Lipid-polymer hybrid nanoparticles loaded with N-acetylcysteine for the modulation of neuroinflammatory biomarkers in human iPSC-derived PSEN2 (N141I) astrocytes as a model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive impairment associated with the accumulation of beta-amyloid protein (Aß). Aß activates glial cells in the brain, increasing the secretion of proinflammatory cytokines, which leads to neuroinflammation and neuronal death. Currently, there are no effective treatments that cure or stop its progression; therefore, AD is considered a global health priority. The main limitations are the low drug bioavailability and impermeability of the blood-brain barrier (BBB). Fortunately, nanomedicine has emerged as a promising field for the development of new nanosystems for the controlled and targeted delivery of drugs to the brain. Therefore, in this work, lipid-polymer hybrid nanoparticles (LPHNPs) conjugated with transferrin (Tf) to facilitate crossing the BBB and loaded with N-acetylcysteine (NAC) for its anti-inflammatory effect were synthesized, and their physicochemical characterization was carried out. Subsequently, an in vitro model involving human astrocytes derived from induced pluripotent stem cells (iPSC) from an AD-diagnosed patient was developed, which was brought to a reactive state by stimulation with lipopolysaccharides (LPSs). The cell culture was treated with either Tf-conjugated LPHNPs loaded with NAC (NAC-Tf-LPHNPs) at 0.25 mg mL-1, or free NAC at 5 mM. The results showed that NAC-Tf-LPHNPs favorably modulated the expression of proinflammatory genes such as interleukin-1ß (IL-1ß), amyloid precursor protein (APP) and glial fibrillary acidic protein (GFAP). In addition, they reduced the secretion of the proinflammatory cytokines interleukin 6 (IL-6), IL-1ß and interferon-gamma (INF-γ). Results for both cases were compared to the group of cells that did not receive any treatment. In contrast, free NAC only had this effect on the expression of IL-1ß and the secretion of the cytokines IL-6 and INF-γ. These results indicate the potential of NAC-Tf-LPHNPs for AD treatment.

Radical Scavenging, Hemocompatibility, and Antibacterial Activity against MDR Acinetobacter baumannii in Alginate-Based Aerogels Containing Lipoic Acid-Capped Silver Nanoparticles.

Retaining the hemocompatibility, supporting cell growth, and exhibiting anti-inflammatory and antioxidant properties, while having antimicrobial activity, particularly against multidrug-resistant bacteria (MDR), remain a challenge when designing aerogels for biomedical applications. Here, we report that our synthesized alginate-based aerogels containing either 7.5 or 11.25 µg of lipoic acid-capped silver nanoparticles (AgNPs) showed improved hemocompatibility properties while retaining their antimicrobial effect against MDR Acinetobacter baumannii and the reference strain Escherichia coli, relative to a commercial dressing and polymyxin B, used as a reference. The differences in terms of the microstructure and nature of the silver, used as the bioactive agent, between our synthesized aerogels and the commercial dressing used as a reference allowed us to improve several biological properties in our aerogels with respect to the reference commercial material. Our aerogels showed significantly higher antioxidant capacity, in terms of nmol of Trolox equivalent antioxidant capacity per mg of aerogel, than the commercial dressing. All our synthesized aerogels showed anti-inflammatory activity, expressed as nmol of indomethacin equivalent anti-inflammatory activity per mg of aerogel, while this property was not found in the commercial dressing material. Finally, our aerogels were highly hemocompatible (less than 1% hemolysis ratio); however, the commercial material showed a 20% hemolysis rate. Therefore, our alginate-based aerogels with lipoic acid-capped AgNPs hold promise for biomedical applications.

Chimeric Peptides from Californiconus californicus and Heterodontus francisci with Antigen-Binding Capacity: A Conotoxin Scaffold to Create Non-Natural Antibodies (NoNaBodies).

Research into various proteins capable of blocking metabolic pathways has improved the detection and treatment of multiple pathologies associated with the malfunction and overexpression of different metabolites. However, antigen-binding proteins have limitations. To overcome the disadvantages of the available antigen-binding proteins, the present investigation aims to provide chimeric antigen-binding peptides by binding a complementarity-determining region 3 (CDR3) of variable domains of new antigen receptors (VNARs) with a conotoxin. Six non-natural antibodies (NoNaBodies) were obtained from the complexes of conotoxin cal14.1a with six CDR3s from the VNARs of Heterodontus francisci and two NoNaBodies from the VNARs of other shark species. The peptides cal_P98Y vs. vascular endothelial growth factor 165 (VEGF165), cal_T10 vs. transforming growth factor beta (TGF-ß), and cal_CV043 vs. carcinoembryonic antigen (CEA) showed in-silico and in vitro recognition capacity. Likewise, cal_P98Y and cal_CV043 demonstrated the capacity to neutralize the antigens for which they were designed.

Neutralizing Ability of a Single Domain VNAR Antibody: In Vitro Neutralization of SARS-CoV-2 Variants of Concern.

Severe Acute Respiratory Syndrome Coronavirus 2 is the causal pathogen of coronavirus disease 2019 (COVID-19). The emergence of new variants with different mutational patterns has limited the therapeutic options available and complicated the development of effective neutralizing antibodies targeting the spike (S) protein. Variable New Antigen Receptors (VNARs) constitute a neutralizing antibody technology that has been introduced into the list of possible therapeutic options against SARS-CoV-2. The unique qualities of VNARs, such as high affinities for target molecules, capacity for paratope reformatting, and relatively high stability, make them attractive molecules to counteract the emerging SARS-CoV-2 variants. In this study, we characterized a VNAR antibody (SP240) that was isolated from a synthetic phage library of VNAR domains. In the phage display, a plasma with high antibody titers against SARS-CoV-2 was used to selectively displace the VNAR antibodies bound to the antigen SARS-CoV-2 receptor binding domain (RBD). In silico data suggested that the SP240 binding epitopes are located within the ACE2 binding interface. The neutralizing ability of SP240 was tested against live Delta and Omicron SARS-CoV-2 variants and was found to clear the infection of both variants in the lung cell line A549-ACE2-TMPRSS2. This study highlights the potential of VNARs to act as neutralizing antibodies against emerging SARS-CoV-2 variants.

Conotoxin Patenting Trends in Academia and Industry.

Sea snails of the genus Conus produce toxins that have been the subjects of numerous studies, projects, publications, and patents over the years. Since Conus toxins were discovered in the 1960s, their biological activity has been thought to have high pharmaceutical potential that could be explored beyond the limits of academic laboratories. We reviewed 224 patent documents related to conotoxins and conopeptides globally to determine the course that innovation and development has taken over the years, their primary applications, the technological trends over the last six years, and the leaders in the field, since the only previous patent review was performed in 2015 and focused in USA valid patents. In addition, we explored which countries/territories protect their inventions and patents and the most relevant collaborations among assignees. We also evaluated whether academia or pharmaceutical companies are the future of conotoxin research. We concluded that the 224 conotoxin patents reviewed in this study have more academic value than industrial value, which was noted by the number of active patents that have not yet been licensed and the contributions to medical research, especially as tools to study neuropathic pain, inflammation, immunology, drug design, receptor binding sites, cancer, neurotransmission, epilepsy, peptide biosynthesis, and depression. The aim of this review is to provide an overview of the current state of conotoxin patents, their main applications, and success based on the number of licensing and products in the market.

Neutralizing antibodies levels are increased in individuals with heterologous vaccination and hybrid immunity with Ad5-nCoV in the north of Mexico.

The coordinated efforts to stop the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) include massive immunization of the population at a global scale. The humoral immunity against COVID-19 is conferred by neutralizing antibodies (NAbs) that occur during the post-infection period and upon vaccination. Here, we provide robust data showing that potent neutralizing antibodies are induced in convalescent patients of SARS-CoV-2 infection who have been immunized with different types of vaccines, and patients with no previous history of COVID-19 immunized with a mixed vaccination schedule regardless of the previous infection. More importantly, we showed that a heterologous prime-boost in individuals with Ad5-nCoV (Cansino) vaccine induces higher NAbs levels in comparison to a single vaccination scheme alone.

Treatment with metformin glycinate reduces SARS-CoV-2 viral load: An in vitro model and randomized, double-blind, Phase IIb clinical trial.

The health crisis caused by the new coronavirus SARS-CoV-2 highlights the need to identify new treatment strategies for this viral infection. During the past year, over 400 coronavirus disease (COVID-19) treatment patents have been registered; nevertheless, the presence of new virus variants has triggered more severe disease presentations and reduced treatment effectiveness, highlighting the need for new treatment options for the COVID-19. This study evaluates the Metformin Glycinate (MG) effect on the SARS-CoV-2 in vitro and in vivo viral load. The in vitro study was conducted in a model of Vero E6 cells, while the in vivo study was an adaptive, two-armed, randomized, prospective, longitudinal, double-blind, multicentric, and phase IIb clinical trial. Our in vitro results revealed that MG effectively inhibits viral replication after 48 h of exposure to the drug, with no cytotoxic effect in doses up to 100 µM. The effect of the MG was also tested against three variants of interest (alpha, delta, and epsilon), showing increased survival rates in cells treated with MG. These results are aligned with our clinical data, which indicates that MG treatment reduces SARS-CoV2-infected patients´ viral load in just 3.3 days and supplementary oxygen requirements compared with the control group. We expect our results can guide efforts to position MG as a therapeutic option for COVID-19 patients.

COVID-19 Neutralizing Antibodies in Breast Milk of Mothers Vaccinated with Three Different Vaccines in Mexico.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the largest pandemic of this century, and all aspects of this virus are being studied. The efforts to mitigate the negative effects associated with the SARS-CoV-2 pandemic have culminated in the development of several vaccines that are effective and safe for use to the general population. However, one aspect that remains relatively underexplored is the efficacy of different vaccines technologies (mRNA and Adenovirus) in providing passive immunity to infants through breastmilk of vaccinated mothers, and whether the antibodies passed through breast milk are functional. In this study, using a Micro-neutralization assay, we evaluate the presence of neutralizing antibodies in breast milk of lactating mothers vaccinated against SARS-CoV-2 with the Pfizer-BioNtech, Johnson & Johnson (J&J)/Janssen, and CanSino Biologics vaccines. Our results show the greatest neutralizing effect in breast milk from mothers vaccinated with Pfizer, followed by mothers vaccinated with J&J. CanSino vaccinations yielded the breast milk with the least neutralizing effects. The results found in this study relating to the neutralizing capacity of breast milk against SARS-CoV-2 highlight the importance of corresponding health authorities recommending vaccination to lactating mothers and of the continuance of breastfeeding to infants due to the potential health benefits.

Distribution of pathogenic vibrios and Vibrio spp. in the water column and sediment samples from the southern Gulf of Mexico.

This study quantified the distribution of Vibrio spp. by qPCR and pathogenic vibrio species by metagenomics, during 2 oceanographic cruises-XIXIMI-04 and XIXIMI-05 -in the southern Gulf of Mexico (GoMex). A total of 708 samples from various levels of the water column and 22 sediment samples were analyzed, according to a designed net of sampling lines. Sampling was focused on reported water masses with distinctive characteristics, to detect the presence-absence of vibrios. The results indicated that the genus Vibrio was detected along the entire water column and in sediments. Pathogenic vibrios, such as V, campbellii, V. parahaemolyticus, V. vulnificus or V. cholerae were also detected in the water column and in sediments, in both oceanographic cruises. Thus, the ecological conditions of the GoMex permit the growth of Vibrio spp. in deep water environments of the GoMex, despite continuous oil input from natural and anthropogenic sources.

Molecular Analysis of Streptomycin Resistance Genes in Clinical Strains of Mycobacterium tuberculosis and Biocomputational Analysis of the MtGidB L101F Variant.

Globally, tuberculosis (TB) remains a prevalent threat to public health. In 2019, TB affected 10 million people and caused 1.4 million deaths. The major challenge for controlling this infectious disease is the emergence and spread of drug-resistant Mycobacterium tuberculosis, the causative agent of TB. The antibiotic streptomycin is not a current first-line anti-TB drug. However, WHO recommends its use in patients infected with a streptomycin-sensitive strain. Several mutations in the M. tuberculosisrpsL, rrs and gidB genes have proved association with streptomycin resistance. In this study, we performed a molecular analysis of these genes in clinical isolates to determine the prevalence of known or novel mutations. Here, we describe the genetic analysis outcome. Furthermore, a biocomputational analysis of the MtGidB L101F variant, the product of a novel mutation detected in gidB during molecular analysis, is also reported as a theoretical approach to study the apparent genotype-phenotype association.

Potential Therapeutic Applications of Synthetic Conotoxin s-cal14.2b, Derived from Californiconus californicus, for Treating Type 2 Diabetes.

The FDA's approval of peptide drugs such as Ziconotide or Exendin for pain relief and diabetes treatment, respectively, enhanced the interest to explore novel conotoxins from Conus species venom. In general, conotoxins can be used in pathologies where voltage-gated channels, membrane receptors, or ligands alter normal physiological functions, as in metabolic diseases such as Type 2 diabetes. In this study, the synthetic cal14.2b (s-cal14.2b) from the unusual Californiconus californicus demonstrated bioactivity on NIT-1 insulinoma cell lines stimulating insulin secretion detecting by high performance liquid chromatography (HPLC). Accordingly, s-cal14.2b increased the CaV1.2/1.3 channel-current by 35 ± 4% with a recovery τ of 10.3 ± 4 s in primary cell culture of rat pancreatic ß-cells. The in vivo results indicated a similar effect of insulin secretion on mice in the glucose tolerance curve model by reducing the glucose from 500 mg/dL to 106 mg/dL in 60 min, compared to the negative control of 325 mg/dL at the same time. The PET-SCAN with radiolabeling 99mTc-s-cal14.2b demonstrated biodistribution and accumulation in rat pancreas with complete depuration in 24 h. These findings show the potential therapeutic use of s-cal14.2b in endocrinal pathologies such as early stages of Type 2 Diabetes where the pancreas's capability to produce insulin is still effective.

Improvement of the wound healing properties of hydrogels with N-acetylcysteine through their modification with methacrylate-containing polymers.

Hydrogels with antioxidant activity have shown to significantly improve the standard of care, because they promote efficient wound healing, i.e. regeneration. N-Acetylcysteine (NAC) is an antioxidant amino acid derivative that promotes complete tissue restoration. However, NAC has anticoagulant properties that may also hinder blood coagulation, which is crucial for hydrogels for wound healing applications. To take advantage of the regenerative activity of NAC while avoiding hampering the hemostasis stage during wound healing, we modified gelatin-NAC with the methacrylate-containing polymers 2-hydroxyethyl methacrylate (H) and poly(ethylene glycol) methyl ether methacrylate (P) to produce Gel-HP-NAC. These hydrogels clotted more blood and faster than Gel and Gel-NAC hydrogels, while maintaining fluid absorption properties adequate to promote wound healing. Similarly, there were more viable human skin fibroblasts after 10 days cultured in Gel-HP-NAC compared with Gel and Gel-NAC. A mouse full-thickness skin wound model demonstrated that Gel-HP-NAC hydrogels improved the wound healing process as compared to the untreated group as proved by the increased wound closure rates and re-epithelialization. Histology of the biopsied tissues indicated more organized collagen deposits on the wounds treated with either Gel-HP-NAC or Gel-NAC than untreated wounds. Our results show that modification of NAC-containing hydrogels through methacrylate-containing polymers improved their wound healing properties, including blood-clotting, and demonstrate the potential of Gel-HP-NAC hydrogels for wound treatment and tissue regeneration.

Smart Nanoformulation Based on Stimuli-Responsive Nanogels and Curcumin: Promising Therapy against Colon Cancer.

Curcumin (CUR) has gained much attention for its widely reported anticancer effect; however, its clinical use is restricted due to its low water solubility and, consequently, its poor bioavailability. Here, we report on the use of a nanoformulation of CUR with cationic nanogels for colon cancer therapy. Cationic stimuli-sensitive nanogels were prepared using a scale-up polymerization methodology based on surfactant-free emulsion polymerization of N,N'-diethylaminoethyl methacrylate (DEAEM) and poly(ethyleneglycol) methacrylate (PEGMA). The obtained nanogels showed a homogeneous size distribution (from 51 to 162 nm, polydispersity index (PDI) < 0.138) and exhibited a spherical form and core-shell morphology as confirmed by dynamic light scattering and electron microscopy, respectively. Nanogels were responsive to and degradable by variations of pH, temperature, or the redox environment, depending on the cross-linker used in the synthesis. Nanogels cross-linked with bis(acryloyl)cystamine incubated in a buffer (pH 7.4) containing 3 mM glutathione degraded in 60 min, while nanogels cross-linked with a divinylacetal cross-linker degraded in 10 min (pH ≤ 6). Nanoformulations of nanogels with CUR were stable as tested up to 30 days at physiological conditions. In vitro studies of the human colon cancer cell line (HCT-116) showed a synergistic effect of CUR and the degradable nanogels. Further, in vivo acute cytotoxicity tests of empty nanogels in mice demonstrate their potential as CUR nanocarriers for colon-anticancer therapies.

Venom components of the scorpion Centruroides limpidus modulate cytokine expression by T helper lymphocytes: Identification of ion channel-related toxins by mass spectrometry.

The study of the effector mechanisms of T helper cells has revealed different phenotypic characteristics that can be manipulated for designing new therapeutic schemes in different pathological scenarios. Ion channels are significant targets in T lymphocyte modulation since they are closely related to their effector activity. Remarkably, some toxins produced by scorpions specifically affect the function of these membrane proteins. For that reason, these toxins are important candidates in the search for new immunomodulators. Here, the effect of two venom fractions of the scorpion Centruroides limpidus was assessed on T lymphocyte proliferation and cytokine production. The venom fractions ClF8 and ClF9 were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and cultured at 25 and 35 µg/ml with murine T lymphocytes. The results indicate that the fraction ClF8 increased both production and secretion levels of IFN-γ, IL-4, IL-17A and IL-10 by CD4+ T cells at 24 h. In contrast, fraction ClF9 only promoted the secretion of IL-17A and IL-10 at its highest concentration (35 µg/ml). Both fractions did not show any effect on T cell proliferation. Subsequent analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed seventeen toxins in the fraction ClF8 and five toxins in the fraction ClF9, most of them with voltage-gated sodium (NaScTx) and potassium (KScTx) channels as molecular targets. These toxins might probably interact with ion channels involved in T lymphocyte activity. Our findings suggest that the difference in composition between the two fractions could be related to the observed effects, and the components identified could be isolated to search for possible immunomodulatory molecules.

Detection of Alcanivorax spp., Cycloclasticus spp., and Methanomicrobiales in water column and sediment samples in the Gulf of Mexico by qPCR.

Water column and sediment samples were collected in the southern Gulf of Mexico (GoMex) during 3 oceanographic cruises: XIXIMI-04 (September 2015), XIXIMI-05 (June 2016), and XIXIMI-06 (August 2017). DNA that was extracted from the samples was analyzed by qPCR to detect and quantify bacterial groups that have been reported to metabolize alkanes (Alcanivorax) and aromatic hydrocarbons (Cycloclasticus) and are involved in methane production (Methanomicrobiales). The results were then analyzed with regard to the water masses that are currently detected in the GoMex. Generally, we observed a decrease in the proportion of Alcanivorax and a rise in those of Cycloclasticus and Methanomicrobiales in samples from the surface to deep waters and in sediment samples. Scatterplots of the results showed that the relative abundance of the 3 groups was higher primarily from the surface to 1000 m, but the levels of Cycloclasticus and Methanomicrobiales were high in certain water samples below 1000 m and in sediments. In conclusion, oil-degrading bacteria are distributed widely from the surface to deep waters and sediments throughout the southern GoMex, representing a potential inoculum of bacteria for various hydrocarbon fractions that are ready for proliferation and degradation in the event of an oil spill from the seafloor or along the water column.

Surface Modification of Polyester-Fabric with Hydrogels and Silver Nanoparticles: Photochemical Versus Gamma Irradiation Methods.

A Gamma irradiation and photochemical crosslinking/grafting of poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(2-hydroxyethyl methacrylate-co-poly(ethylene glycol) methacrylate) (poly(HEMA-co-PEGMA)) hydrogels onto polyethyleneterephtalate fabric (PET) surfaces were evaluated, in order to obtain a hydrophilic homogeneous coating onto PET fabrics. The materials were characterized by FTIR-ATR, SEM, EDS, and thermal analysis. Furthermore, silver nanoparticles (AgNPs) were loaded by in situ reduction of AgNO3, and its antibacterial activity against Staphylococcus aureus and Escherichia coli was determined. Results showed a ticker coating of hydrogel using gamma radiation and stronger in deep modification of the fibers; however, by the photochemical method, a thin coating with good coverage of PET surface was obtained. The differences in hydrophilicity, thermal properties, and antibacterial activity of the coated fabrics by using both methods were rather small.

Transcriptomic and Proteomic Analysis of the Tentacles and Mucus of Anthopleura dowii Verrill, 1869.

Sea anemone venom contains a complex and diverse arsenal of peptides and proteins of pharmacological and biotechnological interest, however, only venom from a few species has been explored from a global perspective to date. In the present study, we identified the polypeptides present in the venom of the sea anemone Anthopleura dowii Verrill, 1869 through a transcriptomic and proteomic analysis of the tentacles and the proteomic profile of the secreted mucus. In our transcriptomic results, we identified 261 polypeptides related to or predicted to be secreted in the venom, including proteases, neurotoxins that could act as either potassium (K+) or sodium (Na+) channels inhibitors, protease inhibitors, phospholipases A2, and other polypeptides. Our proteomic data allowed the identification of 156 polypeptides-48 exclusively identified in the mucus, 20 in the tentacles, and 88 in both protein samples. Only 23 polypeptides identified by tandem mass spectrometry (MS/MS) were related to the venom and 21 exclusively identified in the mucus, most corresponding to neurotoxins and hydrolases. Our data contribute to the knowledge of evolutionary and venomic analyses of cnidarians, particularly of sea anemones.

Cationic versus anionic core-shell nanogels for transport of cisplatin to lung cancer cells.

Stimuli-responsive polymeric nanogels have been proposed as nanocarriers of cisplatin to maximize its effect for cancer treatment. In this work, a comparative study between anionic core nanogels (ACN) and cationic core nanogels (CCN), both with PEGylated shells, has been performed. The nanogels were synthesized with different cross-linked cores: CCN with poly(N,N-diethylaminoethyl methacrylate) (PDEAEMA) and ACN with poly(2-methacryloyloxi benzoic acid) (P2MBA). Cisplatin chelate formation with carboxylic acids (ACN) or metal coordination with the amine groups (CCN) leads to a high loading of cisplatin into the nanocarriers. The nanocarriers ability to contain and modulate the supply of cisplatin was tested according to the pH of the medium, in which ACN efficiently released the drug at a typical pH value of a tumor tissue (pH = 6.8) while CCN only releases the drug at more acidic, endosome like, conditions (pH = 5). The effect of drug-free nanogels on cell lines NCI-H1437 (non-small cell lung carcinoma) was evaluated, showing biocompatibility at all concentrations studied (30-400 µg/mL) for both ACN and CCN. However, the survival percentage of the cells in contact with cisplatin-loaded nanogels were dependent on the dose, the time of contact and the type of nanogel. Cisplatin loaded CCN induced lower cell viability after 48 h of contact. Fluorescence microscopy showed a viable internalization of the CCN nanogels, this was confirmed by flow cytometry in which 37.8% of cells contained drug loaded CCNs after 30 min of contact, representing a more effective nanocarrier for cisplatin to this cell-line.

Marine peptides as immunomodulators: Californiconus californicus-derived synthetic conotoxins induce IL-10 production by regulatory T cells (CD4+Foxp3+).

Context: CD4+ T lymphocytes are able to differentiate into distinct subtypes according to several immunological scenarios, including T helper (Th)1, Th2, Th17 and regulatory T (Treg) cells. CD4+ T cells are phenotypically flexible and have specific ion channels, such as the nicotinic acetylcholine receptors (nAChR) that could be modulated by peptides produced by marine snails, known as conotoxins. Their effect on T lymphocytes has not been explored and emerging evidence suggests that these peptides may have immunomodulatory activities. Objective: This study investigated the effect of two Californiconus californicus-derived synthetic conotoxins on the proliferation and differentiation of T lymphocyte subpopulations Th1, Th2, Th17 and Treg. Methods: Cells from lymph nodes of BALB/c mice were cultured in the presence of conotoxins cal14.1b and cal14.2c (5.5 µM), during 96 h. Cell proliferation and intracellular cytokine production (IFN-γ, IL-4, IL-17 and IL-10) were analyzed by flow cytometry. Results and Discussion: cal14.1b and cal14.2c increased intracellular IL-10 production in Treg (CD3+CD4+Foxp3+) cells and decreased intracellular IL-17 production (CD3+CD4+) after 72 h of culture. Conotoxins did not show any effect on T cell proliferation nor Th1/Th2 balance. Conclusion: These results suggest that synthetic conotoxins exert immunomodulatory activity, especially by regulating specific functions on T lymphocytes.

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry.

BACKGROUND: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. METHODS: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. RESULTS: The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 µg), goat (HU50 = 13.2 ± 0.3 µg), rabbit (HU50 = 34.7 ± 0.5 µg), and human (HU50 = 25.6 ± 0.6 µg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 µg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.