While of significant interest, these latter studies relied on transgenic mice that express human PS1 variant transgenes ubiquitously and at high levels, and the consequences of wild type or mutant PS1 expressed under physiologically relevant levels on EE-mediated AHNPC phenotypes has not yet been tested.Alzheimer’s disease (AD) is characterized by progressive memory loss and impaired cognitive function. Early-onset familial forms of the disease (FAD) are caused by inheritance of mutant genes encoding presenilin 1 (PS1) variants. We have demonstrated that prion promoter (PrP)-driven expression of human FAD-linked PS1 variants in mice leads to impairments in environmental enrichment (EE)-induced adult hippocampal neural progenitor cell (AHNPC) proliferation and neuronal differentiation, and have provided evidence that accessory cells in the hippocampal niche expressing PS1 variants may modulate AHNPC phenotypes, PSEN1 M146V mutation driven by the endogenous PSEN1 promoter (PS1 M146V “knock-in” (KI) mice) to standard or EE-housed conditions. We show that in comparison to wild type PS1 mice, AHNPCs in mice carrying homozygous (PS1M146V/M146V) or heterozygous (PS1M146V/+) M146V mutant alleles fail to exhibit EE-induced proliferation and commitment towards neurogenic lineages. More importantly, we report that the survival of newborn progenitors are diminished in PS1 M146V KI mice exposed to EE-conditions compared to respective EE wild type controls.To assess the impact of mutant PS1 on EE-induced AHNPC phenotypes when expressed under physiological levels, we exposed adult mice that constitutively express the PS1 M146V allele is sufficient to impair EE-induced AHNPC proliferation, survival and neuronal differentiation, in vivo. These results and our finding that microglia expressing a single PS1 M146V allele impairs the proliferation of wild type AHNPCs in vitro argue that expression of mutant PS1 in the AHNPC niche impairs AHNPCs phenotypes in a dominant, non-cell autonomous manner.Our findings reveal that expression at physiological levels achieved by a single  PSEN1, PSEN2 or APP genes, encoding PS1, PS2 or APP variants, respectively, cause early-onset, autosomal dominant forms of familial AD (FAD) [AD is pathologically characterized by the deposition of β-amyloid (Aβ) peptides in senile plaques and neuronal loss that leads to impairments in memory and cognitive function. Aβ peptides are derived from larger amyloid precursor protein (APP) by the concerted action of BACE1 and a multiprotein complex, termed γ-secretase, that catalyzes intramembranous proteolysis of a variety of type I membrane substrates. Inheritance of mutations in AD (FAD) .Adult hippocampal neural stem cells (AHNSCs), a self-renewing cell population in the subgranular layer (SGL) of the dentate gyrus (DG), functionally mature into neurons and integrate into the hippocampal circuitry . This prM146V mutation [We now extend these latter observations and demonstrate that EE-induced AHNPC proliferation and neuronal differentiation is also impaired in PS1 M146V KI mice that harbor a germ line PS1+/+), heterozygous (PS1M146V/+) or homozygous (PS1M146V/M146V) mice were exposed to standard housing (SH) or EE conditions for one month. At the end of one month, animals housed under these two conditions were injected intraperitoneally (i.p) with a single dose of 100 mg/kg of BrdU. Mice were sacrificed 24 hr post injection, and brain sections were subjected to immunofluoresence analysis using a BrdU-specific antibody , or between PS1+/+, PS1M146V/M146V mice , or between PS1M146V/+ and PS1M146V/M146V mice . Exposure to EE lead to a significant increase in BrdU+ cell numbers in all three genotypes when compared to their respective control cohorts exposed to SH conditions . However, and in contrast, with the a prominent ~2.2-fold increase in BrdU+ cell numbers that was detected in EE versus SH PS1+/+ mice, exposure to EE lead to only a modest 1.48- and 1.49-fold increase in BrdU+ cell numbers in PS1M146V/+ and PS1M146V/M146V mice, respectively, compared with their SH cohorts. Moreover, the BrdU+ cell numbers in the hippocampus of PS1M146V/+ and PS1M146V/M146V mice exposed to EE conditions were significantly lower than the numbers observed in enriched PS1+/+ mice .A cohort of one month old male wild type PS+/+, hetePS1+/+, PS1M146V/+ or PS1M146V/M146V mice to SH or EE conditions for one month, and then injected these animals with a single dose of BrdU (100 mg/kg). The cohorts of PS1+/+, PS1M146V/+ or PS1M146V/M146V mice were then returned to respective SH or EE conditions for an additional 2 weeks. We chose the two week time point for analysis because this is an accepted time window to score for neuronal or glial lineage commitment of AHNPCs and their survival efficiency [To assess the impact of the PS1 M146V KI mutation on EE-induced differentiation of newly-born AHNPC into neuronal or glial lineages, we exposed cohorts of ficiency . More spPS1+/+ mice to EE resulted in a 1.366-fold increase, albeit insignificant, in the percentage of BrdU+/βIII tubulin+ cells compared with the SH cohort . Similarly, in comparison with the cohorts maintained in SH conditions, exposure of PS1M146V/+ or PS1M146V/M146V mice to EE conditions resulted in an increase in the percentage of BrdU+/βIII tubulin+ cells by 1.364- and 1.267-fold, respectively .Quantification of BrdU+ cells co-labeled with lineage specific markers showed no significant differences across all the genotypes under SH conditions Figure B.i-vi. EPS1+/+ mice to EE resulted in a significant, 1.474-fold increase in the percentage of BrdU+/Prox1+ cells compared to SH cohorts . In contrast, in comparison with the cohorts maintained in SH conditions, exposure of PS1M146V/+ or PS1M146V/M146V mice to EE conditions resulted in resulted in an insignificant 1.316- and 1.15-fold increase in the percentage of BrdU+/Prox1+ cells, respectively .Exposure of PS1+/+ mice to EE resulted in a significant increase by 1.62- and 1.37-fold in the percentage of BrdU+/NeuroD1+ or BrdU+/NeuN+ double labeled cells, compared with SH cohorts, respectively . On the other hand, exposure of PS1M146V/+ and PS1M146V/M146V mice to EE conditions resulted in a insignificant increase in the percentage of BrdU+/NeuroD1+ or BrdU+/NeuN+ double labeled cells .Analysis of cells colabeled with BrdU and the late neuronal markers, NeuroD1 and NeuN revealed that exposure of PS1+/+ versus PS1M146V/+ or PS1M146V/M146V groups that were exposed to EE conditions . Examination of glial lineages revealed no significant changes in the percentage of BrdU+/GFAP+ or BrdU+/s100β+ double-labeled cells between SH versus EE groups or within EE groups, across all three genotypes  has been knocked-in downstream of the fractalkine receptor Cx3Cr1 promoter [PS1+/+x Cx3Cr1GFP/+ and PS1M146V/+x Cx3Cr1GFP/+ lines were established and eGFP+ microglia fraction was isolated by flow cytometry to establish pure cultures of microglia . These results reveal that the impairments in proliferation of cultured AHNPCs observed earlier with CM from transgenic mice overexpressing FAD-linked PSEN1 variants [PSEN1 promoter.In earlier studies, we reported that both proliferation and neuronal cell fate commitment of AHNPCs from wild type mice was impaired by the addition of CM from IL-4-treated microglia obtained from transgenic mice that express PrP-driven FAD-linked PS1 mutants . To testpromoter , leadingvariants , can be PS1-/- background resulted in an increase in proliferation and decrease in survival of AHNPCs with no apparent reduction in net neuronal differentiation in animals exposed to SH conditions [PSEN1 transgenes reflects the expression patterns of endogenous PSEN1, there remain concerns regarding expression levels and regulation in specific CNS cell types.Earlier studies have shown that hippocampal neurogenesis declines with age and this reduction has been linked with impaired learning, memory, and deterioration of cognitive ability in the elderly -19. In tnditions . On the nditions . In thisnditions , neuronsnditions , cerebranditions , glia annditions ,27. ThusPSEN1 locus, and we now offer several important insights.In order to address these potential concerns, we asked whether the EE-mediated impairments on AHNPC proliferation and neuronal differentiation that we documented in transgenic mice expressing FAD-linked PS1could be validated in “knock-in” mice harboring an FAD-linked PS1 mutation in the endogenous PS1M146V/+ and PS1M146V/M146V animals, similar to our earlier findings in PrP-driven FAD-linked mutant PSEN1 transgenic mice [PS1M146V/+ and PS1M146V/M146V mice compared with wild type animals.First, we show that EE-induced proliferation of AHNPCs is impaired in both nic mice . InteresPS1M146V/+ and PS1M146V/M146V mice. The lack of obvious changes in immature neuronal lineages correlates with the observations reported earlier in FAD-linked PSEN1P264L KI mice [PS1M146V/+ and PS1M146V/M146V mice. It should be noted that we chose a two week time point for these latter analysis in order to score neuronal or glial lineage commitment of AHNPCs and their survival efficiency. The limitation of this analysis is that cells expressing mature neuronal markers may not be functionally integrated into the hippocampal network.Second, we failed to detect significant changes in early immature and late neuronal lineages  or glial lineages following EE. Instead, we observed a prominent reduction in the percentage of new born progenitors that differentiated towards mature neuronal NeuN+ and NeuroD1+ lineages in  KI mice . ConsistPS1M146V/+ and PS1M146V/M146V mice are a reflection of reductions in PS1 “activity”, but earlier studies by Wang and colleagues offer an important insight [PS1M146V/- mice, wherein the mutant allele was placed in a PSEN1 KO background, exhibited significant reductions in both proliferation and neurogenic potential of AHNPC compared with PS1M146V/+ mice [It is presently unclear whether the reduction in AHNPC proliferation and mature neuronal differentiation in  insight . In the V/+ mice . The autV/+ mice , would bV/+ mice .PS1M146V/+ and PS1M146V/M146V mice exposed to EE were significantly reduced compared with PS1+/+mice. While the molecular mechanism(s) underlying this aspect of mutant PS1 function is presently unclear, evidence from several earlier studies have accrued that support our observations [PS1M146V/M146V mice exhibit enhanced susceptibility to kianate-induced necrosis and that primary neurons from these mice showed enhanced sensitivity to glutamate-induced excitotoxicity. These results in PS1M146V/M146V mice were confirmed independently in PrP promoter-driven transgenic mice expressing the L286V variant [PS1M146V/M146V mice exhibit disrupted intracellular Ca2+ signaling in neurons [Third, we report that the survival of BrdU-labeled AHNPCs from rvations -33. For  variant . At a me neurons ). In thi neurons .PS1M146V/+ mice inhibits proliferation of AHNPCs derived from PS1+/+mice. These findings support our earlier observations using CM of microglia from mice expressing PrP-driven PSEN1 transgenes [Finally, we report that CM of microglia from ansgenes . The ideansgenes . In thisansgenes  have offansgenes , and Vilansgenes , sought ansgenes . The natPSEN1 mutation fully recapitulates the findings observed in transgenic mice expressing PrP-driven FAD-linked PSEN1 transgenes. These findings lead us to conclude that the memory deficits and cognitive decline in patients harboring PSEN1 variants is a reflection not just of an increase in the ratio of Aβ42 to Aβ40, but to impairments in the self-renewal, survival and differentiation of AHNPCs.We have documented that the EE-mediated impairments of proliferation, differentiation and survival of AHNPCs in mice with a knock-in of an FAD-linked PS1+/+, PS1M146V/M146V KI mice [Cx3Cr1GFP/GFP[PS1+/+ were crossed to PS1M146V/M146V KI mice to obtain PS1M146V/+ line. The data are reported as mean ± standard error of mean (m ± SEM). Two-way ANOVA test (SPSS ver. 12), were performed for comparisons of quantitative data while analyzing data from multiple groups on AHNPC proliferation or differentiation studies. Tuckey or LSD test were used as a post-hoc analysis. T-test (Unpaired) was performed while comparing data from two independent data sets. Values of p < 0.05 were used as the criterion for statistical significance. Animal experiments were conducted in accordance with institutional and National Institutes of Health guidelines.r1GFP/GFP were maiFor exposure to EE, cohorts of mentioned 1 month old male transgenic, wild type or PS1 M146V KI (heterozygous or homozygous) animals were housed in large cages containing running wheels, tunnels, toys, and chewable materials for 3 h a day for 1 month. Control groups of animals were maintained in standard laboratory housing conditions. Mice received a single i.p. injection of BrdU  on the last day of the enrichment. Half of the mice in each group were sacrificed 1 day after the injection to determine progenitor proliferation as described earlier . For AHNTissue preparation, and immunofluorescent labelling for BrdU, neuronal nuclei (NeuN), β-III tubulin, Prox1, NeuroD1, s100β protein and Glial Fibrillary Acidic Protein (GFAP) were performed as described previously . The ant45 μM coronal sections of the brain were cut on a dry-ice cooled sliding microtome block , serial sections spanning DG were collected in two rows of 24-well dish and stored at -20°C in a cryoprotective buffer and free floating sections were processed for immunohistochemistry or immunofluoresence as described previously , using tPS1+/+ mice as described, previously [5 viable cells/animal), were seeded in serum free culture media at a cell density of 50 cells/μl ). Cultures were maintained at 37°C in 5% CO2/balance air. Proliferating neurospheres started to appear in about 10–12 days. To obtain single cell suspensions, the neurospheres were dissociated by trituration using Neurocult chemical dissociation solution  and cultured through multiple passages. Primary cultures of mixed glia cells were prepared from hippocampus of newborn PS1+/+ or PS1M146V/+ mice according to previously described procedure [7 cells in DMEM, 10% FBS, 1% penicillin/streptomycin and 1% L-glutamine. 5 days later microglial cells were shaken off the primary mixed glial cell cultures (250 rpm for 3 hrs at 37°C). Cells were collected by centrifugation, re-suspended in PBS containing 0.2% FBS and eGFP+ microglia were enriched by flow cytometry . Purified microglial cells were plated in culture media supplemented with 5 ng/ml recombinant mouse macrophage-colony stimulating factor  and used for subsequent studies. 3 × 105 microglial cells/well in a 6 well dish were treated with 10 ng/ml of IL-4  for 24 hrs, later washed and CM was collected in SFM for 24 hrs after initial stimulation. Effect of CM on AHNPC proliferation was determined by BrdU uptake assay as described earlier [AHNPCs that give rise to neurospheres were cultured from dissected hippocampal tissue of 8 weeks old male eviously . In brierocedure . In brieThe authors declare no conflict of interest. The corresponding author (S.S.S) discloses that he is a paid Consultant of Eisai Research Labs Inc, but is not a shareholder in any company that is a maker or owner of a FDA-regulated drug or device.KV, SHC and SSS designed, performed, analyzed and wrote the paper. XZ assisted in maintaining animal colonies used in this study. All authors read and approved the final manuscript.


The broadly-neutralizing anti-HIV antibody 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen, but is inconsistent with splicing factor 3B3. These results demonstrate that 4E10 recognition of liposomes competes with MPER recognition and that HIV antigen and autoepitope recognition may be distinct enough to permit eliciting 4E10-like antibodies, evading autoimmunity through directed engineering. However, 4E10 combining site flexibility, exceptional for a highly-matured antibody, may preclude eliciting 4E10 by conventional immunization strategies. 4E10 is an example of an anti-HIV, broadly neutralizing antibody that is uncommon in infected patients and has not been successfully elicited by any vaccine approach attempted. 4E10 has been proposed to neutralize HIV through a mechanism that requires broad recognition of other antigens, including membrane phospholipids. Such a mechanism would also block the generation of 4E10 during B cell development, confounding vaccination strategies. Analysis of B cell development in 4E10 heavy-chain knock-in mice confirmed that 4E10 does recognize self-antigens. However, a previously proposed autoantigen candidate, the mitochondrial lipid cardiolipin, was not consistent with binding studies which showed that while 4E10 does bind liposomes containing cardiolipin, it does so only weakly and nonspecifically, also binding liposomes without cardiolipin. Using a synthetic human peptidome, 4E10 was shown to be polyreactive, binding peptides from various proteins, but only in a limited manner. Three of the top five hits are from types 1, 2 and 3 inositol trisphosphate receptors, with high scoring peptides sharing a conserved sequence motif. Validation of the top hits was performed by binding analyses and staining of tissue sections, which combined to identify the type 1 inositol trisphosphate receptor as the most likely 4E10 physiological autoantigen. An effective prophylactic AIDS vaccine will need to generate anti-HIV neutralizing antibodies (Abs) that target the HIV envelope glycoprotein (Env) 671NWFDITNWLW680; core epitope: NWFD/NIT), immediately adjacent to the viral membrane in the gp41 membrane proximal external region (MPER) 662ELDKWA667) neighboring the 4E10 epitope in the MPER The HIV envelope protein (Env) consists of gp120 surface subunits and gp41 membrane-anchoring subunits assembled as noncovalent trimers of gp120/gp41 heterodimers to form mature, functional ‘spikes’ on the virion surface. 4E10 recognizes a conserved linear epitope  staining Polyspecificity is the property of recognizing multiple distinct ligands (recognition degeneracy) combined with the specificity with which each of those ligands is recognized In vivo experiments demonstrating functional 4E10 autoreactivity had not been reported when we started these studies. 2F5 and 4E10 were originally concluded to be polyspecific and autoreactive on the basis of binding assays against 11 purified lipidic and nuclear autoantigens KD) in the low micromolar range in one study KDversus ∼2% of uninfected controls) and are strongly linked with the level of viral replication, the level of B cell activation and the level of MPER-specific Abs Abs against phospholipids, including ACLA, are seen in primary antiphospholipid syndrome (APS) or in association with other autoimmune diseases, such systemic lupus erythematosus (SLE). ACLA in this context generally do not recognize CL in isolation, but as complexes with the phospholipid-binding serum protein β2 glycoprotein I (β2GPI) or other proteins with anti-coagulant activity. The presence of ACLA in these conditions can provoke serious complications, including arterial and venous thromboses and recurrent miscarriages. A second class of antiphospholipid antibodies can be generated after certain viral and bacterial infections, such as HIV, hepatitis B and C, syphilis and leprosy. ACLA are found in approximately 50% of people infected with HIV  on murine tissue sections. We determined that 4E10 does not stain mitochondria in HEp-2 cells bona fide 4E10 autoantigens using a synthetic library of 413,611 36-mer peptides spanning the human proteome combined with phage immunoprecipitation sequencing (PhIP-Seq) in vitro binding studies and consistent with IHC results. The PhIP-Seq results were also useful for more finely defining 4E10 polyspecificity beyond previous studies, leading to the conclusion that 4E10 displays limited, highly focused polyspecificity, unexceptional for this class of molecules. Our findings should inform future attempts to generate 4E10-equivalent functionalities by vaccination and determine the precise molecular mechanism of 4E10 neutralization of HIV.Because 4E10 was clearly autoreactive in 4E10 knock-in mice, we sought to identify candidates for +/+ knock-in mice was evaluated by flow cytometry and compared with both wild-type B6 and control KL25H+/+ knock-in mice  locus in B6 embryonic stem cells. B cell development in the derived 4E10H-in mice Fig. 1A.+ B cells could still be found in the spleens and lymph nodes of 4E10H knock-in mice, suggesting that some 4E10H-expressing B cells had escaped deletion during development. To determine whether putative autoreactivity had impaired the function of 4E10H-expressing B cells, we tested the ability of these cells to respond to mitogenic and antigen receptor stimuli in vitro. Splenocytes were purified from 4E10H+/+, KL25H+/+, and WT B6 mice, loaded with cell proliferation dye eFlour670, and cultured overnight in the presence of LPS, CpG DNA, an activating anti-CD40 antibody, anti-IgM F(ab′)2, or in culture media alone (no stimulus). The following day, B cell proliferation was assessed by flow cytometric measurement of dye dilution in B220+ cells  the external leaflet of cellular membranes (PC/cholesterol), (ii) membranes containing CL (PC/cholesterol/CL) or (iii) the HIV viral envelope , confirming the weakness of these interactions in absolute terms.These combined improvements result in SPR sensorgrams that show cleanly negative results for the binding of Abs and Annexin V to control liposomes (PC/cholesterol) but clear, reproducible, positive results for Annexin V binding to liposomes incorporating CL or PS Fig. 2. It had been hypothesized that the HCDR3 of 4E10 makes direct hydrophobic contacts with the viral membrane, facilitating binding through aromatic and apolar amino acid side-chains, particularly W100H and W100bH e. g. CL) predominately through electrostatic interactions, not hydrophobic ones.Biomolecular interactions are mediated by hydrophobic, electrostatic, van der Waals and hydrogen bonds. The widely-held presumption has been that 4E10 binds lipids, liposomes and membranes through predominately hydrophobic interactions mediated by HCDR3 and other combining site residues. In order to determine the overall character of 4E10/liposome binding, SPR analyses were conducted at a series of salt concentrations; increasing the salt concentration drives hydrophobic interactions but ablates electrostatic ones, which is the fundamental principle behind ion exchange and hydrophobic interaction chromatography. SPR analyses showed that 4E10 IgG interactions with both CL-containing and virus-like liposomes were abolished by increasing the salt concentration Fig. 4A,It had previously been suggested that 4E10 contains a phosphate-binding subsite 2SO4 (dmin = 2.4 Å), preliminary phases were determined by molecular replacement, and the structure was rebuilt and refined with good agreement statistics and geometry  or alternate proposed HIV epitopes 10P-value equal to 14.3 and with approximately two dozen peptides rising above the main cluster . The chemical character of these 17 4E10 peptides was distinct, as well: overall moderately hydrophobic or hydrophobic (though none were very hydrophobic), with a range of predicted net charges at neutral pH from −5.7 to +1.9 and greater sequence complexity. None of the 17 high-scoring peptides had sequences related to HIV, as expected, but we were able to identify a conserved motif. Three of the five top-scoring peptides, including the highest scoring peptide, are derived from an ER-resident inositol trisphosphate receptor  within these three peptides quite unrelated to the 4E10 MPER epitope ; b12 library average = 0.33 (σ = 0.45)). The close concordance in library scoring behavior also argues against the presence of very broad, low-avidity binding by 4E10 relative to b12.The 4E10 PhIP-Seq results were distinct from the b12 results 8. Usingdata not shown). The constructs incorporated various purification and epitope-tags, including a C-terminal Strep-tag data not shown) and SPR analyses with an anti-Strep-tag Ab confirmed the presence of full length peptides, with specific activities assayed by SPR of 12% to 34% as coupled, which allowed for normalizing results between SPR experiments  and appropriate secondary Abs. The commercial anti-IP3R1 polyclonal was raised against a synthetic peptide corresponding to C-terminal residues of human IP3R1, cross-reacts with mouse and rat orthologs and strongly stains Purkinje cells per the manufacturer's specifications (LifeSpan Biosciences). 4E10 strongly stained specific nodular substructures in a subset of neurons, identified as Purkinje cells on the basis of morphology, with minimal background staining of other cell types or substructures in these sections  observed when binding to Env proteins We conclude that 4E10 is not specific for CL; rather, 4E10 weakly and non-specifically interacts with phospholipid headgroups or liposomes carrying a net negative charge primarily through electrostatic interactions associated with an electropositive pocket on the unbound conformer of 4E10. As displayed by the absence of binding to control liposomes, 4E10 does not show a general proclivity to indiscriminately bind liposomes, consistent with this conclusion. Such weak, non-specific, electrostatic binding of phospholipids is unlikely to be relevant to B cell tolerance induction nor is likely a requirement for neutralization, consistent with prior studies that showed reducing liposome affinity did not ablate neutralization vs. hydrophilic, aromatic vs. charged vs. zwitterionic), the total conformer space also likely encompasses many peptide mimetics of non-protein species. A caveat with this approach is the difficulty in identifying discontinuous epitopes spanning more than one peptide, though linear segments of discontinuous epitopes may retain detectable binding. However, epitopes inaccessible in folded proteins, but which are exposed by proteolytic cleavage or other antigen processing events during B cell selection, can potentially be identified.Discarding CL from consideration, we utilized an unbiased proteomic approach to identify novel autoantigen candidates and to more fully characterize the polyreactivity of 4E10. A synthetic representation of the complete human proteome, composed of overlapping 36 amino acid peptides, was displayed on T7 phage and coupled with parallel DNA sequencing (PhIP-Seq) 241 peptides were culled from the analysis because of uniform binding across a larger array of distinct Abs, but additional peptides retained in the analysis showed strong binding to more than one Ab in broader analyses not reported here, so binding results do not necessarily reflect just the specificity or polyspecificity of the Ab tested, but may also reflect the polyspecificity of a peptide. b12, an Ab that is presumably not autoreactive or unusually polyreactive, was analyzed to provide a baseline for PhIP-Seq results. The dozen top-scoring b12 peptides rising above the bulk response Fig. 8 s−15P-value threshold set by b12, the top five with P-values ranging from 10−58 to 10−255, consistent with functional autoreactivity and limited, focused polyspecificity quite distinct from b12. There is no evidence that 4E10 displays an exceptional, low-avidity polyreactivity from these results, especially in comparison with b12. The 17 top-scoring 4E10 peptides, constituting novel candidate autoantigens, were moderately hydrophobic to hydrophobic in nature overall, with a range of net charge, consistent with ordered parts of folded proteins. The top-scoring 4E10 peptide is derived from IP3R2, with additional hits from two related IP3R types (IP3R1 and IP3R3) within the top-scoring five peptides , 12 (ras-GEF domain-containing family member 1A), 15 (UBX domain-containing protein 6 isoform 1) and 16 , are from proteins represented on the ProtoArray 5, potentially accounting for the discordance in results between these two approaches. Scoring in PhIP-Seq also reflects internally-controlled peptide binding and not binding relative to a reference antibody, as in 4E10, on the other hand, showed a strikingly different result 8, with 3R were identified as top-scoring PhIP-Seq hits and validated by SPR. In neuronal cell lines, IP3R1 and IP3R3 are expressed predominantly in the ER and perinuclear regions, while IP3R2 is expressed primarily in the nucleus 3R3 shows an evenly diffuse cytoplasmic staining pattern, but the IP3R1 cytoplasmic pattern is more punctate, with strongly-staining nodular structures apparent 3Rs) prevented comparative immunofluorescence analyses. We therefore sought to determine if the PhIP-Seq results identifying IP3R1, IP3R2 and IP3R3 as top candidate autoantigens were consistent with 4E10 recognition in a tissue-specific context by IHC. 4E10 staining of mouse cerebellar sections showed perinuclear staining of nodular structures specifically in Purkinje neurons, mirroring the staining of an anti-IP3R1 control polyclonal antibody. The 4E10 staining pattern observed was most consistent in terms of cell specificity with IP3R1 IHC patterns reported in previous studies 3R1 is the lead candidate for a 4E10 autoantigen. The 4E10 staining pattern observed was also inconsistent with recognition of SF3B3. SF3B3, together with splicing factor 3a and a 12S RNA, forms the U2 small nuclear ribonucleoprotein complex (U2 snRNP) which is exclusively localized to the nucleus and widely-expressed across cell types Three types of IPe. g. Abs CR6261, F10, FI6 and C179) often interact with a hydrophobic groove in the membrane proximal stalk region of hemagglutinin (HA) delineated by helix A and HA2 through hydrophobic HCDR residues Confirmation that 4E10 is autoreactive helps to identify the reasons vaccination strategies have failed, as tolerance mechanisms would limit the generation of anti-HIV Abs equivalent to 4E10. Our findings have important implications for future strategies to induce MPER-specific bNAbs. While lipid cross-reactivity mediated by HCDR3 had been suggested to play a role both in inducing B cell tolerance and in increasing neutralization potency through interactions with viral membranes, mutual functionalities precluding an engineered solution, our results alternately suggest that viral membrane binding is so weak and nonspecific, and competes with HIV epitope binding, that it is unlikely to be involved in the induction of tolerance mechanisms or to play a key role in neutralization. Weak but detectable binding of HCDR3 ubiquitin fusions to gp140s suggests that HCDR3 may make interactions to Env, rather than membrane, outside of the core 4E10 epitope. Neutralizing anti-influenza Abs . 4E10H knock-in B6 ES cells were microinjected into FVB strain blastocysts, which were implanted into pseudo-pregnant female mice. Chimeric knock-in pups were identified by coat color and PCR genotyping, and crossed with B6 mice to generate pure B6 strain 4E10H knock-in mice. Germline transmission of the targeted 4E10H allele to progeny was confirmed by PCR genotyping (data not shown). Knock-in mice were inter-bred to establish homozygosity of the knock-in allele as assessed by PCR genotyping (data not shown). B6 background TgH(KL25) knock-in mice (referred to here as KL25H mice), expressing the mouse LCMV-neutralizing antibody KL25 rearranged IgH VDJ antigen-binding domain from the mouse IgH locus 4E10H targeted transgenic (knock-in) mice were generated by targeted insertion of a DNA construct encoding a mouse immunoglobulin heavy chain (IgH) promoter upstream of the rearranged Ab 4E10 IgH VDJ antigen-binding domain plus an mRNA splice donor sequence. The transgene was targeted to the mouse IgH genomic locus upstream of the immunoglobulin constant domains utilizing homologous recombination in C57BL/6 (B6) strain mouse embryonic stem (ES) cells. Transgene targeting was confirmed in ES cell clones by Southern blot , and analyzed on a FACS Canto II flow cytometer  using a syringe and 18 g needle. Red blood cells were lysed with ACK lysing buffer (Gibco) and live cells were purified by density gradient centrifugation using Histopaque 1119 (Sigma-Aldrich). 5×106 cells per well were incubated overnight in round-bottom 96-well plates with or without B cell stimuli 2) in B cell media, in a 37°C CO2 cell culture incubator. The following day, cells were stained for cell surface markers (B220 and Thy1.2) and B cell proliferation was assessed by flow cytometry . Purified cells were loaded with cell proliferation dye eFluor670 (eBioscience) according to the manufacturer's instructions. 1×10ytometry Fig. 1B.The activities involving the use of vertebrate animals described in herein have been reviewed and approved by the Fred Hutchinson Cancer Research Center (FHCRC) Institutional Animal Care and Use Committee (IACUC), file #1695. FHCRC has an approved Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (OLAW), Assurance number A3226-01.Lipids were purchased from Avanti Polar Lipids: bovine heart CL, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), egg L-α-phosphatidylcholine or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine (PS), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), ovine wool cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-biotinyl (biotinylated PE). Control liposomes (4∶1 w/w PC∶cholesterol), CL liposomes (1∶7∶2 w/w CL∶PC∶cholesterol), and viral mimic liposomes (3.2∶5.4∶5.1∶4.8∶10.2 w/w cholesterol∶PC∶SM∶PE∶PS) 6-purification tags were engineered, cytoplasmically expressed in E. coli BL21(DE3) RIL cells (Invitrogen), and purified employing the methodology described in data not shown).HCDR3-grafted Ubq fusion constructs Fig. 3 w2, all at 300 nM . Liposome experiments 4 were r2SO4 buffered with 0.1 M NaOAc (pH = 4.6). Crystals grew within one week and were cryopreserved in a mother liquor of well solution plus 15% v/v glycerol. Diffraction data were collected with both CuKα radiation on an in-house R-AXIS IV++ image plate detector with HR optics (Rigaku) at −170°C and at the Advanced Light Source beamline 5.0.2  and reduced using HKL-2000 4 placement and the initial correctness of the molecular replacement solution. Crystallographic statistics are reported in Crystals of ligand-free 4E10 Fv 4, pH 7.4. Beads were rocked at ambient for 24 hrs with 60 µg of either 4E10 or b12 IgG in 1 M (NH4)2SO4 and then washed with 10 mM glycine in PBS to cap unreacted epoxy groups. Activity of 4E10 and b12 beads was confirmed by epitope-scaffold 10P-values.For each of the two Abs analyzed, 3 mg of magnetic beads (Invitrogen Dynabeads M-270 Epoxy) were resuspended in 60 µL 0.1 M NaPO3R1 staining: a protein block step consisting of 0.05M Tris, 0.15M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6 (TCT Buffer) was performed for 10 minutes. Tissues were then stained with polyclonal rabbit anti-human IP3R1 IgG (LifeSpan Biosciences) at a dilution of 1∶200 (2.5 µg/mL) or rabbit IgG matched to the protein concentration of the primary Ab for 60 minutes. Mach 2 Rabbit HRP-polymer  was applied for 30 minutes. 4E10 staining: avidin block  was applied for 10 minutes, followed by biotin block  for 10 minutes. Next, a protein block step (TCT Buffer plus 15% goat serum) was performed for 10 minutes. Tissues were then stained with 4E10 IgG (Polymun AB004) at a dilution of 1∶100 (10 µg/mL) or normal human serum matched to the protein concentration of the primary Ab for 60 minutes. The Fab of a biotinylated goat anti-human secondary Ab (Jackson Laboratory 109-067-993) was applied for 30 minutes followed by incubation with streptavidin-HRP (Jackson Laboratory 016-030-084) at a dilution of 1∶2000 for 30 minutes. Finally, a DAB+ substrate-chromogen incubation was done (Dako K3468) for two applications of 4 min each. The slides were counterstained with Automation Hematoxylin (Dako S3301) for 2 minutes and then coverslipped.Four micron sections of formalin-fixed and paraffin-embedded tissues were cut and baked for 1 hr at 60°C, deparaffinized with xylene and rehydrated in a series of graded ethanol solutions  followed by distilled water. Antigen retrieval in pH 6.0 Citrate Buffer  was then performed for 5 min in a pressure cooker. All of the following incubations were performed at room temperature on a Dako Autostainer Plus. Wash buffer  was used between all subsequent steps except between the protein block step and application of the primary Ab. A 3% v/v hydrogen peroxide block step was done for 8 min. IP


C1QTNF6 (rs229541) predicted increased IA risk  but not progression to T1D . SNP (rs10517086) appears to exhibit an age-related effect on risk of IA, with increased risk before age 2 years  but not older ages . C1QTNF6 (rs229541), SNP (rs10517086), and UBASH3A (rs3788013) were associated with development of T1D. This prospective investigation of non-HLA T1D candidate loci shows that some SNPs may exhibit stage- and age-related heterogeneity in the etiology of T1D.Previously, we examined 20 non-HLA SNPs for association with islet autoimmunity (IA) and/or progression to type 1 diabetes (T1D). Our objective was to investigate fourteen additional non-HLA T1D candidate SNPs for stage- and age-related heterogeneity in the etiology of T1D. Of 1634 non-Hispanic white DAISY children genotyped, 132 developed IA ; 50 IA positive children progressed to T1D. Cox regression was used to analyze risk of IA and progression to T1D in IA positive children. Restricted cubic splines were used to model SNPs when there was evidence that risk was not constant with age.  Type 1 diabetes (T1D) is a chronic autoimmune disease in which the insulin-producing beta cells of the pancreas are destroyed. There is typically a preclinical phase of circulating autoantibodies, called islet autoimmunity (IA), which precedes the clinical diagnosis of T1D. T1D is widely believed to be caused by an environmental factor on a susceptible genetic background. The major susceptibility locus for T1D maps to the HLA class II genes at chromosome 6p21. These HLA class II alleles account for 30–50% of the familial clustering of T1D [INS [CTLA4 [PTPN22 [IL2RA [IFIH1 [PTPN22, UBASH3A, INS, and IFIH1 associated with IA and/or progression to T1D [More than 50 non-HLA T1D susceptibility gene markers have been confirmed. The major non-HLA loci include INS , CTLA4 [S [CTLA4 , PTPN22 A [IFIH1 . The DAIn to T1D –11.VDR) and protein tyrosine phosphatase, nonreceptor type 2 gene (PTPN2) with progression to T1D in IA positive children, but not with development of IA [Prospective birth cohorts have the unique ability to study two stages in the natural history of T1D: development of IA and progression to T1D in IA positive children. Different exposures have been associated with one or both stages. For instance, DAISY recently identified an association between a gene-gene interaction involving the vitamin D receptor gene (nt of IA . This wont of IA . The purn = 815). The second group consists of infants born at St. Joseph's Hospital in Denver, CO, whose umbilical cord blood was screened for diabetes-susceptibility HLA-DR, DQ genotypes (n = 819). Details of the newborn screening, sibling and offspring recruitment, and followup of both cohorts have been published previously [The Diabetes Autoimmunity Study in the Young (DAISY) is a prospective study composed of two groups of children at increased risk for T1D who were recruited between 1993 and 2004 and are being followed prospectively for the development of IA and T1D. One group is made up of first degree relatives of patients with T1D, identified and recruited between birth and eight years of age, mainly through the Barbara Davis Center for Childhood Diabetes , as previously described –19, withCases of IA were defined as those children positive for at least one islet autoantibody  on two or more consecutive visits. T1D was diagnosed by a physician and defined as random blood glucose >200 mg/dL and/or HbA1c (A1C) >6.5% with clinical symptoms of T1D.C1QTNF6 (rs229541), C6orf173 (rs9388489), C14orf181 (rs1465788), IL2 (rs2069762), IL2 (rs4505848), IL2RA (rs12722563), IL2RA (rs2104286), IL7R (rs6897932), PRKCQ (rs947474), SKAP2 (rs7804356), SMARCE1 (rs7221109), TLR8 (rs5979785), UBASH3A (rs3788013), and SNP (rs10517086). Thirteen of the fourteen SNPs were chosen from three GWAS meta-analyses [UBASH3A (rs3788013) was chosen based on its strong LD with UBASH3A (rs876498), which was discovered for its association with T1D by Concannon et al. [DAISY children were genotyped for fourteen non-HLA T1D candidate SNPs: ), IL2 rs69762, IL1QTNF6 rs9541, C6on et al. .The SNPs were genotyped utilizing the Taqman SNP genotype based OpenArray platform . Custom designed 48-sample arrays and normalized genomic DNA were loaded using the OpenArray AccuFill system and cycling was performed on a GeneAmp 9700 PCR system , all according to manufacturer protocol. Alleles were analyzed using the OpenArray SNP genotyping analysis software v.1.0.3 and Taqman Genotyper Software 2.0 . All fourteen SNPs had a 95% call rate or higher.χ2 goodness-of-fit test with a P value of 0.05 considered as evidence of a departure from Hardy-Weinberg equilibrium; all fourteen SNPs were in Hardy-Weinberg equilibrium. Linkage disequilibrium (LD) was tested in our population using Haploview version 4.2 as measured by r2 and D', with r2 = 0.257 and D' = 0.862 for the two IL2RA SNPs and r2 = 0.222 and D' = 1.0 for the two IL2 SNPs.Each SNP was tested for consistency with Hardy-Weinberg proportions using a 1-degree of freedom We obtained genetic data on at least one of the fourteen non-HLA T1D candidate SNPs for 1634 non-Hispanic white children in the DAISY cohort. This included 132 children who developed IA, of whom 50 went on to develop T1D. Fifteen IA cases were positive for autoantibodies on their first clinic visits; these left-censored cases were removed from the development of IA analysis cohort but retained in the progression from IA to T1D cohort. The same 50 IA positive children who went on to develop T1D are the same 50 T1D cases in the development of T1D analyses. However, not all IA positive children went on to develop T1D. All statistical analyses were limited to non-Hispanic whites in the DAISY cohort.P value < 0.20 indicating possible departure from proportional hazards [TLR8 (rs5979785) was additionally adjusted for sex because it is on the X chromosome. The significance threshold was defined as α = 0.05. Because our analyses were based on a priori hypotheses with SNPs previously found to be associated with T1D, P values were not corrected for multiple testing. We analyzed each non-HLA SNP in separate, covariate adjusted models. In the analyses examining development of IA, all of the SNPs were treated additively, except IL2RA (rs12722563) and PRKCQ (rs947474), which were dichotomized on the minor allele due to small sample sizes. Additionally, in the progression to T1D analyses, SNPs were treated additively, except C14orf181 (rs1465788), IL2RA (rs12722563), IL2RA (rs2104286), IL7R (rs6897932), PRKCQ (rs947474), SKAP2 (rs7804356), and SNP (rs10517086), which were dichotomized on the minor allele due to small sample sizes.SAS version 9.3  statistical software package was used for all statistical analyses. SNPs were tested for violation of the proportional hazards assumption using a supremum test, with a  hazards . If a SN hazards . AnalyseWe first examined whether non-HLA variants were associated with development of IA. The mean age at first IA positive visit was 6.2 years, and the mean age at last follow-up visit in children who did not develop IA was 9.9 years . IA posihttp://dx.doi.org/10.1155/2013/417657). Adjusting for HLA-DR3/4, DQB1*0302 and first degree relative with T1D, C1QTNF6 (rs229541) was associated with development of IA ) (P = 0.003).Unadjusted SNP association analyses are presented in ) . SNP ) . ChildreP = 0.003). Children with IA who developed T1D were more likely to have the HLA-DR3/4, DQB1*0302 genotype compared to children with IA who did not progress to T1D . Adjusting for HLA-DR3/4, DQB1*0302, first degree relative with T1D, and age at first IA positive visit, none of the fourteen non-HLA T1D candidate SNPs was associated with progression to T1D in IA positive children  and SNP (rs10517086), were also associated with development of IA, but not with progression to T1D in IA positive children. The other SNP associated with development of T1D, UBASH3A (rs3788013), was not associated with development of IA nor progression to T1D in IA positive children.In order to evaluate the same outcome as previous GWAS to see if similar associations could be seen using a time-to-event analysis in a prospective birth cohort and to better understand the role these SNPs play in the natural history of T1D, we examined whether these non-HLA variants were associated with T1D in our population of 1619 children. All 50 children who developed T1D had developed IA previously, so the same 50 T1D cases were included in both the progression from IA to T1D (presented in C1QTNF6 (rs229541) predicts IA but not progression to T1D, demonstrating stage-related heterogeneity. Moreover, SNP (rs10517086) demonstrates age-related heterogeneity with predicting IA only in the youngest ages. These two SNPs were also associated with development of T1D in our cohort, as well as UBASH3A (rs3788013). It is possible that the observed associations between both C1QTNF6 (rs229541) and SNP (rs10517086) and development of T1D were driven by their association with development of IA. Given that all of our T1D cases developed IA prior to clinical diagnosis, it was not possible to determine whether a gene was associated with T1D via a pathway other than IA.In exploring associations between fourteen previously discovered non-HLA T1D candidate SNPs and the development of IA and progression to T1D in the prospective DAISY cohort, we found that C1QTNF6 (rs229541), which was first identified through a meta-analysis of data from three genome-wide association studies (GWAS)  [C1QTNF6 (C1q and tumor necrosis factor related protein 6) and SSTR3 (somatostatin receptor 3). We found that C1QTNF6 (rs229541) is associated with development of IA but not associated with progression to T1D in IA positive children, suggesting that this gene may play a role early in the development of T1D related to the initial appearance of autoimmunity. × 10−8) , is an iSNP (rs10517086) exhibits an age-related effect with development of IA, with an increased risk of developing IA before the age of two or in younger ages, and a null effect in older ages. Children carrying a risk allele for SNP (rs10517086) developed IA significantly earlier than children without a risk allele. Based on these epidemiologic analyses, future studies should investigate mechanisms as to how this SNP influences risk of early autoimmunity. SNP (rs10517086), which was first discovered through another GWAS and meta-analysis of T1D, is located within a gene desert on chromosome 4. Loci in or near genes without a known function or in regions not containing annotated genes may indicate involvement of long-range gene expression regulatory elements and/or nonprotein-coding RNA genes .UBASH3A (rs3788013) was associated with development of T1D, but not with either of the stages preceding this (development of IA and progression from IA to T1D). UBASH3A (rs3788013) is an intronic SNP located on chromosome 21q22 in the UBASH3A (ubiquitin associated and SH3 domain containing A) gene. UBASH3A is expressed predominantly in T cells suppressing T-cell receptor signaling [UBASH3A has also been associated with other autoimmune diseases, such as celiac disease and rheumatoid arthritis [UBASH3A SNP, UBASH3A (rs11203203), was found to be associated with both development of IA and development of T1D in a previous DAISY analysis [UBASH3A (rs3788013) and UBASH3A (rs11203203) is r2 = 0.491 and D' = 0.801. DAISY uses two definitions of IA, one that defines IA as the presence of at least one islet autoantibody on two consecutive visits  and the other that further requires that the children still be autoantibody positive or diabetic on their most recent visit . The definition used in the previous study is closer to T1D, which makes our UBASH3A (rs3788013) association with T1D consistent with what was previously found with UBASH3A (rs11203203).ignaling . UBASH3Arthritis . AnotherBASH3A rs203203, wPTPN22 (rs2476601) associated with development of IA, but not progression from IA to T1D, and CTLA4 (rs231775) associated with progression from IA to T1D, but not development of IA [PTPN2 (rs1893217) was only associated with development of IA, while UBASH3A (rs11203203) was associated with development of IA and development of T1D [INS (rs689) was not associated with development of IA nor progression from IA to T1D but was associated with development of T1D [PTPN22 (rs2476601) was also associated with development of T1D [C1QTNF6 (rs229541) associated with development of IA and development of T1D, but not progression from IA to T1D. UBASH3A (rs3788013) was associated with development of T1D, but not with either of the stages preceding this (development of IA and progression from IA to T1D) and SNP (rs10517086) was associated with development of T1D, while exhibiting an age-related effect with IA risk but was not associated with progression from IA to T1D. The SNPs investigated in these three studies are summarized in triggering islet autoimmunity versus epitope spreading and progressive loss of beta-cell mass leading to overt diabetes.In combination with those presented in this paper, DAISY has now investigated 34 non-HLA T1D candidate SNPs for association with development of IA, progression from IA to T1D, and/or development of T1D with multiple examples of stage-related heterogeneity, which are presented in nt of IA . PTPN2  [TCF7 (rs5742913) [Due to the cost associated with following a large group of children from birth into adulthood, our sample sizes are much smaller than those obtained for GWAS. Our lack of association for many of these SNPs is not evidence against their association with T1D but may result from limited power, especially in the progression from IA to T1D stage. We also have a very unique population comprised of children with high risk HLA genotypes and it is possible that the effect of these SNPs differs based on one's HLA risk status. The risk for non-HLA loci appears to be lower in individuals carrying high-risk HLA genotypes, as has been seen with 2476601) , 29 and 5742913) , 31.We believe that taking these GWAS identified candidate regions and studying them in the context of the natural history of T1D are central to better understanding the disease process and where in the disease process genetic loci may be important. This will allow us to create more accurate risk prediction models for both stages in the natural history of the disease, as well as inform the design of targeted interventions to prevent or slow the progression of IA and subsequent development of T1D.The effect of a SNP may act nonlinearly, with an effect at early ages but not later ages or vice versa. Our results provide evidence that SNP (rs10517086) is acting on early risk of IA, with the age at onset of IA occurring significantly earlier in children with a minor allele compared to children with no minor alleles. By ignoring heterogeneity in the etiology of disease, valuable associations may be missed that could aid in better understanding complex diseases, such as T1D.Unadjusted SNP association analyses are presented in Supplemental Table 1. In the unadjusted SNP analyses, C1QTNF6 (rs229541) was associated with development of IA. None of the SNPs were associated with progression to T1D in IA positive children. Four SNPs, C1QTNF6 (rs229541), IL2RA (rs12722563), UBASH3A (rs3788013), and SNP (rs10517086) were associated with development of T1D.Click here for additional data file.


Implication of pharmacogenetic variations and efavirenz pharmacokinetics in concomitant efavirenz based antiviral therapy and anti-tubercular drug induced liver injury (DILI) has not been yet studied. We performed a prospective case-control association study to identify the incidence, pharmacogenetic, pharmacokinetic and biochemical predictors for anti-tubercular and antiretroviral drugs induced liver injury (DILI) in HIV and tuberculosis (TB) co-infected patients.NAT2, CYP2B6, CYP3A5, ABCB1, UGT2B7 and SLCO1B1 genes were done. The incidence of DILI and identification of predictors was evaluated using survival analysis and the Cox Proportional Hazards Model. The incidence of DILI was 30.0%, or 14.5 per 1000 person-week, and that of severe was 18.4%, or 7.49 per 1000 person-week. A statistically significant association of DILI with being of the female sex (p = 0.001), higher plasma efavirenz level (p = 0.009), efavirenz/8-hydroxyefavirenz ratio (p = 0.036), baseline AST (p = 0.022), ALT (p = 0.014), lower hemoglobin (p = 0.008), and serum albumin (p = 0.007), NAT2 slow-acetylator genotype (p = 0.039) and ABCB1 3435TT genotype (p = 0.001).Newly diagnosed treatment naïve TB-HIV co-infected patients (n = 353) were enrolled to receive efavirenz based ART and rifampicin based anti-TB therapy, and assessed clinically and biochemically for DILI up to 56 weeks. Quantification of plasma efavirenz and 8-hydroxyefaviernz levels and genotyping for NAT2, CYP2B6 and ABCB1 genes determines susceptibility to DILI in TB-HIV co-infected patients. Close monitoring of plasma efavirenz level and liver enzymes during early therapy and/or genotyping practice in HIV clinics is recommended for early identification of patients at risk of DILI.We report high incidence of anti-tubercular and antiretroviral DILI in Ethiopian patients. Between patient variability in systemic efavirenz exposure and pharmacogenetic variations in  Tuberculosis (TB) is the most common opportunistic fatal infection in HIV infected patients Among the first-line TB drugs pyrazinamide, isoniazid and rifampicin have all been associated with DILI CYP2B6, to a minor extent by CYP3AUGT2B7ABCB1 genetic variation on efavirenz pharmacokinetics OATP1B1, coded by SLCO1B1, is a liver-specific uptake transporter important in hepatic drug disposition. RIF is both substrate and inhibitor of OATP1B1CYP2B6, CYP3A4, UGT2B7, ABCB1 and SLCO1B1CYP3AMetabolic pathway of efavirenz is complex as being a substrate inducer and/or inhibitor of its own metabolism involving several CYP enzymes with varying activities CYP2B6 and CYP3A5th in the WHO high TB-burden country list. Biochemical variables, efavirenz pharmacokinetics as well as pharmacogenetic variations in six candidate genes relevant for metabolism and transport of ARV and anti-TB drugs; namely; CYP2B6, CYP3A5, UGT2B7, NAT2, ABCB1 and SLCO1B1 (OATP1B1) were investigated.Plasma concentrations of HIV and TB drugs display wide inter-individual variability partly due to genetic variations in the respective drug metabolizing enzymes or transporter proteins. Genetic variation is believed to play an important role in DILI The study protocol was approved by the Regional Ethical Review Board in Stockholm at Karolinska Institutet, Sweden; Institutional Review Board (Faculty. Research and Publication Committee) at Faculty of Medicine, Addis Ababa University; The National Ethics Review Committee at Ethiopian Science and technology Ministry as well as by the Food and Drug Administration and Control Authority of Ethiopia. Written informed consent was obtained from each subject before the start of this study.Newly diagnosed ART and anti-TB treatment naïve adult TB and HIV co-infected patients (n = 373) were recruited and enrolled prospectively and followed up to one year during June 2007 to January 2011. The eligibility criteria were age ≥18 years, CD4 count <200 cells/UL, not pregnant and not on other known hepatotoxic drugs concurrently . None of the participants received isoniazid prophylaxis and treatment for tuberculosis two years before enrolment and during the study period.st, 2nd, 4th, 8th, 12th, 24th, 48th, and 56th weeks after initiation of anti-TB treatment.All study participants received RIF based short-course chemotherapy for TB following the national TB treatment guideline. ART was then initiated with 600 mg efavirenz based HAART containing stavudine/lamivudine/efavirenz (D4T/3TC/EFV) or zidovudine/lamivudine/efavirenz (AZT/3TC/EFV) or tenofovir/lamivudine/efavirenz (TDF/3TC/EFV). A complete history and physical examination were taken before enrolment and at the scheduled and unscheduled visits. Laboratory tests were performed before anti TB initiation included complete and differential blood counts, platelet count, CD4 count, HIV RNA determination, hepatitis B surface antigen, anti-hepatitis C antibody, serum albumin, renal function tests, liver tests including; aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and direct and total bilirubin. Follow-up for liver enzymes were performed at before and on the 1Blood sample was obtained from 201 patients for genotype analysis. Genomic DNA was isolated from peripheral blood leukocytes using QIAamp DNA Maxi Kit (QIAGEN GmbH. Hilden. Germany). Genotyping was carried out at the division of clinical pharmacology, Department of laboratory medicine. Karolinska University Hospital-Huddinge, Karolinska Institute Stockholm, Sweden.ABCB1 3435C>T rs1045642, C__11711730_20 for CYP2B6 516G>T rs3745274 [CYP2B6*6] , C__30720663_20 for UGT2B7-372G>A rs7662029 , C__26201809_30 for CYP3A5 6986A>G rs776746 [CYP3A5*3], C__30203950_10 for CYP3A5 14690G>A g.14690G>A [CYP3A5*6], C__32287188_10 for CYP3A5 g.27131_27132insT rs241303343 [CYP3A5*7], C___1901697_20 for SLCO1B1 388A>G rs2306283 (*1b) and C__30633906_10 for SLCO1B1 521T>C rs4149056 (*5) on ABI 7500 FAST . The final volume for each reaction was 10 µl, consisting of 2× TaqMan Universal PCR Master Mix® (Applied Biosystems), 20× drug metabolising genotype assay mix and 10 ng genomic DNA. The PCR profile consisted of an initial step at 50°C for 2 min and 50 cycles with 95°C for 10 minutes and 92°C for 15 sec.Allelic discrimination reactions were performed using TaqMan®  genotyping assays: (C__7586657_20 for 5′- GTCACACGAGGAAATCAAATGC-3′) and a reverse primer (5′-GTTTTCTAGCATGAATCACTCTGC-3′) as described previously.5′-GGATGAAAGTATTTGATGTTTAGG-3′). Sequencing reaction was done using the ABI PRISM™ BigDye® terminator cycle sequencing ready reaction kit v3.1 , and analyzed on an ABI Prism 377 DNA sequencer. NAT2 sequence chromatograms were visually inspected and analyzed using software program FinchTV version.1.4.0 (htpp://www.geospiza.com) and aligned with the NAT2 reference sequence  for SNPs identification.The coding regions of NAT2 gene was amplified by PCR using a forward primer  from 212 patients, centrifuged (1700 g for 20 min), and 2 mL plasma aliquot was stored at −80°C for determination of efavirenz and its metabolite concentrations. Plasma samples were sent in dry ice to the Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg. Germany. Plasma efavirenz and 8-hydroxyefavirenz concentration were determined by LC/MS/MS as described previously 2>0.99. Accuracy and precision (within-batch and batch-to-batch) of the assay fulfilled all recommendations of FDA guidelines.On the 4Patients with DILI were identified according to the CIOMS  criteria, which are based on selected laboratory liver parameters (CIOMS laboratory criteria and the exclusion of any disease-related causes of liver injury) Baseline demographic and laboratory parameters were described as median and interquartile range (IQR) for continuous variables and as percentages for categorical variables. Chi-square test was used to compare the observed and expected allele frequencies according to Hardy-Weinberg equilibrium. Haplotype analysis was done using Haploview v.4.1 software. The efavirenz metabolic ratio (EFV MR) was calculated by dividing concentrations of efavirenz by 8-hydroxyefavirenz. Normality of kinetic data was assured by transforming the data to Log 10 values before statistical analysis. Independent t-test was used to compare log transformed plasma efavirenz, 8-hydroxyefavirenz and efavirenz/8-hydroxyefvarinz ratio between patients with and without DILI. Univariate and multivariate Cox proportional Hazards Model were performed to identify potential predictors of DILI and Kaplan-Meier curves was performed to estimate the incidence of DILI over time. Incidence of DILI was calculated as the number of episodes per 1000 persons exposed per week. SPSS version 19.0 for complete data analysis and Statistica version 10 for graphical data presentation were employed. Variables with p<0.05 were considered potential predictors for DILI.A total of 373 newly diagnosed antiretroviral and antitubercular treatment naïve HIV-TB co-infected patients among which 199 (53.4%) women and 174 (46.6%) men were enrolled prospectively and followed up for development of DILI for up to 56 weeks after initiation of antiTB treatment. Twenty patients (20.4%) were excluded from the analysis and put on standard treatment as per the national guideline for they had elevated transaminases (>5×UNL) before starting efavirenz therapy. The remaining 353 patients were initiated with the four fixed dose combination anti TB drugs namely rifampicin, ethambutol, pyrazianmide and isoniazid for the intensive phase (2-month) followed with isoniazid and rifampicin for the continuation phase (4-month). All patients were also initiated with either AZT/3TC/EFV or D4T/3TC/EFV or TDF/3TC/EFV within the first 8 weeks after starting anti-TB treatment.The median age of participants was 35 years (range 18–72) and 49.2% of them had a BMI of <18.5. Seventy eight patients were diagnosed smear positive TB; while 62, 36, and 177 smear negative, disseminated and extra pulmonary TB, respectively. Screening of participants for Hepatitis B and C showed that 33 (9.3%) were positive for Hepatitis B surface antigen, while 5 (1.4%) was positive for Hepatitis C virus antibody. Association of socio demographic parameters, sex and type of HAART with DILI using Cox-regression analysis is presented in From a total of 353 patients, 106 (30.0%) or 14.5 per 1000 person-week developed DILI. The median time for development of DILI was 2 weeks and the majority (91.6%) of the DILI occurred during the first 8 weeks. Severe DILI, i.e., elevation of transaminases >5 times the upper normal limit was observed in 65 participants (18.4%) or 7.49 per 1000 person-week. During the follow up period 12.7% died among which 44.7% had DILI and only 5% had severe DILI before they died but none of the deaths in our cohort were secondary to liver failure. From the total of 65 patients who developed severe DILI 26 (40.4%) were taking D4T30/3TC/EFV , There wAnalysis of baseline biochemical characteristics and efavirenz kinetics with development of DILI is presented in NAT-2, CYP2B6, CYP3A5, ABCB1, UGT2B7*2, and SLCO1B1 genotype was done for 201. Association of each genotype with development of sever DILI is presented in NAT2 haplotypes were determined using Haploview following the nomenclature described in http://louisville.edu/medschool/pharmacology/NAT.html. According to the NAT2 genotypes, all participants were stratified into rapid  and slow . NAT2 genotypic analysis of the different SNPs showed that 138 (68.7%) patients were slow acetylators, while 63 (31.3%) were rapid acetylators. NAT2 rapid acetylator genotype was a significantly reduced the risk of developing DILI. None of the patients who developed DILI were homozygous for functional NAT2 variant allele (p<0.05). Cox-regression analysis showed that there was a statistically significant association DILI with CYP2B6*6 and ABCB1 3435TT genotype  Isoniazid is inactivated by ABCB1 3435TT genotype with increased risk for development of DILI. Interestingly the proportion of patients homozygous for ABCB1 3435TT genotype was three fold higher in the patients who developed DILI compare to those who did not. ABCB1 is responsible for the transport of many antiretroviral and anti tuberculosis drugs including rifampicin and ethambutol. ABCB1 3435T variant allele is reported to lower expression level and protein folding thereby altering the structure of substrate binding sites to and decreased transport activity Several anti TB drugs and antiretroviral drugs and are substrates of P-glycoprotein, coded by ABCB1 gene. We found association of Apart from pharmacogenetic and pharmacokinetic predictors, our result indicates a strong association between development of DILI and baseline elevation in serum aminotransferases as seen in other studies indicating that pretreatment liver enzymes are good predictors for development of DILI. The median time for development of DILI was two weeks, and 91.6% occurred during the first 8 weeks of follow up. This finding has clinical importance in providing information when to frequently assess this group of patients for development of DILI. Five of the patients who discontinued treatment because of severe DILI restarted their anti-TB treatment successfully (none discontinued HAART).CYP2B6*6/*6 and ABCB13435TT genotype, elevated baseline liver aminotransferases, high plasma efavirenz concentration, lower hemoglobin and albumin levels. Our result demonstrates that inter-patient variability in systemic efavirenz exposure and pharmacogenetic variation in NAT2, CYP2B6 and ABCB1 gene determines susceptibility to efavirenz induced liver injury in HIV patients and patients. Close follow up and regular monitoring of plasma efavirenz concentration and liver enzymes during early therapy is recommended, particularly in patients with the described underlying risk factors for early diagnosis and management of efavirenz-based HAART induced liver injury. Therapeutic drug monitoring may not be feasible in resource limited setting. Therefore genotyping practice for common functional variants of NAT2, CYP3A5 and CYP2B6 gene in HIV clinics before initiation of therapy is recommended to identify susceptible individuals and optimize the safety of concomitant rifampicin based anti-TB and efavirenz based antiretroviral therapy in co-infected patients.In summary, we report higher incidence of concomitant anti-tubercular and efavirenz based ARV DILI among Ethiopian TB-HIV co-infected patient The identified predictors include; slow acetylation status,


Single-nucleotide polymorphism typing found missing link between human strains in strain from deer. Escherichia coli (EHEC) O157 cause a spectrum of clinical signs that include diarrhea, bloody diarrhea, and hemolytic uremic syndrome. The current evolutionary model of EHEC O157:H7/H– consists of a stepwise evolution scenario proceeding from O55:H7 to a node  that then branches into sorbitol-fermenting (SF) O157:H– and non-SF (NSF) O157:H7. To identify this hypothetical intermediate, we performed single nucleotide polymorphism analysis by sequencing of 92 randomly distributed backbone genomic regions of 40 O157:H7/H– isolates. Overall, 111 single nucleotide polymorphisms were identified in 75/92 partial open reading frames after sequencing 51,041 nt/strain. The EHEC O157:H7 strain LSU-61 from deer occupied an intermediate position between O55:H7 and both O157 branches (SF and NSF O157), complementing the stepwise evolutionary model of EHEC O157:H7/H–. The animal origin of this intermediate emphasizes the value of nonhuman reservoirs in the clarification of the evolution of human pathogens.Highly pathogenic enterohemorrhagic  Escherichia coli (EHEC) belongs to the Shiga toxin–producing E. coli group and causes clinical signs ranging from watery to bloody diarrhea for most symptomatically infected patients  are the most frequently isolated from patients with severe EHEC-associated diseases, such as bloody diarrhea and hemolytic uremic syndrome. Infections caused by EHEC O157:H7/H– are major public health threats and require considerable resources for control and prevention  diverged  data  enabled precise reconstruction of this model  and had a distinct MLST sequence type  and serotyped by using antiserum against E. coli O antigens 1–181 and H antigens 1–56  was inoculated into a liquid culture of nutrient broth medium (Heipha) and incubated overnight at 37°C. The liquid culture was used to prepare DNA as described (fimH–locus) have been shown to be cluster specific , 1,460,599 , 2,370,797 , and 5,404,166  (E. coli MLST website (http://mlst.ucc.ie/mlst/dbs/Ecoli).As a first classification, we used MLST to determine the sequence type (ST)for all prototype strains of each subgroup and cluster by sequencing internal fragments of 7 housekeeping genes (http://frodo.wi.mit.edu/primer3), we developed 93 primer pairs that generated PCR products of backbone genomic regions ranging from 600 to 700 bp , ≈6 ng DNA, and 1.5 µL each forward and reverse primer, with a final concentration of 10 µmol/L. The cycling reaction conditions were initial denaturation (2 min at 94°C), 35 cycles of denaturation (45 s at 94°C), annealing (60 s at 60°C), and extension (90 s at 72°C), followed by a final extension (10 min at 72°C). PCR products were purified by using the exonuclease I  and shrimp alkaline phosphatase  according to methods modified from  and 2 µL (10 µmol/L) from the sequencing primer  and combined annealing and extension (4 min at 60°C). Finally, the sequencing reaction products were purified by using an alcohol precipitation method as recommended by the manufacturer and loaded onto a 3130xl Genetic Analyzer (Applied Biosystems) for capillary sequencing.For sequencing of both strands, 2 µL of the purified amplicons was mixed with 0.5 µL premix from the ABI Prism BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit  plus 1.8 µL Tris-HCl-MgClstx-phage integration sites. We used the draft genome sequence of the O157 strain LSU-61 (GenBank accession no. AEUC00000000)  and corresponding Kyoto Encyclopedia of Genes and Genomes  assignments. Overall, genes were grouped into 3 functional categories: metabolism/housekeeping, putative metabolism/housekeeping, and hypothetical protein. If no KEGG phenotype assignment was found, a putative metabolism/housekeeping function was predicted on the basis of BLAST (http://blast.ncbi.mlm.nih.gov/Blast.cgi) results.Sequence trace files were analyzed and stored by using SeqSphere software version 0.9 beta ; a minimum-spanning tree was constructed with the integrated minimum-spanning tree algorithm. Gene functions were categorized by using the Pathosystems Resource Integration Center database  (The 50 strains of serotypes O157:H7/H genome) . Sequenc genome) . DeletioOf the 111 SNPs, 53 (47.7%) were synonymous SNPs (sSNPs) and 58 (52.3%) were nonsynonymous SNPs (nsSNPs); 78 (70.3%) SNPs were transitions, and 33 (29.7%) were transversions. Concatenation of all loci resulted in an average of 1 SNP every 460 bp; sSNPs occurred every 963 bp, and nsSNPs every 880 bp. On the level of analyzed partial ORFs, 1 SNP was found in 45 partial ORFs, 2 SNPs in 25 partial ORFs, 3 SNPs in 4 partial ORFs, and 4 SNPs in 1 partial ORF.To further elucidate the SNP distribution, we categorized the 92 loci into 3 functional groups. Most loci belonged to (putative) metabolism or housekeeping genes because these were chosen solely from backbone regions. If no KEGG assignment was possible, we estimated the function of the corresponding fragment on the basis of BLAST homologies. Defined annotation information regarding the function in metabolism or housekeeping was determined for 25 partial ORFs. A housekeeping/metabolism function was predicted for 58 loci. The remaining 9 loci were hypothetical proteins only .–– (subgroup B) or NSF O157:H7/H– . Strain LSU-61 interlinked all 3 subgroups, thereby substituting for the unknown intermediate  exhibited 8 SNP genotypes.On the basis of the 111 SNPs, the 50 strains were clustered into 27 SNP genotypes . The 111rmediate . Moreovestx-negative LSU-61 as a potential intermediate, we investigated each known potential stx insertion site in silico to determine the presence or absence of a Shiga toxin–carrying bacteriophage. We conducted BLAST searches within the recently published draft genome sequence of LSU-61 (stx1 (yehV) and stx2  were intact.To further validate the role of –, 2 O55:H7, and LSU-61) based on the 53 sSNPs and 22 nsSNP genotypes  based on the 58 nsSNPs. This excludes strong selection bias of the different loci.To investigate the effect of selective pressure on some loci and potential selecting biases, we analyzed sSNP and nsSNP types separately. In each scenario, the phylogenetic reconstruction resulted in comparable branching, with distinct lineages for SF and NSF O157 and strain LSU-61 as an intermediate. Only the number of SNP genotypes differed slightly: 19 sSNP genotypes  and enterohemolysin active  as white-tailed deer (North America), with comparable phenotypic and genotypic traits. Some of these were SF O157:H7 strains   shows a certain persistence of these O157:H7 clusters   and single nucleotide polymorphisms (SNPs)  of all 50 strains of Enterohemorrhagic Escherichia coli O157 investigated in this study.