Cell therapy for the treatment of lower limb lymphedema. Case report Terapia celular en el tratamiento de linfedema de miembros inferiores. Presentación de un caso

Although lymphedema is a common disabling disease causing significant morbidity for affected patients, treatment for this condition remains limited and largely ineffective. Some reported data suggest that some bone-marrow derived cells may play a role in lymphangiogenesis. It appears that blood vessels and lymphatic vessels might use the same population of cells for vasculogenesis and lymphangiogenesis. Therefore, adult stem cell therapy could be a new useful strategy for the treatment of lymphedema. We report a resolution of a severe lower limb bilateral lymphedema after implantation of autologous adult stem cells derived from bone marrow. As far as we know, this is the first reported case with chronic lower limb lymphedema treated successfully with autologous cell therapy. This procedure is a low-cost, relatively simple and easy to perform option that opens new ways for the treatment of lymphedema.<br>Aunque el linfedema es una enfermedad crónica inhabilitante común que causa morbilidad significativa en los pacientes afectados, el tratamiento para esta enfermedad se mantiene muy limitada y en la mayor parte de los casos resulta ineficaz. Algunos datos reportados sugieren que algunas de las células madre derivadas de la medula ósea pueden intervenir en la linfangiogénesis. Al parecer, los vasos sanguíneos y los vasos linfáticos podrían usar la misma población celular para la vasculogénesis y la linfangiogénesis. Por consiguiente, la terapia con células madre adultas podría ser una nueva estrategia útil para el tratamiento de linfedema. En el presente trabajo se informa la resolución de un linfedema bilateral severo de miembros inferiores después de la implantación de células madre autólogas derivadas de la médula ósea. Hasta donde sabemos, este es el primer caso de linfedema crónico de los miembros inferiores tratado exitosamente con células madre autólogas. Este método de tratamiento es económico, relativamente simple, fácil de realizar y una opción que abre nuevas vías para el tratamiento del linfedema

Drug Development in Conformational Diseases: A Novel Family of Chemical Chaperones that Bind and Stabilise Several Polymorphic Amyloid Structures

<div><p>The increasing prevalence of conformational diseases, including Alzheimer's disease, type 2 Diabetes Mellitus and Cancer, poses a global challenge at many different levels. It has devastating effects on the sufferers as well as a tremendous economic impact on families and the health system. In this work, we apply a cross-functional approach that combines ideas, concepts and technologies from several disciplines in order to study, <i>in silico</i> and <i>in vitro</i>, the role of a novel chemical chaperones family (NCHCHF) in processes of protein aggregation in conformational diseases. Given that Serum Albumin (SA) is the most abundant protein in the blood of mammals, and Bovine Serum Albumin (BSA) is an off-the-shelf protein available in most labs around the world, we compared the ligandability of BSA:NCHCHF with the interaction sites in the Human Islet Amyloid Polypeptide (hIAPP):NCHCHF, and in the amyloid pharmacophore fragments (Aβ17–42 and Aβ16–21):NCHCHF. We posit that the merging of this interaction sites is a meta-structure of pharmacophore which allows the development of chaperones that can prevent protein aggregation at various states from: stabilizing the native state to destabilizing oligomeric state and protofilament. Furthermore to stabilize fibrillar structures, thus decreasing the amount of toxic oligomers in solution, as is the case with the NCHCHF. The paper demonstrates how a set of NCHCHF can be used for studying and potentially treating the various physiopathological stages of a conformational disease. For instance, when dealing with an acute phase of cytotoxicity, what is needed is the recruitment of cytotoxic oligomers, thus chaperone F, which accelerates fiber formation, would be very useful; whereas in a chronic stage it is better to have chaperones <b>A</b>, <b>B</b>, <b>C</b>, and <b>D</b>, which stabilize the native and fibril structures halting self-catalysis and the creation of cytotoxic oligomers as a consequence of fiber formation. Furthermore, all the chaperones are able to protect and recondition the cerebellar granule cells (CGC) from the cytotoxicity produced by the hIAPP_20–29 fragment or by a low potassium medium, regardless of their capacity for accelerating or inhibiting <i>in vitro</i> formation of fibers. <i>In vivo</i> animal experiments are required to study the impact of chemical chaperones in cognitive and metabolic syndromes.</p></div

Meta-structure of pharmacophore.

<p>A structural alignment using Pymol, whereby the interaction zones between β-amiloyd_17–42 (Aβ_17–42), IAPP and Eisenberg’s pharmacophore molecules and the chaperons were set in position with the interaction zone between BSA and the chaperons. BSA is shown in white at 70% transparency, the interaction zone between the Aβ_17–42 and the chaperons is shown in red, the one corresponding to the Eisenberg pharmacophore in blue, with IAPP in yellow and with BSA in green.</p

Values of molar ellipticity differences of the BSA fibrilogenesis assays at 70°C, in absence and presence of chaperones A, D, E, B, F and C, calculated from the CD spectra.

<p>*Differences of molar ellipticity at 240 and zero min. of incubation time.</p><p>Values of molar ellipticity differences of the BSA fibrilogenesis assays at 70°C, in absence and presence of chaperones A, D, E, B, F and C, calculated from the CD spectra.</p

Cell Apoptosis of CGC exposed to hIAPP_20–29, in monomeric and aggregated form, with and without N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine D; methyl (2-{[4-(1-naphthylamino)-4-oxobutanoyl]amino}ethyl) dithiocarbamate B and H used as reference; at different molar ratio hIAPP_20–29:chaperones.

<p>In all assays caspase-3 levels were measured by immunofluorescence.</p

Cell Apoptosis of CGC exposed to pro-apoptotic stimulus as low potassium concentration (K5) during 4 h and later, the chaperones N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine D; methyl (2-{[4-(1-naphthylamino)-4-oxobutanoyl]amino}ethyl) dithiocarbamate B and C (reference), were inoculated.

<p>In all assays caspase-3 levels were measured by inmunofluorescence.</p

Visualization of binding site of chaperones (A, B, C, D, E, F, G and H) into IIA and IIIA regions of BSA, within 5Å distance.

<p>Visualization of binding site of chaperones (A, B, C, D, E, F, G and H) into IIA and IIIA regions of BSA, within 5Å distance.</p

ThT fluorescence kinetics during amyloid fibrillation of IAPP_20–29 (100 μmol/L).

<p>Experiments were carried out at 25°C in PBS buffer (pH 7.4; 10 mmol/L; NaCl 100 mmol/L), in presence or absence of the selected chaperones at molar relation 1:1 of IAPP_20–29: Chap. (ThT: 24 μM). Time dependent changes in ThT intensity was fitted by sigmoidal function (solid line). The experiments were carried out in triplicate. t_lag of fibril formation of the tested chaperones * p < 0,05 show significant differences with regard to control.</p

Cell Viability of CGC exposed to hIAPP_20–29, in monomeric and aggregated form, through MTT assay, with and without N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine D; methyl (2-{[4-(1-naphthylamino)-4-oxobutanoyl]amino}ethyl) dithiocarbamate B and H used as reference; at different molar ratio hIAPP_20–29:chaperones.

<p>All the experiments have the same concentration of hIAPP_20–29</p

Structures and LogP of the chemical chaperones.

<p><i>N</i>-(2-aminoethyl)-<i>N</i>'-1-naphthylsuccinamide <b>A</b>; methyl (2-{[4-(1-naphthylamino)-4-oxobutanoyl]amino}ethyl) dithiocarbamate <b>B</b>; (2<i>R</i>)-2-(6-methoxy-2-naphthyl)propanoic acid (<i>Naproxen</i>) <b>C</b>; <i>N</i>-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine <b>D</b>; 6-{[4-(1-naphthylamino)-4-oxobutanoyl]amino} hexanoic acid <b>E</b>; <i>N</i>³,<i>N</i>³'-ethane-1,2-dyilbis(<i>N</i>¹-1-naphthylsuccinamide) <b>F</b>; <i>N</i>-(4-aminobutyl)-<i>N</i>'-1-naphthylsuccinamide <b>G</b>; (1<i>E</i>,6<i>E</i>)-1,8-bis(4-hydroxy-3-methoxyphenyl)octa-1,6-diene-3,5-dione (<i>Curcumine</i>) <b>H</b>; 7-hydroxy-8-[(Z)-phenyldiazenyl]naphthalene-1,3-disulfonic acid (<i>Orange G</i>) <b>I</b>; (2Z)-3-[6-(dimethylamino)-2-naphthyl]-2-isocyanobut-2-enenitrile (<i>DDNP</i>) <b>J</b>. *The logP was calculated using <i>ACD/Log P software (ACD/1-Lab Service</i>, <i>Toronto</i>, <i>Ontario</i>, <i>Canada)</i>. <i>** Value</i> estimated <i>for uncharged compound</i>.</p