During gamete formation, crossover recombination must occur on replicated DNA to ensure proper chromosome segregation in the first meiotic division. We identified a Mec1/ATR- and Dbf4-dependent replication checkpoint in budding yeast that prevents the earliest stage of recombination, the programmed induction of DNA double-strand breaks (DSBs), when pre-meiotic DNA replication was delayed. The checkpoint acts through three complementary mechanisms: inhibition of Mer2 phosphorylation by Dbf4-dependent Cdc7 kinase, preclusion of chromosomal loading of Rec114 and Mre11, and lowered abundance of the Spo11 nuclease. Without this checkpoint, cells formed DSBs on partially replicated chromosomes. Importantly, such DSBs frequently failed to be repaired and impeded further DNA synthesis, leading to a rapid loss in cell viability. We conclude that a checkpoint-dependent constraint of DSB formation to duplicated DNA is critical not only for meiotic chromosome assortment, but also to protect genome integrity during gametogenesis.DOI:http://dx.doi.org/10.7554/eLife.00844.001 Most cells in an organism contain two sets of chromosomes, one inherited from the mother and the other from the father. However, sexual reproduction relies on the production of gametes—eggs and sperm—which contain only one set of chromosomes. These are produced through a specialized form of cell division called meiosis.Meiosis begins with a cell replicating its entire genome. Maternal and paternal versions of each chromosome then pair up and swap sections of their DNA through a process known as homologous recombination. This gives rise to chromosomes with new combinations of maternal and paternal genes. Finally, the cell undergoes two successive rounds of division—the first to produce a cell with two nuclei containing two sets of chromosomes each, and the second to produce four gametes, each containing a single set of chromosomes.Homologous recombination requires the formation of double-strand breaks in the DNA, but it is essential that these do not form before DNA replication is complete. Now, Blitzblau and Hochwagen have used yeast, which is easy to maintain in the lab and to manipulate genetically, to reveal the molecular components of a checkpoint that controls this process.Blitzblau and Hochwagen first used an inhibitor called hydroxyurea to block DNA replication in yeast cells, and confirmed that this treatment also suppressed the formation of double-strand breaks. By selectively inhibiting the activity of individual proteins, it was shown that break formation was controlled by a checkpoint that relies on two conserved proteins, the checkpoint kinase Mec1 (homologous to the human tumour suppressor ATR) and the cell-division kinase DDK. Moreover, when double-strand breaks were allowed to form on partially replicated chromosomes, DNA replication stalled and meiosis could not proceed normally, with lethal results for the yeast.These results explain how DNA replication and recombination are coordinated during meiosis in yeast. Moreover, because the genes that control meiosis are highly conserved from yeast to humans, they have implications for research into human fertility.DOI:http://dx.doi.org/10.7554/eLife.00844.002 During meiosis, a single round of DNA replication is followed by two nuclear divisions to produce haploid gametes from diploid progenitor cells. In most organisms, the faithful segregation of homologous chromosome pairs in meiosis I relies on physical connections between homologs produced by meiotic recombination. In budding yeast, DNA exchanges between homologs are the result of the repair of ∼160 DNA double-strand breaks (DSBs), which occur immediately following pre-meiotic S phase (meiS) and are distributed across all 16 chromosome pairs. DSB formation requires the concerted activity of 10 proteins, including the catalytic subunit Spo11 and its partner Ski8, the Mre11/Rad50/Xrs2 (MRX) complex, and the meiosis-specific proteins Mer2, Rec114, Mei4, Rec102 and Rec104 (reviewed in ). AdditiUnder normal circumstances, meiotic DSBs are introduced after DNA replication. This temporal separation is necessary because the crossover-mediated linkages between homologs require sister-chromatid cohesion distal to the crossover site. Thus, only crossovers formed after DNA replication serve to hold homologous chromosomes together in metaphase I. Moreover, replication forks are unable to cross a DSB , so the The mechanisms that coordinate pre-meiotic DNA replication and DSB formation are not well understood. DNA replication and DSB formation are coordinated at the local level, because delayed replication of a single chromosome arm delays DSB formation on that arm . HoweverThe replication checkpoint couples DNA replication and cell cycle progression by sensing and coordinating the response to delayed replication forks . Because we previously observed significantly delayed meiotic entry when cells were treated with high concentrations of HU  and measured the kinetics of DNA replication and DSB formation , consistent with our previous report that high levels of HU can inhibit meiotic cell cycle entry , indicating that the massive accumulation of this form of the protein is checkpoint-dependent in meiotic cells as it is in mitotic cells  to support viability, formed high levels of DSBs in the presence of HU , ultimately leading to a reduction in steady-state RNA levels of at least 5-fold  treated with HU; Mre11 association with core meiotic chromosomal binding sites was substantially lower than in untreated cells, and the protein instead accumulated close to every pre-meiotic replication origin (mec1Δ cells treated with HU (t test). We believe this to be the result of Mre11 recruitment to the aberrant DNA damage structures that form at replication forks when the replication checkpoint is impaired , low levels of DNA replication were observed in this strain   . Becauserevented .cdc6-mn cells at the well-characterized HIS4-LEU2 locus on chromosome 3 , as well as in prophase-arrested cells (ndt80Δ)  . TogetheThe data presented here demonstrate that the replication checkpoint functions during budding yeast meiosis to maintain genome stability and viability. The checkpoint employs multiple strategies to inhibit DSB factors while DNA replication is ongoing. Without this restraining mechanism, cells form high levels of DSBs on partially replicated chromosomes, and lose the ability to complete either DNA replication or DSB repair.In this study we have defined the core components of a pre-meiotic replication checkpoint in budding yeast. Meiotic cells detect replication stress using the canonical replication checkpoint machinery, which then regulates meiosis-specific processes. As during mitotic S phase, detection of HU-induced replication inhibition relies on Mec1 and Ddc2. Mec1 activates the Rad53 effector kinase, which, in turn, inhibits DDK activity. The essential roles of Mec1 and Rad53 in maintaining replication fork stability appear to be equally important in meiotic cells, as removing either protein is lethal when cells are exposed to HU in meiS. Similar to the mitotic replication checkpoint, redundant and separable Mec1- and Rad53-dependent mechanisms maintain genome stability and prevent precocious cell-cycle progression in the presence of replication inhibition during meiosis . HoweverSPO11 transcript levels. Although Spo11 production is not completely prevented, we believe that downregulation of Spo11 has functional consequences because genetically decreasing Spo11 activity was able to significantly rescue DNA replication in the cdc6-mn strain  DNA, consistent with previous observations . Therefoormation . HoweverS. pombe, where Rad3/ATR/Mec1 and Cds1/CHK2/Rad53 are similarly activated in response to HU treatment. Some mechanistic differences exist, which could be due to the lack of identity of the DSB factors themselves. First, unlike budding yeast, fission yeast lacking the Rad53 homolog Cds1 form DSBs in the presence of HU .FACS analysis for total DNA content was performed as in .2) in a polystyrene culture dish. DNA was isolated via standard Southern protocol. All time course experiments for Southern analysis were repeated at least twice with similar results.Clamped-homogeneous electric field (CHEF) gel electrophoresis and Southern blotting for small chromosomes (including chromosome 8) were performed as described . Large cWhole cell protein extracts were prepared by TCA precipitation as in Northern blot analysis was performed as in 25 ml of cells were harvested after 3 hr in SPO. Chromatin immunoprecipitation (ChIP) for genome-wide location analysis was performed as described . One tenTotal genomic DNA extraction for CGH analysis was performed as described in For ChIP experiments, one half of the immunoprecipitated DNA and one tenth of the input DNA were labeled. Samples were labeled and hybridized as in 2 ratios for each experiment and scale normalizations across each set of biological replicates were calculated using the sma package (http://www.r-project.org). The value of the feature closest to each potential pre-meiotic replication origin (http://www.ncbi.nlm.nih.gov/geo/), accession number GSE46841.For each co-hybridization, Cy3 and Cy5 levels were calculated using Agilent Feature Extractor CGH software. Background normalization, log package  in R, a n origin  was usedMeiotic nuclear spreads were performed as described . In brie review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  “We do not know the cause of this repair defect, but one possibility is that allelic DSBs accumulate that have no unbroken sister-chromatid template from which to be repaired. Whatever the cause, cells that form DSBs on partially replicated chromosomes are unable to complete DSB repair.” All sorts of defects could be occurring in these cells, e.g., defective cohesin function (known to cause HR defects). Maybe DSBs continue to be made – indeed, DSB levels in cdc6-mn cells, so strand exchange can clearly occur. Also, crossover products form at appreciable levels – please show quantification. Does this mean that non-crossover recombination is more defective? Maybe a sister chromatid is important for non-crossover formation? We also wondered whether Holliday structures between sister chromatids are detected in cdc6-mn. This is a good control to confirm the replication block in this mutant.2) 3) There is a lack of quantification throughout the paper, both for Southern and Western analysis. None of the figures have error bars and there's no indication that any of the experiments were done more than once. The authors need to explain how they ensure the reproducibility of their observations.mec1 strains but this is not clear from the gel that is shown. There is definitely a shift compared to no HU or even rad53 with HU.4) mec1 and cdc6-mn mutants but this is not apparent in the gel that is shown. Quantitation and some loading controls would clarify this issue.5) S. pombe Rec12 transcription was not altered in S. pombe).6) The relevance of reduced Spo11 levels with respect to inhibition of DSBs is unclear and claims otherwise should be toned down. DSBs form in wild type cells when Spo11 levels are relatively low so it's not clear that effects detected here are relevant. The authors should look at Spo11 transcript levels , then it is impossible to tell whether the rescue that they see in the mec1 delete is rad53-dependent. Some Rad53 functions are preserved in a mec1Δ and others are not.7) We are also quite suspicious of the rad53-independent effect of mec1Δ is very modest. However, it also seems that all of the HU treated lanes are underloaded. The authors never quantify this, but it seems that if you compared to the upper band (or better still integrated over the lane), the rescue by mec1 would seem better. In fact, the data in 8) The data in cdc7ts, mec1Δ, rad53Δ, mek1Δ, and the non-phosphorylated dbf4 alleles.9) There are a lot of molecular details in the summary figure that are never tested in the manuscript. In fact, the only data that are clear are the ones that are previously known (Dbf4 is a Rad53 substrate and Mer2 is a Cdc7/Dbf4 substrate). The authors should examine the phosphorylation of Rec104 in 10) In the section “Mec1 signals to inhibit Mer2 phosphorylation by DDK” subsection of Results:dbf4 heterozygous allele. However there are no data showing this.A) The authors cite dbf4-Δ71-221 bypassing the replication checkpoint shows Southern data. This needs to be FACS data, as we’re not sure how the data presented addresses this.B) Citation of cdk mutant to control this? A phosphosite mutant is not as good, since nonspecific phosphoantibodies still often recognize the exact phosphosite in the unphosphorylated state (but not a mutant site).11) The phospho-S30 Mer2 data in 12) Unless the authors can override, with specific manipulations (specific mutations etc), the various inhibitory branches of the checkpoint, they cannot formally state that any of them are important to inhibit DSB formation. Therefore, they should be more cautious in their claims about the targets of the checkpoint.cdc6-mn strain, so how is full SC measured?13) mec1 + HU scenarios? It would be useful to see a FACS profile for mec1 + HU.14) What is the role of late origin firing for the increased DSB levels seen in WT + HU versus  1) “We do not know the cause of this repair defect, but one possibility is that allelic DSBs accumulate that have no unbroken sister-chromatid template from which to be repaired. Whatever the cause, cells that form DSBs on partially replicated chromosomes are unable to complete DSB repair.” All sorts of defects could be occurring in these cells, e.g., defective cohesin function (known to cause HR defects). Maybe DSBs continue to be made – indeed, DSB levels inare 40% higher in cdc6-mn cells. So, additional deregulation is occurring. Moreover, unlike most mutants with DSB repair defects, checkpoint arrest is not robust in these cells. In addition, with respect to viability loss, surely partially replicated chromosomes are a much bigger problem than unrepaired DSBs? The differences in the amount of replication between cdc6-mn and cdc6-mn spo11 mutants make it impossible to tease out the relative contributions of DSBs and partial replication to cell death. Therefore, it seems that the specific contribution of DSBs made on partially replicated chromosomes to cell death in cdc6-mn cells is completely unclear.cdc6-mn mutants is likely a compound effect of the fact that these mutants have a severe underlying defect in origin firing and the fact that they form DSBs on unreplicated chromosomes. To address this point, we now included viability analysis of dbf4-ΔN221/DBF4 cells. The dbf4-ΔN221 mutation confers a dominant checkpoint bypass . As shown in the revised dbf4-ΔN221/DBF4 cells exhibit a loss of viability specifically in HU. This loss of viability was entirely dependent on DSB formation, indicating that these mutants form DSBs in HU that are not properly repaired. We therefore concluded that inappropriate DSB formation on replicating chromosomes contributes to the loss of cell viability in checkpoint mutants.The reviewers raise an important point: the viability loss of t bypass . Thus, ucdc6-mn mutants with regards to DSB repair and we aimed to convey this in our original statement. The reason we highlighted allelic breaks is that these would be an obvious problem that would uniquely affect unreplicated chromosomes and could explain why some DSBs (at the same locus) were efficiently repaired and others were not. However, we understand that there are many other possible reasons for the repair defect in cdc6-mn cells. Thus, we have removed the discussion of speculatory models from this section.We certainly agree with the reviewers’ point that “all sorts of defects could be occurring” in the cdc6-mn cells load Rec8, build axes and form DSBs comparably to wild type cells. By contrast, loss of cohesin causes a very specific loss of axis structure and decreased DSB formation. We also do not have evidence that cdc6-mn cells continue to form more DSBs than wild type cells, at least under the conditions that we assayed in this study (rad50S mutants), as we do not see the levels of DSBs continually increasing during the time course shown in To address the specific possibilities suggested by the reviewers: we do not favor the hypothesis of defective cohesin function, as we have shown previously that 2); interhomolog double Holliday structures are detected in cdc6-mn cells, so strand exchange can clearly occur. Also, crossover products form at appreciable levels – please show quantification. Does this mean that non-crossover recombination is more defective? Maybe a sister chromatid is important for non-crossover formation? We also wondered whether Holliday structures between sister chromatids are detected in cdc6-mn. This is a good control to confirm the replication block in this mutant.cdc6-mn mutants are capable of completing homologous recombination, as evidenced by both recombination intermediates and full recombinant products that can be seen in this one-dimensional gel analysis. However, further analysis of repair pathways is far from straightforward because of the significant and asynchronous DNA replication in this strain , which essentially renders the analysis of these intermediates impossible in standard 2D-gel analysis. As this manuscript is primarily a characterization of the meiotic replication checkpoint, we feel that the in-depth analysis that would be necessary to further dissect DSB repair on partially replicated chromosomes is beyond the scope of this study. Therefore, in this manuscript, we state that there are problems in repair but refrain from making further claims about the types of repair that might be affected.We agree that rain see , which s3) There is a lack of quantification throughout the paper, both for Southern and Western analysis. None of the figures have error bars and there's no indication that any of the experiments were done more than once. The authors need to explain how they ensure the reproducibility of their observations.mec1 and rad53 mutants, were carried out more than 10 times using independent strains and experiments, a subset of which are shown in this paper.Although we acknowledge the concern raised by this reviewer, we would like to stress (and now included a note) that all of the Southern and Western blots in this study were performed at least twice on independent experiments with highly reproducible results. Some of the experiments, e.g., DSB levels in For each Southern blot, we measured relative DSB levels  for each time point in the time course, as shown in To address the lack of quantification of Western data, we now also included quantitative analysis of the Spo11 protein levels. All Spo11 Westerns include titrations of protein examined, to provide a more reliable estimate of relative protein abundance.4); the authors claim that Dbf4 is not hyperphosphorylated in mec1 strains but this is not clear from the gel that is shown. There is definitely a shift compared to no HU or even rad53 with HU.mec1 and rad53 mutant cells, much less of the protein accumulates in this form. Therefore, the accumulation of such a large abundance of the total Dbf4 protein in this form is dependent on the checkpoint. We have reworded this section to clarify this point.We realize that our use of the term “hyperphosphorylation” was confusing in this context. We intended to refer to the extent of phospho-Dbf4 accumulation, e.g., the relative amounts of the protein that run in each band. Dbf4 migrates as multiple bands in all strains, representing varying levels of phosphorylation. When HU is added to wild type cells, more of the Dbf4 protein accumulates in the upper, hyperphosphorylated form than is observed in untreated cells. Although the band representing the hyperphosphorylated protein is still observed in 5): the authors claim that the decreased levels of Spo11 observed in the presence of HU are restored in in mec1 and cdc6-mn mutants but this is not apparent in the gel that is shown. Quantitation and some loading controls would clarify this issue.SPO11 Transcript accumulation abruptly stops at the time of replication onset, ultimately resulting in a roughly 5-fold reduction of SPO11 RNA. From Western titrations, we estimate that this translates to a >10-fold decrease of Spo11 protein. Furthermore, the new quantifications confirm that this effect is indeed dependent on both Mec1 and Cdc6.We have now included quantitative Western and Northern analysis of Spo11 protein and RNA levels new . We find6) The relevance of reduced Spo11 levels with respect to inhibition of DSBs is unclear and claims otherwise should be toned down. DSBs form in wild type cells when Spo11 levels are relatively low so it's not clear that effects detected here are relevant. The authors should look at Spo11 transcript levels .SPO11 RNA levels with and without HU to complement our protein analysis. We found that SPO11 transcript levels are also decreased, indicating that this regulation occurs at the level of transcription. Therefore, the regulation of Spo11 in budding yeast is distinct from that of Rec12 in fission yeast.As indicated in the answer to point 5, we have carried out Northern analysis of cdc6-mn cells containing hypomorphic alleles of SPO11 exhibited substantially rescued DNA replication as observed by FACS  We are also quite suspicious of the rad53-independent effect of mec1 for the following reason. Rad53 mutants are sicker than mec1 mutants. This is likely because Rad53 still has a bit of activity from Tel1 activation. If the authors are seeing a non-specific loss of DSBs due to the effect of a loss of all Rad53 activity (in the 53 delete or the mec1tel1 double), then it is impossible to tell whether the rescue that they see in the mec1 delete is rad53-dependent. Some Rad53 functions are preserved in a mec1Δ and others are not.mec1Δ tel1Δ mutants in this study  The data indid not initially strike us as overwhelming. The effect of HU is strong and obvious, but the rescue by mec1Δ is very modest. However, it also seems that all of the HU treated lanes are under loaded. The authors never quantify this, but it seems that if you compared to the upper band (or better still integrated over the lane), the rescue by mec1 would seem better. In fact, the data inlook better. We think that quantifying this might be helpful. mec1Δ cells. We also substituted a gel in which the +HU lanes are more equally loaded to compensate for the ∼2-fold reduction in DNA due to the lack of DNA replication in cells treated with HU.We have now included quantification of the Southern blots in this figure to clarify the extent of DSB formation in The quantifications in 9) There are a lot of molecular details in the summary figure that are never tested in the manuscript. In fact, the only data that are clear are the ones that are previously known (Dbf4 is a Rad53 substrate and Mer2 is a Cdc7/Dbf4 substrate). The authors should examine the phosphorylation of Rec104 in cdc7ts, mec1Δ, rad53Δ, mek1Δ, and the non-phosphorylated dbf4 alleles.We have shown that in HU-treated cells, Mec1 is responsible for reducing Spo11 transcript and protein levels, Mer2 phosphorylation and Rec114 and Mre11 DNA loading. All of these are novel observations from this study that we believe belong in the model figure.We marked the potential phosphorylation of Rec104 by Cdc7 with a ‘?’ to indicate that it was a hypothesis. We have removed this arrow completely, as this suggestion is not important to this study and we did not intend to imply that we have shown that Rec104 is a direct target of DDK, which would require mapping and mutating phosphorylation sites on the protein.10) In the section “Mec1 signals to inhibit Mer2 phosphorylation by DDK” subsection of Results:A) The authors citeregarding the dbf4 heterozygous allele. However there are no data showing this.We apologize for this confusion. The wrong version of this figure was uploaded. It has now been replaced by the proper version containing the data referred to in the text.B) Citation of regarding dbf4-Δ71-221 bypassing the replication checkpoint shows Southern data. This needs to be FACS data, as we’re not sure how the data presented addresses this.In this paper we investigate the role of the meiotic replication checkpoint in preventing DSBs, which is what we refer to as a “checkpoint bypass.” For this reason, we show Southern data in what is now 11) The phospho-S30 Mer2 data inmirrors the total, and therefore it isn't clear if this is just nonspecific. Can the authors use a cdk mutant to control this? A phosphosite mutant is not as good, since nonspecific phosphoantibodies still often recognize the exact phosphosite in the unphosphorylated state (but not a mutant site).clb5Δ clb6Δ cells lacking prophase CDK activity was carried out and is included in the new The control experiment comparing wild type and 12) Unless the authors can override, with specific manipulations (specific mutations etc), the various inhibitory branches of the checkpoint, they cannot formally state that any of them are important to inhibit DSB formation. Therefore, they should be more cautious in their claims about the targets of the checkpoint.cdc6-mn cells (see point 6 above). It is not currently clear in the field whether Mre11 and Rec114 must be on chromosomes to allow for DSB formation, however this is a likely prediction and prevailing model in the field ; the data are not very convincing. The SC is presumably abnormal in cdc6-mn strain, so how is full SC measured?cdc6-mn cells occurs relatively efficiently, and previous analysis actually failed to detect defective SC assembly in these cells  What is the role of late origin firing for the increased DSB levels seen in WT + HU versus mec1 + HU scenarios? It would be useful to see a FACS profile for mec1 + HU.mec1Δ cells that was already in mec1 and MEC1 cells. As mentioned above, we performed FACS analysis on all of the strains studied in this paper to ensure that DSBs in HU formed in the absence of increased DNA replication.We have now included the FACS profile for all of the strains in mec1Δ cells +HU, we also probed a late replicating chromosomal region that would not be expected to initiate replication even in mec1Δ cells, and saw the same checkpoint bypass as at the yCR047 locus shown in mec1Δ cells are due to increased DNA replication in these cells. This experiment is currently not included in the revised manuscript as it appears redundant with our PFGE analysis of mec1Δ cells +HU, which revealed that DSBs formed across the genome without significant DNA replication (To further address the question of whether increased DNA replication contributes to the DSBs formed in lication .


To compare the risk of vascular disease, HbA1c and weight change, between first prescribed insulins in people with type 2 diabetes.1c (HbA1c) and weight change.People included in THIN United Kingdom primary care record database who began insulin (2000–2007) after poor control on oral glucose-lowering agents (OGLD) were grouped by the number of OGLDs in their treatment regimen immediately before starting insulin . Within OGLD group, Cox regression compared macrovascular  and microvascular disease  between insulin type  while ANCOVAs compared haemoglobin A1c had reduced less in basal versus pre-mix or NPH at 6–8 and at 9–11 months, and versus pre-mix at 12–14 months.Mean follow-up was 3.6 years. Rates of incident macrovascular events were similar when basal insulin was compared to pre-mix or NPH, adjusted hazard ratio versus basal: pre-mix 1.08 ; NPH 1.00  after two OGLDs, and pre-mix 0.97 ; NPH 0.77  after three OGLDs. An increased risk of microvascular disease in NPH versus basal after 3 OGLDs, adjusted hazard ratio1.87 , was not seen after two agents or in comparisons of basal and pre-mix. At one year, after two OGLDs, weight increase was less with basal compared with pre-mix. After three OGLDs, mean HbA1c compared to other insulins.We found no difference in the risk of macrovascular events between first insulins in the medium term when started during poor glycaemia control. The increased risk of microvascular events with NPH warrants further study. In certain groups, first use of basal insulin was associated with less gain in weight and decrease in HbA A short-term aim of type 2 diabetes management is good glycaemic control with a longer-term objective of reducing complications. People with known type 2 diabetes have a two- to four-fold increased risk of ischaemic heart disease (IHD) and an increased risk of microvascular disease 1c levels, body weight change and subsequent vascular disease. Clinical trials in type 2 diabetes have reported less weight increase and less reduction in HbA1c but fewer hypoglycaemic episodes during treatment with basal insulin 1c and body weight, was different depending on which insulin type was prescribed first.There is little published data from routine care as to the effect of the choice of insulin on HbAThe manuscript describes a non-interventional study so informed consent was not required. The protocol was approved by the Cambridgeshire 4 Research Ethics Committee (UK).http://www.thin-uk.com/) All data came from The Health Information Network (THIN), an observational database containing information collected in computerized primary care practices throughout the UK , were selected. Beginning insulin was defined as more than one insulin prescription as short-term insulin is commonly used during periods of ill health and we were interested in the effects of long-term treatment. In that population, the duration of each OGLD type  was calculated from 1st January 2000 up to beginning insulin. The number of OGLD types still prescribed immediately prior to starting insulin (baseline) was counted. It was assumed that a treatment stopped as a new drug was started if there were no further prescriptions for the first OGLD within a time period which was double that of the duration of the last prescription for this drug. OGLD duration was calculated from the number of items and dosage prescribed. When insufficient dosage information was recorded for the calculation of duration the most common dose for that form and strength of the OGLD was used.People (n = 105 845) identified were those who had received at least one prescription for an OGLD after 11c record in the three months prior to index date, the most recent HbA1c <7.5% (58 mmol/mol) or no body weight record in the previous 15 months, were excluded. The HbA1c criteria were chosen to ensure that only people in poor blood glucose control were included. Comparisons of those who started insulin from one baseline OGLD are not reported. This was because commencing insulin after one agent was uncommon with few vascular events reported and a parallel study comparing OGLD and insulin found that patients who initiated insulin treatment from a regimen of one oral therapy differed from other patients in terms of both medical history and data recording Those who started insulin were initially grouped into nine cohorts depending on the combination of the number of baseline therapies  and whether basal, NPH or pre-mix was started. The number of baseline therapies was used in the groupings as the stage at which insulin is started can be related to diabetes duration, severity and concomitant disease. Patients initiating other insulins or combinations of insulins and those restarting insulin therapy were not included. Consequently analyses were always completed for mutually exclusive groups. The date of the first insulin prescription was taken as the index date. People with more than one year of electronic record pre- or post-index date, aged between 18 and 80 were included. The one year pre-insulin period gave sufficient medical record length to identify new insulin users and those with a history of the study outcomes. People with no interpretable HbA1c and body weight in the year after index date were secondary outcomes.The primary cardiovascular endpoint was a composite of all-cause mortality, myocardial infarction, acute coronary syndrome, or stroke. Angina and coronary procedures were added to the primary cardiovascular endpoint in sensitivity analyses. The microvascular endpoint was a composite of peripheral neuropathy, diabetic nephropathy, and retinopathy. The first event was taken for the composite endpoints. Patients with events prior to the index date were excluded from the relevant analysis. Amputations were identified but in insufficient numbers to perform comparative statistical analysis. Change in HbA1c, year of starting insulin, OGLD and cardiovascular therapies, history of vascular disease and its risk factors. OGLD  and cardiovascular therapies  were described. Comparisons were made for basal versus NPH and basal versus pre-mix with the same number of baseline OGLDs. Follow-up started on the date of the first insulin prescription plus 12 months and was censored on the date of the first outcome for that analysis, transfer out of the practice or last data collection. Outcomes in the first 12 months of treatment were not included in the analysis as the insulin was unlikely to have an effect on vascular outcomes in a short time scale. All events from the year after insulin was started were included, regardless of further treatment changes or inclusion in additional cohorts, in a similar manner to intention to treat analysis in randomised clinical trials. This design was chosen because of the time for the effects of poor glycaemic control on vascular outcomes to become evident and because censoring when a patient joined a second cohort could have introduced bias with shorter follow-up in patients with worse control.Adjusted hazard ratios of the association between our treatment categories and incident (first event) microvascular and macrovascular outcomes were compared using Cox regression models. People with a pre-insulin history of any disease within that composite outcome, or with outcomes in the first year or who transferred out during this time, did not contribute to that analysis. Each baseline covariate was included as a variable in the model for the primary macrovascular outcomes with the microvascular conditions included separately and year of first insulin treated as a continuous variable. Angina and coronary procedures were removed as covariates when these were added to the composite outcomes in sensitivity analyses. All baseline variables except microvascular disease itself were included in the microvascular disease models.1c in the year after insulin initiation were investigated with the treatment groups compared as outlined above adjusting for baseline age, sex, duration of diabetes, year of escalation, BMI, HbA1c, and oral treatment regimen. The changes in weight from baseline to approximately one year (10–15 months) and HbA1c over three month intervals (4–14 months) were compared using ANCOVA least squares means. All data analysis was performed using SAS software .The association of treatment category with body weight change and HbA1c record over 7.5% (58 mmol/mol) available in the 90 days before insulin was started; 558 had no baseline weight reading and 623 were <18 or >80 years of age. After all exclusions, 2537 people began insulin after two OGLDs and 890 after three. NPH was the least common first insulin being prescribed to less than 20% of people in any group  and 1.03  respectively . No diff1c after beginning insulin but the mean HbA1c remained above the target of 7.0–7.5% (53–58 mmol/mol) in all groups  and NPH (6 to 11 months after index date).In all cohorts and at all time periods there was a decrease in HbAl groups . There wBody weight increased after commencing insulin in all cohorts . The incOur data suggest that, in routine practice, people with type 2 diabetes and initially poor glycaemic control have a similar risk of major macrovascular events  in the medium term regardless of what type of insulin is started. The mean follow-up was 3.6 years although events in the first year were not included. Fewer patients started insulin after three oral agents resulting in a small number of outcomes and upper 95% confidence intervals close to two. The sensitivity analysis comparing macrovascular disease plus angina in premix insulin versus basal analogue after two OGLDs had a hazard ratio of 1.49 and a lower 95% confidence interval of 0.99 although the same comparison after three oral agents did not show a difference in risk between the insulins with a hazard ratio of 0.76 .1c levels but a statistically significant greater improvement in glycaemic control from 6 to 11 months after starting NPH rather than basal insulin. A sharp increase in both testing for diabetes complications 1c after a shorter duration of diabetes, which might impact on the development of these complications. This finding therefore warrants further investigation. Differences in weight gain and glycaemic control were identified between insulin groups in the first year of treatment. Starting basal insulin was associated with less reduction in HbA1c than other insulins after three oral agents but less weight gain than pre-mix after two oral agents.Rates of incident microvascular conditions were similar in the treatment groups except in the comparison between basal insulin and NPH after a regimen of three oral agents. The increased rate after NPH is unlikely to be due to better short-term glycaemic control as there was no difference in baseline HbA1c in basal insulin versus NPH treated patients was reported in that trial and in the 4-T study when basal insulin (detemir) was compared to both biphasic and prandial treatment 1c levels later converged in the 4-T study after usual addition of other insulins, 1c ≤7.0% (53 mmol/mol) without nocturnal hypoglycaemia No observational studies or clinical trials were found which specifically aimed to compare the rate of macrovascular disease between first insulins. The Treating to Target in Type 2 diabetes (4-T) clinical trial found no significant difference in death rate between basal, biphasic and prandial insulin regimens over 3 years, but a higher rate of death from cardiovascular disease in the prandial than the biphasic and basal groups 1 c as primary care records only capture episodes known to the physician rather than events treated by family, paramedics or the patient themselves.Less weight gain in the basal group in routine care was consistent with several clinical trials of basal compared to other insulins 1c 9.9% (85 mmol/mol) (after exclusion of 8% with baseline HbA1c <7.5% (58 mmol/mol)). This mean is higher than many clinical trials, which often have an upper as well as lower inclusion limit 1c levels were high, control to target was not achieved in the majority of people even after one year.Insulin was often started when glycaemic control was very poor, with mean HbA1c may have been measured more frequently in those whose glycaemic control was poor resulting in higher mean levels during follow-up than were actually the case. As all patients should have levels measured after insulin introduction there should be no difference between insulin types. The finding that the number of patients who have a value measured remains relatively consistent over time, across treatment groups, is also reassuring. The first year of follow-up was excluded from all groups in the analysis of vascular events as the distribution of insulin use in terminally ill patients may not be even, causing bias, and any effect of the choice of therapy on our outcomes is likely to be in the medium- to long-term. The design was an intention-to-treat type analysis so we did not censor follow-up at the next change of glucose lowering treatment. As a result, the study did not investigate the association of individual insulins and cardiovascular outcomes but rather the mid-term consequences of one therapeutic decision when starting insulin. Later changes in treatment could be expected to relate to the efficacy and problems of the first insulin regimen.This study has a number of limitations. While we were able to adjust for many potential confounding variables, severity of prevalent vascular disease (such as angina), prior long term glycaemic control, socioeconomic status and ethnicity were not routinely available on all patients in this observational dataset in the study years and recorded duration of diabetes has not been validated. While these variables are likely to be non-differential between the insulin types, we cannot exclude the presence of some residual confounding. Confounding by indication should be decreased as all subjects initiated insulin when they had poor glycaemic control, although different insulins may be started in different clinical circumstances such as ambulatory care versus in-patient treatment. We chose to include all-cause mortality rather than cardiovascular mortality because the recording of cause of death has been shown to be incomplete in general practice databases 1c could not be interpreted rather than because it was not measured. The study covered a time of HbA1c unit change in the UK.Overall, while the number of patients lost due to inclusion/exclusion criteria appears high, the criteria for the initial data cut were untypically wide due to the two stage identification process (OGLD therapy and then insulin). For example, the requirement for a year of data recording pre- and post-insulin resulted in the removal of 29% of the total potential population but allowed time for recording of information on prevalent events as well as laboratory measurements. Many of those who had no baseline HbA1c also had less than one year of data pre-insulin. Many of the remaining exclusions occurred because the HbA1c in the first year compared to other insulins in some treatment groups. Glycaemic control was poor at baseline and, while mean HbA1c decreased during insulin therapy, control remained poor across all groups.In conclusion, in routine clinical practice no significant difference in the rate of incident macrovascular disease was found between first insulin type started during poor glycaemic control over a period of 1 to 3.6 years. The increased risk of microvascular disease between users of NPH and basal insulin was found in those who escalated from three but not two agents and so warrants further investigation. First time use of basal insulin was associated with a smaller increase in weight gain and a smaller decrease in HbA


Prior research has shown that removing occupational asthmatics from exposure does not routinely lead to significant improvements in respiratory impairment. These studies were of limited duration and factors determining recovery remain obscure. Our objective was to evaluate residual respiratory impairment and associated sputum and blood biomarkers in subjects with Western red cedar asthma after exposure cessation.20 ) sputum cellularity and select Th1/Th2 (T helper cells 1 and 2) cytokine concentrations in peripheral blood were determined. The asthma impairment class was determined and multivariate analyses were performed to determine its relationship with sputum cell counts and serum cytokines.Subjects previously diagnosed with cedar asthma, and removed from exposure to cedar dust for at least one year, were recruited. Subjects completed a questionnaire and spirometry. PC20 had increased by 2.0 mg/ml; the FEV1 decreased by 1.5 L, with greater decrease in those with greater impairment. Higher impairment was associated with serum interferon-gamma , mainly due to the FEV1 component .40 non-smoking males (mean age 62) were examined at a mean interval of 25 years from cedar asthma diagnosis and 17 years from last cedar exposure. 40% were in impairment class 2/3. On average, the PCYears after exposure cessation, patients with Western red cedar asthma have persistent airflow obstruction and respiratory impairment, associated with systemic inflammation. Individuals who continue to work with red cedar dust following disease diagnosis will near-uniformly continue to have respiratory symptoms and require medications to control their asthmatic symptomsThe finding, that longer exposure duration is a risk for long-standing impairment but that removal from exposure only occasionally leads to complete resolution of impairment, has been seen with other occupational asthmagens such as toluene diisocyanateAccording to the protocol approved by the ethics board at the University of British Columbia, we attempted to contact never-smoking individuals previously diagnosed with Western red cedar asthma by specific inhalational challenge and last known to be living in the lower mainland of British Columbia. Written consent was given by all participants.Peripheral blood was drawn by standard venipuncture procedure.Spirometry was carried out according to ATS criteria20. Those whose FEV1 did not fall 20% even with the 16 mg/ml dose of methacholine were assigned a PC20 of 16. An interviewer trained in the language of the subject's choice (English or Punjabi) administered a questionnaire modified from that used in the 2003-4 studyA methacholine challenge test was carried out according to the method of Cockroft et al1 , NSBH , and the medications used to control asthma  lead to the class of impairment, expressed as Class 0 , class 1 , Class 2 , Class 3  and Class 4  and Class 5 (asthma not controlled despite maximum treatment).The respiratory impairment rating for each subject was determined according to ATS guidelineset al.1 and FVC were measured three times before sputum induction. The best FEV1 value was used as the baseline. The Fisoneb ultrasonic nebulizer  with an output of 0.87 ml/min and an aerodynamic mass median diameter of 5.58 µm was used to deliver saline. Concentrations of saline at 3.00, 4.00 and 5.00% (each for 7 min) were given by inhalation through a mouth piece without a valve or noseclip. After each 7 min inhalation period, spirometry was repeated. The subject was asked to rinse his mouth with water and blow his nose  before coughing the sputum sample into a sterile container. If the FEV1 fell by >10% from the baseline the same concentration of saline was used for the next inhalation. If the FEV1 fell by >20%, the procedure was discontinued and salbutamol given.Sputum was induced using an aerosol of inhaled hypertonic saline by a modification of the method of Pin 6 cells/ml, and two cytospins were prepared with 50 ul of the cell suspension. These slides were air-dried, fixed and stained with Wright's stain. At least 400 non-squamous cells were counted and a differential cell count was made. The supernatant was used for cytokine analyses, as described below.Sputum was analyzed within 2 hours of sample collection using the method described by Pizzichini et alConcentrations of the cytokines in serum  and sputum  were determined using MesoScaleDiscovery  with limits of detection reported as 0.68 pg/mL (IFN-gamma), 0.14 pg/mL (IL-10), 0.50 pg/mL (IL-12), 0.23 pg/mL (IL-5), and 0.064 pg/mL (TGF-beta).Comparison to a prior study populationThe main outcome measure, impairment, was defined by ATS criteria-based impairment classesThe risk of having a higher impairment class (IC) based on demographic characteristics and lung function was determined by multivariate logistic regression. The risk of having a higher impairment class (IC) based on elevated sputum or serum cytokines (dichotomized at the median) was determined by multivariate logistic regression.From our database, there were 46 individuals who met the aforementioned criteria for contact attempt who remained living and were physically able to make the trip to our study centre. Of these, 40 never-smoking male cedar asthmatics successfully completed the study; of these 65% were Caucasian, 41% were atopic, and 40% were currently on inhaled steroids. 6 subjects started the study but did not complete one of the essential study components .1. However, the average PC20-FEV1  in the present study, 6.8 (range 0.1–16.0), was significantly higher than that in our prior studyAs noted in 1 percent predicted and PC20 were each lower in those with moderate impairment relative to those with mild impairment ; the loss of FEV1 per year was greater in those with moderate impairment relative to those with mild impairment . In the case of FEV1, the change since the time of diagnosis was also greater in those with moderate impairment relative to those with mild impairment  and there was a similar trend in the change in PC20 . Years from initial diagnosis showed a trend to being fewer in those with moderate versus mild impairment .As noted in As noted in post hoc analyses were performed to assess the relationship between serum TGF-beta and PC20. Those whose PC20 had decreased, from the time of diagnosis to the time of the current reassessment, had a significantly higher serum TGF-beta than those whose PC20 had increased over that interval Given the literature suggesting the importance of TGF-beta as a mediator of airway remodeling leading to airway reactivity, the longest follow-up of occupational asthma, to our knowledge, in the literature; given the mean follow-up of 25 years since diagnosis and 17 years since removal from exposure, the size of our reported cohort is considerable. Another notable distinction of our work is that we excluded all smokers. Other follow-up studies of occupational asthma, including our ownPrior work from our groupNeither sputum eosinophils nor neutrophils were significantly associated with impairment in our study. This was somewhat surprising since, in a prior study of patients with WRCA who were removed from WRC dust for a mean of approximately 10 years20-FEV1 of 2.0 mg/ml methacholine. Interestingly, this is greater than the improvement noted by Lemiere and colleaguesIt is revealing to compare the improvement in non-specific airway responsiveness in our study, covering an average of 17 years since removal from exposure, to that from prior studies. We found a mean improvement in PCWe are limited by not having data on potential coincident cardiovascular disease in our subjects; though it seems unlikely that such non-respiratory disease would confound the association between asthma-related impairment and interferon-gamma, the possibility of such confounding exists given the likely association between chronic respiratory disease and cardiac diseaseIn any case, our data supports the consensus that minimizing exposure both in terms of exposure intensity and duration remains critical and that it is naïve to expect that time alone will ‘cure’ the disease; while our subjects on average improved in terms of impairment and steroid usage, approximately two-thirds still had a positive methacholine challenge test and reduced HRQL scores. Thus, it remains imperative to provide effective, long-term and individualized asthma management strategies for WRCA patients removed from exposure. For many WRCA patients, even after a long time removed from exposure, reducing AHR by appropriate pharmacology should remain central to asthma management and yet our study suggests that the use of inhaled corticosteroids diminishes over time in spite of ongoing AHR. Care providers should stringently resist this trend and compensation systems should continue to pay for such medications so long as AHR persists. Studies have shown that patients with occupational asthma who left work suffered considerable economic hardship. The income of many patients was reduced by at least half if they were unable to find alternative employment. Understanding the dynamics of asthma persistence and severity in those with WRCA will ultimately help us better control this disease and also minimize the financial impact of WRCA.In summary, we found that significant asthma-related respiratory impairment persists in 40% of cedar asthmatic even long since  removal from exposure and is associated with a classical marker of circulating inflammation (interferon-gamma). This suggests that even many years after exposure cessation, non-specific inflammation persists in the blood of impaired individuals.


Imaging the eye with magnetic resonance imaging (MRI) has proved difficult due to the eye’s propensity to move involuntarily over typical imaging timescales, obscuring the fine structure in the eye due to the resulting motion artifacts. However, advances in MRI technology help to mitigate such drawbacks, enabling the acquisition of high spatiotemporal resolution images with a variety of contrast mechanisms. This review aims to classify the MRI techniques used to date in clinical and preclinical ophthalmologic studies, describing the qualitative and quantitative information that may be extracted and how this may inform on ocular pathophysiology. The eye acts like a camera as it can refract light and produce a focused image, which then stimulates the neural responses that give rise to vision. According to statistics of the World Health Organization in 2002, there were 161 million people affected by visual impairment worldwide, of whom about 37 million were blind , in the form of potassium or sodium dichromate, is thought to alter heterogeneous tissue oxidation by diamagnetic dichromate [Cr(VI)], which creates paramagnetic Cr species [Cr(V) and Cr(III)] that shorten the Tal layer .Both USPIO- and Cr-based contrast agents are toxic and thus can be used only in animal research studies . Further2-weighted MRI techniques with macrophage-specific intracellular contrast agents [Macrophages play an important role during development of autoimmune diseases, including autoimmune ocular diseases. They can be visualized in vivo using high-resolution Tt agents .A USPIO-based contrast agent and a two-turn single-loop surface coil were used for macrophage-specific MRI of the rat eye affected by EAU, an autoimmune inflammatory disease . Infiltr1-weighted MRI images was maximized between 1 h and 3 days and dropped at 1 week and 2 weeks post-Cr(VI) injection. The MRI signal intensity was also enhanced in the region of the rat vitreous humor on T1-weighted MRI images at 1 h but unbound Cr was cleared from the vitreous humor within 12 h after intravitreal Cr injection. Higher spatial resolution images (50×50×50 μm3 voxel size) were acquired ex vivo and allowed visualization of five different Cr-labeled layers in the region of the rat retina. Significant signal averaging was used to achieve reasonable SNR at this higher spatial resolution, resulting in an acquisition time of 16 h [Enhancement of the MRI signal in the region of the rat retina on T of 16 h .2) and hypercapnic (mixture of O2 and CO2 in different concentrations) conditions, with the air breathing condition used as a baseline in each case. The blood flow calculated in the avascular rat eye structures was extremely low compared to that calculated in the vascular rat eye structures. Hyperoxia was found to decrease the blood flow due to vasoconstriction, whereas hypercapnia increased blood flow due to vasodilation. Rat blood flow in the retina was significantly diminished in degenerated retina and robust hypercapnia- and hyperoxia- induced blood flow changes were observed in normal retinas. Hyperoxia/hypercapnic breathing reduced/increased the total blood flow in the retina of normal animals by 12% and 14%, respectively. Basal blood flow in disease-affected rat retinas was markedly reduced compared to those of control rat retinas. Blood flow is tightly coupled to basal metabolic activity and the reduced metabolism of degenerated retinas of diseased rats may have caused the reduction of retinal blood flow. Hypercapnia- and hyperoxia-induced absolute blood flow changes were not statistically different between normal and diseased rat retinas [Rat ocular blood flow has been quantitatively evaluated by fMRI in several studies during hyperoxic  showed a 12% increase of retinal/choroidal blood flow relative to air inhalation conditions [The literature on hypercapnic responses and on basal blood flow in the retina is inconsistent, placing these animal studies some distance from likely clinical implementation -69. Despnditions .19F MR spectroscopy experiments have been performed in the eye [19F techniques are not sensitive enough for imaging in vivo due to the low signal from the significantly lower concentration of 19F nuclei in the body compared to 1H; hence, only localized ocular 19F NMR spectroscopy of large voxels can be used to detect the presence of 19F nuclei. In addition, broadband capabilities, which are necessary to detect NMR signals at frequencies other than that for 1H, are not common features on most clinical MRI systems. Even if a clinical scanner is equipped with a broadband RF channel for nonproton MRI/magnetic resonance spectroscopy, some hardware development is generally required, such as developing a suitable RF coil [ the eye  and in a the eye . However RF coil .Among the MRI techniques for ocular imaging in research and/or clinical environments described in the previous sections, two additional MRI techniques, which do not require the use of contrast agents, show some promise for this field. These techniques are magnetic transfer contrast and magnetic resonance elastography (MRE).1H nuclei in water molecules. The 1H NMR signals can be emitted by mobile 1H nuclei in water molecules, by less mobile nuclei in lipids, or by rigid 1H nuclei in the tissue macromolecules or membranes. The signal emitted by the rigid nuclei is not easily observed directly because of its large bandwidth relative to the free water 1H nuclei. However, 1H nuclei in the rigid pool can be selectively labeled by applying an off-resonance RF pulse. If the 1H nuclei in the mobile and rigid pools are interacting through magnetization exchange , then the magnetic labeling of 1H nuclei in the rigid pool  can be transferred to the 1H nuclei in the mobile pool (water). Effective coupling between the 1H nuclei in the two pools is visualized on the MRI image by a decrease in signal intensity [1H nuclei in the two pools can be quantified by calculating magnetic transfer ratios on the acquired MRI images.Clinical MRI images mainly ntensity . It has ntensity . The magLens opacifications must be accompanied by alterations in tissue compositions, including the hydration state. MTC-enhanced MRI has been successfully used to document lens changes in longitudinal studies of galactosemic dogs during sugar cataract formation . These MMTC-enhanced MRI was also used to quantitatively evaluate the changes produced in the optic nerve in patients with optic neuritis. The quantitative evaluation of the optic nerve using MTC-enhanced MRI may also be useful in the investigation of multiple sclerosis. Magnetic transfer ratio values measured on MRI images in the region of the optic nerve of patients with optic neuritis were significantly different than that measured in corresponding regions of normal eyes . The patThe mechanical properties of the eye and its anatomic components are believed to be of central importance in many pathologic conditions, such as age-related macular degeneration, glaucoma, Graves’ disease, and myopia. The accurate quantification of these mechanical properties can thus be used to improve the basic understanding of eye mechanics and to enhance patient care, diagnosis, and management of disease .Historically, the mechanical properties of the eye have been a challenge to assess and have proved difficult to quantify, especially noninvasively. Ocular elasticity has been called one of the most confused areas of ophthalmology. The eye comprises multiple tissue types and various anatomic structures that exhibit characteristics of heterogeneity, nonlinearity, anisotropy, viscoelasticity, physiologic accommodation, and extreme sensitivity to hydration—mechanical characteristics that are difficult to account for with a single measurement technique .Conventional mechanical testing methods, including axial strip testing and dynamic mechanical analysis, have been applied to various tissues of the eye, although these techniques are generally limited in their accuracy because testing requires dissection and flattening of doubly curved tissue samples. In addition, these methods are restricted to research applications due to their invasive nature .More recently, experimental image-based approaches for studying eye mechanics have been employed with some limited success by directly measuring displacements (strain) in the eye under inflation conditions. These approaches include techniques such as two-point spot scanning, holographic interferometry, optical imaging of treated corneal surfaces, wave speed analysis using ultrasound, and MRE. Of these techniques, ultrasonography and MRE are the only viable techniques for in vivo applications . MRE is The acoustic shear waves in the tissue are generated by external driver devices. The electrical signal for these devices is created by a signal generator triggered by and synchronized to the MRI pulse sequence and is amplified by an audio amplifier before being fed into the mechanical driver. Over the years, several driving mechanisms have been developed, each with their own advantages and limitations. Spin-echo, gradient-recalled echo, steady-state free precession, and echo planar imaging-based pulse sequences can be designed to be sensitive to motion with a specific frequency, a particular multiple of the specific frequency, or a broad range of frequencies . The resDue to flexibility, noninvasiveness, and potential clinical applications, the field of MRE has been rapidly evolving with new applications emerging for various organs, including the eye . The elaAppendix 1.To date, most ocular MRI studies reported in the literature have been performed without the use of contrast agents, while the most successful techniques allowed for the visualization of the eye and optic nerve anatomy of animals ,38,41,423 in rat [3 in human [The smallest reported voxel sizes of the ocular MRI images acquired in vivo range between 23×23×680 μm3 in rat  and 100×in human  eye studin human ,66. In tin human ,42 and bin human  were quain human . The thrin human . Three rin human . The sigin human ,59 and ain human  eye studHuman retinal abnormalities, including retinal detachments, have also been successfully visualized in vivo in MRI images. The pattern of such retinal detachments in the rat eye is similar to that detected in the human eye see . Figure 5 μm2 (0.1 mm2) were measured on MRI images of rat eyes at the early stages of EAU [Quantitative information on the mouse ,45 and hs of EAU .1 [2 [1 and T2 relaxation times measured for different eye structures might provide quantitative information that can give insights into the mechanisms of eye diseases. Diffusion anisotropy in three different mouse retinal layers can quantitatively be monitored in vivo using MRI techniques without contrast agents [T1 ,32,42 an1 [2 ,32 and mt agents .1 and T2 relaxation times in that region. If the cells produce changes of the physical properties of 1H nuclei in water molecules over distances much larger than the vacuole itself, smaller numbers of macrophages can be visualized in vivo.Macrophages were visualized on MRI images of gel phantoms and anesthetized rat eyes at the peak of EAU. A minimum number of 12 to 286 unlabelled macrophages, each having a radius of 29 μm to 10 μm, were detected in the human macrophage gel phantom MRI studies . These eMRI techniques which use contrast agents provide more quantitative information, but only Gd-DTPA-based compounds are routinely used clinically at present. Perfusion of the rat retina  and physUnwanted spatial variations of the signal intensity  can be gThe accuracy of geometric measurements is limited by the spatial resolution of MRI images, which is dependent primarily on the SNR and hence acquisition time when multiple signal averaging is used to improve the SNR . This ma1 contrast tends to be minimized and T2 contrast tends to be emphasized, while the chemical shift separation is more pronounced [Higher magnetic field strengths improve the SNR of MRI images, allowing images with higher spatial and/or temporal resolution to be acquired. However, the tissues imaged are exposed to greater specific absorption rates in higher magnetic field strengths, since the specific absorption rate scales approximately with the square of the static magnetic field. In a study performed on over 500 human subjects, it was shown that measured vital sign changes related to a sitting or supine position are larger than those associated with exposure to a static magnetic field of up to 8 T . Increasonounced .Another possibility of increasing the SNR of MRI images is the special design of the transmit/receive RF coils. The design and use of cryo-cooled eye-shaped coils or multichannel phased-array coils would allow further increase in the SNR of MRI images compared to that obtained at the moment clinically or in animal research. In conclusion, high spatial and temporal resolution MRI techniques with and without contrast agents provide qualitative and quantitative information with clinical potential in ophthalmology.


De novo tumors (DNT) after liver transplantation (LT) represent a growing concern.We analyzed the incidence of DNT, type, time of onset, risk factors and mortality (as of 2010) in 494 adult patients transplanted in the last 26 years (1983–2009).DNT occurred in 41 (8.3%) of the patients. The Standardized Incidence Ratio (SIR) compared with the Italian population was 1.8. There was a higher incidence in males (SIR 2.0), an expected extremely high rate of Kaposi’s sarcoma (SIR 127.95) and unexpected higher rates of tumors of the bladder in males (SIR 3.3). The incidence of DNT was higher within the first two years of LT (SIR 2.7) for Kaposi’s sarcoma (SIR 393.3) and after 10 years (SIR 1.7) for bladder tumors (SIR 10.6). Multivariate analysis identified alcoholic cirrhosis  and sclerosing cholangitis  in the recipient as main risk factors for the occurrence of DNT.Surveillance protocols for DNT must be specifically oriented to patients transplanted for alcoholic cirrhosis and sclerosing cholangitis. They should focus on early detection of Kaposi’s sarcomas, and more remarkably, on late development bladder tumors in men after LT. Liver transplantation (LT) is presently the gold standard therapy for patients with end-stage liver disease. In recent years, the results of this procedure have shown a significant and steady improvement in long term survival The main aim of this study was to compare the rates of different DNTs among patients who have had a liver transplant with those of the general population and to evaluate the DNT risk based on time from transplantation.The Ethics Committee of our institution –Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico of Milan - was notified of this study. According to Italian law  no specific approval is needed for retrospective studies.Informed consent was obtained as usual for medical, surgical, radiological treatments, not specifically for this retrospective study. Patients gave written consent for every procedure performed in the hospital including treatment of data for medical purposes.Data from 571 adult patients with 644 liver grafts spanning a period from 1983 to 2009 were analysed. The detection of some tumors occurred as early as the second month after LT and thus we excluded 57 patients (with 67 LTs) with a post-operative follow-up time ≤30 days.Patients transplanted for primary hepatic tumors (i.e. hepatomas- HCC) were included, assuming that these tumors did not influence the occurrence of DNTs.Cyclosporine was the main immunosuppressive drug from 1983 through march 1993. Thereafter it was often replaced by Tacrolimus except for HCV patients. OKT3 was sometimes used. The policy of our Center was to treat HCV patients with Cyclosporine.No specific protocol for detecting de novo tumors was employed. In the post-operative period patients underwent physical, radiological  and biochemical assessment. In suspected cases of DNT measurement of tumor markers  were included as part of the biochemical investigation. All data was entered into a retrospective database specifically for this study. Cancer incidence and mortality follow-up was performed by collecting clinical information during periodic pre-scheduled visits from patients/relatives or by periodic active phone/fax contact.The follow-up ended on April 30, 2010. Twenty patients were lost to follow-up bringing the final cohort of patients included in the study to 557 grafts in 494 adult patients: 437 patients were transplanted once, 51 twice, and 6 three times.New primary cancer diagnoses were coded according to the International Classification of Diseases and Related Health Problems, 10th revision (ICD-10). Non-melanoma skin cancers were excluded from analysis in the study. The recurrence of HCC was not considered an event in our study. For patients residing in Milan, completeness of cancer identification was checked by record-linkage with official hospital admissions databases in the period 2000–2008 and with the Milan cancer registry.http://www.registri-tumori.it/cms/) specific for gender, calendar period, and age. Person-years at risk for cancer incidence were computed from 30 days after the date of transplant to the date of cancer diagnosis, date of death, date of last follow-up visit or to the date of termination of the study , whichever came first. For each cancer site, we calculated Standardized Incidence Ratios (SIR) as the ratio of observed (Obs) to expected (Exp) number of cancer cases; 95% CI of SIRs were calculated using the exact method based on the Poisson distribution The overall and tumor-specific cancer incidence in the cohort of transplanted patients was compared with the expected incidence according to national cancer incidence rates  using mortality rates specific for calendar period (5 year categories), gender, and age (5 year classes). Statistical analyses were performed using Stata 11  The average follow-up time in the 494 pts was 7.2 years (standard deviation 5.6 years), (range from 33 days to 22 years). Overall, the person-years at risk from LT to de-novo occurrence or censoring were 3,761 Years.Recipient characteristics are reported in In 494 liver transplant recipients, 50 DNTs (10.1%) were found, including 9 (1.8%) non-melanoma skin cancers. ExcludiThe highest incidence of DNT (SIR 2.7) was found within the first two years of LT . Five ofThere was also a periodic shift in the occurrence of a predominant malignancy. From 1986–1991 there was a higher rate of colon cancer (SIR 57.8), from 1992–1996 of lung (SIR 6.22) cancer and lymphomas (SIR 13.6) and from 2006 a higher rate of Kaposi’s sarcomas (SIR 270.7) and bladder tumors (SIR 6.69).Among five patients with urothelial  bladder cancers, two tumors were discovered after the onset of gross hematuria whereas the others during routine abdominal ultrasound exams. Three patients had early stage tumors, whereas two had T2 and T3N2 tumors, respectively. Two patients underwent a Transurethral Resection of a Bladder Tumor (TURBT), and two a radical cystectomy with extended pelvic lymph node dissection and ileal conduit urinary diversion. One patient underwent a TURBT for a T2 cancer, but then was scheduled for major surgery. Unfortunately he died before the surgical treatment.The cumulative risk (Kaplan-Meier failure function) of DNT after LT was 8.9% (95% CI 6.2–12.7%) at 10 years and 22.8% (95% CI 15.2–33.3%) at 20 years. In the univariate Cox regression analyses only male gender, alcoholic cirrhosis, and sclerosing cholangitis were found to be predictors for development of DNT.The covariates of priori interests and those emerging from univariate analyses (age and sex of recipient and indications for LT) were then included in a multiple Cox model to calculate adjusted HRs. Alcoholic cirrhosis  and sclerosing cholangitis  were confirmed as independent risk factors.There were 14 deaths among patients with DNT and 119 among patients without DNT. Survival was significantly different . The genCompared to the general Italian population, there was a dramatic reduction in mortality over time in transplanted patients both without , and with DNT (SMR from 269.2 in 1983–1989 to 5.2 in 2005–2010) compared to the Italian Mortality Rates (data not shown).This is the largest and longest single centre Italian collection of DNT after LT series to date.DNT represents a complication that has increasingly gained importance, partly due to improvements of liver transplantation Distinct from most other European studies The incidence of DNT in patients undergoing liver transplantation varies between 3 and 26% In the few studies that have calculated SIRs for DNT the reported figures ranges from 2.2 to 4.0 The incidence of DNT in our cohort of patients was 8.3%. The SIR compared to the Italian cancer registries was 1.8. Our rates and SIR were slightly lower compared to other reported Italian series In the published literature males appear to be more affected than females (ratio 3.29 vs 2.35) The age of onset of cancers has been reported to be similar between transplanted patients and the general population In our series, the age range associated with a higher frequency of tumors was 55–59 years. This is consistent with the fact that in the general population the incidence of cancer increases with age. However, if we consider the SIR, the greatest increase if compared to the general population, is between 20 and 24 years - in relation to Kaposi's sarcoma - and between 40 and 44 years.Viral infections appear to contribute to the development of DNT. In immunocompetent subjects, cervical cancer has been linked to Human Papilloma Virus infections, and Kaposi's sarcoma with Human Herpes Virus 8. Infection with Epstein Barr Virus is the main risk factor for developing lymphoproliferative disorders All studies Delcò With regard to immunosuppressive drugs, though there are some confirmations of the association between the use of cyclosporin and non-melanoma skin cancers We also observed an higher rate of lymphomas in the early years of transplantation (1991–1996), but of kaposi’s sarcoma and bladder tumors after 2006. No specific correlation with initial immunosuppressive therapy concerning calcineurin inhibitors was found even though treatment with cyclosporine was more frequent in the early years. The influence of other immunosuppressive drugs such as Mycofenolate, azathioprine and boluses of corticosteroids could not be studied.In general, the published literature Some authors agree that the highest rate of onset of tumors is in the first year after transplantation, probably due to a greater initial immunosuppression. Our study confirms the expected early appearance of viral/lymphatic tumors. It can be postulated that early viral/lymphatic tumors may be stimulated by early immunosuppression whereas other tumors may need a longer time of stimulation by immunosuppression: so some Authors The study has some limitations. The analysis of the influence of immunosuppressive drugs had to be limited to calcineurine inhibitors and to monoclonal antibodies, only. Indeed amount of steroids, azatiophrine and mycophenolate doses were initially calculated but that study soon resulted too complicated because of dose and timing variations occurred through the years, as it often happens after LT. Such a study can be easily done for short periods, but not for long-term follow-ups.The usual blood target levels for calcineurine inhibitors were applied . The calculation of the entire load of Cyclosporine or Tacrolimus that each patients received all along the years was attempted but this analysis is very complex. We considered OKT3, as that is a very powerful immunosuppressive drug that is expected to influence the onset of tumors as it influences the onset of septic complications. However, in our analysis no directly correlation was shown between OKT3 and DNT. This result can be explained by a poor influence of an “acute” immunosuppressive load on the onset of DNT after LT.As already reported before, an other limitation of this study is that we were unable to evaluate smoking history in our patients. In fact, due to study design concerning data even from the ‘80s and ‘90s, some patients’ characteristics of the medical history could not be retrieved. In Italy the percentage of daily smokers is 24,2% and higher rates can be found among patients with alcoholic cirrhosis. As tobacco consumption in our patients is similar to that reported in other Western countries, and that the medical literature never mentioned specific observations on bladder tumors to date, we can argue that smoking history is probably not involved as ethiologic factor of DNT. Thus our finding of high rates of late onset bladder cancers remains unexplained.In recent years, there has been a dramatic decrease in mortality in transplanted patients with and without DNT, probably due to an improvement in treatments of liver transplantation and cancer. However, the onset of a DNT still heavily influences the patient outcome. The analysis of post LT survival according to different DNT failed to show any statistical significance among them in this study (p = 0.71). It is the authors’ opinion that the best approach to DNT appears to be a very early detection through appropriate screening protocols. Specifically, whereas detection of Kaposi’s sarcoma and lymphomas should be the focus in the early years after LT, 10 years after LT screening should also focus on the detection of urological tumors.This is the largest and longest Italian monocentric study published so far on DNT occurrence after LT. Males were more affected. As expected, we observed increased risks of Kaposi’s sarcoma (in particular in the first 2 years after LT) and of tumors of the lymphatic system. Interestingly, we observed an increased incidence of bladder tumors in males.Multivariate analysis of risk factors identified alcoholic cirrhosis and sclerosing cholangitis in the recipient as the main risk factors for the onset of DNT. The use of tacrolimus and cyclosporine as initial therapy showed no differences with regard to appearance of DNT.The risk of mortality at the onset of DNT adjusted for recipient age and sex, was 4–5 times increased. No differences in survival was demonstrated among different DNT.In conclusion, screening protocols for DNT should be specifically oriented to patients transplanted for alcoholic cirrhosis and sclerosing cholangitis. The focus should be, as expected, on Kaposi’s sarcomas early after LT, and, more remarkably, as never specifically mentioned before, on bladder tumors in men after 10–15 years from LT.