UNIVERSIDAD POLITÉCNICA DE MADRID E SCUELA T ÉCNICA S UPERIOR DE I NGENIERÍA
A GRONÓMICA, A LIMENTARIA Y DE B IOSISTEMAS
GRADO EN BIOTECNOLOGÍA
D EPARTAMENTO DE BIOTECNOLOGÍA Y BIOLOGÍA VEGETAL
Caracterización de una inmunoterapia basada en
nanopartículas virales para el tratamiento de la alergia al melocotón / Characterization of an immunotherapy based
on viral nanoparticles for the treatment of peach allergy
TRABAJO FIN DE GRADO Autor/a: Marina Amores Borge
Tutor/a: Jaime Tomé Amat
Junio de 2021
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UNIVERSIDAD POLITÉCNICA DE MADRID
ESCUELA TÉCNICA SUPERIOR DE INGENIERÍA AGRONÓMICA, ALIMENTARIA Y DE BIOSISTEMASGRADO DE BIOTECNOLOGÍA
CARACTERIZACIÓN DE UNA INMUNOTERAPIA BASADA EN NANOPARTÍCULAS VIRALES PARA EL TRATAMIENTO DE LA ALERGIA
AL MELOCOTÓN / CHARACTERIZATION OF AN IMMUNOTHERAPY BASED ON VIRAL NANOPARTICLES FOR THE TREATMENT OF PEACH
ALLERGY
TRABAJO FIN DE GRADO
Marina Amores Borge MADRID, 2021
Director: Jaime Tomé Amat Investigador Postdoctoral Dpto. de Biotecnología y Biología Vegetal
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TITULO DEL TFG- CARACTERIZACIÓN DE UNA INMUNOTERAPIA BASADA EN NANOPARTÍCULAS VIRALES PARA EL TRATAMIENTO DE
LA ALERGIA AL MELOCOTÓN / CHARACTERIZATION OF AN IMMUNOTHERAPY BASED ON VIRAL NANOPARTICLES FOR THE
TREATMENT OF PEACH ALLERGY
Memoria presentada por Marina Amores Borge para la obtención del título de Graduado en Biotecnología por la Universidad Politécnica de Madrid
Fdo: Alumno
VºBº Tutor
Firmado por TOME AMAT JAIME MARIA - 53549451T el día 21/06/2021 con un certificado emitido por AC FNMT Usuarios
D. Jaime Tomé Amat Investigador Postdoctoral
Dpto. de Biotecnología y Biología Vegetal ETSIAAB – Universidad Politécnica de Madrid
Madrid, 24 de junio de 2021
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Acknowledgements.
I would like to thank the people that have guided and accompanied me during the process of making this work.
Jaime, you have been my major guide and this work is here thanks to you. You have trusted me and given me the confidence to believe in my own progress. I hope you find in these pages all the wisdom you have transmitted me.
Ali, thank you so much for your constant motivation and trust in me. You opened my eyes to the world of immunology and gave me the opportunity to explore it.
María, your support and advices have been extremely helpful. Thanks for always being willing to lend me a helping hand.
And to all my laboratory colleagues: Zu, Diego, Guada and Jano, you made this work much easier and filled it with guidance and encouragement. You have provided me an enormous help and cheer in and outside the lab.
To my friends, you have emotionally supported me during this year and always trusted in my success. I hope I can one day give you back all the love you put on me.
To my family, especially my mom and sister: you have warmed my heart every single day. You are my fuel to keep aiming further and further.
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TABLE OF CONTENTS.
1. Introduction. ... 1
Objectives... 3
2. Materials and methods. ... 4
2.1. Biochemical characterization of the IT formula. ... 4
2.1.1. Immunodetection. ... 4
2.1.2. Transmission electron microscopy. ... 4
2.2. Confirmation of the VNP-Complex formation. ... 5
2.2.1. Thin layer chromatography. ... 5
2.2.2. Absorbance at 254 nm and 260 nm. ... 5
2.3. Immunological characterization of the IT formula. ... 6
2.3.1. NF-κB/AP-1 activation analysis. ... 6
2.3.2. PBMCs proliferation analysis. ... 7
2.4. Transportation analysis of the IT formula through a simulated gastrointestinal epithelium. ... 7
2.4.1. Caco-2 cell culture. ... 7
2.4.2. Dichlorofluorescein transportation analysis. ... 8
2.4.3. IT formula transportation assay. ... 8
2.4.4. Quantification of the IT formula transportation by ELISA. ... 9
2.4.5. Immunohistochemistry. ... 9
2.5. In vitro toxicity analysis of the IT formula. ... 10
2.5.1. Cytotoxicity analysis of the IT formula in THP1-XBlueTM cells. ... 10
2.6. In vivo toxicity analysis of the IT formula. ... 10
2.5.2. Hepatotoxicity analysis of the IT formula in mouse serum. ... 11
2.5.3. Nephrotoxicity analysis of the IT formula in mouse serum... 11
3. Results. ... 12
3.1. Biochemical characterization of the IT formula. ... 12
3.2. The IT formula could stimulate NF-κB/AP-1 activation and PBMCs proliferation...14
3.3. The IT formula could cross through a Caco-2 cell monolayer. ... 15
3.4. The IT formula did not show any toxicity in vitro or in vivo. ... 18
4. Discussion. ... 21
5. Conclusions. ... 24
6. References. ... 25
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FIGURES.
Fig. 1 Transwell® culture plates. ... 8
Fig. 2 Characterization of VNP and VNP-Pru p 3 ... 12
Fig. 3 TEM imaging of VNP and VNP-Pru p 3 ... 13
Fig. 4 VNP-Complex formation. ... 13
Fig. 5 NF-κB/AP-1 activation using the transfected cell line THP1-XBlueTM... 14
Fig. 6 Proliferation assay of human PBMCs of control and allergic patients. ... 15
Fig. 7 Dichlorofluorescein transportation assay. ... 16
Fig. 8 Transportation of IT formula and its components through a simulated gastrointestinal epithelium. ... 16
Fig. 9 Analysis of the integrity of the Caco-2 monolayers. ... 17
Fig. 10 Immunolocalization of Pru p 3 and VNP inside Caco-2 cells after 2 hours of incubation. ... 17
Fig. 11Cytotoxicity analysis in THP1-XBlueTM cells... 18
Fig. 12 Variation of mice body weight during the six-week period of treatment... 19
Fig. 13 Hepatotoxicity analysis of the IT mouse model. ... 20
Fig. 14 Nephrotoxicity analysis of the IT mouse model. ... 20
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Keywords
Allergy / immunotherapy / Pru p 3 / VNP
Abbreviations
ALP: alkaline phosphatase APC: antigen-presenting cell BSA: bovine serum albumin BUN: blood urea nitrogen CP: capsid protein
DMEM: Dulbecco´s modified Eagle´s medium ELISA: enzyme-linked immunosorbent assay FBS: fetal bovine serum
HBSS: Hanks´ Balanced Salt Solution IgE: immunoglobulin E
IgG: immunoglobulin G IT: immunotherapy LPS: lipopolysaccharide LTP: lipid transfer protein OH-CPT: hydroxy-camptothecin
PBMCs: peripheral blood mononuclear cells PBS: phosphate-buffered saline
PI: propidium iodide
pNPP: para-nitrophenylphosphate RPMI: Roswell Park Memorial Institute RT: room temperature
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis SEAP: secreted embryonic alkaline phosphatase
SI: stimulation index
TEER: transepithelial electric resistance TEM: transmission electron microscopy TLC: thin layer chromatography TLR: toll-like receptor
TMB: 3,3´,5,5´-tetramethylbenzidine TuMV: turnip mosaic virus
VLP: virus-like particle VNP: viral nanoparticle
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RESUMEN
La alergia es una de las enfermedades más prevalentes a nivel mundial y su incidencia se está incrementando sin que dispongamos a día de hoy de tratamientos curativos efectivos, más allá de los paliativos que tratan los síntomas, pero sin dirigirse a la raíz del problema. Por este motivo, actualmente se investigan nuevas formas de inmunoterapia que puedan ser efectivas y seguras.
En este trabajo se presenta una fórmula de inmunoterapia dirigida al tratamiento de la alergia alimentaria al melocotón. Esta fórmula consiste en el alérgeno principal del melocotón, la proteína Pru p 3, en complejo con su ligando (Complex) y en combinación con una nanopartícula viral (VNP) basada en el virus del mosaico del nabo (TuMV) de plantas. De esta forma se obtiene un ensamblado donde el alérgeno es expuesto de forma repetitiva en la superficie de la partícula.
El objetivo principal del proyecto ha consistido en la realización de una primera caracterización de esta fórmula, a la que nos referimos como VNP-Complex.
Los estudios bioquímicos de su composición confirmaron la correcta síntesis de VNP-Complex, y los análisis de microscopía electrónica indicaron la presencia de ensamblados helicoidales.
Además, se analizó su capacidad para interactuar con el sistema inmune, y para ello se usó una línea celular derivada de monocitos humanos, así como células mononucleares de sangre periférica. Los resultados obtenidos indicaron que VNP-Complex presenta una gran capacidad inmunogénica y de estimulación de la proliferación celular.
A continuación, se analizó la capacidad de la fórmula para ser transportada a través de un epitelio gastrointestinal, para lo que se realizó un modelo celular en 3D. Los primeros indicios de este experimento nos indicaron que la fórmula era transportada hacia el lado basolateral, aunque la integridad de VNP-Complex tras el transporte deberá ser analizada en futuros ensayos.
Por último, llevamos a cabo varios ensayos preliminares acerca de la posible toxicidad de VNP- Complex sobre el organismo. Los estudios in vitro e in vivo en modelo de ratón no mostraron ningún signo de daño sobre células inmunes e intestinales, crecimiento o función hepática y renal.
Por lo tanto, podemos considerar la nueva fórmula como un compuesto no tóxico.
Los siguientes pasos en este proyecto deberán incluir nuevos estudios de transporte y toxicidad, incluyendo nuevos biomarcadores, así como el estudio in vivo de la inhibición de la desgranulación de células efectoras y los cambios inmunológicos generados por VNP-Complex en la respuesta de anticuerpos.
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ABSTRACT
Allergy is one of the most prevalent diseases globally and its incidence is increasing with no effective curative treatments available today, beyond palliative treatments that treat symptoms, but do not address the root of the problem. For this reason, there is current investigation of new forms of immunotherapy that can be effective and safe.
In this work, we present an immunotherapy formula for the treatment of food allergy to peach.
This formula consists of the major peach allergen, the protein Pru p 3, with its ligand (Complex) in combination with a viral nanoparticle (VNP) based on the plant turnip mosaic virus (TuMV).
In this way, we obtain an assembly where the allergen is repeatedly exposed on the surface of the particle.
The main objective of this project has been to carry out a first characterization of this formula, which we refer to as VNP-Complex.
The biochemical studies of its composition confirmed the correct synthesis of VNP-Complex, and the electron microscopy analysis indicated the presence of helical arrangements. In addition, its ability to interact with the immune system was analyzed using a cell line derived from human monocytes, as well as peripheral blood mononuclear cells. The results obtained indicated that VNP-Complex shows great capacity for immunogenicity and cell proliferation stimulation.
Furthermore, the ability of the formula to be transported through a gastrointestinal epithelium was analyzed, for which a 3D cell model was carried out. The first indications of this experiment showed that the formula was transported towards the basolateral side, although the integrity of VNP-Complex after transport should be analyzed in future studies.
Finally, we carried out several preliminary assays on the possible toxicity of VNP-Complex. In vitro and in vivo mouse model studies did not show any signs of damage to intestinal and immune cells, growth or liver and kidney function. Thus, we can consider the new formula as a non-toxic compound.
The next steps in this project should include new transport and toxicity studies, including new biomarkers, as well as the in vivo study of the inhibition of effector cell degranulation and the immunological changes generated by VNP-Complex in the antibody response.
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CHAPTER 1. Introduction.
Allergic diseases can seriously affect the physical and psychological health of patients and thus exert a huge burden on their quality of life, as well as on society as a whole. Recent epidemiological studies have revealed new risk factors for allergy development and indicate that its prevalence is increasing in several countries (Simon, 2018). As an example, food allergy has an estimated prevalence of 10% in the developed world (Savage and Johns, 2015). However, there are still a number of unmet needs regarding, for example, the role of genetics and its interaction with the environment or the prevention of allergic diseases (Simon, 2018).
For overall health, it is essential that the immune system distinguishes pathogenic antigens from innocuous environmental antigens. However, sensitization to allergens results in inappropriate inflammatory responses. During an inflammatory state, antigen-presenting cells (APC) that transport allergens to secondary lymphoid tissues for antigen presentation, deviate the immune system towards Th2 allergic responses, rather than tolerogenic. This leads to immunoglobulin E (IgE) class-switching and production by B cells. Consequently, upon future exposures, allergen- derived epitopes can attach to IgE molecules bound to FcεRI receptors on the surface of immune effector cells, such as mast cells and basophils. This event leads to cell degranulation and release of preformed histamine and other inflammatory mediators, which results in the rapid manifestation of symptoms. After this immediate phase of response, the de novo production of leukotrienes, platelet activating factor and cytokines maintains the allergic inflammation (Yu et al., 2016).
Peach allergy consist of one representative example of food allergy. In this case, Pru p 3, the major peach allergen, forms part of the lipid transfer protein (LTP) family and represents the most common food allergen to which patients in the Mediterranean area are sensitized (van Winkle and Chang, 2014). The characterization of the ligand of Pru p 3 showed that it was formed by a derivative of camptothecin bound to a long hydrophobic tail, identified as phytosphingosine (Cubells-Baeza et al., 2017). This ligand could act as an adjuvant in the sensitization process as it has the ability to modulate the immune system towards a Th2 response (Gonzalez-Klein et al., 2021).
Currently, no definitive therapies apart from allergen avoidance and injectable epinephrine exist for the treatment of acute allergic reactions; along with antihistamines, steroids or H1 receptor blockers for localized allergy symptoms. Moreover, these treatments do not address the underlying immune disorder, but only the symptoms associated, presenting several side effects such as nutritional deficiencies and impaired growth in children with avoidance diets (Wood, 2017; Yu et al., 2016).
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Hence, allergen-specific immunotherapy (IT) for the desensitization of individuals to potential allergens represents a tremendous progress in the treatment of allergy (Yu et al., 2016). It is currently the only specific and disease‐modifying treatment for allergic conditions, having the capacity to improve symptoms, reduce the need for medications, induce specific tolerance beyond the duration of the treatment and prevent the development of new allergic conditions (Alvaro- Lozano et al., 2020).
The immune changes that accompany IT are thought to occur as a progression of several elements:
decreasing mast cell and basophil responsiveness to degranulation; increasing levels of regulatory T cells; deletion of allergen-specific Th2 cells; declining levels of allergen-specific IgE and growing numbers of regulatory B cells, which secrete regulatory interleukin-10 and allergen- specific immunoglobulin G (IgG) (Yu et al., 2016).
Desensitizing IT is generally delivered sublingually, orally or through the skin. In any case, daily allergen doses begin in the submilligram range and increase gradually over a period of days, weeks or months until a maintenance dose is reached (Yu et al., 2016). Unfortunately, only few molecular IT approaches have been moved successfully into clinical evaluation, and there are currently only allergen extract-based vaccines available (Valenta et al., 2018). However, the use of standardized allergen extracts has shown several drawbacks such as duration of administrations, high risks of threatening anaphylactic reactions or fluctuating quality of allergen extracts due to proteases, unknown nonallergic materials or contaminants (Schmitz et al., 2009;
Soongrung et al., 2020; Valenta et al., 2018).
This shows the necessity to find different IT formulas that alleviate allergic symptoms upon few injections while being nonreactogenic. Therefore, alternative approaches consisting of allergens coupled to viral nanoparticles (VNPs) are being investigated and, more precisely, the use of certain subclass of VNPs, the virus-like particles (VLPs), which are the genome-free counterparts of VNPs (Schmitz et al., 2009; Yildiz et al., 2011). It has been observed that the resultant display of repetitive arrays of allergens on VNP surfaces leads to hypoallergenic and highly immunogenic IT formulas. Apparently, these formulas show drastic reductions of the binding capacity of the allergen to immobilized IgE, together with potent inhibition of basophil and mast cell activation by supra-optimal allergen concentrations. Moreover, the high allergen density on these formulas could boost B-cell activation through strong B cell receptor signaling to optimize the production of allergen-specific blocking IgG antibodies (Soongrung et al., 2020).
The present work has been focused on the preliminary study and characterization of a VNP-based IT formula for the treatment of peach allergy. This IT formula consists of the genetic fusion of the coat protein (CP) from turnip mosaic virus (TuMV) with the allergen Pru p 3, which is
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simultaneously forming a Complex with its ligand; resulting in the formation of a VNP displaying multi-copies of Complex (Pru p 3 + ligand). We refer to this formula as VNP-Complex.
Drug development is a slow process since it must proceed through several stages in order to create a product that is safe, efficacious and has passed all regulatory requirements. The first steps of this process include drug discovery, which in this case was based on several previous studies for both molecules forming the immunotherapy: Complex and TuMV VNPs. Thus, the analysis of this therapy starts with the product characterization, which typically includes the study of its size, composition and shape, along with its toxicity, bioactivity and bioavailability. Following this step, the preclinical testing analyzes the safety and efficacy of the formulated drug product using both mammalian cells and different mammalian species, and should be designed to support the clinical studies on humans that will follow (Pacific Biolabs, 2021).
The study of this IT formula would provide us with new insights into the understanding of the mechanism underlaying allergy desensitization and add more information about the design of effective formulas for immunotherapy.
Objectives
The presented work has been carried out with the aim of performing an initial characterization of the VNP-based immunotherapy formula addressed to the treatment of peach allergy. For this purpose, we have focused our attention in the following objectives regarding the formula:
Biochemical characterization of its composition and assembly.
Immunological effect on the activation of the NF-κB/AP-1 pathway and on the proliferation of peripheral blood mononuclear cells.
Transportation analysis through a model of gastrointestinal epithelium.
In vitro and in vivo toxicological analysis.
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CHAPTER 2. Materials and methods.
2.1. Biochemical characterization of the IT formula.
To elucidate the composition of the IT formula, VNP samples alone and fused with Pru p 3 (VNP- Pru p 3) were analyzed with different approaches.
VNP concentration in the samples was determined by considering an absorption coefficient (A0.1%, 1 cm at 260nm) of 2.65 (Cuesta et al., 2019). Pru p 3 concentration was elucidated by the assumption that one protein of Pru p 3 (9 kDa) is bounded to one CP from TuMV (35 kDa), leading to a 1:4 ratio.
2.1.1. Immunodetection.
VNP and VNP-Pru p 3 samples (Pru p 3, 1.25 μg; VNP, 5 μg) were separated by 12%
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were stained with Coomassie Blue or electrotransferred onto methanol-activated 0.45 µm polyvinylidene difluoride membranes (IMMOBILON, Millipore, USA) using Tris-Boric buffer (50mM; pH 8.3) with a constant intensity of 70V during an hour.
Membranes were then blocked with blocking buffer (Sigma-Aldrich, Germany) and incubated with rabbit polyclonal antibody against Pru p 3 (1:1000) or rabbit polyclonal antibody against TuMV (1:500) at room temperature (RT) with agitation. Subsequently, membranes were incubated with alkaline phosphatase-conjugated anti-rabbit IgG (Sigma- Aldrich, 1:20000) or horseradish peroxidase-conjugated anti-rabbit IgG (Sigma-Aldrich, 1:30000), for Pru p 3 or TuMV detection respectively. Membrane development was performed with an alkaline phosphatase substrate solution, NitroBlue tetrazolium/5- bromo-4-chloro-3´-indolyl phosphate (Sigma-Aldrich) or a horseradish peroxidase substrate solution, SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher, USA).
2.1.2. Transmission electron microscopy.
In order to confirm the assembly of VNP and VNP-Pru p 3 into helical arrangements, VNP and VNP-Pru p 3 samples (Pru p 3, 0.025 mg/mL; VNP, 0.1 mg/mL) were resuspended in borate buffer (0.05M; pH 8.1). Microscopy grids were incubated for 10 minutes with rabbit polyclonal antibody against TuMV or rabbit polyclonal antibody against Pru p 3 at a 1:1000 dilution, for VNP and VNP-Pru p 3 samples respectively.
After washing with milli-Q water, grids were incubated with 10 μL of VNP or VNP-Pru p 3 for 10 minutes and washed in milli-Q water. Rabbit polyclonal antibody against TuMV and rabbit polyclonal antibody against Pru p 3 were used for decoration at a 1:50
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dilution for VNP and VNP-Pru p 3 samples, respectively. After washing in milli-Q water, grids were incubated for 3 minutes with uranyl acetate 2% and sent to the ICTS Centro Nacional de Microscopia Electrónica (Madrid, Spain) for their visualization with transmission electron microscopy (TEM). Samples were visualized using the microscope JEOL JEM 1400.
2.2. Confirmation of the VNP-Complex formation.
In order to clarify if the ligand was still able to bind to Pru p 3 in the presence of the VNP genetically fused to it, we boarded two strategies.
For the preparation of Complex and VNP-Complex samples, the ligand was lyophilized and then resuspended with the corresponding amount of Pru p 3 and VNP-Pru p 3, respectively. This was achieved by maintaining the 1:10 ratio between the ligand and Pru p 3 (Gonzalez-Klein et al., 2021). These samples were then dialyzed with ammonium acetate 0.5M using 3.5 kDa dialysis membranes (Spectrum Spectra/Por, ThermoFisher).
2.2.1. Thin layer chromatography.
A thin layer chromatography (TLC) was performed on Silica gel-coated 60 F254 plates (Merck) for the separation of the lipid fraction of the different samples: ligand, Pru p 3, Complex, VNP, VNP-Pru p 3 and VNP-Complex (ligand, 1 μg; Pru p 3, 10 μg; VNP, 40 μg). The TLC was developed using ethanol: acetic acid: ethyl acetate (6:3:1 v/v) as the mobile phase in a saturated chromatography chamber. It was then visualized under UV light (365nm) in order to assess the presence of the ligand, which has the capacity to emit under UV light due to its camptothecin derivate head (Cubells-Baeza et al., 2017).
2.2.2. Absorbance at 254 nm and 260 nm.
Samples absorbances at 254nm were measured in a NanoDropTM spectrophotometer (ThermoFisher), since the ligand presents the capacity to absorb under this wavelength (Cubells-Baeza et al., 2017). In addition, absorbance values at 260nm were also measured, since proteins, and specially VNPs from TuMV, present high absorbance values at this wavelength and could interfere with the absorbance at 254nm. Blank samples used were ammonium acetate 0.5 M for Pru p 3 and Complex, and VNP in ammonium acetate 0.5 M for VNP-Pru p 3 and VNP-Complex. Statistically significant differences were analyzed by GraphPad 6 (GraphPad Software Inc, La Jolla, USA) using 2-way ANOVA test, and P-values <.05 were considered positive.
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2.3. Immunological characterization of the IT formula.
Immunological characterization of VNP, VNP-Pru p 3 and VNP-Complex was carried out by using two different types of immune cells: immortalized THP1-XBlueTM cells and peripheral blood mononuclear cells (PBMCs). For this purpose, Pru p 3 and Complex were used as controls, since its immunological characterization has already been studied (Tordesillas et al., 2017).
On the one hand, THP1-XBlue™ (Invivogen, France) is a transfected cell line derived from the human monocytic THP-1 cell line. These cells express a reporter gene, the secreted embryonic alkaline phosphatase (SEAP), under the control of an inducible promoter by the transcription factors NF-κB and AP-1. These transcription factors are activated when stimulation through toll- like receptors (TLRs) present on the cell surface occurs. SEAP levels can then be detected and measured using the reagent QUANTI-Blue™ (Invivogen), which produces a colorimetric reaction in the presence of SEAP.
On the other hand, six voluntary healthy donors and four patients with a clear clinical history of peach allergy were recruited for peripheral blood donation at the Fundación Jiménez Díaz Hospital (Madrid, Spain). All individuals provided informed consent and the experiments were conducted in accordance with the latest revision of the Declaration of Helsinki (World Medical Association, 2013).
For this immunological study VNP, VNP-Pru p 3 and VNP-Complex samples were resuspended in phosphate-buffered saline (PBS) using Amicon® Ultra 2 mL filters.
2.3.1. NF-κB/AP-1 activation analysis.
THP1-XBlueTM cells were seeded, according to the manufacturer’s instructions, in a Petri dish (15x100 mm) until a concentration of 1x106 cells/mL using Roswell Park Memorial Institute (RPMI) 1640 medium (Lonza, Basel, Switzerland), supplemented with L- glutamine (Sigma-Aldrich), heat-inactivated fetal bovine serum (FBS) (GE HealthCare Life Sciences, Little Chalfont, UK) and penicillin-streptomycin (Sigma-Aldrich). Cells were positively selected with 200 μg/mL of Zeocin (Invivogen).
The assay was carried out in a flat-bottom 96-well plate (Costar, New York, USA) containing 200 μL of cell suspension (~200,000 cells per well). Pru p 3, Complex, VNP, VNP-Pru p 3 and VNP-Complex samples (ligand, 0.1 μg; Pru p 3, 1 μg; VNP, 4 μg) were added to the cells and incubated for 24 hours at 37ºC and 5% CO2. Lipopolysaccharide (LPS) (0,03 μg) (Sigma-Aldrich) and PBS were used as positive and negative controls, respectively. After incubation, 20 μL of each cell suspension were added to 180 μL of QUANTI-Blue™ reagent in a flat-bottom 96-well plate and incubated at 37ºC and 5% CO2. After 2 hours of incubation, SEAP levels were measured at 600nm with a SPECTROstar
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Nano spectrophotometer (BMG LABTECH, Ortenberg, Baden-Wuerttember, Germany).
Results were expressed as the ratio between the absorbance values of the stimulated cells and the absorbance value of the negative control, expressed as stimulation index (SI):
𝑆𝐼 = 𝑂. 𝐷. 𝑤𝑖𝑡ℎ 𝑠𝑡𝑖𝑚𝑢𝑙𝑢𝑠 𝑂. 𝐷. 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑠𝑡𝑖𝑚𝑢𝑙𝑢𝑠
SI values above 1.5 were considered positive. The assay was performed in triplicates and statistically analyzed by GraphPad 6 using Kruskal–Wallis test. P-values <.05 were considered significant.
2.3.2. PBMCs proliferation analysis.
PMBCs were isolated from peripheral blood using a density gradient centrifugation on Lymphoprep (Axis-Shield, Oslo, Norway). Cell cultures (~200,000 cells per well) were seeded in triplicates in flat-bottom 96-well plates in a total volume of 200 μL of supplemented RPMI 1640 medium. Cells were incubated with Pru p 3, Complex, VNP, VNP-Pru p 3 and VNP-Complex samples (ligand 8.325 ng; Pru p 3, 83.25 ng; VNP, 333 ng). LPS (0,2 μg) was used as positive control. Cell cultures were incubated for 5 days at 37°C, in a 5% CO2 humidified atmosphere. PBMCs proliferation was quantified by flow cytometry using a BD AccuriTM C6 Flow Cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA). Results were represented as the percentage of proliferation and SI of allergic patients. For this assay, SI of allergic patients was calculated by dividing their percentage of proliferation by the percentage of proliferation of control patients stimulated with the same stimulus:
𝑆𝐼 =𝑃𝑟𝑜𝑙𝑖𝑓𝑒𝑟𝑎𝑡𝑖𝑜𝑛 (%) 𝑜𝑓 𝑎𝑙𝑙𝑒𝑟𝑔𝑖𝑐 𝑝𝑎𝑡𝑖𝑒𝑛𝑡𝑠 𝑃𝑟𝑜𝑙𝑖𝑓𝑒𝑟𝑎𝑡𝑖𝑜𝑛 (%) 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑝𝑎𝑡𝑖𝑒𝑛𝑡𝑠
Percentages of proliferation were statistically analyzed with GraphPad 6 using a 2-way ANOVA test and SI of allergic patients, with a Kruskal-Wallis test. P-values <.05 were considered positive.
2.4. Transportation analysis of the IT formula through a simulated gastrointestinal epithelium.
2.4.1. Caco-2 cell culture.
Intestinal epithelium Caco-2 cells (ATCC® HTB-37TM, Manassas, USA) were seeded in 24-well Transwell® inserts (Fig. 1) of 8 µm pore diameter (Corning® Inc., Sigma- Aldrich) at a density of ~8x104 cells per well. In order to let Caco-2 cells differentiate into a complete monolayer, cells were incubated for 21 days at 37ºC and 5% CO2, having the
8
medium replaced every 2 days. The medium employed was Dulbecco’s modified Eagle’s medium (DMEM) 4.5 g/L glucose (Lonza), supplemented with L-glutamine, FBS, antimycotic (Sigma-Aldrich) and penicillin-streptomycin. For measuring the integrity of the Caco-2 cell monolayers, the transepithelial electrical resistance (TEER) was checked using a Millicell-ERS device (Merck). Monolayers showing TEER values above 300 Ωcm2 were considered adequate for transport analysis (Tordesillas et al., 2013).
Fig. 1 Transwell® culture plates.
The porous membrane favors the formation, polarization and differentiation of a monolayer by the
creation of a space
compartmentalization that simulates the gastrointestinal tract, forming an apical and basolateral side for cells.
2.4.2. Dichlorofluorescein transportation analysis.
Dichlorofluorescein transportation through a Caco-2 cell monolayer was subsequently performed as an additional approach for confirming the integrity of the monolayers. For this assay, 100 μg of 2´,7´-Dichlorofluorescein (Sigma-Aldrich) were added to the apical side of an empty and a Caco-2 seeded Transwell® insert. The basolateral sides were filled with Hanks' Balanced Salt Solution (HBSS) (Lonza) and the medium was retired and equally replaced after 10, 20, 60, 90, 120 and 240 minutes. During the assay, cells were maintained in incubation at 37ºC and 5% CO2. Dichlorofluorescein transported values were detected by measuring the fluorescence of the collected media at 492nm using a SPECTROstar Nano spectrophotometer. Statistically significant differences were analyzed by GraphPad 6 using 2-way ANOVA test, and P-values <.05 were considered positive.
2.4.3. IT formula transportation assay.
For this assay, all samples employed were previously resuspended in PBS. Complex, VNP and VNP-Complex samples (ligand 0.1 μg; Pru p 3, 1 μg; VNP, 4 μg) were added to the apical side of the Transwell® inserts in 200 μL of supplemented DMEM 4.5 g/L glucose. The basolateral sides were filled with culture medium, but in this case, using 600 μL of FBS-free supplemented DMEM 4.5 g/L glucose. Basolateral samples were collected after 0.5, 1, 2, 6 and 24 hours of incubation and an equal volume of FBS-free supplemented DMEM 4.5 g/L glucose was replaced immediately after each sampling.
During the assay, cells were maintained in incubation at 37ºC and 5% CO2. The integrity
9
of the Caco-2 monolayers at the end of the transportation assay was checked by measuring their TEER values.
2.4.4. Quantification of the IT formula transportation by ELISA.
Collected basolateral samples of the IT formula transportation assay were dialyzed in ammonium acetate 10mM overnight at 4º using 3.5 kDa dialysis membranes. Samples were then lyophilized and resuspended in PBS.
An enzyme-linked immunosorbent assay (ELISA) was then performed using basolateral samples to coat polystyrene 96-well microtiter plaques (Corning® Costar, Sigma- Aldrich) overnight at 4 ºC. Coated wells were next blocked with hydrolyzed casein blocking buffer for an hour at RT with agitation and incubated with rabbit polyclonal antibody against Pru p 3 (1:1000) or rabbit polyclonal antibody against TuMV (1:1000).
Likewise, both antibodies were used for the wells coated with samples of VNP-Complex.
After one hour of incubation, wells were washed and incubated with horseradish peroxidase-conjugated anti-rabbit IgG. After newly washing the wells, 3,3′,5,5′- tetramethylbenzidine (TMB) (ThermoFisher) was used to develop the ELISA. Reaction was stopped with HCl 2N and the absorbance was measured at 450nm using a SPECTROstar Nano spectrophotometer. Tests were performed in triplicate. Statistically significant differences were analyzed by GraphPad 6 using 2-way ANOVA test, and P- values <.05 were considered positive.
2.4.5. Immunohistochemistry.
This technique was performed to detect the presence of Pru p 3 and VNP inside Caco-2 cells. These cells were seeded on poly-L lysine (ThermoFisher) treated round coverslips of 2 cm2 (Paul Marienfeld, Lauda-Königshofen, Germany) by adding ~200,000 cells per coverslip in 250 μL of supplemented DMEM 4.5 g/L glucose. After 48 hours of incubation at 37ºC and 5% CO2, VNP-Complex was added (ligand 0.45 μg; Pru p 3, 4.5 μg; VNP, 18 μg) to cells in 500 μL of supplemented DMEM 4.5 g/L glucose. After 2 hours of incubation, coverslips were washed three times with PBS and fixed with paraformaldehyde 4% (ThermoFisher) for ten minutes at RT. Nucleus of Caco-2 cells were stained with DAPI by a seven-minutes incubation with 0.1% Triton - 0.72mM DAPI solution. After washing in PBS, coverslips were blocked for 45 minutes with bovine serum albumin (BSA) blocking buffer (PBS-BSA 1%) and incubated at RT with mouse monoclonal antibody against TuMV and rabbit polyclonal antibody against Pru p 3, both at a 1:50 dilution with PBS-BSA 0.1%, for 60 and 40 minutes respectively. Antibody excess was removed by washing three times with PBS, followed by incubation with secondary antibodies for 45 minutes at RT in dark. In this case, secondary antibodies were
10
Alexa Fluor® 546-conjugated anti-mouse IgG and Alexa Fluor® 647-conjugated anti- rabbit IgG for VNP and Pru p 3 recognition, respectively. Finally, coverslips were washed 3 times with PBS before being mounted with ProLongTM Gold Antifade Mountant (ThermoFisher). Images were obtained using 488, 561 and 633nm laser excitations with a Zeiss LSM 880 confocal microscope (Zeiss, Oberkochen, Germany). Image treatment was done using ZEN 3.3 (ZEN Lite) software.
2.5. In vitro toxicity analysis of the IT formula.
2.5.1. Cytotoxicity analysis of the IT formula in THP1-XBlueTM cells.
THP1-XBlueTM cells were seeded in a flat-bottom 96-well plate containing 100 μL of cell suspension (~100,000 cells per well). After resuspending in PBS, Complex, VNP, VNP- Pru p 3 and VNP-Complex samples (ligand, 50 ng; Pru p 3, 500 ng; VNP, 2 μg) were added to cells and incubated for 24 hours at 37ºC and 5% CO2. Hydroxy-camptothecin (OH-CPT) (50 ng) and PBS were used as positive and negative controls, respectively. As for the cytotoxicity study, annexin V-FITC and propidium iodide (PI) were used as biomarkers for apoptosis and necrosis, respectively. Hence, 50 μL of cell suspension where mixed with 5 μL of 1:10 diluted annexin-V-FITC (Sigma-Aldrich) and 10 μL of 1:10 diluted propidium iodide (Sigma-Aldrich). After protecting samples from light for ten minutes at RT, cells were analyzed by flow cytometry using a BD AccuriTM C6 Flow Cytometer. Fluorescence was detected at the proper channels, FL-1 for annexin V-FITC and FL-3 for PI. Results were represented as the percentage of dead cells. The assay was performed in triplicates and statistically significant differences were analyzed by GraphPad 6 using a Kruskal-Wallis test. P-values <.05 were considered positive.
2.6. In vivo toxicity analysis of the IT formula.
In order to study the efficacy and toxicity of the VNP-based IT formula in vivo, a murine model using C3H/HeOuJ mice (Charles River, Barcelona, Spain) was developed at the Fundación Jiménez Díaz Hospital (Madrid, Spain) following a previous anaphylactic mouse model against Pru p 3 (Pazos-Castro et al., 2021). Control and allergic mice were treated weekly for a total of 6 weeks with VNP, VNP-Pru p 3 and VNP-Complex (ligand, 10 μg; Pru p 3, 100 μg; VNP, 400 μg). A non-treated group was also carried out for both control and allergic mice. Each group was formed by 5 rodents and body weight was measured weekly. After the six-week period of treatment, mice serum was collected and preserved at -80ºC until its analysis. Finally, two weeks after the end of the treatment period, mice were intraperitoneally challenged with 100 µg of Pru p 3 and sacrificed. All the project was positively evaluated by the Institutional Animal Care and Use Committee from the Community of Madrid (Ref. PROEX 392/15) and developed in accordance with the European Union Directive 2010/63/EU.
11
2.5.2. Hepatotoxicity analysis of the IT formula in mouse serum.
Alkaline phosphatase (ALP) levels in mice sera were measured as a biomarker for hepatotoxicity. A 25 μL pool of sera from the five mice composing each group was mixed with 215 μL of carbonate buffer (0.1M; pH 10.2), 30 μL of para-nitrophenylphosphate (pNPP) substrate (10 mM) (Sigma-Aldrich) and 30 μL of Cl2Mg (3mM) in a flat-bottom 96-well plate. Samples were incubated at 37ºC in dark and measured every 10 minutes at 405nm using a SPECTROstar Nano spectrophotometer. As positive controls, 1 μL of ALP-conjugated anti-rabbit IgG (Sigma-Aldrich, 1:20,000) was used at a dilution 1:1000.
Tests were performed in duplicate. Mice serum data was statistically analyzed with GraphPad 6 using a Kruskal-Wallis test and a Mann-Whitney test. P-values <.05 were considered positive.
2.5.3. Nephrotoxicity analysis of the IT formula in mouse serum.
Blood urea nitrogen (BUN) was measured in mice sera as a biomarker for nephrotoxicity using the Urea Nitrogen Colorimetric Detection Kit (Invitrogen, Frederick, USA). Mouse serum was diluted 1:20 with PBS and processed according to the manufacturer’s instructions in a clear 96-well plate (Invitrogen). After 30 minutes of incubation, BUN levels were measured at 450nm using a SPECTROstar Nano spectrophotometer. Tests were performed in duplicate. Data was statistically analyzed with GraphPad 6 using a Kruskal-Wallis test. P-values <.05 were considered positive.
12
CHAPTER 3. Results.
3.1. Biochemical characterization of the IT formula.
VNP and VNP-Pru p 3 samples were analyzed by 12% SDS-PAGE, stained with Coomassie Blue or incubated with anti-Pru p 3 or with anti-TuMV specific antibodies. For VNP samples, a band corresponding to the molecular weight of the CP (35kDa) (black arrow), could be observed in Coomassie and in immunodetection with anti-TuMV, but not with anti-Pru p 3 (Fig. 2). In the other hand, a band of 44kDa (black arrow) corresponding to the sum of the molecular weights of the CP and Pru p 3 (9kDa) was detected for VNP-Pru p 3 samples in Coomassie and in both immunodetections with anti-Pru p 3 and anti-TuMV (Fig. 2).
Fig. 2 Characterization of VNP and VNP-Pru p 3. VNP and VNP-Pru p 3 samples (Pru p 3, 1.25 μg; VNP, 5 μg) were separated by 12% SDS-PAGE and stained with Coomassie Blue (Coomassie) or incubated with specific polyclonal antibodies against Pru p 3 or TuMV (Anti-Pru p 3 and Anti-TuMV, respectively).
The TEM analysis of VNP and VNP-Pru p 3 revealed that both types of samples formed thin filamentous structures (Fig. 3). It was notable that VNP assembled into larger helical arrangements of ~600nm (Fig. 3A), whereas VNP-Pru p 3 assemblies were of ~400nm (Fig. 3B).
13
Fig. 3 TEM imaging of VNP and VNP-Pru p 3. Panels show TEM images for (A) VNP and (B) VNP-Pru p 3.
Since the ligand of Pru p 3 is suggested to be essential as an adjuvant (Gonzalez-Klein et al., 2021), its presence in VNP-Complex was analyzed by developing a TLC and observing it under UV light (365nm). A band corresponding to the ligand could be detected in ligand, Complex and VNP-Complex, but not in Pru p 3, VNP or VNP-Pru p 3 samples (Fig. 4A). Yet, the ligand band in VNP-Complex showed a lower retention factor than the ligand alone or in Complex.
To confirm this result, absorbance values at 254 and 260nm were measured and a significant increase (Fig. 4B; P <.05) could be observed for Complex and VNP-Complex in comparison with their ligand-free counterparts: Pru p 3 and VNP-Pru p 3, respectively.
Fig. 4 VNP-Complex formation. A) Thin layer chromatography (TLC) of ligand, Pru p 3, Complex, VNP, VNP-Pru p 3 and VNP-Complex samples (lipid, 1 μg; Pru p 3, 10 μg; VNP, 40 μg) in an ethanol: acetic:
ethyl acetate (6:3:1 v/v) atmosphere using silica gel-coated plates and observed under UV light (365nm).
B) Pru p 3, Complex, VNP-Pru p 3 and VNP-Complex absorbance values at 254 and 260nm. Blanks used were ammonium acetate 0.5M for Pru p 3 and Complex, and VNP in ammonium acetate 0.5M for VNP- Pru p 3 and VNP-Complex. *P-values <.05.
Absorbance (AU)
Pru p 3
Complex
VNP-Pru p 3 VNP-C
omplex 0 .0
0 .5 1 .0 1 .5
2 5 4 2 6 0
*
*
14
3.2. The IT formula could stimulate NF-κB/AP-1 activation and PBMCs proliferation.
The immunological activity of the IT formula and its components was studied using THP1- XBlue™ cells (human monocyte derived cell line) and human PBMCs obtained from healthy (n=6) and allergic donors (n=4).
As can be seen in Fig. 5, all samples except for Pru p 3 presented immunological activity, with a stimulation index higher than 1.5. Additionally, VNP-Pru p 3 showed a lower stimulation index (SI=10.2) than VNP alone (SI=20.9), but this value increased with the binding of the ligand, as can be seen for VNP-Complex (SI=14.2). All stimuli containing VNPs showed higher SI than Complex alone (SI=1.8).
Fig. 5 NF-κB/AP-1 activation using the transfected cell line THP1-XBlueTM. Cells were incubated with Pru p 3, Complex, VNP, VNP-Pru p 3 and VNP-Complex (ligand, 0.1 μg; Pru p 3, 1 μg; VNP, 4 μg). LPS (0,03 µg) and PBS were used as positive and negative controls, respectively. Activation of NF-κB/AP-1 pathway was revealed with QUANTI-Blue™. Stimulation index was calculated as the ratio between stimulated cells and negative control. An SI > 1.5 (black dotted line) was considered positive. Means and SE (bars) are shown. *P-value <.05 **P-value <.01.
In the same way, PBMCs proliferation after 5 days of stimulation was quantified by flow cytometry and the percentage of proliferation was calculated for every stimulus in comparison with the negative control. Allergic patients showed higher percentages of proliferation than controls when stimulated with samples containing either Pru p 3 or Complex. In accordance with the data obtained from THP1-XBlueTM cells, strong proliferation under stimulation with VNP (Fig. 6A; P <.001) was observed in comparison with the rest of stimuli. Also, VNP-Pru p 3 and VNP-Complex showed lower percentages of proliferation than VNP alone, but higher than Pru p 3 and Complex.
15
As can be seen in Fig. 6B, allergic patients showed SI > 1.5 for VNP-Pru p 3 and VNP-Complex, but not for VNP. Significant differences (Fig. 6B; P<.05) were found between VNP and VNP- Complex.
Fig. 6 Proliferation assay of human PBMCs of control and allergic patients. Cells were incubated with Pru p 3, Complex, VNP, VNP-Pru p 3 and VNP-Complex (ligand, 8.3 ng; Pru p 3, 83 ng; VNP, 333 ng).
LPS (0,2 µg) was used as positive control. After 5 days of incubation, proliferation of PBMCs was measured by flow cytometry. Results were represented as A) the percentage of proliferation of stimulated cells regarding the negative control and as B) the stimulation index of allergic patients, calculated as the ratio between proliferation of allergic and control patients for the same stimulus. An SI > 1.5 (black dotted line) was considered positive. Means and SE (bars) are shown. *P-value<.05 ** P-value<.01 *** P-value
<.001.
3.3. The IT formula could cross through a Caco-2 cell monolayer.
To study if the IT formula could cross through a gastrointestinal epithelium, Caco-2 cell monolayers were used as model in a Transwell® format.
In order to check if monolayers were fully differentiated, with correctly formed tight junctions, a dichlorofluorescein transportation assay was carried out. Dichlorofluorescein was added to the apical side of an empty and a Caco-2 seeded Transwell® insert and basolateral medium was recovered during 4 hours. Dichlorofluorescein concentration was measured by fluorescence. As can be seen in Fig. 7, the percentage of dichlorofluorescein transported after this time was lower and significantly different (Fig. 7; P<.0001) through the Caco-2 cell monolayer (34.5%) than through the empty Transwell® insert (76.6 %), demonstrating that transport was impaired by cell monolayer.
Stimulation Index (SI)
VNP
VNP-Pr u p 3
VNP-Complex 0
1 2 3 4
5 *
16
Fig. 7 Dichlorofluorescein transportation assay. Dichlorofluorescein (100 μg) was added to the apical side of empty and Caco-2 seeded Transwell® inserts. Basolateral medium was recovered at different times and cumulative transport was measured by fluorescence.
**P<.01 ****P<.0001.
Complex, VNP and VNP-Complex transportation was then analyzed by adding these samples into the apical sides of Caco-2 monolayers and incubated for 24 hours. Basolateral media was collected at different time points and protein concentration was measured by ELISA with anti- Pru p 3 and anti-TuMV specific antibodies. When VNP-Complex was added to the apical side, Pru p 3 and VNP transport through the monolayer showed significantly different (Fig. 8; P<.0001) mismatched values (29.2% for Pru p 3 and 47.3% for VNP). In addition, the percentage of Pru p 3 transported in VNP-Complex was lower (29.2%) than when transported in Complex alone (51.2%). Conversely, in the case of VNP only a difference of 6.9% was observed between the transport in the form of VNP-Complex (47.3%) and VNP alone (54.2%).
Fig. 8 Transportation of the IT formula and its components through a simulated gastrointestinal epithelium. Complex, VNP and VNP-Complex (ligand, 0.1 μg;
Pru p 3, 1 μg; VNP, 4 μg) were added to the apical sides of Transwell® inserts seeded with Caco-2 monolayers, and the basolateral medium was recovered at different times. Cumulative transport of proteins in the basolateral medium was measured by ELISA. Means and SD (bars) are shown. ****P<.0001.
C o m p l e x
T im e ( h o u r s )
0 5 1 0 1 5 2 0 2 5
0 2 0 4 0 6 0 8 0 1 0 0
Transported protein (%)
V N P
T im e ( h o u r s )
0 5 1 0 1 5 2 0 2 5
0 2 0 4 0 6 0 8 0 1 0 0
Transported protein (%)
V N P - C o m p le x
T im e ( h o u r s )
0 5 1 0 1 5 2 0 2 5
0 2 0 4 0 6 0 8 0 1 0 0
P r u p 3 V N P
Transported protein (%)
* * * *
* * * *
* * * *
* * * *
T im e ( m in u t e s )
0 1 0 0 2 0 0 3 0 0
0 2 0 4 0 6 0 8 0 1 0 0
S e e d e d E m p ty
Transported dichlorofluorescein (%)
* *
* * * *
* * * *
* * * *
* * * *
* * * *
17
TEER values were measured in order to check if the integrity of the monolayers was maintained after treatment (TEER >300 Ωcm2). TEER values before and after the IT formula transportation assay were all higher than 300 Ωcm2 and without statistically significant differences (2-way ANOVA, P>.05), indicating that monolayers were not damaged by any stimulus (Fig. 9).
Fig. 9 Analysis of the integrity of the Caco-2 monolayers. Transepithelial electric resistance (TEER) was measured before and after the transportation assay of Complex, VNP and VNP-Complex. Monolayers with TEER values >300 Ωcm2 (black dotted line) were considered to maintain their integrity. Means and SD (bars) are shown.
In addition, Caco-2 cells incubated for 2 hours with VNP-Complex were observed at the confocal microscope (Fig. 10). Pru p 3 and VNP were observed to co-localize inside cells, although VNP could also be observed alone.
Fig. 10 Immunolocalization inside Caco-2 cells after 2 hours of incubation. Immunolocalization of DAPI (blue), Pru p 3 (red) and VNP (green).
TEER ( x cm2 )
C-
Complex
VNP
VNP-Complex 0
5 0 0 1 0 0 0 1 5 0 0 2 0 0 0
B e fo r e A f te r
18
3.4. The IT formula did not show any toxicity in vitro or in vivo.
In order to analyze the cytotoxicity of the IT formula, an in vitro study was carried out using the cell line THP1-XBlueTM. After 24 hours of incubation with Complex, VNP, VNP-Pru p 3 and VNP-Complex, cell viability was measured by flow cytometry using annexin-V-FICT and propidium iodide as apoptosis and necrosis biomarkers, respectively. It was observed that none of the stimuli caused more than a 1.6% of cell death (Fig. 11A). Likewise, significant differences (Fig. 11A; P <.01) could be detected between the positive control and every stimulus. Moreover, stimulated cell populations were very similar to that of the negative control and different from the one of the positive control (Fig. 11B).
Fig. 11 Cytotoxicity analysis in THP1-XBlueTM cells. Cells were incubated with Complex, VNP, VNP-Pru p 3 and VNP-Complex (ligand, 50 ng; Pru p 3, 500 ng; VNP, 2 μg) and marked with annexin-V-FICT and propidium iodide. Hydroxy-camptothecin (50 ng) and PBS were used as positive and negative controls, respectively. A) Percentage of dead cells. Means and SD (bars) are shown. *P-value<.05 **P-value<.01
***P-value<.001 ****P-value<.0001. B) Flow cytometry of THP1-XBlueTM cells marked with annexin-V- FITC (FL1-A) and propidium iodide (FL3-A).
19
Complementary to the in vitro cytotoxicity analysis, an in vivo toxicity analysis was developed using a murine model designed for the study of the IT formula. This model was formed by control and allergic mice that were treated with VNP, VNP-Pru p 3, VNP-Complex or untreated.
Mouse body weight of treated mice was measured every week during the six-week period of treatment. It was observed (Fig. 12) that all mice were in the normal weight range (18-35 g) corresponding to their sex and age (Johns Hopkins University, 2015).
Fig. 12 Variation of mice body weight during the six-week period of treatment.
In addition, mice sera was collected in order to analyse the presence of hepatotoxic and nephrotoxic biomarkers: alkaline phosphatase and blood urea nitrogen, respectively.
Alkaline phosphatase (ALP) levels shown by all mice were either undetectable or below their normal range values (126-240 U/L) (Mazzaccara et al., 2008) (Fig. 13A). Significant differences were found (Fig. 13A; P<.05) between the positive control and untreated control and allergic mice, as wells as with VNP and VNP-Complex treated allergic mice. In order to test if the inner components of the sera were blocking the detection of the real ALP levels, 100 U/L of outer ALP were added to serum samples. This assay showed that only ~50% of outer ALP (58.5 U/L in ALP+ serum and 6.2 U/L in ALP- serum) was being detected with our method (Fig. 13B). This was translated as if mice truly presented hepatotoxic damage, we should have observed the presence of at least 120 U/L (half of the maximum normal value) of ALP in their serum.
W e e k s
Weight (g)
1 2 3 4 5 6
1 0 1 5 2 0 2 5 3 0 3 5
C o n tr o l V N P A lle r g ic V N P
C o n tr o l V N P - P r u p 3 A lle r g ic V N P - P r u p 3 C o n tr o l V N P - C o m p le x A lle r g ic V N P - C o m p le x
20
Fig. 13 Hepatotoxicity analysis of the IT mouse model. A) Alkaline phosphatase (ALP) levels were measured in serum of control and allergic mice treated with VNP, VNP-Pru p 3 and VNP-Complex (ligand, 10 µg; Pru p 3, 100 µg; VNP, 400 µg), as well as in untreated mice. ALP-conjugated anti-rabbit IgG was used as positive control. Black-dotted lines represent the minimum and maximum normal ALP values for mice (126 and 240 U/L, respectively). B) Mice serum was measured alone and in the presence of 100 U/L of outer ALP. Means and SE (bars) are shown. *P-value<.05.
In the case of blood urea nitrogen (BUN), it can be observed in Fig. 14 that all mice showed BUN values below the minimun toxic concentration for impaired renal function (50 mg/dL) (Bao et al., 2002). Significant differences (Fig. 14; P <.01) were only found between control untreated mice and allergic VNP-treated mice.
Fig. 14 Nephrotoxicity analysis of the IT mouse model. Blood urea nitrogen (BUN) levels were measured in serum from control and allergic mice treated with VNP, VNP-Pru p 3 and VNP-Complex (ligand, 10 µg;
Pru p 3, 100 µg; VNP, 400 µg), as well as in untreated mice. Red-dotted line represents the minimum toxic concentration (50 mg/dL). Means and SE (bars) are shown. ** P-value< 0.01.
Alkaline Phosphatase (U/L)
- ALP
+ ALP 0
2 0 4 0 6 0 8 0 1 0 0
A L P - +
*
Control Control VNP
Control VNP-Pru p 3 Control VNP-C
omplex Allergic
Allergic VNP Allergic VNP-Pp3
Allergic VNP-C omplex 0
1 0 2 0 3 0 4 0 5 0 6 0
* *
C o n tro l A lle r g ic
VNP VNP-Pru p 3 VNP-Complex
-
VNP VNP-Pru p 3 VNP-Complex
- Blood Urea Nitrogen (mg/dL)
Alkaline Phosphatase (U/L)
C+ Control
Control VNP Control VNP Pp3
Control VNP Complex Alérgico
Alérg ico VNP
Alérgico VNP-Pp3 Alérgico VNP-C
omplex 0
5 0 1 0 0 1 5 0 2 0 0 2 5 0
- V N P V N P
P ru p 3 V N P C o m p le x
C o n t r o l
V N P C o m p le x V N P
P ru p 3 V N P
-
A lle r g ic C+
M o u s e s e r u m
*
*
*
*
