Biomedical subjects

Heping Xu

Publications and source records attributed to Heping Xu.

16 recordsLinked to original sources

PubMed2006

Application of the modified spherical harmonics method to some problems in biomedical optics.

It is demonstrated that a semi-analytical method, modified spherical harmonics (MSH), is a fast and rigorous solution for the radiative transport equation in an infinite medium containing an isotropic source. The optical properties of the medium can be determined using an inverse scheme based on fluence data (including phase if a modulated source is used) without the drawbacks inherent in the diffusion approximation. The MSH method can also be used to solve for the fluorescence fluence due to a point excitation source in an infinite medium.

Algorithms↗

PubMed2005

Involvement of CCR5 in the passage of Th1-type cells across the blood-retina barrier in experimental autoimmune uveitis.

Although the recruitment of T helper cell type 1 (Th1)/Th2 cells into peripheral tissues is essential for inflammation and the host response to infection, the traffic signals that enable the distinct positioning of Th1/Th2 cells are unclear. We have determined the role of CC chemokine receptor 5 (CCR5) in this using experimental autoimmune uveitis (EAU) as a model system. In EAU, Th1-like cells are preferentially recruited into the retina across the blood-retina barrier, partly as a result of expression of the adhesion molecules P-selectin glycoprotein ligand 1 and lymphocyte function-associated antigen-1 on these cells. CD3+ T cells, infiltrating the retina, also expressed the chemokine receptor CCR5, and CCR5 ligands, macrophage-inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and regulated on activation, normal T expressed and secreted (RANTES), were strongly expressed in the retina at peak EAU. Th1-like cells, polarized in vitro, expressed high levels of CCR5. The trafficking of these CCR5+ cells was examined by tracking them after adoptive transfer in real time in vivo at an early disease stage using scanning laser ophthalmoscopy. Treatment of the cells with antibody against CCR5 prior to transfer resulted in a reduction in their infiltration into the retina. However, rolling velocity, rolling efficiency, and adherence of the cells to retinal endothelium were not reduced. CCR5 is clearly important for Th1 cell recruitment, and this study demonstrates for the first time in vivo that CCR5 may act at the level of transendothelial migration rather than at the earlier stage of rolling on the endothelium.

Adoptive Transfer↗

PubMed2005

Differentiation to the CCR2+ inflammatory phenotype in vivo is a constitutive, time-limited property of blood monocytes and is independent of local inflammatory mediators.

It is proposed that CCR2+ monocytes are specifically recruited to inflammatory sites, whereas CCR2- monocytes are recruited to normal tissue to become resident macrophages. Whether these subsets represent separate lineages, how differential trafficking is regulated and whether monocytes undergo further differentiation is uncertain. Using a mouse model of autoimmune uveoretinitis we examined monocyte trafficking to the inflamed retina in vivo. We show that bone marrow-derived CD11b+ F4/80- monocytes require 24 to 48 h within the circulation and lymphoid system before acquiring the CCR2+ phenotype and trafficking to the inflamed retina is enabled. This phenotype, and the capacity to traffic were lost by 72 h. Monocyte CCR2 expression followed a similar time course in normal mice indicating that differentiation to an inflammatory phenotype is a constitutive, time-limited property, independent of local inflammatory mediators. Phenotypic analysis of adoptively transferred cells indicated that circulating inflammatory monocytes also differentiate into CD11c+ and B220+ dendritic cells and F4/80+ tissue macrophages in vivo. Our data supports the hypothesis of continuous extravasation and progressive differentiation over time of inflammatory monocytes in the circulation rather than replication within the actively inflamed tissue, and supports the concept of myeloid dendritic cell differentiation from trafficking monocytes under physiological conditions in vivo.

Adoptive Transfer↗

PubMed2005

The keratan sulfate disaccharide Gal(6S03) beta1,4-GlcNAc(6S03) modulates interleukin 12 production by macrophages in murine Thy-1 type autoimmune disease.

It has been reported that disaccharides of the glycosaminoglycans (GAGs), heparin, or heparan sulfate suppress the production of cytokines. Therefore, we examined the effects of GAGs (keratan sulfate, hyaluronan, chondroitin, chondroitin sulfate, and heparin sulfate) disaccharides on production of interleukin (IL)-12, a pivotal cytokine in the Th-1 type immune system. Among the GAG disaccharides, only a keratan sulfate disaccharide, Gal(6-SO(3))-GlcNAc(6-SO(3)) (L4), suppressed IL-12 production in macrophages stimulated with lipopolysaccharides and interferon-gamma. Neither keratan sulfate chains nor keratan sulfate tetrasaccharides elicited any change in the IL-12 production. N-Acetyl-lactosamine, Gal-GlcNAc (LacNAc), also did not change IL-12 production. These results indicated that a certain size, i.e. disaccharide and sulfate, are essential to suppress IL-12 production. L4 was then applied to MRL-lpr/lpr mice, a Th-1 type autoimmune disease model. The treatment of MRL-lpr/lpr mice with L4 1) decreased in serum IL-12, 2) induced apoptosis in T cells in lymph nodes thereby suppressing lymphoaccumulation, and 3) suppressed hypergammaglobulinemia and glomerulonephritis. We showed previously that IL-12 suppresses cell death of T cells, thereby enhancing the lymphoaccumulation in MRL-lpr/lpr mice. Moreover, it has been reported that IL-12 deficiency in MRL-lpr/lpr mice diminishes lymphoaccumulation and delays glomerulonephritis. The treatment with L4 suppressed phosphoprotein kinase C and phosphoinositide 3-kinase expression in macrophages, suggesting that L4 suppresses IL-12 production by inhibiting phosphoprotein kinase C and phosphoinositide 3-kinase pathways.

Animals↗

PubMed2005

Leukocyte diapedesis in vivo induces transient loss of tight junction protein at the blood-retina barrier.

PURPOSE: Although much is now understood about the molecular structure of tight junctions (TJs), little is known about the regulation of their function during neural inflammatory disease processes in vivo. The mechanisms by which leukocytes transmigrate the blood-retina barrier (BRB) without affecting endothelial permeability are controversial. METHODS: Confocal immunofluorescence microscopy of ex vivo retinal wholemounts was used to study BRB integrity during leukocyte adhesion and migration during experimental autoimmune uveoretinitis (EAU). Western blot analysis was used to measure levels of TJ proteins in EAU retina and RPE and in normal retina or RPE cultured with cytokines or chemokines. RESULTS: No evidence for discontinuity or other weakness of the endothelial or epithelial barrier at tricellular corners was observed, and maximum disruption of TJ protein expression was focused in retinal venules correlating with sites of leukocyte extravasation. Areas of maximum TJ protein loss in vivo also correlated with redistribution or loss of ensheathing astrocyte processes on venules but not adjacent capillaries or arterioles. Exposure of normal choroidal and retinal explants ex vivo to cytokines and chemokines alone did not downregulate total occludin-1 or claudin-3 TJ protein expression. CONCLUSIONS: The data presented herein support an active role for leukocytes in TJ disruption and blood-retina barrier (BRB) breakdown during retinal inflammation and further implicate venule microenvironment as a key factor in leukocyte recruitment to retinal tissue in vivo.

Animals↗

PubMed2004

Recruitment of IFN-gamma-producing (Th1-like) cells into the inflamed retina in vivo is preferentially regulated by P-selectin glycoprotein ligand 1:P/E-selectin interactions.

Although there is evidence that altering the Th1/Th2 balance toward Th2 cells may be important in the resolution of Th1-type autoimmune disease, adoptive transfer of Th2 cells is not effective in protecting against Th1-type disease and may cause disease. Therefore, we examined the recruitment of Th1- and Th2-like cells into the retina in the murine autoimmune disease experimental autoimmune uveoretinitis. CD4 T cells were polarized in vitro to IFN-gamma-producing Th1-like cells and non-IFN-gamma-producing Th2-like cells, labeled, and adoptively transferred. Trafficking to the retina in vivo was evaluated by scanning laser ophthalmoscopy and infiltration by confocal microscopy. There were more rolling and adherent Th1-like cells and they rolled more slowly than did Th2-like cells. Th1-like cells were preferentially recruited into the retinal parenchyma at both initiation and resolution. Surface P-selectin glycoprotein ligand 1 (PSGL-1) and LFA-1 were up-regulated on both populations but were expressed at higher levels on Th1-like cells. Up-regulation of CD44 expression was higher on Th2-like cells. P-selectin, E-selectin, and ICAM-1 are up-regulated on postcapillary venules in the retina. Pretreatment of Th1-like cells with anti-PSGL-1 inhibited rolling and infiltration of Th1-like cells but not Th2-like cells, providing direct in vivo evidence for the inability of Th2 to respond to P/E-selectin despite increased expression of PSGL-1. Anti-LFA-1 pretreatment inhibited infiltration of both Th1- and Th2-like cells, but more so Th-1. We suggest that random trafficking of activated T cells (both Th1 and Th2) across the blood-retina barrier is mediated by CD44:CD44R and LFA-1:ICAM-1, whereas preferential recruitment of Th1 cells is mediated by PSGL-1:P/E-selectin.

Animals↗

PubMed2003

Reduction in shear stress, activation of the endothelium, and leukocyte priming are all required for leukocyte passage across the blood--retina barrier.

The passage of leukocytes across the blood-retina barrier at the early stages of an inflammatory reaction is influenced by a complex series of interactions about which little is known. In particular, the relationship between hydrodynamic factors, such as shear stress and leukocyte velocity, to the adherence and subsequent extravasation of leukocytes into the retina is unclear. We have used a physiological method, scanning laser ophthalmoscopy, to track labeled leukocytes circulating in the retina, followed by confocal microscopy of retinal flatmounts to detect infiltrating cells at the early stage of experimental autoimmune uveitis. This has shown that retinal vessels are subjected to high shear stress under normal circumstances. During the inflammatory reaction, shear stress in retinal veins is reduced 24 h before leukocyte infiltration. This reduction is negatively correlated with leukocyte rolling and sticking in veins and postcapillary venules, the sites of leukocyte extravasation. Activation of vascular endothelial cells is also a prerequisite for leukocyte rolling and infiltration. In addition, antigen priming of leukocytes is influential at the early stage of inflammation, and this is seen clearly in the reduction in rolling velocity and adherence of the primed leukocytes in activated retinal venules, 9 days postimmunization.

Animals↗

PubMed2003

Effect of anti-macrophage inflammatory protein-1alpha on leukocyte trafficking and disease progression in experimental autoimmune uveoretinitis.

This study has enabled us to identify the influence of the chemokine, macrophage inflammatory protein-1alpha (MIP-1alpha), on leukocyte behavior at the blood-retina barrier in vivo and its link with the inflammatory process and disease pathogenesis. MIP-1alpha has not previously been thought to be effective under conditions of physiological shear flow. However, short-term anti-MIP-1alpha treatment inhibited leukocyte slowing and accumulation and subsequent extravasation of leukocytes at the blood-retina barrier in animals with experimental autoimmune uveoretinitis. This was effective predominantly in the post-capillary venules which have been shown to be the main site of passage of leukocytes across the blood-retina barrier. Long-term anti-MIP-1alpha treatment also prevented decreased leukocyte velocity and reduced disease severity as measured clinically, histologically and in terms of blood-retina barrier breakdown.

Animals↗

PubMed2003

Requirements for passage of T lymphocytes across non-inflamed retinal microvessels.

Although activated T lymphocytes can migrate through unstimulated neural endothelium to perform immune surveillance or initiate inflammation, the precise mechanism by which this occurs is not clear. In this study, we have used intravital scanning laser ophthalmoscopy to show that circulating, activated T cells induce early changes in the retinal venules that enable T cell diapedesis in the absence of cell rolling, and without any reduction in shear stress within the venules. Concanavalin A (Con A)-activated T cells, but not naive T cells, were able to penetrate the normal blood-retinal barrier (BRB) 8-16 h after adoptive transfer. A minimum number (> or =1 x 10(5) cells/mouse) of Con A-activated T cells needed to be transferred before lymphocytes crossed the normal BRB. Cell rolling and reduction of shear stress did not occur in normal retinal venules and post-capillary venules. In contrast, in mice with experimental autoimmune uveoretinitis (EAU), in which the BRB has broken down, 45% of blast cells were rolling in retinal venules. Cell rolling correlated with significantly reduced shear stress. Both naive and Con A-activated T cells could cross the disabled barrier, with Con A-activated T cells migrating faster and in greater numbers than naive cells. Adoptive transfer of Con A-activated cells into normal recipient mice induced limited and transient breakdown of the BRB and up-regulation of ICAM-1 but not P-selectin. Pretreatment of Con A-activated cells with anti-LFA-1 significantly suppressed T cell infiltration in normal recipient mice. Our data indicate that critical to immune surveillance in the central nervous system (CNS) is the ability of activated T cells to interact with the endothelium, up-regulating ICAM-1 and inducing transient breakdown of the barrier.

Adoptive Transfer↗

PubMed2003

Leukocyte trafficking in experimental autoimmune uveitis: breakdown of blood-retinal barrier and upregulation of cellular adhesion molecules.

PURPOSE: To clarify the order of events occurring in the breakdown of the blood-retinal barrier (BRB) in experimental autoimmune uveoretinitis (EAU) and in particular to study the relationships between increased vascular permeability, upregulation of endothelial cell adhesion molecules, and leukocyte adhesion and infiltration during EAU. METHODS: B10.RIII mice were immunized with human interphotoreceptor retinoid binding protein (IRBP) peptide 161-180. Changes in the retinal microvasculature were examined on days 3, 6, 7, 8, 9, 10, 16, and 21 postimmunization (pi). Evans blue dye was administered intravenously to assess vascular permeability. Expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, P-selectin, E-selectin, and platelet endothelial cell adhesion molecule (PECAM)-1 was evaluated by in vivo administration of antibody and subsequent immunostaining of retinal wholemounts. Lymphocytes from inguinal lymph nodes of normal and chicken ovalbumin (OVA)- or IRBP peptide-immunized mice at day 5, 6, 7, 8, and 15 pi were labeled in vitro with calcein-AM (C-AM) and infused intravenously into syngeneic recipient mice, which had been immunized with peptide at the same corresponding time point. Wholemount preparations of retinas were observed 24 hours later by confocal microscopy to determine the adhesion and infiltration of lymphocytes. RESULTS: The first observation of an increase in vascular permeability occurred at day 7 pi and was restricted to focal areas of the retinal postcapillary venules of the inner vascular plexus. This progressively extended to the outer vascular plexus at day 9 pi. Specific adhesion of leukocytes to the endothelium of retinal venules of the inner vascular plexus was first observed at day 6 pi. Leukocyte extravasation into the retinal parenchyma from these vessels began at day 8 pi and extended to the outer vascular plexus at day 9 pi. The expression of adhesion molecules increased progressively during the development of EAU. In particular, the adhesion molecules ICAM-1, P-selectin, and E-selectin were expressed predominately in retinal venules, the sites of BRB breakdown, cell adhesion, and extravasation, from day 7 pi. The increases in expression of ICAM-1 and P-selectin were associated both spatially and temporally with breakdown of the BRB, cell adhesion, and extravasation. No increase in expression of P-selectin and ICAM-1 was observed in either the mesenteric vessels of EAU mice or the retinal vessels of OVA-immunized mice. CONCLUSIONS: The sequence of events in EAU appears to be focal adhesion of leukocytes to discrete sites on postcapillary venules, followed by upregulation of adhesion molecules, especially ICAM-1 and P-selectin, and breakdown of the BRB, leading to transendothelial migration of leukocytes and recruitment of large numbers of cells to the retinal parenchyma. These changes occur over a short period of 6 to 9 days pi and initiate the process of tissue damage during the following 2 to 3 weeks.

Animals↗

PubMed2002

Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72.

We have previously shown that intraarticular treatment with a hyaluronan (HA) preparation (840 kDa), HA84, up-regulates heat shock protein 72 (Hsp72) expression and suppresses degeneration of synovial cells in an arthritis model. In that study, the HA84 administered was degraded into HA oligosaccharides in the synovial tissue, suggesting that HA84 or degradation products of HA may up-regulate Hsp72 expression. Thus, in the present study, we examined the effects of HA of various molecular sizes on Hsp72 expression and cell death in stressed cells. Western blotting analysis showed that treatment of K562 cells with HA tetrasaccharides up-regulated Hsp72 expression after exposure to hyperthermia. On the other hand, treatment of the cells with HA of other sizes (di-, hexa-, deca-, dodecasaccharides), HA84, or tetrasaccharides of keratan sulfate did not elicit any change in expression of the Hsp72 protein. Treatment of the cells with tetrasaccharides of HA up-regulated not only expression of the Hsp72 protein but also Hsp72 mRNA expression and enhanced activation of HSF1, a transcription factor controlling Hsp72 expression, after exposure to hyperthermia. Because the level of Hsp72 protein was not affected by tetrasaccharides of HA when the K562 cells were kept at 37 degrees C without any stress, it is evident that tetrasaccharides of HA did not act as a stress factor. In addition, tetrasaccharides of HA suppressed cell death in the case of K562 cells exposed to hyperthermia and of PC12 cells under serum deprivation. These results suggest that a certain size of oligosaccharides, i.e. the tetrasaccharides of HA, up-regulates Hsp72 expression by enhancing the activation of HSF1 under stress conditions and suppresses cell death.

Adjuvants, Immunologic↗

PubMed2002

Improved leukocyte tracking in mouse retinal and choroidal circulation.

The purpose of this study is to develop a new method with which to visualize leukocyte dynamics in murine choroidal and retinal circulation. Both pigmented (B10.RIII) and non-pigmented (BALB/c) mice were used in this study. One hundred microl of 0.05% sodium fluorescein was injected via the mice tail vein to outline the vessel, followed by 150 microl (10(7) cells) C-AM labelled leukocytes. Fundus images were obtained with a confocal scanning laser ophthalmoscope. The dynamic image sequences were recorded simultaneously on videotape (S-VHS) and digitally at 25 frames per sec. The digital images were later analysed with a custom-made personal computer-based image analysis system. Both the choroidal and retinal circulation can be visualized in non-pigmented mice in the first few seconds of fluorescein angiography. However, the view of the choroidal and the retinal capillary circulation is soon blurred due to the rapid fluorescein leakage in the choroid. In contrast, in pigmented mice, retinal circulation is clear against the dark background of the choroid, while choroidal circulation is masked behind the pigment epithelial layer and cannot be seen at all. C-AM labelled leukocytes were clearly seen in the retinal circulation of all experimental mice and in the choroidal circulation of non-pigmented mice for as long as 30 min. The number of labelled circulating cells decreased as time clasped. Cells moved rapidly in the retinal arteries, slowing down or even stopping for a few seconds in the capillary system, and then moved slightly faster again through the postcapillary venules and veins. In non-pigmented mice, significant number of cells were seen to have arrested in the choroidal circulation. There was no difference between B10.RIII mice and BALB/c mice in vessel diameters, leukocyte velocities and shear stresses. This method allows the visualization of leukocytes and provides data on their behavior as they move through the choroidal and retinal circulation of non-pigmented mice, and in the retinal circulation of pigmented mice. It provides a valuable new tool for the investigation of real time leukocyte dynamics in murine retinal and choroidal microcirculations both under physiological conditions and during the development of ocular disease.

Animals↗

PubMed2002

Secretion of interleukin-10 or interleukin-12 by LPS-activated dendritic cells is critically dependent on time of stimulus relative to initiation of purified DC culture.

Dendritic cells (DC) are key regulators of adaptive immunity with the potential to induce T cell activation/immunity or T cell suppression/tolerance. DC are themselves induced by "maturation" signals such as bacterial lipopolysaccharide (LPS). We demonstrate here that LPS can stimulate DC to display similar maturation phenotypes but to differentiate toward an interleukin (IL)-10(high)- or IL-12(high)-secretor profile depending on the timing of maturation signal induction. Immediate/early administration of LPS induced purified bone marrow-derived DC (BMDC) to differentiate as IL-10(high)IL-12(low)-secreting cells, termed early DC (eDC). Conversely, delayed administration of LPS altered the DC cytokine profile to IL-10(low)IL-12(high), termed later DC (lDC). The presence of IL-4 enhanced the yield and maturation of BMDC but inhibited LPS-induced IL-10 production by eDC. In contrast, interferon-gamma reduced the yield of DC but promoted the level of LPS-induced IL-10 production by lDC. Our data provide new evidence that ex vivo manipulation and the cytokine environment regulate DC maturation status and cytokine-secretor phenotype with implications for the control of T cell differentiation and function via DC-based immunotherapeutic strategies.

Animals↗

PubMed2002

Involvement of CD44 in leukocyte trafficking at the blood-retinal barrier.

In the present study, we investigated the involvement of CD44 in leukocyte trafficking in vivo at the blood-retinal barrier using experimental autoimmune uveoretinitis (EAU) as a model system. Leukocyte trafficking was evaluated using adoptive transfer of calcein-AM (C-AM)-labeled spleen cells harvested from syngeneic mice at prepeak severity of EAU to mice at a similar stage of disease. CD44 and its ligand hyaluronan were up-regulated in the eye during EAU. CD44-positive leukocytes were found sticking in the retinal venules and postcapillary venules but not in the retinal arterioles nor in mesenteric vessels. Preincubation of in vitro C-AM-labeled leukocytes with anti-CD44 monoclonal antibodies (mAb; IM7) or high molecular weight hyaluronic acid (HA) before transfer significantly suppressed leukocyte rolling but not sticking in retinal venules and also reduced cell infiltration in the retinal parenchyma. Administration of the HA-specific enzyme hyaluronidase to mice before cell transfer also reduced leukocyte infiltration, suggesting that CD44-HA interactions are involved in leukocyte recruitment in EAU. This was further supported by the observation that disease severity was reduced by administration of anti-CD44 mAb (IM7) at the early leukocyte-infiltration stage. Further studies also indicated that CD44 activation was associated with increased levels of apoptosis, and this may also be in part responsible for the reduction in disease severity. These findings demonstrate that CD44 is directly involved in leukocyte-endothelial interaction in vivo and influence the trafficking of primed leukocytes to the retina and their overall survival.

Animals↗

PubMedDate not supplied

Imaging ocular immune responses by intravital microscopy.

The eye offers excellent opportunities to observe cellular interactions in vivo. This applies especially to the immune response in which discrete events can be studied, including cell trafficking, transendothelial migration, adhesion, antigen presentation, and T cell activation. Intravital microscopy has allowed study of immune cell interactions in tissues such as the conjunctiva, the inflamed cornea, and the iris. Thus the realtime observation of presentation of antigen injected into the anterior chamber of the eye can be imaged using fluorescently labelled antigen and cells. Application of the scanning laser ophthalmoscope to the rat and mouse eye allows analysis of leukocyte-endothelial interactions in the retinal and choroidal circulations. These studies have provided important information on rolling and adhesion of leukocytes in real time in different microvascular beds that have not been manipulated in any way and has, for instance, provided quantitation to the effects of shear stress on leukocyte-endothelial adhesion. In addition, the model permits an accurate analysis of the timing of trafficking of T cells into the eye and the possibility of determining which cells, if any, may be responsible for antigen presentation in the tissues as opposed to the secondary lymphoid organs. Finally, these experimental methods are now being applied to the human eye and should prove valuable in determining the nature of tissue damage events in the eye as well as evaluating the response to treatments.

Animals↗

PubMedDate not supplied

Investigation of light propagation models to determine the optical properties of tissue from interstitial frequency domain fluence measurements.

Four models, standard diffusion approximation (SDA), single Monte Carlo (SMC), delta-P1, and isotropic similarity (ISM), are developed and evaluated as forward calculation tools in the estimation of tissue optical properties. The inverse calculation uses the ratio of the fluences and phase difference at two locations close to an intensity modulated isotropic source to recover the reduced scattering coefficient mus' and the absorption coefficient mua. Diffusion theory allows recovery of optical properties (OPs) within 5% for media with mus'mua>10. The performance of the delta-P1 model is similar to SDA, with limited enhanced accuracy. The collimation approximation may limit the use of the delta-P1 model for spherical geometry, and/or the fluence may not be accurately calculated by this model. The SMC model is the best, recovering OPs within 10% regardless of the albedo. However, the necessary restriction of the searched OPs space is inconvenient. The performance of ISM is similar to that of diffusion theory for media with mus'mua>10, and better for 1 5.

Algorithms↗