Potentiation of drug toxicity through virus latency reversal promotes preferential elimination of HIV infected cells

Eliminating latently infected cells is a highly challenging, indispensable step towards the overall cure for HIV/AIDS. We recognized that the unique HIV protease cut site (Phe-Pro) can be reconstructed using a potent toxin, monomethyl auristatin F (MMAF), which features Phe at its C-terminus. We hypothesized that this presents opportunities to design prodrugs that are specifically activated by the HIV protease. To investigate this, a series of MMAF derivatives was synthesized and evaluated in cell culture using latently HIV-infected cells. Cytotoxicity of compounds was enhanced upon latency reversal by up to 11-fold. In a mixed cell population, nanomolar concentrations of the lead compound depleted predominantly the HIV-infected cells and in doing so markedly enriched the pool with the uninfected cells. Despite expectation, mechanism of action of the synthesized toxins was not as HIV protease-specific prodrugs, but likely through the synergy of toxicities between the toxin and the reactivated virus.

Autologous Non-Cultured Epidermal Cellular Grafting in the Surgical Treatment of Stable Vitiligo: The Skin Hospital Protocol

Autologous non-cultured epidermal cellular grafting is the treatment of choice for patients with stable refractory vitiligo. Recently, studies have shown cost-effective alternatives for this procedure, superseding previous techniques that required large research facilities or expensive pre-packaged kits. We provide modifications to current techniques, including the use of individual Petri dishes to allow for processing larger skin grafts, hyfrecation instead of conventional manual dermabrasion of the recipient site to reduce scar formation as well as better margin delineation, and an intravenous giving set with a filter for improved filtration of the mixed cell population. These modifications facilitated sufficient skin repigmentation in a cost-effective outpatient setting.

Strong mutator phenotype drives faster adaptation from growth on glucose to growth on acetate in Salmonella

The metabolic adaptation of strong mutator strains was studied to better understand the link between the strong mutator phenotype and virulence. Analysis of the growth curves of isogenic strains of Salmonella, which were previously grown in M63 glucose media, revealed that the exponential phase of growth was reached earlier in an M63 acetate medium with strong mutator strains (mutated in mutS or in mutL) than with normomutator strains (P<0.05). Complemented strains confirmed the direct role of the strong mutator phenotype in this faster metabolic adaptation to the assimilation of acetate. In a mixed cell population, proliferation of strong mutators over normomutators was observed when the carbon source was switched from glucose to acetate. These results add to the sparse body of knowledge about strong mutators and highlight the selective advantage conferred by the strong mutator phenotype to adapt to a switch of carbon source in the environment. This work may provide clinically useful information given that there is a high prevalence of strong mutators among pathogenic strains of Salmonella and that acetate is the principal short chain fatty acid of the human terminal ileum and colon where Salmonella infection is localized.

Augmentation of Murine Vasculogenesis by Injected Human Hematopoietic Stem Cells in Response to Ischemia Follows Early Activation of Innate Immune Response

Although NOD.SCID mice lack an adaptive immune response, their innate immune system is intact. NOD.SCID mice are the model system used for both allo- and xeno-graft cell therapies to in vivo neovascularization of ischemic beds. As most previous studies have used undefined or mixed cell population, we tested the hypothesis that selected human umbilical cord blood (UCB) CD133+ stem cells (human HSC) would promote in vivo neovascularization, thereby conferring a therapeutic advantage over the mixed cell population found in mononuclear cell (MNC) preparations. These experiments focused on the murine response within the ischemic bed to human HSC cells as evaluated by gene array analyses. Gene array data was mined for inflammation, innate immunity and wound repair genes, with a focus on factors favorable to regeneration, angiogenesis and limits collagen deposition (scar formation). Normal mammalian wound repair occurs in 3 overlapping stages inflammation, new tissue formation, and remodeling. To evaluate these cell populations, NOD.SCID mice underwent femoral ligation and were divided into 3 study groups: controls (media alone), human selected UCB CD133+ HSC, or human non-selected UCB MNC. Previously, laser Doppler studies after femoral ligation showed significantly improved blood flow ratio in animals treated with human HSC vs. controls, 0.55±0.06 (n=9) and 0.37±0.03 (n=12, p&lt;0.05), respectfully, and histology confirmed increased capillary density in animals treated with human HSC compared to controls, (320±18 and 131±6.9 cells/mm2; respectively p&lt;0.0001). To compare the treatment effects on the early transcriptome and to wound repair, murine gene expression microarrays (Affymetrix murine GeneChip 430 2.0) of RNA extracted from tissues harvested from the ischemic limbs 3 days following femoral ligation and injection of cells (n=3 each experimental group) were utilized. In comparing control mice to those receiving human selected CD133+ HSC, 414 genes with 2-fold or greater change was observed. Several genes were differentially regulated between study groups. The level of IL12a mRNA was 2-fold lower in the hindlimb tissue of mice treated with CD133+ HSC than in the untreated controls: IL12 is a known regulator of the TH1-associated cytokine INF-g and TNF-a, a primary regulator of immune cells and cytolytic stimuli. While there was no observed change in the levels of INF-g in the hindlimb, 17 interferon induced genes were observed to be significantly lower in the human HSC-treated hindlimb compared to controls. Genes induced by TGF-b were lower including IL-12a, Ras-related genes, and the inhibitor latent transforming growth factor beta binding protein 2 was 3-fold higher in the hindlimbs treated with CD133+ HSC cells compared to controls. Innate immune cytokine receptors IL4 and IL13 mRNAs were expressed at a lower level than that seen in control mice. However, the IL6 receptor increased 3-fold in the human selected CD133+ HSC-treated cells as well as the downstream genes TIMP-1 (tissue inhibitor of matrix metalloprotease) and Pim1 gene, a stressed induced serine-threonine kinase which promotes cell survival. These results confirm prior observations in IL-6−/− knockout mice demonstrating delayed responses in leukocyte infiltration, angiogenesis and tissue remodeling. In addition, there was significant increases in genes regulated by the Wnt signaling pathway, compared to hindlimbs treated with UCB MNC cells including: Jun (2-fold higher), Sox4 (2–5 fold higher), Frizzle homolog 8 (2-fold), and MAPKKK4 (2-fold higher). The modulation of these factors and the observation that the inhibitor of Wnt signaling dickkopf was reduced 2.3-fold may indicate enhanced regenerative mechanisms as Wnt functions during embryogenesis and also during adult limb formation during metamorphosis. In summary, NOD.SCID treated with human selected CD133+ HSC after femoral ligation demonstrate significantly improved capillary density and blood flow recovery at later time points (day 28–42). Receptors and cytokines regulating innate immunity are significantly higher at an early time point (day 3) in mice treated with human CD133+ HSC suggesting an important regulatory role for innate inflammatory responses contributing to augmentation of vasculogenesis after acute vascular injury.

Significance of the inital cytomorphological and immunocytochemical findings and the correlation with the international prognostic index for the survival in patients with non-Hodgkin’s lymphoma

Background/Aim. Fine-needle aspiration biopsy is a quick, economical, and safe initial method in managing a patient with suspected lymphoma. According to a few reports on this preoblem, the aim of this study was to compare histological findings to cytomorphological ones in needle aspirates. We also compared these findings to the overal survival (OS) time. Methods. We analyzed the fine-needle aspiration biopsies of peripheral lymph nodes, and the International Prognostic Index (IPI) in 81 patients with non-Hodgkin?s lymphoma (NHL). We put these findings into correlation with OS time. Results. According to the International Working Formulation (IWF) criteria, the dominant cell population was as follows: 18 patients had the small cell population, 21 patients had small cleaved cells, 18 patients had the mixed cell population, 21 patients had large cell population, 2 patients had Burkitt lymphoma type, and 1 patient had the dominant lymphoblasts. On presentation, 32 patients had a low IPI index, 32 patients had a low intermediate, and 17 patients had a high intermediate IPI. We confirmed the statistical significance (Kaplan-Mayer) of cytomorphology (p = 0.013) and IPI index (p = 0.016) for survival time. During a 48-month follow-up, OS was 37.2 months for the patients with the dominant small cells, and 32 months for the patients with small cleaved cells (PH equivalent to indolent NHL). For the patients with the dominant mixed cell population, large cell population and Burkitt limphoma cell, OS were 17, 14.4, and 9.3 months, respectively (PH equivalent to aggressive NHL). Patients with low IPI had the highest OS, 36 months for the low intermediate and only 11.6 months for the high intermediate IPI index. Conclusion. We concluded that an initial cytological and clinical profile of patients with NHL, might give a quick and relevant information for planning an adequate therapy.

Multiparameter in situ analysis of trophoblast cells in mixed cell populations by combined DNA and RNA in situ hybridization.

We developed a non-radioactive assay for simultaneous detection of cytoplasmic mRNA and nuclear genomic DNA in fetal trophoblast cells by sequential in situ hybridization. Trophoblast-specific mRNA is detected with a digoxigenin-labeled RNA probe complementary to HLA-G, followed by visualization through the generation of stable contrast-rich DAB/Ni complexes. Genomic target DNA is subsequently visualized in labeled cells by fluorescent in situ hybridization using biotin-labeled chromosome-specific DNA probes. Simultaneous visualization of both targets is made possible using a fluorescence microscope with FITC filter and conventional brightfield light. This method allows detection of trophoblast cells within a mixed cell population and, at the same time, analysis of chromosome anomalies in the trophoblast cells identified. For prenatal diagnosis of fetal cells enriched from maternal peripheral blood during pregnancy, this multiparameter in situ analysis of immobilized fetal trophoblast cells will be very useful.