A substantial fraction of the genome is transcribed in a cell-type-specific manner, producing long non-coding RNAs (lncRNAs), rather than protein-coding transcripts. Here, we systematically characterize transcriptional dynamics during hematopoiesis and in hematological malignancies. Our analysis of annotated and de novo assembled lncRNAs showed many are regulated during differentiation and mis-regulated in disease. We assessed lncRNA function via an in vivo RNAi screen in a model of acute myeloid leukemia. This identified several lncRNAs essential for leukemia maintenance, and found that a number act by promoting leukemia stem cell signatures. Leukemia blasts show a myeloid differentiation phenotype when these lncRNAs were depleted, and our data indicates that this effect is mediated via effects on the MYC oncogene. Bone marrow reconstitutions showed that a lncRNA expressed across all progenitors was required for the myeloid lineage, whereas the other leukemia-induced lncRNAs were dispensable in the normal setting. HOTAIR has been shown to promote metastasis through re-location of PRC2  have emerged as an additional layer of regulation of gene expression . Althoug of PRC2 , and PVTtability . In T ceof IGF1R .lncRNA-EPS promotes survival and inhibits apoptosis in murine fetal erythroblasts  and B-cell lymphoma. This transcriptome analysis revealed a large number of lncRNAs that are tightly regulated during hematopoietic cell-fate choices. As a first approach to identify functionally relevant lncRNAs, we decided to focus on an in vivo model of murine AML.HOTTIP and HoxBLinc, have been shown to activate expression of Hox genes by mediating recruitment of histone methyltransferase complexes WDR5-MLL and Setd1a/MLL1, respectively  using 11 cell types representing different stages of hematopoietic differentiation, ranging from long-term hematopoietic stem cells (LT-HSC) to differentiated cell types and blood cancers . Each liWe observed a substantial overlap between our lncRNA genes and GENCODE lncRNAs, as well as the lncRNA catalogs from megakaryocyte-erythroid progenitors (MEP) differentiated in vitro , erythroLncRNAs can have a number of different relationships to their neighboring protein coding genes. They can fall in intergenic regions, be divergently or convergently transcribed, they can overlap in antisense orientation (interspersed), or they can have the same orientation as the neighboring gene, upstream or downstream. To address the possibility that the open chromatin signatures we observed were exclusively the result of regulatory regions being shared between lncRNAs and neighboring protein-coding genes, we performed our analysis independently for each category of lncRNA defined above. Irrespective of their relationship to surrounding protein-coding genes, our assembled lncRNAs showed enrichment in ATACseq signal at their presumed TSS in at least one cell type .Using DESeq2 , we perfMDS1 and EVI1 complex locus (Mecom). These are known to regulate hematopoietic stem cell self-renewal . MLL-AF9/NRASG12D AML cells were infected at low multiplicity to minimize the probability of double infection, and were Neomycin-selected to eliminate non-infected cells. Infected AML cells were transplanted into mice, and hairpin expression was subsequently induced. To ensure a good representation of every hairpin during the 14 days that the cells proliferate in vivo, we performed virus production, infections and injections using pools of 50 shRNAs  or one hairpin significantly depleted and a second hairpin depleted more than twofold  but consistent knockdown across hairpins and primer pairs. We also examined the relationships between lncRNAs and their closest genes, with a particular interest in whether down-regulation of the lncRNA would lead to any expression changes. We did not observe any consistent trend, with most lncRNA knockdowns failing to induce any reproducible change of expression in either of their two flanking coding genes .lncRNA_041249 and lncRNA_097790) and one lncRNA annotation overlaps, in the antisense direction, with its closest coding neighbor (lnc_166788). We looked particularly at whether these might function by controlling the level of expression of their linked divergent coding transcript, as previously reported for ES cell differentiation , lnc_104449, or lnc_177417, while leukemia stem cell signatures  showed an enrichment for Macrophage Differentiation genes  upon knockdown of gnatures  showed egnatures . Additiognatures . This regnatures  and suggRpa3 was required for proliferation of 4T1 cells in vitro, none of our lncRNAs showed depletion even after 14 days in culture  and lnc_10449 Pilna (progenitor-induced lncRNA neighboring Ak3). lnc_071255, which corresponds to the previously annotated lncRNA Pvt1, was recently shown to correlate with MYC protein, but not Myc mRNA levels. In human breast cancer, Pvt1 has been proposed to act by stabilizing MYC protein  in the MLL/ENL model (similar to the MLL/AF9). In a different model, where Myc is one of the drivers, we observed a less striking phenotype. This is unsurprising since enforced Myc expression rescues the phenotype of Lilam, Pilna and Pvt1  as compared to their most similar normal progenitors, GMPs, or HSCs  in leukemia, only Pilna showed an effect in normal reconstitution of the myeloid lineage. This is consistent with the progenitor-wide expression of Pilna and suggests a general role in hematopoiesis for this lncRNA. Expression of lncRNAs Pvt1 and Lilam is highly induced in the leukemia context and they could, on the other hand, be dispensable for normal myelogenesis.Long non-coding RNAs are emerging as a recently recognized class of regulators that have broad impact in biology. To enable the study of this impact and breadth of function in a well-characterized developmental model, we have produced a de novo catalog of lncRNAs from a representative sampling of cell types throughout the hematopoietic lineage and from two models of hematological malignancies. To globally characterize the likely impact of lncRNAs in normal and malignant hematopoiesis, we chose a subset of RNAs with a range of expression levels and patterns and tested whether these had a function in a murine model of AML. We performed a loss-of-function screen of 120 candidates and found that 20 of these were required for in vivo leukemia progression. We further characterized nine lncRNAs, all of which were required for leukemia cell proliferation in vitro but were dispensable in at least one other unrelated cancer model. A subset of these lncRNAs functioned in maintaining leukemia stem cell signatures; the leukemic blasts acquired a myeloid differentiated phenotype upon lncRNA knockdown. Amongst such lncRNAs was ctor MYC . This alExpression analysis of our annotated lncRNAs revealed that differential regulation between cell types and at different stages of differentiation. Much attention has been focused on noncoding RNAs transcribed in the opposite orientation from the same promoter region as coding transcripts, known as divergent transcripts. This raises questions regarding whether these noncoding transcripts are truly functional, or whether they are simply a byproduct of transcription from an activated promoter. Expression of divergent lncRNAs has been shown to correlate with their corresponding protein-coding counterparts, and even to regulate their counterparts in pluripotent stem cells .cis regulation of their corresponding divergent gene.We indeed find a correlation between lncRNA expression and that of the closest protein-coding gene; however, this is not an exclusive property of divergent transcripts. We also observe correlation for other lncRNA-gene genomic orientations, as well as for gene-gene pairs. We therefore hypothesize that this correlation in expression arises from shared regulatory mechanisms or common regulatory environments. For the two divergent lncRNAs amongst the ones we characterized, we did not find evidence for We probed the functional relevance of lncRNAs using AML as a model. By performing an shRNA screen targeting 120 lncRNAs, we identified 20 lncRNAs whose expression confers a proliferative advantage to leukemia cells in vivo. This was striking particularly because we did not solely target AML-enriched lncRNAs but rather cast our net broadly. Thus, our findings emphasize the general importance for lncRNAs in leukemia and possibly normal hematopoiesis. We tested whether the lncRNAs that impacted AML were also required for proliferation of the breast cancer cell line 4T1. In this model, we observed no significant effect of knockdown. This could be due to cell type-restricted expression or function but can be taken as support for the specificity of the effects observed in AML.Pvt1, a lncRNA previously reported to be a marker of poor prognosis in colorectal cancer    or the M4 (Brd4) . lncRNA Considered as a whole, we have produced an extensive resource of lncRNA expression in the hematopoietic system and demonstrated that a substantial percentage of these are functional, at least in a model of malignant development. One of these lncRNAs, expressed across most hematopoietic progenitors, is additionally required in myeloid reconstitution. The data and tools that we have produced should serve a useful purpose in promoting studies in both the normal and leukemic context.a/BoAiTac  were purchased from Taconic . All these experiments were approved by the Cold Spring Harbor Animal Care and Use Committee.Tissue extraction for transcriptome studies and the in vivo shRNA screen were performed in Cold Spring Harbor Laboratories . RNAseq libraries for transcriptomic assembly and differential expression were prepared from female C57BL/6 (6–12 weeks old) purchased from Charles River (Wilmington MA). For the in vivo shRNA screen, female B6.SJL-PtprcBone marrow transplantations of modified HSCs and subsequent analysis of peripheral blood from these mice were performed at the Cancer Research UK Cambridge Institute . C57BL6J (6–12 weeks old) were purchased from Charles River . C57BL/6-LY5.1 females were purchased from Charles River  and used at 9–12 weeks old. These animal procedures were conducted in accordance with project and personal licenses issued under the United Kingdom Animals (Scientific Procedures) Act, 1986.Low input libraries were prepared from C57BL6 mice for all normal hematopoietic cell types depicted in RNAseq libraries enriched for low-abundance transcripts were prepared for AML cell lines, lymphomas, and FACS-sorted PreB and ProB cells as follows. RNA was extracted using Trizol reagent (Thermo Fisher Scientific), DNase-treated, then polyadenylated transcripts were isolated using the Dynabead mRNA purification kit (Thermo Fisher Scientific). Purified mRNA was fragmented by heating to 98°C for 30 min in RNA storage buffer (Ambion), then converted to cDNA with random primers using the Superscript III RT kit (Invitrogen). Second-strand synthesis was performed in the presence of dUTP, then Illumina adapters were ligated onto the dsDNA fragments. To preserve strand identity, the uracil-containing cDNA strand was digested using USER enzyme (NEB), then cDNA was amplified using adapter-specific PCR primers and purified using Agencourt AMPure XP beads (Beckman Coulter). The samples were subsequently enriched for low-abundance transcripts by Duplex-Specific Nuclease (DSN) treatment (Illumina) followed by low-cycle PCR, then gel-purified and cleaned up using Agencourt AMPure XP beads . Libraries were sequenced by paired-end 76 bp on an Illumina HiSeq2500.http://broadinstitute.github.io/picard). Transcriptome assembly was performed individually for each library with cufflinks  and were kept in the same culture conditions as RN2. Platinum-A packaging  were cultured in DMEM containing 4.5 g/L glucose, 4 nM L-Glutamine and 110 mg/L Sodium Pyruvate, supplemented with 10% FBS (HyCLone) and 1% Penicillin/Streptomycin (Gibco) under 5% CO2 culture conditions. 293 FT (purchased from Thermo Fisher Scientific) were cultured as per manufacturer’s instructions. The mouse mammary tumour cell line 4T1  was cultured in DMEM high glucose (Life Technologies) supplemented with 5% fetal bovine serum (HyCLone), 5% fetal calf serum (HyCLone), non-essential amino acids (Life Technologies) and penicillin-streptomycin (Life Technologies). All cell lines tested negative for mycoplasma contamination RNA-capture ELISA.MLL-AF9;NRASboratory  and cultshRNAs were predicted using the shERWOOD computation algorithm . To sele2 was prepared and brought to 500 μl with H2O. This mixture was vigorously bubbled into 500 μl of 2X HBS solution  for 30–60 s and added to cells in 9 ml of supplemented DMEM. After 16 hr, media was changed to supplemented RPMI and then collected 24 hr, 36 hr and 48 hr after media change and filter through a 0.45-μm filter (EMD Millipore). When necessary, virus was concentrated using Retro-X concentrator (Clontech).Virus production for was performed as previously described . In brie2 brought to 1250 μl with H2O and bubbled into 1250 μl 2X HBS. Media was changed to IMDM supplemente with 10% heat-inactivated FBS right before transfection and collected in 16 ml of the same media. 38 ml of viral supernant was ultracentrifuged for 2.5 hr at 25,000 rpm at 4°C, and resuspended in 100 μl of D-PBS (Gibco).For third generation lentiviruses, virus was prepared in 15 cm dishes using 293FT cells (Thermo Fisher Scientific). The transfection mixture contained 32 μg of DNA vector, 12.5 μg of pMDL, 6.25 μg of CMV-Rev, 9 μg of VSV-G, 200 μg of Pasha siRNA, 125 2.5M of CaCl−1 G-418  from 2 days after infection until fully selected. 1 × 106 fully selected AML cells were injected in the tail vein of sublethally irradiated  B6.SJL (CD45.1) female mice 6–8 weeks of age. For shRNA induction, animals were treated with doxycycline in the food . Leukemic mice were euthanized 14 days after transplantation, at terminal disease stage, by CO2. Cells were extracted from the bone marrow by flushing tibias and femurs and filtered (0.45 μm) to obtain a single cell suspension. For each pool, we required a minimum of three mice at the 14-day timepoint.Pools of 50 shRNA vectors were used to produce virus and transduce AML cells at a low multiplicity of infection to minimize double infections. Cells were treated with 500 μg mlLibrary preparation was performed as previously described . In brieCells cells were transduced with retroviral supernatant. Infection was assessed by flow cytometry analysis of a fluorescent reporter and kept to <30%. The percentage of cells expressing the fluorescent reporter over time was used to determine whether cells harboring the shRNA were being outcompeted by their uninfected counterparts.RN2 cells harboring inducible shRNAs were cultured in complete media containing 1 μg/mL doxycycline (Clontech) for 4 days. Cells were stained in MACS Buffer (Miltenyi Biotech) with CD11b/Mac-1 PE-Cy7 and cKit APC for 30 min on ice and analyzed by flow cytometry. Plots were produced using FlowJo software and Geometric Mean of the appropriate channel was extracted for fold change calculations.Cells were resuspended to 50,000 cells in 100 μl in MACS Buffer (Miltenyi Biotec). Of this buffer, 100 μl was first spun on the slides, followed by spinning of the cells for 5 min at 500 RPM. Slides were stained using the Kwik-Diff three step stain  from Thermo Fisher Scientific and imaged using the Aperio XT system at 40X.20. Primers were designed using IDT PrimerQuest tool or chosen from IDT’s pre-designed set when available. Fast SYBR Green (ThermoFisher Scientific) was used for qPCR. Primer pair efficiency was assessed using serial dilutions of cDNA from untreated RN2 cells, and melting curves were examined to ensure the presence of only one amplicon. Gapdh was used as a housekeeping normalization control in the delta-delta-Ct analysis. For lnc_104449, we did not obtain a CT value upon knockdown with sh4 (sh1 in the heatmap) in 14 out of the 18 technical replicates after 40 cycles of qPCR. Therefore, we set those values to 40, if at all underestimating the actual knockdown with this hairpin.Analysis of knockdown efficiency and relative gene expression changes was performed after inducing shRNA expression for 2 days. RNA was extracted using the RNeasy Mini Kit (Qiagen), including treatment with the DNase Set (Qiagen). Reverse transcription was performed using Superscript II (ThermoFisher Scientific), with 4 μg of RNA and 1 ul of 50 μM oligo(dT)6 aligned reads per sample. HTSeq-count . RNA sequencing libraries were prepared using TruSeq Stranded Total RNA Library Prep Kit (Illumina) and run on an Illumina HiSeq 2000. Reads were mapped to the mm10 genome assembly yielding at least 10eq-count  was usedeq-count  for qualWhole cell lysates were prepared by resuspending cell pellets in SDS-PAGE loading buffer and run in a NuPAGE Novex 4–12% Bris-Tris Protein Gel. Transfer was performed using iBlot2.0 into PVDF membranes. Membranes were incubated with primary antibodies against MYC  and b-actin (Abcam Ab6276) and secondary LiCor fluorescent antibodies. Membranes were imaged using the semi-quantitative LiCor Odyssey CLx system. MYC values are internally normalized to the B-ACTIN within each lane.−4 M β–mercaptoethanol (Gibco) plus 20 ng/mL interleukin-11  and 300 ng/mL Steel factor (R&D Systems)). Ultracentrifuged viral supernatant was added aiming at a final concentration of ~2×107 IU/ml following the sorting. Each well was used to inject four animals; cells were washed prior to injecting to remove remaining viral particles.Bone marrow from C57BL6 mice was extract by flushing, filtered through a 0.30 μm filter and lineage depleted . Cells were stained with EPCR-PE, CD45-APC, CD150-PE/Cy7 and CD48-FITC. DAPI or LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Thermo Scientific L34963) was used for dead cell exclusion. Sorting of highly pure E-SLAM HSC and sort-term (<24 hr) culture was performed as previously described . In shorBlood was analyzed starting from 4 weeks after transplantation and every 4 weeks thereafter. 50–75 μl of blood was extracted from the animals’ tail vein into heparin coated capillary tubes. Red blood cell lysis was performed using Ammonium Chloride Solution (Stem Cell Technologies). Samples were then stained with a pre-mix antibody cocktail and analysed in a LSR Fortessa (BD Biosciences). Flow data analysis was performed using FlowJo and statistical analysis using R Studio.http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The resulting FASTQ files were mapped with bowtie2 (http://broadinstitute.github.io/picard); alignments to mitochondrial chromosome and unplaced contigs were also removed. The coverages were calculated with bedtools  and three reviewers, one of whom, Juan Valcárcel (Reviewer #1), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Jan-Henning Klusmann (Reviewer #3).Thank you for submitting your article "lncRNA requirements for mouse acute myeloid leukemia and normal differentiation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Joint review:In their study, Delas et al. systematically characterize lncRNAs expression during mouse hematopoiesis and in a well-established mouse model for AML (MLL-AF9/NRAS G12D). Their analysis of previously annotated and de novo assembled lncRNAs led them to uncover several lncRNAs with a specific expression pattern during differentiation and in AML. To investigate the function of 120 lncRNAs with such a specific expression pattern they performed a shRNA in vivo screening, using the same MLL-AF9/NRAS G12D mouse model. They identified 20 lncRNAs whose knock down by at least two hairpins reduces leukemia cell proliferation in vivo, possibly related to the maintenance of a leukemic stem cell state (and inhibition of a myeloid differentiated phenotype). 14 out of these were further validated in one cell line in in vitro competition assays. Mechanistically they linked the function of three of their candidate lncRNAs  to the expression of MYC.These data provide a useful resource for scientists working on hematopoiesis, leukemias and lncRNA biology and provide a proof of principle that specific lncRNAs can play distinct roles in leukemia cell maintenance, in addition to more general functions during hematopoiesis. There have been of course previous efforts to map lncRNAs in both AML and hematopoietic stem cells, as well as reports arguing for a crucial regulatory role for pvt1 in MYC-driven cancers, affecting novelty of some of the data. Nevertheless, the study remains relevant, as it can facilitate understanding the function of lncRNAs in normal and malignant hematopoiesis, which is to date poorly understood. Technically the in vivo screening was thoroughly conducted (including the use of pools of 50 shRNAs) and the candidates well chosen.Major points:The manuscript will benefit from addressing issues of validation and generality of the results and from additional functional characterization of the lncRNAs. Specifically:1) Validation issues.a) Previous studies have demonstrated that lncRNA prediction based on de novo assembly of RNAseq data has a high frequency of false predictions. A recent study showed that the accuracy greatly improved by combining the RNAseq data with CAGE data . Thus, alignment of the newly identified lncRNAs with the CAGE signals would greatly increase the confidence of their resource.b) A major caveat for shRNA targeting of lncRNAs is the inefficiency for targeting nuclear lncRNAs due to the fact that the RNAi machinery is mostly cytoplasmic. It would be important to further document the general validity of their shRNA approach to efficiently knockdown nuclear lncRNAs (e.g. are most of the lncRNA candidates biased towards cytoplasmic lncRNA function compared to nuclear roles?). Given the high isoform complexity, the authors should comment to what extent the levels of different regions/isoforms of key lncRNAs are depleted when targeted by shRNAs directed against regions of highest RNAseq coverage.c) As suggested previously, RNAi has some disadvantages in knocking down lncRNAs, and relying on a single methodology for all the experiments increases the likelihood of false positives. Therefore, the authors should provide an alternative approach to validate the loss of function of these lncRNA candidates, for example antisense oligonucleotides, CRISPRi and paired-guide RNA CRISPR-Cas9 .2) Generality issues.a) They only utilized a single murine cell line model, and it will be beneficial to include another murine AML cell line to exclude cell line-specific bias in these studies. Also, in this paper, the authors claim that Lilam and Pvt1 are AML-specific but have only demonstrated the specificity with one breast cancer cell line; however other groups have shown crucial roles of Pvt1 in ovarian, cervical and colorectal cancer. To claim specificity, the authors should test if knockdown of Lilam and PVT1 is required in other hematopoietic lineages or cell types.b) PVT1, as previously mentioned is also annotated in human cancers and it would be great to see if this phenomenon is translated into human AMLs (e.g. targeting PVT1 or/and the other lncRNAs in a panel of human MLL-AF9 cell lines). Major points:The manuscript will benefit from addressing issues of validation and generality of the results and from additional functional characterization of the lncRNAs. Specifically:1) Validation issues.a) Previous studies have demonstrated that lncRNA prediction based on de novo assembly of RNAseq data has a high frequency of false predictions. A recent study showed that the accuracy greatly improved by combining the RNAseq data with CAGE data . Thus, alignment of the newly identified lncRNAs with the CAGE signals would greatly increase the confidence of their resource.CAGE data is unfortunately not available for our mouse cell types. Producing these data and incorporating it into the catalog assembly pipeline would likely take at least a year and would be possible only for a subset of cell types. Others, particularly stem/progenitor populations, are too rare to enable us to obtain sufficient material for conventional CAGE protocols.Regarding the confidence in our current lncRNA catalog, our assembly is based on two types of RNA sequencing libraries, one of them produced using template switching RT . This strategy is specifically designed to capture full-length cDNA. Additionally, we required each individual transcript model to be independently assembled in different samples, dramatically decreasing the likelihood of false predictions.b) A major caveat for shRNA targeting of lncRNAs is the inefficiency for targeting nuclear lncRNAs due to the fact that the RNAi machinery is mostly cytoplasmic. It would be important to further document the general validity of their shRNA approach to efficiently knockdown nuclear lncRNAs (e.g. are most of the lncRNA candidates biased towards cytoplasmic lncRNA function compared to nuclear roles?). Given the high isoform complexity, the authors should comment to what extent the levels of different regions/isoforms of key lncRNAs are depleted when targeted by shRNAs directed against regions of highest RNAseq coverage.We appreciate the reviewers’ concerns regarding the potential bias towards cytoplasmic lncRNAs. The main focus of our work was to identify functional lncRNAs where depletion of the RNA had a robust, penetrant phenotype. We therefore felt RNAi was the best approach available, even if we could potentially be biased to cytoplasmic RNAs. Nonetheless, we have tried to address the sub cellular localization of our top candidate lncRNAs by single molecule RNA FISH and sub-cellular fractionations. However, being suspension cells with large nuclei and very little cytoplasma, the RNA FISH was inconclusive, as we could not distinguish perinuclear from true cytoplasmic or nuclear signals. Our multiple attempts at sub cellular fractionation of AML cells, with several protocols, have been unsuccessful, perhaps also due to their unusual nuclear/cytoplasmic ratio.Regarding the second point, directing the shRNAs towards areas of highest coverage was meant to capture as many isoforms as possible. Consequently, we tend to have more shRNAs in areas that would affect most predicted isoforms. When looking both at qRT-PCR or transcriptome coverage, we can see different transcript areas equally affected. For example, for Pvt1, both hairpins are located in the same area, with shRNA 1 having a stronger knockdown than shRNA 9. However, the level of knockdown is relatively consistent between primer pairs, even though primer set 2 is located spanning two exons ~ 10kb upstream .LncRNA_096443 (aka 7358) has a higher expression so we could also look at the overall transcriptome coverage upon knockdown with either shRNA 5 or shRNA 8 . Althougc) As suggested previously, RNAi has some disadvantages in knocking down lncRNAs, and relying on a single methodology for all the experiments increases the likelihood of false positives. Therefore, the authors should provide an alternative approach to validate the loss of function of these lncRNA candidates, for example antisense oligonucleotides, CRISPRi and paired-guide RNA CRISPR-Cas9 .We value the reviewers’ concerns regarding false positives. For that reason, we evaluated every phenotype with two independent shRNAs and we carefully checked knock down with different primer sets.We have additionally made a considerable effort in trying to establish CRISPRi to further validate our results, as suggested by the reviewers. To this end, we transduced our AML cells with a Cas9-KRAB expression vector, which also expresses Cyan Fluorescent Protein (CFP) in a bicistronic fashion (using P2A). We then transduced these cells with a vector expressing sgRNAs against the start site of each lncRNA, against a negative control gene , or our positive control Replication Protein A3 (RPA3). For RPA3, we used a published sgRNA from Liu et al. Science 2017.Even after 14 days post sgRNA transduction, the% of sgRNA and dCas9-KRAB expressing cells remained above 50% for the positive control, RPA3 . This isMost of our targeted lncRNAs show a slight depletion trend. Given the very mild effect on our positive control, this does not seem surprising. It is possible, though uncertain, that we might eventually optimize CRISPRi in our cell types, but as of now, we have been unsuccessful at applying this approach as a complement to shRNA.2) Generality issues.a) They only utilized a single murine cell line model, and it will be beneficial to include another murine AML cell line to exclude cell line-specific bias in these studies. Also, in this paper, the authors claim that Lilam and Pvt1 are AML-specific but have only demonstrated the specificity with one breast cancer cell line; however other groups have shown crucial roles of Pvt1 in ovarian, cervical and colorectal cancer. To claim specificity, the authors should test if knockdown of Lilam and PVT1 is required in other hematopoietic lineages or cell types.We have performed proliferation assays following the same set up as Regarding the AML-specificity of Lilam and Pvt1, we did not mean to imply that these lncRNAs had a function specific to AML versus other cancers, rather that their expression was higher in AML than in the rest of the hematopoietic lineages (b) PVT1, as previously mentioned is also annotated in human cancers and it would be great to see if this phenomenon is translated into human AMLs (e.g. targeting PVT1 or/and the other lncRNAs in a panel of human MLL-AF9 cell lines).The potentially conserved function of Pvt1 and/or other leukemia lncRNAs in human leukemias is highly interesting. However, establishing these models in the lab would require quite some time. Additionally, lncRNA conservation is not straightforward and these experiments would require identifying the human lncRNAs, designing and validating knockdown tools.


Sir, Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder which is characterized by oculocutaneous albinism, bleeding, and lysosomal ceroid storage resulted from deficiencies in multiple cytoplasmic organelles including melanosomes, platelet-dense granules, and lysosomes . A wide HPS1 gene associated with HPS1 (OMIM #203300) which encodes a transmembrane protein that is believed to be a component of multiple cytoplasmic organelles and is critical for their normal development and function . A homozygous mutation was detected in HPS1 gene (c.255+5G>A) which has not been reported in generalist polymorphism databases (ExaC or exome variant server (EVS)). The results were confirmed by Sanger sequencing in patient. To evaluate the significance of this novel splice site mutation, the full-length HPS1-cDNA was synthesized from RNAs extracted from lymphocytes and then amplified by PCR. A fragment of amplified cDNA in the region of exons 4-5 was sequenced using the ABI Prism3130 Genetic Analyzer . This novel splice site mutation has been shown to result in a 17 bp deletion and a frameshift afterwards (p.Y81Lfs*38)  . Other aHPS1 gene in an Iranian family suffering from HPS1. The defined mutation has been shown to cause a deletion in the transcribed RNA and subsequent frameshift and truncated protein. Of note, the observed phenotypes were different in affected family members from a severe pulmonary involvement in a single patient which started in her twenties to a relatively normal pulmonary function in others with some of them being beyond such age. In addition, pigmentation degree was different between affected relatives. Such variable phenotypes could be partly attributed to the age of affected individuals but also gene-gene and gene-environment interactions and should be assessed in future studies. Other studies have also reported phenotypic variability in the HPS1 symptoms including colitis (Here we reported a novel splice site mutation in  colitis , pulmona colitis  as well  colitis .HPS1 have been found in Puerto Rican, Swiss, Irish, and Japanese HPS patients in 1996. The different clinical phenotypes associated with different HPS1 frameshifts implied that differentially truncated HPS1 polypeptides may have fairly different significances in subcellular function (HPS1 mutation in Europeans and suggested the presence of a founder effect in Puerto Rican patients. Although other mutations have been reported, a frameshift hotspot has been detected at codons 321-322 (The first homozygous frameshifts in function . Previou 321-322 . Althoug


Tertiary lymphoid organs (TLOs) are HEV containing LN-like structures that develop inside organized tissues undergoing chronic immune-mediated inflammation. In autoimmune lesions, the development of TLOs is thought to exacerbate disease. In cancerous tissues, the development of HEVs and TLOs is associated with improved patient outcomes in several cancers. Therefore, it is important to understand what drives the development of HEVs and TLOs and how these structures contribute to pathology. In several human diseases and experimental animal models of chronic inflammation, there are some similarities between the development and function of HEVs within LN and TLOs. This review will summarize current knowledge of how hemopoietic cells with lymphoid tissue-inducing, HEV-inducing, and HEV-maintaining properties are recruited from the bloodstream to induce the development and control the function of lymphoid organs.The blood vasculature regulates both the development and function of secondary lymphoid organs by providing a portal for entry of hemopoietic cells. During the development of lymphoid organs in the embryo, blood vessels deliver lymphoid tissue inducer cells that initiate and sustain the development of lymphoid tissues. In adults, the blood vessels are structurally distinct from those in other organs due to the requirement for high levels of lymphocyte recruitment under non-inflammatory conditions. In lymph nodes (LNs) and Peyer’s patches, high endothelial venules (HEVs) especially adapted for lymphocyte trafficking form a spatially organized network of blood vessels, which controls both the type of lymphocyte and the site of entry into lymphoid tissues. Uniquely, HEVs express vascular addressins that regulate lymphocyte entry into lymphoid organs and are, therefore, critical to the function of lymphoid organs. Recent studies have demonstrated important roles for CD11c Secondary lymphoid organs (SLOs) are sites in which immune responses are initiated and maintained in order to generate protective immunity against exogenous pathogens and tolerance to self-antigens and commensal organisms , 2. ThesHelicobacter pylori-infected humans and mice, TLO development precedes carcinogenesis but whether TLOs promote the development of gastric cancer is not clear (Tertiary lymphoid organs (TLOs) are lymph node-like immune cell clusters that develop inside non-lymphoid organs in response to chronic immune-mediated inflammation stimulated by persistent infections, chronic graft rejection, autoimmunity, and cancer . Defininot clear , 13. The+ fibroblasts and are precursors of lymphoid stromal cells such as fibroblast reticular cells (FRCs) and FDCs. Lymphotoxin (LT)-αβ on hemopoietic LTi cells engaging lymphotoxin β receptor (LTβR) on LTo cells plays a dominant role in differentiating LTo into chemokine-secreting, adhesion molecule expressing fibroblasts that are able to recruit and retain LTis in the developing lymphoid tissue. An equally important, but unanswered question, is how embryonic blood vessels deliver LTi cells from their site of generation in fetal liver to predetermined sites where LNs are to develop. A role for inflamed blood vessels in initiating TLO development is implied by the finding that myeloid cells recruited into inflamed tissues drive the differentiation of gp38+ fibroblasts that share properties with lymphoid stromal cells and are likely precursors of LTo cells  cells in driving the differentiation of local mesenchyme into lymphoid tissue organizer (LTo) cells during the development of LNs and PPs in mice –16. LTo To cells . However+ DCs in the vascular addressin switch  cells, which can be divided into distinct stages of rolling and activation-induced arrest. In general, selectins mediate rolling and chemokines immobilized on the endothelial cell (EC) surface activate leukocyte integrins to arrest rolling cells. How hemopoietic cells with LT-inducing, HEV-inducing, and HEV-maintaining properties are recruited into lymphoid organs is central to understanding both the development of lymphoid tissues and the mechanisms regulating adaptive immune responses and disease pathologies.+ CD4+ CD3− IL7Rα+ RORγt+, Id2+ lymphoid tissue-inducing (LTi) cells (members of the group 3 category of innate lymphoid cells) are recruited into the anlagen and drive the development of gp38+ LTo cells from local mesenchyme (The earliest event in LN development is the formation of lymphatic vasculature around embryonic day 10.5 (E10.5) by budding from larger veins and establishing a primordial lymph sac or anlagen Figure . CD45+ Csenchyme . CXCL13 senchyme . In the senchyme . LTαβ issenchyme .During embryogenesis, LTi cells are generated in the fetal liver so how are they recruited to the sites at which LNs and PPs are to develop? A key finding is that all embryonic venous blood vessels express MAdCAM-1 . LTis exLymph nodes and PPs do not develop in LTα ko mice . Adminisvia Iκκβ and p50/RelA as well as non-classical signaling via Iκκα and p52/RelB, and there is considerable interplay between these two pathways , sacral, and lumbar LN  develop pathways . LN and and RelB , 47. In and RelB . It is sand RelB , 49. Thex on extended core 1 O-linked oligosaccharides and detected by monoclonal antibody MECA79 , which is required to generate apically expessed PNAd, is restricted to HEV ECs, whereas GlcNAc6ST-1 (CHST2), which generates basolaterally expressed PNAd, is more widely expressed in stromal cells   and MAdC+ DCs in the induction, as well as the maintenance, of PNAd expression by HEV.A unique feature of HEV is the connection with afferent lymph. HEVs are supported by a perivascular sheath composed of FRCs that are connected to the FRC coated conduit system within LN. The conduit system allows communication between afferent lymph and HEV whereby incoming lymph-borne soluble factors, such as chemokines and cytokines, are delivered directly to the basal lamina of HEV Figure  56). Ea. Ea56). +, CD11b+, CD103+ DCs mobilized from the gut lamina propria in response to microbial colonization induce the switch from mucosal to peripheral addressin expression by HEV in peripheral LN  lined with continuous ECs are the preferred sites of lymphocyte extravasation in LNs and other organs such as the skin and GI tract. The increase in diameter as blood vessels transition from capillaries to PCV alters hemodynamics such that leukocytes move to the outer stream or margin of flowing blood adjacent to the inner surface of the vessel  into the postcapillary venular network, which extends throughout the paracortex (T cell area) of the LN; HEVs form part of this postcapillary network  stromal cells are induced locally by CD11b+ myeloid cells recruited to inflamed skin of mice, recapitulating the earliest stages of lymphoid stromal cell development, but whether CD11b+ myeloid cells drive the development HEV and/or TLOs in chronic inflammation is not known , inflammatory (Th17 cells in autoimmunity), or tolerance inducing (Foxp3+ regulatory T cells), and this may be regulated by the activation status of PNAd-expressing HEVs and additional factors such as chemokines. Further studies are required to understand exactly how ectopic HEVs are formed and their impact on different types of chronic diseases. Such studies may reveal therapeutic targets for intervention in autoimmune diseases and cancers.Evidence of ongoing immune responses are seen inside TLOs; activation-induced cytidine deaminase is active in ectopic germinal centers in salivary glands of Sjorgren’s syndrome patients  and T cevia PNAd+ ligands indicating they are not completely immature  and PNAd expression was located to the basolateral EC surface where, in LN, it does not support high levels of lymphocyte recruitment. Structurally distinct, PNAd-expressing blood vessels, similar to HEVs in LN, were only formed when LTα and LTβ were co-expressed in pancreatic islets . Interesimmature .LTα and LTαβ activate the classical NF-κB pathway characterized by nuclear translocation of p50–RelA complexes. LTαβ also activates the alternative, non-canonical NF-κB pathway of NF-κB-inducing kinase (NIK)-dependent activation of IκB kinase (IKK)-α and nuclear translocation of p52–RelB complexes . Non-canIt is proposed that the development of HEVs is dissected into at least two distinct stages based on NF-κB signaling in blood vessel ECs Figure . The fir+ Tregs or myeloid-derived suppressor cells are depleted allowing effector T cells to exit TLO, infiltrate, and kill cancerous tissues (If HEVs in TLO are critical to exacerbation of autoimmune diseases by allowing activation of tissue destroying lymphocytes within target tissues, can the development of HEVs be prevented? Since HEVs in SLOs are important for generating protective immunity to infection any such therapy would need to be targeted to TLO. Intuitively, this will require the identification of markers or signaling pathways in ectopic HEVs that are not shared by HEVs in LN. One candidate is TNFR signaling that is not required for the development or maintenance of HEVs in LN  but has  tissues , 150. ItThe author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PASMC from patients with idiopathic PAH (IPAH) have increased resting [Ca2+]cyt and enhanced Ca2+ influx. Enhanced Ca2+ entry into PASMC due to upregulation of membrane receptors and/or Ca2+ channels may contribute to PASMC contraction and proliferation and to pulmonary vasoconstriction and pulmonary vascular remodeling. We have shown that the extracellular Ca2+-sensing receptor (CaSR), which is a member of G protein-coupled receptor (GPCR) subfamily C, is upregulated, and the extracellular Ca2+-induced increase in [Ca2+]cyt is enhanced in PASMC from patients with IPAH in comparison to PASMC from normal subjects. Pharmacologically blockade of CaSR significantly attenuate the development and progression of experimental pulmonary hypertension in animals. Additionally, we have demonstrated that dihydropyridine Ca2+ channel blockers , which are used to treat PAH patients but are only effective in 15–20% of patients, activate CaSR resulting in an increase in [Ca2+]cyt in IPAH-PASMC, but not normal PASMC. Our data indicate that CaSR functionally couples with transient receptor potential canonical (TRPC) channels to mediate extracellular Ca2+-induced Ca2+ influx and increase in [Ca2+]cyt in IPAH-PASMC. Upregulated CaSR is necessary for the enhanced extracellular Ca2+-induced increase in [Ca2+]cyt and the augmented proliferation of PASMC in patients with IPAH. This review will highlight the pathogenic role of CaSR in the development and progression of PAH.Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary vascular resistance (PVR) leading to right heart failure and premature death. The increased PVR results in part from pulmonary vascular remodeling and sustained pulmonary vasoconstriction. Excessive pulmonary vascular remodeling stems from increased pulmonary arterial smooth muscle cell (PASMC) proliferation and decreased PASMC apoptosis. A rise in cytosolic free Ca Under normal physiological conditions, the pulmonary circulation is maintained in a high flow, low pressure, and low resistance state. During conditions of increased blood flow or cardiac output , vasodilation and recruitment (opening of closed blood vessels) of pulmonary arteries are two important mechanisms for reducing pulmonary vascular resistance (PVR). However, under pathological conditions, increased PVR is a major cause for the development of pulmonary hypertension. Elevated PVR over time leads to right ventricular hypertrophy and right heart failure.Pulmonary hypertension is defined by a mean pulmonary arterial pressure (PAP) ≥25 mmHg at rest, as measured by right heart catheterization. Pulmonary arterial hypertension (PAH) describes a subpopulation of patients with pulmonary hypertension, and is hemodynamically defined by a resting mean PAP ≥25 mmHg, pulmonary arterial wedge pressure ≤ 15 mmHg, and PVR >3 Wood units   is upregulated in PASMC and lung tissue from patients with IPAH , and pulmonary arterial endothelial cells is essential for maintaining normal structural and functional integrity of the pulmonary vasculature. If the balance is tipped in favor of cell proliferation, thickening of the wall, luminal narrowing, and eventual obliteration can occur. These structural changes that lead to hypertrophy and/or luminal occlusion are referred to as pulmonary vascular remodeling  and a member of family C of the GPCR acids. It has a large extracellular domain that is approximately 600 amino acids long. This large extracellular domain is composed of a bi-lobed Venus-flytrap-like domain which is connected to the seven-transmembrane-domain by a cysteine rich region. The intracellular domain of CaSR contains the COOH domain which is 216 amino acids long  resulting in conversion of phosphatidylinositol 4,5 bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) , which then activates the mitogen-activated protein kinase (MAPK) signaling cascade ultimately resulting in phosphorylation and activation of ERK1/2  leading to decreased conversion of ATP to cAMP and decreased protein kinase A activity  activity. RhoGEF then activates RhoA by catalyzing the exchange of GDP for GTP  on the sarcoplasmic reticulum (SR) releasing Ca2+ from the SR to the cytosol. In PASMC, depletion of Ca2+ from the SR induces store-operated Ca2+ entry (SOCE) through store-operated Ca2+ channels (SOC). DAG activates receptor-operated Ca2+ channels (ROC) in the plasma membrane resulting in receptor-operated Ca2+ entry (ROCE). A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation. Several studies have demonstrated that resting [Ca2+]cyt, SOCE, and ROCE are all increased in PASMC isolated from IPAH patients  compared to vehicle injected control rats. Additionally, resting [Ca2+]cyt. The increased [Ca2+]cyt results from Ca2+ release from intracellular stores and Ca2+ influx through ROC and SOC. Transient receptor potential conical (TRPC) channels have been shown to function as both ROC and SOC in PASMC   to chronic hypoxia results in attenuated RVSP, Fulton's Index, and pulmonary arterial wall thickness compared to chronically hypoxic wildtype mice  have been successfully administered as a long term therapy to a number of PAH patients who respond positively to acute vasodilator challenge , we have identified several miRNAs that may potentially target CASR. Further efforts should focus on in vitro and in vivo approaches to examine the potential involvement of upregulated/downregulated miRNAs in the upregulation of CASR shown in PASMC from IPAH patients and animals with experimental PH.While it has been demonstrated that both CaSR and TRPC6 are upregulated in PASMC from IPAH patients and animals with experimental pulmonary hypertension and play a critical role in the pathogenesis of PAH, it remains unknown how this is achieved. The human CaSR gene, CASR, can be transcriptionally regulated by transcription factors, post-transcriptionally regulated by microRNAs and epigenetically regulated by DNA methylation .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purpose: The size of polymeric nanoparticles is considered as an effective factor in cancer therapy due to enterance into tumor tissue via the EPR effect. The purpose of this work was to investigate the effective parameters on poly(lactic-co-glycolic acid)-paclitaxel (PLGA –PTX) nanoparticles size.Methods: We prepared PLGA-PTX nanoparticles via single emulsion and precipitation methods with variable paremeters including drug concentration, aqueous to organic phase volume ratio, polymer concentration, sonication time and PVA concentration.Results: PLGA-PTX nanoparticles were characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM). The results exhibited that the diameter of nanoparticles enhanced with increasing drug, polymer and PVA concentrations whereas organic to aqueous phase volume ratio and sonication time required to the optimization for a given size.Conclusion: The precipitation method provides smaller nanoparticles compared to emulsion one. Variable parameters including drug concentration, aqueous to organic phase volume ratio, polymer concentration, sonication time and PVA concentration affect diameter of nanoparticles. In addition, chemotherapy is easier to administer and less invasive compared to other clinical cancer treatment modalities such as surgical removal and radiotherapy. Unfortunately, since the efficacy of most chemotherapeutic drugs is dose dependent, severe chemo-induced side events have been observed at higher doses.8 Thus targeted delivery of drugs with minimum non-specific exposure is essential for successful chemotherapy. Tumor targeting chemotherapy can be accomplished by exploiting the diseases’ pathophysiology such as unique or overexpressed molecules9 and leaky tumor vasculature.10Cancer is a major global cause of morbidity and mortality which is estimated that the incidence of all new cancer cases will reach 22 million by 2030 in worldwide.12 This unusual extravasation, accumulation and retention of expediently sized therapeutic molecules within tumor tissue is called enhanced permeability and retention (EPR) effect.13 In addition, nano-sized drug carriers can simultaneously deliver higher amounts of drugs with lower unspecific toxicity, without loss in therapeutic activity. Examples of these biocompatible nano-scale drug carriers include solid lipid nanoparticles (SLN),14 liposomes,15 micelles,16 nanobubbles17 and polymers.19 Among these, an extensively studied family of materials in the fabrication of biocompatible nanostructures is polymers.21 Polymeric nanostructures possesses several advantages such as simple synthesis techniques, ability to carry a wide payload of therapeutic agents and biodegradability.22 A wide range of synthetic and natural polymers have been investigated for a variety of biomedical applications such as tissue engineering,24 bioimaging25 biosensors28 and drug delivery.30 Synthetic polymers, notably, PLGA and its co-monomer PLA are widely used in the synthesis of nano-sized drug delivery systems. Flexible synthesis techniques enable the tailoring of nanoparticle properties such as size,31 drug loading32 and in-vivo drug release.33Therapeutics can be passively targeted to the hyper-permeable tumor vasculature commonly observed on most cancers. Moreover, the absence of lymphatic drainage in tumors leads to retention of accumulated therapeutic agents within the tumor tissue.34 glioblastoma35 and pancreatic cancer36 have yielded promising results. However, a major clinical limitation of paclitaxel is the drugs’ poor solubility in water. Therefore biocompatible nano-sized colloidal structures offer safer alternative paclitaxel delivery vehicles.40Among a legion of anti-cancer drugs, paclitaxel is a potent chemo-agent used in the treatment of several solid tumors including ovarian cancer, breast cancer, AIDS related kaposi sarcoma and lung cancer. Further investigations on the efficacy of paclitaxel against gastrointestinal cancer,41 whilst sizes larger than 250 nm are ideal candidates for phagocytosis.42 Therefore, the effective therapeutic window for EPR targeting may be considered between 50 and 200 nm. Thus the aim of this work was investigate the various parameters which affect the diameter of PTX loaded PLGA nanoparticles for effective EPR targeting. PLGA nanoparticles were chosen for this study due the simplicity and flexibility of synthesis techniques such as nanoprecipitation and emulsion/solvent evaporation.44Since most synthesis nanoparticle and drug loading techniques are well established, current scientific focus is increasingly being directed towards optimization of various parameters to obtain effective formulations. Therefore, for EPR targeting, it is of great interest to determine the various input parameters which affect the diameter of nanoparticles. Meanwhile, it is also important to be cognizant of the rate limiting size-dependent physiological processes which may affect the intra-tumoral accumulation of nanoparticles. Nanoparticle sizes less than 30 nm are prone to renal filtration−1) was bought from Shenzhen Esun Industrial Co., China. Dichloromethane (DCM) and acetone (99%) supplied by Carol Erba. Polyvinyl alcohol (PVA), fully hydrolized (MW 60000 g mol−1) was obtained from Merck (Germany). All solutions were prepared using deionized water.Paclitaxel was purchased from sigma. PLGA  method. The PVA polymer was dissolved in deionized water as aqueous phase under continuous magnetic stirring at 40 °C for 5 h to obtain a homogenous solution. Different amounts of paclitaxel and PLGA were dissolved in DCM and stirred for 1 h at room temperature. Then the organic phase poured in PVA solution (30 ml) and stirred for 30 min at room temperature. Then emulsion was sonicated using probe sonication  for 4 minute. After evaporation of the organic solvent overnight, the nanoparticles were collected by centrifugation (Eppendorf centrifuge) at 12000 rpm for 20 min at room temperature and washed twice with deionized water.The homogenous solutions of PVA were prepared. Specific amount of PLGA and paclitaxel was dissolved in acetone (5 ml) as the organic phase and stirred for 30 min at room temperature. Next, organic phase was added to the 20 ml of PVA solution while stirring. Afterward, organic phase was evaporated overnight and nanoparticles were collected by centrifugation at 12000 rpm for 20 min at room temperature and washed twice with deionized water.The morphology and diameter of nanoparticles were carried out using SEM as an accelerating voltage of 20.0 kv (Philips XL-30) after sputtering with gold. The diameter of nanoparticles was measured by randomly choosing 30 nanoparticles by SemAfore  software as shown in The hydrodynamic diameter and the median nanoparticles size were obtained using DLS (Scatter Scope) as shown in et al45 and Fessi et al,46 respectively. In an study PLGA incorporated with procaine nanoparticles was prepared via nanoprecipitaion technique with the size less than 210 nm.32 In another study protein encapsulated in PLGA nanoparticles were prepared by phase separation method with the size around 300 nm for biomedical application.47 Commercial success of the various polymeric nanoformulation products developed by emulsion and nanoprecipitation were documented by Nava-Arzaluz et al48 and Minost et al.49 We investigated the effect of two methods of single emulsion and precipitation on nanoparticles size. The size of polymeric nanoparticles is particularly important in cancer therapy as drug delivery vehicles can enter into the tumor tissue via the EPR effect. Our results indicated that paclitaxel-loaded nanoparticles prepared with precipitation had smaller size (50 Therefore, precipitation method is suggested for medical applications.ler size . No. 11 ler size , No. 19.51The function of PVA concentration as an effective factor on PLGA diameter is shown in 53 Although some reports presented lack of relationship between size of nanoparticles and drug concentration.54The effect of the amount of drug was also examined on nanoparticles diameter. In this study the different amounts of drug were used whilst other parameters were constant by both emulsion (55 In this experiment, for nanoparticles prepared by emulsion method, probe sonicator was used for 2, 4, 5 and 6 min (56 or agglomeration.57 In a study, size of drug loaded PLGA nanoparticles was optimized by varing sonication time.58 Therefore, sonication time can be considered as a parameter for optimized size of nanoparticles.Polymeric nanoparticles can be synthesized via two approaches; “bottom up” or “top down”. In “bottom up”, the nanoparticles are formed from continuous deposition of molecular growth species on a nanoparticle nuclei, that is, polymer aggregation. In “Top down” synthesis, external energy sources are used to break down colloidal polymer complex structures into nanoemulsions. A commonly applied technique in the “top down” synthesis is the use of ultra-sound homogenization.60 In precipitation method, frequency of collisions increase which leads to fusion of nanoparticles as concentration of polymer enhances.61In this experiment, by increasing the PLGA concentration from 10 to 60 mg, the mean size of nanoparticles enhanced from about 250 to 414 nm . In bothIn our experiments, the nanoparticles size demonstrated an initial decrease from 478 nm to 300 nm as the amount of the organic phase enhanced from 1 to 3 ml (as shown in This study compared two methods for preparation of paclitaxel loaded PLGA nanoparticles including single emulsion and precipitation. The results indicated that precipitation method results in smaller nanoparticles compared to emulsion one. In addition, the effect of the various parameters on the size of the nanoparticles was investigated The results demonstrated that the concentration of the PLGA polymer, drug and PVA had a direct relationship with the size of nanoparticles whereas sonication time and organic phase to aqueous volume ratio needed to the optimization to obtain the nanoparticles with smaller sizes.This work was supported by Tehran University of Medical Sciences, Grant No. 96-01-87-34138.Not applicable.The authors declare no conflict of interests.


Acute and chronic liver injury results in hepatocyte death and turnover. If injury becomes chronic, the continuous cell death and turnover leads to chronic inflammation, fibrosis and ultimately cirrhosis and hepatocellular carcinoma. Controlling liver cell death both in acute injury, to rescue the liver from acute liver failure, and in chronic injury, to curb secondary inflammation and fibrosis, is of paramount importance as a therapeutic strategy. Both apoptosis and necrosis occur in the liver, but the occurrence of necroptosis in the liver and its contribution to liver disease is controversial. Necroptosis is a form of regulated necrosis which occurs in certain cell types when caspases (+/−cIAPs) are inhibited through the RIPK1-RIPK3 activation of MLKL. The occurrence of necroptosis in the liver has recently been examined in multiple liver injury models with conflicting results. The aim of this review is to summarize the published data with an emphasis on the controversies and remaining questions in the field. Hepatocytes do not express RIPK3 under basal conditions.Blocking apoptosis in hepatocytes protects from cell death (with no switch to necroptosis).Necrostatin-1 (RIPK1 kinase inhibitor) has been shown to protect against many models of acute liver injury such as acetaminophen toxicity.versus RIPK1 kinase functions?What are the RIPK1 platform functions in hepatocytes Does RIPK1 participate independent of the necrosome in hepatocyte death pathways?Does necroptosis occur in non-parenchymal cells (NPCs)?Can RIPK3 be induced in hepatocytes in chronic liver disease, and if so does this sensitize to necroptosis?Does RIPK3-independent, but MLKL-dependent necrosis exist in hepatocytes in liver diseases?2 Hepatocytes are the main parenchymal cells making up 80% of liver mass, with the other epithelial cell type, the cholangiocytes lining the bile ducts, being the minority. The NPCs despite being high in number  contribute to only 7% of liver volume because of their small size. These cells include the resident liver macrophages or Kupffer cells (KC), the liver sinusoidal endothelial cells (LSEC), the peri-sinusoidal hepatic stellate cells (HSC) formerly known as fat storing Ito cells, as well as liver lymphocytes particularly enriched in natural killer (NK) and natural killer T (NKT) cells. NPCs participate in normal physiologic liver functions and are also contributors to liver disease.The liver is a complicated vital organ responsible for metabolic processes, protein synthesis, and clearance of toxins and xenobiotics. The liver is the largest solid organ and has an unparalleled regenerative capacity, high vascular capacitance (receiving 30% of cardiac output) and is a site of immune-privilege.3 Chronic liver injury is the result of long standing and ongoing liver damage and hepatitis from inflammation or intracellular stress responses . Common examples of chronic liver injury include steatohepatitis , autoimmune-hepatitis (AIH), primary biliary cholangitis, and chronic viral hepatitis . Acute and chronic liver injury results in hepatocyte death and turnover, which is commonly detected by an increase in the serum aminotransaminases (AST and ALT). If injury becomes chronic, the continuous cell death and turnover leads to chronic inflammation, activation of HSC into myofibroblasts and tissue repair responses, which result in fibrosis and ultimately cirrhosis and hepatocellular carcinoma. Controlling liver cell death both in acute injury to rescue the liver and in chronic injury to curb secondary inflammation and fibrosis is of paramount importance as a therapeutic strategy. When studying liver cell death, it is important to draw a distinction between acute and chronic liver disease models, since much of the controversy in the liver cell death field is because of these various contexts of injury under study. Different liver injury models differ in the type of signaling pathways activated, the role and the type of inflammatory cells involved, which ultimately dictate the cell death subroutine. For example, viral hepatitis results in cell death via death receptors (DR) and is executed mainly by adaptive cytotoxic T-cell activity resulting in liver cell apoptosis, whereas cell death from acetaminophen (APAP) hepatotoxicity, a hepatocyte intrinsic toxin, is largely necrotic and caspase and TNFR independent.4–7 This brings us to another point, and that is, although hepatocytes do express DR on their surface, not all liver cell death is DR-mediated and when comparing liver injury models one must take into account intrinsic death pathways activated by damage to the ER, mitochondria or nucleus, versus cytokine/DR ligand-activated DR signaling resulting in cell death.8Liver injury is classified by duration into acute and chronic. Acute injury in its most extreme form results in acute liver failure (ALF), which is defined as an acute loss of hepatocyte mass (by necrosis or apoptosis) resulting in coagulopathy (elevated INR) and any degree of mental alteration  in a patient without preexisting liver disease.9 In the past few years, much attention has been directed towards the role of necroptosis in human disease. This has also led to an increasing number of studies to explore this cell death mode in the liver using various animal models of acute and chronic liver injury; however, a wide range of conflicting conclusions have been generated  are inhibited. In certain cells when these conditions are met, RIPK1 and RIPK3 interact via their RIP homology interaction motif (RHIM) domains to form a complex recruiting the psudokinase mixed-lineage kinase domain-like (MLKL). MLKL is activated and phosphorylated by RIPK3 and subsequently translocates to the cell membrane resulting in its rupture .9 In theenerated ,2,3. Bel10 Activated MLKL (p-MLKL) forms a tetramer that translocates to the cell membrane where it executes necroptosis by a mechanism, which is still under debate.11–15 MLKL driven rupture of cell membrane is the necessary end point of necroptosis and the only known activator of MLKL at present is RIPK3. Therefore, RIPK3 and MLKL are necessary for the induction of necroptotic cell death.16As briefly described above, necroptosis is a form of programmed cell death often mediated by TNFR1 and other DR engagement with their ligands. It occurs through the RIPK1-RIPK3-mediated activation of MLKL in certain cell types when there is inhibition of caspases +/−cIAPs.17 RIPK3, the third member of the receptor interacting protein kinase family was first described in 1999.18 RIPK3 mRNA distribution was examined using Northern blotting and its presence in the liver was not conclusive in the original report.18 Kasof et al.19 further dissected the role of RIPK3 in apoptosis in 2000 and reexamined its tissue distribution and failed to detect RIPK3 mRNA under basal conditions in the liver. RIPK3 protein is weakly present in whole liver homogenate (mixed cell types) but this in fact may be because of its presence in the NPC compartment. Much of the controversy lies with its induction during injury, which has been based mainly on western blotting and immunohistochemistry (IHC) of RIPK3 protein using commercially available non-specific polyclonal antibodies on injured liver tissue. Interestingly, in most reports, the area of RIPK3 staining corresponds to the area of necrotic or damaged liver. Injured liver tissue is highly immunoreactive, we have observed positive staining with non-specific normal rabbit and mouse IgG.17 Furthermore, we have observed positive RIPK3 staining in livers of RIPK3−/− mice using commercially available polyclonal antibodies, confirming the lack of specificity of these polyclonal antibodies.17 To determine if hepatocytes express RIPK3, we isolated primary mouse hepatocytes (PMH) and immediately measured RIPK3 mRNA and performed western blotting on freshly isolated PMH using a monoclonal antibody to RIPK3 provided to us by Dr. Newton of Genentech. We could not detect RIPK3 in hepatocytes under basal conditions nor after cell culture with APAP or TNF at different time points. Nor could we detect an increase in RIPK3 in PMH isolated from mice treated in vivo with APAP for 3 h.17 Recently others have confirmed this data, reporting much lower RIPK3 expression in whole liver homogenate compared with other organs such as spleen and intestine and undetectable RIPK3 in isolated mouse hepatocytes.20 Whether hepatocytes express RIPK3 or not is of critical importance since, as far as we know now, cells that do not express RIPK3 do not undergo necroptosis.21 Of course, theoretically in some unique circumstances it is possible that MLKL can be activated in a RIPK3-independent fashion and this has been suggested to occur in immune-mediated liver disease, see ConA section for more details.22Although hepatocytes clearly express MLKL, the presence of RIPK3 in liver cells has been controversial.24 Direct DR activation under experimental conditions by TNF, FASL, TRAIL or DNA damage , or growth factor withdrawal have been shown to result in apoptotic cell death.23 Liver cells express DR , likely because of an evolutionary pressure to eradicate hepatotropic viruses. Indeed, during viral hepatitis T cells kill hepatocytes by DR-mediated apoptosis, which eliminates virally infected cells.24 Hepatocytes are type II cells and require the participation of the intrinsic mitochondrial apoptosis pathway for cell death completion.26 Only certain primary cells and cell lines such as L929 fibrosarcoma cells, Jurkat T cells, HT29 cells, MEF, keratinocytes express RIPK3 and have been shown to switch to an alternate form of cell death with DR signaling under conditions where caspases +/−cIAPs are inhibited.21 This has not been the case for all cells; for example, MCF-7, Hela cells and Hek293 cells do not undergo necroptosis because of a lack of RIPK3 expression.21 In fact, absence of RIPK3 in cells predicts whether they can undergo necroptosis.27 Under numerous apoptosis-inducing experimental conditions in hepatocytes we and others have observed a protection afforded by caspase inhibition without a switch to necroptosis.28–35 In addition, liver-specific ablation of caspase-8 has no effect on the normal liver and is in fact protective against Fas and TNF-mediated apoptosis.36 Therefore, the lack of a switch to a necroptotic form of cell death with caspase inhibition would argue against necroptosis occurring in liver cells. Necrosis of hepatocytes does occur in certain contexts but appears to be mediated by the mitochondrial permeability transition that can be considered a form of regulated necrosis. The MPT responds to ROS production and cyclophilin D controls pore opening.Hepatocytes can die by necrosis and or apoptosis. In the liver, apoptosis is a common event in liver injury as hepatocytes robustly express DRs and many disease states have been shown to result in hepatocyte apoptosis, such as viral hepatitis (Councilman bodies), NASH (apoptotic bodies), alcoholic hepatitis, and autoimmune liver disease (adaptive immune-mediated).37–42 APAP-induced cell death has long been recognized to be necrotic and caspase and TNFR independent.4–7 It is a form of cell death which results in mitochondrial dysfunction and ROS generation, ATP depletion, MPT and necrotic oncosis. Given the involvement of signaling molecules, APAP hepatocyte death is a regulated form of cell death. However, as it pertains to necroptosis, it is important to point out that APAP cell death is independent of receptor signaling.43 Many investigators have evaluated the role of Necrostatin-1 (Nec-1) in APAP toxicity and have shown protection with the RIPK1 kinase inhibitor  at 6 h but this protection did not extend to 24 h. We were unable to see any protection with APAP (300 mg/kg) using strain-matched controls at early or late times points.17 Deutsch et al.46 reported a robust protection against APAP (700 mg/kg) with a 75% survival advantage compared with C57BL/6j controls. In our hands 700 mg/kg of APAP results in 100% mortality in all WT and transgenic mice models (C57BL/6n).Acetaminophen (APAP) toxicity is a valuable model of liver cell death, hepatotoxicity and fulminant liver failure. The murine APAP model closely mimics human disease and is an excellent early translational model to study necrosis of hepatocytes. Once glucuronidation and sulfation pathways are saturated (in instances of overdose) APAP is metabolized to NAPQI via the Cyp450 system. NAPQI is highly electrophilic and covalently binds intracellular proteins and is thereby intrinsically toxic to hepatocytes. Inhibition or knockdown of many signaling molecules and MPT including components of the MAP Kinase pathway , GSK3b, PKCa, as well as cyclophilin D have been shown to protect against APAP.nhibitor .17,44–4849 Finally, given the controversy with the RIPK3 knockout mice and the fact that MLKL is indispensable for the execution of necroptosis, we examined the effect of APAP 300 mg/kg on MLKL−/− mice (compared with j sub-strain-matched controls) and found no attenuation of injury.17 This demonstrates that cell death in the APAP model is MLKL independent and thus most likely not necroptosis.17 Gunther et al.20 have independently obtained similar results, MLKL−/− mice were not protected against APAP compared with WT controls (j sub-strain).20 Although these results seem confirmatory, more rigorous studies using littermate controls and inducible liver cell-specific knockout are needed for conclusive results.22 Furthermore, we could find no change in RIPK3 expression in the short time course APAP toxicity (up to 24 h) in vitro or in vivo.The discrepancy in the reports may be in part because of experimental variations (such as APAP preparation methods) as well as strain matching. The Genentech generated RIPK3−/− mice are on an n sub-strain , whereas WT control mice used in the Ramachandran paper were j sub-strain and the C57BL/6 mice in the Deutsche paper were purchased from Jackson (likely j sub-strain unless otherwise requested). C57BL/6 substrains n and j have different susceptibility to APAP liver injury.50–54 Subsequent activation of macrophages/KC and recruitment of leukocytes into the liver significantly contributes to the liver injury.56 The mode of cell death in ConA is largely necrotic, although some reports have observed DNA fragmentation and apoptotic bodies as early as 5 h after ConA injection, suggesting that apoptosis of hepatocytes occurs early in the injury phase followed by massive cell lysis and necrosis in the later hours.58 TNF is presumed to be the major cytokine activating hepatocyte DR as interference with TNF (treatment with anti-TNF antibodies) has been shown to be protective against ConA-induced hepatitis and hepatocellular death.57–59 TNF is directly capable of inducing hepatocyte apoptosis via TNF-receptor signaling inducing caspase-8 activation and involvement of the mitochondrial intrinsic death pathway, leading to mitochondrial cytochrome C release and caspase-3 activation. However, as TNF signaling also activates NF-κB, which in turn switches on pro-survival gene expression such as cFLIP, BclXL among others, in order for TNF to mediate hepatocyte death, the deactivation or inhibition of the NF-κB pathway is necessary. However, this does not seem to be the case with ConA as the lectin alone can result in hepatitis and cell death without the need for transcriptional or translational inhibition.57 In addition to TNF, other major cytokines such as IFN-γ, IL4, IL6 and other DR pathways  have also been implicated in ConA hepatotoxicity, although to a lesser extent than TNF.60–65 The ConA model is very complex. In addition to immune cells, LSECs have also been suggested to have a pivotal role in the injury by mediating a hypercoagulative state in the liver.66The plant lectin concanavalin A (ConA) is widely used to induce ‘immune-mediated’ acute liver injury in mice. It is often used as a model of hepatitis as the hepatocellular injury in ConA is primarily initiated by the direct activation of T cells, particularly CD4+ T cells and NKT cells and targeting of hepatocytes by the immune system in an antigen TCR-independent fashion.γ signaling, liver cell death appears to be necrosis morphologically and not apoptosis. This is further supported by the fact that no significant activation of caspase activity has been observed with ConA and no protection from liver injury is afforded by caspase inhibition.68 Given the involvement of the TNF DR pathway and the late appearance of necrotic cell death in ConA model, multiple laboratories have investigated whether necroptosis and the RIPKs contribute to ConA-induced hepatitis  administration 15 min before on ConA  induced hepatitis on liver damage using female C57BL6 mice and reported a decrease of about 50% in ALT and histologic injury versus PBS pre-treatment. Nec-1 is dissolved in DMSO, which is known to be hepatocyte protective, may have some confounding effects especially if not controlled for. They demonstrated that ConA-activated PARP-1 in the liver of WT mice and that the PARP-1 inhibitor, PJ-34, protected to the same extent as Nec-1. Nec-1 not only resulted in less hepatotoxicity as measured by ALT but also decreased PARP-1 activation, suggesting that PARP-1 may be downstream of RIPK1 kinase activation in ConA.69 Recently, the same group published a follow up paper, this time using 12 mg/kg of ConA and the same pre-treatment protocol with Nec-1 125 µg, 15 min before ConA and they concluded that protection by RIPK1 inhibitor and PARP inhibitor was because of lower levels of IL-33.70 Zhou et al.,71 performed similar experiments on male C57BL/6 mice. They administered Nec-1 (1.8 mg/kg) or DMSO via tail vein 1 hour before ConA (20 mg/kg) and observed less AST and ALT as well as a survival benefit with Nec-1 pre-treatment . Deutsch et al. while studying necroptosis in the ConA model observed that although RIPK3−/− mice were mildly protected as measured by ALT compared with WT C57BL/6, this did not translate to a survival advantage. However, interestingly, simultaneous treatment of WT mice with Nec-1 (1.6 mg/kg) and ConA (20 mg/kg) compared with ConA (20 mg/kg) alone exacerbated liver injury, more than doubling the ALT and resulting in 50% mortality from Nec-1 treatment.46In spite of the immune-mediated features of ConA toxicity, such as the requirement for TNF and IFN-epatitis . Jouan-Let al. investigated the mode of cell death in ConA using a global knockout of RIPK3. Even though they interpret RIPK3 deletion as being protective, there was only a modest (<10%) decrease in transaminases.46 These minor differences in in vivo experiments using possibly non-strain-matched mice that are not littermates should be interpreted with caution. It is interesting that Deutsche and colleagues observed a robust aggravation of injury with Nec-1 and ConA co-treatment compared with ConA alone in contrast to findings by Zhou et al.,71 Jouan-Lanhouet et al.69 and Arshad et al.70 The protection reported with Nec-1 should also be further scrutinized given the possible off target effects of Nec-1.72 Interestingly, Weinlich et al.73 have also examined the effect of ConA on RIPK3−/− mice compared with WT littermates and noted that the knockout animals displayed the same level of liver damage as control littermates, strongly arguing against RIPK3-dependent necroptosis in ConA liver injury. Recently Gunther et al.20 have reported protection against ConA (25 mg/kg) in global MLKL−/− mice. Interestingly, whereas Nec-1 s (400 µg i.p.) was also protective in their experiments, RIPK3 knockout was not. In addition, they noted an increase in both RIPK1 and MLKL protein after ConA, whereas RIPK3 protein levels, which are very weak in whole liver and absent in hepatocytes did not change or increase after ConA liver injury. It is unclear in these studies how MLKL is activated in the absence of RIPK3, but an unidentified kinase downstream of RIPK1 is suggested to contribute to this non-canonical necroptosis pathway which seems to be MLKL-dependent.22 Investigation of conditional and liver-specific knockout models is still warranted.Deutsche α-galactosylceramide , a specific activator for invariant NKT cells resulting in immune-mediated liver injury and induction of regulatory and inflammatory cytokines. In WT mice α-GalCer activation of NKT cells results in increased TNF, IFN-γ, IL4, and subsequent activation of innate immune cells in the liver, causing a mild to moderate liver injury (ALT ~300–1000 IU) at the peak time of 16–24 h. This immune-mediated model is quite similar to the ConA model, but relatively less severe and initiated by specific activation of NKT cells, which are uniquely abundant in the liver. Kang et al.28 have reported that both α-GalCer injury and ConA-induced liver damage is attenuated in global RIPK3−/− mice (ALT 200) accompanied by less TNF and IFNγ mRNA expression. To confirm that the RIPK3 deletion in NKT cells was attenuating the injury, they performed bone marrow (BM) transplants between RIPK3−/− and WT mice to generate chimeric animals. They showed that RIPK3 in BM cells was essential for liver injury to occur as ConA-induced liver injury was significantly attenuated in the WT animals that had RIPK3−/− BM. They provided evidence showing that the reduced liver injury in RIPK3−/− mice is not related to necroptosis of hepatocytes, and most likely because of an impairment of production of cytokines (TNF and IFN-γ) by RIPK3-deficient NKT cells in a necroptosis-independent fashion. Thus, these findings are very interesting but do not support the hypothesis of necroptosis occurring in hepatocytes as the RIPK3 deletion in NKT cells seems to be the limiting factor.28 We have also been interested in the α-GalCer model of liver injury and have examined the role of RIPK3 and MLKL deletion as well as RIPK1 knockdown via antisense oligonucleotide in NKT-mediated immune injury from α-GalCer. Contrary to the findings by Kang et al., we found no difference in liver injury between global RIPK3−/−, MLKL−/− mice and strain-matched controls.74 However, we found that knockdown of RIPK1 markedly exacerbated α-GalCer-mediated liver injury and induced lethality as early as 6 h after treatment. Unexpectedly, despite the massive hepatocyte death and the effective RIPK1 knockdown (>95%) the exacerbated liver injury was not because of an impaired hepatic NF-κB activation. As we observed no difference with RIPK3−/− or MLKL−/− mice, we concluded that necroptosis was not involved. We observed increased TUNEL staining and caspase-3 activation in the RIPK1 knockdown animals with severe liver injury and we suspected the mode of death was in fact apoptosis. On further investigation, we observed that the exacerbation of liver injury with RIPK1 knockdown was because of TNF-and caspase-dependent massive apoptosis of hepatocytes as neutralization TNF or inhibition of caspase by pan-caspase inhibitor zVAD.fmk conferred an almost complete protection.74 Interestingly, ZVAD protection was not accompanied by a switch to necroptosis.Another model of immune cell activation is in vivo animal models. However, studies of hepatitis C in cell culture have to a large extent concluded the mode of cell death as apoptotic.75–77 One study has reported an additive protection with in vitro utilization of Nec-1 to the pan-caspase inhibitor QVD, although this was examined on Huh7 cells, and not primary hepatocytes and there was no mechanistic exploration as to how Nec-1 was exerting its effects.78Alcoholic and non-alcoholic steatohepatitis (ASH and NASH) along with models of viral hepatitis are the most commonly studied chronic liver injury models. Studies of mechanisms of hepatocyte cell death in viral hepatitis have been somewhat restricted by the limited availability of 79 Chronic alcohol use also results in steatohepatitis and is an important cause of cirrhosis worldwide. Long standing steatohepatitis results in inflammation, cell death, fibrosis, leading to cirrhosis and hepatocellular carcinoma. Therefore, therapeutic strategies other than weight loss and abstinence from alcohol are sorely needed.79 So far, the mode of cell death in NASH is presumed to be apoptosis as interfering with apoptosis, caspase inhibition and liver-specific caspase-8 knockout greatly protect.80 However, studying animal models of steatohepatitis has proven to be challenging. Unlike the APAP model which very closely mimics human pathology, it is difficult to study alcoholic steatohepatitis (ASH) and NASH by replicating robust steatohepatitis in mice using alcohol or a high fat diet (HFD) alone. Mice do not develop the same degree of inflammation and fibrosis as humans which may be a function of the duration and intensity of alcohol or HFD feeding or normal physiologic metabolic differences.81 In humans, fatty liver and NASH are accompanied by visceral adiposity, insulin resistance, type II diabetes and metabolic syndrome, which has proven somewhat difficult to recapitulate in animal models. The same is true for alcohol, existing rodent models of alcoholic liver disease result in less liver injury from alcohol  in mice and rats compared with the effect of alcohol on human liver.82 Nevertheless, using the available animal models the role of necroptosis has been investigated in ASH and NASH  are a leading cause of liver disease and obesity related mortality is the US with increasing prevalence worldwide.and NASH .et al.83 examined the effects of RIPK3 in mice fed alcohol. They reported an increase in RIPK3 expression by IHC using a commercially available polyclonal antibody with the Lieber de Carli model of free access to alcohol as well as chronic binge feeding. The RIPK3 staining was more prominent around the central vein. In addition, the authors report RIPK3 induction in human liver biopsy samples of patients with alcoholic liver disease using IHC. In RIPK3−/− mice (back-crossed to j sub-strain) they reported a modest attenuation of liver injury compared with WT C57BL6/j sub-strain mice . They also reported less liver triglycerides, TNF and liver inflammatory cell numbers by IHC. Wang and colleagues used the Gao-binge model of alcohol feeding and reported an increased expression of RIPK3 using a  with alcohol and protection from global RIPK3 deletion (ALT and steatosis) but no difference in hepatitis and neutrophil infiltration in the RIPK3−/− mice. They could not detect a transcriptional change (mRNA) in RIPK3 and attributed the increased protein levels of RIPK3 to impaired hepatic proteasome function because of less protein turnover resulting from alcohol feeding.85 Roychowdhury et al.86 have recently published a follow up paper to using a HFD model and comparing RIPK3−/− and WT controls. They find no protection against HFD-induced steatohepatitis with global RIPK3 knockout. In fact, they report worsening of injury compared with controls. The RIPK3−/− animals had basal insulin resistance, which significantly worsened on a HFD leading to steatosis, inflammation and fibrosis. It is interesting that RIPK3 knockout had diametrically opposite results in the alcohol model and HFD model experiments performed on the same mice by the same group. Even more intriguing is that in both instances the authors report increased RIPK3 in the liver by immunostaining.86 The chow-fed wild-type mice in the present study expressed no basal RIPK3 or p-MLKL, but after HFD feeding showed increased expression of both proteins by IHC. However, despite the high expression of these necroptosis proteins the mice had very little injury. Therefore it is highly unlikely for necroptosis to be activated in this model.86Roychowdhury 88 Gautheron et al.88 demonstrate an increase in RIPK3 expression using IHC and western blotting . They also reported that liver injury is worsened in the animals by caspase-8 liver-specific knockout. Interestingly, in the liver-specific caspase-8 KO increased hepatic RIPK3 expression was observed even in the control diet, which may be because of compensatory mechanisms that occur in embryonic knockout animal models, making them difficult to interpret. Alfonso et al.87 have used IHC on human livers with various diseases and have demonstrated an increase in RIPK3 staining in liver sections from humans with NASH, ASH and hepatitis B and C. One could interpret this one of two ways, either all of these human liver diseases result in RIPK3 upregulation and as the authors suggest an involvement of necroptosis, or the antibody is non-specifically binding injured liver tissue. The authors offer no explanation as to how caspases are inhibited in these disease states for necroptosis to ensue. In mice using the high fat choline-deficient and methionine deficient models, Alfonso and colleagues demonstrated an increase in RIPK3 and p-MLKL in the insoluble liver fraction and a protection from RIPK3 knockout. Curiously, although TNF  is known to induce apoptosis in hepatocytes,89–91 the authors were unable to prevent cell death from TNF/CHX by caspase inhibition. Both of these studies used the methionine and or choline-deficient models which are not ideal models of NASH because of a lack of insulin resistance and weight gain in the animals.93 Furthermore, the technical limitations and non-specific binding of polyclonal antibodies used in IHC of the liver, in our opinion, limits the conclusions one can reach from these descriptive studies.17Two groups have investigated the role of RIPK3 in liver injury, using the methionine choline-deficient model, which is used as a NASH model.92 One must also take into account that these models are global RIPK3 knockouts and it is not clear whether the observed protection from lack of RIPK3 is because of a hepatocyte intrinsic mechanisms or an effect on the immune system. In certain disease models, which exhibit hemorrhage or severe inflammation in the liver, the inflammatory cells and RBC infiltration to the liver could theoretically increase total RIPK3 levels because of the mixed cell population. Nevertheless, although we do not believe that RIPK3 is upregulated in in acute liver injury, we recognize that context is important and it is possible that in chronic models of liver injury RIPK3 is indeed induced in hepatocytes. Whether this has any relevance or correlation with necroptosis in the absence of caspase inhibition remains to be determined. Furthermore, one has to consider cell death-independent functions of RIPK3 expression in hepatocytes.Regardless, assuming the RIPK3 induction in hepatocytes is indeed robust and relevant in chronic disease models, we are left with the dilemma of understanding how physiologic necroptosis occurs in the presence of caspases, as all models of necroptosis require the genetic deletion or inhibition of caspases +/− IAPs.et al.15 while testing a phospho-MLKL antibody as a specific necroptosis marker had reported positive staining in liver biopsy samples from patients with drug-induced liver injury (DILI). The authors however do not specify what kind of DILI samples they used and what drug or toxicant was the culprit. Drug-induced liver injury is a complicated clinical diagnosis with a wide range of presentations and a lack of objective diagnostic test, making it largely a diagnosis of exclusion.94 In the study by Wang et al.15 no clinical data or patient characteristics, pattern of injury (hepatocellular or cholestatic) or identification of suspected drugs was provided. It is extremely important to emphasize that although some drugs such as acetaminophen are direct hepatotoxins, most drug toxicities are idiosyncratic and HLA/adaptive immune-mediated.95 DILI presents in a diverse and heterogeneous fashion, some drugs target hepatocytes and cause a hepatocellular pattern of injury, whereas others result in cholestasis. Some drugs result in hepatocyte necrosis, some activate immune cells to target DR-mediated apoptotic cell death, whereas other drugs result in vanishing bile ducts and are mainly toxic to cholangiocytes. The authors report patchy p-MLKL staining of dead hepatocytes around the central vein area of the 14 reported ‘DILI patients’.15 This is an intriguing descriptive finding. However, there is not a mechanistic explanation. Furthermore, if this implicates necroptosis activation, one would have to assume that in these 14 patients, caspase activation was somehow inhibited or defective for the hepatocytes to not die of apoptosis as they are very prone to do and instead default to necroptosis. This scenario has yet to be shown in physiologic conditions.Wang α-GalCer. These two models are vastly different. The former is a cell intrinsic toxin to hepatocytes, which mediates a cyclophilin D and MPT-dependent necrotic form of cell death and the latter a DR-mediated apoptosis of hepatocytes induced by activated-NKT cells. It should not be surprising that RIPK1, which is a key molecule in inflammatory pathways, functions both as a survival protein and a death-inducing protein in a context-dependent manner.97 Furthermore, RIPK1 has been suggested as a participant in ER stress-induced apoptosis,98 and it is likely that RIPK1 has additional functions beyond mediating apoptosis and necroptosis.9 We should note that although we did observe a protection against APAP with RIPK1 knockdown, others have observed no protection with embryonic RIPK1 liver-specific knockout.99 Although the antisense may have off target effects, there are also issues with embryonic knockout, namely the capacity of living organisms to compensate in unexpected ways for the loss of vital proteins and alterations in normal homeostasis. It remains to be seen if adult inducible liver-specific knockout of RIPK1 protects against any of the acute and chronic liver injury models and hepatocyte death.Multiple groups have studied the role of Nec-1 in models of acute liver injury in particular APAP, and have observed protection. Given the off target effects of Nec-1, we examined the effect of RIPK1 knockdown using an IONIS antisense oligonucleotide which concentrates in the liver and knocks down RIPK1 with about 95% efficiency. As RIPK3−/− and MLKL−/− mice were not protected but knockdown of RIPK1 in these mice afforded partial protection against APAP necrosis, we believe RIPK1 may have a novel and independent function in hepatocyte death, which appears to be upstream of JNK. One of our most striking observations in the past few years was that knockdown of RIPK1 protected against hepatocyte death in the APAP toxicity model while worsening injury from immune-mediated liver injury caused by 17 As NPC participate in normal liver functions and have all the required machinery for the execution of necropoptosis, it is intriguing to hypothesize that NPC necropoptosis may contribute to some forms of liver disease.As discussed in the introduction, hepatocytes are not the only liver cell type. In fact, the liver has a rich and diverse microenvironment harboring multiple types of specialized cells. Hepatocytes function mainly to regulate metabolism, detoxify xenobiotics, synthesize critical circulating proteins, and generate and secrete bile acids. The NPCs, including the LSECs, KC, HSC, NK, NKT, are in constant cross talk with hepatocytes and each other. We have observed a robust expression of RIPK3 and MLKL in isolated LSECs, KCs and liver leukocytes.RIPK3 is not expressed under basal conditions in hepatocytes.As MLKL is expressed in hepatocytes there may yet be a RIPK3-independent mechanism for activation of MLKL and necroptosis in these cells.RIPK3 may be induced in certain disease conditions of the liver, but if so, necroptosis-independent pathways must always be considered as well.Preventing apoptosis in hepatocytes after DR engagement does not result in a switch to necroptosis; rather it prevents cell death.Human liver disease conditions in which caspases are inhibited (a requirement for necroptosis) have yet to be reported.More conclusive littermate controlled studies are needed using cell-specific inducible knockouts of MLKL, and the RIPKs and valid antisera and reagents that are specific and suitable for immunoblotting to resolve controversies and uncertainties.