Dose-response of cytokine secretion by PBMCs to pentoxifylline and anti-leishmanial-pentoxifylline combinations.

<p>Cells from 10 patients were infected at a parasite: monocyte ratio of 10:1. Cytokine levels (IL-10, IL-13, TNF-α, IFN-γ) were determined after exposure for 96 h to different concentrations of pentoxifylline (PTX), (A) alone, combined with (B) 8 μg Sb^V/ml meglumine antimoniate or (C) 4 μM miltefosine. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.0001, **** p ≤ 0.0001.</p

Kinetics of parasite survival and cytokine secretion in response to 4 μM miltefosine and 8 μg Sb^V/mL as meglumine antimoniate in the <i>ex vivo</i> PBMC model.

<p>(A) Parasite survival and (B to E) cytokine secretion (IL-13, TNF-α, IL-10, IFN-γ) over 96 h. Mean values ± SEM for PBMCs from 5 patients. ** p ≤ 0.01, control vs both drugs.</p

Kinetics of parasite and immunologic responses in the absence of drugs.

<p>(A) Kinetics of infection evaluating the parasites: monocyte ratios of 10:1 and 20:1 (left panel), and the parasite burden (right panel) in PBMCs vs macrophages alone (infection ratio 10:1) are shown. Parasite survival is expressed as bioluminescence produced by luciferase activity in relative light units (RLU). Mean signal of uninfected cells: 135.3 ± 23.7 RLU. (B) Kinetics of cytokine secretion (TNF-α, IL-10, IL-13, IFN-γ) over the 72 hours of treatment. Data are based on at least 4 patients and expressed as means ± SEM.</p

Effect of anti-leishmanial drugs, immunomodulators and their combinations on parasite burden.

<p>(A, C and E) Dose response for pentoxifylline alone, combined with 8 μg Sb^V/ml meglumine antimoniate or 4 μM miltefosine. (B, D and F) Dose response for CpG alone, combined with 8 μg Sb^V/ml meglumine antimoniate or 4 μM miltefosine. Data are presented as means ± SEM of the parasite burden compared to infected control cultures without drugs for PBMCs from 10 patients. * p< 0.05.</p

Dose-response of cytokine secretion by PBMCs to CpG, and anti-leishmanial- CpG combinations.

<p>Cells from 10 patients were infected at a parasite: monocyte ratio of 10:1. Cytokine levels (IL-10, IL-13, TNF-α, IFN-γ) were determined after exposure for 96 h to different concentrations of CpG (A) alone, combined with (B) 8 μg Sb^V/ml meglumine antimoniate or (C) 4 μM miltefosine. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01.</p

Concentration-dependent effect of miltefosine and meglumine antimoniate on parasite survival and cytokine secretion.

<p>(A) Parasite survival and (B) Cytokine secretion after 96 hours of exposure to increasing concentrations of miltefosine (HePC) and meglumine antimoniate (Sb^V). TNFα, IL-10, IFNγ and IL-13 were evaluated in supernatants of PBMCs infected with <i>L</i>. <i>(V) panamensis</i>. Data are based on at least 6 patients and presented as mean ± SEM of the parasite burden or cytokine secretion compared to infected control without drug. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.</p

Long Circulating Self-Assembled Nanoparticles from Cholesterol-Containing Brush-Like Block Copolymers for Improved Drug Delivery to Tumors

Amphiphilic brush-like block copolymers composed of polynorbonene-cholesterol/poly­(ethylene glycol) (P­(NBCh9-<i>b</i>-NBPEG)) self-assembled to form a long circulating nanostructure capable of encapsulating the anticancer drug doxorubicin (DOX) with high drug loading (22.1% w/w). The release of DOX from the DOX-loaded P­(NBCh9-<i>b</i>-NBPEG) nanoparticles (DOX-NPs) was steady at less than 2% per day in PBS. DOX-NPs were effectively internalized by human cervical cancer cells (HeLa) and showed dose-dependent cytotoxicity, whereas blank nanoparticles were noncytotoxic. The DOX-NPs demonstrated a superior <i>in vivo</i> circulation time relative to that of free DOX. Tissue distribution and <i>in vivo</i> imaging studies showed that DOX-NPs preferentially accumulated in tumor tissue with markedly reduced accumulation in the heart and other vital organs. The DOX-NPs greatly improved survival and significantly inhibited tumor growth in tumor-bearing SCID mice compared to that for the untreated and free DOX-treated groups. The results indicated that self-assembled P­(NBCh9-<i>b</i>-NBPEG) may be a useful carrier for improving tumor delivery of hydrophobic anticancer drugs

Fluorescent, Bioactive Protein Nanoparticles (Prodots) for Rapid, Improved Cellular Uptake

A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (Prodots) and their facile uptake into the cytoplasm of cells is described here. Prodots made from bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) were found to be 15–50 nm wide and have been characterized by gel electrophoresis, transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and optical microscopic methods. Data showed that the secondary structure of the protein in Prodots is retained to a significant extent and specific activities of nGO, nHRP, nCatalase, and nLipase were 80%, 70%, 65%, and 50% of their respective unmodified enzyme activities. Calorimetric studies indicated that the denaturation temperatures of nGO and nBSA increased while those of other Prodots remained nearly unchanged, and accelerated storage half-lives of Prodots at 60 °C increased by 4- to 8-fold. Exposure of nGO and nBSA+ nGO to cells indicated rapid uptake within 1–3 h, accompanied by significant blebbing of the plasma membrane, but no uptake has been noted in the absence of nGO. The presence of nGO/glucose in the media facilitated the uptake, and hydrogen peroxide induced membrane permeability could be responsible for this rapid uptake of Prodots. In control studies, FITC alone did not enter the cell, BSA-FITC was not internalized even in the presence of nGO, and there has been no uptake of nBSA-FITC in the absence of nGO. These are the very first examples of very rapid cellular uptake of fluorescent nanoparticles into cells, particularly nanoparticles made from pure proteins. The current approach is a simple and efficient method for the preparation of bioactive, fluorescent protein nanoparticles of controllable size for cellular imaging, and cell uptake is under the control of two separate chemical triggers