Bioassays for the Evaluation of Target Neutralization and Complement-Dependent Cytotoxicity (CDC) of Therapeutic Antibodies.

Therapeutic monoclonal antibodies (mAbs) are complex bioengineered proteins that require to be routinely characterized with robust and reliable bioassays. Infliximab was the first anti-TNFα mAb approved for use in humans and its use has revolutionized the treatment TNF-mediated inflammatory disorders. The mechanism of action (MOA) of infliximab involves its binding to soluble (s) and membrane (m) TNFα. Here, we describe two simple in vitro bioassays for the assessment of key activities of infliximab. First, a bioassay for TNFα neutralization, which evaluates the Fab binding to sTNFα and the consequent reduction in the activation of TNFα receptors and TNFα-induced expression of adhesion molecules on endothelial cells. A second bioassay evaluates the triggering of Complement-Dependent Cytotoxicity (CDC) in cells expressing mTNFα, which requires the interaction of infliximab-Fc with proteins of the complement system. In both cases, the biological responses are measured by flow cytometry, which is accessible for most laboratories. The methods reported here can be easily adapted to other therapeutic mAbs with similar MOA.

Reverse Docking for the Identification of Molecular Targets of Anticancer Compounds.

Molecular docking is a useful and powerful computational method for the identification of potential interactions between small molecules and pharmacological targets. In reverse docking, the ability of one or a few compounds to bind a large dataset of proteins is evaluated in silico. This strategy is useful for identifying molecular targets of orphan bioactive compounds, proposing new molecular mechanisms, finding alternative indications of drugs, or predicting drug toxicity. Herein, we describe a detailed reverse docking protocol for the identification of potential targets for 4-hydroxycoumarin (4-HC). Our results showed that RAC1 is a target of 4-HC, which partially explains the biological activities of 4-HC on cancer cells. The strategy reported here can be easily applied to other compounds and protein datasets.

Early Differentiation of Human CD11c+NK Cells with γδ T Cell Activation Properties Is Promoted by Dialyzable Leukocyte Extracts.

Reconstitution of the hematopoietic system during immune responses and immunological and neoplastic diseases or upon transplantation depends on the emergent differentiation of hematopoietic stem/progenitor cells within the bone marrow. Although in the last decade the use of dialyzable leukocyte extracts (DLE) as supportive therapy in both infectious and malignant settings has increased, its activity on the earliest stages of human hematopoietic development remains poorly understood. Here, we have examined the ability of DLE to promote replenishment of functional lymphoid lineages from CD34+ cells. Our findings suggest that DLE increases their differentiation toward a conspicuous CD56+CD16+CD11c+ NK-like cell population endowed with properties such as IFNy production, tumor cell cytotoxicity, and the capability of inducing γδ T lymphocyte proliferation. Of note, long-term coculture controlled systems showed the bystander effect of DLE-stromal cells by providing NK progenitors with signals to overproduce this cell subset. Thus, by direct effect on progenitor cells and through activation and remodeling of the supporting hematopoietic microenvironment, DLE may contribute a robust innate immune response by promoting the emerging lymphopoiesis of functional CD11c+ NK cells in a partially TLR-related manner. Unraveling the identity and mechanisms of the involved DLE components may be fundamental to advance the NK cell-based therapy field.

Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions.

BACKGROUND: In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs) from bone marrow and other "classic" sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs) on effector T lymphocytes through the purinergic pathway. METHODS: We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs). We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+) lymphocyte function through the generation of adenosine (Ado). RESULTS: We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. CONCLUSIONS: This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.

IL-8 and IL-6 primarily mediate the inflammatory response in fibromyalgia patients.

Fibromyalgia (FM) is a chronic disease that has been linked to inflammatory reactions and changes in the systemic levels of proinflammatory cytokines that modulate responses in the sympathetic nervous system and hypothalamic-pituitary-adrenal axis. We found that concentrations of IL-6 and IL-8 were elevated in FM patients. Both cytokines correlated with clinical scores, suggesting that IL-6 and IL-8 have additive or synergistic effects in perpetuating the chronic pain in FM patients. These findings indicate that IL-6 and IL-8 are two of the most constant inflammatory mediators in FM and that their levels correlate significantly with the severity of symptoms.

Herpes murine model as a biological assay to test dialyzable leukocyte extracts activity.

Human dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that are released on disruption of peripheral blood leukocytes from healthy donors. DLEs improve clinical responses in infections, allergies, cancer, and immunodeficiencies. Transferon is a human DLE that has been registered as a hemoderivate by Mexican health authorities and commercialized nationally. To develop an animal model that could be used routinely as a quality control assay for Transferon, we standardized and validated a murine model of cutaneous HSV-1 infection. Using this model, we evaluated the activity of 27 Transferon batches. All batches improved the survival of HSV-1-infected mice, wherein average survival rose from 20.9% in control mice to 59.6% in Transferon-treated mice. The activity of Transferon correlated with increased serum levels of IFN-γ and reduced IL-6 and TNF-α concentrations. Our results demonstrate that (i) this mouse model of cutaneous herpes can be used to examine the activity of DLEs, such as Transferon; (ii) the assay can be used as a routine test for batch release; (iii) Transferon is produced with high homogeneity between batches; (iv) Transferon does not have direct virucidal, cytoprotective, or antireplicative effects; and (v) the protective effect of Transferon in vivo correlates with changes in serum cytokines.

Immunomodulatory effects mediated by serotonin.

Serotonin (5-HT) induces concentration-dependent metabolic effects in diverse cell types, including neurons, entherochromaffin cells, adipocytes, pancreatic beta-cells, fibroblasts, smooth muscle cells, epithelial cells, and leukocytes. Three classes of genes regulating 5-HT function are constitutively expressed or induced in these cells: (a) membrane proteins that regulate the response to 5-HT, such as SERT, 5HTR-GPCR, and the 5HT3-ion channels; (b) downstream signaling transduction proteins; and (c) enzymes controlling 5-HT metabolism, such as IDO and MAO, which can generate biologically active catabolites, including melatonin, kynurenines, and kynurenamines. This review covers the clinical and experimental mechanisms involved in 5-HT-induced immunomodulation. These mechanisms are cell-specific and depend on the expression of serotonergic components in immune cells. Consequently, 5-HT can modulate several immunological events, such as chemotaxis, leukocyte activation, proliferation, cytokine secretion, anergy, and apoptosis. The effects of 5-HT on immune cells may be relevant in the clinical outcome of pathologies with an inflammatory component. Major depression, fibromyalgia, Alzheimer disease, psoriasis, arthritis, allergies, and asthma are all associated with changes in the serotonergic system associated with leukocytes. Thus, pharmacological regulation of the serotonergic system may modulate immune function and provide therapeutic alternatives for these diseases.

Chronic deep brain stimulation of the hypothalamic nucleus in wistar rats alters circulatory levels of corticosterone and proinflammatory cytokines.

Deep brain stimulation (DBS) is a therapeutic option for several diseases, but its effects on HPA axis activity and systemic inflammation are unknown. This study aimed to detect circulatory variations of corticosterone and cytokines levels in Wistar rats, after 21 days of DBS-at the ventrolateral part of the ventromedial hypothalamic nucleus (VMHvl), unilateral cervical vagotomy (UCVgX), or UCVgX plus DBS. We included the respective control (C) and sham (S) groups (n = 6 rats per group). DBS treated rats had higher levels of TNF-α (120%; P < 0.01) and IFN-γ (305%; P < 0.001) but lower corticosterone concentration (48%; P < 0.001) than C and S. UCVgX animals showed increased corticosterone levels (154%; P < 0.001) versus C and S. UCVgX plus DBS increased IL-1ß (402%; P < 0.001), IL-6 (160%; P < 0.001), and corsticosterone (178%; P < 0.001 versus 48%; P < 0.001) compared with the C and S groups. Chronic DBS at VMHvl induced a systemic inflammatory response accompanied by a decrease of HPA axis function. UCVgX rats experienced HPA axis hyperactivity as result of vagus nerve injury; however, DBS was unable to block the HPA axis hyperactivity induced by unilateral cervical vagotomy. Further studies are necessary to explore these findings and their clinical implication.

Hormone multifunctionalities: a theory of endocrine signaling, command and control.

A theory is presented outlining how organisms can function and benefit from multifunctionality of hormones in order to enhance greatly the information-carrying potential of endocrine signaling. Hormones are produced continuously as micropulses, and intermittently as larger pulses. It is generally believed that micropulses generate fluctuating basal hormone concentrations, which may consistently elicit particular responses among diverse variables. Evidence is discussed suggesting that in contrast to the hormone micropulses, the larger endogenous hormone pulses may elicit responses which may differ from one pulse to another and may therefore serve different physiological functions. In this paper we postulate that an endogenous hormone pulse is a specific form of a multisignal message that serves a certain physiological function. Different pulses of a hormone may be signals of diverse multisignal messages that serve different functions. A multisignal message may elicit congruous responses by selectively enhancing some actions and suppressing other actions of the component signals. Various roles of signals of multisignal messages are discussed, as well as processes that may be involved in the diversity and selectivity of actions of different pulses of a hormone. Hormones also are converted into other hormones; we analyze how precursor and derived hormones may function independently of each other, and how precursor hormones may give rise to permissive effects. Mechanisms involved in therapeutic and adverse effects of hormone administrations are analyzed, and a strategy is suggested for developing more selective hormonal therapies.

4-Hydroxycoumarin disorganizes the actin cytoskeleton in B16-F10 melanoma cells but not in B82 fibroblasts, decreasing their adhesion to extracellular matrix proteins and motility.

This study determined the in vitro effects of 4-hydroxycoumarin (4-HC) employing the melanoma cell line B16-F10 and the non-malignant fibroblastic cell line B82. 4-HC disorganized the actin cytoskeleton in B16-F10 cells, but not in B82 fibroblasts. Cytoskeletal disorganization correlated with reductions in cell adhesion to four extracellular matrix proteins and inhibition of random motility. 4-HC did not modify cell viability or actin expression, but decreased tyrosine phosphorylation of several proteins in melanoma cells. Because adhesion of tumor cells to extracellular matrix is required during the metastatic process, 4-HC might be useful as an adjuvant therapy for melanoma.

Macrophage--Mycobacterium tuberculosis interactions: role of complement receptor 3.

Tuberculosis is the leading infectious disease in the world. Mycobacterium tuberculosis, the causal agent of this disease, invades macrophages and can replicate inside them. Because invasion of macrophages is a critical step for establishing a mycobacterial infection, there is much interest in understanding the mechanisms for M. tuberculosis entry into macrophages. Complement receptor 3 (CR3) is a heterodimeric surface receptor with multiple binding sites, which can mediate complement-opsonized as well as nonopsonic entrance of M. tuberculosis into macrophages. Here, we describe and discuss the role of CR3 in macrophage[bond]M. tuberculosis interactions. The actual information suggests that CR3 mediates a substantial amount of M. tuberculosis binding to macrophages, but CR3 is not related to the mechanisms that allow mycobacteria to survive and replicate intracellularly. Understanding the mechanisms of macrophage[bond]M. tuberculosis interaction will help developing more effective methods to prevent and treat tuberculosis in the future.

Anormalidades en el sistema adhesivo de neoplasias pulmonares / Abnormalities in adhesión system of pulmonary neoplasms

La progresión de una neoplasia pulmonar requiere alteraciones en la expresión de moléculas de adhesión en la célula tumoral. Un decremento en la expresión de E-cadherina y de las cateninas a y b disminuye la adhesión homotípica e incrementa el número de células neoplásicas liberadas del tumor primario. Las integrinas presentan cambios complejos en su expresión; la capacidad invasiva se ve favorecida por el aumento en la expresión de unas integrinas, como la a 2b 1 y por la disminución en la expresión de otras, como la a 3b 1. Los cambios en la expresión de ICAM-1 favorecen la evasión de la respuesta inmune. La disminución en la densidad de ICAM-1 en la superficie de células tumorales disminuye la posibilidad de contacto célula-célula. El aumento en la concentración de la isoforma soluble de ICAM-1 bloquea los contrarreceptores presentes en las células inmunológicas. También se han identificado alteraciones en moléculas que modulan la adhesión, como FAK y paxilina. Las moléculas de adhesión y los componentes regulatorios de la adhesión, pueden ser blancos farmacológicos para el desarrollo de nuevas terapias adyuvantes para el tratamiento del cáncer.

Ciclo celular y sus anormalidades en el cáncer de pulmón / Cell cycle and its abnormalities in lung cancer

En esta revisión se describen los eventos celulares que ocurren normalmente en cada fase del ciclo celular. Se explica el papel de las proteínas que promueven la progresión del ciclo celular y las que lo detienen. En el cáncer de pulmón, los tipos de genes que presentan mutaciones con mayor frecuencia son: 1) los oncogenes c-ras y c-myc, 2) los genes supresores de tumores, RB y p53, y 3) el gen de la proteína inhibidora de Cdk p16. Las proteínas codificadas por estos genes mutados sufren cambios funcionales, lo que origina la pérdida del control del ciclo celular. La acumulación gradual de errores en genes conducen a tumores malignos

Integrinas y moléculas asociadas a integrinas: blancos para el desarrollo de terapias antimetastásicas / Integrins and integrin-associated molecules: targets for the development of antimetastasic therapies

Las integrinas son receptores que median la adhesión celular y regulan la formación de complejos de señalización. Se requieren modificaciones en la expresión de integrinas durante las siguientes etapas de la generación de metástasis: a) angiogénesis intratumoral; b) desprendimiento del tumor primario; c) interacción de células tumorales con plaquetas; d) adhesión al endotelio vascular y e) proliferación. Existe, también, correlación entre la capacidad invasiva y la expresión alterada de algunas proteínas que se agregan en los sitios de adhesión focal, como la cinasa de adhesión focal (FAK), CD82, CD9 o CD63. Tanto el bloqueo de integrinas (utilizando anticuerpos o péptidos que contienen la secuencia RGD) como modificaciones inducidas en la expresión de moléculas asociadas a integrinas pueden inhibir la formación de metástasis. El descubrimiento y caracterización de moléculas que regulen la capacidad adherente de la células tumorales conducirá al desarrollo de tarapias antimetastásicas. En la búsqueda de inhibidores de la diseminación tumoral, las integrinas y algunas moléculas asociadas a integrinas son importantes blancos farmacológicos

Expresión de moléculas de adhesión en el melanoma metastásico murino B16-F10 y su modificación farmacológica in vitro con el empleo de cumarina / Adhesion molecules expression in murine B16-F10 metastatic melanoma and its pharmacological modification in vitro by the use of coumarin

Recientemente se han demostrado correlaciones significativas entre la expresión de algunas moléculas de adhesión y la capacidad de células de producir metástasis. Por ejemplo, se ha observado una correlación entre la expresión de la integrina a6/ß1 en células cancerosas pulmonares y la producción de metástasis. También se ha observado correlación entre la expresión de algunas moléculas de adhesión en células de melanoma maligno y su capacidad de producir metástasis pulmonares. En este trabajo estudiamos la acción in vitro de la cumarina en el melanoma murino B16-F10, productor de metástasis pulmonares, sobre la expresión de dos moléculas de adhesión, ICAM-1 y LFA-1. No se observó disminución en la expresión de la molécula de adhesión LFA-1, y la expresión de ICAM-1 disminuyó de manera uniforme con todas las concentraciones de cumarina estudiadas. Estos resultados no explican los efectos antimetastásicos producidos por la cumarina en modelos animales de metástasis pulmonares experimentales y espontáneas, ni los efectos antimetastásicos en humanos. Es necesario, por tanto, estudiar el efecto de la cumarina en la expresión de otras integrinas. Este tipo de estudios permite el desarrollo de nuevas estrategias en la búsqueda de mejores agentes antineoplásicos que disminuyan en mayor grado el número y tamaño de metástasis, y retarden importantemente su producción; contribuye, adenomás, al conocimiento de la fisiopatogenia de estos tumores malignos