Category Archives: Immunotherapy

IgM antibody and fungal load impacts of P. scutellarioides

J Pharm Pharmacogn Res 6(1): 27-34, 2018.

Original Article | Artículo Original

IgM antibody and colony fungal load impacts of orally administered ethanol extract of Plectranthus scutellarioides on mice with systemic candidiasis

[Impacto de la administración oral del extracto etanólico de Plectranthus scutellarioides sobre anticuerpos IgM y la carga fúngica en ratones con candidiasis sistémica]

Marni Br. Karo1,2*, Mochammad Hatta3, Ilhamjaya Patellongi4, Rosdiana Natzir5, Titus Tambaip6

1Midwifery Program of Medistra Health Higher School, Bekasi City, West Java, 17113, Indonesia.
2School of Post Graduate Faculty of Medicine, Hasanuddin University, Makassar City, South Sulawesi, 90245, Indonesia.
3Molecular Biology and Immunology Laboratory for Infection Diseases, Faculty of Medicine, Hasanuddin Makassar, Makassar City, South Sulawesi, 90245, Indonesia.
4Department of Physiology, Faculty of Medicine, Hasanuddin Makassar, Makassar City, South Sulawesi, 90245, Indonesia.
5Department of Biochemistry, Faculty of Medicine, Hasanuddin Makassar, Makassar City, South Sulawesi, 90245, Indonesia.
6Midwifery Program of Yaleka Maro School, Merauke City, Papua, 99613, Indonesia.

*E-mail: marnikaro.stikesmi@gmail.com

Abstract

Context: Candida albicans is a pathogenic fungus that causes vulvovaginal candidiasis (VVC), which presents significant clinical problems in immunocompromised patients. There are no studies that correlate IgM antibody responses and fungal loads following the use of ethanol leaf extract of Plectranthus scutellarioides (L.) R.Br. (ELEP) on VVC.

Aims: To examine colony fungi loads, IgM antibody responses, and their correlation in Balb/c mice infected with C. albicans treated with orally administered ELEP.

Methods: Twenty-five female Balb/c mice were randomly divided into five groups (five mice in each group): normal group (oral ELEP + not infected), VVC control (VCC) group (infected + oral 0.2 mL saline), ketoconazole-treated in VCC group (infected + oral antifungal agents), and two treatment groups (infected + oral ELEP with 500 and 750 mg/kg b.w.). Treatments were administered for 14 days after infection with C. albicans. Colony fungal loads and IgM antibodies were measured by counting colony forming units and flow cytometry using anti-C. albicans antibody kits, respectively.

Results: Ingestion of the ELEP significantly affected fungal load clearance and decreased IgM antibody levels compared to that in the VCC group (p<0.001). Furthermore, ingestion of the ELEP was associated with significantly lower concentrations of IgM antibodies and colony forming units (p<0.001).

Conclusions: These data strongly suggest that IgM antibody response is involved in of the mechanism by which ELEP decreases C. albicans infection. Furthermore, these results provide important information for the potential application of ELEP in the treatment of Balb/C mice with systemic candidiasis.

Keywords: anti-candida; immune response; miana leaves; vulvovaginal candidiasis.

Resumen

Contexto: Candida albicans es un hongo patógeno que causa candidiasis vulvovaginal (VVC), que presenta problemas clínicos significativos en pacientes inmunocomprometidos. No existen estudios que correlacionen las respuestas de anticuerpos IgM y cargas fúngicas después del uso del extracto etanólico de hoja de Plectranthus scutellarioides (L.) R.Br. (ELEP) en VVC.

Objetivos: Examinar la carga fúngica de colonias, la respuesta de anticuerpos IgM y su correlación en ratones Balb/c infectados con C. albicans tratados con ELEP administrado por vía oral.

Métodos: Veinticinco ratones Balb/c hembras se dividieron en cinco grupos (cinco ratones/grupo): grupo normal (ELEP oral no infectado), grupo control VVC (infectado + 0,2 mL salino oral), tratado con ketoconazol en el grupo VCC (VVC + agentes antifúngicos orales) y dos grupos de tratamiento (VVC + ELEP oral con 500 y 750 mg/kg p.c.). Los tratamientos se administraron durante 14 días después de la infección con C. albicans. Las cargas fúngicas y los anticuerpos IgM se midieron contando unidades formadoras de colonias y citometría de flujo usando kits de anticuerpos anti-C. albicans, respectivamente.

Resultados: La ingestión de ELEP afectó el aclaramiento de la carga fúngica y la disminución de los niveles de anticuerpos IgM en comparación con el grupo VCC (p<0,001). Además, la ingestión de ELEP se asoció con concentraciones más bajas de anticuerpos IgM y unidades formadoras de colonias (p<0,001).

Conclusiones: Estos datos sugieren que la respuesta de anticuerpos IgM está implicada en el mecanismo por el cual ELEP disminuye la infección por C. albicans. Además, estos resultados proporcionan información importante para la aplicación potencial de ELEP en el tratamiento de ratones Balb/c con candidiasis sistémica.

Palabras Clave: anti-candida; candidiasis vulvovaginal; hojas de miana; respuesta inmune.

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Citation Format: Karo M, Hatta M, Patellongi I, Natzir T, Tambaip T (2018) IgM antibody and colony fungal load impacts of orally administered ethanol extract of Plectranthus scutellarioides on mice with systemic candidiasis. J Pharm Pharmacogn Res 6(1): 27–34.

© 2018 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

Antibiotics, cytokine production and cross-talk with cancer cells

J Pharm Pharmacogn Res 4(4): 134-143, 2016.

Original Article | Artículo Original

The effect of antibiotics on cytokine production by mononuclear cells and the cross-talk with colon cancer cells

[Efecto de antibióticos sobre la producción de citocinas por células mononucleares y la interacción con células de cáncer de colon]

Meir Djaldetti1*, Nimrod Nachmias2, Hanna Bessler1

1Laboratory for Immunology and Hematology Research, 2Department of Orthopedics, Rabin Medical Center, Hasharon Hospital, 7, Keren Kayemet St Petah Tiqva, and the Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel.

*E-mail: meird@clalit.org.il

Abstract

Context: Antibiotics belong to the powerful weapons applied against microbial infections. It is notable that in addition to their antimicrobial effect they express immunomodulatory and anti-cancer activities.

Aims: To explore the effect of four antibiotics on the immune cross-talk between peripheral blood mononuclear cells (PBMC) and colon carcinoma cells from two human lines.

Methods: Cefotaxime, meropenem, ampicillin and vancomycin were separately added to PBMC co-incubated with cells from two human colon carcinoma cell lines, i.e. HT-29 and RKO. After 24 hours, the level of the following cytokines produced by PBMC was evaluated: IL-6, IL-1ra, IL-1β, TNFα, IFNγ and IL-10.

Results: All four antibiotics did not affect the generation of IL-6 and IL-1ra in both co-cultures. On the other hand all of them restrained the production of IL-1β by PBMC incubated with HT-29 cells. In the same incubation mixture cefotaxime, vancomycin and meropenem decreased IFNγ and IL-10 production, while ampicillin and vancomycin inhibited TNFα. As for PBMC incubated with RKO carcinoma cells, cefotaxime inhibited the production of IL-1β, IFNγ and mildly of IL-10, whereas vancomycin repressed that of IL-1β, TNFα and IFNγ. Notably, vancomycin increased the production of IL-1β and decreased that of TNFα and IFNγ. The results indicate that the four antibiotics examined exert a modulatory effect on the immune cross-talk between PBMC and human colon cancer cells from two lines expressed by a different impact on pro-and anti-inflammatory cytokines generation.

Conclusions: These findings support the conception that antibiotics may express not only an anti-microbial effect, but also possess an anti-cancer activity that may be considered for integration to the therapeutic arsenal against cancer.

Keywords: Ampicillin; cefotaxime; colon carcinoma cells; cytokines; meropenem; mononuclears; vancomycin.

Resumen

Contexto: Los antibióticos son armas poderosas aplicadas contra las infecciones microbianas. Además de su efecto antimicrobiano expresan actividades inmunomoduladoras y contra el cáncer.

Objetivos: Explorar el efecto de cuatro antibióticos sobre la interacción inmune entre las células mononucleares de sangre periférica (PBMC) y células de carcinoma de colon de dos líneas humanas.

Métodos: Cefotaxima, meropenem, ampicilina y vancomicina se añadieron por separado a PBMC co-incubadas con células de dos líneas de carcinoma de colon humano (HT-29 y RKO). Después de 24 horas, se evaluaron las concentraciones de las siguientes citocinas producidas por PBMC: IL-6, IL-1ra, IL-1β, TNFα, IFNγ e IL-10.

Resultados: Los cuatro antibióticos no afectaron la generación de IL-6 e IL-1ra en ambos co-cultivos. Por otra parte, todos contuvieron la producción de IL-1β por PBMC incubadas con células HT-29. En la misma mezcla de incubación cefotaxima, vancomicina y meropenem disminuyeron la producción de IFNγ e IL-10, mientras que la ampicilina y vancomicina inhibieron TNFα. Cuando PBMC se incubaron con células RKO, cefotaxima inhibió la producción de IL-1β, IFNγ y ligeramente de IL-10, mientras que la vancomicina reprimió las de IL-1β, TNFα e IFNγ. En particular, la vancomicina aumentó la producción de IL-1β y disminuyó las de TNFα e IFNγ. Los resultados indican que los cuatro antibióticos examinados ejercieron un efecto modulador sobre la interacción inmune entre PBMC y las células de cáncer de colon humano a partir de dos líneas, expresadas por un impacto diferente sobre la generación de citocinas pro- y anti-inflamatorias.

Conclusiones: Estos hallazgos apoyan la idea de que los antibióticos pueden presentar no sólo un efecto anti-microbiano, también poseen una actividad anti-cancerígena que pudiera ser considerada para la integración de éstos al arsenal terapéutico contra el cáncer.

Palabras Clave: Ampicilina; cefotaxima; células de carcinoma de colon; citocinas; meropenem; mononucleares; vancomicina.

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Citation Format: Meir Djaldetti, Nimrod Nachmias, Hanna Bessler (2016) The effect of antibiotics on cytokine production by mononuclear cells and the cross-talk with colon cancer cells. J Pharm Pharmacogn Res 4(4): 134-143.
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Krishnananthasivam S, Sathkumara HD, Corea E, Natesan M, De Silva AD (2017) Gene expression profile of human cytokines in response to Burkholderia pseudomallei infection. mSphere 2: e00121-17. DOI: 10.1128/mSphere.00121-17

© 2016 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

CO 071: EARLY PHASE OF SYSTEMIC REACTION TO ORAL ASPIRIN CHALLENGE IS RELATED TO AN INCREASE IN THE LEVELS OF IL-6 AND THE PERCENTAGE OF EFFECTORS MEMORY T-CELLS

J Pharm Pharmacogn Res 2(Suppl. 1): S43, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 071: EARLY PHASE OF SYSTEMIC REACTION TO ORAL ASPIRIN CHALLENGE IS RELATED TO AN INCREASE IN THE LEVELS OF IL-6 AND THE PERCENTAGE OF EFFECTORS MEMORY T-CELLS

Pérez-Novo CA1, Świerczyńska-Krępa M2,3, Niżankowska-Mogilnicka E2, Sanak M2, De Ruyck N2, Holtappels G1, Szczeklik A2, Bachert C1.

1Upper Airways Research Laboratory, Dept. of Otorhinolaryngology, Ghent University Hospital, Belgium.
2Dept. of Medicine, Jagiellonian University School of Medicine, Cracow, Poland.
3Out-patient Allergy ClinicMedex, Bielsko-Biała, Poland.
Abstract

Introduction: Systemic reactions have been suggested to be linked to the hypersensitivity reactions occurring in aspirin exacerbated respiratory disease; however the mechanism has not been elucidated yet. In this study we aim to study the T- cell response and release of inflammatory mediators at systemic level following oral aspirin challenge and their possible link with clinical reactions in patients with aspirin exacerbated respiratory disease (AERD).

Methods: Patients with nasal polyposis and asthma with (n=20) and without AERD (n=18) were orally challenged with aspirin in a double placebo controlled study. Peripheral blood mononuclear cells were isolated and serum samples were collected before and after placebo and aspirin oral challenges. Cells were analyzed for CD4, CD8, CD25, CD127, CD45RA and CD45RO surface markers by Flow cytometry. Serum levels of inflammatory mediators: cytokines, chemokines and lipid mediators were assayed by using the Bioplex (Luminex) technology and ELISA. Urinary LTE4, 9α, 11β-PGF2 were analysed by ELISA.

Results: At baseline, AERD patients showed significant higher levels of sIL5Ra, uLTE4 and percentage of CD4+, CD4+CD25+CD127pos and CD4+CD45RA-CD45RO+ but decreased levels of TGF-β1 and number of CD4+CD25+CD127neg cells. Serum concentrations of IL-8 and sIL5R-α were significantly elevated in patients reacting to a dose of aspirin lower than 250 mg (half of the cumulative dose of 500 mg) when compared to non-AERD subjects and patients reacting to the highest doses of aspirin administrated (500 mg). Oral aspirin challenge resulted in the release of IL-6, urinary LTE4 and in the increased of the number of CD4+CD45RA-CD45RO+ memory T-cells only in AERD patients but failed to reduce the levels of sCD40L as occurred in nonAERD subjects. The release of IL-6 was elevated in patients with bronchial and nasal reactions when compared to subjects showing only bronchial reactions.

Conclusion: In conclusion, we demonstrated that early phase of systemic reaction after oral administration of aspirin is characterized by the release of IL-6 and the recruitment of effector memory T-cells. This points out to a novel mechanism that has been already discussed in allergy, asthma but not in AERD.

Citation Format: Pérez-Novo CA, Świerczyńska-Krępa M, Niżankowska-Mogilnicka E, Sanak M, De Ruyck N, Holtappels G, Szczeklik A, Bachert C (2014) EARLY PHASE OF SYSTEMIC REACTION TO ORAL ASPIRIN CHALLENGE IS RELATED TO AN INCREASE IN THE LEVELS OF IL-6 AND THE PERCENTAGE OF EFFECTORS MEMORY T-CELLS. [abstract]. In: Proceedings of the LATINFARMA 2013; 2013 October 21–25; La Habana, Cuba: SCF; J Pharm Pharmacogn Res 2(Suppl. 1): S43.

© 2014 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

CO 070: IMMUNOMODULATING AND ENHANCING ACTIVITIES OF SOLUBLE GLUCANS FROM Sacharomyces cerevisiae ASSESED BY MONOCYTE ACTIVATION TEST

J Pharm Pharmacogn Res 2(Suppl. 1): S43, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 070: IMMUNOMODULATING AND ENHANCING ACTIVITIES OF SOLUBLE GLUCANS FROM Sacharomyces cerevisiae ASSESED BY MONOCYTE ACTIVATION TEST

Pardo-Ruiz Z, Perdomo-Morales R, Pacios-Michelena A.

Centro de Investigación y Desarrollo de Medicamentos (CIDEM). Ave. 26 No. 1605. Plaza de la Revolución. La Habana, Cuba. E-mail: zenia.pardo@infomed.sld.cu
Abstract

Introduction: Beta-glucans are homopolymers of glucose that have shown immunomodulating effects depending on their structural characteristics and could be potential contaminants of some parenterals that are clarified through cellulose filters. The ability of glucans to develop an immune response or enhance the immune response induced by other pathogens is overlooked by the conventional pyrogen tests. Thus, the objective of this work was to determine the enhancing immune effect of glucans from the yeast Sacharomyses cerevisiae when they are incubated concomitantly with LPS and to confirm the significance of Monocyte Activation Test (MAT) for detecting such activities.

Material and methods: Soluble glucans were purified from S. cerevisiae and were partially characterized. The content of hexoses was determined by phenol-sulphuric colorimetric method and endotoxin contamination was evaluated by the Limulus test. Both immunostimulating and enhancing effects were determined by employing MAT. As monocyte source was used human whole blood that was incubated in polypropilene tubes with glucans, either alone or concomitantly with LPS. The releasing of Interleukin-6, -1β and Tumor Necrosis Factor-α was determined in the supernatant by ELISA.

Results: Glucans induced the releasing of all proinflammatory cytokines assayed, TNF-α was the most released cytokine, followed by IL-6 and IL-1β. In addition, the LPS induced-immunostimulating effect was powerfully enhanced by glucans.

Conclusions: Glucans showed enhancing and immunomodulating effects that became evident through MAT. It was shown that MAT could be an appropriate test to evaluate glucans activities.

CO 069: LEVELS OF SOLUBLE IL-15, IL-6, TNFΑ AND IL-15RΑ IN SYNOVIAL FLUID AND MEMBRANE BOUND IL-15 ON SYNOVIAL CELLS FROM PATIENTS WITH RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS

J Pharm Pharmacogn Res 2(Suppl. 1): S42, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 069: LEVELS OF SOLUBLE IL-15, IL-6, TNFΑ AND IL-15RΑ IN SYNOVIAL FLUID AND MEMBRANE BOUND IL-15 ON SYNOVIAL CELLS FROM PATIENTS WITH RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS

Machado-Díaz AC1, Arrieta-Aguero C1, Chico-Capote A2, RodriguezAlvarez Y1, García-del Barco Herrera D1, Raices-Cruz I1, Falcón-Cama V1, Chacón-Quintero Y1, Guillen-Nieto G1, Santos-Savio A1.

1Centro de Ingeniería Genética y Biotenología. La Habana, Cuba. E-mail: ana.machado@cigb.edu.cu
2Hospital Hermanos Amejeiras. La Habana, Cuba.
Abstract

Introduction: Cytokines are a family of proteins involved in regulation of the immune system and the intercellular communication. Many autoimmune diseases such as Rheumatoid arthritis (RA) are related to deregulation of cytokine expression. High levels of pro-inflammatory cytokines IL-15, IL-6 and TNF-α have been described in synovial fluid from RA patients.

Material and methods: In this study we quantified these cytokines levels in synovial fluid from 18 RA and 17 osteoarthritis (OA) patients, from Rheumatology service at B. Ameijeiras hospital, who presented inflammation and abundant synovial fluid in damaged joints. We also quantified levels of soluble IL-15Rα, a private receptor for IL-15, using an ELISA designed by our group. We also studied expression of membrane-bound IL-15 on synovial cells by FACS and EM.

Results: Current study confirmed presence of soluble IL-15Rα in synovial fluids from RA and OA patients. Although we detected expression of membrane IL-15 on cells from synovial fluids of RA patients. Also we determined that IL-15 is present as a membrane bound ligand. Acidic treatment produced a slight decrease in the amount of membrane-bound IL-15. Indicating that, in addition to transmembrane IL-15, a certain number of IL-15 molecules are bound to membrane IL-15Rα. Interestingly, we found a positive correlation between high levels of IL-6 and high levels of IL-15Rα in RA but not in OA.

Conclusions: The presence of soluble IL-15Rα and membrane IL-15 suggested that soluble IL-15Rα could induce IL-6 through a reverse signaling pathway and then contributing to a proinflammatory medium in RA.

CO 068: THE LONG PENTRAXIN PTX3: A NON-REDUNDANT COMPONENT OF THE HUMORAL INNATE IMMUNITY AND A PROMISING BIOMARKER IN INFLAMMATORY CONDITIONS

J Pharm Pharmacogn Res 2(Suppl. 1): S42, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 068: THE LONG PENTRAXIN PTX3: A NON-REDUNDANT COMPONENT OF THE HUMORAL INNATE IMMUNITY AND A PROMISING BIOMARKER IN INFLAMMATORY CONDITIONS

Garlanda C, Jaillon S, Barbati E, Bottazzi B, Mantovani A.

Humanitas Clinical and Research Center, via Manzoni 113, Rozzano, 20089, Italy. E-mail: cecilia.garlanda@humanitasresearch.it
Department of Translational Medicine, University of Milan, Milan, Italy.
Abstract

Pentraxins are a family of evolutionarily conserved multifunctional pattern-recognition proteins characterized by a cyclic multimeric structure. Based on the primary structure of the subunit, the pentraxins are divided into two groups: short pentraxins and long pentraxins. C-reactive protein (CRP) and serum amyloid P-component (SAP) are the two short pentraxins. The prototype protein of the long pentraxin group is pentraxin 3 (PTX3). CRP and SAP are produced primarily in the liver in response to IL-6, while PTX3 is produced by a variety of tissues and cells and in particular by innate immunity cells in response to pro-inflammatory signals and Toll-like receptor (TLR) engagement.

Through in vitro and in vivo studies performed with original tools generated by this group (recombinant human and mouse PTX3, its domains and mutated variants, original anti-PTX3 antibodies and PTX3 gene targeted mice), it has been shown that PTX3 interacts with several ligands, including growth factors, extracellular matrix components and selected pathogens, playing a role in complement activation and pathogen recognition by phagocytes, in tuning inflammation and in tissue remodelling. In addition, data obtained so far with ELISA assays on human plasma or serum suggest that PTX3 may represent a useful marker of different inflammatory conditions including cardiovascular pathology complementary to CRP: being directly produced by damaged tissues, its increase precedes CRP and rapidly reflects the vascular involvement by inflammatory process. The combination of PTX3 and classical biomarkers showed an incremental diagnostic and prognostic value in several conditions, including sepsis, acute coronary syndromes and chronic heart failure.

Thus, the prototypic long pentraxin PTX3 is a multifunctional soluble pattern recognition receptor acting as a non-redundant component of the humoral arm of innate immunity and a novel promising biomarker to provide useful prognostic information for clinical outcomes in inflammatory conditions.

CO 067: ENHANCEMENT OF THE INHIBITORY EFFECT OF AN IL-15 ANTAGONIST PEPTIDE BY ALANINE SCANNING

J Pharm Pharmacogn Res 2(Suppl. 1): S41, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 067: ENHANCEMENT OF THE INHIBITORY EFFECT OF AN IL-15 ANTAGONIST PEPTIDE BY ALANINE SCANNING

Rodríguez Y1, Reyes O1, Gerónimo H1, Chico A2, Garay H1, Ojeda M1, Arrieta C1, Estévez M2, Guillén G1 and Santos A1.

1Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana City 10600, Cuba. E-mail: yunier.rodriguez@cigb.edu.cu
2Hermanos Ameijeiras Hospital, San Lazaro 701, Havana 10300, Cuba.
Abstract

Introduction: IL-15 is a proinflammatory cytokine that acts early in the inflammatory response and has been associated with several autoimmune diseases including Rheumatoid Arthritis (RA), where it had been proposed as a therapeutic target. We recently reported an IL15 antagonist peptide corresponding to sequence 36–45 of IL-15 (KVTAMKCFLL) named P8, which specifically binds to IL-15Rα and inhibits IL-15 biological activity with a half maximal inhibitory concentration (IC50) of 130 μM in CTLL-2 proliferation assay.

Material and methods: In order to improve binding of peptide P8 to the receptor IL-15Rα, we used an Ala scan strategy to study contribution of each individual amino acid to the peptide’s antagonist effect. To evaluate the effects of the peptides, CTLL-2 cells were incubated with serial dilutions of peptides in presence of 300 pg of IL15 during 72 hours. Proliferation was measured by MTT mitochondrial staining. Cells from patients with RA were incubated with 50 µg/mL of peptide or 60 ng/mL of IL-15, or a combination of both. After 48 h incubation, supernatants were collected and TNF-α concentration was determined by ELISA.

Results: Here, we found that Phe and Cys are important for peptide binding to IL-15Rα. We also investigated other single site mutations and replaced the second Lys in the sequence by the polar non-charged amino acid threonine. The resulting peptide [K6T]P8 exhibited a higher activity than P8 with an IC50 of 24 μM. CTLL-2 proliferation assays showed that the dimeric form of [K6T] P8 had a higher inhibitory activity (IC50 8 μM) than the monomeric form. We also found that this peptide was more active than peptide P8 in the inhibition of TNFα secretion by synovial cells from RA patients.

Conclusion The peptide [K6T]P8 described in this work is a new type of IL-15 antagonist and constitutes a potential therapeutic agent for RA.

CO 066: CIGB-814, A PEPTIDE AS IMMUNOMODULATOR FOR THE TREATMENT OF RHEUMATOID ARTHRITIS

J Pharm Pharmacogn Res 2(Suppl. 1): S41, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 066: CIGB-814, A PEPTIDE AS IMMUNOMODULATOR FOR THE TREATMENT OF RHEUMATOID ARTHRITIS

Domínguez MC1, Lorenzo N1, Barberá A1, Ancizar JA1, Torres AM2, Hernandez MV2, Hernandez I2, Gil R2, Altruda F3, Silengo L3, Padrón GR1.

1Centro de Ingeniería Genética y Biotecnología, La Habana, Cuba. E-mail: mcarmen.dominguez@cigb.edu.cu
2Servicio Nacional de Reumatología, La Habana, Cuba.
3Universidad de Turín, Italia.
Abstract

Induction of immune tolerance as therapeutic approach for autoimmune diseases constitutes a current research focal point. In this sense, we aimed to evaluate an Altered Peptides Ligand (APL) for induction of peripheral tolerance in patients with Rheumatoid Arthritis (RA). A novel T cell epitope from human heat-shock protein 60 (Hsp60), an autoantigen involved in the pathogenesis of RA, was identified by bioinformatics tools and an APL was design from this epitope (CIGB-814). Firstly, we investigated the ability of this peptide for inducing regulatory T cells (Treg cells) in mice. CIGB-814 induced an increase of the proportions of Treg cells in the draining lymph nodes of the injected site in mice. On other hand, this peptide increases the proportions of the CD4+CD25highFoxP3+ Treg cells in ex vivo assays using PBMC isolated from RA patients. In addition, we evaluated the therapeutic effect of this APL in two animal models: adjuvant induced arthritis (AA) in Lewis rat and collagen induced arthritis (CIA) in DBA/1 mice. Our approach was compared to metotrexate (MTX), the gold standard treatment for RA, in CIA model. Clinical score, Treg, TNFα levels and histopathology were monitored. CIGB-814 efficiently inhibited the course of AA and CIA, with significant reduction of the clinical and histopathology score. The therapeutic effect induced by CIGB-814 is mediated by an increase of the proportions of Treg cells and a decrease of TNFα levels. These results indicate a therapeutic potentiality of this peptide and support further investigation of this candidate drugs for treatment of RA. Interference of the pathogenic T cell function in a specific manner using an APL derived from an autoantigen that can induce tolerance mediated by activation of Tregs as shows here, represents an attractive therapeutic approach for autoimmune diseases.

Citation Format: Domínguez MC, Lorenzo N, Barberá A, Ancizar JA, Torres AM, Hernandez MV, Hernandez I, Gil R, Altruda F, Silengo L, Padrón GR (2014) EARLY PHASE OF SYSTEMIC REACTION TO ORAL ASPIRIN CHALLENGE IS RELATED TO AN INCREASE IN THE LEVELS OF IL-6 AND THE PERCENTAGE OF EFFECTORS MEMORY T-CELLS. [abstract]. In: Proceedings of the LATINFARMA 2013; 2013 October 21–25; La Habana, Cuba: SCF; J Pharm Pharmacogn Res 2(Suppl. 1): S41.

© 2014 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

CO 054: AFFINITY MATURATION AND FINE FUNCTIONAL MAPPING OF AN ANTIBODY FRAGMENT AGAINST A NOVEL NEUTRALIZING EPITOPE ON HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR

J Pharm Pharmacogn Res 2(Suppl. 1): S33, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 054: AFFINITY MATURATION AND FINE FUNCTIONAL MAPPING OF AN ANTIBODY FRAGMENT AGAINST A NOVEL NEUTRALIZING EPITOPE ON HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR

Lamdan Ha, Gavilondo JVa, Muñoz Ya, Pupo Ab, Huerta Va, Musacchio Aa, Pérez La, Ayala Ma, Rojas Gb, Balint RFc, Larrick JWd.

aCenter for Genetic Engineering and Biotechnology, La Habana 10600, Cuba. Email: humberto.lamdan@cigb.edu.cu
bCenter of Molecular Immunology, La Habana 11600, Cuba.
cCytoDesign, Inc., Palo Alto, CA 94306, USA.
dPanorama Research Inc., Sunnyvale, CA 94089, USA.
Abstract

Vascular endothelial growth factor (VEGF) is a major mediator of pathological angiogenesis. VEGF antagonists represent attractive candidates as therapeutic agents in the treatment of tumors and other angiogenesis-dependent diseases. Despite the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between VEGF and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Soluble 2H1 inhibited VEGF biological activity but exhibited a moderate binding affinity. We performed the affinity maturation of 2H1 antibody fragment. Two phage-displayed libraries were constructed by diversification of the third complementarity-determining regions (CDRs) of the light (VL) and heavy (VH) chain variable domains of 2H1 using parsimonious mutagenesis. A competitive phage-selection strategy in the presence of 2H1 as a competitor was used to eliminate low affinity binders. High affinity variants were retrieved from both libraries. An optimized VL variant was constructed by combining recurrent replacements found among selected variants, resulting in an additional affinity increase. Further affinity improvements were achieved by combining this optimized VL with the best VH variants. The final variant, L3H6, showed an overall affinity improvement of 18-fold over the parental antibody and exhibited an enhanced potency to block the binding of VEGF to its receptor. Functional mapping studies of L3H6 using phage display and extensive mutagenesis of VEGF revealed a novel neutralizing epitope on human VEGF.

CO 038: FREQUENCY OF USE OF THE MONOCLONAL ANTIBODY RITUXIMAB ON THE NATIONAL CANCER INSTITUTE OF MEXICO: ONE YEAR EXPERIENCE

J Pharm Pharmacogn Res 2(Suppl. 1): S20, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 038: FREQUENCY OF USE OF THE MONOCLONAL ANTIBODY RITUXIMAB ON THE NATIONAL CANCER INSTITUTE OF MEXICO: ONE YEAR EXPERIENCE

López-Gamboa M1,2, Aguilar-Ponce JL2, Espinoza-Zamora JR2, DavalosFiesco M2, Mena-Rodríguez FJ2, Castañeda-Hernández G1.

1Departamento de Farmacología, Centro de Estudios Avanzados del Instituto Politécnico Nacional, Av. Instituto Politécnico Nacional 2508, San Pedro Zacatenco, C.P. 07360, México D.F.
2Instituto Nacional de Cancerología; Av. San Fernando No. 22, Sol. Sección XVI, C.P. 14080, México D.F. E-mail: dralopezg@gmail.com
Abstract

Introduction: For the health care professionals and for the maker decisions staff on the hospital is very important have a real panorama about the use of the drugs in order to improve the patient attention. In Mexico for the oncology therapeutic segment, Rituximab the chimeric, anti-CD20 monoclonal antibody is registered for: a) the treatment of Non-Hodgking´s Lymphoma (CD20+); b) also combined with chemotherapy for previously untreated patients with relapsed/refractory of chronic lymphocytic leukemia.

Methods: With the aim of determine the Rituximab frequency usage on the National Cancer Institute of Mexico we made a retrospective analysis of the rituximab dispensation data from march 2012 to march 2013. Our collected data were: the pharmaceutical presentation and vials quantity of Rituximab dispensed per patient; and from the clinical record the data recorded included: clinical indication (diagnostic) result of CD20 test, weigh, height, age, and Rituximab dosage prescribed.

Results: During the twelve-month period, a total of 2663 Rituximab vials where dispensed from the Institute pharmacy, 1353 were 500 mg vials, and 1308 were 100 mg vials, distributed on 304 patients. The list of primary diagnostics included 75 different pathologies. All patients had immunohystochemical test, the 95% of the cases were positive to the CD 20 test, 4.7% of the patients had not report and 0.3% were negative to the CD20 test.

Conclusion: Even when Rituximab was prescribed for a diverse range of clinical conditions, the use of CD20 test was a standard followed on the patients, this practice showed the specific use of Rituximab.