Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.

Transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent CCl4-treated mice

Chronic liver injury and inflammation lead to hepatic fibrosis, cirrhosis, and liver failure. Embryonic and mesenchymal stem cells have been shown to reduce experimental liver fibrosis but have potential limitations, including the formation of dysplastic precursors, tumors, and profibrogenic cells. Other stem-like cells may reduce hepatic inflammation and fibrosis without tumor and profibrogenic cell formation. To test this hypothesis we transplanted human amnion epithelial cells (hAEC), isolated from term delivered placenta, into immunocompetent C57/BL6 mice at week 2 of a 4-week regimen of carbon tetrachloride (CCl4) exposure to induce liver fibrosis. Two weeks following hAEC infusion, intact cells expressing the human-specific markers inner mitochondrial membrane protein and human leukocyte antigen-G were found in mouse liver without evidence of host rejection of the transplanted cells. Human albumin, known to be produced by hAEC, was detected in sera of hAEC-treated mice. Human DNA was detected in mouse liver and also spleen, lungs, and heart of some animals. Following hAEC transplantation, CCl4-treated animals showed decreased serum ALT levels and reduced hepatocyte apoptosis, compared to controls. hAEC-treated mouse liver had lower TNF-α and IL-6 protein levels and higher IL-10 compared to animals given CCl4 alone. Compared to CCl4 controls, hAEC-treated mice showed fewer activated collagen-producing hepatic stellate cells and less fibrosis area and collagen content. Reduced hepatic TGF-β levels in conjunction with a twofold increase in the active form of the collagen-degrading enzyme matrix metalloproteinase-2 in hAEC-treated mice compared to CCl4 controls may account for the reduction in fibrosis. hAEC transplantation into immunocompetent mice leads to cell engraftment, reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis.

PSMA-targeting iron oxide magnetic nanoparticles enhance MRI of preclinical prostate cancer

Aim Evaluate potential of newly-developed, biocompatible iron oxide magnetic nanoparticles (MNPs) conjugated with J591, an antibody to an extracellular epitope of prostate specific membrane antigen (PSMA), to enhance MRI of prostate cancer (PCa). Materials & Methods Specific binding to PSMA by J591-MNP was investigated in vitro. MRI studies were performed on orthotopic tumor-bearing NOD.SCID mice 2h and 24hr after intravenous injection of J591-MNPs, or non-targeting MNPs. Results and Conclusions In vitro, MNPs did not affect PCa cell viability, and conjugation to J591 did not compromise antibody specificity and enhanced cellular iron uptake. In vivo, PSMA-targeting MNPs increased MR contrast of tumors, but not by non-targeting MNPs. This provides proof-of-concept that PSMA-targeting MNPs have potential to enhance MR detection/localization of PCa.,

Enhancing MRI of prostate cancer using PSMA-targeting iron oxide magnetic nanoparticles

Introduction Novel imaging techniques for prostate cancer (PCa) are required to improve staging and real-time assessment of therapeutic response. We performed preclinical evaluation of newly-developed, biocompatible magnetic nanoparticles (MNPs) conjugated with J591, an antibody specific for prostate specific membrane antigen (PSMA), to enhance magnetic resonance imaging (MRI) of PCa. PSMA is expressed on ∼90% of PCa, including those that are castrate-resistant, rendering it as a rational target for PCa imaging. Materials and Methods The specificity of J591 for PSMA was confirmed by flow cytometric analysis of several PCa cell lines of known PSMA status. MNPs were prepared, engineered to the appropriate size, labeled with DiR fluorophore, and their toxicity to a panel of PC cells was assessed by in vitro Alamar Blue assay. Immunohistochemistry, fluorescence microscopy and Prussian Blue staining (iron uptake) were used to evaluate PSMA specificity of J591-MNP conjugates. In vivo MRI studies (16.4T MRI system) were performed using live immunodeficient mice bearing orthotopic LNCaP xenografts and injected intravenously with J591-MNPs or MNPs alone. Results MNPs were non-toxic to PCa cells. J591-MNP conjugates showed no compromise in specificity of binding to PSMA+ cells and showed enhanced iron uptake compared with MNPs alone. In vivo, tumour targeting (significant MR image contrast) was evident in mice injected with J591-MNPs, but not MNPs alone. Resected tumours from targeted mice had an accumulation of MNPs, not seen in normal control prostate. Conclusions Application of PSMA-targeting MNPs into conventional MRI has potential to enhance PCa detection and localization in real-time, improving patient management.

Establishing prostate cancer patient derived xenografts: Lessons learned from older studies

Background Understanding the progression of prostate cancer to androgen-independence/castrate resistance and development of preclinical testing models are important for developing new prostate cancer therapies. This report describes studies performed 30 years ago, which demonstrate utility and shortfalls of xenografting to preclinical modeling. Methods We subcutaneously implanted male nude mice with small prostate cancer fragments from transurethral resection of the prostate (TURP) from 29 patients. Successful xenografts were passaged into new host mice. They were characterized using histology, immunohistochemistry for marker expression, flow cytometry for ploidy status, and in some cases by electron microscopy and response to testosterone. Two xenografts were karyotyped by G-banding. Results Tissues from 3/29 donors (10%) gave rise to xenografts that were successfully serially passaged in vivo. Two, (UCRU-PR-1, which subsequently was replaced by a mouse fibrosarcoma, and UCRU-PR-2, which combined epithelial and neuroendocrine features) have been described. UCRU-PR-4 line was a poorly differentiated prostatic adenocarcinoma derived from a patient who had undergone estrogen therapy and bilateral castration after his cancer relapsed. Histologically, this comprised diffusely infiltrating small acinar cell carcinoma with more solid aggregates of poorly differentiated adenocarcinoma. The xenografted line showed histology consistent with a poorly differentiated adenocarcinoma and stained positively for prostatic acid phosphatase (PAcP), epithelial membrane antigen (EMA) and the cytokeratin cocktail, CAM5.2, with weak staining for prostate specific antigen (PSA). The line failed to grow in female nude mice. Castration of three male nude mice after xenograft establishment resulted in cessation of growth in one, growth regression in another and transient growth in another, suggesting that some cells had retained androgen sensitivity. The karyotype (from passage 1) was 43–46, XY, dic(1;12)(p11;p11), der(3)t(3:?5)(q13;q13), -5, inv(7)(p15q35) x2, +add(7)(p13), add(8)(p22), add(11)(p14), add(13)(p11), add(20)(p12), -22, +r4[cp8]. Conclusions Xenografts provide a clinically relevant model of prostate cancer, although establishing serially transplantable prostate cancer patient derived xenografts is challenging and requires rigorous characterization and high quality starting material. Xenografting from advanced prostate cancer is more likely to succeed, as xenografting from well differentiated, localized disease has not been achieved in our experience. Strong translational correlations can be demonstrated between the clinical disease state and the xenograft model

N,N'-Carbonyldiimidazole-mediated functionalization of superparamagnetic nanoparticles as vaccine carrier

Particulates with specific sizes and characteristics can induce potent immune responses by promoting antigen uptake of appropriate immuno-stimulatory cell types. Magnetite (Fe3O4) nanoparticles have shown many potential bioapplications due to their biocompatibility and special characteristics. Here, superparamagnetic Fe3O4 nanoparticles (SPIONs) with high magnetization value (70emug-1) were stabilized with trisodium citrate and successfully conjugated with a model antigen (ovalbumin, OVA) via N,N'-carbonyldiimidazole (CDI) mediated reaction, to achieve a maximum conjugation capacity at approximately 13μgμm-2. It was shown that different mechanisms governed the interactions between the OVA molecules and magnetite nanoparticles at different pH conditions. We evaluated as-synthesized SPION against commercially available magnetite nanoparticles. The cytotoxicity of these nanoparticles was investigated using mammalian cells. The reported CDI-mediated reaction can be considered as a potential approach in conjugating biomolecules onto magnetite or other biodegradable nanoparticles for vaccine delivery.

Microparticle-mediated gene delivery for the enhanced expression of a 19-KDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP119) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP119 is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h1. High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 lm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA-mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.

Process considerations related to the microencapsulation of plasmid DNA via ultrasonic atomization

An effective means of facilitating DNA vaccine delivery to antigen presenting cells is through biodegradable microspheres. Microspheres offer distinct advantages over other delivery technologies by providing release of DNA vaccine in its bioactive form in a controlled fashion. In this study, biodegradable poly(D,L-lactide-coglycolide) (PLGA) microspheres containing polyethylenimine (PEI) condensed plasmid DNA (pDNA) were prepared using a 40 kHz ultrasonic atomization system. Process synthesis parameters, which are important to the scale-up of microspheres that are suitable for nasal delivery (i.e., less than 20 μm), were studied. These parameters include polymer concentration; feed flowrate; volumetric ratio of polymer and pDNA-PEI (plasmid DNA-polyethylenimine) complexes; and nitrogen to phosphorous (N/P) ratio. PDNA encapsulation efficiencies were predominantly in the range 82-96%, and the mean sizes of the particle were between 6 and 15 μm. The ultrasonic synthesis method was shown to have excellent reproducibility. PEI affected morphology of the microspheres, as it induced the formation of porous particles that accelerate the release rate of pDNA. The PLGA microspheres displayed an in vitro release of pDNA of 95-99% within 30 days and demonstrated zero order release kinetics without an initial spike of pDNA. Agarose electrophoresis confirmed conservation of the supercoiled form of pDNA throughout the synthesis and in vitro release stages. It was concluded that ultrasonic atomization is an efficient technique to overcome the key obstacles in scaling-up the manufacture of encapsulated vaccine for clinical trials and ultimately, commercial applications.

Rapid production of a plasmid DNA encoding a malaria vaccine candidate via amino-functionalized poly(GMA-co-EDMA) monolith

Malaria is a global health problem; an effective vaccine is urgently needed. Due to the relative poverty and lack of infrastructure in malaria endemic areas, DNA-based vaccines that are stable at ambient temperatures and easy to formulate have great potential. While attention has been focused mainly on antigen selection, vector design and efficacy assessment, the development of a rapid and commercially viable process to manufacture DNA is generally overlooked. We report here a continuous purification technique employing an optimized stationary adsorbent to allow high-vaccine recovery, low-processing time, and, hence, high-productivity. A 40.0 mL monolithic stationary phase was synthesized and functionalized with amino groups from 2-Chloro-N,N- diethylethylamine hydrochloride for anion-exchange isolation of a plasmid DNA (pDNA) that encodes a malaria vaccine candidate, VR1020-PyMSP4/5. Physical characterization of the monolithic polymer showed a macroporous material with a modal pore diameter of 750 nm. The final vaccine product isolated after 3 min elution was homogeneous supercoiled plasmid with gDNA, RNA and protein levels in keeping with clinical regulatory standards. Toxicological studies of the pVR1020-PyMSP4/5 showed a minimum endotoxin level of 0.28 EU/m.g pDNA. This cost-effective technique is cGMP compatible and highly scalable for the production of DNA-based vaccines in commercial quantities, when such vaccines prove to be effective against malaria. © 2008 American Institute of Chemical Engineers.

Inorganic-organic composite particle as a staged delivery platform for plasmid DNA-based biopharmaceuticals

Infectious diseases such as SARS, influenza and bird flu may spread exponentially throughout communities. In fact, most infectious diseases remain major health risks due to the lack of vaccine or the lack of facilities to deliver the vaccines. Conventional vaccinations are based on damaged pathogens, live attenuated viruses and viral vectors. If the damage was not complete, the vaccination itself may cause adverse effects. Therefore, researchers have been prompted to prepare viable replacements for the attenuated vaccines that would be more effective and safer to use. DNA vaccines are generally composed of a double stranded plasmid that includes a gene encoding the target antigen under the transcriptional directory and control of a promoter region which is active in cells. Plasmid DNA (pDNA) vaccines allow the foreign genes to be expressed transiently in cells, mimicking intracellular pathogenic infection and inducing both humoral and cellular immune responses. Currently, because of their highly evolved and specialized components, viral systems are the most effective means for DNA delivery, and they achieve high efficiencies (generally >90%), for both DNA delivery and expression. As yet, viral-mediated deliveries have several limitations, including toxicity, limited DNA carrying capacity, restricted target to specific cell types, production and packing problems, and high cost. Thus, nonviral systems, particularly a synthetic DNA delivery system, are highly desirable in both research and clinical applications.

Autologous human kidney proximal tubule epithelial cells (PTEC) modulate dendritic cell (DC), T cell and B cell responses

This is a comprehensive study of human kidney proximal tubular epithelial cells (PTEC) which are known to respond to and mediate the pathological process of a range of kidney diseases. It identifies various molecules expressed by PTEC and how these molecules participate in down-regulating the inflammatory process, thereby highlighting the clinical potential of these molecules to treat various kidney diseases. In the disease state, PTEC gain the ability to regulate the immune cell responses present within the interstitium. This down-regulation is a complex interaction of contact dependent/independent mechanisms involving various immuno-regulatory molecules including PD-L1, sHLA-G and IDO. The overall outcome of this down-regulation is suppressed DC maturation, decreased number of antibody producing B cells and low T cell responses. These manifestations within a clinical setting are expected to dampen the ongoing inflammation, preventing the damage caused to the kidney tissue.

Pan-Plasmodium band sensitivity for Plasmodium falciparum detection in combination malaria rapid diagnostic tests and implications for clinical management

Background: Malaria rapid diagnostic tests (RDTs) are appropriate for case management, but persistent antigenaemia is a concern for HRP2-detecting RDTs in endemic areas. It has been suggested that pan-pLDH test bands on combination RDTs could be used to distinguish persistent antigenaemia from active Plasmodium falciparum infection, however this assumes all active infections produce positive results on both bands of RDTs, an assertion that has not been demonstrated. Methods: In this study, data generated during the WHO-FIND product testing programme for malaria RDTs was reviewed to investigate the reactivity of individual test bands against P. falciparum in 18 combination RDTs. Each product was tested against multiple wild-type P. falciparum only samples. Antigen levels were measured by quantitative ELISA for HRP2, pLDH and aldolase. Results: When tested against P. falciparum samples at 200 parasites/μL, 92% of RDTs were positive; 57% of these on both the P. falciparum and pan bands, while 43% were positive on the P. falciparum band only. There was a relationship between antigen concentration and band positivity; ≥4 ng/mL of HRP2 produced positive results in more than 95% of P. falciparum bands, while ≥45 ng/mL of pLDH was required for at least 90% of pan bands to be positive. Conclusions: In active P. falciparum infections it is common for combination RDTs to return a positive HRP2 band combined with a negative pan-pLDH band, and when both bands are positive, often the pan band is faint. Thus active infections could be missed if the presence of a HRP2 band in the absence of a pan band is interpreted as being caused solely by persistent antigenaemia.

Evaluation of intra- and extra-epithelial secretory IgA in chlamydial infections

IgA is an important mucosal antibody that can neutralize mucosal pathogens by either preventing attachment to epithelia (immune exclusion) or alternatively inhibit intraepithelial replication following transcytosis by the polymeric immunoglobulin receptor (pIgR). Chlamydia trachomatis is a major human pathogen that initially targets the endocervical or urethral epithelium in women and men, respectively. As both tissues contain abundant SIgA we assessed the protection afforded by IgA targeting different chlamydial antigens expressed during the extra and intraepithelial stages of infection. We developed an in vitro model utilizing polarizing cells expressing the murine pIgR together with antigen-specific mouse IgA, and an in vivo model utilizing pIgR-/- mice. SIgA targeting the extraepithelial chlamydial antigen, the major outer membrane protein (MOMP), significantly reduced infection in vitro by 24 % and in vivo by 44 %. Conversely, pIgR-mediated delivery of IgA targeting the intraepithelial inclusion membrane protein A (IncA) bound to the inclusion but did not reduce infection in vitro or in vivo. Similarly, intraepithelial IgA targeting the secreted protease Chlamydia protease-like activity factor (CPAF) also failed to reduce infection. Together, these data suggest the importance of pIgR-mediated delivery of IgA targeting extra but not intraepithelial chlamydial antigens for protection against a genital tract infection.

Visualization of complex biological systems: An immune response model using OpenGL

In this paper we present an update on our novel visualization technologies based on cellular immune interaction from both large-scale spatial and temporal perspectives. We do so with a primary motive: to present a visually and behaviourally realistic environment to the community of experimental biologists and physicians such that their knowledge and expertise may be more readily integrated into the model creation and calibration process. Visualization aids understanding as we rely on visual perception to make crucial decisions. For example, with our initial model, we can visualize the dynamics of an idealized lymphatic compartment, with antigen presenting cells (APC) and cytotoxic T lymphocyte (CTL) cells. The visualization technology presented here offers the researcher the ability to start, pause, zoom-in, zoom-out and navigate in 3-dimensions through an idealised lymphatic compartment.

Variation in the complex carbohydrate biosynthesis loci of Acinetobacter baumannii genomes

Extracellular polysaccharides are major immunogenic components of the bacterial cell envelope. However, little is known about their biosynthesis in the genus Acinetobacter, which includes A. baumannii, an important nosocomial pathogen. Whether Acinetobacter sp. produce a capsule or a lipopolysaccharide carrying an O antigen or both is not resolved. To explore these issues, genes involved in the synthesis of complex polysaccharides were located in 10 complete A. baumannii genome sequences, and the function of each of their products was predicted via comparison to enzymes with a known function. The absence of a gene encoding a WaaL ligase, required to link the carbohydrate polymer to the lipid A-core oligosaccharide (lipooligosaccharide) forming lipopolysaccharide, suggests that only a capsule is produced. Nine distinct arrangements of a large capsule biosynthesis locus, designated KL1 to KL9, were found in the genomes. Three forms of a second, smaller variable locus, likely to be required for synthesis of the outer core of the lipid A-core moiety, were designated OCL1 to OCL3 and also annotated. Each K locus includes genes for capsule export as well as genes for synthesis of activated sugar precursors, and for glycosyltransfer, glycan modification and oligosaccharide repeat-unit processing. The K loci all include the export genes at one end and genes for synthesis of common sugar precursors at the other, with a highly variable region that includes the remaining genes in between. Five different capsule loci, KL2, KL6, KL7, KL8 and KL9 were detected in multiply antibiotic resistant isolates belonging to global clone 2, and two other loci, KL1 and KL4, in global clone 1. This indicates that this region is being substituted repeatedly in multiply antibiotic resistant isolates from these clones.