Toxin Fragment Research Articles

Transcriptomic Analysis of the Response of the Dioryctria abietella Larva Midgut to Bacillus thuringiensis 2913 Infection.

Dioryctria abietella Denis Schiffermuller (Lepidoptera: Pyralidae) is an oligophagous pest that mainly damages Pinaceae plants. Here, we investigated the effects of the Bacillus thuringiensis 2913 strain (Bt 2913), which carries the Cry1Ac, Cry2Ab, and Vip3Aa genes, on the D. abietella midgut transcriptome at 6, 12, and 24 h after infection. In total, 7497 differentially expressed genes (DEGs) were identified from the midgut transcriptome of D. abietella larvae infected with Bt 2913. Among these DEGs, we identified genes possibly involved in Bt 2913-induced perforation of the larval midgut. For example, the DEGs included 67 genes encoding midgut proteases involved in Cry/Vip toxin activation, 74 genes encoding potential receptor proteins that bind to insecticidal proteins, and 19 genes encoding receptor NADH dehydrogenases that may bind to Cry1Ac. Among the three transcriptomes, 88 genes related to metabolic detoxification and 98 genes related to immune defense against Bt 2913 infection were identified. Interestingly, 145 genes related to the 60S ribosomal protein were among the DEGs identified in the three transcriptomes. Furthermore, we performed bioinformatic analysis of zonadhesin, GST, CYP450, and CarE in the D. abietella midgut to determine their possible associations with Bt 2913. On the basis of the results of this analysis, we speculated that trypsin and other serine proteases in the D. abietella larval midgut began to activate Cry/Vip prototoxin at 6 h to 12 h after Bt 2913 ingestion. At 12 h after Bt 2913 ingestion, chymotrypsin was potentially involved in degrading the active core fragment of Vip3Aa toxin, and the detoxification enzymes in the larvae contributed to the metabolic detoxification of the Bt toxin. The ABC transporter and several other receptor-protein-related genes were also downregulated to increase resistance to Bt 2913. However, the upregulation of 60S ribosomal protein and heat shock protein expression weakened the resistance of larvae to Bt 2913, thereby enhancing the expression of NADH dehydrogenase and other receptor proteins that are highly expressed in the larval midgut and bind to activating toxins, including Cry1Ac. At 24 h after Bt 2913 ingestion, many activated toxins were bound to receptor proteins such as APN in the larval midgut, resulting in membrane perforation. Here, we clarified the mechanism of Bt 2913 infection in D. abietella larvae, as well as the larval immune defense response to Bt 2913, which provides a theoretical basis for the subsequent control of D. abietella using B. thuringiensis.

Transcriptional evidence for transient regulation of muscle regeneration by brown adipose transplant in the rotator cuff.

Chronic rotator cuff(RC) injuries can lead to a degenerative microenvironment that favors chronic inflammation, fibrosis, and fatty infiltration. Recovery of muscle structure and function will ultimately require a complex network of muscle resident cells, including satellite cells, fibro-adipogenic progenitors (FAPs), and immune cells. Recent work suggests that signaling from adipose tissue and progenitors could modulate regeneration and recovery of function, particularly promyogenic signaling from brown or beige adipose (BAT). In this study, we sought to identify cellular targets of BAT signaling during muscle regeneration using a RCBAT transplantation mouse model. Cardiotoxin injured supraspinatus muscle had improved mass at 7 days postsurgery (dps) when transplanted with exogeneous BAT. Transcriptional analysis revealed transplanted BAT modulates FAP signaling early in regeneration likely via crosstalk with immune cells. However, this conferred no long-term benefit as muscle mass and function were not improved at 28 dps. To eliminate the confounding effects of endogenous BAT, we transplanted BAT in the "BAT-free" uncoupling protein-1diphtheria toxin fragment A (UCP1-DTA) mouse and here found improved muscle contractile function, but not mass at 28 dps. Interestingly, the transplanted BAT increased fatty infiltration in all experimental groups, implying modulation of FAP adipogenesis during regeneration. Thus, we conclude that transplanted BAT modulates FAP signaling early in regeneration,but does not grant long-term benefits.

An epidermal growth factor receptor‐targeting immunotoxin based on IgG shows potent antitumor activity against head and neck cancer

AbstractThe epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti‐EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR‐targeting immunotoxin consisting of a clinically proven anti‐EGFR IgG (cetuximab; CTX) and a toxin fragment (LR‐LO10) derived from Pseudomonas exotoxin A (PE) using a novel site‐specific conjugation technology (peptide‐directed photo‐crosslinking reaction), as an alternative option. The immunotoxin (CTX‐LR‐LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor‐2. Treatment of EGFR‐positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX‐LR‐LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR‐LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX‐LR‐LO10 may serve as a new therapeutic agent targeting EGFR‐positive cancers.

Designing a Multi-epitope Vaccine against the SARS-CoV-2 Variant Based on an Immunoinformatics Approach.

SARS-CoV-2 is a life-threatening virus in the world. Scientific evidence indicates that this pathogen will emerge again in the future. Although the current vaccines have a pivotal role in the control of this pathogen, the emergence of new variants has a negative impact on their effectiveness. Therefore, it is urgent to consider the protective and safe vaccine against all sub-coronavirus species and variants based on the conserved region of the virus. Multi-epitope peptide vaccine (MEV), comprised of immune-dominant epitopes, is designed by immunoinformatic tools and it is a promising strategy against infectious diseases. Spike glycoprotein and nucleocapsid proteins from all coronavirus species and variants were aligned and the conserved region was selected. Antigenicity, toxicity, and allergenicity of epitopes were checked by a proper server. To robust the immunity of the multi-epitope vaccine, cholera toxin b (CTB) and three HTL epitopes of tetanus toxin fragment C (TTFrC) were linked at the N-terminal and C-terminal of the construct, respectively. Selected epitopes with MHC molecules and the designed vaccines with Toll-like receptors (TLR-2 and TLR-4) were docked and analyzed. The immunological and physicochemical properties of the designed vaccine were evaluated. The immune responses to the designed vaccine were simulated. Furthermore, molecular dynamic simulations were performed to study the stability and interaction of the MEV-TLRs complexes during simulation time by NAMD (Nanoscale molecular dynamic) software. Finally, the codon of the designed vaccine was optimized according to Saccharomyces boulardii. The conserved regions of spike glycoprotein and nucleocapsid protein were gathered. Then, safe and antigenic epitopes were selected. The population coverage of the designed vaccine was 74.83%. The instability index indicated that the designed multi-epitope was stable (38.61). The binding affinity of the designed vaccine to TLR2 and TLR4 was -11.4 and -11.1, respectively. The designed vaccine could induce humoral and cellular immunity. In silico analysis showed that the designed vaccine is a protective multi-epitope vaccine against SARS-CoV-2 variants.

Intradermal Naked DNA Vaccination by DNA Tattooing for Mounting Tumor-Specific Immunity in Stage IV Melanoma Patients: A Phase I Clinical Trial

Introduction: Naked DNA vaccination could be a powerful and safe strategy to mount antigen-specific cellular immunity. We designed a phase I clinical trial to investigate the toxicity of naked DNA vaccines encoding CD8+ T-cell epitope from tumor-associated antigen MART-1 in patients with advanced melanoma. Methods: This dose escalating phase Ia clinical trial investigates the toxicity and immunological response upon naked DNA vaccines encoding a CD8+ T-cell epitope from the tumor-associated antigen MART-1, genetically linked to the gene encoding domain 1 of subunit-tetanus toxin fragment C in patients with advanced melanoma (inoperable stage IIIC-IV, AJCC 7th edition). The vaccine was administrated via intradermal application using a permanent make-up or tattoo device. Safety was monitored according to CTCAE v.3.0 and skin biopsies and blood samples were obtained for immunologic monitoring. Results: Nine pretreated, HLA-A*0201-positive patients with advanced melanoma expressing MART-1 and MHC class I, with a good performance status, and adequate organ function, were included. With a median follow-up of 5.9 months, DNA vaccination was safe, without treatment-related deaths. Common treatment-emergent adverse events of any grade were dermatologic reactions at the vaccination site (100%) and pain (56%). One patient experienced grade 4 toxicity, most likely related to tumor progression. One patient (11%) achieved stable disease, lasting 353 days. Immune analysis showed no increase in vaccine-induced T cell response in peripheral blood of 5 patients, but did show a MART-1 specific CD8+ T cell response at the tattoo administration site. The maximum dose administered was 2 mg due to lack of clinical activity. Conclusion: We showed that the developed DNA vaccine, applied using a novel intradermal application strategy, can be administered safely. Further research with improved vaccine formats is required to show possible clinical benefit of DNA vaccination.

Structural basis of the activation of MARTX cysteine protease domain from Vibrio vulnificus.

The multifunctional autoprocessing repeat-in-toxin (MARTX) toxin is the primary virulence factor of Vibrio vulnificus displaying cytotoxic and hemolytic properties. The cysteine protease domain (CPD) is responsible for activating the MARTX toxin by cleaving the toxin precursor and releasing the mature toxin fragments. To investigate the structural determinants for inositol hexakisphosphate (InsP6)-mediated activation of the CPD, we determined the crystal structures of unprocessed and β-flap truncated MARTX CPDs of Vibrio vulnificus strain MO6-24/O in complex with InsP6 at 1.3 and 2.2Å resolution, respectively. The CPD displays a conserved domain with a central seven-stranded β-sheet flanked by three α-helices. The scissile bond Leu3587-Ala3588 is bound in the catalytic site of the InsP6-loaded form of the Cys3727Ala mutant. InsP6 interacts with the conserved basic cleft and the β-flap inducing the active conformation of catalytic residues. The β-flap of the post-CPD is flexible in the InsP6-unbound state. The structure of the CPD Δβ-flap showed an inactive conformation of the catalytic residues due to the absence of interaction between the active site and the β-flap. This study confirms the InsP6-mediated activation of the MARTX CPDs in which InsP6-binding induces conformational changes of the catalytic residues and the β-flap that holds the N terminus of the CPD in the active site, facilitating hydrolysis of the scissile bond.

The Ablation of Sensory Neurons Expressing the Nav1.8 Sodium Channel Improves Glucose Homeostasis and Amplifies the GLP-1 Signaling in Obese Female Mice.

Sensory neurons expressing the sodium channel Nav1.8 contain a repertoire of receptors for nutrient, hormonal, and inflammatory ligands. However, their function in key regulators of energy homeostasis control is not well understood and is completely unexplored in females. Mice lacking neurons expressing the sodium channel Nav1.8 were generated using an ablation strategy based on cre recombinase-mediated expression of diphtheria toxin fragment A (DTA) (Nav1.8-cre/DTA mice) to investigate whether these neurons modulate body weight, food intake, gut hormone secretion, gastrointestinal transit, and glucose tolerance in response to nutrient challenges in a sex-dependent manner. Male Nav1.8-cre/DTA mice show resistance to gain weight in response to high-fat high-sugar diet (HFHSD), whereas females lacking Nav1.8+ neurons have improved oral glucose tolerance accompanied by higher insulin levels and attenuated glucagon secretion after an oral glucose load. Female Nav1.8-cre/DTA mice also show higher fasting and postprandial glucagon like peptide-1 (GLP-1) levels with an increased number of GLP-1-positive cells. Finally, ablation of Nav1.8-expressing neurons accelerates the gastrointestinal transit in female mice under HFHSD. This data demonstrates sex-dependent differences in the Nav1.8-mediated regulation of energy metabolism, and provides new insights that may help in the design of sex-specific neuromodulation therapies for metabolic disorders induced by diets rich in fats and simple sugars.

Structural and functional insights into the delivery of a bacterial Rhs pore-forming toxin to the membrane

Bacterial competition is a significant driver of toxin polymorphism, which allows continual compensatory evolution between toxins and the resistance developed to overcome their activity. Bacterial Rearrangement hot spot (Rhs) proteins represent a widespread example of toxin polymorphism. Here, we present the 2.45 Å cryo-electron microscopy structure of Tse5, an Rhs protein central to Pseudomonas aeruginosa type VI secretion system-mediated bacterial competition. This structural insight, coupled with an extensive array of biophysical and genetic investigations, unravels the multifaceted functional mechanisms of Tse5. The data suggest that interfacial Tse5-membrane binding delivers its encapsulated pore-forming toxin fragment to the target bacterial membrane, where it assembles pores that cause cell depolarisation and, ultimately, bacterial death.

Synthetic Peptide Fragments of the Wtx Toxin Reduce Blood Pressure in Rats under General Anesthesia

Previously, it was shown that the non-conventional toxin WTX from the venom of the cobra Naja kaouthia, when administered intravenously, caused a decrease in blood pressure (BP) and an increase in heart rate (HR) in rats [13]. To identify the site of the toxin molecule responsible for these effects, we studied the influence of synthetic peptide fragments of the WTX on BP and HR in normotensive male Sprague–Dawley rats under general anesthesia induced by Telazol and Xylazine. It was found that peptides corresponding to the WTX central polypeptide loop, stabilized by a disulfide bond, at intravenous injection at concentrations from 0.1 to 1.0 mg/mL caused a dose-dependent decrease in BP, with the HR increasing only in the first 5–10 min after administration. Thus, WTX fragments corresponding to the central polypeptide loop reproduce the decrease in blood pressure caused by the toxin.

Improving the cytotoxicity of immunotoxins by reducing the affinity of the antibody in acidic pH

BackgroundImmunotoxins are antibody-toxin conjugates that bind to surface antigens and exert effective cytotoxic activity after internalization into tumor cells. Immunotoxins exhibit effective cytotoxicity and have been approved by the FDA to treat multiple hematological malignancies, such as hairy cell leukemia and cutaneous T-cell lymphoma. However, most of the internalized immunotoxin is degraded in lysosomes, and only approximately 5% of free toxin escapes into the cytosol to exert cytotoxicity. Many studies have improved immunotoxins by engineering the toxin fragment to reduce immunogenicity or increase stability, but how the antibody fragment contributes to the activity of immunotoxins has not been well demonstrated.MethodsIn the current study, we used 32A9 and 42A1, two anti-GPC3 antibodies with similar antigen-binding capabilities and internalization rates, to construct scFv-mPE24 immunotoxins and evaluated their in vitro and in vivo antitumor activities. Next, the antigen-binding capacity, trafficking, intracellular protein stability and release of free toxin of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 were compared to elucidate their different antitumor activities. Furthermore, we used a lysosome inhibitor to evaluate the degradation behavior of 32A9 scFv-mPE24 and 42A1 scFv-mPE24. Finally, the antigen-binding patterns of 32A9 and 42A1 were compared under neutral and acidic pH conditions.ResultsAlthough 32A9 and 42A1 had similar antigen binding capacities and internalization rates, 32A9 scFv-mPE24 had superior antitumor activity compared to 42A1 scFv-mPE24. We found that 32A9 scFv-mPE24 exhibited faster degradation and drove efficient free toxin release compared to 42A1 scFv-mPE24. These phenomena were determined by the different degradation behaviors of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 in lysosomes. Moreover, 32A9 was sensitive to the low-pH environment, which made the 32A9 conjugate easily lose antigen binding and undergo degradation in lysosomes, and the free toxin was then efficiently produced to exert cytotoxicity, whereas 42A1 was resistant to the acidic environment, which kept the 42A1 conjugate relatively stable in lysosomes and delayed the release of free toxin.ConclusionsThese results showed that a low pH-sensitive antibody-based immunotoxin degraded faster in lysosomes, caused effective free toxin release, and led to improved cytotoxicity compared to an immunotoxin based on a normal antibody. Our findings suggested that a low pH-sensitive antibody might have an advantage in the design of immunotoxins and other lysosomal degradation-dependent antibody conjugate drugs.

A template-based approach to the synthesis of 1,7-dioxaspiro[5.5]undecane spiroketal derivatives

This work investigates the preparation of 1,7-dioxaspiro [5.5]undecane derivatives via the application of the template compound 2,8-bis(bromomethyl)-1,7-dioxaspiro [5.5]undecane and a series of catalysed carbonyl-ene reactions introducing aromatic and non-aromatic linkers to the heterocyclic system, providing an alternative approach to various marine toxin fragments.

Cardiac Inflammation As an Underlying Trigger for the Pathological Deficits Associated with Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disorder that causes cardiomyocyte loss, resulting in myocardial fibro-fatty deposition leading to cardiac dysfunction and sudden cardiac death, particularly in young patients and athletes, with no available treatments. The genetics of ARVC follow an autosomal dominant pattern, although severe cases (especially in pediatric populations) have been reported as recessive. Mutations in genes encoding components of desmosomes (specialized cell-cell junction that act as mechanical anchor between cardiac muscle cells) resulting in loss of desmosomal proteins underlie approximately 40-60% genetic cases of ARVC. Studies highlight that cardiac inflammation may be a critical and early component of ARVC as patients harboring mutations in the desmosomal gene desmoplakin (DSP) present with recurrent myocardial episodes (observed as myocarditis); however, the mechanisms and whether inflammation is a driver of ARVC remain unclear. We hypothesize that desmosomal gene mutations/loss/byproducts alter the acute inflammatory response on the cardiomyocyte cell surface leading to chronic immune intolerance and triggering of ARVC disease. To test our hypothesis, we injected DSP heterozygous deficient mice (harboring 50% loss of DSP without overt signs of ARVC) with a fragment of α-myosin and pertussis toxin to stimulate the immune system to assess its impact on ARVC pathogenesis. We show that DSP heterozygous mice exposed to acute immunological challenge triggered a destructive cardiomyocyte immune response in DSP heterozygous deficient hearts, reminiscent of fulminant myocarditis. Furthermore, we show that this acute immunological challenge was sufficient to trigger classic ARVC disease features in DSP heterozygous deficient mice as exemplified by: (i) enlarged ventricular (mostly right ventricular) chambers and heart size, (ii) fibrotic remodeling, with more clearly striking effects in the right ventricle (RV), (iii) epicardial fat tissue accumulation, (iv) electrophysiological deficits, such as premature ventricular contractions and QRS prolongation as well as (v) right and left ventricular physiological deficits associated with the bi-ventricular form of ARVC, although the RV appeared to be more significantly impacted. Our studies highlight the development of a novel DSP-related model of myocarditis. Using this model, we show that immunological stress is sufficient to lead to loss of cardiomyocyte immune tolerance in DSP heterozygous deficient mice with compromised desmosomal integrity to drive ARVC pathogenesis. More broadly these studies suggest that the immune system maybe an important driver of ARVC pathogenesis in patients with underlying desmosomal gene mutations/heterozygous loss, especially in the setting of autosomal dominant genetics. Work in the laboratory of FS is supported by grants from the NIH (HL142251, HL162369) and industry (LEXEO Therapeutics Inc.). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Multiomic Approach for Bioprospection: Investigation of Toxins and Peptides of Brazilian Sea Anemone Bunodosoma caissarum.

Sea anemones are sessile invertebrates of the phylum Cnidaria and their survival and evolutive success are highly related to the ability to produce and quickly inoculate venom, with the presence of potent toxins. In this study, a multi-omics approach was applied to characterize the protein composition of the tentacles and mucus of Bunodosoma caissarum, a species of sea anemone from the Brazilian coast. The tentacles transcriptome resulted in 23,444 annotated genes, of which 1% showed similarity with toxins or proteins related to toxin activity. In the proteome analysis, 430 polypeptides were consistently identified: 316 of them were more abundant in the tentacles while 114 were enriched in the mucus. Tentacle proteins were mostly enzymes, followed by DNA- and RNA-associated proteins, while in the mucus most proteins were toxins. In addition, peptidomics allowed the identification of large and small fragments of mature toxins, neuropeptides, and intracellular peptides. In conclusion, integrated omics identified previously unknown or uncharacterized genes in addition to 23 toxin-like proteins of therapeutic potential, improving the understanding of tentacle and mucus composition of sea anemones.

Electroacupuncture Ameliorates Depression-Like Behaviors Comorbid to Chronic Neuropathic Pain via Tet1-Mediated Restoration of Adult Neurogenesis.

Although electroacupuncture (EA) stimulation is a widely used therapy for chronic pain and comorbid psychiatric disorders, its long-term effects on chronic neuropathic pain-induced depression and the underlying mechanisms remain elusive. In the present study, we found that EA stimulation was able to restore adult neurogenesis in the ventral dentate gyrus (DG), by both increasing neuronal differentiation and restoring the normal morphology of newborn dendrites, in mice with spared nerve injury surgery. By ablating the Nestin+ neural stem cells (NSCs) via diphtheria toxin fragment A expression, we further proved that neurogenesis in the ventral DG was crucial to the long-term, but not the immediate antidepressant effect of EA, nor was it associated with nociception. Furthermore, we found that the restoration of neurogenesis was dependent on Tet1-mediated epigenetic modification upon EA treatment. Tet1 could bind to the promoter of the Prox1 gene, thus catalyzing its demethylation and facilitating its expression, which finally contributed to the restoration of neurogenesis and amelioration of depression-like behaviors induced by chronic neuropathic pain. Thus, we conclude that EA stimulation restores inhibited Tet1 expression in hippocampal NSCs of mice with chronic neuropathic pain, and increased Tet1 expression ameliorates hypermethylation of Prox1 and restores normal adult neurogenesis in the ventral DG, which contributes to the long-term antidepressant effect of EA.

Tendon Cells Root Into (Instead of Attach to) Humeral Bone Head via Fibrocartilage-Enthesis.

Large joints are composed of two closely linked cartilages: articular cartilage (AC; rich in type II collagen, a well-studied tissue) and fibrocartilaginous enthesis (FE; rich in type I collagen, common disorder sites of enthesopathy and sporting injuries, although receiving little attention). For many years, both cartilages were thought to be formed by chondrocytes, whereas tendon, which attaches to the humeral bone head, is primarily considered as a completely different connective tissue. In this study, we raised an unconventional hypothesis: tendon cells directly form FE via cell transdifferentiation. To test this hypothesis, we first qualitatively and quantitatively demonstrated distinct differences between AC and FE in cell morphology and cell distribution, mineralization status, extracellular matrix (ECM) contents, and critical ECM protein expression profiles using comprehensive approaches. Next, we traced the cell fate of tendon cells using ScxLin (a tendon specific Cre ScxCreERT2; R26R-tdTomato line) with one-time tamoxifen induction at early (P3) or young adult (P28) stages and harvested mice at different development ages, respectively. Our early tracing data revealed different growth events in tendon and FE: an initial increase but gradual decrease in the ScxLin tendon cells and a continuous expansion in the ScxLin FE cells. The young adult tracing data demonstrated continuous recruitment of ScxLin cells into FE expansion during P28 and P56. A separate tracing line, 3.2 Col 1Lin (a so-called "bone-specific" line), further confirmed the direct contribution of tendon cells for FE cell formation, which occurred in days but FE ECM maturation (including high levels of SOST, a potent Wnt signaling inhibitor) took weeks. Finally, loss of function data using diphtheria toxin fragment A (DTA) in ScxLin cells demonstrated a significant reduction of ScxLin cells in both tendons and FE cells, whereas the gain of function study (by stabilizing β-catenin in ScxLin tendon cells via one-time injection of tamoxifen at P3 and harvesting at P60) displayed great expansion of both ScxLin tendon and FE mass. Together, our studies demonstrated that fibrocartilage is an invaded enthesis likely originating from the tendon via a quick cell transdifferentiation mechanism with a lengthy ECM maturation process. The postnatally formed fibrocartilage roots into existing cartilage and firmly connects tendon and bone instead of acting as a simple attachment site as widely believed. We believe that this study will stimulate more intense exploring in this understudied area, especially for patients with enthesopathy and sporting injuries.

An electrochemiluminescence assay for quantification of Denileukin Diftitox and its anti-drug antibodies in rat serum

Denileukin Diftitox (DD), comprising fragments of diphtheria toxin (DT) and interleukin-2 (IL2), was developed for the treatment of lymphoma and has been approved for marketing in Japan. Toxicological evaluation including pharmacokinetics and immunogenicity in preclinical animals is important for drug development and thus the assays of DD and anti-drug antibody (ADA) were developed by electrochemiluminescence (ECL) detection. For the DD assay, ruthenium-labeled anti-DT Ab and biotinylated anti-IL2 Ab were mixed with serum samples and the mixture was captured by streptavidin-coated wells for ECL detection. For the ADA assay, signals of immuno-complex of biotinylated DD, ruthenium-labeled DD, and ADA, bound to streptavidin plate were determined. DD was quantifiable from 10 ng/mL. Accuracy and precision of quality control samples were within ±20% and 20%, respectively, and stability of DD in rat serum was successfully assessed. Precision of positive control samples of ADA was within the acceptance criteria and cut point values for ADA detection in the screening and confirmatory assay were determined by statistical analysis. Drug-induced ADA was detected by screening assay followed by confirmatory assay. The developed method was successfully applied to assess pharmacokinetics and immunogenicity to support toxicity studies in rats.

Safety and Tolerability of E7777 (improved purity Denileukin diftitox [ONTAK]) in Patients with Relapsed or Refractory Cutaneous T-Cell Lymphoma: Results from Pivotal Study 302

Introduction: Denileukin diftitox (Dd), a recombinant fusion protein composed of diphtheria toxin fragments and human interleukin-2 was approved and marketed (as ONTAK) in the US from 1999-2014 for the treatment of relapsed/refractory CTCL. Manufacturing improvements (to decrease the presence of misfolded and aggregated proteins) resulted in a new, more bioactive formulation, E7777. It is considered a new drug by the FDA and required a new registrational clinical trial. Study 302 (NCT01871727) is a multicenter, open-label, single-arm registrational trial in which the primary efficacy and safety of E7777 were assessed. Here, we report the safety results of E7777. Methods: E7777 was given at 9 mcg/kg/day for 5 days every 21 days up to 8 cycles in patients with relapsed/refractory CTCL. Key inclusion/exclusion criteria: histopathologic diagnosis of CTCL; ECOG PS 0 or 1; no prior exposure to ONTAK. Evaluation of safety included incidence and severity of treatment-emergent AEs (TEAEs), and adverse events of special interest (AESIs). Key AESIs include capillary leak syndrome (CLS), infusion reaction, visual impairment (these three AEs were listed as Box warnings in the ONTAK label); and hepatotoxicity. Results: The Primary Efficacy endpoint, ORR (95% CI) by IRC, was 36.2% (95% CI: 25.0%, 48.7%), with 8.7% achieving a CR; detailed efficacy data will be presented in a separate abstract. In the subset of patients with Stage I-III disease (n=69), the median age was 64 years and the median number of E7777 cycles received was 6 (range 1 to 42); 66 patients had mycosis fungoides and 3 had Sezary syndrome, and 39 (57%) had disease of stage IIb or worse. The most common TEAEs were nausea (43.5%); fatigue (31.9%); and increased ALT, chills, and peripheral edema (27.5% each). Thirty patients (43.5%) had a Grade ≥3 TEAE (90% Grade 3; 10% Grade 4)). The most common serious adverse events (≥ 5%) were capillary leak syndrome (10%) and infusion reactions (9%). Overall, the mean numbers of TEAEs per subject were higher in the first 1 to 2 treatment cycles. Most patients [92.8%] had at least 1 AESI (mostly Grade 1/2). Overall, 22 patients (31.9%) experienced AESIs that were Grade ≥3; (7.2%) had drug discontinuation; (4.3%) had drug dose reduction, and (26.1%) had drug dose interruption. Fourteen patients (20.3%) had CLS. CLS was defined as the occurrence of at least 2 of the following: hypotension, edema, or serum albumin < 3.0 g/dL. CLS was Grade 1 in 2.9%, Grade 2 in 11.6%, Grade 3 in 4.3%, and Grade 4 in 1.4%. Nine patients (13.0%) underwent drug modification: (4.3%) discontinued E7777; and (10.1%) had either dose reduction or temporary dose interruption. CLS typically occurred in the first 1/2 cycles. Risk/severity of CLS was mitigated by fluid management; confirmation of serum albumin levels (≥ 3.0 g/dl); close monitoring of weight, edema, and BP; early drug interruption; and rapid initiation of diuretic therapy on recovery. Fifty-one patients (73.9%) had an AESI related to infusion reaction. Grade 1 in 43.5%, Grade 2 in 26.1%, and Grade 3 in 4.3%. One patient (1.4%) had study drug discontinuation, and (11.6%) had either study drug dose reduction or interruption. In the event of infusion-related reactions, systemic corticosteroids may be added to premedication for subsequent E7777 infusions. Nine patients (13.0%) had an event related to visual impairment; all were AEs of blurred vision, which was Grade 1 in 11.6% and Grade 2 in 1.4%; no Grades 3, 4, or 5. One patient (1.0%) had drug interruption; no events led to study drug dose reduction. Twenty-five patients (36.2%) had AESI related to hepatoxicity. The TEAEs were Grade 1 in 17.4%, Grade 2 in 7.2%, Grade 3 in 11.6%, and no Grade 4/5. The majority of hepatic adverse events were elevations in transaminases that occurred within the first or second cycle, resolved without medical intervention, and did not require treatment discontinuation. Conclusion: No new safety signals were observed with E7777 when compared to the safety profile of ONTAK. There is no evidence of cumulative toxicity. Most patients had at least 1 TEAE which were mostly Grade 1/2. Specific AESIs: CLS, infusion reactions, and visual impairment (prior ONTAK Box warnings) were mostly Grade 1/2 and effectively managed. Overall, E7777 was well-tolerated with the use of pre-medications, close patient monitoring, and prompt initiation of supportive measures and drug management. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Production of a Novel Multi-Epitope Peptide Vaccine against Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the prevalent cancers in the world with a high recurrence rate. In recent years, different researches have focused on designing efficient multi-epitope peptide vaccines against HCC. In designing these vaccines, over-expressed antigens in HCC patients, such as α- fetoprotein (AFP) and glypican-3 (GPC-3), have been employed. In our previous study, a multi-epitope peptide vaccine for HCC was designed by in-silico methods. The designed vaccine construct included the AFP, GPC-3, and aspartyl-β-hydroxylase (ASPH) as CytoLoxic T cell Lymphocytes (CTL), one epitope from Tetanus Toxin Fragment C (TTFrC) as Helper T cell Lymphocytes (HTL), and a segment of microbial heat shock protein (HSP70) peptide407-426 as an adjuvant. All the mentioned parts were connected by appropriate linkers. The aim of this study is the production of the designed vaccine. This research is experimental and was carried out in Fasa, Iran, in 2017. The designed vaccine construct gene was transformed to the Escherchia coli BL21 (DE3) strain and expressed in different isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations (0.6 and 1 mM), times (4, 6, 8, 16 hours), and temperatures (25 and 37 °C). Then, the expressed protein was analyzed by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot methods. The best conditions for protein expression were obtained in the Super Optimal Broth (SOB) medium at 37 °C after the induction of expression by 1 mM IPTG for six hour. The recombinant HCC vaccine was produced with a proper concentration.

Heterologous expression of Ts8, a neurotoxin from Tityus serrulatus venom, evidences its antifungal activity

In this study we expressed the Ts8, a neurotoxin from Tityus serrulatus scorpion venom, in Pichia pastoris yeast. We evaluated the peptide expression in different conditions, such as pH, temperature, and addition of casamino acids supplement. Analyses of expressed products by mass spectrometry and Edman degradation showed that rTs8 has sites that allow its cleavage by yeast proteases released into the culture medium. The casamino acids addition was favourable for toxin expression, however, was not sufficient to minimize proteolytic degradation. Functional assays with recombinant toxin fragments and native toxins have demonstrated the release of cytokines such as TNF-α and IL-1β in some peptides tested. In addition, the toxins were shown to inhibit the Pichia pastoris growth in antifungal test and were not toxic to alveolar macrophages cells at the concentrations analyzed The electrophysiological screening, by voltage clamp technique, showed that the rTs8 fragment with the highest molecular weight inhibited the Kv1.3 channel, whereas the N-terminal fragment had no activity on the ion channels tested.

Molecular characterization of Virulence genes Shiga-like, Heat-labile Toxins and Antibiotics resistance in multidrug-resistant Escherichia coli

Escherichia coli is a part of human intestinal flora and considered the second most common death factor between children younger than five years. Escherichia coli Shiga-like, Heat-labile toxins and multidrug-resistant are important virulence factors. Therefore, the main targets of this work were molecular diagnosis and characterization of toxine genes Shiga-like and Heat-labile and resistance genes of ciprofloxacin and gentamicin on molecular basis for Escherichia coli isolated from children stool sample in Egypt. The gene detection was carried out using two different approaches which were using chromosome and plasmid. Escherichia coli isolates were resistant to 24 antibiotics including four categories based on their functions. For example, ciprofloxacin, gentamicin, novobiocin and vancomycin. All of tested strains showed band near 300 bp that represents of shiga-like toxin (SLT) gene in their genomic DNA. Heat-labile toxin (LT) fragment ~ 200 bp was detected in plasmid of strain 8H and 8G. Moreover, gentamycin resistance fragment (aac C2) was detected in chromosomal DNA of all strains as a single robust fragment of molecular weight ~ 856 bp. In the case of ciprofloxacin-resistance gene a slight amplification was detected in strain 8G as well as 8H at about 1 kb and 800 bp in genomic, while it was absent in plasmids of tested strains.