Abstract Lower-extremity diabetic ulcers are responsible for 80% of annual worldwide nontraumatic amputations. Epidermal growth factor (EGF) reduction is one of the molecular pillars of diabetic ulcer chronicity, thus EGF administration may be considered a type of replacement therapy. Topical EGF administration to improve and speed wound healing began in 1989 on burn patients as part of an acute-healing therapy. Further clinical studies based on topically administering EGF to different chronic wounds resulted in disappointing outcomes. An analysis of the literature on unsuccessful clinical trials identified a lack of knowledge concerning: (I) molecular and cellular foundations of wound chronicity and (II) the pharmacodynamic requisites governing EGF interaction with its receptor to promote cell response. Yet, EGF intra- and perilesional infiltration were shown to circumvent the pharmacodynamic limitations of topical application. Since the first studies, the following decades of basic and clinical research on EGF therapy for problem wounds have shed light on potential uses of growth factors in regenerative medicine. EGF’s molecular and biochemical effects at both local and systemic levels are diverse: (1) downregulation of genes encoding inflammation mediators and increased expression of genes involved in cell proliferation, angiogenesis and matrix secretion; (2) EGF intervention positively impacts both mesenchymal and epithelial cells, reducing inflammation and stimulating the recruitment of precursor circulating cells that promote the formation of new blood vessels; (3) at the subcellular level, upregulation of the EGF receptor with subsequent intracellular trafficking, including mitochondrial allocation along with restored morphology of multiple organelles; and (4) local EGF infiltration resulting in a systemic, organismal repercussion, thus contributing to attenuation of circulating inflammatory and catabolic reactants, restored reduction-oxidation balance, and decreased toxic glycation products and soluble apoptogenic effectors. It is likely that EGF treatment may rearrange critical epigenetic drivers of diabetic metabolic memory.

In previous work, we identified the peptide comprising amino acids 32-51 on the antimicrobial protein limulus anti-LPS factor (LALF) from Limulus poliphemus, based on its capacity to bind to the bacterial lipopolysaccharide (LPS). In this study, we designed a chemical peptide library by alanine scanning from the sequence of the LALF32-51 peptide. Four new peptides, named L-2, L-8, L-12 and L-20 by the Ala residue substituted in the given position of the LALF32-51 peptide, were identified as retaining their penetrating activity but also showing antitumor effects when subcutaneously administered in female C57Bl/6 mice after the implantation of malignant TC-1 lung epithelial cells. They significantly increased animal survival (p < 0.05) as compared to LALF32-51. The substitution of Tyr residue to Ala at position 2 in the peptide sequence enhanced the cytotoxic effect, while residues Phe8, Lys12 and Trp20 were essential for the antitumor activity. Moreover, the administration of the L-2 peptide significantly reduced tumor growth in comparison to PBS- or L-20 peptide-treated lung TC-1 cells in C57Bl/6 mice. L-2 was found to deregulate the tumor cell cycle and induces apoptosis, through mechanisms affecting glycolytic and protein biosynthesis pathways. It also significantly increased survival in human colon cancer LS-174T xenotransplanted nude mice for up to 32 days, two of the animals being tumor free. The L-2 peptide could serve as peptide-based prototype drug with potential to reduce tumor load or could be coadministered with conventional chemotherapy. This research granted the 2013 Award of the Cuban National Academy of Sciences.

En este estudio se diseñó una biblioteca química de péptidos mediante el barrido de alanina del péptido LALF32-51, derivado de la proteína antimicrobiana factor anti-LPS de Limulus poliphemus (LALF) con capacidad de unión al lipopolisacárido bacteriano (LPS). Se identificaron cuatro nuevos péptidos, L-2, L-8, L-12 y L-20 con el residuo Ala en la posición indicada en cada caso, que retuvieron la penetrabilidad celular original y mostraron actividad antitumoral al ser administrados en ratones C57Bl/6 a los que previamente se implantó tumores malignos TC-1 de células epiteliales de pulmón. Los péptidos incrementaron significativamente la supervivencia de los animales en comparación con el LALF32-51 (p < 0.05). La sustitución por Ala en la posición 2 incrementó el efecto citotóxico, y en sustitución de los residuos Phe8, Lys12 y Trp20, evidenció la importancia de estos para la actividad antitumoral del péptido. La administración del L-2 redujo significativamente el crecimiento tumoral respecto al tratamiento con PBS o con L-20 en células TC-1 de ratones C57Bl/6. Este péptido desreguló el ciclo celular e indujo apoptosis en las células tumorales, mediante mecanismos que afectaron a las rutas de la glicólisis y la biosíntesis de proteínas, e incrementó significativamente a 32 días la supervivencia de ratones desnudos xenotrasplantados con células LS-174T de cáncer de colon humano. El péptido L-2 puede ser un prototipo de fármaco peptídico con potencial para reducir la carga tumoral o que se pueda coadministrar con la quimioterapia convencional. Este trabajo mereció el Premio Anual de la Academia de Ciencias de Cuba para el año 2013.

Experimental autoimmune encephalomyelitis (EAE), when induced in syngenic C57BL/6 mice by using myelin oligodendrocyte glycoprotein (MOG), usually exhibits a chronic progressive pattern. This model mimics many of the symptoms and clinical signs typical of multiple sclerosis (MS) in humans. The present work describes specific adjustments to the experimental parameters described in the literature that were necessary when implementing this model under our conditions, demonstrating the presence of EAE in experimental animals by means of clinical evaluations, molecular assays and ultrastructural studies. The disorder was induced by active immunization with peptide MOG35-55 emulsified in Freund's Incomplete Adjuvant supplemented with Mycobacterium tuberculosis, together with two additional administrations of Pertussis toxin. All immunized animals exhibited the typical clinical signs of EAE, with a severity that increased progressively from day 8 post-induction to the end of the evaluation, at day 28 post-induction. The maximum clinical score was 3.5, and the disorder was not reversible. Body weight loss was associated with clinical deterioration at the initial stages of the experiment. From a molecular perspective, it was shown that effector cytokines (mainly IL-17) were positively regulated in the brain of diseased animals. The characteristic ultrastructural changes of MS were also detected; namely, demyelination and axonal damage. The methodology described here enabled the implementation of an animal model that reproduces fundamental aspects of the pathogenesis of MS and is, therefore, highly useful for the study of physiopathological mechanisms, the identification of new pharmacological targets and the evaluation of specific biomolecules with therapeutic purposes.

La inducción de la encefalomielitis autoinmune experimental (EAE) mediante la glicoproteína de la mielina del oligodendrocito (MOG) en ratones C57BL/6 isogénicos y homocigóticos, se caracteriza por un patrón crónico progresivo. Este modelo remeda un amplio espectro de síntomas y signos clínicos típicos de la esclerosis múltiple (EM) en humanos. Se describe el ajuste de las condiciones experimentales con respecto a las referidas en la literatura, y se demuestra la generación del modelo mediante evaluaciones clínicas, moleculares y ultraestructurales. La enfermedad se indujo por inmunización activa con el encefalitógeno constituido por el péptido MOG35-55 emulsificado en adyuvante incompleto de Freund, suplementado con Mycobacterium tuberculosis y dos administraciones adicionales de toxina pertussis. Los animales inmunizados mostraron signos clínicos de EAE, cuya severidad aumentó en forma progresiva y ascendente a partir del día 8 después de la inducción, con un puntaje clínico máximo de 3.5, sin reversión hasta el día 28, en que finalizó el periodo de evaluación. La disminución del peso corporal estuvo asociada con el inicio del deterioro clínico. Desde el punto de vista molecular, se identificó una regulación positiva de citocinas efectoras en el cerebro de los animales enfermos, principalmente de la IL-17. Se observaron daños ultraestructurales característicos de la EM en la mielina y el axón. La metodología permitió la generación de un modelo animal con elementos esenciales de la patogénesis de la EM y, por tanto, útil para el estudio de mecanismos fisiopatológicos, la identificación de nuevos blancos farmacológicos y la evaluación de biomoléculas con fines terapéuticos.