Molecular Mechanisms in Pathophysiology of Mucopolysaccharidosis and Prospects for Innovative Therapy.

Mucopolysaccharidoses (MPSs) are a group of inborn errors of the metabolism caused by a deficiency in the lysosomal enzymes required to break down molecules called glycosaminoglycans (GAGs). These GAGs accumulate over time in various tissues and disrupt multiple biological systems, including catabolism of other substances, autophagy, and mitochondrial function. These pathological changes ultimately increase oxidative stress and activate innate immunity and inflammation. We have described the pathophysiology of MPS and activated inflammation in this paper, starting with accumulating the primary storage materials, GAGs. At the initial stage of GAG accumulation, affected tissues/cells are reversibly affected but progress irreversibly to: (1) disruption of substrate degradation with pathogenic changes in lysosomal function, (2) cellular dysfunction, secondary/tertiary accumulation (toxins such as GM2 or GM3 ganglioside, etc.), and inflammatory process, and (3) progressive tissue/organ damage and cell death (e.g., skeletal dysplasia, CNS impairment, etc.). For current and future treatment, several potential treatments for MPS that can penetrate the blood-brain barrier and bone have been proposed and/or are in clinical trials, including targeting peptides and molecular Trojan horses such as monoclonal antibodies attached to enzymes via receptor-mediated transport. Gene therapy trials with AAV, ex vivo LV, and Sleeping Beauty transposon system for MPS are proposed and/or underway as innovative therapeutic options. In addition, possible immunomodulatory reagents that can suppress MPS symptoms have been summarized in this review.

Cancer Cell-Derived Exosomes Promote HCC Tumorigenesis Through Hedgehog Pathway.

PURPOSE: Hepatocellular carcinoma (HCC) accounts for more than 80% of primary liver cancers and is one of the leading causes of cancer-related death in many countries. Cancer cell-derived exosomes are shown to mediate communications between cancer cells and the microenvironment, promoting tumorigenesis. Hedgehog signaling pathway plays important roles in cancer development of HCC. METHODS: Exosomes were isolated from culture medium of HCC cell lines PLC/PRF/5 and MHCC-97H and were found to promote cancer cell growth measured with cell proliferation and colony formation assay. HCC cells cultured with cancer cell-derived exosome had increased cancer stem cell (CSC) population demonstrated by increased cell sphere formation CSC marker expressions. Hedgehog protein Shh was found to be highly expressed in these two HCC cell lines and preferably carried by exosomes. When Shh was knocked down with shRNA, the resulting exosomes had a reduced effect on promoting cancer cell growth or CSC population increase compared to normal cell-derived exosomes. RESULTS: The ability of PLC/PRF/5 cells to form tumor in a xenograft model was increased by the addition of the exosomes from control cancer cells but not the exosomes from Shh knocked down cancer cells. Finally, the higher plasma Exo-Shh levels were associated with later tumor stages, higher histological grades, multiple tumors, and higher recurrence rates. CONCLUSION: This study demonstrated that HCC cells secreted Shh via exosome and promote tumorigenesis through the activated Hedgehog pathway.

[New Design of Revivent TC System].

Left ventricular aneurysm (LVA) is a common complication of myocardial infarction. Traditional medical and surgical treatments are not effective or require high doctors' operational skills and patients' physical fitness. With the development of minimally invasive medical devices, it becomes possible for revivent TC system to treat LVA and reconstruct the left ventricle. This study introduces an existing product and its defect when used. From the perspective of clinical needs, we propose a new design of revivent TC system which realizes accurate force measurement and simplifies surgery.

The Influence of Tool Rake Surface Geometry on the Hard Turning Process of AISI52100 Hardened Steel.

The hard turning process has been widely used in the field of hard material precision machining because of its high efficiency, low processing residual stress, and low environmental pollution. Due to its undesirably processing quality, it is still not a substitute for traditional grinding, so many studies have reported that the process has been optimized. However, there has been little research on the geometry optimization of hard cutting tools, which have a great influence on the traditional machining process. In this paper, two tools with different rake face shapes are designed. The finite element analysis method is used to compare their performance with a conventional plane tool while turning hardened steel. The results show that the cutting performance of the designed tool T1 and T2 (chip morphology, cutting force, and cutting temperature) and the quality of the machined surface are improved compared with the tool. The cutting force decreased by 12.72% and 14.74%, the cutting temperature decreased by 7.56% and 9.01%, respectively, and the surface residual stress decreased by 26.56% and 28.66%.

Poly (propylene fumarate)/ß-calcium phosphate composites for enhanced bone repair.

A composite based on poly (propylene fumarate) (PPF) was investigated as a potential bone repair material for clinical use and it showed low heat release, suitable mechanical property and good biocompatibility. The in situ curing process would finish in less than 10 min. Compared with PMMA, PPF/TCP showed great decrease in heat release as the maximum temperature during curing process was 54.7 °C ± 1.69 °C. The compressive strength was between 109 ± 2 and 133 ± 6 MPa and the compressive modulus was 146 ± 11 to 161 ± 27 MPa, which were believed to be compatible and further supportive to surrounding bone. Besides, the surface morphology and hydrophilicity could be tailored by adjusting the content of ß-calcium phosphate (ß-TCP). Relatively stable pH value during degradation in PBS solution implied that it would not bring about acidification when implanted in vivo. In addition, PPF/TCP would boost mineralization and the apatite-like deposits on surface may advance the integrity of bone and materials. Moreover, the PPF/TCP obviously degraded and new bone formed especially when loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) in vivo. In summary, PPF/TCP composites showed suitable physical and chemical properties as well as good bioactivity and may therefore be a promising material for bone repair.

Chimeric Antigen Receptors Based on Low Affinity Mutants of FcεRI Re-direct T Cell Specificity to Cells Expressing Membrane IgE.

IgE is the key mediator of allergic responses. Omalizumab, an IgE-specific monoclonal antibody that depletes IgE, is effective for treating severe allergic asthma. The need for frequent administration of the expensive drug, however, limits its applications. Taking advantage of T cell memory, adoptive T cell therapy (ACT) targeting IgE-producing cells has the potential to achieve long-term suppression of IgE and relief of symptoms for severe allergic diseases. The transmembrane form of IgE (mIgE), which is present on all IgE-producing cells, serves as an excellent molecular target for ACT that employs chimeric antigen receptors (CARs). Here, we designed and tested CARs that use the extracellular domain of high affinity IgE receptor, FcεRIα, for mIgE recognition. When expressed on Jurkat T cells, FcεRIα-based CARs mediated robust responses in terms of CD69 upregulation to U266 myeloma cells expressing low levels of mIgE. FcεRIα-based CARs specifically recognized cells expressing mIgE, but not cells with secreted IgE captured through Fcε receptors. CAR+ Jurkat cells did not respond to LAD2 mast cells with secreted IgE bound through FcεRI or Ramos cells with secreted IgE bound through FcεRII. Co-culture of CAR+ Jurkat cells and LAD2 mast cells with IgE bound did not trigger LAD2 cell degranulation. The activity of CAR using wild type FcεRIα for mIgE binding was inhibited by the presence secreted IgE, which likely blocked CAR-mIgE interaction. The activities of CARs using low affinity mutants of FcεRIα, however, tolerated secreted IgE at relatively high concentrations. Moreover, primary human CD8+ T cells expressing a low affinity mutant CAR responded to U266 cells with INFγ production and cytotoxicity despite the presence of secreted IgE. The potency, specificity, and robustness of our CAR design, combined with repaid advances in the safety of ACT, hold promise for novel and highly effective cell-based therapies against severe allergic diseases.

Improved osteogenesis and angiogenesis of magnesium-doped calcium phosphate cement via macrophage immunomodulation.

Immune responses are vital for bone regeneration and play an essential role in the fate of biomaterials after implantation. As a kind of plastic cell, macrophages are central regulators of the immune response during the infection and wound healing process including osteogenesis and angiogenesis. Magnesium-calcium phosphate cement (MCPC) has been reported as a promising candidate for bone repair with promoted osteogenesis both in vitro and in vivo. However, relatively little is known about the effects of MCPC on immune response and the following outcome. In this study, we investigated the interactions between macrophages and MCPC. Here we found that the pro-inflammatory cytokines including TNF-α and IL-6 were less expressed and the bone repair related cytokine of TGF-ß1 was up-regulated by macrophages in MCPC extract. Furthermore, the enhanced osteogenic capacity of BMSCs and angiogenic potential of HUVECs were acquired in vitro by the MCPC-induced immune microenvironment. These findings suggest that MCPC is able to facilitate bone healing by endowing favorable osteoimmunomodulatory properties and influencing crosstalk behavior between immune cells and osteogenesis-related cells.

TCR triggering by pMHC ligands tethered on surfaces via poly(ethylene glycol) depends on polymer length.

Antigen recognition by T cells relies on the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) at the interface between the T cell and the antigen presenting cell (APC). The pMHC-TCR interaction is two-dimensional (2D), in that both the ligand and receptor are membrane-anchored and their movement is limited to 2D diffusion. The 2D nature of the interaction is critical for the ability of pMHC ligands to trigger TCR. The exact properties of the 2D pMHC-TCR interaction that enable TCR triggering, however, are not fully understood. Here, we altered the 2D pMHC-TCR interaction by tethering pMHC ligands to a rigid plastic surface with flexible poly(ethylene glycol) (PEG) polymers of different lengths, thereby gradually increasing the ligands' range of motion in the third dimension. We found that pMHC ligands tethered by PEG linkers with long contour length were capable of activating T cells. Shorter PEG linkers, however, triggered TCR more efficiently. Molecular dynamics simulation suggested that shorter PEGs exhibit faster TCR binding on-rates and off-rates. Our findings indicate that TCR signaling can be triggered by surface-tethered pMHC ligands within a defined 3D range of motion, and that fast binding rates lead to higher TCR triggering efficiency. These observations are consistent with a model of TCR triggering that incorporates the dynamic interaction between T cell and antigen-presenting cell.

The impact of Nucleofection® on the activation state of primary human CD4 T cells.

Gene transfer into primary human CD4 T lymphocytes is a critical tool in studying the mechanism of T cell-dependent immune responses and human immunodeficiency virus-1 (HIV-1) infection. Nucleofection® is an electroporation technique that allows efficient gene transfer into primary human CD4 T cells that are notoriously resistant to traditional electroporation. Despite its popularity in immunological research, careful characterization of its impact on the physiology of CD4 T cells has not been documented. Herein, using freshly-isolated primary human CD4 T cells, we examine the effects of Nucleofection® on CD4 T cell morphology, intracellular calcium levels, cell surface activation markers, and transcriptional activity. We find that immediately after Nucleofection®, CD4 T cells undergo dramatic morphological changes characterized by wrinkled and dilated plasma membranes before recovering 1h later. The intracellular calcium level also increases after Nucleofection®, peaking after 1h before recovering 8h post transfection. Moreover, Nucleofection® leads to increased expression of T cell activation markers, CD154 and CD69, for more than 24h, and enhances the activation effects of phytohemagglutinin (PHA) stimulation. In addition, transcriptional activity is increased in the first 24h after Nucleofection®, even in the absence of exogenous stimuli. Therefore, Nucleofection® significantly alters the activation state of primary human CD4 T cells. The effect of transferred gene products on CD4 T cell function by Nucleofection® should be assessed after sufficient resting time post transfection or analyzed in light of the activation caveats mentioned above.

Strength of PD-1 signaling differentially affects T-cell effector functions.

High surface expression of programmed death 1 (PD-1) is associated with T-cell exhaustion; however, the relationship between PD-1 expression and T-cell dysfunction has not been delineated. We developed a model to study PD-1 signaling in primary human T cells to study how PD-1 expression affected T-cell function. By determining the number of T-cell receptor/peptide-MHC complexes needed to initiate a Ca(2+) flux, we found that PD-1 ligation dramatically shifts the dose-response curve, making T cells much less sensitive to T-cell receptor-generated signals. Importantly, other T-cell functions were differentially sensitive to PD-1 expression. We observed that high levels of PD-1 expression were required to inhibit macrophage inflammatory protein 1 beta production, lower levels were required to block cytotoxicity and IFN-γ production, and very low levels of PD-1 expression could inhibit TNF-α and IL-2 production as well as T-cell expansion. These findings provide insight into the role of PD-1 expression in enforcing T-cell exhaustion and the therapeutic potential of PD-1 blockade.

T cell receptor triggering by force.

Antigen recognition through the interaction between the T cell receptor (TCR) and peptide presented by major histocompatibility complex (pMHC) is the first step in T cell-mediated immune responses. How this interaction triggers TCR signalling that leads to T cell activation is still unclear. Taking into account the mechanical stress exerted on the pMHC-TCR interaction at the dynamic interface between T cells and antigen presenting cells (APCs), we propose the so-called receptor deformation model of TCR triggering. In this model, TCR conformational change induced by mechanical forces initiates TCR signalling. The receptor deformation model, for the first time, explains all three aspects of the TCR triggering puzzle: mechanism, specificity, and sensitivity.

Complement receptor 3 ligation of dendritic cells suppresses their stimulatory capacity.

To activate T cells effectively, dendritic cells (DCs) must provide three separate signals, MHC-Ag, costimulatory molecules (such as CD80 and CD86), and proinflammatory cytokines (such as IL-12). These three signals are up-regulated in the presence of "danger signals" such as LPS or viral nucleic acids. Evidence suggests that DCs providing only the first two of these signals cannot successfully stimulate T cells. Apoptotic cells have been proposed to suppress DC immunogenicity through the ligation of apoptotic cell receptors. Complement receptor 3 (CR3) and CD36 have been suggested to be important in this process, although the mechanism by which this modulation occurs is still unclear. We demonstrate that ligation of CR3, but not CD36, directs DCs to increase surface MHC and costimulatory molecules, while suppressing inflammatory cytokine release. CR3 modulation of DCs does not require a type I IFN response, does not involve the specific regulation of the MyD88- or Toll/IL-1R domain-containing adaptor-inducing IFN-beta-dependent TLR signaling pathways, and occurs even in the absence of danger signals. The functional outcome of this process is poor Ag-specific stimulation of CD4 and CD8 T cells by CR3-ligated DCs both in naive response as well as upon subsequent challenge with normal DCs. We propose that CR3 provides a "nondanger" signal that suppresses the stimulatory capacity of DCs.

Ligation of CD28 by its natural ligand CD86 in the absence of TCR stimulation induces lipid raft polarization in human CD4 T cells.

Stimulation of resting CD4 T cells with anti-CD3/CD28-coated beads leads to rapid polarization of lipid rafts (LRs). It has been postulated that a major role of costimulation is to facilitate LR aggregation. CD86 is up-regulated or expressed aberrantly on immune cells in a wide array of autoimmune and infectious diseases. Using an Ig fusion with the extracellular domain of CD86 (CD86Ig) bound to a magnetic bead or K562 cells expressing CD86, we demonstrated that ligation of CD28 by its natural ligand, but not by Ab, induced polarization of LRs at the cell-bead interface of fresh human CD4 T cells in the absence of TCR ligation. This correlated with activation of Vav-1, increase of the intracellular calcium concentration, and nuclear translocation of NF-kappaB p65, but did not result in T cell proliferation or cytokine production. These studies show, for the first time, that LR polarization can occur in the absence of TCR triggering, driven solely by the CD28/CD86 interaction. This result has implications for mechanisms of T cell activation. Abnormalities in this process may alter T and B cell tolerance and susceptibility to infection.