Modulation of Signaling and Intracellular Trafficking Pathways by Surface-Engineered Hydrogel Nanoparticles in Tumor Cells.

Surface engineering of a polyacrylamide (PAA) hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKKC), confers binding specificity toward Nucleolin overexpressing tumor cells (9L rat gliosarcoma, and MDA-MB-435 human breast adenocarcinoma). In this study, the endocytic internalization, and intracellular trafficking of the non-targeted PAA-NPs (NTNPs), and F3-targeted PAA-NPs (F3NPs) in the above-mentioned cell lines, was investigated. Caveolae-mediated internalization of both types of PAA-NPs peaked at 2 hours post-delivery, although internalization of the NTNPs was ~2-fold greater than for the F3NPs. In contrast, clathrin-mediated internalization of both types of PAA-NPs was markedly faster; the NTNPs and F3NPs both reached similar peak colocalization levels with early endosome antigen-1 (EEA1, ~32%) at 30 minutes post-delivery. However, at 60 minutes post-delivery, the NTNPs exhibited faster egress from the early endosomes than the F3NPs, with a concomitant, sharp increase in trafficking to the lysosomes (acidic, degradative vesicles), whereas the F3NPs largely evaded trafficking to the lysosomes. Furthermore, the F3 peptides alone exhibited significantly higher accumulation within the lysosomes than both the NTNPs, and the F3NPs. The p38 Mitogen-Activated Protein Kinases (MAPKs), upon activation, promote (i) internalization of caveolae from the cell membrane, and (ii) rapid trafficking of early endosomes to the lysosomes by directly phosphorylating Caveolin1 and EEA1, respectively. Phospho-proteomic analyses, in MDA-MB-435 cells, revealed that the peak levels of activated p38β and p38δ MAPKs (at 2 hours post-delivery) elicited by the F3 peptides alone, and the NTNPs was ~2-fold greater than by the F3NPs. These data therefore provide compelling evidence that the intracellular trafficking behavior of the F3 peptides, NTNPs and F3NPs are attributable to their differential activation of the p38 MAPKs. Further analysis of the ERK MAPK, JNK MAPK, and Akt pathways revealed that the NTNPs elicit a pro-apoptotic signaling profile, whereas the F3 peptides, and F3NPs elicit proliferative profiles. The findings of this thesis suggest that the design of tumor-targeting nanoparticles also need to consider the MAPK signaling profiles that they elicit on the intended target cell type, due to the influence of the p38 MAPKs, in particular, on endocytic trafficking, and the survival status of the target tumor cell.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/110348/1/leshernk_1.pd

Apoptosis in peripheral blood mononuclear cells of human immunodeficiency virus (HIV) infected patients undergoing highly active antiretroviral therapy.

Thesis (M.Med.Sci.)--University of KwaZulu-Natal, 2008.Highly active antiretroviral therapy (HAART) is currently the only treatment that effectively reduces the morbidity and mortality of individuals infected with Human Immunodeficiency Virus-1 (HIV-1). Standard HAART regimens typically comprise 2 nucleoside reverse transcriptase inhibitors and either one non-nucleoside reverse transcriptase inhibitor or a protease inhibitor. These drugs bind to and inhibit the HIV-1 Reverse Transcriptase and Protease enzymes respectively, thereby suppressing viral replication. The nucleoside reverse transcriptase inhibitors promote mitochondrial (mt) dysfunction by strongly inhibiting mt polymerase gamma (Pol-y) and subsequently, mtDNA replication. In contrast, the non-nucleoside reverse transcriptase inhibitors, efavirenz (EFV) and nevirapine (NVP) do not inhibit Pol-y although EFV has been shown to induce mt depolarisation ( mlow) in vitro at supra-therapeutic concentrations. However, the capacity of non-nucleoside reverse transcriptase inhibitor drugs to induce mt toxicity in vivo previously remained undetermined. The objective of this study was to determine the influence of EFV and NVP on peripheral lymphocyte mt transmembrane potential (Avj/m) and apoptosis in HIV-1-infected patients treated with these non-nucleoside reverse transcriptase inhibitors. Thirty-two HIV-1-infected patients on HAART between 4 and 24 months (12 on EFV, 20 on NVP) and 16 HAART-naive HIV-1-infected patients were enrolled into this study. All participants were black South African patients. Spontaneous peripheral lymphocyte apoptosis and mlow were measured ex vivo by flow cytometry for all patients. CD4 T-helper apoptosis for the EFV and NVP cohorts was 19.38% ± 2.62% and 23.35% ± 1.51% (mean ± SEM), respectively, whereas total lymphocyte mlow was 27.25% ± 5.05% and 17.04% ± 2.98%, respectively. Both parameters for each cohort were significantly lower (P < 0.05) than that of the HAART-naive patients. The NVP cohort exhibited both a significant time dependent increase in peripheral lymphocyte ö¿mlow (P = 0.038) and correlation between Thelper apoptosis and low (P = 0.0005). These trends were not observed in the EFV cohort. This study provides evidence that both EFV and NVP induce peripheral lymphocyte ö¿ m low in HIV-1-infected patients on non-nucleoside reverse transcriptase inhibitor-based HAART, which in the case of NVP is sufficient to induce the apoptosis cascade

Association of -308 TNF-alpha promoter polymorphism with viral load and CD4 T-helper cell apoptosis in HIV-1 infected black South Africans

Objective. To determine whether the -308 TNF-α promoter polymorphism is associated with markers of HIV progression in the South African population. Methods. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the -308 TNF-α polymorphism in 75 patients and 76 healthy controls. Serum TNF-α concentrations were measured using ELISA in each cohort. CD4+ T cell apoptosis and HIV-1 RNA viral load were determined using Annexin-V-FITC assay and Nuclisens Easy Q HIV-1 assay respectively. CD4 + T cell counts were measured flow cytometrically. Results. The frequency of -308 G allele was similar in the HIV-1 and control cohorts. The -308GG genotype was associated with lower TNF-α concentrations and markers of increased HIV progression indicated by higher TH lymphocyte apoptosis, lower TH lymphocyte count and higher plasma viral load, irrespective of treatment. Conclusion. The presence of the TNF-α -308 G allele in HIV-1 patients may be associated with increased risk of HIV-1 progression. Further research is required to investigate the nature of this association. S Afr J HIV Med 2012;13(2):72-77