Complement Factor H and Apolipoprotein E Participate in Regulation of Inflammation in THP-1 Macrophages

The alternative pathway (AP) of complement is constantly active in plasma and can easily be activated on self surfaces and trigger local inflammation. Host cells are protected from AP attack by Factor H (FH), the main AP regulator in plasma. Although complement is known to play a role in atherosclerosis, the mechanisms of its contribution are not fully understood. Since FH via its domains 5-7 binds apoliporotein E (apoE) and macrophages produce apoE we examined how FH could be involved in the antiatherogenic effects of apoE. We used blood peripheral monocytes and THP-1 monocyte/macrophage cells which were also loaded with acetylated low-density lipoprotein (LDL) to form foam cells. Binding of FH and apoE on these cells was analyzed by flow cytometry. High-density lipoprotein (HDL)-mediated cholesterol efflux of activated THP-1 cells was measured and transcriptomes of THP-1 cells using mRNA sequencing were determined. We found that binding of FH to human blood monocytes and cholesterol-loaded THP-1 macrophages increased apoE binding to these cells. Preincubation of fluorescent cholesterol labeled THP-1 macrophages in the presence of FH increased cholesterol efflux and cholesterol-loaded macrophages displayed reduced transcription of proinflammatory/proatherogenic factors and increased transcription of anti-inflammatory/anti-atherogenic factors. Further incubation of THP-1 cells with serum reduced C3b/iC3b deposition. Overall, our data indicate that apoE and FH interact with monocytic cells in a concerted action and this interaction reduces complement activation and inflammation in the atherosclerotic lesions. By this way FH may participate in mediating the beneficial effects of apoE in suppressing atherosclerotic lesion progression.Peer reviewe

Thyroid Cancer Imaging In Vivo by Targeting the Anti-Apoptotic Molecule Galectin-3

Background The prevalence of thyroid nodules increases with age, average 4-7% for the U.S.A. adult population, but it is much higher (19-67%) when sub-clinical nodules are considered. About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information (cold or hot nodules). The expression of the anti-apoptotic molecule galectin-3 is restricted to cancer cells and this feature has potential diagnostic and therapeutic implications. We show here the possibility to obtain thyroid cancer imaging in vivo by targeting galectin-3. Methods The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific 99mTc-radiolabeled mAb. A position-sensitive high-resolution mini-gamma camera was used as imaging capture device. Human galectin-3 positive thyroid cancer xenografts (ARO) and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 ?Ci of 99mTc-labeled mAb to galectin-3 (30 ?g protein/in 100 ?l saline solution). Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera. Findings Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared. Conclusions These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting. In vivo imaging of thyroid cancer may allow a better selection of patients referred to surgery. The possibility to apply this method for imaging and treatment of other galectin-3 expressing tumors is also discussed

Magnetic fluids under applied field studied by light scattering and microscopy

The optical response of several magnetic nanoparticle dispersions is studied by angle resolved transmittance at wavelength of 600 nm in zero-field and under an applied magnetic field of 0.1 T, with the purpose of exploring to what extent this could be used as a method of monitoring the colloidal dispersions. In an external magnetic field, magnetic particles will start to order into needle-like structures aligned with the field, which was observed by microscopy. This will change the light scattering caused by the particles. Results showed a clear effect from applying the magnetic field by a dip in the transmitted intensity at angles around 3-5 degrees. The scattering is compared to that of a homogeneous infinite cylinder and theoretical explanations to the effect are discussed. Spectrophotometry at wavelengths between 300-1100 nm is performed and compared with calculations from Kubelka-Munk approximation to give a first characterization of the particle dispersions. Particles used in this project are magnetite, Fe3O4, and maghemite, gamma-Fe2O3, nanoparticles with sizes ranging from 5-250 nm

Temperature dependence in human Rhinovirus infection of human MRC-5

Temperature has been known to be an important factor for in vitro studies where human cell cultures are infected with HRV (human Rhinovirus). The mechanisms behind the temperature effect on the struggle between virulence and cellular defense, are still largely unknown and may be a crucial part in finding a treatment to the common cold. In this study we focused on a few cellular key elements in this struggle and observed behavior changes in regards to the pre-infection growth temperature and the temperature during the viral infection. Past studies have focused mainly on the temperature post inoculation, but here we also wanted to correlate virulence to the growth temperatures preceding the viral infection. We found that the growth temperature of the cell did indeed affect its response to the HRV. If the cells had been growing in an optimal body temperature of 37°C before getting virally infected at 33°C, the viability of the cells did decrease in comparison to cells that had been growing in 33°C from before the viral infection. We could also observe a significant temperature dependence regarding IL-8 release upon HRV inoculation. HRV strive to block induction of inflammatory cytokines such as interferons and IL-1. It may be that impaired IL-8 release at lower temperatures will prevent important danger signals alerting the immune system when cytokine signaling is otherwise hampered by viral intervention

Temperature dependence in human Rhinovirus infection of human MRC-5

Temperature has been known to be an important factor for in vitro studies where human cell cultures are infected with HRV (human Rhinovirus). The mechanisms behind the temperature effect on the struggle between virulence and cellular defense, are still largely unknown and may be a crucial part in finding a treatment to the common cold. In this study we focused on a few cellular key elements in this struggle and observed behavior changes in regards to the pre-infection growth temperature and the temperature during the viral infection. Past studies have focused mainly on the temperature post inoculation, but here we also wanted to correlate virulence to the growth temperatures preceding the viral infection. We found that the growth temperature of the cell did indeed affect its response to the HRV. If the cells had been growing in an optimal body temperature of 37°C before getting virally infected at 33°C, the viability of the cells did decrease in comparison to cells that had been growing in 33°C from before the viral infection. We could also observe a significant temperature dependence regarding IL-8 release upon HRV inoculation. HRV strive to block induction of inflammatory cytokines such as interferons and IL-1. It may be that impaired IL-8 release at lower temperatures will prevent important danger signals alerting the immune system when cytokine signaling is otherwise hampered by viral intervention

Sialylation of the B lymphocyte molecule CD22 by alpha 2,6-sialyltransferase is implicated in the regulation of CD22-mediated adhesion.

The B cell surface receptor CD22 binds several sialoglycoproteins containing sialic acid in alpha 2,6 linkage, on the surface of B and T lymphocytes. Because lymphocytes adhere tightly to fibroblasts transfected with CD22 cDNA, it would appear reasonable to suggest that regulatory mechanisms might have evolved which prevent undesired CD22-mediated leukocyte aggregation. Here we provide evidence for the existence of at least one mechanism that might regulate CD22 interaction with ligands on adjacent cells. We demonstrate that sialylation of CD22 by beta-galactoside alpha 2,6-sialyltransferase abrogates CD22-mediated lymphocyte adhesion, and that adhesion can be restored by removal of alpha 2,6-linked sialic acid residues from the CD22 molecule. Taken together, our results suggest that alpha 2,6-sialyltransferase can both promote and inhibit CD22-ligand interactions. These observations provide the first direct evidence that receptor-ligand interactions mediated by an Ig superfamily molecule are under the control of a specific glycosyltransferase

ApoD Mediates Binding of HDL to LDL and to Growing T24 Carcinoma

<div><p>Apolipoprotein (Apo) D is an important protein produced in many parts of the body. It is necessary for the development and repair of the brain and protection from oxidative stress. The purpose of this study was to investigate the extent to which apoD interacts with lipoproteins in human plasma. By using detergent-free ELISA, we show that immobilized monoclonal antibodies against apoD very efficiently bind to low density lipoprotein (LDL) from plasma; this binding is as equally efficient as binding to an anti-apoB monoclonal antibody. Adding detergent to the plasma inhibited the binding, suggesting that the binding is dependent on the presence of intact lipoprotein particles. Reversing the system by using immobilized anti-apoB revealed that the affinity of apoD for LDL is rather low, suggesting that multiple bindings are needed for a durable connection. Biosensor experiments using purified lipoproteins also showed that purified apoD and high density lipoprotein 3 (HDL3), a lipoprotein fraction rich in apoD, were both able to bind LDL very efficiently, indicating that the HDL3-LDL interaction may be a physiological consequence of the affinity of apoD for LDL. Furthermore, we found that apoD increases the binding of HDL to actively growing T24 bladder carcinoma cells but not to quiescent, contact-inhibited, confluent T24 cells. This result is especially intriguing given that the T24 supernatant only contained detectable levels of apoD after growth inhibition, raising the possibility that alternating the expression of apoD and a putative apoD-receptor could give direction to the flow of lipids. In the current paper, we conclude that apoD mediates binding of HDL to LDL and to growing T24 carcinomas, thereby highlighting the importance of apoD in lipid metabolism.</p></div