain volume loss over time already over the short term. Thus, assessment of CSF NFs in CIS may be of clinical importance to help stratifying patients with more aggressive disease already at the earliest stage of the disease. Although there is good evidence for CSF NAA to serve as a marker for axonal damage in established and progressive MS,


The pulmonary artery (PA) plays an essential role in smoothing the transition from right-ventricular pulsatile flow to the nearly steady-flow at capillary level. The loss of PA compliance has considerable influence on elevated right-ventricle workload. MRI velocity-encoding is an effective technique for assessing pulse-wave-velocity (PWV) by measuring the disturbances in flow or vessel diameter the pressure wave causes . Two metTwenty-five volunteersTable  were scaThe MRI exam lasted for 15-20 minutes. Image analysis lasted for 1 minute and 4 minutes for the TT and QA methods, respectively. The TT and QA methods showed good agreement (P>0.1). The Bland-Altman analysis resulted in mean±SD of 0.13±0.35m/s for the measurement differences. All the differences lied within the ±2SD limit. The correlation coefficient between the two methods was r=0.93. The repeated measurements showed low inter-observer and intra-observer variabilities (Figure The TT and QA techniques showed good agreement in estimating PWV, although the QA method resulted in larger variabilities than in TT. Long processing time was required in the QA method, mostly for identifying vessel cross-sections. However, the TT method required double the imaging time as in QA due to the acquisition of two slices. The use of 3.0-Tesla allowed for improving the temporal and spatial resolutions in the TT and QA sequences, respectively. In conclusion, each technique has its own advantages and disadvantages. The choice depends on patient condition, heart rate, and required image quality.


Non-medical prescription opioid use (NMPOU) and prescription opioid (PO) related harms have become major substance use and public health problems in North America, the region with the world’s highest PO use levels. In Ontario, Canada’s most populous province, NMPOU rates, PO-related treatment admissions and accidental mortality have risen sharply in recent years. A series of recent policy interventions from governmental and non-governmental entities to stem PO-related problems have been implemented since 2010.We compared the prevalence of NMPOU in the Ontario general adult population (18 years+) in 2010 and 2011 based on data from the ‘Centre for Addiction and Mental Health (CAMH) Monitor’ (CM), a long-standing annual telephone interview-based representative population survey of substance use and health indicators. While ‘any PO use’ (in past year) changed non-significantly from 26.6% to 23.9% , NMPOU decreased significantly from 7.7% to 4.0%  between 2010 and 2011. Over-time changes varied by age group but not by sex.The observed substantial decrease in NMPOU in the Ontario adult population could be related to recent policy interventions, alongside extensive media reporting, focusing on NMPOU and PO-related harms, and may mean that these interventions have shown initial effects. However, other casual factors could have been involved. Thus, it is necessary to systematically examine whether the observed changes will be sustained, and whether other key PO-related harm indicators  change correspondingly in order to more systematically assess the impact of the policy measures. Since about 2000, non-medical use and harms  related to prescription opioids (POs) have emerged as a major substance use and public health problem in North America, including Canada,2. WhileThe most comprehensive Canadian data on NMPOU and PO-related harms come from Ontario, its most populous province. There are two reasons for this: overall PO consumption rates (a known determinant of levels of PO-related harms) are higher in Ontario than in most other provinces, and it has the most comprehensive monitoring data available,6. SpeciWhile Canada, unlike countries like the US or Australia, yet remains without a national strategy targeting the problem of NMPOU and PO-related harms, various governmental and non-governmental entities in Ontario have implemented a number of large-scale interventions aiming to reduce these problem phenomena in the past couple of years. For example, in 2010, the Ontario College of Physicians and Surgeons (CPSO) issued a report containing numerous recommendations for improved regulatory control, clinical practice guidelines, and interventions related to POs with the aim of reducing the “opioid public health crisis” in Ontario. In NoveThe above interventions mostly occurred on an ad-hoc basis, and commentators have emphasized the need for systematic evaluations of their effects on reducing NMPOU and harms in Ontario, in the interest of science, evidence-based policy and public health,19. ImpoRelevant annual cycles of the CM were based on telephone interviews with randomly selected annual samples of n = 2024 (2010) and n = 1999  adults ages 18 and older, representative of the Ontario general adult population. Specifically, ‘any PO use’ in the past year was assessed by the combined results of the two questions: “In the past 12 months how many times, if at all, have you used any pain relievers  with a prescription or because a doctor told you to take them?” and “In the last 12 months, if at all, how often did you use pain relievers without a prescription or without a doctor telling you to take them?”; NMPOU was assessed by the second question item only [for further CM methods details, see[While ‘any PO use’ in the past year changed non-significantly , the overall rate of NMPOU decreased significantly from 7.7% to 4.0%  among Ontario adults from 2010 to 2011. Examining the changes by subgroups, there was a significant interaction of survey year with age  = 3.62; p = 0.027): reductions occurred among age groups 55+ years  and 30 – 54 years  but not among ages 18 – 29 years . With respect to sex, the interaction was only marginally significant at the 7% level  = 3.45; p = 0.063), indicating that these data offered no evidence that the reductions in NMPOU were more pronounced in women than in men.One possible explanation for the observed substantial  recent decrease in NMPOU among Ontario adults could be that recent policy measures, combined with extensive media coverage and evolving social debate focusing on PO abuse and related harms in Ontario, may have had some initial effects. However, there is no empirical evidence for such causal influence at present which would, for example, require time-series analyses of relevant indicators if available. Alternatively, factors like ecological or natural changes in general awareness about NMPOU, PO-related drug use behaviors or availability could have contributed to the observed NMPOU reductions. The demonstrated sizeable drop in NMPOU among Ontario adults mirrors recent developments in the US. There, NMPOU has declined in the general adult population as measured by the National Survey of Drug Use and Health (NSDUH); specifically, NMPOU (in past year) among adults 18+ years has declined significantly from 4.7% (2010) to 4.2% . The parNotably, the changes in NMPOU observed in Ontario are markedly uneven by socio-demographic subgroups, i.e. sex and age. This deviates from previously observed patterns of a rather even spread of NMPOU across these socio-demographic groups. ImportaWhile it is yet uncertain whether the observed reductions in NMPOU are causally related to the described recent interventions, and whether other key PO-related problems in Ontario are characterized by corresponding decreases, there are examples from other jurisdictions where such reductions have been observed following targeted interventions. For example, the recent implementation of more restrictive PO dosing guidelines has resulted in substantive reduction of PO-related mortality in Washington State. The burThe authors declare that they have no competing interests.BF led the overall study and writing; RM, JR and AI co-led the CM survey and data analyses. All authors contributed substantively to data analyses and interpretation, as well as contributed to manuscript drafting and revisions. All authors read and approved the final manuscript.


The aim of this paper is to reassess the significance of postmortem cerebrospinal fluid pleocytosis. Published articles of CSF changes after death were reviewed, and reanalysis, in the light of modern views on the significance of bacterial postmortem isolates, was undertaken. There is theoretical and experimental evidence that the blood brain barrier to the movement of protein and cells is preserved in the first few hours after death. The number of mononuclear cells in the cerebrospinal fluid does rise in the first 24 hours after death, and this is most probably due to detachment of leptomeningeal lining cells. But the marked increase in lymphocyte counts seen in some cases of sudden infant death syndrome (SIDS) and in other deaths in the paediatric age range could well be a marker of inflammation. In a healthy individual, the cerebrospinal fluid (CSF) is clear and colourless. It has a low concentration of protein and contains very few cells. The composition is different than that of the blood due to a highly selective blood brain barrier. Any increase in cellular content or protein composition of the cerebrospinal fluid (CSF) above the normal range for the specific age group is an absolute indicator of meningeal disease (inflammation) . But aftPlatt and colleagues obtained postmortem specimens of CSF from 26 cases of sudden infant death syndrome (SIDS), 24 paediatric deaths from a hospital setting, and 14 adult deaths [P < 0.005) than in the hospital paediatric group and the adult group. The cells were mononuclear; the authors do not record the presence of neutrophil polymorphs in any of the cases. The typeable cells were 60–70% macrophages and 20–40% lymphocytes, but beyond 12 hours postmortem, the cells became vacuolated and were difficult to type. It is stated that “none of the postmortem cultures grew bacteria, and sections of the brain revealed no inflammation.”The CSF cell count in the SIDS cases varied from 37 to 3250 cells per cubic mm (mean 647). These counts were significantly higher  due to homicidal firearm wounds, stabwounds, or hanging. The mean CSF protein level in these cases was 0.373 g/L with a standard deviation of 0.181 g/L.13 subjects whose “death agony duration lasted between 10 minutes and 6 hours.” No more clinical information is given but these are cases who became acutely unwell and died within 6 hours of the onset of the illness. The CSF protein in this group was 1.546 ± 0.46 g/L (mean ± SD).16 subjects whose “death agony duration was more than 6 hours” and who died in spite of intensive care. The CSF protein was 8.267 ± 6.249 g/L.These results indicate that if death is rapid and the CSF protein is normal at the time of death, then it will remain within the normal range for a few hours after death. In those first few hours there is no significant passive leakage because the endothelial cells and their tight junctions are still intact. However, over a period of 24 hours the endothelial cells will start to lyse, the junctions will start to leak, and passive transfer will gradually ensue. The passive leakage involves proteins to a small extent but not red cells and by inference not white cells. In groups 2 and 3, the subjects were unwell prior to death and the most likely explanation is production of cytokines as part of the illness causing a generalized inflammatory process throughout the body including the blood brain barrier [Osuna and colleagues also studied CSF protein levels in autopsy cases . They exIn the study by Platt et al. , there wIn the publication by Platt et al. , the folStaphylococcus aureus and Escherichia coli from lung, blood, spleen, or CSF was more common in unexplained SUDI . E. coli was found in 6% of cultures from unexplained SUDI compared with 1% of cultures from explained noninfective SUDI (P = 0.003). The authors are cautious in their analysis and emphasise that they have shown an association and not proven a causal link. But other evidence from a number of studies, reviewed in a lead article in the same issue of the Lancet, supports this link [S. aureus and E. coli have been linked to unexplained SUDI in a number of studies extending back to 1987 [The latest in a long line of publications linking bacterial infection and SIDS appeared in the Lancet in 2008 . A grouphis link . The org to 1987 . There i to 1987 –17. S. aureus and E. coli present in the blood, but in the absence of overt inflammation, can cause death in a short interval, the possible mechanisms include the following. The mode of death in unexplained SUDI is not known but we do know that it is rapid in many cases. In the CESDI SUDI study carried out in England between 1993 and 1996, a total of 318 cases of unexplained SUDI were investigated . If bactThe release of toxins, which directly combine with neural and or cardiac membranes and interfere with homeostatic control of cardiorespiratory function. A heightened or abnormal host response to the bacteria with lymphocyte proliferation, cytokine release, and generalized but subtle signs of inflammation. But since lymphocyte proliferation takes time, there would have to be a subclinical prodromal period prior to the dramatic final episode. S. aureus leading to the explosive release of cytokines from lymphocytes primed by previous episodes of staphylococcal bacteraemia. The cytokine cascade causes toxic shock and a transfer of protein and cells across capillary cell walls in both the systemic and cerebral circulations.A specific mechanism that includes both the above possibilities is the release of pyrogenic toxins  by The morphology, physiology, and pathology of the blood-brain barrier has been studied in detail in animal models and in human subjects. Fishman published a comprehensive review in 1992 and since then there have been further reviews by Ballabh et al.  and AbboIn life, a normal specimen of CSF contains no more than 2 to 4 mononuclear cells per cubic mm and no neutrophil polymorphs. If more cells are found, the sample is not normal and CSF pleocytosis is diagnostic of meningeal inflammation as long as an acute CSF bleed can be excluded . HoweverIf death is sudden, the endothelial lining cells of the cerebral blood vessels will remain intact for a few hours at least. This is what we would expect from our knowledge of their anatomy and physiology. It is also what was observed by Mangin et al.  in theirIn summary, if the lymphocyte entry is passive one would expect to see red cells and neutrophil polymorphs as well as mononuclear cells and a much more marked increase in CSF protein than was observed in the Mangin et al.  study. IThere were two reasons for regarding the lymphocytes seen in the SIDS cases as a postmortem artifact. The first was that there was no evidence of meningitis as assessed by histology. The second was that there was no evidence of infection as the cause of death. The first objection is no longer valid because simple calculations indicate that counting cells in the CSF is a much more sensitive indicator of meningitis than is histological assessment of the meninges. The second objection is also not valid because there is now a considerable body of evidence pointing to an infectious aetiology of SIDS –17. It is time to reassess the pathogenic significance of lymphocytes seen in postmortem CSF samples. We need to collect CSF samples in the first few hours after death and examine them urgently as we would in life. A cell count is essential but we should also use the full armory of modern immunohistological techniques with a wide range of cluster differentiation (CD) markers to differentiate lymphocytes from other mononuclear cells. We need to compare unexplained SUDI with cases in which an explained noninfective cause appears likely. We should also investigate deaths at other ages, not just the paediatric age range but also adults. Bacteria are commonly found at autopsy and too readily dismissed as contaminants or as the consequence of agonal events . It coulStudies are planned, and in some cases underway, to investigate unexplained SUDI using the full array of modern molecular techniques. These include microarray analysis to identify genetic signatures of the host response to infection as well as genetic signatures to mark the presence of pathogens. Proteomic analysis of fluids for bacterial toxins is another area of considerable promise. But we should not forget something which is much simpler-counting lymphocytes in the CSF.


In this study we quantified changes in [NAD(H)] and markers of inflammation and oxidative damage  in the cerebrospinal fluid (CSF) of healthy humans across a wide age range (24–91 years). CSF was collected from consenting patients who required a spinal tap for the administration of anesthetic. CSF of participants aged >45 years was found to contain increased levels of lipid peroxidation (F2-isoprostanes) (p = 0.04) and inflammation (IL-6) (p = 0.00) and decreased levels of both total antioxidant capacity (p = 0.00) and NAD(H) (p = 0.05), compared to their younger counterparts. A positive association was also observed between plasma [NAD(H)] and CSF NAD(H) levels (p = 0.03). Further analysis of the data identified a relationship between alcohol intake and CSF [NAD(H)] and markers of inflammation. The CSF of participants who consumed >1 standard drink of alcohol per day contained lower levels of NAD(H) compared to those who consumed no alcohol (p<0.05). An increase in CSF IL-6 was observed in participants who reported drinking >0–1 (p<0.05) and >1 (p<0.05) standard alcoholic drinks per day compared to those who did not drink alcohol. Taken together these data suggest a progressive age associated increase in oxidative damage, inflammation and reduced [NAD(H)] in the brain which may be exacerbated by alcohol intake.An extensive body of evidence indicates that oxidative stress and inflammation play a central role in the degenerative changes of systemic tissues in aging. However a comparatively limited amount of data is available to verify whether these processes also contribute to normal aging within the brain. High levels of oxidative damage results in key cellular changes including a reduction in available nicotinamide adenine dinucleotide (NAD In 1956 Harman proposed the oxidative stress theory of aging suggesting that the accumulation of unrepaired oxidative damage results in the typical aging phenotype The brain is particularly vulnerable to oxidative damage as a consequence of its high oxygen demand, high level of both polyunsaturated fatty acids and transition metals, and poor antioxidant defenses Both normal brain aging and neurodegenerative disease are characterized by increased inflammation associated with microglial over activation and a subsequent rise in pro-inflammatory cytokines +) has been observed in multiple organs of the rat + is a ubiquitous molecule that is required for a number of vital cellular processes. In addition to its role in cellular energy and metabolism there are several enzymes, including poly(ADP-ribose) polymerase 1 (PARP) and silent information regulators (e.g. SIRT1), that use NAD+ as their substrate + resulting in the formation of poly(ADP-ribose) subunits. Under conditions of mild-to-moderate DNA damage this process facilitates DNA repair + depletion + must be sustained.As inflammation and oxidative damage rise with age a decrease in available nicotinamide adenine dinucleotide  a gradual increase in oxidative damage burden in many of the body’s tissues. If experienced in the brain, neurodegenerative changes are likely. While age is the major risk factor for the development of most neurodegenerative disorders + availability) within the central nervous system. While evidence from cell culture, animal and limited postmortem brain tissue studies support this hypothesis, to date no study has investigated this in a healthy human cohort.Collectively these reports indicate that certain lifestyle choices may accelerate the development of an age associated oxidative-inflammatory state leading to increased tissue damage and reduced DNA repair capacity . We further correlated changes in CSF oxidative/inflammatory markers and [NAD(H)] with specific modifiable lifestyle factors.This study was conducted in accordance with the Helsinki declaration. Ethical approval was obtained from the Human Research Ethics Committee, Sydney Adventist Hospital (HREC# 2011-005). Written informed consent was obtained from all participants prior to commencement.Male (n = 20) and female (n = 50) participants, who required a spinal tap for the administration of anesthetic as part of routine care, were recruited at Sydney Adventist Hospital, Australia. The average age of participants was 53 years . Participants were excluded from the cohort if they were smokers or had a confirmed diagnosis of a neurological/neurodegenerative disorder or central nervous system infection. In total 70 CSF and 38 matched blood samples were collected from consenting participants considered in general good health.Fasting (≥10 h) blood and CSF samples were collected by an accredited anesthetist no longer than 30 minutes apart. CSF samples were collected, prior to injection of spinal anesthetics, via standard lumber puncture. Blood samples were collected into heparinized tubes from an intravenous cannula inserted into a superficial vein on an upper limb, prior to the administration of fluids or anesthetics. Samples were prepared by centrifuging at 1800 rpm for 10 minutes and stored within 1 hour of collection, at −194 degrees Celsius until analysis. Samples intended for F2-isoprostane analysis where stored in the presence of a glutathione/butylated hydroxytoluene preservative.Do you drink alcohol.’ If this was affirmative participants were asked to provide the number of standard drinks consumed per day.Alcohol consumption was assessed via questionnaire upon hospital admission. Specifically participants were asked ‘Total NAD(H) concentrations in plasma and CSF samples were measured spectrophotometrically using a thiazolyl blue microcycling assay established by Bernofsky and Swan (1973) CSF total antioxidant capacity was measured using a standardized commercial kit . This assay relies on the ability of antioxidants to inhibit oxidation of 2,2′-Azino-di-[3-ethylbenzthiazoline sulphonate] (ABTS) by metmyoglobin. Briefly, 10 µL of metmyoglobin was added to 10 µL of diluted sample. 150 µL of chromagen, containing ABTS, was subsequently added and the reaction initiated by adding 40 µL of 441 µM hydrogen peroxide. The plate was incubated on a shaker for 5 minutes and the amount of oxidized ABTS was measured spectrophotometrically, at an absorbance of 750 nm, using a Model 680XR microplate reader .2SO4 was added to the wells to stop the enzyme-substrate reaction. The intensity of this colored product is directly proportional to IL-6 concentration. Absorbance was measured at 450 nm using a Model 680XR microplate reader .IL-6 was measured using a standardized commercial solid phase sandwich enzyme linked-immuno-sorbent assay (ELISA) . Briefly, 100 µL of CSF was added to a plate pre-coated with a monoclonal antibody specific for IL-6. 50 µL of biotinylated anti-IL6 was then added and the plate incubated for 3 hours. After incubation, the plate was washed before the addition of the enzyme, horseradish peroxidase. The plate was incubated for 30 minutes and then washed again to remove any unbound enzymes. The 3,3′,5,5′-Tetramethylbenzidine substrate was added and the plate was incubated in the dark for 12–15 minutes, after which H8-OHdG was measured in CSF samples using a standardized commercial competitive ELISA . Briefly 50 µL of sample or standard and 50 µL monoclonal antibody was adsorbed onto a 96-well plate precoated with 8 OHdG. Following an overnight incubation at 4 degrees Celsius the plate was washed and incubated with a secondary antibody for 1 hour. The plate was washed again before the addition of a chromatic solution for 15 minutes, after which the reaction was terminated and absorbance was measured at 450 nm using a Model 680XR microplate reader .Total F2-Isoprostanes were measured in CSF by gas chromatography–mass spectrometry (GC-MS) using electron capture negative ionization according to a modification in the method of Mori et al (1999) Statistical analyses were performed using SPSS version 16.0 and GraphPad Prism version 5 for Windows. Data is presented as means ± standard deviation unless otherwise stated. Multiple linear regression, controlling for age, was used to identify significant relationships between CSF [NAD(H)], plasma [NAD(H)], and markers of oxidative damage and inflammation. The Independent T Test or Mann-Whitney U Test was employed to analyze the effect of age on markers of oxidative damage, inflammation and metabolism. The Wilcoxon Signed Ranks Test was used to identify the association between mean plasma and CSF NAD(H) levels. Kruskal-Wallis with Dunn’s post-hoc test was used to assess the effect of alcohol consumption on markers of oxidative damage, inflammation and metabolism. Both the Kolmogorov-Smirnov, Shapiro-Wilk and histogram analysis was used to check normality of the variables. When required the Levene’s Test of Equality was used to check homogeneity of variances between groups. If the variances of the groups were found to be either not homogenous and/or normality tests for the variables were not significant then further investigation with graphical displays was performed to assess the distributions of the variables. Both adjusted and non-adjusted P-values are provided throughout with test significance set at P value ≤0.05.A number of studies have shown that lifestyle behaviors in midlife (i.e. around 45–50 years) are associated with reduced cognitive function in later life In this cohort older age was associated with an increase in a number of CSF oxidative and inflammatory markers . SpecifiAssessing these trends in each gender revealed that the CSF of females >45 years contained significantly higher levels of IL-6  and lower TAC levels  than their younger counterparts. Due to low number of male participants ≤45 years, valid comparisons were not possible.CSF NAD(H) levels were significantly lower in participants aged >45 years compared to those aged ≤45 years; 75.88±30.14 vs. 88.59±21.07 µg/mL respectively (p = 0.05) . AssessiAfter controlling for age, a significant inverse association was observed between CSF log TAC and CSF 8 OHdG  . A signiAfter controlling for age, a significant positive relationship was observed between plasma and CSF NAD(H) concentrations  . An incrThe data was further analyzed to identify possible relationships between alcohol intake and CSF [NAD(H)] and markers of inflammation and oxidative damage. For this analysis, participants were divided into three groups, those who consumed zero (n = 32), >0–1 (n = 14) and >1 (n = 8) standard alcoholic drinks per day. CSF NAD(H) levels were significantly different between the groups (p = 0.02). Specifically the CSF of participants who consumed >1 standard drink of alcohol per day contained significantly lower levels of NAD(H) compared to those who reported consuming zero drinks per day; 62.39±19.93 vs. 86.93±25.32 µg/mL respectively (p<0.05) . A signiIn this cohort, the CSF of male participants (n = 18) contained significantly higher levels of IL-6 compared to females (n = 42) (p = 0.00); 2.82±2.23 vs. 1.03±0.91 pg/mL respectively. However when the data was stratified into age groups, this observation did not remain statistically significant . No signConsiderable evidence now indicates that both inflammation and oxidative stress contribute to the development of various neuropathologies including Alzheimer’s and Parkinson’s disease + depletion It is well established, that oxidative DNA damage activates the NAD-dependent DNA repair enzyme, PARP, which is involved in base excision repair + as the sole substrate for their activities + it has been suggested that depletion of NAD+ results in reduced SIRT1 deacetylase activity + depletion resulting in neuronal death + depletion facilitates cell death is supported by observations in rodent models of brain ischemia and Alzheimer’s disease where significantly reduced levels of total cellular NAD+ occur prior to neuronal death + levels are therefore required to maintain cellular energy and robust SIRT1 activity. However further functional studies are required to determine the level of biochemical impact the relatively modest (∼14%) decrease in NAD(H) levels would have on cell metabolism.Adequate levels of NAD(H) are required to maintain normal cellular functions. In addition to its role in cellular energy metabolism there are several enzymes, in addition to PARP, that use the oxidized form, NAD+/NADH dependant mechanisms’ in both the central nervous system and periphery is apparent, it was not previously known whether peripheral stores of NAD(H) influence central NAD(H) levels. This study is the first to show a positive correlation between matched CSF and plasma NAD(H) levels in a healthy human cohort. This is consistent with a previous study by Rex and colleagues (2002) who observed that NAD(H) in both its oxidized and reduced forms is capable of crossing the blood brain barrier in rats +While the physiologic and pathologic importance of NADAlthough age is the major risk factor for the development of most neurodegenerative disorders The suggested pathways by which alcohol may damage the brain are numerous and include, but are not limited to, disruption of neural cell adhesion molecules Drosophila that NAD+ levels decrease by at least 20% in response to ethanol stress We also report for the first time an inverse relationship between alcohol consumption and CSF [NAD(H)]. Specifically the CSF of participants who consumed greater than one glass of alcohol per day had significantly lower levels of NAD(H) compared to those who did not drink alcohol. While research investigating the effect of alcohol on NAD(H) is scarce our results are consistent with a very early report by McElfresh and McDonald (1983) who also observed in While the observations reported in this study are statistically valid it is recognized that these associations have been obtained from a modest number of participants (38 plasma/CSF matched and 70 CSF). Though both genders were well represented in the older age groups, due to the difficulty in obtaining CSF samples from essentially healthy individuals greater numbers of females were represented in the younger age range. The disproportionately low number of younger male participants, as well as the small number of participants for which alcohol consumption data was available, prevented a comprehensive analysis on how gender may influence our findings. Self-reported alcohol consumption was also relied upon in this study and may have introduced some error into our analysis. However while such error may cause the levels of alcohol reported to slightly differ from the number of glasses actually consumed, it is unlikely to significantly affect the rank order of participants. Finally the restricted volume of sample collected as part of this study limited our analytical profile negating comparisons with other important molecular species such as the range of anti-inflammatory cytokines. Future studies overcoming these limitations are required to verify the consistency of our observations.An extensive body of evidence indicates that oxidative stress and inflammation play a central role in the physiology of aging. However, comparatively limited data are available to verify whether these processes also contribute to normal aging within the brain. This study reports for the first time a potential link between aging, increased oxidative stress, inflammation and alcohol consumption and a decline in the essential pyridine nucleotide [NAD(H)], in the CSF of a healthy human population. We also provide evidence of a relationship between peripheral and central NAD(H) stores.Taken together these data suggest a progressive age associated increase in oxidative damage, inflammation and reduced NAD(H) in the brain which may be exacerbated by certain lifestyle choices such as regular alcohol consumption.As reduced NAD(H) levels impact at least PARP and SIRT1 activity the observed decrease in NAD(H) availability within the aging brain may facilitate cell metabolic and genomic instability increasing an individuals’ susceptibility to degenerative disease. However further follow-up of the participants characterized in this study is required to confirm this hypothesis.


Purpose. To evaluate the confocal microscopy findings of a 46-year-old male with bilateral biopsy proven argyrosis. Materials and Methods. Besides routine ophthalmologic examination, anterior segment photography and confocal microscopy with cornea Rostoch module attached to HRT II  were performed. Findings. Squamous metaplastic changes on conjunctival epithelium and intense highly reflective extracellular punctiform deposits in conjunctival substantia propria were detected. Corneal epithelium was normal. Highly reflective punctiform deposits starting from anterior to mid-stroma and increasing through Descemet's membrane were evident. Corneal endothelium could not be evaluated due to intense stromal deposits. Conclusion. Confocal microscopy not only supports diagnosis in ocular argyrosis, but also demonstrates the intensity of the deposition in these patients. Prolonged exposure to silver might cause irreversible pigmentation of the skin (argyria) and/or the eyes (argyrosis) . Hands, Confocal microscopy provides high-resolution, high-contrast in vivo images and is a powerful tool for studying the ultrastructure of the cell, its molecular components, and their functions. The Rostock cornea module is an option of the Heidelberg Retina Tomograph  introduced as an improvement over older confocal microscopes. The module consists of a monochrome laser radiation source which avoids chromatic aberrations and provides extremely sharp images and a high-powered lens that allows the operator to change the confocal plane within the cornea to capture images at different depths without losing sharpness .The aim of this study is to demonstrate the location of conjunctival and corneal silver deposits by confocal microscopy and to evaluate the correlation between conjunctival histopathology and confocal microscopy findings in a case of occupational argyrosis. To the best of our knowledge this is the first biopsy proven argyrosis case to be evaluated by confocal microscopy. A 46-year-old long-standing silver worker who was diagnosed as ocular argyrosis 4 years earlier was evaluated with confocal microscopy. His visual acuity was 10/20 and intraocular pressures were normal bilaterally. The periocular skin and ocular surface displayed diffuse black-gray pigmentation in both eyes . Patchy On confocal microscopy, squamous metaplastic changes on conjunctival epithelium and diffuse highly reflective extracellular punctiform deposits in conjunctival substantia propria were detected bilaterally . CornealGray discoloration of the conjunctiva and scattered gray opacities in the cornea are clinical ocular manifestations of argyrosis –3. Our p Confocal microscopy is a noninvasive tool for in vivo examination of diseases affecting the ocular surface. It provides images of histologic sections of tissue giving a better sense of the three-dimensional nature of the pathology . The HRTIn vortex keratopathy—either due to Fabry disease or due to amiodarone use—the corneal deposits are intracellular due to systemic retention . HoweverConfocal microscopy is a noninvasive diagnostic tool in ocular argyrosis. This technique provides high quality images which display high correlation with histopathologic results. Therefore, in patients opposing surgical intervention, confocal microscopy can be a safe and a reliable approach for diagnosis.


The tolerogenic properties of the liver have long been recognised, especially in regard to transplantation. Spontaneous acceptance of liver grafts occurs in a number of experimental models and also in a proportion of clinical transplant recipients. Liver graft acceptance results from donor antigen-specific tolerance, demonstrated by the extension of tolerance to other grafts of donor origin. A number of factors have been proposed to be involved in liver transplant tolerance induction, including the release of soluble major histocompatibility (MHC) molecules from the liver, its complement of immunosuppressive donor leucocytes, and the ability of hepatocytes to directly interact with and destroy antigen-specific T cells. The large tissue mass of the liver has also been suggested to act as a cytokine sink, with the potential to exhaust the immune response. In this review, we outline the growing body of evidence, from experimental models and clinical transplantation, which supports a role for large tissue mass and high antigen dose in the induction of tolerance. We also discuss a novel gene therapy approach to exploit this dose effect and induce antigen-specific tolerance robust enough to overcome a primed T cell memory response. From the very first experimental liver transplants, it was clear that livers were less likely to be rejected than other transplanted organs . In animSpontaneous acceptance of a transplanted liver leads rapidly to liver donor-specific tolerance in many models , 2, 5, 6There have been many proposed mechanisms for the ability of the transplanted liver to be accepted by the recipient. Initially, it was thought that the high levels of soluble major histocompatibility (MHC) molecules produced by the donor liver were responsible for liver tolerance . This ha+ T cells and hepatocytes [A recently proposed mechanism for the ability of the liver to induce tolerance is based on the unique vascular architecture of the liver which allows intimate contact of circulating T cells with hepatocytes. This is facilitated by the fenestrated endothelium of the liver sinusoids, where small endothelial pores (fenestrae) permit contact between recirculating T cells and hepatocytes. The intact lining and tight junctions of the endothelium in other organs prevents contact with parenchymal cells. The slow rate of blood flow in liver sinusoids further aids the establishment of contact between circulating CD8atocytes . Contactatocytes  or to thatocytes . Both ofatocytes –29. DespA further basis of liver transplant acceptance is the large size of the liver, approximately 10 times greater than that of a heart or a kidney. This mass of tissue can function as a cytokine sink and/or dilute the finite clones of alloreactive T cells and thus potentially exhaust the recipient's immune response. There is mounting experimental evidence that the volume of allogeneic tissue transplanted is an important contributor to tolerance, as increasing the mass of transplanted tissue prolongs survival. Of considerable interest, in clinical transplantation, there is convincing evidence from many studies that multiple organ transplants from the same donor to a single recipient have a better outcome than single organs alone. As there has been no previous review of the dose effect in organ transplantation, the following sections will examine the experimental and clinical evidence for high dose tolerance and describe a gene therapy approach that can exploit it as a potential means to induce antigen-specific tolerance.The first description of a dose effect in transplantation was Medawar's landmark study on the immunological basis of skin graft rejection, part of which described more rapid rejection of larger compared to smaller skin grafts in rabbits . In thisSubsequent studies in mouse skin transplant models with minor antigen mismatches gave rise to similar findings. The first of these showed that if the donor and recipient were incompatible at loci other than H-2, larger grafts demonstrated prolonged survival. They also observed that while small secondary grafts underwent accelerated rejection, larger secondary grafts did not . SimilarSince then, studies in the fully histoincompatible rat transplant model of PVG donor to DA recipient have shown that increasing antigen load, by transplanting multiple organs, increases allograft survival rates. Transplantation of one heart or kidney in this model led to rejection in 9 and 8.5 days, respectively. Administration of donor leucocytes alone could not increase survival of a cardiac graft, but transplanting two hearts and two kidneys, with donor leucocytes, led to spontaneous acceptance and indefinite survival of the grafts . Two or This dose effect was also observed in an inbred miniature swine model where single MHC class I mismatched heart allografts were rejected within 55 days after transplanting into cyclosporine-treated recipients . In contOne possible explanation for the prolonged survival of very large grafts is nonspecific immunosuppression due to the increased trauma associated with the surgery involved in transplanting massive or multiple grafts . Alternab, expressed on the donor grafts. T cell-depleted mice were immunocompromised and were unable to reject H-2Kb-expressing heart, skin, or islet grafts without the addition of the alloreactive T cells. Adoptive transfer of the H-2Kb-specific T cells resulted in rejection of all three types of graft although many more cells (~6000-fold) were required to mediate rejection of the heart grafts than the skin and islet transplants, confirming that the latter two are more susceptible to rejection. This suggested that a threshold number of donor-reactive T cells is required for graft rejection and that this threshold is higher in heart grafts compared to skin or islets.Over the years, a number of rodent studies have demonstrated that there is a hierarchy of susceptibility between different graft types and sizes with skin and intestine being the most prone to rejection while islets, hearts, kidneys, and livers are progressively more easy to tolerize –49. It hThese findings were confirmed in a polyclonal system by a study which assessed the T cell response following heart and skin grafting in a mouse model of HY minor antigen mismatch . Female b-specific T cells, to examine the response in mice following transplantation of H-2Kb-expressing heart, kidney, and liver grafts [A more recent study used the model described above , in whicr grafts . Transfeγ chain cytokines and their receptors in the PVG to DA rat model of transplantation [γc, IL-2Rα, IL-2Rβ/IL-5Rβ, IL-4Rα, IL-7Rα, IL-9Rα, IL-15Rα, and IL-21Rα) were assessed by qPCR at days 3, 5, and 7 following grafting and, except for IL-21, the levels of the γ chain cytokines and receptors were lower in transplanted livers than in hearts or kidney. As γ chain-signalling is crucial for T cell survival, this indicated that the tolerance seen in this model may be due to a low level of signalling, reflecting the “dilution” of a fixed number of alloreactive T cells in a large organ or tissue mass. Some recent work has examined whether this tolerance is due to exhaustion by examining the expression of various antation . In thisThe observations made in animal models relating to the tolerogenicity of the liver have also been supported by clinical transplantation data. There have been a number of anecdotal reports of tolerant patients, resulting from a variety of treatments, and up to 15% of liver transplant recipients have been shown to be able to completely discontinue immunosuppressive therapy  mice were injected with rAAV-H-2Kb to induce specific expression of H-2Kb on their hepatocytes [b on a k haplotype background. Therefore, we were able to express the single, mismatched MHC antigen, found on the donor skin graft, in the recipient's liver prior to transplantation. Uninjected B10.BR mice rejected their 178.3 skin grafts with an MST of 18.5 days, while mice injected with rAAV-H-2Kb accepted their grafts long term. Injection with low doses of rAAV-H-2Kb did not produce the same result. This confirmed that high-level, liver-directed donor MHC antigen expression was able to promote tolerance to donor-strain skin grafts.We employed a minimally immunogenic recombinant adenoassociated virus (rAAV) vector, designed to specifically express high levels of antigen in host livers , 90 to aatocytes . These rb-expressing skin graft which they rejected in the expected time. Uninjected mice rejected secondary 178.3 skin grafts with an accelerated tempo, as expected. However, primed mice that were then treated with rAAV-H-2Kb accepted their second skin grafts in the majority of cases, reminiscent of the effect observed previously where skin grafts onto rats that had received a liver transplant from the same donor survived for long term [b led to a significant reduction in the number of cells expressing IFNγ in response to H-2Kb. However, we did not see deletion of H-2Kb-reactive cells and we were also unable to demonstrate any linked epitope suppression following gene transfer, which might have indicated development of regulatory cells. Therefore, we suggest that the mechanism of tolerance induction in this case is a form of functional silencing of alloreactive T cells, mediated by high-level expression of antigen in hepatocytes.We were able to achieve this tolerance even in animals previously primed to produce a memory response to the antigen. Mice were primed with an H-2Kong term . This waong term . We founExpression of donor MHC, following gene transfer to different recipient cell types in rodents, has previously produced variable results regarding allograft survival. A study of the ability of MHC class I or II expression to induce unresponsiveness to cardiac allografts in mice showed that pretreating recipients with cells (expressing a variety of MHC antigens) before grafting was able to prolong survival. However, this was dependent on the immunogenicity of the antigen and the load delivered . SimilarWe have previously suggested a mechanism of tolerance based on the idea that there is a relationship between antigen dose and tolerance induction . At low Overall, the evidence from experimental and clinical transplantation seems to support this type of model. Initially, it seemed that the liver had a unique ability to be accepted and could protect other organs against rejection. However, following the confirmation, from both animal models and clinical analyses, that other organs and multiple organs can exert a similar affect, it seems more likely that antigen dose plays a larger role in this process than simply the type of graft. From our work with liver-directed expression of donor MHC mediated by the AAV vector system, we have been able to confirm that protection to other organs can be induced solely by expression of high doses of donor antigen in the liver. This work has not delineated the relative contributions of liver-specific expression and high-level expression to tolerance induction. Further studies using this approach to achieve high-level expression of donor MHC in nonliver tissues should yield this information.


Granulocyte-colony stimulating factor (G-CSF) is one of the most important pharmacologically used proteins. Potential uses beyond the stimulation of neutrophilic granulocytes are the treatment of CNS disorders. Disadvantages of the G-CSF protein as a drug are its moderate plasma half-life time and considerable production costs. We therefore conducted a screen for peptide agonists derived from the sequence of human G-CSF.Despite of the high sensitivity of our screening system we could not detect any positive hits in a single peptide approach. In a multiplex approach using a permutation of any combination of 10 different peptides we could also not detect a positive block.We conclude that larger coherent parts of the protein or dimerising peptides may be needed to achieve activation of the receptor. Granulocyte-colony stimulating factor (G-CSF) is one the most widely employed protein drugs. It is mostly used for counteracting neutropenia in patients receiving chemotherapy , but alsRecently we and others have defined a novel spectrum of G-CSF activities in the central nervous system where it acts on neurons. For example, it was shown that G-CSF can reduce the infarct size in animal stroke models -11 and tDisadvantages of the G-CSF protein for a potential lifetime continuous therapy as in the case of chronic neurodegenerative conditions are its limited plasma half-life time (~4 h), the relatively high costs, and chronic effects on the hematopoietic system. Pegylated forms of G-CSF are available that have a much extended half-life, however, it is unclear at present if those modifications hinder passage of the blood-brain-barrier (BBB).We therefore decided to conduct a screen searching for peptides derived from the human G-CSF peptide sequence with agonistic activity. Peptides would be considerably cheaper to produce, be potentially suitable for delivery methods other than subcutaneous injection, and might also show improved neuronal selectivity.It was previously shown for a number of protein receptors that this is a feasible concept in principle. For example, peptides derived from the NCAM  sequence, a cell surface glycoprotein that belongs to the Ig superfamily, act agonistically on the fibroblast growth factor (FGF) receptor -17. NCAMMoreover, agonistic peptides have been found for the EPO receptor (EPO-R), functionally very close to the G-CSF receptor (G-CSF-R), both derived from the EPO sequence  or fullyIn 1998 the nonpeptidyl small molecule SB 247464 was identified as a ligand of the murine G-CSF receptor in a high-throughput cell based screening approach by Tian and colleagues . Like G-Thus we conducted a sensitive screen for peptide agonists derived from the sequence of human G-CSF employing the murine myeloblastic cell line NFS-60. This suspension cell line expands rapidly in culture and is dependent on G-CSF of either murine or human origin, or alternatively on M-CSF (Macrophage colony-stimulating factor) or IL-3 (Interleukin-3) . Our ass® Library Design Tool [For the design of the peptide library the protein sequence of human G-CSF [NCBI: NP_000750] was used. The first 29 amino acids of the G-CSF protein  were not considered for the library. For the remaining 178 amino acids of the G-CSF sequence we designed an overlapping peptide library for G-CSF with the help of the PepScreenign Tool . We chos2 fraction of 5%.The murine myeloblastic cell line NFS-60 expands rapidly in culture and is dependent on G-CSF of either murine or human origin, or alternatively on M-CSF (Macrophage colony-stimulating factor) or IL-3 (Interleukin-3) . The cel4/well in 96-well plates. Subsequently human recombinant G-CSF  was added to the cells at increasing concentrations, and cells were incubated for 48 h at 37°C. Viability of the cells was detected using the MultiTox-Fluor Multiplexicity Assay  as described by the provider. Fluorescence was measured using the plate reader FLUOstar  at 390 nm/520 nm.NFS-60 cells were washed twice with cell culture medium containing no G-CSF and were then plated at a total density of 1 × 1096-well plates were prepared with 8 peptides with 8 replicates/plate, 2 negative controls ) at 8 replicates/plate, and two positive controls, one at the lower end of sensitivity, and one at a 100% viability level (each at 8 replicates). In order to avoid evaporation of the edging wells and to have the same conditions for all probes the samples with their eight replicates were pipetted in a diagonal pattern on a 96-well plate automatically by the MultiProbeII EX from Packard [Additional file 4/well. The peptide plate was then transferred by the Liquidator96 (Steinbrenner Laborsysteme GmbH) to this plate and incubated at 37°C for 48 hours. Final DMSO concentrations were 0.2% for the 1 μM screen, and 2% for the 10 μM screen. For detection of viability the MultiTox-Fluor Multiplexicity Assay from Promega was used as described by the provider. The fluorescence of the plate was measured in a fluorescence reader (FLUOstar) at 390 nm and 520 nm.NFS-60 cells were washed twice with cell culture medium containing no G-CSF and were then plated in a 96-well plate at a total cell number of 1 × 10Up to 20 peptides per well were tested in a multiplex approach. The 80 peptides of the library were arranged in eight groups per ten peptides. Permutations of all combinations of two peptide groups were tested.All results were evaluated in EXCEL. For analysis of the data a student's t-test against both negative controls was performed. A p-value <0.05 was considered significant.We used the G-CSF-dependent cell line NFS-60 for screening a library of G-CSF-derived peptides. First we established a survival curve over a wide concentration range of G-CSF Figure  with a r4-fold higher molarity than the lower positive control. Results for the 80 peptides are given in Figure We designed a 20-mer peptide library derived from the human G-CSF sequence with a sliding overlap of 2 amino acids. We first conducted a screen at a concentration of 1 μM per peptide, a 10 We conducted a second screen at a concentration of 10 μM per peptide. This resulted in an increase of the DMSO concentration to 2%, the highest level tolerated by the NFS-60 cell line as established before (data not shown). While at a 2% DMSO concentration the upper positive control remained at 0.1 nM as above, the lower positive control had to be elevated to 3.4 pM to show clear increase in viability. These concentrations were chosen as lower and upper positive control in the screen. Results are shown in Figure As both screening approaches of single peptides with molarities of 1 μM and 10 μM were unsuccessful, we finally tried a multiplex approach, based on possibly necessary cooperative effects of different protein epitopes in order to activate the G-CSF receptor. As the cell line NFS-60 showed high sensitivity towards higher DMSO concentrations the maximum number of peptides to be tested per well were 20 at a total molarity of 10 μM (single concentration 0.5 μM). This resulted in a DMSO content of 2%.Figure 7-fold less potent than the parent protein, we could not detect any positive hits. Also in a multiplex approach using a permutation of any combination of 10 different peptides we could not detect a positive block. A post-hoc power analysis of our screen revealed that it should have been possible to detect an increase in viability down to 10% with a power of 80%.In the present study we have utilized the G-CSF dependence of the cell line NFS-60 to screen for agonistic peptides derived from the G-CSF human sequence. Although the screening system was highly sensitive and would have allowed the detection of an agonistic peptide at least 3.4 × 10 Reasons for this failure may either be technical problems, or principal in nature. We believe that the latter is true. The coverage of the G-CSF sequence was certainly dense with a frame shifted by 2 amino acids over the entire sequence. The purity of the synthesized peptides was reasonable with a minimum content of pure and intact peptide of 30%, and the peptides were all fully dissolved at the assayed concentrations.It is possible that the length of the peptides was not sufficient. However, other agonistic peptides described for the EPO (Erythropoietin) receptor or NCAM  have 20 or less amino acids. Screening much longer peptides would also make less sense in view of their pharmaceutical advantage over the full-length protein.An interesting point is the potential need for dimerising peptides. G-CSF itself dimerizes when binding to its receptor, and induces receptor dimerization -6. Indee(G-CSF): Granulocyte-colony stimulating factor; (G-CSF-R): Granulocyte-colony stimulating factor receptor; (JAK): Janus kinase; (ALS): Amyotrophic lateral sclerosis; (BBB): blood-brain-barrier; (NCAM): neural cell adhesion molecule; (FGF): fibroblast growth factor, (EPO-R): Erythropoietin receptor; (M-CSF): Macrophage colony-stimulating factor; (IL-3): Interleukin-3.The authors are employees of SYGNIS Bioscience. This does not interfere with any aspects of this study.NC established, designed, and carried out the screening assay and analysis, and wrote the manuscript. AS conceived the study, contributed to analysis of the data, and wrote the manuscript. Both authors read and approved the final manuscript.Schematic design of the screening plates. Description: 96-well plates were filled in a diagonal way to avoid evaporation effects and to get equal conditions for every sample. (A and B) Exemplary design of plates for 1 μM and 10 μM screening. Plates contained two negative controls, two positive controls and eight peptides with arising numbering. Second positive control had to be adapted due to increased DMSO content in the 10 μM screening. (C) Exemplary graphic of plates for the multiplex screening. Layout of controls was analogue to the 10 μM screening.Click here for file


Moreover, these mutations occur exclusively in the male germ line and older men have a higher probability of having an affected child than younger ones, known as the paternal age effect (PAE). The classic example of a genetic disorder exhibiting a PAE is achondroplasia, caused predominantly by a single-nucleotide substitution (c.1138G>A) in FGFR3. To elucidate what mechanisms might be driving the high frequency of this mutation in the male germline, we examined the spatial distribution of the c.1138G>A substitution in a testis from an 80-year-old unaffected man. Using a technology based on bead-emulsion amplification, we were able to measure mutation frequencies in 192 individual pieces of the dissected testis with a false-positive rate lower than 2.7 × 10−6. We observed that most mutations are clustered in a few pieces with 95% of all mutations occurring in 27% of the total testis. Using computational simulations, we rejected the model proposing an elevated mutation rate per cell division at this nucleotide site. Instead, we determined that the observed mutation distribution fits a germline selection model, where mutant spermatogonial stem cells have a proliferative advantage over unmutated cells. Combined with data on several other PAE mutations, our results support the idea that the PAE, associated with a number of Mendelian disorders, may be explained primarily by a selective mechanism.There are certain  Within the last decade, it was shown for Apert syndrome –24 and m−6. We then measured the c.1138G>A mutation frequency in 192 testis pieces from an unaffected 80-year-old donor and analyzed the data using computational modeling of spermatogonial stem-cell division schemes.It has previously been suggested that the ACH mutation in FGFR3 is driven by selection mechanisms similar to those seen for Apert mutations in FGFR2 . The number of mutant beads divided by the total number of wild-type and mutant beads was used to estimate the ACH mutation frequency of the original testis piece.The number of mutant ACH molecules c.1138G>A) was assessed by a highly sensitive methodology based on the amplification of millions of single DNA molecules with BEA ,29, foll138G>A waE. coli DNA spiked with 330 000 copies of a plasmid containing a wild-type ACH insert . We found no c.1138G>A mutations after genotyping 369 000 beads , demonstrating a false-positive rate <2.7 × 10−6. To assess the accuracy and reproducibility of the assay, we used a series of positive controls with varying amounts of genomes with the ACH mutation spiked into wild-type DNA at mutation frequencies of 1:10, 1:100, 1:1000 and 1:10 000. We found a good correspondence between the input ratio of ACH copies to wild-type copies with respect to the actual measured ratio . We also performed technical replicates on 18 testis pieces with low mutant counts for which a second aliquot of the initial pre-BEA PCR was analyzed . For these pieces, the number of mutants per million genomes (pmg) was consistent and not significantly different between measurements. Two pieces with one of the highest measured frequencies (Slice 1-Piece 1 and Slice 1-Piece 3) were re-amplified from testis genomic DNA and measured again by one or multiple BEA replicates. In this case, the measurements were also reproducible .We established the false-positive rate of our technology using as a proxy for the testis piece 1 μg of Supplementary Material, Table 4. The mutations are not uniformly distributed throughout the testis but are highly clustered. In fact, there are a small number of pieces with mutation frequencies that are orders of magnitude greater than the remaining pieces. The average mutation frequency (Av) for the whole testis is 122 mutants pmg. The piece with the maximum mutation frequency (Mx) is 3072 pmg (0.31%). This piece is adjacent to a piece with no mutants. Next, we introduce several summary statistics to quantify the amount of mutation clustering. The ratio of the maximum piece frequency to the testis average frequency (Mx/Av) is 25. If the mutations were uniformly distributed and there were no clusters, this ratio would be closer to 1. Most of the pieces have low mutation frequencies: the fraction of the pieces with mutation frequencies <50 pmg (F < 50) is 76% . As described previously –21, the In order to understand the unexpectedly high frequency of the c.1138G>A mutation and its highly clustered distribution in the testis, we consider several computational models. We have previously proposed these models based on what is known about human germline development and maturation. More details on these models including the computer code used for the model simulations can be found in the original publications –21.All the models have two phases: a growth phase and an adult phase. The growth phase models the testis from zygote formation to puberty. During the growth phase, the germ cells divide symmetrically and increase in numbers exponentially. Since the daughter cells share all the mutations of their parent cells (and possible add a few more) and remain physically close to their parent cells, mutations in the growth phase will produce mutation clusters. The growth phase is the same in all the models, but the models differ in terms of the details of the adult phase. The adult phase models the testis after puberty, when the germ cells originating in the growth phase have formed the adult self-renewing Ap spermatogonial stem cells (SrAp). We tested four models: (i) a hot spot model which predicts a uniform distribution of the mutation throughout the testis (asymmetric hot spot model); (ii) a variant of the hot spot model called the symmetric hot spot model; (iii) a selection model which predicts mutational clusters caused by the expansion of mutated SrAp (selection model) and (iv) a variant of the selection model that also incorporates cell-death at an advanced age (selection model with cell death).−8  and (iii) the N95 statistic in the testis is 27%, but in 95% of simulations this fraction is 95%. All three summary statistics are significantly different than measured in our data (P < 10−6) as summarized in Table In this model –21, the Rejecting not just the asymmetric hot spot model, but the idea that the elevated c.1138G>A mutation frequency is due to hypermutation at this site, is a strong conclusion. So, we have also considered a model based on a different SrAp division scheme motivated by work in non-human primates  and miceF < 50 and N95 summary statistics (Table P < 10−6).As shown in Table cs Table . BecauseF < 50 and N95 summary statistics are also similar between the simulated and the observed data  , but the selection parameter p set to zero, so as to model a neutral site rather than a site subject to germline selection, have an average mutation frequency of 2.7 × 10 × 10−8) –18. This−8, which is now within a factor of two of the estimate for transitions at CpG sites derived from genome wide data. We originally introduced cell death into the selection model to explain the observation that for MEN2B some of the oldest donors do not have mutation clusters  throughout an entire testis of an 80-year-old unaffected man. We used a technology based on BEA to get a highly accurate and sensitive measurement of the c.1138G>A mutation in each of the 192 pieces of a whole testis. This method allowed us to measure mutation frequencies as low as 108G>A thrositivity , which rP < 10−6). Our testis data are, however, consistent with a selection model in which mutant SrAp cells have a proliferative advantage over unmutated SrAp. All that is required is a slight chance of undergoing a symmetric division . Similar clustering patterns in the testes of middle-aged and older men were also observed for Apert syndrome and MEN2B, but not in young (<21 years) testes, indicating that the clonal expansion occurs in the adult phase of spermatogenesis (In spite of the high frequency of sporadic cases of achondroplasia (1/15 000–1/30 000) with new c.1138G>A transition mutations at a CpG site, an explanation evoking hypermutation at this nucleotide is not compatible with the observed clustering of the ACH mutation we detected in the testis. Both the asymmetric and symmetric hot spot models were rejected  on ACH was also analyzed previously for both the Apert syndrome (c.755C>G) and MEN2B c.2943T>C) mutations . For theT>C mutatde novo ACH mutation than fathers in their twenties  or thanatophoric dysplasia II (K650E), for which either no correlation with age or a positive correlation was detected in sperm analysis, respectively , could hThe testis, frozen 10–12 h after death, was obtained from the National Disease Research Interchange  from a donor free of drug treatments that interfere with normal spermatogenesis. The sample was collected from an anonymous organ donor and complied with the ethical regulations for collection of human samples approved by the Institutional Review Board of the University of Southern California and certified as exempt (46.101 b4). Testis dissection and DNA isolation was performed as published ,21.m each dNTP (Roche Applied Science), 2U of Phusion Hot start DNA polymerase (NEB) and 0.2 µm of each primer F-ACH-88 and R-SNP. The reactions were held at 98°C for 2 min followed by 20 cycles of 98°C for 15 s, 68°C for 10 s and 72°C for 30 s and a final extension step at 72°C for 2 min.The number of amplifiable genomes in the DNA from each testis piece was determined by real-time PCR using primers F-ACH-88 (GAGCTGGTGGAGGCTGACGA) and R-SNP (GGTCAGCAGGGGCAGGTTGG) that included the c.1138 site. Then 1 µg of amplifiable DNA from each individual testis piece was pre-amplified for 20 cycles, which served as template DNA for the subsequent steps of emulsion PCR (BEA). Each 100 µl pre-BEA PCR reaction included 1× Phusion HF buffer (NEB), 0.2 m6 beads covered with the dual biotinylated primer R-ACH-bead primer (MWG/Operon) [bioteg][bio-on]CAGAGCGTCACAGCCGCCACCACCAGGATGAACAGGAAG, in 1× Titanium buffer and 16 mm MgCl2. The last 20 bp of this primer are complementary to the 3′ end of the pre-BEA PCR product. Hybridizations were carried out for 2 min at 94°C, followed by 15 min at 68°C and cooling down to room temperature. The beads were washed once with water and mixed with the aqueous PCR phase  and the oil phase prepared as described previously  were aligned by computing the normalized cross-correlation matrix of pairs of images in Matlab (Mathworks) as described previously . A mask E. coli DNA was spiked with 330 000 copies  of a linearized plasmid with a 550 bp FGFR3 insert containing the wild-type c.1138 locus and amplified by ‘pre-BEA PCR’ (using the same conditions as used for 1 µg of genomic testis DNA). A dilution was further analyzed with BEA as described earlier. The plasmid was constructed by cloning a PCR product derived from blood genomic DNA amplified for 35 cycles using the ‘Pre-BEA PCR’ conditions into a pJET1.2/blunt Cloning Vector (Fermentas) of the CloneJet PCR Cloning Kit (Fermentas). Clones with an insert were verified by PCR and sequencing. The purified plasmid was linearized by digestion with XhoI or sonication for 5 s and quantified spectrophotometrically and by qPCR using human genomic DNA as standards. We also confirmed that the amplification efficiency of the plasmid was equivalent to 330 000 human white blood cell genomes.For experiments used to determine the false-positive mutation frequency of the assay, 1 µg of E. coli DNA spiked with 330 000 copies of wild-type plasmid (described earlier).In order to examine the efficiency of detecting the ACH mutation, different ratios of mutant to wild-type were constructed and processed by the assay (Pre-BEA PCR and BEA using the same conditions as for the testis DNA). For mutant to wild-type FGFR3 ratios of 1:10, 1:100 or 1:1000, triplicate samples contained 1 µg of human blood genomic DNA (Clontech) and varying amounts of genomic DNA derived from an achondroplasia patient (Coriell Cell Repositories). For the 1:10 000 ratio, we measured two samples which contained genomic DNA derived from an achondroplasia patient (30 genomes) and 1 µg of Supplementary Material, Table S4. To compute the average mutation frequency for the entire testis, the mutation frequency for each piece was weighted by the number of genomes in each piece. The total number of genomes per testis piece was estimated previously by real time PCR  measured in the testis. We then simulate the models using these parameters and only consider those simulations with Av within 5% of the Av observed for the testis. We repeat until we have one million such simulations (for each model), and then we test whether the summary statistics measuring clustering  in the testis match the values of these summary statistics in the simulations. If the summary statistics for the testis are within the range of summary statistics for the simulations then the model is consistent with the data; conversely, if the summary statistics for the testis are outliers for the range of summary statistics in the simulations then the model is rejected (F < 50 fraction went from 76 to 66% and the fraction of the testis necessary to capture 95% of mutants (N95) went from 27 to 41%. These changes in the statistics had no effect on the conclusions about the models.The hot spot and selection models have been proposed previously –21. Linkrejected –21. We aHMG online.Supplementary Material is available at ‘Austrian Science Fund’ (FWF) (P25525-B13 to I.T-B.); and National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM36745 (N.A. and P.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS or NIH. Funding to pay the Open Access publication charges for this article was provided by the Austrian Science Fund (FWF).This work was supported by the