Diphtheria toxin-derived, anti-PD-1 immunotoxin, a potent and practical tool to selectively deplete PD-1 cells.

Programmed death-1 (PD-1), an immune checkpoint receptor, is expressed on activated lymphocytes, macrophages, and some types of tumor cells. While PD-1 cells have been implicated in outcomes of cancer immunity, autoimmunity, and chronic infections, the exact roles of these cells in various physiological and pathological processes remain elusive. Molecules that target and deplete PD-1 cells would be instrumental in defining the roles unambiguously.

Antibody-mediated depletion of programmed death 1-positive (PD-1) cells.

PD-1 immune checkpoint has been intensively investigated in pathogenesis and treatments for cancer and autoimmune diseases. Cells that express PD-1 (PD-1 cells) draw ever-increasing attention in cancer and autoimmune disease research although the role of PD-1 cells in the progression and treatments of these diseases remains largely ambiguous. One definite approach to elucidate their roles is to deplete these cells in disease settings and examine how the depletion impacts disease progression and treatments.

Promotion of CTL epitope presentation by a nanoparticle with environment-responsive stability and phagolysosomal escape capacity.

Vaccines that induce cytotoxic T lymphocyte (CTL)-mediated immune responses constitute an important class of medical tools to fend off diseases like infections and malignancy. Epitope peptides, as a format of CTL vaccines, are being tested preclinically and clinically. To elicit CTL responses, epitope vaccines go through an epitope presentation pathway in dendritic cells (DCs) that has multiple bottleneck steps and hence is inefficient.

Depletion of PD-1-positive cells ameliorates autoimmune disease.

Targeted suppression of autoimmune diseases without collateral suppression of normal immunity remains an elusive yet clinically important goal. Targeted blockade of programmed-cell-death-protein-1 (PD-1)-an immune checkpoint factor expressed by activated T cells and B cells-is an efficacious therapy for potentiating immune activation against tumours. Here we show that an immunotoxin consisting of an anti-PD-1 single-chain variable fragment, an albumin-binding domain and Pseudomonas exotoxin targeting PD-1-expressing cells, selectively recognizes and induces the killing of the cells.

Direct loading of CTL epitopes onto MHC class I complexes on dendritic cell surface in vivo.

Dendritic cell (DC)-based cytotoxic T lymphocyte (CTL) epitope vaccines are effective to induce CTL responses but require complex ex vivo DC preparation and epitope-loading. To take advantage of DC-based epitope vaccines without involving the ex vivo procedures, we aimed to develop carriers to directly load CTL epitopes onto DCs in vivo. Here, we first engineered a carrier consisting of a hydrophilic polypeptide, immune-tolerant elastin-like polypeptide (iTEP) and a substrate peptide of matrix metalloproteinases-9 (sMMP).

An Albumin-binding Polypeptide Both Targets Cytotoxic T Lymphocyte Vaccines to Lymph Nodes and Boosts Vaccine Presentation by Dendritic Cells.

Albumin-binding carriers have been shown to target cytotoxic T lymphocyte (CTL) vaccines to lymph nodes (LNs) and improve the efficacy of the vaccines. However, it was not clear whether the improved efficacy is solely due to the LN targeting, which prompted this study. First, we generated a fusion protein consisting of an albumin-binding domain (ABD) and an immune-tolerant elastin-like polypeptide (iTEP).

Direct Loading of iTEP-Delivered CTL Epitope onto MHC Class I Complexes on the Dendritic Cell Surface.

Cytotoxic T lymphocyte (CTL)-mediated immune responses are the primary defense mechanism against cancer and infection. CTL epitope peptides have been used as vaccines to boost CTL responses; however, the efficacy of these peptides is suboptimal. Under current vaccine formulation and delivery strategies, these vaccines are delivered into and processed inside antigen-presenting cells such as dendritic cells (DCs).

Engineering of a self-adjuvanted iTEP-delivered CTL vaccine.

Cytotoxic T lymphocyte (CTL) epitope peptide-based vaccines are widely used in cancer and infectious disease therapy. We previously generated an immune-tolerant elastin-like polypeptides (iTEPs)-based carrier to deliver a peptide CTL vaccine and enhance the efficiency of the vaccine. To further optimize the vaccine carrier, we intended to potentiate its function by designing an iTEP-based carrier that was able to deliver adjuvant and a vaccine epitope as one molecule.

An Anti-Programmed Death-1 Antibody (αPD-1) Fusion Protein That Self-Assembles into a Multivalent and Functional αPD-1 Nanoparticle.

Cancer immune checkpoint therapy has achieved remarkable clinical successes in various cancers. However, current immune checkpoint inhibitors block the checkpoint of not only the immune cells that are important to cancer therapy but also the immune cells that are irrelevant to the therapy. Such an indiscriminate blockade limits the efficacy and causes the autoimmune toxicity of the therapy.

Correction: Degradation of YRA1 Pre-mRNA in the Cytoplasm Requires Translational Repression, Multiple Modular Intronic Elements, Edc3p, and Mex67p.

[This corrects the article DOI: 10.1371/journal.pbio.

An iTEP-salinomycin nanoparticle that specifically and effectively inhibits metastases of 4T1 orthotopic breast tumors.

Cancer stem cell (CSC) inhibitors are a new category of investigational drugs to treat metastasis. Salinomycin (Sali) is one of most studied CSC inhibitors and has reached clinical tests. Several drug carriers have been developed to improve efficacy of Sali.

A Comparison Study of iTEP Nanoparticle-Based CTL Vaccine Carriers Revealed a Surprise Relationship between the Stability and Efficiency of the Carriers.

Vaccine carriers have been shown to enhance cytotoxic T lymphocyte (CTL) epitope peptide vaccines by addressing intrinsic limitations of the vaccines. We have previously developed an immune-tolerant elastin-like polypeptide (iTEP)-based nanoparticle (NP) as an effective and unique CTL vaccine carrier. The NP is unique for its humoral immune tolerance, flexible structure, and ability to deliver CTL vaccines as polypeptide fusions.

iTEP nanoparticle-delivered salinomycin displays an enhanced toxicity to cancer stem cells in orthotopic breast tumors.

Salinomycin (Sali) has selective toxicity to cancer stem cells (CSCs), a subpopulation of cancer cells that have been recently linked with tumor multidrug resistance (MDR). To utilize its selective toxicity for cancer therapy, we sought to devise a nanoparticle (NP) carrier to deliver Sali to solid tumors through the enhanced permeability and retention effect and, hence, to increase its exposure to CSCs. First, hydrophobic Sali was conjugated to a hydrophilic, immune-tolerant, elastin-like polypeptide (iTEP); the amphiphilic iTEP-Sali conjugates self-assemble into NPs.

Treatment of type 1 myotonic dystrophy by engineering site-specific RNA endonucleases that target (CUG)(n) repeats.

Myotonic dystrophy type 1 (DM1) is caused by the expansion of (CTG)n in the 3' untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, which is transcribed as (CUG)n repeats that accumulate in the nucleus. The RNA repeats specifically sequester or change the expression levels of several RNA-binding proteins, leading to aberrant splicing of many target genes. In this study, we developed artificial site-specific RNA endonucleases (ASREs) that specifically bind and cleave (CUG)n repeats RNA.

Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains.

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system.

A chemical-genetic approach for precise spatio-temporal control of cellular signaling.

Recently we have perfected a chemical-genetic approach to gain precise spatio-temporal control of cellular signaling. This approach entails the cell-type specific expression of mutant G-protein coupled receptors which have been evolved to be activated by the pharmacologically inert drug-like small molecule clozapine N-oxide. We have named these mutant GPCRs DREADDs (Designer Receptors Exclusively Activated by Designer Drugs).

Degradation of YRA1 Pre-mRNA in the cytoplasm requires translational repression, multiple modular intronic elements, Edc3p, and Mex67p.

Intron-containing pre-mRNAs are normally retained and processed in the nucleus but are sometimes exported to the cytoplasm and degraded by the nonsense-mediated mRNA decay (NMD) pathway as a consequence of their inclusion of intronic in-frame termination codons. When shunted to the cytoplasm by autoregulated nuclear export, the intron-containing yeast YRA1 pre-mRNA evades NMD and is targeted by a cytoplasmic decay pathway mediated by the decapping activator Edc3p. Here, we have elucidated this transcript-specific decay mechanism, showing that Edc3p-mediated YRA1 pre-mRNA degradation occurs independently of translation and is controlled through five structurally distinct but functionally interdependent modular elements in the YRA1 intron.

Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs.

G protein-coupled receptors (GPCRs) and their downstream signaling cascades contribute to most physiological processes and a variety of human diseases. Isolating the effects of GPCR activation in an in vivo experimental setting is challenging as exogenous ligands have off-target effects and endogenous ligands constantly modulate the activity of native receptors. Highly specific designer drug-designer receptor complexes are a valuable tool for elucidating the effects of activating particular receptors and signaling pathways within selected cell types in vivo.

Generation of designer receptors exclusively activated by designer drugs (DREADDs) using directed molecular evolution.

G protein-coupled receptors (GPCRs) and their signal transductions are important for both physiological and pathological processes in neuron systems. Neuronal GPCRs activated by synthetic ligands have been created by designed mutagenesis for studying their functions and signal pathways. However, these engineered GPCRs have problems, such as their high constitutive activity.

[Effects of taurine on TNF-alpha and NF-kappaB expression of liver injury after limbs ischemia/reperfusion in rats].

Aim: To investigate the effect of pretreatment with taurine on liver injury changes and the change of tumor necrosis factor alpha and NFkappaB expression following rats limbs ischemia/reperfusion.

Methods: The model of limbs ischemia/reperfusion injury on rats was adopted in the experiment. Wistar rats were randomized into 4 groups (n = 10): Control group, T group, I/R group and TR group.

[Effect of ischemic preconditioning on lung injury induced by ischemia/reperfusion in the hind limbs of rats].

Aim: To study the effect of ischemic preconditioning on lung injury following ischemia/reperfusion (I/R) in the hind limbs of rats.

Methods: Wistar rats were randomly divided into four groups (n=8): control group,limbs ischemia/reperfusion (LI/R) group, ischemia preconditioning (IPC) group and L-NAME group. At the end of the experiment, blood/gas analysis and the contents of serum MDA, NO, ET and lung tissue MDA, NO, ET, MPO were measured.

[The affection and significance of NO on the expression of P-selectin in renal injury following hind limb ischemia/reperfusion in rats].

Aim: To probe into the affection and significance of NO on the expression of P-selectin in renal injury following hind limb ischemia/reperfusion in rats.

Methods: In accordance with the conventional approaches of our department, the model rats were prepared after they were made to undergo 4 hours or ischemia followed by 4 hours of reperfusion of hind limbs. The Wistar rats were divided into four groups randomly: Control group, LI/R group, L-Arg group and L-NAME group.

[Effect of MK801 on apoptosis in the development of brain injury after hind limbs ischemia/reperfusion in rats].

Aim: To evaluate development of brain injury after hind limbs ischemia/reperfusion (LI/R) in rats, and the effect of MK801 on the brain injury following LI/R.

Methods: The limbs ischemia/reperfusion model was established in rats. The MDA contents were evaluated in each group, apoptotic cells were detected with TUNEL, the expression of apoptosis-associated protein, such as bcl-2, cytoC and caspase-3 were determined with immunohistochemistry and Western-blot.