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Clostridioides difficile causes a range of debilitating intestinal symptoms that may be fatal. It is particularly problematic as a hospital-acquired infection, causing significant costs to the health care system. Antibiotics, such as vancomycin and fidaxomicin, are still the drugs of choice for C. difficile infections, but their effectiveness is limited, and microbial interventions are emerging as a new treatment option. This paper focuses on alternative treatment approaches, which are currently in various stages of development and can be divided into four therapeutic strategies. Direct killing of C. difficile (i) includes beside established antibiotics, less studied bacteriophages, and their derivatives, such as endolysins and tailocins. Restoration of microbiota composition and function (ii) is achieved with fecal microbiota transplantation, which has recently been approved, with standardized defined microbial mixtures, and with probiotics, which have been administered with moderate success. Prevention of deleterious effects of antibiotics on microbiota is achieved with agents for the neutralization of antibiotics that act in the gut and are nearing regulatory approval. Neutralization of C. difficile toxins (iii) which are crucial virulence factors is achieved with antibodies/antibody fragments or alternative binding proteins. Of these, the monoclonal antibody bezlotoxumab is already in clinical use. Immunomodulation (iv) can help eliminate or prevent C. difficile infection by interfering with cytokine signaling. Small-molecule agents without bacteriolytic activity are usually selected by drug repurposing and can act via a variety of mechanisms. The multiple treatment options described in this article provide optimism for the future treatment of C. difficile infection.
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Clostridioides difficile , Infecciones por Clostridium , Microbioma Gastrointestinal , Humanos , Antibacterianos/uso terapéutico , Antibacterianos/farmacología , Trasplante de Microbiota Fecal , Vancomicina/farmacología , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/prevención & controlRESUMEN
BACKGROUND: Clinical and experimental analyses indicate a pathognomonic role for allergen IgE crosslinking through epitope-paratope interactions as a major initial step in the cascade leading to effector cell activation and clinical manifestations of lgE-mediated food allergies. We aimed to undertake the initial development and assessment of Ara h 2-specific IgE epitope-like peptides that can bind to allergen-specific IgE paratopes and suppress effector cell activation. METHODS: We performed biopanning, screening, IgE binding, selection and mapping of peptides. We generated synthetic peptides for use in all functional experiments. ImmunoCAP inhibition, basophil and mast cell activation tests, with LAD2 cells, a human mast cell line were performed. Twenty-six children or young adults who had peanut allergy were studied. RESULTS: We identified and selected three linear peptides (DHPRFNRDNDVA, DHPRYGP and DHPRFST), and immunoblot analyses revealed binding to lgE from peanut-allergic individuals. The peptide sequences were aligned to the disordered region corresponding to the loop between helices 2 and 3 of Ara h 2, and conformational mapping showed that the peptides match the surface of Ara h 2 and h 6 but not other peanut allergens. In ImmunoCAP inhibition experiments, the peptides significantly inhibit the binding of IgE to Ara h 2 (p < .001). In basophil and mast cell activation tests, the peptides significantly suppressed Ara h 2-induced effector cell activation (p < .05) and increased the half-maximal Ara h 2 effective concentration (p < .05). Binding of the peptides to specific IgEs did not induce activation of basophils or mast cells. CONCLUSIONS: These studies show that the indicated peptides reduce the allergenic activity of Ara h 2 and suppress lgE-dependent basophil and mast cell activation. These observations may suggest a novel therapeutic strategy for food allergy based on epitope-paratop blocking.
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Hipersensibilidad a los Alimentos , Hipersensibilidad al Cacahuete , Niño , Adulto Joven , Humanos , Epítopos , Antígenos de Plantas , Glicoproteínas , Péptidos , Inmunoglobulina E , Alérgenos , Arachis , Albuminas 2S de PlantasRESUMEN
Difficulties in obtaining and maintaining the desired level of the critical quality attributes (CQAs) of therapeutic proteins as well as the pace of the development are major challenges of current biopharmaceutical development. Therapeutic proteins, both innovative and biosimilars, are mostly glycosylated. Glycans directly influence the stability, potency, plasma half-life, immunogenicity, and effector functions of the therapeutic. Hence, glycosylation is widely recognized as a process-dependent CQA of therapeutic glycoproteins. Due to the typically high heterogeneity of glycoforms attached to the proteins, control of glycosylation represents one of the most challenging aspects of biopharmaceutical development. Here, we explored a new glycoengineering approach in therapeutic glycoproteins development, which enabled us to achieve the targeted glycoprofile of the Fc-fusion protein in a fast manner. Coupling CRISPRi technology with lectin-FACS sorting enabled downregulation of the endogenous gene involved in fucosylation and further enrichment of CHO cells producing Fc-fusion proteins with reduced fucosylation levels. Enrichment of cells with targeted glycoprofile can lead to time-optimized clone screening and speed up cell line development. Moreover, the presented approach allows isolation of clones with varying levels of fucosylation, which makes it applicable to a broad range of glycoproteins differing in target fucosylation level.
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Biosimilares Farmacéuticos , Animales , Células CHO , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cricetinae , Cricetulus , Glicoproteínas/genética , PolisacáridosRESUMEN
Development of targeted treatment for colorectal cancer is crucial to avoid side effects. To harness the possibilities offered by microbiome engineering, we prepared safe multifunctional cancer cell-targeting bacteria Lactococcus lactis. They displayed, on their surface, binding proteins for cancer-associated transmembrane receptors epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2) and co-expressed an infrared fluorescent protein for imaging. Binding of engineered L. lactis to tumour antigens EpCAM and HER2 was confirmed and characterised in vitro using soluble receptors. The proof-of-principle of targeting was demonstrated on human cell lines HEK293, HT-29 and Caco-2 with fluorescent microscopy and flow cytometry. The highest L. lactis adhesion was seen for the HEK293 cells with the overexpressed tumour antigens, where colocalisation with their tumour antigens was seen for 39% and 67% of EpCAM-targeting and HER2-targeting bacteria, respectively. On the other hand, no binding was observed to HEK293 cells without tumour antigens, confirming the selectivity of the engineered L. lactis. Apart from cell targeting in static conditions, targeting ability of engineered L. lactis was also shown in conditions of constant flow of bacterial suspension over the HEK293 cells. Successful targeting by engineered L. lactis support the future use of these bacteria in biopharmaceutical delivery for the treatment of colorectal cancer.
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Neoplasias Colorrectales , Lactococcus lactis , Antígenos de Neoplasias/genética , Células CACO-2 , Proteínas Portadoras , Neoplasias Colorrectales/terapia , Células HEK293 , Humanos , Lactococcus lactis/genéticaRESUMEN
The two most important bacterial phyla in the gastrointestinal tract, Firmicutes and Bacteroidetes, have gained much attention in recent years. The Firmicutes/Bacteroidetes (F/B) ratio is widely accepted to have an important influence in maintaining normal intestinal homeostasis. Increased or decreased F/B ratio is regarded as dysbiosis, whereby the former is usually observed with obesity, and the latter with inflammatory bowel disease (IBD). Probiotics as live microorganisms can confer health benefits to the host when administered in adequate amounts. There is considerable evidence of their nutritional and immunosuppressive properties including reports that elucidate the association of probiotics with the F/B ratio, obesity, and IBD. Orally administered probiotics can contribute to the restoration of dysbiotic microbiota and to the prevention of obesity or IBD. However, as the effects of different probiotics on the F/B ratio differ, selecting the appropriate species or mixture is crucial. The most commonly tested probiotics for modifying the F/B ratio and treating obesity and IBD are from the genus Lactobacillus. In this paper, we review the effects of probiotics on the F/B ratio that lead to weight loss or immunosuppression.
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Several plant polyphenols have been shown to reduce osteoarthritis symptoms due to their antioxidant, anti-inflammatory and immunomodulatory properties. We investigated the effects of two different polyphenolic extracts (Belinal, Pycnogenol) and two different polyphenols (resveratrol, quercetin) on the chondrogenic potential of bone-derived mesenchymal stem/stromal cells (MSCs) from healthy donors and patients with osteoarthritis. Our main aim was to determine whether Belinal, a commercially available polyphenolic extract from silver fir (Abies alba L.) branches, has comparable chondrogenic potential with the other tested extract and the polyphenols under inflammatory and non-inflammatory conditions. In our study, Belinal promoted significantly greater chondrogenesis compared to the untreated (p = 0.0289) and resveratrol-treated (p = 0.0468) MSCs from patients with hip osteoarthritis under non-inflammatory conditions. Under inflammatory conditions, chondrogenesis was significantly enhanced for MSCs treated with Belinal compared to the control (p = 0.0483). The other extract and the polyphenols did not show any significant effects on chondrogenesis under non-inflammatory or inflammatory conditions. None of the tested extracts and polyphenols showed significant effects on chondrogenesis in healthy donors, under either non-inflammatory or inflammatory conditions. Our data show that Belinal can boost the chondrogenesis of MSCs derived from patients with osteoarthritis, under both non-inflammatory and inflammatory conditions.
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Abies/química , Lipopolisacáridos/farmacología , Extractos Vegetales/farmacología , Polifenoles/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Condrogénesis/efectos de los fármacos , Citometría de Flujo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Osteoartritis de la Cadera/tratamiento farmacológico , Osteoartritis de la Cadera/metabolismo , Extractos Vegetales/químicaRESUMEN
Recently, growing interest is devoted to investigation of bioactive secondary metabolites of endophytic fungi. Thus, as an extension to our previous achievements related to antimicrobial potential of endophytic fungi, Phomopsis species isolated from conifer needles was selected as appropriately promising natural source for drug discovery. Its dichloromethane and ethanol extracts considerably inhibited growth of Escherichia coli and Staphylococcus aureus. Moreover, the individual compounds of dichloromethane extract have been separated, collected and purified using semi preparative liquid chromatographic analysis and comprehensively characterized using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). Based on their antimicrobial activity and unique structural characteristics in comparison with well-established drugs from the same therapeutic category, two dominant compounds (Z)-(Z)-2-acetoxyprop-1-en-1-yl-3-(3-((E)-3,4-dihydroxypent-1-en-1-yl)oxiran-2-yl)acrylate (denoted as 325-3) and (Z)-(Z)-2-acetoxyprop-1-en-1-yl 3-(3-((E)-4-hydroxy-3-oxopent-1-en-1-yl)oxiran-2-yl)acrylate (denoted as 325-5) were recognized as valuable leading structures for future discovery of novel antibiotics.
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Acrilatos/farmacología , Antibacterianos/farmacología , Phomopsis/química , Acrilatos/química , Acrilatos/aislamiento & purificación , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/efectos de los fármacosRESUMEN
The display of recombinant proteins on bacterial surfaces is a developing research area with a wide range of potential biotechnological applications. The lactic acid bacterium Lactococcus lactis is an attractive host for such surface display, and a promising vector for in vivo delivery of bioactive proteins. Surface-displayed recombinant proteins are usually anchored to the bacterial cell wall through anchoring domains. Here, we investigated alternatives to the commonly applied lactococcal lysine motif (LysM)-containing surface anchoring domain, the C-terminus of AcmA (cAcmA). We screened 15 anchoring domains of lactococcal or phage origins that belong to the Pfam categories LPXTG, LysM, CW_1, Cpl-7, WxL, SH3, and ChW, which can provide non-covalent or covalent binding to the cell wall. LPXTG, LysM, the duplicated CW_1 and SH3 domains promoted significant surface display of two model proteins, B domain and DARPin I07, although the display achieved was lower than that for the reference anchoring domain, cAcmA. On the other hand, the ChW-containing anchoring domain of the lactococcal phage AM12 endolysin (cAM12) demonstrated surface display comparable to that of cAcmA. The anchoring ability of cAM12 was confirmed by enabling non-covalent heterologous anchoring of the B domain on wild-type bacteria, as well as anchoring of CXCL8-binding evasin-3, which provided potential therapeutic applicability; both were displayed to an extent comparable to that of cAcmA. We have thereby demonstrated the effective use of different protein anchoring domains in L. lactis, with ChW-containing cAM12 the most promising alternative to the established approaches for surface display on L. lactis.
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PURPOSE: There is no clear evidence on whether sunscreens and personal care products containing UV-filters like titanium dioxide (TiO2) are protective against or may be a contributing factor in melanoma development. Extensive studies have shown that TiO2 can cause cell toxicity under in vitro and in vivo conditions. The transmembrane protein ABCB5 is closely linked to tumorigenicity, progression and disease recurrence of diverse human malignancies, including melanoma. Accordingly, the aim of the present study was to investigate in vitro any potential influence of nanosized TiO2 (nano-TiO2) on metastatic melanoma cells' metabolic activity, cytotoxicity and ABCB5 mRNA expression. METHODS: The human metastatic melanoma cell line WM-266-4 (ATCC) was used to obtain dose- and time-dependent responses. We used the MTT and LDH assays to measure metabolic activity of selected cells and cytotoxicity. Real-time quantitative PCR (RT-qPCR) was performed and gene expression ratios were calculated for the target (ABCB5) and the reference (LDHA) gene. Standard statistical tests were used for analysis in SPSS and Excell. RESULTS: Our results suggest decreased metastatic melanoma cells' metabolic activity, increased cytotoxicity and increased ABCB5 mRNA expression after 24 and 48 hrs as compared to control (untreated) cells (p<0.05). Thus, we showed that nano-TiO2 might influence cells' invasiveness and aggressiveness. CONCLUSION: We show for the first time that ABCB5 expression in metastatic melanoma cells might be affected by nano-TiO2 exposure. In addition, nano-TiO2 as a sunscreen ingredient might play a role in metastatic melanoma progression.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Melanoma/metabolismo , ARN Mensajero/biosíntesis , Neoplasias Cutáneas/metabolismo , Titanio/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Melanoma/genética , Melanoma/patología , Persona de Mediana Edad , Nanopartículas/administración & dosificación , Nanopartículas/química , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Titanio/químicaRESUMEN
Dysbiosis of intestinal microbiota and aberrant inflammatory responses in gastrointestinal mucosa plays important roles in the development of inflammatory bowel disease (IBD). The purpose of this study was to demonstrate the probiotic activity of Lactococcus lactis and the ability of TNF-α-binding by recombinant L. lactis bearing TNF-α-binding affibodies. Various concentrations of recombinant L. lactis were exposed to TNF-α and its binding measured by ELISA. Mucosal biopsies of patients with active IBD were incubated with various L. lactis strains or E. coli DH5α strain and concentrations of TNF-α, IL-23, and IL-10 in the supernatants determined by ELISA. Recombinant L. lactis, at 1 × 109 and 1 × 108 CFU/mL, bound 22.6% and 18.4%, respectively of TNF-α (p < 0.05). When IBD-mucosa was incubated with any L. lactis strain at 1 × 109 CFU/mL, levels of TNF-α and IL-23 were significantly decreased and that of IL-10 increased relative to that for the sterile culture. Opposite trends were observed with E. coli cultures. Recombinant L. lactis at 1 × 108 CFU/mL bound as much as 62.8% (p = 0.026) of TNF-α in IBD-mucosa supernatants compared with the control strain. L. lactis strains are reported, for the first time, to induce an ex vivo anti-inflammatory cytokine profile in IBD inflamed mucosa. L. lactis could therefore constitute a promising alternative approach for treating IBD.
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Antiinflamatorios/metabolismo , Citocinas/metabolismo , Ingeniería Genética , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Mucosa Intestinal/microbiología , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Probióticos/farmacología , Adolescente , Antiinflamatorios/uso terapéutico , Niño , Citocinas/genética , Citocinas/uso terapéutico , Disbiosis , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Microbioma Gastrointestinal , Humanos , Enfermedades Inflamatorias del Intestino/microbiología , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Masculino , Proteínas Recombinantes/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/uso terapéutico , Adulto JovenRESUMEN
The diet rich in fruits and vegetables reduces the risk of metabolic syndrome, including diabetes development by various mechanisms of action, mainly due to the presence of polyphenolic compounds. Extracts from different conifer species are known to be a rich source of various polyphenols. In the present study we elucidated the in vitro mechanism of anti-diabetic activity of silver fir (Abies alba) wood and bark extracts and compared their activity to non-coniferous sweet chestnut wood extract and standardized maritime pine bark extract. Extracts and lignans were tested for their inhibitory activity of enzymes involved in the regulation of blood glucose in vitro. The ability of extracts to protect against oxidative stress in high glucose environment was tested on mouse myoblast cell line. Silver fir wood and bark extracts were shown to be effective inhibitors of α-glucosidase, α-amylase and dipeptidyl peptidase 4, three enzymes involved in the regulation of blood glucose levels. Coniferous extracts also showed protection against oxidative stress generated in high glucose environment. Lignans, particularly pinoresinol diglucoside, isolariciresinol and secolariciresinol were shown to be important contributors of antihyperglycemic activity through inhibition of dipeptidyl peptidase 4. This corroborates previously published in vivo results on blood glucose level obtained with silver fir wood extract and supports the use of silver fir wood and bark extracts as food supplements or functional foods in borderline diabetes.
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Abies/química , Glucemia/efectos de los fármacos , Hipoglucemiantes/farmacología , Extractos Vegetales/farmacología , alfa-Amilasas/antagonistas & inhibidores , Animales , Línea Celular , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/aislamiento & purificación , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Inhibidores de Glicósido Hidrolasas/aislamiento & purificación , Inhibidores de Glicósido Hidrolasas/farmacología , Humanos , Hipoglucemiantes/aislamiento & purificación , Ratones , Estrés Oxidativo/efectos de los fármacos , Corteza de la Planta/química , Extractos Vegetales/aislamiento & purificación , Madera/química , alfa-Glucosidasas/metabolismoAsunto(s)
Alérgenos/inmunología , Venenos de Abeja/inmunología , Epítopos de Linfocito B/inmunología , Mordeduras y Picaduras de Insectos/inmunología , Proteínas de Insectos/inmunología , Fosfolipasas A/inmunología , Adulto , Anciano , Secuencias de Aminoácidos , Animales , Abejas/inmunología , Mapeo Epitopo , Femenino , Humanos , Inmunoglobulina E/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Ragweed is a prominent cause of seasonal allergies. Thus far, information on IgE-binding sites of major allergen in ragweed pollen, Amb a 1, is very limited. A powerful experimental method to gain insights on the allergen epitopes is the selection of peptides from biological libraries that bind to anti-allergen antibodies. In this work, we aimed to map IgE epitopes of Amb a 1 using epitope-mimicking short peptides - mimotopes that were affinity-selected from phage-displayed random peptide libraries. The peptides weakly aligned with the Amb a 1 primary sequence, thus suggesting that the epitopes are conformational. When the peptides were mapped onto the surface of Amb a 1 homology model, the EpiSearch analysis predicted the location of four potential epitopic sites on surface patches centred at residues K104, S110, H214, and W312. The peptides matching to the predicted epitopes bound selectively to the IgE from pool of ragweed-allergic patients' sera and therefore represent mimetics of Amb a 1 IgE epitopes. The knowledge of IgE epitopes is a prerequisite for the rational design of molecular-based approaches to diagnosis and immunotherapy of allergic diseases.
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Orexigenic peptide ghrelin and its receptor have been extensively investigated as potential therapeutic targets, primarily because of their role in feeding initiation and growth hormone (GH) release. However, no specific ghrelin targeting anti-obesity or cachexia therapeutics are available for clinical use thus far and further efforts in this direction are warranted. The present study aimed to find new peptide drug leads modulating ghrelin signal transduction. By targeting neutralising antibodies against ghrelin with phage display libraries, we aimed to identify peptides binding to the cognate receptor. Four synthetic peptides were selected and tested using calcium screening assays. The most effective competitive antagonist FSFLPPE was further tested in vivo. Administration of the peptide produced no significant effect on either food intake or GH release. Surprisingly, when co-administered with ghrelin, the peptide significantly enhanced GH secretion and c-Fos expression. The evidence obtained in the present study indicates that FSFLPPE might act as an ago-allosteric modulator.
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Ghrelina/farmacología , Hormona del Crecimiento/sangre , Imitación Molecular , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Animales , Anticuerpos/inmunología , Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Núcleo Arqueado del Hipotálamo/metabolismo , Calcio/metabolismo , Células Cultivadas , Ingestión de Alimentos/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Ghrelina/antagonistas & inhibidores , Ghrelina/inmunología , Humanos , Masculino , Ratones , Oligopéptidos/farmacología , Péptidos/antagonistas & inhibidores , Péptidos/síntesis química , Péptidos/inmunología , ConejosRESUMEN
Lactic acid bacteria (LAB) are attractive hosts for the expression of heterologous proteins and can be engineered to deliver therapeutic proteins or peptides to mucosal surfaces. The gastric stable pentadecapeptide BPC-157 is able to prevent and treat gastrointestinal inflammation by reducing the production of reactive oxygen species (ROS). In this study, we used LAB Lactococcus lactis as a vector to deliver BPC-157 by surface display and trypsin shedding or by secretion to the growth medium. Surface display of BPC-157 was achieved by fusing it with basic membrane protein A (BmpA) or with the peptidoglycan binding domain of AcmA and Usp45 secretion signal. While the expression of BmpA-fusion proteins was higher than that of AcmA/Usp45-fusion protein, the surface display ability of BPC-157 was approximately 14-fold higher with AcmA/Usp45-fusion protein. Release of BPC-157 from the bacterial surface or from isolated fusion proteins by trypsinization was demonstrated with anti-BPC-157 antibodies or by mass spectrometry. The concentration of BPC-157 delivered by surface display via AcmA/Usp45-fusion was 30 ng/ml. This increased to 117 ng/ml by Usp45 signal-mediated secretion, making the latter the most effective lactococcal delivery approach for BPC-157. Secreted BPC-157 significantly decreased ROS production in 149BR fibroblast cell model, suggesting its potential benefit in the treatment of intestinal inflammations. Additionally, a comparison of different modes of small peptide delivery by L. lactis, performed in the present study, will facilitate the future use of L. lactis as peptide delivery vehicle.
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Sistemas de Liberación de Medicamentos , Lactococcus lactis , Fragmentos de Péptidos/administración & dosificación , Proteínas/administración & dosificación , Línea Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/terapia , Microorganismos Modificados Genéticamente , Estrés Oxidativo , Fragmentos de Péptidos/farmacología , Plásmidos , Ingeniería de Proteínas , Proteínas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: Development of new recombinant biotechnology products has greatly expanded in the field of modern pharmacy and medicine. Since biological recombinant molecules are sensitive, simple or composed proteins, their function is heavily dependent on their structure. In addition to their efficacy, biological medicinal products could show side effects such as immunogenicity. Therefore, detection and characterization of protein structural variants is essential during development and quality control of therapeutic proteins that might trigger immunogenic response in organism. METHODS: This article includes proposed detection and characterization of aggregated, as well as other modified forms of monoclonal antibodies (mAb), by using selected chromatographic and spectrometric methods. Additionally, selected mAb's aggregates and modified structural variants of monoclonal antibodies were subjected to the immature monocyte-derived dendritic cells' (DC) examination experiment for monitoring of activated DC cells in order to determine potential immunogenicity of mAb structural variants. Furthermore, potential innate immunogenic response of peripheral blood mononuclear cells (PBMC) cultures to mAb aggregates was also evaluated by measuring pro-inflammatory cytokine response during early exposure of PBMCs to different mAb samples and by determining the effect of mAb aggregates on PBMC proliferation during long-term cultures. RESULT AND CONCLUSION: All developed and proposed analytical methods and immunological in vitro DC and PBMC assays, could be used as platform for complementary analytical characterization and determination of potential for immunogenicity for all biopharmaceutical products which contain monoclonal antibodies as active pharmaceutical ingredients.
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Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Células Cultivadas , Cromatografía en Gel , Citocinas/biosíntesis , Células Dendríticas/inmunología , Descubrimiento de Drogas , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoglobulina G/inmunología , Leucocitos Mononucleares/inmunología , Peso MolecularRESUMEN
Diabetes mellitus is one of the leading world's public health problems. Therefore, it is of a huge interest to develop new antidiabetic drugs. Apart from traditional therapy of diabetes, nowadays, importance is given to natural substances with antidiabetic potential. Fomes fomentarius is a mushroom widely used for different purposes, due to its range of already confirmed activities. Fomentariol is a constituent of Fomes fomentarius, responsible for its antidiabetic potential. In that respect, it is important to develop a method for isolation and quantification of fomentariol from fungal material, which will be simple and efficient. Multistep, complex extraction applied in the previously reported studies was avoided with ethanol, providing rapid single-step extraction. The presence of fomentariol in ethanolic extract was confirmed by high-resolution mass spectrometry. Semipreparative HPLC method was developed and applied for isolation from ethanol extract and purification of the active compound fomentariol. It was a gradient reversed-phase method with a mobile phase consisting of acetonitrile and 0.1% formic acid in water and total run time of 15 minutes. The amount of 6.5 mg of high-purity fomentariol was determined by quantitative NMR with toluene as internal standard. The isolated and determined amount of substance can be further used for the quantitative estimation of activity of fomentariol.
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Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed. Genes for the infrared fluorescent protein and for the human IgG-binding DARPin were cloned in all possible combinations to assess the protein yield. The dual promoter plasmid pNZDual enabled balanced expression of the two model proteins. It was exploited for the development of a single-plasmid inducible CRISPR-Cas9 system (pNZCRISPR) by using a nisin promoter, first to drive Cas9 expression and, secondly, to drive single guide RNA transcription. sgRNAs against htrA and ermR directed Cas9 against genomic or plasmid DNA and caused changes in bacterial growth and survival. Replacing Cas9 by dCas9 enabled CRISPR interference-mediated silencing of the upp gene. The present study introduces a new series of plasmids for advanced genetic modification of lactic acid bacterium L. lactis.
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Regulación Bacteriana de la Expresión Génica , Ingeniería Genética/métodos , Genoma Bacteriano , Lactococcus lactis/genética , Plásmidos/química , Transgenes , Antibacterianos/farmacología , Sistemas CRISPR-Cas , Clonación Molecular , Fermentación , Edición Génica/métodos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Lactococcus lactis/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Nisina/farmacología , Plásmidos/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Chemokines are key signals in the immune system and play an important role as proinflammatory mediators in the pathology of inflammatory bowel disease and colorectal cancer, making them an important target for therapy. Recombinant lactic acid bacteria (LAB) were engineered to bind CC and CXC chemokines by displaying chemokine-binding proteins evasin-1, evasin-3 and evasin-4 on their surface. Evasin genes were cloned into lactococcal surface display vector and overexpressed in L. lactis NZ9000 and NZ9000ΔhtrA in fusion with secretion signal and surface anchor. Evasin-displaying bacteria removed from 15% to 90% of 11 different chemokines from the solution as determined with ELISA and Luminex multiplexing assays, whereby L. lactis NZ9000ΔhtrA proved more efficient. Lactobacillus salivarius ATCC 11741 was coated with L. . lactis-expressed evasin fusion protein, and its ability to bind chemokines was also confirmed. Evasin-3-displaying L. lactis removed 76.0% of IL-1ß-induced CXCL8 from the supernatant of Caco-2 epithelial cells. It also prevented secretion of CXCL8 from Caco-2 cells in a time-dependent manner when added before induction with IL-1ß. Evasin-displaying LAB have the ability to bind multiple chemokines simultaneously and exert synergistic activity. This innovative treatment approach therefore has the potential for mucosal therapy of inflammatory bowel disease or colorectal cancer.