Cytolytic Protein Research Articles

Glycogen Synthase Kinase-3 (GSK-3) Regulation of Inhibitory Coreceptor Expression in T-cell Immunity.

The serine/threonine kinase, glycogen synthase kinase 3 (GSK-3) has been implicated in immune cell activation and function. Our recent studies have shown that the abrogation of GSK-3 activity down-regulates the expression of key inhibitory receptors PD-1 and LAG-3. It also regulates the expression of the transcription factor NFAT which, in turn, is responsible for inhibiting PD-1/LAG-3 transcription as well as activating the expression of cytolytic effector proteins such as perforin and granzyme B. The role of components of the Wnt signaling pathway in these events remains to be fully uncovered. This mini-review discusses the recent discoveries that have elucidated the role of the GSK-3 signaling pathway in cancer immunotherapy.

Determination and Quantitation of Cytotoxic T Cell-Mediated Cell Death.

Cytotoxic T cell-induced cell death is well documented. Cytotoxic T cell releases various cytolytic proteins. The cytolytic proteins induce target cell death. T cell-induced cell death can be measured by the lytic assay. One of the well-known lytic assays uses radioactive tracer, Chromium-51 (51Cr), and detects the amount of 51Cr released from target cells. This assay can detect cell death and the efficiency of the T cell-induced cell death by coculture effector cells (T cells) and target cells. This assay can determine the kinetics of the cell lysis. The issue of this approach is the use of radioactive material. This chapter describes measuring T cell-induced cell death by determining the epigenetic remodeling and the release of cytolytic proteins. Determine the efficiency of T cell-induced cell death by using a flow cytometry-based detection method.

Abstract PO-007: The Class I/IV HDAC Inhibitor Mocetinostat Augments Anti-Tumor Immune Responses in Melanoma Patients

Abstract While immune checkpoint inhibitor (CPI) therapies have revolutionized the treatment of metastatic melanoma and other malignancies, many patients fail to respond, necessitating to explore combination therapies to improve response rates. Histone deacetylases (HDACs) are epigenetic modifying proteins that remove acetyl groups from histones and non-histone proteins. HDAC inhibitors (HDACi) have shown success in the treatment of several hematologic malignancies. Beyond direct tumor cytotoxicity, HDACi have also demonstrated immunomodulatory effects. We previously published data showing the anti-tumor efficacy of the class I/IV HDACi mocetinostat in pre-clinical murine models through upregulation of tumor antigen presentation machinery and downregulation of Tregs. As a result of these and other data, a phase Iβ clinical trial (NCT03565406) was initiated to evaluate combination of mocetinostat with nivolumab and ipilimumab for the treatment of unresectable stage III/IV metastatic melanoma. A total of 10 patients were treated. Objective responses by RECIST 1.1 criteria were seen in 7/10 patients; 2 patients had complete responses, 5 had partial responses and 3 had progressive disease. To further understand the impact of mocetinostat on immune function, we evaluated serum and peripheral blood (PBMC) samples from patients prior to and on-treatment, in addition to in vitro treatment of treatment-naive patient PBMC samples with mocetinostat. Both in vitro treated and in vivo paired patient samples demonstrated a decrease in M2-like monocytes (CD80-CD86-CD163+CD206+) and an increase in M1-like monocytes (CD80+CD86+CD163-CD206-). We also observed a decrease in myeloid derived suppressor cells (MDSC, CD14+CD11b+HLA-DRlow) and expression of NOS2, an enzyme involved in MDSC mediated T-cell suppression, in this subset in both treated patients in vivo and in vitro. CD4+ and CD8+ T-cells showed increased levels of the proliferation marker Ki67 on-treatment relative to baseline in vivo in treated patients. Analysis of patient serum levels showed an increase in Granzyme A, Granzyme B, IFNγ and TNF. Similar upregulation of inflammatory and cytolytic proteins were seen in vitro. Assessment of histone 3 acetylation levels showed robust increases in all patients assessed on-treatment except 1 patient (progressive disease). Combining the results of in vitro assays with patient sample analyses, these data suggest that mocetinostat decreases immunosuppressive immune cell phenotypes while augmenting anti-tumor phenotypes, demonstrating potential of combination therapy with CPI. Citation Format: David M. Woods, Andressa S. Laino, Melinda Vassallo, Jeffrey Weber. The Class I/IV HDAC Inhibitor Mocetinostat Augments Anti-Tumor Immune Responses in Melanoma Patients [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-007.

Genomic characterization of methicillin-susceptible Staphylococcus aureus – An emerging aetiologic agent of concurrent bacteraemia in dengue patient

Background: Concurrent bacteraemia in dengue patient is uncommon but most often will result in severe adverse clinical outcome. The major bacterial aetiologic agents of dengue dual infections (DDIs) are members of the Enterobacteriaceae family. During recent years, Staphylococcus aureus (S. aureus) has emerged as an important bacterial pathogen causing DDIs. In cases of staphylococcal co-infection in dengue patients, the methicillin-susceptible phenotype seems to predominate over the resistant phenotype, although the reason behind this phenomenon remains obscured. Therefore, we aim to investigate the genomic features of a methicillin-susceptible S. aureus (MSSA) strain isolated from a dengue patient, in order to elucidate the possible genetic drives for the pathogenicity of this organism. Methods and materials: A methicillin-susceptible S. aureus strain (referred as HS-MSSA) was isolated from the blood culture of a patient initially diagnosed with dengue fever, but later succumbed to septic shock within 24 h post-admission. Antimicrobial susceptibility test showed that the HS-MSSA strain was pan-susceptible to all tested antibiotics. The genomic DNA of the HS-MSSA strain was extracted and subjected to whole genome sequencing and genomic analyses. Results: The draft genome of the HS-MSSA strain is 2.78 Mb in size, harbouring 2643 coding sequences (CDSs). A comparative analysis with the S. aureus reference genome NCTC8325 showed 2415 common genes. A total of 118 CDSs were found unique to the HS-MSSA genome, comprising of mainly phage proteins, mobile genetic elements, and efflux pumps. Majority of the unique CDSs were found in two genomic regions with considerable genetic variation when aligned to the reference genome NCTC8325. One of the genomic regions was found showing greater genetic similarity to methicillin-resistant S. aureus (MRSA) genomes in the NCBI Genbank repository, and harboured multiple phage-associated virulence factors (β-hemolysin, cytolytic pore-forming proteins, etc.). Another genomic region (contig-13) resembled an insertion on which multiple exotoxins and super-antigenic enterotoxins (SEC3 and SElL) were found encoded by two pathogenicity islands, namely SaPI and SaPIn2. Conclusion: We infer that the gaining of multiple virulence determinants during the evolution of the MSSA population might have caused the organism to be more invasive and pathogenic when compared to its antimicrobial-resistant counterpart.

MR1-Restricted T Cells with MAIT-like Characteristics Are Functionally Conserved in the Pteropid Bat Pteropus alecto.

SummaryBats are reservoirs for a large number of viruses which have potential to cause major human disease outbreaks, including the current coronavirus disease 2019 (COVID-19) pandemic. Major efforts are underway to understand bat immune response to viruses, whereas much less is known about their immune responses to bacteria. In this study, MR1-restricted T (MR1T) cells were detected through the use of MR1 tetramers in circulation and tissues of Pteropus alecto (Pa) bats. Pa MR1T cells exhibited weak responses to MR1-presented microbial metabolites at resting state. However, following priming with MR1-presented agonist they proliferated, upregulated critical transcription factors and cytolytic proteins, and gained transient expression of Th1/17-related cytokines and antibacterial cytotoxicity. Collectively, these findings show that the Pa bat immune system encompasses an abundant and functionally conserved population of MR1T cells with mucosal-associated invariant T-like characteristics, suggesting that MR1 and MR1T cells also play a significant role in bat immune defense.

Development of a Molecular Imprinting-Based Surface Plasmon Resonance Biosensor for Rapid and Sensitive Detection of Staphylococcus aureus Alpha Hemolysin From Human Serum.

Stapylococcus aureus is a common infectious agent in e.g. sepsis, associated with both high mortality rates and severe long-term effects. The cytolytic protein α-hemolysin has repeatedly been shown to enhance the virulence of S. aureus. Combined with an unhindered spread of multi drug-resistant strains, this has triggered research into novel anti virulence (i.e. anti α-hemolysin) drugs. Their functionality will depend on our ability to identify infections that might be alleviated by such. We therefore saw a need for detection methods that could identify individuals suffering from S. aureus infections where α-hemolysin was a major determinant. Molecular imprinted polymers were subsequently prepared on gold coated sensor chips. Used in combination with a surface plasmon resonance biosensor, α-hemolysin could therethrough be quantified from septic blood samples (n = 9), without pre-culturing of the infectious agent. The biosensor recognized α-hemolysin with high affinity (KD = 2.75 x 10-7 M) and demonstrated a statistically significant difference (p < 0.0001) between the α-hemolysin response and potential sample contaminants. The detection scheme proved equally good, or better, when compared to antibody-based detection methods. This novel detection scheme constitutes a more rapid, economical, and user-friendly alternative to many methods currently in use. Heightening both reproducibility and sensitivity, molecular imprinting in combination with surface plasmon resonance (SPR)-technology could be a versatile new tool in clinical- and research-settings alike.

Natural killer cell-derived extracellular vesicles in cancer therapy.

Cancer is a major cause of death in the industrialized world. New therapies are constantly being developed in order to reduce morbidity and mortality. NK cell-based cellular therapies have shown effect against haematological malignancies, but it has been difficult to target solid tumours due to low NK cell infiltration of the tumour and efficient tumour evasion strategies. NK cells release extracellular vesicles that naturally contain cytolytic proteins and tumour-targeting molecules. These vesicles can directly interact with and kill malignant cells, and their small size could allow more efficient extravasation into the tumour tissue. Extracellular vesicles are also less sensitive to the hostile tumour microenvironment compared to cells. Based on their features, NK cell-derived extracellular vesicles represent promising novel tools in oncology. In this review, we summarize the current available literature on NK cell-derived extracellular vesicles and discuss how they may be utilized in therapy for solid tumours.

Tobacco smoke and morphine alter peripheral and CNS inflammation following HIV infection in a humanized mouse model

Tobacco smoking is common in HIV-infected patients, and is prevalent among intravenous opiate abusers. Conversely, intravenous opiate abusers are more likely HIV-infected, and opiate abuse is associated with more severe neuroinflammation. Given the coincident use of tobacco smoking among HIV-infected intravenous drug users (IVDUs), we set out to study the effects of smoke exposure, chronic morphine administration, and HIV infection using the NSG humanized mouse model. Our results show that smoke, morphine, and the combination promotes the decline in CD4+ T cells in HIV-infected mice. Further, chronic morphine administration increases the numbers of circulating CD8+ T cells which express the inhibitory receptor PD-1, as well as the cytolytic proteins perforin and granzyme B in the infected mice. We also found that the combination of smoke and morphine inhibited the expression of IL-1α, IL-4 and IL-17A. Finally, the combination of smoke and morphine exposure induces microglial activation following infection, as well as in the absence of HIV infection. To our knowledge, this is the first report to assess the combined effects of smoke and chronic morphine exposure on the inflammation associated with HIV infection, and demonstrate that these two insults exert significant neuroinflammatory activity.

Abstract LB-145: Immunomodulatory activity of selective DRD2 antagonist ONC201, an imipridone, in metastatic endometrial cancer (mEC)

Abstract ONC201 is a small molecule DRD2 antagonist and ClpP agonist that has prolonged antitumor efficacy and immunomodulatory properties in preclinical models. The enhanced immune modulatory nature of ONC201 was observed in a Phase I study that revealed increased infiltration of granzyme B+ and CD56+ cells, corroborating preclinical reports of NK cell intratumoral infiltration in response to ONC201. A broad induction of immune cytokines and effector molecules was observed in patients treated with ONC201, in particular among patients who experienced at least stable disease by RECIST for 12 or more weeks. Patients who had a &amp;gt; 50% induction in serum perforin, a cytolytic protein found in the granules of cytotoxic T lymphocytes (CTLs) and NK cells, upon ONC201 administration had better outcomes. Regressions in individual metastatic lesions were noted in a small number of EC patients. A multi-center Phase 2 study to explore the efficacy of weekly ONC201 in recurrent or metastatic Type II EC enrolled 5 patients with recurrent EC who underwent paired tumor biopsies before and 9 days after ONC201 initiation. Four of the 5 patients exhibited a 2-5-fold increase in CD4+ Thelper cells in their tumors and the majority of these patients also had CD4+FoxP3+ cells, indicating a significant population of Tregulatory cells (Treg). One patient exhibited an 80% increase in CD8+ T-cells in their tumor 8 days after initiating ONC201. Two of the patients had &amp;gt;2 fold induction of intratumoral granzyme B expression that was surprisingly not accompanied by NK cell infiltration, as assessed by CD56 staining. These results may be consistent with innate immune system activation by ONC201 that results in T-cell modulation. The multi-fold increase in Treg expression could be suppressing tumor cell death. As such, ONC201 dose intensification or combination with immunotherapy are being evaluated for ONC201 in the context of endometrial cancer. Citation Format: Rohinton S. Tarapore, Varun V. Prabhu, Samuel C. Mok, Chilam Au Yeung, Lorna Rodriguez, Eugenia Girda, Gina Mantia-Smaldone, Victoria Bae-Jump, Guangrong Lu, Wolfgang Oster, Joshua E. Allen. Immunomodulatory activity of selective DRD2 antagonist ONC201, an imipridone, in metastatic endometrial cancer (mEC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-145.

Targeted Delivery of CXCL9 and OX40L by Mesenchymal Stem Cells Elicits Potent Antitumor Immunity

Major obstacles in immunotherapies include toxicities associated with systemic administration of therapeutic agents, as well as low tumor lymphocyte infiltration that hampers the efficacies. In this study, we report a mesenchymal stem cell (MSC)-based immunotherapeutic strategy in which MSCs specifically deliver T/natural killer (NK) cell-targeting chemokine CXCL9 and immunostimulatory factor OX40 ligand (OX40L)/tumor necrosis factor superfamily member 4 (TNFSF4) to tumor sites in syngeneic subcutaneous and azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced spontaneous colon cancer mouse models. This approach generated potent local antitumor immunity by increasing the ratios of tumor-infiltrating CD8+ T and NK cells and production of antitumor cytokines and cytolytic proteins in the tumor microenvironment. Moreover, it improved the efficacy of programmed death-1 (PD-1) blockade in a syngeneic mouse model and significantly suppressed the growth of major histocompatibility complex class I (MHC class I)-deficient tumors. Our MSC-based immunotherapeutic strategy simultaneously recruits and activates immune effector cells at the tumor site, thus overcoming the problems with toxicities of systemic therapeutic agents and low lymphocyte infiltration of solid tumors.

Targeting of Perforin Inhibitor into the Brain Parenchyma Via a Prodrug Approach Can Decrease Oxidative Stress and Neuroinflammation and Improve Cell Survival

The cytolytic protein perforin has a crucial role in infections and tumor surveillance. Recently, it has also been associated with many brain diseases, such as neurodegenerative diseases and stroke. Therefore, inhibitors of perforin have attracted interest as novel drug candidates. We have previously reported that converting a perforin inhibitor into an L-type amino acid transporter 1 (LAT1)-utilizing prodrug can improve the compound’s brain drug delivery not only across the blood–brain barrier (BBB) but also into the brain parenchymal cells: neurons, astrocytes, and microglia. The present study evaluated whether the increased uptake into mouse primary cortical astrocytes and subsequently improvements in the cellular bioavailability of this brain-targeted perforin inhibitor prodrug could enhance its pharmacological effects, such as inhibition of production of caspase-3/-7, lipid peroxidation products and prostaglandin E2 (PGE2) in the lipopolysaccharide (LPS)-induced neuroinflammation mouse model. It was demonstrated that increased brain and cellular drug delivery could improve the ability of perforin inhibitors to elicit their pharmacological effects in the brain at nano- to picomolar levels. Furthermore, the prodrug displayed multifunctional properties since it also inhibited the activity of several key enzymes related to Alzheimer’s disease (AD), such as the β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), acetylcholinesterase (AChE), and most probably also cyclooxygenases (COX) at micromolar concentrations. Therefore, this prodrug is a potential drug candidate for preventing Aβ-accumulation and ACh-depletion in addition to combatting neuroinflammation, oxidative stress, and neural apoptosis within the brain.Graphical abstract

Immunogenicity of Immunotoxins Containing Pseudomonas Exotoxin A: Causes, Consequences, and Mitigation.

Immunotoxins are cytolytic fusion proteins developed for cancer therapy, composed of an antibody fragment that binds to a cancer cell and a protein toxin fragment that kills the cell. Pseudomonas exotoxin A (PE) is a potent toxin that is used for the killing moiety in many immunotoxins. Moxetumomab Pasudotox (Lumoxiti) contains an anti-CD22 Fv and a 38 kDa portion of PE. Lumoxiti was discovered in the Laboratory of Molecular Biology at the U.S. National Cancer Institute and co-developed with Medimmune/AstraZeneca to treat hairy cell leukemia. In 2018 Lumoxiti was approved by the US Food and Drug Administration for the treatment of drug-resistant Hairy Cell Leukemia. Due to the bacterial origin of the killing moiety, immunotoxins containing PE are highly immunogenic in patients with normal immune systems, but less immunogenic in patients with hematologic malignancies, whose immune systems are often compromised. LMB-100 is a de-immunized variant of the toxin with a humanized antibody that targets mesothelin and a PE toxin that was rationally designed for diminished reactivity with antibodies and B cell receptors. It is now being evaluated in clinical trials for the treatment of mesothelioma and pancreatic cancer and is showing somewhat diminished immunogenicity compared to its un modified parental counterpart. Here we review the immunogenicity of the original and de-immunized PE immunotoxins in mice and patients, the development of anti-drug antibodies (ADAs), their impact on drug availability and their effect on clinical efficacy. Efforts to mitigate the immunogenicity of immunotoxins and its impact on immunogenicity will be described including rational design to identify, remove, or suppress B cell or T cell epitopes, and combination of immunotoxins with immune modulating drugs.

Microglia complement astrocytes in neuromyelitis optica.

Neuromyelitis optica (NMO) is a central nervous system (CNS) inflammatory autoimmune disease caused by antibodies against aquaporin-4 (AQP4) expressed on astrocytes. Binding of AQP4-specific antibodies (NMO-IgG) triggers activation of the complement cascade, which is responsible for astrocyte loss and secondary demyelination. Although the role for the cytolytic complement proteins in astrocyte destruction in NMO is well established, little is known regarding the initial phase of astrocyte injury. In this issue of the JCI, Chen and colleagues evaluated the precytolytic phase when NMO-IgG binds astrocytes in vivo in the absence of exogenous complement. NMO-IgG alone caused astrocyte activation and AQP4 loss. Surprisingly, microglia, CNS-resident innate immune cells that produce endogenous complement, were required for clinical manifestations of disease, a finding that suggests microglia may serve as a therapeutic target in NMO.

Insecticidal Activity of Bacteria from Larvae Breeding Site with Natural Larvae Mortality: Screening of Separated Supernatant and Pellet Fractions.

Mosquitoes can transmit to humans devastating and deadly pathogens. As many chemical insecticides are banned due to environmental side effects or are of reduced efficacy due to resistance, biological control, including the use of bacterial strains with insecticidal activity, is of increasing interest and importance. The urgent actual need relies on the discovery of new compounds, preferably of a biological nature. Here, we explored the phenomenon of natural larvae mortality in larval breeding sites to identify potential novel compounds that may be used in biological control. From there, we isolated 14 bacterial strains of the phylum Firmicutes, most of the order Bacillales. Cultures were carried out under controlled conditions and were separated on supernatant and pellet fractions. The two fractions and a 1:1 mixture of the two fractions were tested on L3 and early L4 Aedes albopictus. Two concentrations were tested (2 and 6 mg/L). Larvae mortality was recorded at 24, 48 and 72 h and compared to that induced by the commercialized B. thuringiensis subsp. israelensis. Of the 14 strains isolated, 11 were active against the A. albopictus larvae: 10 of the supernatant fractions and one pellet fraction, and mortality increased with the concentration. For the insecticide activity prediction in three strains of the Bacillus cereus complex, PCR screening of the crystal (Cry) and cytolytic (Cyt) protein families characteristic to B. thuringiensis subsp. israelensis was performed. Most of the genes coding for these proteins’ synthesis were not detected. We identified bacterial strains that exhibit higher insecticidal activity compared with a commercial product. Further studies are needed for the characterization of active compounds.

Human MAIT cell cytolytic effector proteins synergize to overcome carbapenem resistance in Escherichia coli.

Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1-restricted antimicrobial activity against Escherichia coli clinical strains in a manner dependent on the activity of cytolytic proteins but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to T cell receptor (TCR)-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living, extracellular forms of E. coli. Furthermore, MAIT cell-mediated bacterial control extends to multidrug-resistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli.

Mechanism of Candida pathogenesis: revisiting the vital drivers.

Candida is the most implicated fungal pathogen in the clinical setting. Several factors play important roles in the pathogenesis of Candida spp. Multiple transcriptional circuits, morphological and phenotypic switching, biofilm formation, tissue damaging extracellular hydrolytic enzymes, metabolic flexibility, genome plasticity, adaptation to environmental pH fluctuation, robust nutrient acquisition system, adherence and invasions (mediated by adhesins and invasins), heat shock proteins (HSPs), cytolytic proteins, escape from phagocytosis, evasion from host immune system, synergistic coaggregation with resident microbiota, resistance to antifungal agents, and the ability to efficiently respond to multiple stresses are some of the major pathogenic determinants of Candida species. The existence of multiple connections, in addition to the interactions and associations among all of these factors, are distinctive features that play important roles in the establishment of Candida infections. This review describes all the underlying factors and mechanisms involved in Candida pathogenesis by evaluating pathogenic determinants of Candida species. It reinforces the already available pool of data on the pathogenesis of Candida species by providing a clear and simplified understanding of the most important factors implicated in the pathogenesis of Candida species. The Candida pathogenesis network, an illustration linking all the major determinants of Candida pathogenesis, is also presented. Taken together, they will further improve our current understanding of how these factors modulate virulence and consequent infection(s). Development of new antifungal drugs and better therapeutic approaches to candidiasis can be achieved in the near future with continuing progress in the understanding of the mechanisms of Candida pathogenesis.

NATURAL KILLER CELL KILLING OF EXTRACELLULAR PSEUDOMONAS AERUGINOSA

Abstract Pseudomonas aeruginosa is an opportunistic pathogen that commonly infects individuals with the genetic illness, Cystic Fibrosis and contributes to airway blockage and loss of lung function. NK cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. NK cells, in addition to their cytotoxic effects on cancer and virally infected cells have been shown to play a role in an innate defense against microbes. Hypothesis NK cells will kill P. aeruginosa using cytolytic effector protein(perforin, granulysin or granzymes) alone or working synergistically. Results Live-cell imaging of a co-culture of YT cells, a human NK cell line, incubated with GFP P. aeruginosa in the presence of the viability dye, propidium iodide, demonstrated that YT cell killing of P. aeruginosa is contact-mediated. CRISPR knockout of granulysin or perforin in YT cells had no significant affect on NK cell killing of P. aeruginosa, as determined by CFU counts. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-Dichloroisocoumarin (DCI) to inhibit granzymes, resulted in an inhibition of killing. CRISPR knockout of granzyme B in YT cells, in addition to treatment with the granzyme A inhibitor Futhan, significantly inhibited killing of P. aeruginosa, as determined by CFU counts. These results suggest that NK cells induce membrane damage in P. aeruginosa through a contact dependent process, and that killing requires granzymes.

Isolation, Structure Determination, and Synthesis of Cyclic Tetraglutamic Acids from Box Jellyfish Species Alatina alata and Chironex yamaguchii.

Cubozoan nematocyst venoms contain known cytolytic and hemolytic proteins, but small molecule components have not been previously reported from cubozoan venom. We screened nematocyst extracts of Alatina alata and Chironex yamaguchii by LC-MS for the presence of small molecule metabolites. Three isomeric compounds, cnidarins 4A (1), 4B (2), and 4C (3), were isolated from venom extracts and characterized by NMR and MS, which revealed their planar structure as cyclic γ-linked tetraglutamic acids. The full configurational assignments were established by syntheses of all six possible stereoisomers, comparison of spectral data and optical rotations, and stereochemical analysis of derivatized degradation products. Compounds 1–3 were subsequently detected by LC-MS in tissues of eight other cnidarian species. The most abundant of these compounds, cnidarin 4A (1), showed no mammalian cell toxicity or hemolytic activity, which may suggest a role for these cyclic tetraglutamates in nematocyst discharge.

Pharmacological protection of the detoxification activity of animals’ liver with the help of phospholipid and polysaccharide nature substances complex

Currently, among chemical pollutants a significant danger to the health of animals and humans represent oxygen-containing nitrogen compounds nitrates and nitrites, which are widely used as mineral fertilizers. The article presents data on the study of chronic nitrate intoxication, reproduced in laboratory animals and its pharmacological correction with a complex of substances of phospholipid and polysaccharide nature. The introduction of sodium nitrate to non-linear rats for 30 days at a dose of 3.8 mg per animal leads to the development of intoxication in rats with dominant signs of liver damage. On this background, the use of the complex of beet fiber and rapeseed lecithin leads to a weakening of nitrate toxicosis, which is demonstrated by an increase in rats’ body weight, weakening of the clinical manifestations of intoxication, a decrease in cytolytic syndrome, intrahepatic cholestasis and impaired protein synthesis function of liver.

P28-Fur-Grb, A Novel Fusion Protein with Enhanced Targeted Cytotoxicity Against Breast Cancer

Targeting tumor cells via multiple pathways promises the emergence of a new era in cancer therapy. Consisting of a cell-binding ligand and a cytotoxic moiety, cytolytic fusion proteins can selectively bind and kill target cells with minimal adverse effects. We designed a novel immunoproapoptotic fusion protein, p28-fur-GrB, composed of the cancer-specific azurin-derived cell penetrating peptide, p28, and a mutant version of human serine protease granzyme B. The two moieties were genetically fused by a furin sensitive linker, allowing in vivo cleavage and activation of the immunotoxin after cell entry. Synthesized coding gene of the recombinant protein was cloned and expressed in HEK293T cells, and nickel chromatography was applied for protein purification. After in vitro furin cleavage and primary analyses of SDS-PAGE, Western blotting, GrB activity and ELISA binding assay, the fusion protein was tested for its cytotoxicity on various breast cancer cell lines. Suppression of cell proliferation and viability was evaluated using the WST-1 assay. Furthermore, DNA fragmentation was measured as an indication of apoptotic effects of the fusion protein on treated cells. Based on our results, p28-fur-GrB was efficiently cleaved by furin and showed high GrB activity and binding affinity after cleavage. Following 72h of incubation with IC50 values of the fusion protein, significant cytotoxic effects of 80.6%, 77.1%, 74% and 69.6% were recorded for BT474, MCF7, SK-BR-3 and MDA-MB-231 tumor cells, respectively. Proliferative potential of MCF 10A normal cells was not affected by the treatment. Analysis of the rate of apoptosis in treated cells confirmed our cytotoxicity results. We concluded that p28-fur-GrB is a potent anti-tumor agent with high cytotoxicity against breast cancer cells.