Kinetics of ocular and systemic antigen-specific T-cell responses elicited during murine cytomegalovirus retinitis

Cytomegalovirus (CMV) reactivation in the retina of immunocompromized patients is a cause of significant morbidity as it can lead to blindness. The adaptive immune response is critical in controlling murine CMV (MCMV) infection in MCMV-susceptible mouse strains. CD8(+) T cells limit systemic viral replication in the acute phase of infection and are essential to contain latent virus. In this study, we provide the first evaluation of the kinetics of anti-viral T-cell responses after subretinal infection with MCMV. The acute response was characterized by a rapid expansion phase, with infiltration of CD8(+) T cells into the infected retina, followed by a contraction phase. MCMV-specific T cells displayed biphasic kinetics with a first peak at day 12 and contraction by day 18 followed by sustained recruitment of these cells into the retina at later time points post-infection. MCMV-specific CD8(+) T cells were also observed in the draining cervical lymph nodes and the spleen. Presentation of viral epitopes and activation of CD8(+) T cells was widespread and could be detected in the spleen and the draining lymph nodes, but not in the retina or iris. Moreover, after intraocular infection, antigen-specific cytotoxic activity was detectable and exhibited kinetics equivalent to those observed after intraperitoneal infection with the same viral dose. These data provide novel insights of how and where immune responses are initiated when viral antigen is present in the subretinal space.

Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors

Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

Long-term cell-mediated protein release from calcium phosphate ceramics

Efficient delivery of growth factors from carrier biomaterials depends critically on the release kinetics of the proteins that constitute the carrier. Immobilizing growth factors to calcium phosphate ceramics has been attempted by direct adsorption and usually resulted in a rapid and passive release of the superficially adherent proteins. The insufficient retention of growth factors limited their bioavailability and their efficacy in the treatment of bone regeneration. In this study, a coprecipitation technique of proteins and calcium phosphate was employed to modify the delivery of proteins from biphasic calcium phosphate (BCP) ceramics. To this end, tritium-labeled bovine serum albumin ([(3)H]BSA) was utilized as a model protein to analyze the coprecipitation efficacy and the release kinetics of the protein from the carrier material. Conventional adsorption of [(3)H]BSA resulted in a rapid and passive release of the protein from BCP ceramics, whereas the coprecipitation technique effectively prevented the burst release of [(3)H]BSA. Further analysis of the in vitro kinetics demonstrated a sustained, cell-mediated release of coprecipitated [(3)H]BSA from BCP ceramics induced by resorbing osteoclasts. The coprecipitation technique described herein, achieved a physiologic-like protein release, by incorporating [(3)H]BSA into its respective carriers, rendering it a promising tool in growth factor delivery for bone healing.

Kinetic assessment of persistent halogenated xenobiotics in cell culture models: comparison of mono- and poly-halogenated compounds

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3h to (14)C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 microM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (=low accumulation compounds). DBP and DDE showed much higher cellular accumulations (=high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.

The impact of different nanoparticle surface chemistry and size on uptake and toxicity in a murine macrophage cell line

This study investigated the uptake, kinetics and cellular distribution of different surface coated quantum dots (QDs) before relating this to their toxicity. J774.A1 cells were treated with organic, COOH and NH2 (PEG) surface coated QDs (40 nM). Model 20 nm and 200 nm COOH-modified coated polystyrene beads (PBs) were also examined (50 microg ml(-1)). The potential for uptake of QDs was examined by both fixed and live cell confocal microscopy as well as by flow cytometry over 2 h. Both the COOH 20 nm and 200 nm PBs were clearly and rapidly taken up by the J774.A1 cells, with uptake of 20 nm PBs being relatively quicker and more extensive. Similarly, COOH QDs were clearly taken up by the macrophages. Uptake of NH2 (PEG) QDs was not detectable by live cell imaging however, was observed following 3D reconstruction of fixed cells, as well as by flow cytometry. Cells treated with organic QDs, monitored by live cell imaging, showed only a small amount of uptake in a relatively small number of cells. This uptake was insufficient to be detected by flow cytometry. Imaging of fixed cells was not possible due to a loss in cell integrity related to cytotoxicity. A significant reduction (p<0.05) in the fluorescent intensity in a cell-free environment was found with organic QDs, NH2 (PEG) QDs, 20 nm and 200 nm PBs at pH 4.0 (indicative of an endosome) after 2 h, suggesting reduced stability. No evidence of exocytosis was found over 2 h. These findings confirm that surface coating has a significant influence on the mode of NP interaction with cells, as well as the subsequent consequences of that interaction.

Red blood cell lifespan, erythropoiesis and hemoglobin control

Erythropoietin (EPO) and iron deficiency as causes of anemia in patients with limited renal function or end-stage renal disease are well addressed. The concomitant impairment of red blood cell (RBC) survival has been largely neglected. Properties of the uremic environment like inflammation, increased oxidative stress and uremic toxins seem to be responsible for the premature changes in RBC membrane and cytoskeleton. The exposure of antigenic sites and breakdown of the phosphatidylserine asymmetry promote RBC phagocytosis. While the individual response to treatment with EPO-stimulating agents (ESA) depends on both the RBC's lifespan and the production rate, uniform dosing algorithms do not meet that demand. The clinical use of mathematical models predicting ESA-induced changes in hematocrit might be greatly improved once independent estimates of RBC production rate and/or lifespan become available, thus making the concomitant estimation of both parameters unnecessary. Since heme breakdown by the hemoxygenase pathway results in carbon monoxide (CO) which is exhaled, a simple CO breath test has been used to calculate hemoglobin turnover and therefore RBC survival and lifespan. Future research will have to be done to validate and implement this method in patients with kidney failure. This will result in new insights into RBC kinetics in renal patients. Eventually, these findings are expected to improve our understanding of the hemoglobin variability in response to ESA.

Accelerated hypertrophic chondrocyte kinetics in GDF-7 deficient murine tibial growth plates

The Growth/Differentiation Factors (GDFs) are a subgroup of the Bone Morphogenetic Proteins (BMPs) well known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling molecules, GDF-5, have recently been shown to exhibit a decreased rate of endochondral bone growth in the proximal tibia due to a significantly longer hypertrophic phase duration. GDF-7 is a related family member, which exhibits a high degree of sequence identity with GDF-5. The purpose of the present study was to determine whether GDF-7 deficiency also alters the endochondral bone growth rate in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5-week-old female GDF-7 -/- mice and wild type control littermates were examined. GDF-7 deficiency resulted in a statistically significant increase in growth rate (+26%; p = 0.0084) and rate of cell loss at the chondrosseous junction (+25%; p = 0.0217). Cells from GDF-7 deficient mice also exhibited a significantly shorter hypertrophic phase duration compared to wild type controls (-27%; p = 0.0326). These data demonstrate that, in the absence of GDF-7, the rate of endochondral bone growth is affected through the modulation of hypertrophic phase duration in growth plate chondrocytes. These findings further support a growing body of evidence implicating the GDFs in the formation, maturation, and maintenance of healthy cartilage.

Altered hypertrophic chondrocyte kinetics in GDF-5 deficient murine tibial growth plates

The growth/differentiation factors (GDFs) are a subgroup of the bone morphogenetic proteins best known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling proteins, GDF-5, exhibit numerous skeletal abnormalities, including shortened limb bones. The primary aim of this study was determine whether GDF-5 deficiency would alter the growth rate in growth plates from the long bones in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5-week-old female GDF-5 -/- mice and control littermates were examined. GDF-5 deficiency resulted in a statistically significant reduction in growth rate (-14%, p=0.03). The effect of genotype on growth rate was associated with an altered hypertrophic phase duration, with hypertrophic cells from GDF-5 deficient mice exhibiting a significantly longer phase duration compared to control littermates (+25%, p=0.006). These data suggest that one way in which GDF-5 might modulate the rate of endochondral bone growth could be by affecting the duration of the hypertrophic phase in growth plate chondrocytes.

Class-switzched B cells display response to therapeutic B-cell depletion in rheumatoid arthritis

Introduction Reconstitution of peripheral blood (PB) B cells after therapeutic depletion with the chimeric anti-CD20 antibody rituximab (RTX) mimics lymphatic ontogeny. In this situation, the repletion kinetics and migratory properties of distinct developmental B-cell stages and their correlation to disease activity might facilitate our understanding of innate and adaptive B-cell functions in rheumatoid arthritis (RA). Methods Thirty-five 'RTX-naïve' RA patients with active arthritis were treated after failure of tumour necrosis factor blockade in an open-label study with two infusions of 1,000 mg RTX. Prednisone dose was tapered according to clinical improvement from a median of 10 mg at baseline to 5 mg at 9 and 12 months. Conventional disease-modifying antirheumatic drugs were kept stable. Subsets of CD19+ B cells were assessed by flow cytometry according to their IgD and CD27 surface expression. Their absolute number and relative frequency in PB were followed every 3 months and were determined in parallel in synovial tissue (n = 3) or synovial fluid (n = 3) in the case of florid arthritis. Results Six of 35 patients fulfilled the European League Against Rheumatism criteria for moderate clinical response, and 19 others for good clinical response. All PB B-cell fractions decreased significantly in number (P < 0.001) after the first infusion. Disease activity developed independently of the total B-cell number. B-cell repopulation was dominated in quantity by CD27-IgD+ 'naïve' B cells. The low number of CD27+IgD- class-switched memory B cells (MemB) in the blood, together with sustained reduction of rheumatoid factor serum concentrations, correlated with good clinical response. Class-switched MemB were found accumulated in flaring joints. Conclusions The present data support the hypothesis that control of adaptive immune processes involving germinal centre-derived, antigen, and T-cell-dependently matured B cells is essential for successful RTX treatment.

The kinetics of feline leukaemia virus shedding in experimentally infected cats are associated with infection outcome

Feline leukaemia virus (FeLV) infection in felids results mainly from oronasal exposure to infectious saliva and nasal secretions, but the potential for viral transmission through faeces and urine has not been completely characterized. In order to assess and compare potential FeLV transmission routes, we determined the viral kinetics in plasma, saliva, faeces and urine during early experimental FeLV infection (up to week 15 post-exposure) in specific pathogen-free cats. In addition to monitoring p27 antigen levels measured by ELISA, we evaluated the presence of infectious particles by cell culture assays and quantified viral RNA loads by a quantitative real-time TaqMan polymerase chain reaction. RNA load was associated with infection outcome (high load-progressive infection; low load-regressive infection) not only in plasma, but also in saliva, faeces and urine. Infectious virus was isolated from the saliva, faeces and urine of infected cats with progressive infection as early as 3-6 weeks post-infection, but usually not in cats with regressive infection. In cats with progressive infection, therefore, not only saliva but also faeces and to some extent urine might represent potential FeLV transmission routes. These results should be taken into account when modelling FeLV-host interactions and assessing FeLV transmission risk. Moreover, during early FeLV infection, detection of viral RNA in saliva may be used as an indicator of recent virus exposure, even in cats without detectable antigenaemia/viraemia. To determine the clinically relevant outcome of FeLV infection in exposed cats, however, p27 antigen levels in the peripheral blood should be measured.

Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra.

Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe(2+) transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe(2+) or Cd(2+) influx could be reliably monitored in calcium 5-loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K0.5 value of 1.4 µM and 3.5 µM for Fe(2+) and Cd(2+), respectively. In addition, Cd(2+) was used as a substrate for DMT1, and we find a Ki value of 2.1 µM for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z' factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation.

INTRODUCTION Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. METHODS Bone marrow-derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4(+) T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. RESULTS The frequency of PS particle-positive CD11c(+)/CD11b(+) BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4(+) T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. CONCLUSION These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4(+) T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.

Persistent and compartmentalised disruption of dendritic cell subpopulations in the lung following influenza A virus infection.

Immunological homeostasis in the respiratory tract is thought to require balanced interactions between networks of dendritic cell (DC) subsets in lung microenvironments in order to regulate tolerance or immunity to inhaled antigens and pathogens. Influenza A virus (IAV) poses a serious threat of long-term disruption to this balance through its potent pro-inflammatory activities. In this study, we have used a BALB/c mouse model of A/PR8/34 H1N1 Influenza Type A Virus infection to examine the effects of IAV on respiratory tissue DC subsets during the recovery phase following clearance of the virus. In adult mice, we found differences in the kinetics and activation states of DC residing in the airway mucosa (AMDC) compared to those in the parenchymal lung (PLDC) compartments. A significant depletion in the percentage of AMDC was observed at day 4 post-infection that was associated with a change in steady-state CD11b+ and CD11b- AMDC subset frequencies and significantly elevated CD40 and CD80 expression and that returned to baseline by day 14 post-infection. In contrast, percentages and total numbers of PLDC were significantly elevated at day 14 and remained so until day 21 post-infection. Accompanying this was a change in CD11b+and CD11b- PLDC subset frequencies and significant increase in CD40 and CD80 expression at these time points. Furthermore, mice infected with IAV at 4 weeks of age showed a significant increase in total numbers of PLDC, and increased CD40 expression on both AMDC and PLDC, when analysed as adults 35 days later. These data suggest that the rate of recovery of DC populations following IAV infection differs in the mucosal and parenchymal compartments of the lung and that DC populations can remain disrupted and activated for a prolonged period following viral clearance, into adulthood if infection occurred early in life.

Lymph node stromal cells negatively regulate antigen-specific CD4+ T cell responses

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.