ABSTRACT INTRODUCTION Diabetic foot ulcers are a chronic complication in patients with diabetes mellitus. They appear as a result of the combination of diabetic polyneuropathy and angiopathy, and in many cases require amputation of the affected extremity. Clinical trials have demonstrated that repeated local infiltration with Heberprot-P can improve healing of chronic diabetic foot ulcers. Although there is evidence of its effects as a granulation stimulator and on cell migration and proliferation, genetic control mechanisms explaining its anti-inflammatory and oxidative stress reduction properties are not yet thoroughly understood. OBJECTIVE Analyze changes in expression of genes involved in healing after treatment of diabetic foot ulcers with Heberprot-P. METHODS Biopsies were collected from diabetic foot ulcers of 10 responding patients before and after 2 weeks’ treatment with Heberprot-P (75-μg applied intralesionally 3 times per week). Total RNA was obtained and quantitative PCR used to determine expression of 26 genes related to inflammation, oxidative stress, cell proliferation, angiogenesis and extracellular matrix formation. Genetic expression was quantified before and after treatment using REST 2009 v2.0.13. RESULTS After treatment, there was a statistically significant increase in expression of genes related to cell proliferation, angiogenesis and formation of extracellular matrix (PDGFB, CDK4, P21, TP53, ANGPT1, COL1A1, MMP2 and TIMP2). A significant decrease was observed in gene expression related to inflammatory processes and oxidative stress (NFKB1, TNFA and IL-1A). CONCLUSIONS Our findings suggest that Heberprot-P’s healing action on diabetic foot ulcers is mediated through changes in genetic expression that reduce hypoxia, inflammation and oxidative stress, and at the same time increase cell proliferation, collagen synthesis and extracellular matrix remodeling. The kinetics of expression of two genes related to extracellular matrix formation needs further exploration.

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.

Resumen. La Troponina I cardiaca es considerada el marcador bioquímico de elección para el infarto agudo del miocardio; que es una urgencia médica y es necesario su diagnóstico rápido. En este artículo se estudió la posibilidad de diagnosticar esta entidad nosológica de una manera cualitativa a través de un ensayo inmunocromatográfico de un solo paso de detección de Troponina I cardiaca elaborado en el laboratorio y otro ensayo inmunocromatográfico cualitativo de detección de Troponina I cardiaca Cardiac STATus™. Se evaluaron retrospectivamente 76 plasmas de pacientes con infarto agudo del miocardio y 50 plasmas de donantes sanos. El inmunoensayo del laboratorio no presentó reactividad cruzada con la isoforma esquelética de la Troponina I. Esta prueba detectó 1 ng/mL o mayor de Troponina I cardiaca en forma de complejo terciario en plasma y también reconoció la molécula libre. La sensibilidad clínica del inmunoensayo del laboratorio en pacientes con infarto agudo del miocardio de tipo Q fue 100% y para el inmunoensayo comercial, 85,7% en el período de 6 h a 24 h de inicio del dolor en el pecho. Para ese tipo de infarto se detectó señal hasta las 148 h y la sensibilidad clínica osciló entre 84,2% y 90,9% para ambos sistemas. En el caso de pacientes con infarto agudo del miocardio de tipo no-Q la sensibilidad clínica fue 70% en los dos inmunoensayos. La especificidad clínica del inmunocromatográfico de Troponina I cardiaca preparado en el laboratorio con muestras de donantes sanos fue 90.4% y para el inmunocromatográfico comercial, 100%. Los dos sistemas inmunocromatográficos de un solo paso para la detección de Troponina I cardiaca evaluados en este trabajo diagnostican de una manera rápida y fácil la muerte celular miocárdica importante y en menor grado, aquella necrosis más pequeña, sin signos electrocardiográficos concluyentes y con posibilidades de la ocurrencia de complicaciones a corto y mediano plazo en el paciente con síndrome coronario agudo.

Abstract. The cardiac Troponin I is considered the biochemical marker of election to detect acute myocardial infarction, a medical urgency that requires a rapid diagnosis. In this article, the diagnosis of this condition was studied qualitatively through an immunochromatographic assay of a single step detection of cardiac Troponin I elaborated in the laboratory comparing it with another, commercially available, qualitative immunochromatographic assay of detection of cardiac Troponin I, Cardiac STATUS TM. The plasmas of 76 patients with acute myocardial infarction and 50 plasmas obtained from healthy donors were evaluated retrospectively. The laboratory’s immunoassay did not present cross reactivity with the skeletal isoform of Troponin I. This test detected 1 ng/mL or more of cardiac Troponin I in the form of a tertiary complex in plasma and it also recognized the free molecule. The clinical sensitivity of the immunoassay of the laboratory in patients with Q wave type acute myocardial infarction was 100% and for the commercial immunoassay was 85.7% in the period of 6 h to 24 h following the onset of chest pain. For this type of infarction, the signal was detected up to 148 h after the onset of symptoms and the clinical sensitivity oscillated between 84.2% and 90.9% for both assays. The clinical sensitivities of the two immunoassays were 70% in the case of patients with non-Q wave acute myocardial infarction. With healthy donor’s samples, the clinical specificity of the immunochromatographic assay prepared in the laboratory was of 90.4% and for commercial immunoassay was 100%. The immunochromatographic immunoassays of a single step for the detection of cardiac Troponin I evaluated in this work, diagnosed in a quick and easy way, important myocardial cell death and to lesser extent smaller necrosis, in patients without concluding electrocardioghraphic signs and with the possibility of the occurrence of complications.