Quantification of cell-free DNA (cfDNA) in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD) quantifies donor-derived cfDNA (dd-cfDNA) by taking advantage of single-nucleotide polymorphisms (SNPs) distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM) of identity-by-descent (IBD) states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD). These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application. More than 180,000 people live with organ transplants in the US. Monitoring the health of the allograft is a critically important component of post-transplant therapy. Recently, we have demonstrated that cell-free DNA (cfDNA) enables diagnosis of post-transplant rejection. In this approach, genotyping of the recipient and the donor together with shotgun sequencing of cfDNA are used to quantify the proportion of donor-derived cfDNA (dd-cfDNA). This approach offers key advantages over the current practice of organ biopsy, which is invasive, costly, and risky. However, the difficulty and cost of establishing a pure reference donor genotype is a major barrier to its widespread clinical implementation. Here, we developed a computational approach that enables an accurate estimation of dd-cfDNA levels without the need for a donor genotype. In addition, we provide a solution of closely related recipients and donors—a scenario which is common in bone marrow and kidney transplants—by accounting for the identity-by-descent between the recipient and the donor. Last, we demonstrate a new application of cfDNA in the noninvasive diagnosis of Graft Versus Host Disease, a serious and difficult-to-diagnose complication of allogeneic bone marrow and stem cell transplantation. Solid-organ transplantation is now a common practice . HoweverHowever, the current implementation of shotgun based GTD, which we will refer to as the “two-genomes” method, requires genotyping of both the donor and recipient. In contrast to the recipient genotype that is easy to obtain, the donor genotype is often unavailable. We therefore set out to develop a method that enables dd-cfDNA monitoring using shotgun sequencing without donor genotype information—a “one-genome” method. We apply the method to lung and heart transplant recipient cohort data and demonstrate that the performance of a one-genome method approaches the performance of the two-genomes method.As in solid organ, cfDNA examination may inform about the status of a bone marrow transplant. More than 10,000 patients receive life-saving allogeneic bone or stem cell transplants in the United States each year, yet complications due to acute Graft-Versus Host Disease (GVHD) occur frequently (in up to 50% of patients), cause morbidity and mortality, and limit the therapeutic value of allogeneic bone or stem cell transplants . DiagnosAs opposed to heart and lung transplants, in which the donor and recipient are not related, in bone marrow transplantation  close relations are common. Therefore, we refined the “one-genome” approach to robustly handle the scenario where the donor and recipient share a recent common ancestor. Chromosomes of such donor-recipient share long stretches of DNA that are Identical By Descent (IBD) , which mWe developed a statistical model that quantifies donor- and recipient-derived cfDNA fragments in the absence of donor genotype information , S1 Fig To calculate the probability of the observed cfDNA, we first calculate the probability of each possible donor and recipient genotype. The likelihood of the recipient genotype is a function of the measured genotype and the genotyping error rate. Vital organ transplants are rarely closely related. Therefore, for heart and lung transplants, our model assumes that the donor genotype is randomly selected from a human population. Given this assumption, the probability of a specific donor allele is its frequency in the population. Our algorithm iterates over 1000 Genomes Project populations and super-populations  to detecPutting it together, the probability of observing a specific allele in a cfDNA fragment is computed by integrating over all possible recipient and donor genotypes and depends on the sequencing error rate, the fraction of dd-cfDNA in the recipient plasma and the probabilities of observing the allele conditioning on it being donor- or recipient-derived . Finally2 = 0.996, Spearman’s ρ = 0.97, mean absolute error = 0.002; 2 = 0.990, Spearman’s ρ = 0.82, mean absolute error = 0.001; To assess the performance of the one-genome model, we directly compared estimates of dd-cfDNA for the one and two-genome methods. We find that the dd-cfDNA predictions based on the two models are highly correlated for lung transplants . The abundance and length of the IBD segments depend on the number of meioses separating the two chromosomes and the recombination rate ,18–21. I2 = 0.998, Spearman’s ρ = 0.99, mean absolute error = 0.004; To evaluate the performance of the refined one-genome method, we applied it to 76 samples from 8 bone marrow transplant recipient patients . Two of The success of bone marrow transplants is often impaired by cancer relapses and graft versus host disease (GVHD) . DiagnosWhat are potential explanations for an increase in the level of cfDNA from recipient origin? In the case of a cancer relapse, the fraction of lymphocytes of recipient origin increases. The cfDNA will therefore reflect increasing levels of recipient-origin lymphocytes. On the other hand, in the case of GVHD, the fraction of lymphocytes from recipient origin does not increase. In this case, the increase in dd-cfDNA is caused by injury to recipient tissues. We therefore hypothesized that differences in the recipient-origin DNA in the cellular and plasma (cell-free) fractions can distinguish between relapse and GVHD. As a proof of principle, we sequenced both the cfDNA and the cellular fraction in patient I8. In agreement with our hypothesis, the two values match until the onset of the acute GVHD (since most cfDNA originates from lymphocytes) and then diverge—after the onset of GVHD, the cellular fraction remains low and cfDNA level increases . This “NTo further assess the robustness and accuracy of the one-genome method described here, we performed extensive simulations . To thisIn general, we found an excellent agreement between the simulated and predicted dd-cfDNA across relatedness . Given the population from which the donor was drawn, the prior probability of observing each allele in the donor is exactly the allele frequency in the population (assuming Hardy-Weinberg equilibrium). Since the donor population is unknown, the optimization function iterates over 1000 Genomes project populations and super-population  and selei where i ∈ {1,2,…,N}; i; i; i; i in population m; i allele in a cfDNA fragment that contains it; and i from a sequencing read of this fragment. The observed data  is therefore the recipient measured genotype at N SNPs and the observed allele of these SNPs in cfDNA sequencing reads.Formally, let N be the number of bi-allelic SNP that were genotyped in the recipient; A and B denote the two possible alleles for SNPd ∈  is the fraction cfDNA fragments that are donor-derived (dd-cfDNA); es ∈  is the sequencing error rate; eg ∈  is the genotyping error rate; and Popm ∈ {1,…,M} is one of M ancestral population and super populations of 1000 genomes project from which the donor is randomly drawn. The model sequencing and genotyping error rates were bound to technically realistic range. The goal of our model is to estimate d–the fraction of dd-cfDNA.Lets also define the following model parameters (θ): i on the true genotype involves the genotyping error rate. So, for example:In our model, of the dependency of the observed recipient genotype of SNPi on the true allele of SNPi in the cfDNA fragment that was sequenced involves the sequencing error rate :Similarly, the dependency of the observed allele in a sequencing read that map to SNPi depends on SNPi alleles frequencies in the population and therefore on which ancestral population is used to achieve the SNPi alleles frequencies estimates:Following the assumption that the donor was randomly drawn from a population, the genotype of SNPi contains a specific allele of SNPi depends of the true genotypes of the recipient and the donor and the fraction of dd-cfDNA (d); for example:Lastly, the probability that a cfDNA sequence that maps to SNPi is:Putting it together the likelihood of observing the recipient genotype and the sequencing reads that map to SNPAlthough it is possible to model the probability of the recipient genotype , the likelihood function is:plink method  is the marginal probability of the pair to be in IBD state. We define lb,b+1 to be the genetic distance between two neighboring loci b,b+1 . The probability of an odd number of recombination events is y1 = m−2 and y2 = 2 + θb,b+12. The transition matrix for two haploids is:i ∈ {1,2}. The transition matrix for the IBD states of the two pairs of haploids is a simple combination of the two haploid pairs transition matrices and depends on their two IBD parameters: In contrast to lung and heart, bone marrow and other organs such as kidney, are often donated by individuals that are closely related to the recipient. Therefore, the assumption that the donor is drawn randomly from the population is no longer valid. Closely-related individuals share stretches of identical haplotypes that were inherited from a recent common ancestor, a phenomenon known as Identity By Descent (IBD). For each pair of chromosomes, IBD segments’ length distribution and total length depend on the number of meioses from their Most Recent Common Ancestor (MRCA). The model accounts for IBD using a non-homogenous Hidden Markov Model (HMM) in which each position in the genome can be in one of three states IBD = 0, IBD = 1 or IBD = 2. The three states correspond to 0,1, or 2 pairs of chromosomes being identical by descent . For effk method . In shorThe emissions probabilities of each SNP in each IBD state are similar to the likelihood function above with one difference—the probability of the donor genotype depends also on the recipient genotype :i, which has two possible alleles: A and B that are occur with frequency fA and fB in Popm respectively, for IBDi = 0,1,2.Tables d—the fraction of dd-cfDNA, and the nuisance parameters are es—sequencing error probability, eg—genotyping probability, popm—donor's ancestral population and Putting it together, the parameters of interest of the model is To assess how well each method dd-cfDNA predictions can be used to discriminate between different levels of heart and lung rejection, we computed the area under the curve (AUC) of the receiver operating characteristic (ROC) similar to how this was done in our previous publications ,9: the dTo simulate organ transplant recipient samples, we used the pre-transplant samples from the bone marrow cohort of patients I2, I3, and I8. In each simulation, we considered one sample as the recipient and one sample as the donor. We merged the data from the two samples and filtered SNPs that were clear genotyping errors . We then mixed randomly sampled reads from each of the two samples at a specific ratio. We considered the fraction of the second sample as the simulated fraction of dd-cfDNA. To simulate IBD we used the same HMM transition matrix as used for the inference. In each transition between genetics blocks, we randomly selected one of the states for the next block using the transition probabilities matrix. If a state has an IBD state of 1, for each “donor” read we randomly selected with probability 0.5 read from the first sample and with probability 0.5 read from the second sample. In IBD state 2 all the “donor” reads were selected from the first sample and in IBD state 0 all “donor” reads were from the second sample. We down sampled the SNPs by taking K SNPs in each genetic block with the highest alternative allele frequency in 1000 genomes project . We randomly down sampled the reads to a desired average coverage . To run many simulations, we saved running time by limiting the inference to a single population. We used the population that was inferred for the donor, since for these cases  the donor was a sibling of the recipient. We show in S1 TableClinical and experimental information and predicted dd-cfDNA levels using two- and one-genome methods of samples drawn from lung transplant recipients.(XLSX)Click here for additional data file.S2 TableClinical and experimental information and predicted dd-cfDNA levels using two- and one-genome methods of samples drawn from heart transplant recipients.(XLSX)Click here for additional data file.S3 TableExperimental information and predicted dd-cfDNA levels using two- and one-genome methods of samples drawn from bone marrow transplant recipients.(XLSX)Click here for additional data file.S4 TableEngraftment, GVHD, repalse and VOD diagnosis dates of marrow transplant recipients.(XLSX)Click here for additional data file.S5 TableMethod performances in various simulated levels of dd-cfDNA, genotyped SNPs and cfDNA sequencing depth.(XLSX)Click here for additional data file.S6 TableComparing predictions of percent of dd-cfDNA using all 1000 genomes populations and a single population that was inferred for the simulated donor sibling in the real data.(XLSX)Click here for additional data file.S1 FigIllustration of the pipeline used to retrieve allele counts in cfDNA fragments for each recipient-genotyped SNP from the raw cfDNA sequencing and genotyping measurements.(PDF)Click here for additional data file.S2 FigAssuming the two-genomes model is the gold standard, we assess the absolute error of the one-genome method in lung (a) heart (b) and bone marrow (c) recipients. Note that this may be an over estimated error, since the two-genomes method is probably not completely accurate.(PDF)Click here for additional data file.S3 Fig(a) and (b) show a comparison of one- and two-genomes methods predictability of organ rejection. In opposed to (PDF)Click here for additional data file.S4 FigPatients I1, I2 and I4 the recipient are males with female donors; patient I8 is a female with a male donor.(PDF)Click here for additional data file.S5 FigEach panel shows results for a single patient. Dotted-dash line marks day in which engraftment was detected (absolute neutrophil count (ANC) > 500 for three consecutive days). Purple dashed lines mark clinical diagnoses. Notice that the predictions of one-genome method that learns IBD are similar to the prediction of the two-genomes method, while fixing the one-genome method to non-related recipient and donor state (IBD = 0) underestimate the dd-cfDNA fraction.(PDF)Click here for additional data file.S6 FigA comparison of the predicted and simulated levels of dd-cfDNA (a) and the corresponding absolute error (b) for different levels of relatedness of the donor and the recipient. dd-cfDNA levels were estimated using 640-700K SNPs and cfDNA sequencing depth of 1.76–1.78x (corresponding to 26M 100bp paired ends reads or 210 USD in our academic sequencing center). Larger markers the median result for a simulated dd-cfDNA level, smaller, unconnected markers show independent simulations. Lines show spline interpolation of the median values.(PDF)Click here for additional data file.S7 FigA comparison of the predicted and simulated levels of dd-cfDNA (a) and the corresponding absolute error (b) for different levels of relatedness of the donor and the recipient. dd-cfDNA levels were estimated using 640-700K SNPs and cfDNA sequencing depth of 1x (corresponding to 15M 100bp paired ends reads or 120 USD in our academic sequencing center). Larger markers the median result for a simulated dd-cfDNA level, smaller, unconnected markers show independent simulations. Lines show spline interpolation of the median values.(PDF)Click here for additional data file.S8 FigA comparison of the predicted and simulated levels of dd-cfDNA (a) and the corresponding absolute error (b) for different levels of relatedness of the donor and the recipient. dd-cfDNA levels were estimated using 157K SNPs and cfDNA sequencing depth of 1x (corresponding to 15M 100bp paired ends reads or 120 USD in our academic sequencing center). Larger markers the median result for a simulated dd-cfDNA level, smaller, unconnected markers show independent simulations. Lines show spline interpolation of the median values.(PDF)Click here for additional data file.S9 FigA comparison of the predicted and simulated levels of dd-cfDNA  and the corresponding absolute error  for different levels of relatedness of the donor and the recipient. dd-cfDNA levels were estimated using 640-700K SNPs and cfDNA sequencing depth of 0.5x  or 0.33x . Larger markers the median result for a simulated dd-cfDNA level, smaller, unconnected markers show independent simulations. Lines show spline interpolation of the median values.(PDF)Click here for additional data file.S10 FigA comparison of the predicted and simulated levels of dd-cfDNA (a) and the corresponding absolute error (b) for different levels of relatedness of the donor and the recipient. dd-cfDNA levels were estimated using 640-700K SNPs and cfDNA sequencing depth of 0.1–1.78x. Larger markers the median result for a simulated dd-cfDNA level, smaller, unconnected markers show independent simulations. Lines show spline interpolation of the median values.(PDF)Click here for additional data file.S11 FigA comparison of the predicted and simulated levels of dd-cfDNA (a) and the corresponding absolute error (b) for different levels of relatedness of the donor and the recipient. dd-cfDNA levels were estimated using 640-700K SNPs and cfDNA sequencing depth of 0.1–1.78x. Larger markers the median result for a simulated dd-cfDNA level, smaller, unconnected marker show independent simulations. Lines show spline interpolation of the median values.(PDF)Click here for additional data file.S12 Fig2 = 0.995). The single population was the population that was inferred for the simulated “donor” sibling in real samples.Comparing predicted percent cf-ddDNA using all 1000 genomes project populations (x-axis) to using the single population (y-axis) over 93 simulations (Pearson’s R(PDF)Click here for additional data file.


Chronic inflammation is thought to be a major driving force for the development of colitis-associated colorectal cancer (CAC). As one member of proinflammatory cytokine family, granulocyte macrophage colony-stimulating factor (GM-CSF) has been identified to play a key role in CAC pathogenesis recently. The underlying mechanisms, however, remain largely unknown. In this study, we found that myeloid-derived suppressor cells (MDSCs) accumulated increasingly in the lesions during the progression from colitis to cancer, which was critical for CAC formation. Importantly, this MDSC accumulation was controlled by GM-CSF. MDSC number decreased significantly in GM-CSF-deficient mice suffering from CAC induction, and transfusion of MDSCs from wild-type CAC-bearing mice into GM-CSF-deficient counterparts led to recurrence of CAC. Furthermore, the supernatants of CAC lesions or GM-CSF alone was sufficient to differentiate hematopoietic precursors into MDSCs. Addition of neutralizing anti-GM-CSF antibody impaired the MDSC-differentiating effects of the supernatants of CAC lesions. Overall, these findings shed new insights into the mechanisms of GM-CSF underlying CAC development, by inducing/recruiting CAC-promoting MDSCs. Blocking GM-CSF activity or MDSC function may represent new therapeutic strategies for CAC in clinic. Although the molecular mechanisms underlying transition from colitis to colorectal cancer remain elusive, a set of proinflammatory factors secreted by immune and non-immune cells has been shown to contribute to tumor growth in chronic intestinal inflammation.5It is well established that chronic unresolved inflammation represents a major driving force for the initiation and progression of malignancies in many organs.7 In accordance with this, GM-CSF-deficient mice were more susceptible to DSS-induced colitis, as shown by more weight loss and severe diarrhea and by upregulation of proinflammatory mediator production.8 The role of GM-CSF in CAC development, however, is largely unknown. Recently, Wang et al.9 have reported that blockade of GM-CSF bioactivity using neutralizing specific monoclonal antibodies dramatically repressed CAC formation, through stimulating vascular endothelial growth factor (VEGF) release of colonic epithelial cells (CECs). Given that GM-CSF has a great impact on host intestinal immunity under physiological and pathological conditions,10 the regulation of immune microenvironment (e.g. MDSCs) in CAC settings by GM-CSF is deserved to be further addressed.Granulocyte macrophage colony-stimulating factor  is one of hematopoietic cytokine family and its levels significantly elevated in the lesions of patients and rodents with colitis.11 Tumor-derived factors mobilize MDSCs from bone marrow into peripheral blood, and then MDSCs suppress anti-tumor immunity and thereby favor tumor growth.1214 The inhibitory properties of MDSCs have been proposed to be attributed to the expression of inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1), which are both involved in the metabolism of l-arginine.15MDSCs represent a heterogeneous population of myeloid cells, which comprise immature macrophages, granulocytes, dendritic cells (DCs), and other myeloid cells at earlier stages of differentiation that can be identified in mice by expression of CD11b and Gr-1.17 These cells suppress cytotoxic T-cell responses and promote CAC development, whereas the regulation of MDSC generation and accumulation in this setting is still elusive. We hypothesized that GM-CSF plays an important role in MDSC accumulation in CAC, considering the function of this cytokine on MDSC generation and recruitment reported in several tumor models.18 Herein, we showed that MDSC accumulation was profoundly impaired in CSF2−/− mice subjected to CAC induction. Supplement of MDSCs from CAC-bearing wild-type (WT) mice into CSF2−/− littermates resulted in recurrence of CAC. Furthermore, the supernatants of tumor explant culture or GM-CSF alone enabled differentiation of hematopoietic progenitors into MDSCs. Blocking GM-CSF activity remarkably abrogated this effect.Recently, massive accumulation of MDSCs in the lesions of inflammatory bowel disease (IBD) and CAC has been observed.C57BL/6 WT and CSF2−/− mice were purchased originally from Jackson Laboratory . Animals were housed in specific pathogen-free conditions with an alternating light/dark cycle. All experiments were performed using 6- to 8-week-old male mice. Care, use and treatment of mice in this study were in strict agreement with international guidelines for the care and use of laboratory animals and approved by Animal Ethics Committee of Jilin University.19 with mild modification  and CD8α (eBiosciences) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 10% goat serum. Biotinylated secondary antibody (Dako) was added and incubated at room temperature for 1 h. Streptavidin–horseradish peroxidase (HRP) (BD Pharmingen) was added, and after 40 min the sections were stained with DAB  substrate and counterstained with hematoxylin.20 with the purity >80%, identified by CK-18 staining. For isolation of lamina propria (LP) immune cell subsets, the entire length of colon was opened longitudinally, washed with PBS, washed with PBS containing 100 µg/mL gentamycin, and cut into small pieces. The dissected mucosa was incubated with Ca2+, Mg2+-free Hank’s Balanced Salt Solution (HBSS) supplemented with 10% fetal bovine serum, 5 mM ethylenediaminetetraacetic acid (EDTA), 15 mM HEPES, 100 µg/mL gentamycin under shaking at 37°C for 20 min then repeated for four times. The supernatants containing CECs were depleted and the remaining was washed with HBSS, followed by digestion in RPMI 1640 medium containing 100 U/mL collagenase D (Roche Diagnostics) under shaking at 37°C for 1 h then repeated for two times. The total of digested supernatants was centrifuged at 800g for 10 min at 4°C and washed with PBS. The pellet was layered on a 40%/100% Percoll gradient (Pharmacia) and spun at 1800 r/min for 5 min to collect the mononuclear immune cell–enriched population at the 40%/100% Percoll interface.CECs were fractionated as described previouslyA single cell suspension prepared from colonic tissues as described above was fixed using PBS containing 1% paraformaldehyde and stained on ice for 25 min with antibodies in PBS containing 2% heat-inactivated fetal calf serum. The antibodies used were fluorescein isothiocyanate (FITC)-labeled anti-murine Gr-1 , and allophycocyanin-labeled anti-murine CD11b . Data collection and analysis were performed on a fluorescence-activated cell sorting (FACS) Calibur flow cytometer using CellQuest software (Becton Dickinson). For cell sorting, a single cell suspension from the colon was stained with antibodies and sorted on a FACS AriaII (Becton Dickinson). The antibodies used for sorting were those described above plus FITC-labeled anti-murine T-cell receptor (TCR)-β , Ly-6G , CD19 (1D3. eBioscience) or CD3 , PE-labeled anti-murine CD49b , CD11c , or F4/80 .g at 4°C for 5 min and stored at −80°C until use.Colon tissue (200–300 mg) was washed in cold PBS containing penicillin and streptomycin and cut into small pieces (0.5 cm × 0.5 cm), which were cultured (three pieces per mouse) in 24-well flat bottom culture plates in serum-free RPMI 1640 medium (Gibco) at 37°C for 24 h. The culture supernatants were then centrifuged at 90005 splenocytes isolated from naïve C57BL/6 mice, in round-bottom 96-well plates. These cocultures were promptly stimulated with 5 µg/mL Con A for 72 h. Cultures were pulsed with 1 µCi/well [3H] thymidine for the last 16 h, and then the cells were harvested and counted by standard liquid scintillation. Inhibitors of Arg-1 and/or iNOS included N-hydroxy-l-arginine , an inhibitor of iNOS and Arg-1 , the specific Arg-1 inhibitor N-hydroxy-nor-l-arginine , and the NOS inhibitor l-N-iminoethyl-lysine  were added from the beginning of the culture.FACS-sorted MDSCs were added in various amounts to 5 × 106 FACS-sorted MDSCs or non-MDSCs (Gr-1−CD11b+) were intravenously injected into one CSF2-deficient mouse twice a week from the beginning of DSS treatment to the end of the experiments.A total of 3 × 10+CD11b+ cells from bone marrow progenitors was done as described previously,22 with mild modifications. In brief, bone marrow cells were obtained from the femurs and tibias. C-kit+lineage− progenitors were sorted by FACS and cultured in 24-well plates in medium conditioned by the supernatants from colon explants or recombinant murine GM-CSF , supplemented with 10% fetal calf serum (Gibco) and interleukin (IL)-4 . In some experiments, purified neutralizing anti-GM-CSF monoclonal antibody  was added into the culture. Cells were collected 7 days later, and the proportion of Gr-1+CD11b+ cells was analyzed by flow cytometry.Induction of Gr-1GM-CSF was examined in whole colon culture supernatants by ELISA kits obtained from R&D Systems, according to the manufacturer’s instructions.23 The supernatants were deproteinized and derivatized with 0.2 M ophthalaldehyde/β-mercaptoethanol. A total of 50 µL was injected per sample. Standards of l-arginine in methanol were run with each experiment.High-performance liquid chromatography (HPLC)-electrochemical detector was performed using an ESA CoulArray Model 540, with an 80 × 3.2 column with 120A pore size as described previously.To deplete MDSCs, mice received intraperitoneal (i.p.) injections of 250 µg of purified monoclonal anti-Gr-1 antibody  or a control isotype on days −3 and −2 before initiation of DSS drinking, then twice a week (days 2 and 5 each week) throughout the entire experimental period to ensure depletion. Depletion efficiency was confirmed at the end of experiments using flow cytometry (>80% MDSCs were depleted).RNA extracted from colon epithelial cells or cell lines was reverse-transcribed into complementary DNA (cDNA) using the SuperScriptIII First Strand cDNA synthesis system (Invitrogen). cDNA was synthesized from 0.5 µg RNA using random hexamer primers and SuperScriptIII (Invitrogen). Real-time reverse-transcriptase-polymerase chain reaction (RT-PCR) was performed on a Bio-Rad iCycler to quantify messenger RNA (mRNA) levels. The primers are as follows: GM-CSF—sense: 5′-TCT CAG CAC CCA CCC GCT CA-3′, anti-sense: 5′-GCC CCG TAG ACC CTG CTC GAA-3′; Arg-1—sense: 5′-CTC CAA GCC AAA TAC AAG A-3′, anti-sense: 5′-AGG AGC TGT CAT TAG GGA CAT C-3′; iNOS—sense: 5′-GTT CTC AGC CCA ACA ATA CAA GA-3′, anti-sense: 5′-GTG GAC GGG TCG ATG TCA C-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)—sense: 5′-TCT TGG GCT ACA CTG AGG AC-3′, anti-sense: 5′-CAT ACC AGG AAA TGA GCT TGA-3′. All reactions were performed in triplicate. The data were analyzed using Q-Gene software and expressed as fold change mean normalized expression (MNE) from control value. MNE is directly proportional to the amount of RNA of the target gene relative to the amount of RNA of the reference gene, GAPDH.t-test (two-tailed) was used to determine significance, with P < 0.05 considered significant. Statistics were performed using SPSS 10.0 for Macintosh, and graphs were made on Deltagraph (SPSS).Data are presented as mean ± standard deviation (SD). Student’s 9 GM-CSF was predominantly secreted by CECs as well as mononuclear phagocytes (MPs) residing in the LP of the colon, although the abundance of this cytokine in the latter population was much lower than that in CECs  residing in LP also increased significantly following CAC induction  or MDSCs from lesions of AOM/DSS-induced CSF2−/− mice had no effect  were fractionated from bone marrow and treated with GM-CSF or the supernatants of colon explant culture in vitro (here named CAC CM). The results showed that GM-CSF alone sufficiently induced progenitors to differentiate into Gr-1+CD11b+ cells, which resembled CAC CM , their levels have not been detected in this study, but previous studies have shown that the increase in their production in CAC lesions was observed at the late stage instead of the early stage of CAC.9 It is thus reasonable that G-CSF and M-CSF may be dispensable for the early-stage accumulation of MDSCs in CAC lesions and possibly be involved in the late-stage accumulation of these cells. Recently, another proinflammatory mediator prostaglandin E2 (PGE2) has been demonstrated to be linked to MDSC accumulation in CAC model.17 Therefore, we can make a conclusion that GM-CSF, with synergistic action of other factors (e.g. PGE2), recruits MDSCs into lesions and eventually promotes CAC growth.Of note, a set of cytokines and growth factors other than GM-CSF has been implicated in the pathophysiology of MDSCs in several mouse models including IL-1β, IL-6, and VEGF.33 It should be noted that CSF2−/− mice have relatively normal hematopoiesis in bone marrow.35 So, the decrease in MDSC number in lesions of CSF2−/− mice appears to be due to the impairment of differentiation of progenitors into MDSCs and/or the inhibition of MDSC recruitment into lesions.Furthermore, we have shown that compared with WT MDSCs, the equal number of MDSCs from CSF2−/− mice cannot recover CAC in CSF2−/− recipients subjected to AOM/DSS. This indicates that the suppressive capacity of MDSC was seriously blunted in the absence of GM-CSF. In support of this viewpoint, GM-CSF alone is sufficient to induce the generation of immunosuppressive MDSCs in vitro, which is consistent with previous observations in a spontaneous model of pancreatic ductal adenocarcinoma.36 or their effect on angiogenesis,37 further investigations in the details of MDSC function on CAC in the future are warranted. Overall, our findings shed new insights on the understanding of mechanisms underlying CAC pathogenesis. Targeting GM-CSF or blocking MDSC generation and/or recruitment using appropriate inhibitors may represent new regimens for the treatment of CAC in clinic.In conclusion, our study confirms the pathogenic role of GM-CSF in CAC development. GM-CSF favors tumor-permissive microenvironment by inducing MDSC generation and recruiting them into colonic tissues. MDSCs thereafter actively suppress anti-tumor immune responses and support CAC growth. Considering a direct function of MDSCs on cancer cells


India ink microscopy on cerebrospinal fluid is still utilized in resource limited settings for the diagnosis of cryptococcal meningitis despite its poor sensitivity. We hypothesized that staining fungal nucleic acids with fluorescent dyes instead of the capsule with India ink might improve sensitivity for the diagnosis of cryptococcal meningitis.We enrolled 96 HIV-infected participants with cryptococcal meningitis who provided 194 CSF specimens at serial time points in Kampala, Uganda. Cryptococcosis was diagnosed by cerebrospinal fluid (CSF) cryptococcal antigen (CrAg) test and only positive samples were included. We stained CSF with India ink and acridine orange. We cultured the same samples on standard fungal media. We compared acridine orange to CrAg, India ink and CSF culture.Cryptococcus yeasts. India ink had more negative results (22%) than acridine orange (4%). The sensitivity for India ink increased (86%) while that of acridine orange did not change (97%) when compared to CSF culture. However, both India ink and acridine orange had poor predictive values with reference to culture.Acridine orange was more sensitive (96%) than India ink (79%) with reference to CSF CrAg. Acridine orange and India ink had a statistically significant difference (P<0.001) with a 25% correlation for detection of Acridine orange is a better alternative to India ink in the rapid detection of cryptococcosis among CrAg positive HIV patients. Cryptococcal meningitis (CM) is the most common cause of adult meningitis in Africa accounting for 15%–25% of AIDS-related deaths; with majority of the cases registered in sub-Saharan Africa . CultureIndia ink has over time been used in the rapid diagnosis of cryptococcal meningitis using cerebrospinal fluid (CSF) among AIDS patients . It is sCryptococcus neoformans using three laboratory strains  of CSF sediment added to one drop [40μl] of acridine orange). The concentration of acridine orange solution used was “0.1g acridine orange in 1000ml of 0.5M acetate buffer”. The mixture was left to stand for 5 minutes at room temperature. One drop of the mixture was then loaded onto a glass slide and a cover slip added. The slide was examined on a fluorescent microscope using a blue filter and 40x objectives.For this procedure, whole CSF was span for 5 minutes at 10000g. One drop (40μl) of India ink was put on a clean slide and one drop (40μl) of CSF sediment was added and the slide was examined using a light microscope through 40x objective.Statistical analysis was aimed at comparing the sensitivity, level of agreement and deviation in the levels of positive results obtained using acridine orange compared with India ink in the detection of cryptococcal cells at 95% confidence interval (CI). Data were analyzed using STATA version 13 .2O  while the median CSF white blood cell count was 4 cells/mL  (Table 1).We enrolled 96 study participants with cryptococcal meningitis who provided 194 CSF specimens at serial time points in Kampala, Uganda from March 2015 to December 2015. 62% of participants were males with an overall median age of 35 years . All participants were HIV-positive adult Ugandans with a median CD4 T cell count of 14 cells/μL  at diagnosis. Only 51% (49/96) of the participants were receiving antiretroviral therapy at diagnosis. Among patients reporting a headache (n = 92), the median duration of headache was 14 days . The CSF opening pressures at baseline (n = 95) had a median of 322 mmHOf the 194 CrAg LFA positive CSF samples tested from different time points during antifungal therapy, 152 were positive for India ink, 186 were positive for acridine orange, and 151 were positive for quantitative cultures. 127 samples were positive for all these three tests. The overall (n = 194) median for quantitative cultures was 820 CFU/ml (IQR = 10 to 73000). Acridine orange had a sensitivity of 96% while India ink had a sensitivity of 78% with reference to CSF CrAg (n = 194).Table 2).For both India ink and acridine orange tests, the level of positivity was graded on a scale of zero (0) to +3 where; zero = negative, +1 = 1–9 cells in 100 fields, +2 = 10–99 cells in 100 fields and +3 is ≥100 cells in one field. Using India ink (n = 194), 33% of the samples had a low positivity grade of +1, followed by a +3 (25%), then zero (negative—22%) and the least with a +2 (21%). However, using acridine orange (n = 194), 47% of the samples had a positivity grade of +1, followed by a +2 (29%), then +3 (20%) and the least with zero (negative- 4%)  in the grades of positivity between acridine orange and India ink at a 95% CI. However, there was a 25% correlation between the two tests. The same statistical analysis also revealed that acridine orange was more sensitive (96%) than India ink (79%) with reference to CSF CrAg. This confirmed the analysis done in the previous paragraphs. In addition, since all samples were from known positive cases (using CSF CrAg), this result was also supported by the fact that India ink had more negative results ([42] 22%) than acridine orange ( 4%).Table 2). False negatives were registered more among patients who had received more than seven doses of Amphotericin B. However, with reference to CSF CrAg, CSF culture had a lower sensitivity (78.4%) and only 66% of the samples (n = 194) were positive for all the three tests . Even though culture of CSF is still considered the gold standard for diagnosis of cryptococcal meningitis, recent evidence indicates that detection of the cryptococcal antigen (CrAg) in CSF has a better sensitivity [When compared to quantitative CSF culture, the sensitivity for India ink increased to 86% 130/152) while that of Acridine orange did not change (97%). However, both India ink  and acridine orange  had poor predictive values with reference to culture. 52% (22/42) of the India ink negative samples were positive with culture while 63% (5/8) of the acridine orange negative samples were positive with culture. When comparing both India ink and acridine orange to culture, false positive results were registered more among samples with a (+1) grade of positivity (2 while tMycobacterium tuberculosis among HIV patients indicated that fluorescent microscopy is more sensitive than light microscopy (unpublished). Therefore, at the beginning of the study we stained cryptococcal isolates with auramine-O. The cryptococcal yeasts were clearly visible fluorescing against a pale background. This differential fluorescence of cryptococcal yeasts picked our interest to evaluate acridine orange as a potential replacement for India ink. We thought that its ability to freely enter dead or live cells and bind to nucleic acids would be very vital and increase the sensitivity of determining sterility of cerebrospinal fluid rapidly other than using india ink to stain the capsule ; which sometimes is absent in some strains among our HIV patients being managed for cryptococcal meningitis.These results suggest that fluorescent microscopy maybe more sensitive and reliable in mass screening programs for cryptococcal disease among HIV patients, especially in resource limited setting where the disease remains a burden to the health systems. Our prior utility of auramine-O staining for the detection of Cryptococcus neoformans, by determining the limit of detection of three strains. Results indicated that conventional methods are of comparable sensitivity. However, there was no apparent advantage to the acridine orange method over the other tests [A similar study by Cohen investigated the sensitivity of India ink, acridine orange and latex agglutination for the rapid identification of er tests . Besideser tests . Most ofer tests , 16 and er tests . Howeverer tests  and low er tests , 19, acrThe major study limitation was that we did not include samples that were negative for CSF CrAg. However, based on our findings, we conclude that with the availability of a fluorescent microscope, acridine orange is a better alternative to India ink in the rapid detection of cryptococcosis among CrAg positive HIV patients especially in sub-Saharan Africa where the disease remains a burden to health systems.S1 TableSpreadsheet contains all raw data for the study population.(XLSX)Click here for additional data file.


Palate is a complex structure separating oro- and nasopharynx. However, reconstruction of the defects of palate is much simpler because of the versatile mucoperiosteal flaps. Here, we present our experience of palatal mucoperiosteal flap used in different situations. Fifteen patients of palatal as well as buccal mucosa defects were reconstructed using either free or pedicled mucoperiosteum.All patients recovered well. No flap loss or secondary procedure were required.Success in Reconstruction of the palatal defects depends on creation of good nasal as well as buccal mucosal lining. The rich vascular macronet in the palatal mucosa makes it an ideal donor site for local reconstruction. The mucoperiosteum harvested either as a free graft or as pedicled flap serves the purpose well leaving no donor site deformity. The bare bony frame work epithelises leaving no donor site deformity. All these features make it the best available tissue for reconstruction of local as well as adjacent buccal mucosa.3Palate is a complex structure both anatomically and functionally, comprising of bone, muscle and mucosa. Functionally both hard and soft palate, together serve as one unit to separate nasopharynx and oropharynx. Such separation is important for articulation of speech and deglutition.Ten patients of palatal as well as buccal mucosa defects were reconstructed using either free or pedicled mucoperiosteum. (i) Two patients were case of isolated Group-2 cleft palate, in which Wardill-Kilner-Veau pushback palatoplasty was done. The nasal layer breached during dissection and suturing. Hence, repair of nasal lining at the junction of hard and soft palate could not be achieved. The two mucoperiosteal flaps were intact. The nasal mucosa was reconstructed by flipping the left mucoperiosteal flap with mucosa facing nasal cavity and it was sutured to the bony palate.On top of this layer the right mucoperiosteal flap was transposed and sutured to create the oral mucosal layer  and 2. TFour patients had buccal mucosal defect following excision of early malignancy. In these patients, mucosa was replaced with free grafts  from the intact palate. The free mucoperiosteal graft took very well and functioned as mucosa after the wound healed.Pedicled flaps as well as free grafts healed very well without any morbidity. Palatal donor site healed by epithelisation and had no further deformity.Pedicled flaps as well as free grafts healed very well without any morbidity. Palatal donor site healed by epithelisation and had no further deformity. Initially when pedicled flap was used, there was speech disturbance which improved later.4 towards the in posterior region. This dynamicity is due to the musculature of the soft palate.5 Trauma and scarring of the soft palate mucosa may lead to tethering and hamper speech and deglutition, hence its mucosa can neither be harvested nor be used for any reconstruction.6,7Palate is a complex structure. On either side of palate, there are cavities which have major functions . The separating structure has thin bone, covered on either side by mucosa anteriorly. As we move posteriorly the rigid hard palate becomes dynamic soft palate6,8 Pedicled mucoperiosteal flaps can either be islanded or Peninsular flap.9,10 The Greater palatine artery entering the mucoperiosteum at the palatine foramen forms the major supply and one pedicle can sustain most of the mucoperiosteum over the palate. In two of our cases of complete cleft of secondary palate, during the Wardill-Kilner-Veau (V-W-K) repair, the nasal mucosa breached and resulted in fistula at the junction of hard and soft palate. The thinness of nasal mucosa and less tonicity makes it very difficult to suture and as literature shows fistula is inevitable in inexperienced or even in case of experienced surgeons.8 The nasal and oral mucosa was deficient. Hence the adjacent mucoperiosteal flaps were used to create both nasal and oral mucosa. Creating a good nasal lining is an important step in cleft palate repair and double layered closure is the mainstay for a successful cleft palate repair.8 Every effort should be given to create tough nasal mucosal layer. The thinness of nasal mucosa and less tonicity makes it very difficult to suture and fistula is inevitable in inexperienced/ even in case of experienced surgeons.8The buccal mucosa, as a donor site for grafts in itself is very versatile, as it heals on its own and leaves no donor site deformity. However, the mucoperiosteum of hard palate, is supplied by greater palatine artery is highly vascular. In view of its versatility it can either be harvested as a free graft or pedicled flap.11 But in our case, we used one side palatine mucoperiosteal flap for nasal lining and other for oral lining. Both the flaps used to create palatal separation healed without complication and had good speech outcome.On table nasal mucosal fistula as a small perforation can be left alone to contract and heal but if large, then a salvage procedure to recreate nasal lining is a mandate. Variety of flaps has been used as salvage procedure. Ipsilateral vomerine flap is one. Buccal mucosal flap is another choice.12 In two of our cases of salivary gland tumor, post-excision defect was situated at the junction of hard and soft palate. In one case, the challenge was to recreate the mucosal lining without altering soft palate function. Here we used the whole of mucoperiosteal islanded flap based on one greater palatine artery to cover the area.The vascular capillary network (Vascular macronets) allows whole of the mucoperiosteum to be elevated based on one greater palatine artery and also as islanded pedicle flap.14.But the posterior reach of islanded palatine flap is difficult, due to the bony canal through which the pedicle emerges. But breaking the posterior wall of the palatine foramina makes the reach possible.13 The donor site heals completely in 2-3 weeks without contractures or compromising function of the palate.14,15Since 1977, island flap for palatal reconstruction is used with 95% success rate16 The Results of palatal free grafts were superior to skin grafts. Whereas buccal mucosal grafts as skin grafts resulted in secondary contracture and the area had less pliability. Small defects in oral cavity can be left alone for secondary healing or skin grafted. However these have disadvantages like unpredictable contracture, initial malodorous discharge and pooling of saliva.17 Similarly use of mucoperiosteal flap did not result in donor site deformity and was native to the tissue. But the limitation was the size of graft available. Larger defects definitely need distant/regional flap.18-21 In all our cases, there was no flap loss/necrosis. Even in free grafts, we had excellent outcome. There was absolutely no donor site deformity.Free graft in our case was used for a small carcinoma in situ, where the maximum mucosal defect was 3×4 cm. This has the advantage of being local tissue and hence it does not require any other donor area for harvesting. The donor site also granulates and heals well by epithelisation.In our adult patient there was no donor site morbidity. Free graft has been used in recalcitrant pharyngeal wall stenosis.Palate is a complex structure both functionally and anatomically. Successful reconstruction of palatal defects depends on creation of good nasal as well as buccal mucosal lining. The rich vascular macronet in the palatal mucosa makes it an ideal donor site for local reconstruction. The mucoperiosteum harvested either as a free graft or as pedicled flap serves the purpose well leaving no donor site deformity.The authors declare no conflict of interest.


Supplemental Digital Content is Available in the Text. Cerebrospinal fluid (CSF) viral escape is an increasingly recognized clinical event among HIV-1-infected adults. We analyzed longitudinal data and drug-resistance mutations to characterize profiles of HIV-1-infected patients on antiretroviral therapy with discordant CSF and plasma HIV-1 RNA levels.Forty-one cases of CSF escape defined as detectable CSF HIV-1 RNA when plasma levels were undetectable, or HIV-1 RNA >0.5-log higher in CSF than plasma were identified from Boston Hospitals and National NeuroAIDS Tissue Consortium (NNTC) from 2005 to 2016.3 and CD4 nadir 21 cells/mm3. Neurological symptoms were present in 30 cases; 4 had repeat episodes of CSF escape. Cases were classified into subtypes based plasma HIV-1 RNA levels in the preceding 24 months: high-level viremia (1000 copies/mL), low-level viremia (LLV: 51–999 copies/mL), and plasma suppression with CSF blip or escape (CSF RNA <200 or ≥200 copies/mL). High-level viremia cases reported more substance abuse, whereas LLV or plasma suppression cases were more neurosymptomatic (81% vs. 53%); 75% of repeat CSF escape cases were classified LLV. M184V/I mutations were identified in 74% of CSF samples when plasma levels were ≤50 copies per milliliter.Estimated prevalence of CSF escape in Boston and NNTC cohorts was 6.0% and 6.8%, respectively; median age was 50, duration of HIV-1 infection 17 years, CD4 count 329 cells/mmCharacteristics frequently observed in CSF escape include HIV-1 infection >15 years, previous LLV, and M184V/I mutations in CSF. Classification based on preceding plasma HIV RNA levels provides a useful conceptual framework to identify causal factors and test therapeutics. Although CSF viral suppression is maintained in most HIV-infected individuals taking ART, higher HIV-1 RNA levels in CSF than plasma are observed in some ART-experienced individuals, a discordance referred to as CSF viral escape.4The global prevalence of HIV-1 associated dementia has declined with suppressive antiretroviral therapy (ART), yet milder forms of HIV-1 associated neurocognitive disorders persist in 25%–50% in people with chronic HIV-1 infection.9 Predictors of CSF escape remain poorly defined; potential risk factors include poor CNS penetration by some ART regimens,7 persistent low-level viremia (LLV),9 length of time on ART,6 drug-resistance mutations (DRMs) in CSF,11 and low CD4 nadir.8 The relationship between CSF escape and neurological symptoms is unclear among individuals on suppressive ART regimens because CSF HIV-1 RNA levels do not correlate consistently with neurological symptoms.12 A designation of asymptomatic CSF viral escape has been proposed to classify individuals with CSF HIV-1 RNA up to 200 copies per milliliter, and absence of new or progressive neurological impairment.13 In contrast, several case series 8 and case reports 29 describe individuals presenting with new neurological symptoms, discordant elevation in CSF HIV-1 RNA, and substantial improvement after switching ART regimens; many cases have unique DRMs in the CSF. However, the mechanisms leading to CSF viral escape are not well defined.The prevalence of CSF viral escape is estimated at 4%–21% among ART-experienced HIV+ adults.Given variable prevalence and few published cohorts of CSF viral escape, the aim of this study was to analyze temporal patterns and relationships between CSF viral escape and antecedent plasma HIV-1 RNA levels in a cohort of ART-experienced HIV-1-infected individuals. We also evaluated drug resistance mutations in the viral reverse transcriptase (RT), protease, and integrase genes in plasma and CSF from CSF escape cases from the study cohort and 19 published studies.30 NNTC evaluations yield neuropsychological diagnosis of asymptomatic neurocognitive impairment , mild neurocognitive disorder , HIV-associated dementia (HAD), and neuropsychological impairment due to other factors based on Frascati guidelines.31 The study was reviewed and approved by the institutional review boards at MGH, BWH, Dana Farber Cancer Institute, and Tufts MC. The institutional review board at each of the 4 NNTC clinical sites approved the research, and subjects signed a written statement of informed consent.This is a retrospective case-series of 41 cases from 2 sources: (1) neurology and infectious disease clinics from 5 Boston hospitals ; and (2) NNTC . The NNTC protocol includes neuromedical and cognitive examinations, assessments of comorbidities, and laboratory values for medical, immunologic, and virologic parameters.4The study was restricted to the calendar period between January 2005 and May 2016. Cases were eligible for inclusion if they were on stable ART regimens for at least 6 months and met either of the following criteria: (1) CSF HIV-1 RNA >50 copies per milliliter when paired plasma HIV-1 RNA were <50 copies per milliliter; or (2) CSF HIV-1 RNA >0.5 log higher than paired plasma levels. NNTC cases were identified by searching the database for subjects with paired plasma and CSF HIV-1 RNA values (within 7 days) (n = 426) before applying the above criteria for CSF escape (n = 29). Boston cases were identified by directly querying HIV providers. A neurological examination was performed in all cases; neurologically symptomatic conditions were defined as symptoms or diagnoses attributable to the CNS, as described.http://links.lww.com/QAI/A991). The 2010 CNS penetration effectiveness (CPE) score for drug regimens was calculated for each ART regimen.32HIV-1 RNA levels were measured in cell-free CSF and plasma at local sites; because of variability by hospital and calendar year, levels reported as undetectable were imputed to the lower limit of detection for the assay .Descriptive statistics  were performed based on variable distribution. Univariate/bivariate tests were conducted using Among Boston and NNTC patients, 41 HIV-1-infected adults met inclusion criteria for CSF viral escape and were included in these analyses. Twenty-nine CSF escape cases were identified from 426 cases with paired plasma and CSF HIV-1 RNA values from the NNTC, a prevalence of 6.8%. Twelve CSF escape cases were identified from the Boston hospitals from approximately 200 patients screened for CSF HIV-1 RNA levels during the study period, an estimated prevalence of 6.0%.P = 0.4), sex (P = 0.2), or race (P = 0.5) between the 2 cohorts. The NNTC cohort had higher percentages reporting cocaine and/or amphetamine use (43% vs. 8%) and alcohol use (24% vs. 17%) than the Boston cohort. The median duration of HIV-1 infection in the combined cohort was 17 years (IQR: 13–22), median CD4 T-lymphocyte count 329 cells/mm3 (IQR: 217–388), and median CD4 nadir 21 cells/mm3 (IQR: 2–60). ART regimens at the time of CSF escape consisted of a ritonavir-boosted protease inhibitor (PI/r) in 31 of 41 cases (76%); PI/r regimens had either at least 2 nucleoside reverse transcriptase inhibitors  or an integrase inhibitor .Clinical characteristics for the NNTC and Boston cases are shown in Table http://links.lww.com/QAI/A991).Neurological abnormalities included worsening depression, cognitive symptoms, and/or evidence of HIV-1 associated neurocognitive disorders (n = 24), seizures (n = 3), sensory hypoasthesia and carotid artery dissection (n = 1), and stroke (n = 1); 2 cases had a concurrent diagnosis of progressive multifocal leukoencephalopathy and one had CNS lymphoma .All Boston cases had CSF analysis for neurological symptoms, and on average underwent 2 lumbar punctures. The NNTC collects data for clinical and research purposes; 62% of NNTC cases had neurological symptoms at time of lumbar puncture, and on average, each case underwent 7 lumbar punctures. Median CSF HIV-1 RNA levels among NNTC subjects were 1406 vs. 615 copies per milliliter for Boston cases (http://links.lww.com/QAI/A991); 1 case remained neurologically asymptomatic (No. 29). In case 14 (HAD), consecutive CSF HIV-1 RNA was 1262, 101, and 171,000 copies per milliliter at CSF escape, 3 and 4.5 years later, respectively, whereas case 29 (asymptomatic) had CSF HIV-1 RNA of 141, <50, 180, and 490 copies per milliliter, at CSF escape, 1, 2, and 4 years later : incomplete plasma suppression (n = 24) and durable plasma suppression (n = 17). Among cases with incomplete plasma suppression, those with detectable plasma level ≥1000 copies per milliliter or levels between 51 and 999 copies per milliliter during 6 months before escape were defined as high-level viremia (HLV) or LLV, respectively, a cutoff used to differentiate between persistant LLV and virologic failure as previously described.35 Cases with durable plasma suppression (undetectable plasma HIV-1 RNA levels for 24 monthsbefore escape) were further subdivided as CSF HIV-1 RNA levels <200 copies per milliliter (CSF blip), or levels ≥200 copies per milliliter (CSF escape). Cases with a single viral blip <1000 HIV RNA copies per milliliter 7–24 monthsbefore escape with subsequent return to undetectable levels were considered durably suppressed.36 Plasma and CSF HIV-1 RNA levels and timing of neurological symptoms from representative cases for each CSF viral escape subtype  are depicted in Figure After analyzing all available antecedent plasma HIV-1 RNA levels and their respective relationship to CSF HIV-1 RNA, 4 patterns were evident within 2 broad categories defined by plasma HIV-1 RNA levels , plasma HIV-1 RNA copies at CSF escape , or CD4 nadir ; CD4 cell count was lower in the HLV subgroup (P = 0.04). Although 6 of 9 LLV subjects had multiple visits with plasma HIV-1 RNA levels between 51 and 999 copies per milliliterbefore CSF escape, in 5 cases, CSF HIV-1 RNA levels were >1 log10 higher than the preceding 12 months of plasma HIV-1 RNA levels. Paired plasma and CSF HIV-1 genotypes were available for 3 LLV cases , whereas CSF WBC (P = 0.2) and CSF total protein levels (P = 0.1) were similar. One case classified as CSF escape had multiple thymidine analogue-associated mutations  in the presence of the M184I mutation (Table http://links.lww.com/QAI/A991).Among 17 cases with durable plasma suppression, subgroups classified as CSF blip vs. CSF escape were similar with respect to age, duration of HIV diagnosis, plasma HIV-1 RNA levels, and CD4 cell count Table . CSF HIVon Table  on a reg38 Two studies reported sequencing the integrase gene in CSF39; N155H, L74I, and Q148R mutations were observed as single mutations in the integrase gene, and Y143C was observed in combination with T66I.Given that drug-resistant HIV-1 virus has been frequently associated with CSF viral escape, we analyzed HIV-1 mutations from this cohort and published studies. A total of 172 mutations in the RT gene, 135 mutations in the protease gene and 6 mutations in the integrase gene were identified in CSF from 49 cases (Table 13 Cases in this study cohort were classified into 4 subtypes based on plasma HIV RNA levels in the preceding 24 months: HLV(≥1000 copies/mL), LLV(51–999 copies/mL), and plasma suppression with CSF blip or escape (CSF HIV-1 RNA <200 or ≥200 copies/mL). Temporal analysis of antecedent plasma viral loads revealed that cross-sectional definitions of viral escape based on >0.5 log10 higher levels of CSF HIV-1 RNA will include HLV cases, a subtype with sustained plasma viremia. In HLV cases, discordant CSF viral levels could reflect delayed CSF viral clearance or continuous CSF seeding from plasma,40 and therefore, may not necessarily represent compartmentalized virus. In contrast, definitions requiring plasma suppression will miss LLV cases, a subtype previously shown to harbor DRMs in CSF,8 and here shown to have repeat episodes of viral escape. M184V/I were the most frequently observed mutations in all cases present in 74% of CSF isolates from individuals with plasma levels ≤50 copies per milliliter, and frequently occurring in the presence of other DRMs.This longitudinal analysis of 41 ART-experienced HIV+ individuals with CSF viral escape is one of the largest case-series investigating an uncommon, but increasingly recognized clinical event among ART-treated HIV+ individuals. The estimated prevalence and clinical characteristics of CSF escape were similar between Boston hospitals (6.0%) and NNTC clinical research cohort (6.8%). Four cases had detectable CSF HIV-1 RNA at multiple time points despite plasma suppression, indicative of repeated CSF escape, a phenotype that is not yet well characterized in the literature.41 HLV cases also made up a greater proportion of viral escape casesbefore 2010, whereas durable plasma suppression and detectable CSF HIV-1 RNA classified cases represented most CSF escape cases after 2010. This shift in calendar period could represent a change in clinical practices  because of recent guideline recommendations,44 increased use of ultrasensitive HIV-1 RNA assays and/or decreased prevalence of virologic failure because of improved ART regimens. Although low levels of CSF HIV-1 RNA may reflect variability in assay sensitivity and be unrelated to neurological symptoms, a recent study showed that low CSF HIV-1 RNA levels in viral escape were associated with elevated inflammatory mediators and endothelial adhesion molecule VCAM,45 and may not always be benign. One factor that can account for the association with neuroinflammation is secondary escape, defined as discordant CSF and plasma HIV-1 RNA levels in the context of a new infection.4 In our series, there were 3 candidates for secondary escape; 2 cases with progressive multifocal leukoencephalopathy and one with CNS lymphoma. As the field begins to harmonize clinical definitions for CSF escape, the epidemiology and associated cofactors of escape subtypes based on previous plasma HIV-1 RNA levels will be a relevant indicator of evolving clinical practices, and may also enable future studies to identify biomarkers linked to virologic failure in the CNS.By classifying CSF viral escape based on plasma HIV-1 RNA in the 24 months before CSF discordance, we identified differences in clinical characteristics associated with CSF escape subtypes. HLV cases were more likely to use cocaine, amphetamines, and alcohol, behaviors that are frequently linked to suboptimal ART adherence and reduced health care engagement.46 a 2015 study in ART-experienced patients with plasma HIV-1RNA >50 copies per milliliter showed that 12.5% and 19.8% of individuals had unique CSF resistance mutations to RT and protease, respectively; M41L and T215Y, were statistically more prevalent in the CSF.47 In our study, M184V/I, T215Y, D67N, and M41L were the most commonly detected CSF mutations. Three CSF integrase mutations were identified in CSF . Although these mutations are not known to reduce susceptibility to integrase inhibitors in isolation,38 integrase mutations remain important to monitor.HIV-1 has high genetic diversity and recombination, which can result in DRMs when viral replication is incompletely suppressed because of factors such as poor adherence, persistent LLV, and incomplete ART penetration into the CNS. In the present study, over 80% of individuals had HIV-1 infection for greater than 10 years before CSF escape with long exposures to ART, including older regimens, suggesting duration of HIV-infection, ART use, and potential emergence of DRMs are likely contributing factors to CSF escape. Although there has been a declining trend in resistance mutations among HIV+ individuals in resource-rich settings,9 However, the combination of poor CSF penetration by tenofovir, a widely used NRTI in ART48 and M184V/I mutation leading to emtricitabine or lamuvidine resistance may lower the threshold for subsequent CSF mutations that could lead to NRTI cross resistance.38 The high frequency of M184V/I mutations reported here and in the literature, and simultaneous presence of TAMs in neurologically symptomatic CSF escape raises the possibility that the M184V/I mutations could be a factor influencing CNS viral reservoirs and CSF escape. Future studies that systematically assay CSF DRMs, particularly in neurosymptomatic individuals, will help to understand the impact of M184 and other DRMs on CNS viral reservoir size, their potential impact on neuropathogenesis, and effects on neurological outcomes.A recent study showed that low-level of CSF HIV-1 RNA was associated with better neurocognitive outcomes, and hypothesized that one mechanism was through DRMs such as M184V, which reduce viral fitness but may stimulate a more effective immune response.49 Three of 4 cases in this series with historical evidence of LLV in plasma had recurrent CSF escape; most but not all were associated with neurological symptoms. In a recent series of longitudinal CSF samples from 75 neurologically asymptomatic HIV+ individuals on ART,13 7 cases had a quantifiable CSF HIV-1 RNA in 2 consecutive samples and remained neurologically asymptomatic.13 These data suggest that dynamic CSF viremia is present among symptomatic and asymptomatic individuals, and research efforts to identify individuals with CNS viral reservoirs may require CSF profiling irrespective of neurological symptoms.8 An additional consideration is CNS penetration of ART regimens as calculated by CPE score. An “adjusted” CPE score has been proposed as a more relevant score in CSF escape, as it takes into account resistance profiles for the calculation of CNS ART effectiveness.6 Although CPE score at the time of escape was not a distinguishing feature in this series, 6 cases with genotyping from CSF or plasma isolates had mutations that would result in resistance to at least one prescribed ART drug, resulting in a lower “adjusted” CPE score. Together, these findings support the strategy that changes in treatment regimens for CSF escape should be guided by CSF resistance testing, when possible.44Persistent LLV is associated with plasma virologic failure, and resistance mutations arising from stable HIV-1 reservoirs or ongoing replication cycles may influence viral pathogenesis in the CNS.43 there is considerable heterogeneity in provider practices. As such, neurological symptoms requiring hospitalization or profound cognitive decline are more likely to lead to CSF HIV-1 RNA quantification, whereas milder neurological symptoms may not result in lumbar punctures unless imaging abnormalities are identified.This study has several limitations common to retrospective case-series including selection and recall bias, and limitations in data based on chart reviews. In addition, there was no systematic cataloging of CNS escape in the Boston cohort; thus, identification of these cases relied on current providers' recollection of patients, which has the potential to skew analysis toward more recent years and cases with severe neurological symptoms and may lead to underreporting true prevalence. Given that HIV-1 RNA assays are not FDA-approved for CSF quantification and testing has only recently been adopted in US-based guidelines, 50 The CSF escape subtypes we propose will help to shed light on the true prevalence, and provide a new conceptual framework for future studies of viral and host factors leading to CSF escape and testing of therapeutics aimed at virus eradication.The precise definitions of CSF viral escape, neurological symptoms that require CSF analysis, and when providers should repeat lumbar puncture after CSF viral escape remains debated. Our estimates suggest that individuals with a recent history of LLV or historical virologic failure with M184V/I mutations are potentially at elevated risk for CSF escape. Given that the search for a curative strategy is a top priority, barriers to cure including LLV on ART and replication-competent HIV-1 virus in the CNS require further study.


Drosophila ovarian somatic cells, piRNAs arise from longer single-stranded RNA precursors that are processed in the cytoplasm presumably within the Yb-bodies. piRNA precursors encoded by the flamenco (flam) piRNA cluster accumulate in a single focus away from their sites of transcription. In this study, we identify the exportin complex containing Nxf1 and Nxt1 as required for flam precursor nuclear export. Together with components of the exon junction complex (EJC), it is necessary for the efficient transfer of flam precursors away from their site of transcription. Indeed, depletion of these components greatly affects flam intra-nuclear transit. Moreover, we show that Yb-body assembly is dependent on the nucleo-cytoplasmic export of flam transcripts. These results suggest that somatic piRNA precursors are thus required for the assembly of the cytoplasmic transposon silencing machinery.PIWI-interacting RNAs (piRNAs) are effectors of transposable element (TE) silencing in the reproductive apparatus. In  In Drosophila ovarian follicle cells, piRNAs generated from RNA precursors are processed in cytoplasmic Yb-bodies. The authors identify the exportin and the exon junction complexes as required to transfer precursors to cytoplasm. They also show that Yb-body formation requires piRNA precursor export. Drosophila, piRNAs originate from the processing of piRNA precursors transcribed from discrete genomic regions called piRNA clusters. piRNA clusters are enriched in TEs or their vestiges. In Drosophila melanogaster, their size ranges from 1 to a few hundred kb and they are almost exclusively located within or in close proximity to heterochromatic regionsflam locus, a uni-strand piRNA cluster active in the follicular cellsflam is transcribed by the RNA polymerase II (RNA pol II) into a precursor that is similar to other RNA Pol II transcripts in basic RNA features5flam precursor has been shown to contain a constitutive first exon. Several alternative splicings have also been reportedPiwi-interacting RNAs (piRNAs) are a class of 23–29 nucleotide (nt) small RNAs that are expressed in animal gonads. In the metazoan germline, they play an important role in silencing transposable elements (TEs) and are thus critical for genome integrity12In the follicle cells, RNA precursors transcribed from piRNA clusters are first transferred to cytoplasmic perinuclear granules called Yb-bodies that have been reported to be major sites for piRNA biogenesis791112131415et al.flam precursors channel through the nucleoplasm and first accumulate in a structure called Dot COM within the nucleus. This nuclear structure faces the cytoplasmic Yb-body and has been proposed to be a nuclear site of accumulation of piRNA precursors coming from various piRNA clusters before their export. This nuclear accumulation was not detected by Murota et al., who instead reported a cytoplasmic flam focusflam transcripts before processing, it is still unknown how such a transcript can be distinguished from other mRNAs and find its way to the Yb-bodies. A sequence crucial for piRNA production has been identifiedflam fused to luciferase reporter gene is transfected into the ovarian somatic cells, luciferase RNA is processed via the primary processing machinery, giving rise to thousands of piRNAs. This prompted the authors to suggest that the 5′ end of flam transcript is a potential piRNA-trigger sequence. A cis-acting 100-nt-long fragment bound by Yb—a core component of Yb-bodies, has also been identified in the traffic jam (Tj) 3′ untranslated region that is sufficient for producing artificial piRNAs from unintegrated DNAIn a previous study, Dennis Drosophila revealed factors required for the piRNA pathway21222425262831323321233536Several screens performed on flam nuclear traffic and export to the cytoplasm. We also demonstrate that export of piRNA precursors is the signal for the Yb-bodies to be assembled. In the follicle cells the flam cluster, which gives rise to most piRNAs, acts as a master locus for the formation of Yb-bodies.In the present study, we identify exportins-Nxt1 and Nxf1, EJC components—Mago, Tsu, RnpS1, Acn and UAP56 proteins—as factors required at different steps of flam transcripts in more than 1,700 follicular cells. flam transcripts were revealed by in situ hybridization using the specific flam508 RNA probe that recognizes a unique sequence of the flam locusflam transcripts in wild-type (WT) flies ISO1A. As previously reported, flam precursor transcripts were detected as a single focus per cell although a few cells displayed one or two foci at early stages of oogenesis17n=1,764) had a focus co-localizing with the lamin staining  and the cytoplasm, respectively  using the traffic-jam (tj)-Gal4 driver leads to female sterility and atrophic ovaries, which can however be dissected for analysis. The level of piwi and yb transcripts was examined in the SKD lines. No depletion was detected by quantitative PCR with reverse transcription (RT–qPCR) for both genes. Nevertheless, we found that depletion of Nxt1 and Nxf1 in ovarian somatic cells causes loss of Piwi nuclear localization and leads to TE derepression  compared with WT ISO1A   but not in Nxf1-SKD mutants (52%).n=1,764)  correlatflam nuclear staining detected by larger foci in Nxt1-SKD, suggesting a nuclear retention of flam precursor in this exportin mutant  (flam locus (flam piRNAs was low in Nxf1-SKD flies (37 rpkm) with no significant decrease along the locus (If 41 rpkm) . As expeam locus . The deche locus .flam transcripts are channelled within the nucleus from their site of transcription to the distant Dot COM, thus we decided to investigate whether Nxt1- and Nxf1-SKD also affect this intra-nuclear traffic of piRNA precursors. Double DNA/RNA FISH experiments were performed using flam 508 RNA probe and a DNA probe made from the DIP1 gene, a unique genomic sequence flanking the flam locus. As previously reported, two distinct foci were detected in 93% of the 346 WT ISO1A nuclei examined due to the accumulation of flam transcripts away from the genomic flam piRNA cluster. Surprisingly, in Nxt1- and Nxf1-SKD flies, flam RNA and DNA co-localized in 43% and 38% of 438 and 532 nuclei examined, respectively , maelstrom (mael) or yb  or yb . We founflam export in SKD mutants for EJC components and UAP56. As seen in flam transcripts accumulate in a single focus in all depleted lines. In addition, the proportion of cells having cytoplasmic, transmembrane and nuclear accumulation of flam transcripts in mutant lines was similar in SKD lines and WT  and 38% (n=533) of follicle cells, respectively, while flam transcripts are detected at their site of transcription in only 7% (n=346) of WT cells. To a lesser extent 16% (n=430), of follicle cells have a co-localization between flam RNA and DNA in Tsu-depleted line  of follicle cells, still harboured Yb-bodies compared with 63%  in WT flies. Instead, Armi and Yb proteins were dispersed throughout the cytoplasm of follicular cells giving rise to a diffuse cytoplasmic signal , which, owing to a P-transgene inserted upstream of flam transcription start site, produces no flam transcriptsflamBG, similar to what was seen in Nxt1-SKD follicle cells, suggesting that a fraction of Armi protein fails to accumulate in foci   . Nxt1 plflam transfer to the cytoplasm. Mago and Tsu are known to form a heterodimer required for a tight binding of EJC to the targeted RNAsflam. Their mutation does affect neither flam transcription, nor splicing, nor its nucleo-cytoplasmic export. The only step that is affected in all mutants is the intra-nuclear traffic of flam transcripts and/or, occasionally, their gathering to a single focus of accumulation -GAL4/CyO came from the collection of the GReD. nxt1 [VDRC GD49602] strain was obtained from Vienna Drosophila Resource Center. nxf1/sbr [34945], mago [35453], rnpS1 [36580], tsu [36585], acn [53676] and uap56/hel25E [33666] strains were obtained from the Bloomington Drosophila Stock Center.All flies were kept at 20 °C. Strains flam 508 probe to detect flam transcripts was PCR amplified from the ISO1A line using primers 5′-ATTCTCCTTTCTCAGGATGC-3′ and 5′-GCATTGCTACCTTACGTTTC-3′ and cloned into pGEMT easy vector.The DNA fragment to prepare the specific in vitro transcription using Sp6 or T7 polymerase and digoxygenin labelled UTP (Roche), DNAse I treatment and purification.Riboprobe was synthesized by digestion of pGEMT easy plasmids with NcoI or SpeI enzyme, followed by DNA probe are made of eleven PCR amplifications  labelled−1, heparin 0.25 mg ml−1) at 60 °C. Ovaries were hybridized overnight at 60 °C with 1 μg riboprobe. Ovaries were then rinsed 20 min in HYB- and in HYB-/PBT 1:1 at 60 °C then four times in PBT at RT before blocking 1 h at RT in TNB 0.3% triton (Perkin-Elmer TSA kit) and immunodetection 1 h 30 min at RT with anti-digoxigenin-HRP  in TNB 0.2% tween. Ovaries were rinsed three times in PBT, incubated 10 min with TSA-Cy3 in amplification diluent , and rinsed.RNA FISH was performed on ovaries from 2- to 4-days-old flies dissected in 0.2% Tween 20, phosphate-buffered saline (PBT). Ovaries were fixed with 4% formaldehyde/PBT at room temperature (RT) for 30 min, rinsed three times with PBT, post-fixed 10 min in 4% formaldehyde/PBT and washed in PBT. After permeabilization 1 h in PBS-0.3% Triton, prehybridization was performed as follow: 10 min HYB- /PBT 1:1, 10 min HYB-, 1 h HYB+  and goat anti-Armi antibody . Secondary antibodies coupled to Cy3 or Alexa-488 were used.in situ hybridization, RNA staining was followed by treatment with 200 μg ml−1 RNase A for 2 h after which the ovaries were transferred to FISH hybridization buffer containing 50% formamide, 4 × SSC, 0.1% Tween 20, 0.1 M NaH2PO4. DNA was denatured for 15 min at 80 °C and hybridization with DNA probe was carried out O/N at 37 °C. After washes, the ovaries were first incubated with glycine 0.1 M–0.1% Tween HCL pH 2.2 before washes and DNA staining. DNA staining was performed by blocking for 1 h at RT in TNB, immuno-detection for 1 h 30 min at RT with anti-Digoxigenin-HRP  in TNB 0.3% Triton, washes three times in PBT, incubation for 10 min with TSA-Cy3 in amplification diluent , rinsing three times in PBT and staining with Hoescht.For DNA/RNA −1 RNase A for 2 h. The ovaries were then transferred to FISH hybridization buffer containing 50% formamide, 4 × SSC, 0.1% Tween 20, 0.1 M NaH2PO4. DNA was denatured for 15 min at 80 °C and hybridization with DNA probe was carried out O/N at 37 °C. After washes, the ovaries were treated for DNA staining.For DNA FISH coupled to immunofluorescence, ovaries from 2- to 4-day-old flies were dissected in PBT (PBS-0.2% Tween) fixed with 4% formaldehyde/PBT at RT for 30 min, rinsed three times with PBT, post fixed for 10 min with 4% formaldehyde/PBT at RT and rinsed in PBT. The ovaries were then permeabilized for 1 h in PBS-0.3% Triton, rinsed three times with PBT and incubated for 1 h in TNB (Perkin-Elmer TSA kit) 0.3% Triton prior to staining with mouse anti-Armi antibody (kindly provided by M. Siomi). Incubation with secondary antibodies coupled to Cy3, Cy-5 or Alexa-488 was followed by treatment with 200 μg mlOvaries from 2- to 4-day-old flies were dissected in PBT (PBS-0.2% Tween) fixed with 4% formaldehyde/PBT at RT for 20 min, rinsed three times with PBT, incubated for 1 h in PBS-0.3% Triton, rinsed three times with PBT and incubated for 1 h in TNB (Perkin-Elmer TSA kit) 0.3% Triton prior to staining with mouse anti-lamin antibody , goat anti-Armi antibody , rabbit anti-Yb antibody  or rabbit anti-Piwi antibody . Secondary antibodies coupled to Cy3, Cy-5 or Alexa-488 were used. Three-dimensional images were acquired on Leica SP5 and Leica SP8 confocal microscopes using a × 40 objective.Total RNA was isolated from 10 pairs of ovaries from 2- to 4-day-old flies with Trizol (Ambion). Following DNAse I treatment, cDNA was prepared from 1 μg RNA by random priming of total RNA using Superscript IV Reverse Transcriptase (ThermoFisher Scientific).PCR were performed with Phusion DNA polymerase (New England Biolabs) using e1F 5′-AGTTGCTTTATGACGCCGGGC-3′ and e2R 5′-GCTAGGAAGCTAATTGATCAATGACAAC-3′ primers.rp49 levels. Fold enrichments were calculated in comparison with RNA levels obtained from WT ISO1A line. Primer sequences for RT–qPCR analyses are listed in qPCR were performed using standard techniques. All experiments were conducted in biological triplicates with technical triplicates. Relative RNA levels were normalized to Drosophila genome. Only reads mapping uniquely to the genome were used for further analysis. All libraries were normalized based on the number of mapped reads. Size profile for flam piRNA cluster was obtained by extracting the abundance and read length of 23–29 nt sequences uniquely matching to this locus.Total RNA was isolated from 10 pairs of ovaries from 2- to 4-day-old flies with Trizol (Ambion). Deep sequencing of 18–30 nt small RNAs was performed by Fasteris S.A. (Geneva/CH) on an Illumina Hi-Seq 2500. Illumina reads were matched to release 6 of the All Illumina data sets that support the findings of this study have been deposited at GEO with the accession codes: BioProject ID PRJNA348903. The data that support the findings of this study are available from the corresponding author upon reasonable request.How to cite this article: Dennis, C. et al. Export of piRNA precursors by EJC triggers assembly of cytoplasmic Yb-body in Drosophila. Nat. Commun.7, 13739 doi: 10.1038/ncomms13739 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary TablesTranscripts from Dot COM localize within the nucleus, close to the nuclear membrane. Movie of 3D imaging of ovarian follicle cells stained with flam 508 riboprobe (red) and anti-lamin antibody (green).


This data article contains supporting information regarding the research article entitled “High butter-fat diet and bisphenol A additively impair male rat spermatogenesis”   Specifications TableValue of the data•These data revealing the minimal bisphenol A (BPA) dose that impedes spermatogenesis in the presence of high fat butter diet, may assist in the choice of dietary BPA concentrations for rat studies.•Immuno-histological patterns of expressions of aromatase (Cyp19) and ERα in testis may be useful for future work related to the distribution of these two markers in testis.1in utero to diets with bisphenol A (BPA) and high fat butter (HFB). Details on diets, animal groups and approach are outlined in Fig. 1A. The number of seminiferous tubules (STs) within the testis  with progression of spermatogenesis upto the round spermatids  or upto spermatozoa , was scored and plotted (T+E2 model). The body weights and the weights of testis and spleen were scored (A–C). In a separate work, data is presented for body weight and weight of the testis, epididymis, spleen, and kidney for offspring prenatally exposed to AIN, BPA, HFB, high fat olive oil (HFO), HFB+BPA, or HFO+BPA diets (D–G) and T+E2.We conducted a dose-response analyses to determine the minimal BPA dose that impedes spermatogenesis  in male e scored A–C. In aPA diets D–G and TWe examined the STs of the testis for presence of clusters of cells . All diets were switched to AIN after the pups were born. At postnatal day (PND 70), prenatally exposed pups from each diet group were treated with T+E2 via SilasticTM implants Figs. 1B and C). More details on the T+E2 model, tissue collection and data analyses are outlined in Tarapore et al., 2016 Sprague–Dawley females were fed AIN, high fat butter , 17α-ethinyl estradiol (EE2 (0.5 µg/kg bw-d), or HFB plus four BPA doses  before and during pregnancy A. All di2.2The procedure and antibody sources are as outlined in Tarapore et al. 2.3For