Identification of antibodies cross-reactive with woodchuck immune cells and activation of virus-specific and global cytotoxic T cell responses by anti-PD-1 and anti-PD-L1 in experimental chronic hepatitis B and persistent occult hepadnaviral infection.

Woodchuck (Marmota monax) infected with woodchuck hepatitis virus (WHV) is the most pathogenically compatible naturally occurring model of human hepatitis B virus (HBV) infection, chronic hepatitis B, and HBV-induced hepatocellular carcinoma. This system plays a crucial role in discovery and preclinical evaluation of anti-HBV therapies. Its utilization remains tempered by the relatively narrow range of validated immunologic and molecular tools. We evaluated commercial antibodies against immune cell phenotypic markers and T cell molecules for cross-reactivity with woodchuck antigenic equivalents. The confirmed antibodies against programed cell death protein-1 (PD-1) and its ligand (PD-L1) were examined for ex vivo ability to activate WHV-specific, global and bystander cytotoxic T cells (CTLs) in chronic hepatitis and asymptomatic infection persisting after self-resolved acute hepatitis. Examination of 65 antibodies led to identification or confirmation of 23 recognizing woodchuck T, regulatory T, B and natural killer cells, T cell-associated PD-1, PD-L1, CTLA-4 and TIM-3 molecules, CD25 and CD69 markers of T cell activation, and interferon gamma (IFNγ). Antibodies against woodchuck PD-1 and PD-L1 triggered in vitro highly individualized WHV-specific and global activation of CTLs in both chronic hepatitis and persistent occult infection. WHV-specific CTLs were more robustly augmented by anti-PD-1 than by anti-PD-L1 in chronic hepatitis, while global IFNγ-positive CTL response was significantly suppressed in chronic hepatitis compared to persistent occult infection. Anti-PD-1 and anti-PD-L1 also occasionally activated CTLs to specificities other than those tested suggesting their potency to trigger side effects. This was particularly apparent when T cells from chronic hepatitis were treated with anti-PD-L1. The current findings indicate that inhibition of the PD-1/PD-L1 pathway could reactivate virus-specific and global T cell responses in both chronic hepatitis and asymptomatic persistent infection. They suggest a mechanism of potential reactivation of clinically silent infection during anti-PD-1/PD-L1 treatment and indicate that this therapy may also subdue occult HBV infection.

TractaViewer: a genome-wide tool for preliminary assessment of therapeutic target druggability.

SUMMARY: We present software to characterize and rank potential therapeutic (drug) targets with data from public databases and present it in a user-friendly format. By understanding potential obstacles to drug development through the gathering and understanding of this information, combined with robust approaches to target validation to generate therapeutic hypotheses, this approach may provide high quality targets, leading the process of drug development to become more efficient and cost-effective. AVAILABILITY AND IMPLEMENTATION: The information we gather on potential targets concerns small-molecule druggability (ligandability), suitability for large-molecule approaches (e.g. antibodies) or new modalities (e.g. antisense oligonucleotides, siRNA or PROTAC), feasibility (availability of resources such as assays and biological knowledge) and potential safety risks (adverse tissue-wise expression, deleterious phenotypes). This information can be termed 'tractability'. We provide visualization tools to understand its components. TractaViewer is available from https://github.com/NeilPearson-Lilly/TractaViewer. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant.

Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

Genomic structure and expression of the human serotonin 2A receptor gene (HTR2A) locus: identification of novel HTR2A and antisense (HTR2A-AS1) exons.

BACKGROUND: The serotonin 2A receptor is widely implicated in genetic association studies and remains an important drug target for psychiatric, neurological, and cardiovascular conditions. RNA sequencing redefined the architecture of the serotonin 2A receptor gene (HTR2A), revealing novel mRNA transcript isoforms utilizing unannotated untranslated regions of the gene. Expression of these untranslated regions is modulated by common single nucleotide polymorphisms (SNPs), namely rs6311. Previous studies did not fully capture the complexity of the sense- and antisense-encoded transcripts with respect to novel exons in the HTR2A gene locus. Here, we comprehensively catalogued exons and RNA isoforms for both HTR2A and HTR2A-AS1 using RNA-Seq from human prefrontal cortex and multiple mouse tissues. We subsequently tested associations between expression of newfound gene features and common SNPs in humans. RESULTS: We find that the human HTR2A gene spans ~66 kilobases and consists of 7, rather than 4 exons. Furthermore, the revised human HTR2A-AS1 gene spans ~474 kilobases and consists of 18, rather than 3 exons. Three HTR2A exons directly overlap with HTR2A-AS1 exons, suggesting potential for complementary nucleotide interactions. The repertoire of possible mouse Htr2a splice isoforms is remarkably similar to humans and we also find evidence for overlapping sense-antisense transcripts in the same relative positions as the human transcripts. rs6311 and SNPs in high linkage disequilibrium are associated with HTR2A-AS1 expression, in addition to previously described associations with expression of the extended 5' untranslated region of HTR2A. CONCLUSIONS: Our proposed HTR2A and HTR2A-AS1 gene structures dramatically differ from current annotations, now including overlapping exons on the sense and anti-sense strands. We also find orthologous transcript isoforms expressed in mice, providing opportunities to elucidate the biological roles of the human isoforms using a model system. Associations between rs6311 and expression of HTR2A and HTR2A-AS1 suggest this polymorphism is capable of modulating the expression of the sense or antisense transcripts. Still unclear is whether these SNPs act directly on the expression of the sense or antisense transcripts and whether overlapping exons are capable of interacting through complimentary base-pairing. Additional studies are necessary to determine the extent and nature of interactions between the SNPs and the transcripts prior to interpreting these findings in the context of phenotypes associated with HTR2A.

Gain and loss of function of P2X7 receptors: mechanisms, pharmacology and relevance to diabetic neuropathic pain.

BACKGROUND: Genetic causes of exaggerated or reduced pain sensitivity in humans are well known. Recently, single nucleotide polymorphisms (SNPs) in the gene P2RX7, coding for the ATP-gated ion channel P2X7, have been described that cause gain-of-function (GOF) and loss-of-function (LOF), respectively of this channel. Importantly, P2RX7 SNPs have been associated with more or less severe pain scores in patient suffering of post-mastectomy pain and osteoarthritis. RESULTS: The functional consequences of some P2RX7 SNPs (rs208294 (His155Tyr), rs1718119 (Ala348Thr) and rs3751143 (Glu496Ala)) were studied in recombinant cells in vitro. Our findings suggest a correlation between GOF and LOF of P2X7 and actual channel protein expression. Both channel and pore function for these mutant P2X7 receptors changed in parallel to protein levels. On the other hand, the mutant receptors did not differ in their sensitivity to known P2X7 agonists and antagonists. We further demonstrated that in patients with diabetic peripheral neuropathic pain (DPNP), the presence of the GOF SNPs rs208294 (His155Tyr) and rs1718119 (Ala348Thr) is associated, in females, with higher pain intensity scores. CONCLUSIONS: Our present results confirm the physiological relevance of some of the SNPs in the P2RX7 gene and show that the presence of these genetic variants correlates with pain sensitivity also in a diabetic neuropathic pain patient population.

Pharmacological profiling of the TRPV3 channel in recombinant and native assays.

BACKGROUND AND PURPOSE: Transient receptor potential vanilloid subtype 3 (TRPV3) is implicated in nociception and certain skin conditions. As such, it is an attractive target for pharmaceutical research. Understanding of endogenous TRPV3 function and pharmacology remains elusive as selective compounds and native preparations utilizing higher throughput methodologies are lacking. In this study, we developed medium-throughput recombinant and native cellular assays to assess the detailed pharmacological profile of human, rat and mouse TRPV3 channels. EXPERIMENTAL APPROACH: Medium-throughput cellular assays were developed using a Ca(2+) -sensitive dye and a fluorescent imaging plate reader. Human and rat TRPV3 pharmacology was examined in recombinant cell lines, while the mouse 308 keratinocyte cell line was used to assess endogenous TRPV3 activity. KEY RESULTS: A recombinant rat TRPV3 cellular assay was successfully developed after solving a discrepancy in the published rat TRPV3 protein sequence. A medium-throughput, native, mouse TRPV3 keratinocyte assay was also developed and confirmed using genetic approaches. Whereas the recombinant human and rat TRPV3 assays exhibited similar agonist and antagonist profiles, the native mouse assay showed important differences, namely, TRPV3 activity was detected only in the presence of potentiator or during agonist synergy. Furthermore, the native assay was more sensitive to block by some antagonists. CONCLUSIONS AND IMPLICATIONS: Our findings demonstrate similarities but also notable differences in TRPV3 pharmacology between recombinant and native systems. These findings offer insights into TRPV3 function and these assays should aid further research towards developing TRPV3 therapies.

Multiple regulatory variants modulate expression of 5-hydroxytryptamine 2A receptors in human cortex.

BACKGROUND: The 5-hydroxytryptamine 2A receptor, encoded by HTR2A, is a major postsynaptic target for serotonin in the human brain and a therapeutic drug target. Despite hundreds of genetic associations investigating HTR2A polymorphisms in neuropsychiatric disorders and therapies, the role of genetic HTR2A variability in health and disease remains uncertain. METHODS: To discover and characterize regulatory HTR2A variants, we sequenced whole transcriptomes from 10 human brain regions with massively parallel RNA sequencing and measured allelic expression of multiple HTR2A messenger (m)RNA transcript variants. Following discovery of functional variants, we further characterized their impact on genetic expression in vitro. RESULTS: Three polymorphisms modulate the use of novel alternative exons and untranslated regions (UTRs), changing expression of RNA and protein. The frequent promoter variant rs6311, widely implicated in human neuropsychiatric disorders, decreases usage of an upstream transcription start site encoding a longer 5'UTR with greater translation efficiency. rs76665058, located in an extended 3'UTR and unique to individuals of African descent, modulates allelic HTR2A mRNA expression. The third single nucleotide polymorphism, unannotated and present in only a single subject, directs alternative splicing of exon 2. Targeted analysis of HTR2A in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study reveals associations between functional variants and depression severity or citalopram response. CONCLUSIONS: Regulatory polymorphisms modulate HTR2A mRNA expression in an isoform-specific manner, directing the usage of novel untranslated regions and alternative exons. These results provide a foundation for delineating the role of HTR2A and serotonin signaling in central nervous system disorders.

A cluster of novel serotonin receptor 3-like genes on human chromosome 3.

The ligand-gated ion channel family includes receptors for serotonin (5-hydroxytryptamine, 5-HT), acetylcholine, GABA, and glutamate. Drugs targeting subtypes of these receptors have proven useful for the treatment of various neuropsychiatric and neurological disorders. To identify new ligand-gated ion channels as potential therapeutic targets, drafts of human genome sequence were interrogated. Portions of four novel genes homologous to 5-HT(3A) and 5-HT(3B) receptors were identified within human sequence databases. We named the genes 5-HT(3C1)-5-HT(3C4). Radiation hybrid (RH) mapping localized these genes to chromosome 3q27-28. All four genes shared similar intron-exon organizations and predicted protein secondary structure with 5-HT(3A) and 5-HT(3B). Orthologous genes were detected by Southern blotting in several species including dog, cow, and chicken, but not in rodents, suggesting that these novel genes are not present in rodents or are very poorly conserved. Two of the novel genes are predicted to be pseudogenes, but two other genes are transcribed and spliced to form appropriate open reading frames. The 5-HT(3C1) transcript is expressed almost exclusively in small intestine and colon, suggesting a possible role in the serotonin-responsiveness of the gut.

SEL-10 interacts with presenilin 1, facilitates its ubiquitination, and alters A-beta peptide production.

Mutations in the human presenilin genes (PS1 or PS2) have been linked to autosomal dominant, early onset Alzheimer's disease (AD). Presenilins, probably as an essential part of gamma-secretase, modulate gamma-cleavage of the amyloid protein precursor (APP) to the amyloid beta-peptide (Abeta). Mutations in sel-12, a Caenorhabditis elegans presenilin homologue, cause a defect in egg laying that can be suppressed by loss of function mutations in a second gene, SEL-10. SEL-10 protein is a homologue of yeast Cdc4, a member of the SCF (Skp1-Cdc53/CUL1-F-box protein) E2-E3 ubiquitin ligase family. In this study, we show that human SEL-10 interacts with PS1 and enhances PS1 ubiquitination, thus altering cellular levels of unprocessed PS1 and its N- and C-terminal fragments. Co-transfection of sel-10 and APP cDNAs in HEK293 cells leads to an alteration in the metabolism of APP and to an increase in the production of amyloid beta-peptide, the principal component of amyloid plaque in Alzheimer's disease.

aph-1 and pen-2 are required for Notch pathway signaling, gamma-secretase cleavage of betaAPP, and presenilin protein accumulation.

Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.