Accelerated stem cell labeling with ferucarbotran and protamine

To develop and characterize a clinically applicable, fast and efficient method for stem cell labeling with ferucarbotran and protamine for depiction with clinical MRI. The hydrodynamic diameter, zeta potential and relaxivities of ferucarbotran and varying concentrations of protamine were measured. Once the optimized ratio was found, human mesenchymal stem cells (MSCs) were labeled at varying incubation times (1–24 h). Viability was assessed via Trypan blue exclusion testing. 150,000 labeled cells in Ficoll solution were imaged with T1-, T2- and T2*-weighted sequences at 3 T, and relaxation rates were calculated. Varying the concentrations of protamine allows for easy modification of the physicochemical properties. Simple incubation with ferucarbotran alone resulted in efficient labeling after 24 h of incubation while assisted labeling with protamine resulted in similar results after only 1 h. Cell viability remained unaffected. R2 and R2* relaxation rates were drastically increased. Electron microscopy confirmed intracellular iron oxide uptake in lysosomes. Relaxation times correlated with results from ICP-AES. Our results show internalization of ferucarbotran can be accelerated in MSCs with protamine, an approved heparin antagonist and potentially clinically applicable uptake-enhancing agent

The Effect of Enzymatically Polymerised Polyphenols on CD4 Binding and Cytokine Production in Murine Splenocytes

High-molecular weight polymerised polyphenols have been shown to exhibit anti-influenza virus, anti-HIV, and anti-cancer activities. The purpose of this study was to evaluate the immunomodulating activities of enzymatically polymerised polyphenols, and to clarify the underlying mechanisms of their effects. The cytokine-inducing activity of the enzymatically polymerised polyphenols derived from caffeic acid (CA), ferulic acid (FA), and p-coumaric acid (CoA) was investigated using murine splenocytes. Polymerised polyphenols, but not non-polymerised polyphenols, induced cytokine synthesis in murine splenocytes. Polymerised polyphenols induced several cytokines in murine splenocytes, with interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) being the most prominent. The underlying mechanisms of the effects of the polymerised polyphenols were then studied using neutralising antibodies and fluorescent-activated cell sorting (FACS) analysis. Our results show that polymerised polyphenols increased IFN-γ and GM-CSF production in splenocytes. In addition, the anti-CD4 neutralised monoclonal antibody (mAb) inhibited polymerised polyphenol-induced IFN-γ and GM-CSF secretion. Moreover, polymerised polyphenols bound directly to a recombinant CD4 protein, and FACS analysis confirmed that interaction occurs between polymerised polyphenols and CD4 molecules expressed on the cell surface. In this study, we clearly demonstrated that enzymatic polymerisation confers immunoactivating potential to phenylpropanoic acids, and CD4 plays a key role in their cytokine-inducing activity

Macrophage entry mediated by HIV Envs from brain and lymphoid tissues is determined by the capacity to use low CD4 levels and overall efficiency of fusion

AbstractHIV infects macrophages and microglia in the central nervous system (CNS), which express lower levels of CD4 than CD4+ T cells in peripheral blood. To investigate mechanisms of HIV neurotropism, full-length env genes were cloned from autopsy brain and lymphoid tissues from 4 AIDS patients with HIV-associated dementia (HAD). Characterization of 55 functional Env clones demonstrated that Envs with reduced dependence on CD4 for fusion and viral entry are more frequent in brain compared to lymphoid tissue. Envs that mediated efficient entry into macrophages were frequent in brain but were also present in lymphoid tissue. For most Envs, entry into macrophages correlated with overall fusion activity at all levels of CD4 and CCR5. gp160 nucleotide sequences were compartmentalized in brain versus lymphoid tissue within each patient. Proline at position 308 in the V3 loop of gp120 was associated with brain compartmentalization in 3 patients, but mutagenesis studies suggested that P308 alone does not contribute to reduced CD4 dependence or macrophage-tropism. These results suggest that HIV adaptation to replicate in the CNS selects for Envs with reduced CD4 dependence and increased fusion activity. Macrophage-tropic Envs are frequent in brain but are also present in lymphoid tissues of AIDS patients with HAD, and entry into macrophages in the CNS and other tissues is dependent on the ability to use low receptor levels and overall efficiency of fusion

The expression and signal transduction of CD4, an HIV and interleukin-16 receptor, in monocytic cells.

The down-regulation of CD4 by cultured monocytes has been observed by our group and by other investigators. Flow cytometric analysis was performed to elucidate factors influencing this phenomenon. The addition of LPS, GM-CSF, M-CSF or IL-10 to monocyte cultures failed to inhibit the decrease in monocyte CD4 expression following overnight culture. The down-regulation was an adherence-independent phenomenon since it was not prevented by culture in Teflon vials. The type of anticoagulant into which the peripheral blood was collected did not appear to be a factor, The presence or absence of lymphocytes within the cultures was also inconsequential. The continued culture of monocytes in a whole blood environment resulted in decreased CD4 down-regulation. Experiments in which CD4 was tagged with alpha-CD4-PE mAb prior to cell culture revealed that the down-regulation observed was the result of CD4 internalization. In an attempt to further understand monocyte CD4 function, we investigated the role of monocyte CD4 in signal transduction. Stimulation of Thp-1 monocytic cells with antibody to CD4 resulted in a Ca2+ flux, as well as in the time-dependent tyrosine phosphorylation of various proteins having molecular weights of approximately 180, 140, 120, 110, 85, 65, 55, 50 and 35 kDa. We identified the 140 and 85 kDa proteins as PLC-gamma1, and the regulatory subunit of PI3-K, respectively. Using immunoprecipitation/Western immunoblotting, we were unable, however, to show any direct association between CD4 and PLC-gamma1, PI3-K, or other signaling proteins. In an attempt to identify proteins capable of associating with the cytoplasmic tail of CD4, we generated a GST-CD4cyt fusion protein for use in far Western blots and immunoprecipitation experiments. In both types of experiments, the GST-CD4cyt fusion protein routinely associated with 45 and 55 kDa proteins. In the immunoprecipitation experiments, a 35 kDa protein was also observed. The above results suggest that the expression of monocytic CD4 is regulated during the differentiation process. Furthermore, the cytoplasmic tail of monocytic CD4 is associated with various proteins which we postulate function in signal transduction, and which may also play a role in CD4 down-regulation

Monocyte-derived cytokines in multiple sclerosis

MS is an inflammatory, presumably autoimmune, disease mediated by the activation of T cells, B cells and monocytes (MO). Inflammation is thought to occur early during the relapsing-remitting phase of MS (RRMS), whereas in the later phases of MS such as secondary progressive MS (SPMS), inflammation tends to diminish. Our objective was to compare the types and amounts of proinflammatory and regulatory cytokines produced by MO from relapsing–remitting patients with or without treatment with IFN-β (RRMS(+) therapy, RRMS(−) therapy), respectively, from secondary progressive patients (SPMS) and from healthy controls (HC). MO were isolated by a density-gradient technique and three different techniques (RNase protection assay, ELISA and intracellular cytokine staining) were used to assess cytokine levels. An increase in IL6, IL12 and TNF-α was observed by all three methods for RRMS(−) therapy and for SPMS patients compared to HC and RRMS(+) therapy patients. We conclude that proinflammatory and regulatory monokines can be derived from MO of MS patients and that these levels are modulated by IFN-β therapy. Although it is believed that inflammation tends to diminish in SPMS patients, our data show that inflammatory cytokines continue to be released at high levels, suggesting that IFN-β or IL10 treatment may be beneficial for this group