Co-localization Of Antigens Research Articles

Double Chromogen-based Immunohistochemical Staining: An Efficient Approach for Utilizing Long-term Formalin-fixed Tissue in Biobanks.

The New South Wales Brain Tissue Resource Centre is a human brain bank that provides top-quality brain tissue for cutting-edge neuroscience research spanning various conditions from alcohol use disorder to neurodegenerative diseases. However, the conventional practice of preserving brain tissue in formalin poses challenges for immunofluorescent staining primarily due to the formalin's tendency, over time, to create cross-links between antigens, which can obscure epitopes of interest. In addition, researchers can encounter issues such as spectral bleeding, limitations in using multiple colors, autofluorescence, and cross-reactivity when working with long-term formalin-fixed brain tissue. The purpose of the study was to test chromogen-based double immunolabeling to negate the issues with immunofluorescent staining. Colocalization of antigens was explored using chromogens 3-amino-9-ethylcarbazole (AEC) and 3,3,-diaminobenzidine in a sequential staining procedure where the AEC signal was eliminated by alcohol treatment. Combinations of 2 or 3 primary antibodies from the same or different species were trialed successfully with this protocol. The colocalization of antigens was also demonstrated with pseudocoloring that mimicked immunofluorescence staining. This staining technique increases the utility of archival formalin-fixed tissue samples.

Modeling the kinetics of lymph node retention and exposure of a cargo protein delivered by biotin-functionalized nanoparticles

Generation of protective immunity through vaccination arises from the adaptive immune response developed primarily in the lymph nodes drained from the immunization site. Relative to the intramuscular route, subcutaneous administration allows for direct and rapid access to the lymphatics, but accumulation of soluble protein antigens within the lymph nodes is limited. Subunit vaccines also require immune stimulating adjuvants which may not accumulate in the same lymph nodes simultaneously with antigen. Herein we report the use of biotinylated poly (lactic-co-glycolic acid) nanoparticles (bNPs) to enhance delivery of a model protein antigen to the lymphatics. bNPs provide dual functionality as adjuvant and vehicle to localize antigens with stimulated immune cells in the same draining lymph node. Using streptavidin as a model antigen, which can be loaded directly onto the bNP surface, we evaluated the kinetics of lymph node occupancy and adaptive immune responses in wildtype C57BL/6 mice. Antigen exposure in vivo was significantly improved through surface loading onto bNPs, and we developed a working kinetic model to account for the retention of both particles and antigen in draining lymph nodes. We observed enhanced T cell responses and antigen-specific B cell response in vivo when antigen was delivered on the particle surface. This work highlights the advantage of combining intrinsic adjuvant and antigen loading in a single entity, and the utility of kinetic modeling in the understanding of particle-based vaccines. Statement of significanceDevelopment of safe and effective subunit vaccines depends on effective formulations that render optimized exposure and colocalization of antigens and adjuvants. In this work, we utilize a nanoparticle system which features self-adjuvanting properties and allows for surface loading of recombinant protein antigens. Using in vivo imaging, we demonstrated prolonged co-localization of the antigen and adjuvant particles in draining lymph nodes and provided evidence of B cell activation for up to 21 days following subcutaneous injection. A pharmacokinetic model was developed as a step towards bridging the translational gap between particulate-based vaccines and observed outcomes. The results have implications for the rational design of particle-based vaccines.

Zika virus induced microcephaly and aberrant hematopoietic cell differentiation modeled in novel neonatal humanized mice.

Immunocompetent and immunocompromised murine models have been instrumental in answering important questions regarding ZIKV pathogenesis and vertical transmission. However, mimicking human congenital zika syndrome (CZS) characteristics in these murine models has been less than optimal and does not address the potential viral effects on the human immune system. Here, we utilized neonatal humanized Rag2-/-γc-/- mice to model CZS and evaluate the potential viral effects on the differentiation of human hematopoietic stem cells in vivo. Newborn Rag2-/-γc-/- mice were engrafted with ZIKV-infected hematopoietic stem cells (HSC) and monitored for symptoms and lesions. Within 13 days, mice displayed outward clinical symptoms that encompassed stunted growth, hunched posture, ruffled fur, and ocular defects. Striking gross pathologies in the brain and visceral organs were noted. Our results also confirmed that ZIKV actively infected human CD34+ hematopoietic stem cells and restricted the development of terminally differentiated B cells. Histologically, there was multifocal mineralization in several different regions of the brain together with ZIKV antigen co-localization. Diffuse necrosis of pyramidal neurons was seen with collapse of the hippocampal formation. Overall, this model recapitulated ZIKV microcephaly and CZS together with viral adverse effects on the human immune cell ontogeny thus providing a unique in vivo model to assess the efficacy of novel therapeutics and immune interventions.

Quaternized chitosan-coated liposomes enhance immune responses by co-delivery of antigens and resveratrol

Co-localization of antigens and immunomodulators in the same antigen-presenting cells (APCs) can powerfully activate APCs and enhance immune responses. In this study, the immunomodulator resveratrol (Res) was encapsulated into quaternized chitosan (QCS) - coated liposomes for developing a new nanoparticle delivery system (QCS-Res-LP), and ovalbumin (OVA) was selected as a model antigen and adsorbed on the surface of QCS-Res-LP. The results showed that the particle size of QCS-Res-LP was 96.3 ± 3.52 nm; the PDI value was 0.280 ± 0.010; the Zeta potential was 9.59 ± 0.36 mV. QCS-Res-LP could encapsulate 76.22 ± 1.02 % resveratrol and adsorb 88.2 ± 16.3 % antigen. QCS-Res-LP effectively promoted the co-uptake of antigen and Res by dendritic cells (DCs) with 50-fold greater than resveratrol liposomes (Res-LP). QCS-Res-LP promoted expression levels of CD80, CD86, IL-2, and IL-12 in DCs. QCS-Res-LP did not cause hemolysis. The levels of ovalbumin-specific IgG antibodies and cytokines were significantly increased in mice vaccinated with ovalbumin-absorbed QCS-Res-LP, which induced a mixed Th1/Th2 immune response. In conclusion, these results demonstrated that QCS-coated liposomes enable the co-delivery of antigens and immunomodulators to induce strong and durable immune responses.

STING agonist delivery by tumour-penetrating PEG-lipid nanodiscs primes robust anticancer immunity

Activation of the innate immune STimulator of INterferon Genes (STING) pathway potentiates antitumour immunity, but systemic delivery of STING agonists to tumours is challenging. We conjugated STING-activating cyclic dinucleotides (CDNs) to PEGylated lipids (CDN-PEG-lipids; PEG, polyethylene glycol) via a cleavable linker and incorporated them into lipid nanodiscs (LNDs), which are discoid nanoparticles formed by self-assembly. Compared to state-of-the-art liposomes, intravenously administered LNDs carrying CDN-PEG-lipid (LND-CDNs) exhibited more efficient penetration of tumours, exposing the majority of tumour cells to STING agonist. A single dose of LND-CDNs induced rejection of established tumours, coincident with immune memory against tumour rechallenge. Although CDNs were not directly tumoricidal, LND-CDN uptake by cancer cells correlated with robust T-cell activation by promoting CDN and tumour antigen co-localization in dendritic cells. LNDs thus appear promising as a vehicle for robust delivery of compounds throughout solid tumours, which can be exploited for enhanced immunotherapy.

Quantitative Analysis of Multiplexed Immunofluorescence in T-Cell Lymphoma

IntroductionImmunohistochemistry provides biologically and clinically useful information on protein expression and cellular localization in cancer cells, but quantification of the expression by visual scores on light microscopy is subjective and suffers from poor reproducibility. There is increasing need for multiplex immunohistochemistry to study the co-localization of antigens in cell of interest. However, interpretation and quantification of co-localizing signals using conventional IHC is challenging. Novel methods in automated microscopy using machine learning algorithms have been utilized in epithelial cancers to quantitate the signals from immunohistochemistry. However, these have not been well tested in lymphoid malignancies, where IHC plays a critical role in the diagnosis and classification. Here we describe the application of quantitative analysis of immunofluorescence in lymphomas, using an example of a T- cell lymphoma, which can be challenging in terms of diagnosis and treatment.MethodsWe developed a multiplexed panel of immunofluorescent stains including CD4, CD8 and CD20 (T and B-lymphocyte markers) along with T-follicular helper markers (TFH) such as BCL6 and PD1. These were optimized and validated using benign tonsillar tissue to demonstrate appropriate cellular localization of each of these markers. (Figure1.) We then show the applicability of such a multiplexed panel in Angioimmunoblastic T-cell Lymphoma (AITL), a peripheral T-cell lymphoma characterized by cellular heterogeneity in the tumor microenvironment which often impedes IHC interpretation and quantification (figure not shown)Results and DiscussionIn AITL, we generated a tissue map which allows visualization of the spatial relationship of the different lymphocyte subsets and the quantification of TFH markers in tumor and non-tumor cell populations. We demonstrate the feasibility of an automated and quantitative imaging of this multiplexed panel of stains in formalin-fixed paraffin embedded tissue sections, which allows the study of the distribution of tumor cells in relationship to surrounding immune cells and the quantification of the expression of different biomarkers of interest in tumor compared to non-tumor cells in the microenvironment. Quantitative multiplex immunofluorescence and/or immunohistochemistry is likely to improve diagnostic accuracy and reproducibility compared to visual scoring in conventional single marker IHC. Furthermore, the ability to quantify the expression of multiple immune markers in tumor and surrounding immune cells and correlation with treatment response will play a vital role in the selection of patients for immunotherapy (such as anti PD1 or anti CD30 antibodies) which is becoming an increasingly important modality in lymphoma treatment.Figure legend. (Figure 1)Multiplexed composite immunofluorescence of CD20, CD8, CD4 and BCL6 in benign tonsil tissue (A). Unmixed images of CD20 (B, green, cell membrane), CD8 (C, magenta, cell membrane), CD4 (D, yellow, cell membrane), BCL6 (E, red, nuclear) and CD4/BCL6 (F) demonstrating the normal distribution of B and T cells and localization of BCL6-positive cells within germinal centres. The number of CD4-positive (G, red), BCL6-positive (H, green) and CD4/BCL6 double positive cells (I, yellow) are obtained after the images are segmented and scored using the InForm 2.0 software. [Display omitted] DisclosuresChng:Janssen China R&D: Research Funding.

High-affinity antigen association to cationic liposomes via coiled coil-forming peptides induces a strong antigen-specific CD4+ T-cell response

Liposomes are widely investigated as vaccine delivery systems, but antigen loading efficiency can be low. Moreover, adsorbed antigen may rapidly desorb under physiological conditions. Encapsulation of antigens overcomes the latter problem but results in significant antigen loss during preparation and purification of the liposomes. Here, we propose an alternative attachment method, based on a complementary heterodimeric coiled coil peptide pair pepK and pepE.PepK was conjugated to cholesterol (yielding CPK) and pepE was covalently linked to model antigen OVA323 (yielding pepE-OVA323). CPK was incorporated in the lipid bilayer of cationic liposomes (180 nm in size). Antigen was associated more efficiently to functionalized liposomes (Kd 166 nM) than to cationic liposomes (Kd not detectable). In vivo co-localization of antigen and liposomes was strongly increased upon CPK-functionalization (35% -> 80%). CPK-functionalized liposomes induced 5-fold stronger CD4+ T-cell proliferation than non-functionalized liposomes in vitro. Both formulations were able to induce strong CD4+ T-cell expansion in mice, but more IFN-y and IL-10 production was observed after immunization with functionalized liposomes. In conclusion, antigen association via coiled coil peptide pair increased co-localization of antigen and liposomes, increased CD4+ T-cell proliferation in vitro and induced a stronger CD4+ T-cell response in vivo.

Cationic pH-Responsive Polycaprolactone Nanoparticles as Intranasal Antigen Delivery System for Potent Humoral and Cellular Immunity against Recombinant Tetravalent Dengue Antigen.

Research on amalgamation of an antigen with a delivery system for developing a potent mucosal vaccine that elicits both cellular and humoral response has captured immense attention these days. Cationic delivery systems being the first choice for mucosal antigen delivery, despite being effective, are associated with inherent problems like cytotoxicity. Therefore, the quest for developing a precise system that can effectively deliver antigen to immune cells without adverse toxic effect has led to the use of partial cationic systems which are mostly humoral immune mediators. The art of fine-tuning cationic nature, avoiding side effects, and being immune responsive are the needs of the hour. Herein, we try to optimize the cationic nature of polycaprolactone (USFDA approved) by conjugating it with hydrazine. The polymer was modified using two stoichiometry ratios (5 and 10 equiv) of hydrazine monohydrate and characterized using FTIR and XRD. Free amine quantification of the modified polymers concluded that both the modified polymers had 232 and 457 μM/mg amine groups, respectively. A cytotoxicity assay performed using RAW 264.7 macrophages proved the safety of cationic polymers. In vitro assays for antigen colocalization and cross-presentation have revealed that the modified polymers could effectively execute the anticipated function. In vivo evaluation in BALB/c mice using recombinant dengue antigen for intranasal immunization affirmed that the modified polymer having 457 μM/mg of free amine groups effectively stimulated humoral and potent cellular immune response. The overall data suggests that the modified polymeric nanoparticles-with their cationic, pH-responsive, and adjuvanting characteristics-proved to be a versatile system for effective mucosal antigen delivery.

Critical Analysis of Particle Detection Artifacts in Synaptosome Flow Cytometry.

Flow cytometry and fluorescence-activated sorting are powerful techniques that hold great promise for studying heterogeneous populations of submicron particles such as synaptosomes, but many technical challenges arise in these experiments. To date, most flow cytometry studies of synaptosomes have relied on particle detection using forward scatter (FSC) measurements and size estimation with polystyrene (PS) bead standards. However, these practices have serious limitations, and special care must be taken to overcome the poor sensitivity of conventional flow cytometers in the analysis of submicron particles. Technical artifacts can confound these experiments, especially the detection of multiple particles as a single event. Here, we compared analysis of P2 crude synaptosomal preparations from murine forebrain on multiple flow cytometers using both FSC-triggered and fluorescence-triggered detection. We implemented multicolor fluorescent dye-based assays to quantify coincident particle detection and aggregation, and we assessed the false colocalization of antigens in immunostaining analyses. Our results demonstrate that fluorescence triggering and proper dilution can control for coincident particle detection, but not particle aggregation. We confirmed previous studies showing that FSC-based size estimation with PS beads underestimates biological particle size, and we identified pervasive aggregation in the FSC range analyzed in most synaptosome flow cytometry studies. We found that analyzing P2 samples in sucrose/EDTA/tris (SET) buffer reduces aggregation compared to PBS, but does not completely eliminate the presence of aggregates, especially in immunostaining experiments. Our study highlights challenges and pitfalls in synaptosome flow cytometry and provides a methodological framework for future studies.

Easy Employment and Crosstalk-Free Detection of Seven Fluorophores in a Widefield Fluorescence Microscope.

Immunofluorescence staining has become an essential tool in pathology and biomedical sciences to identify rare cells, cell–cell interactions, and submicroscopic cellular components. Many experimental settings, however, suffer from the fact that traditional widefield fluorescence microscopy is usually restricted to imaging three or four fluorophores only. Due to a lack of morphological information and a high detection limit, even flow cytometry—which is capable of staining 20 or more fluorophores at the same time—is limited in its applicability, especially in areas such as rare cell detection. Other advanced imaging approaches, such as confocal laser scanning microscopy and imaging flow cytometry, may be addressing these shortcomings, but in turn require sophisticated downstream data processing and high capital outlay. Here, we describe a new method and filter set-up to routinely employ up to seven fluorophores on a traditional widefield fluorescence microscope equipped with a standard high-pressure mercury light source. Quantification of crosstalk between channels and actual seven-color imaging of cancer cells spiked into leukocytes demonstrate that there is no need for digital compensation correction algorithms. Our set-up thus permits a detailed analysis of rare cell populations, co-localization of antigens, and cell morphology in a standard research or routine laboratory setting.

Incorporation of Phosphonate into Benzonaphthyridine Toll-like Receptor 7 Agonists for Adsorption to Aluminum Hydroxide.

Small molecule Toll-like receptor 7 (TLR7) agonists have been used as vaccine adjuvants by enhancing innate immune activation to afford better adaptive response. Localized TLR7 agonists without systemic exposure can afford good adjuvanticity, suggesting peripheral innate activation (non-antigen-specific) is not required for immune priming. To enhance colocalization of antigen and adjuvant, benzonaphthyridine (BZN) TLR7 agonists are chemically modified with phosphonates to allow adsorption onto aluminum hydroxide (alum), a formulation commonly used in vaccines for antigen stabilization and injection site deposition. The adsorption process is facilitated by enhancing aqueous solubility of BZN analogs to avoid physical mixture of two insoluble particulates. These BZN-phosphonates are highly adsorbed onto alum, which significantly reduced systemic exposure and increased local retention post injection. This report demonstrates a novel approach in vaccine adjuvant design using phosphonate modification to afford adsorption of small molecule immune potentiator (SMIP) onto alum, thereby enhancing co-delivery with antigen.

Assembly of immune signals into carrier-free capsules for rationale vaccine design

Event Abstract Back to Event Assembly of immune signals into carrier-free capsules for rationale vaccine design Yu-Chieh Chiu1, Joshua M. Gammon1, James I. Andorko1, Lisa H. Tostanoski1 and Christopher M. Jewell1, 2, 3 1 University of Maryland - College Park, Fischell Department of Bioengineering, United States 2 University of Maryland Medical School, Department of Microbiology and Immunology, United States 3 Marlene and Stewart Greenbaum Cancer Center, United States Introduction: New vaccines would benefit from more defined compositions that provide modular control over specific immune pathways. Recent studies show that polymer carriers such as poly(lactide-co-glycolide) can exhibit intrinsic immunogenic characteristics that can complicate rationale design since the carrier itself can alter how the immune system processes vaccines[1]. We hypothesize that self-assembly of immune signals into polyelectrolyte multilayers (PEMs) might provide a route for simple, “carrier-free” vaccines assembled entirely from antigens and adjuvants. The immune-PEMs (iPEMs) are assembled in a layer-by-layer (LBL) manner from peptide antigens modified with cationic amino acids and an anionic, nucleic-acid based toll-like receptor agonist (TLRa) that serves as an adjuvant[2],[3]. iPEM capsules provide a high density of antigens and adjuvants since no carriers are involved, while also mimicking features of polymers such as tunable sizes and co-delivery. Materials and Methods: iPEMs were assembled from PolyIC, a dsRNA TLR3 agonist, and a model antigen, SIINFEKL, modified with arginine residues as a cationic component. PEMs were assembled by alternately exposing CaCO3 to these two vaccine components. EDTA was then used to remove the core. Particle diameters were measured as a function of pH, and flow cytometry was used to assess dendritic cell (DC) activation and T cell proliferation. Mice were immunized intra-dermally and boosted at Day 15 or 28. SIINFEKL-specific T cells was measured in blood using MHC-I tetramer and lymph nodes (LNs) were analyzed by histology. For tumor studies, mice were challenged with 1x106 B16-OVA cells on Day 36. Results and Discussion: iPEM capsules exhibited tunable sizes across nano- and micro-scales by varying the pH of the EDTA solution (Fig. 1A). Importantly, these sizes were maintained upon transfer to PBS (Fig. 1B), and were stable during incubation in PBS, media, and serum-containing media (Figure 1C). These materials offer tunable sizes for vaccination. In mice, iPEM capsules drove a significant expansion of antigen-specific T cells with a potent recall response that was 4.7-fold greater than that induced by admixed vaccines (i.e., free) containing the same dose of antigen and adjuvant (Fig. 2A). These effects were driven by co-localization of antigen and adjuvant in draining LNs (yellow signal, Fig. 2B) and efficient antigen presentation that induce greater proliferation in SIIN-specific CD8+ T during ex-vivo co-culture (Fig. 2C). Tumor challenge in mice revealed a significant increase in median survival, with a value of 25 days for mice immunized with iPEM capsules, and 16 days and 13 days for soluble vaccine and untreated mice, respectively (Figure 2D). Conclusions: iPEMs eliminate carrier and supports but offer many useful features of biomaterials. These structures generate functional, antigen-specific T cell responses in mice and could serve as a simple, modular platform for studying and programming immunity. A. Beaven at the University of Maryland Imaging Core Facility for assistance in confocal microscopy.

IFN-γ ameliorates autoimmune encephalomyelitis by limiting myelin lipid peroxidation

Evidence has suggested both a pathogenic and a protective role for the proinflammatory cytokine IFN-γ in experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying the protective role of IFN-γ in EAE have not been fully resolved, particularly in the context of CNS antigen-presenting cells (APCs). In this study we examined the role of IFN-γ in myelin antigen uptake by CNS APCs during EAE. We found that myelin antigen colocalization with APCs was decreased substantially and that EAE was significantly more severe and showed a chronic-progressive course in IFN-γ knockout (IFN-γ-/-) or IFN-γ receptor knockout (IFN-γR-/-) mice as compared with WT animals. IFN-γ was a critical regulator of phagocytic/activating receptors on CNS APCs. Importantly, "free" myelin debris and lipid peroxidation activity at CNS lesions was increased in mice lacking IFN-γ signaling. Treatment with N-acetyl-l-cysteine, a potent antioxidant, abolished lipid peroxidation activity and ameliorated EAE in IFN-γ-signaling-deficient mice. Taken together the data suggest a protective role for IFN-γ in EAE by regulating the removal of myelin debris by CNS APCs and thereby limiting the substrate available for the generation of neurotoxic lipid peroxidation products.

Combining immunodetection with histochemical techniques: the effect of heat-induced antigen retrieval on picro-Sirius red staining.

Picro-Sirius red is a routine diagnostic stain intended for the histological visualization of collagen fibers (fibrosis) in tissue. Multi-label immunohistochemistry is a powerful tool used by researchers to visualize different cell types and their location within a tissue specimen, and to observe co-localization of antigens. Combining the specificity of immunodetection with the simplicity of Sirius red staining will allow researchers to visualize multi-antigen detection in relation to fibrosis, a common histological feature of injury in many chronic diseases. Pre-treatment of formalin-fixed, paraffin-embedded tissue (FFPE) specimens with antigen retrieval is essential for the work-up of most commercially available antibodies. The most common form of antigen retrieval involves boiling tissue specimens in buffer to break the cross-linkages caused by formalin fixation. However, this method causes tissue modification and collagen fiber shrinkage leading to suboptimal results when counterstaining for Sirius red. Reduced heat and enzymatic digestion are antigen retrieval methods compatible with Sirius red counterstaining. This paper will discuss the difficulties faced when combining these two staining methods, and provide a detailed method for the simultaneous detection of antigen and Sirius red in FFPE tissues.

Imaging flow cytometry for morphologic and phenotypic characterization of rare circulating endothelial cells

Endothelial cells in the peripheral circulation are rare events that require technically rigorous approaches for detection by flow cytometry. Visualization of these cells has been even more demanding, as this has historically required extensive enrichment and processing prior to attempting imaging. As a result, few, if any, examples exist on images of peripheral blood circulating endothelial cells (CECs) that include verification of the cell lineage both phenotypically and genomically. In this study, we have devised a method whereby CECs can be directly visualized after lysis of red blood cells and staining, without pre-enrichment or additional processing. Peripheral blood is stained with CD45, CD146, CD3, Hoechst, and DAPI to permit identification of CD146 positive, nonleukocyte, nucleated, and live cells that fit the description of CECs. These cells are imaged using the Amnis ImageStream(X), an imaging flow cytometer. Genomic verification of the endothelial nature of these cells is accomplished by using an aliquot of the same stained samples for sorting CECs using similar gating strategies. This proof of principle of direct imaging of CECs by imaging flow cytometry will permit studies to be conducted heretofore not possible, as the ImageStream(X) has the capability of detecting additional fluorochromes other than those used to identify the CECs. Such potential investigations include antigen colocalization or capping, autophagy and apoptosis, morphologic changes in response to therapy, and others. Thus, this method will enable a broad range of novel studies to be conducted using CECs as surrogates of the endothelium.

Colocalized Delivery of Adjuvant and Antigen Using Nanolipoprotein Particles Enhances the Immune Response to Recombinant Antigens

Subunit antigen-based vaccines can provide a number of important benefits over traditional vaccine candidates, such as overall safety. However, because of the inherently low immunogenicity of these antigens, methods for colocalized delivery of antigen and immunostimulatory molecules (i.e., adjuvants) are needed. Here we report a robust nanolipoprotein particle (NLP)-based vaccine delivery platform that facilitates the codelivery of both subunit antigens and adjuvants. Ni-chelating NLPs (NiNLPs) were assembled to incorporate the amphipathic adjuvants monophosphoryl lipid A and cholesterol-modified CpG oligodeoxynucleotides, which can bind His-tagged protein antigens. Colocalization of antigen and adjuvant delivery using the NiNLP platform resulted in elevated antibody production against His-tagged influenza hemagglutinin 5 and Yersinia pestis LcrV antigens. Antibody titers in mice immunized with the adjuvanted NLPs were 5-10 times higher than those observed with coadministration formulations and nonadjuvanted NiNLPs. Colocalized delivery of adjuvant and antigen provides significantly greater immune stimulation in mice than coadministered formulations.

Chromogen-based Immunohistochemical Method for Elucidation of the Coexpression of Two Antigens Using Antibodies from the Same Species

The authors established a chromogen-based, double immunolabeling method using antibodies from the same species without any unwanted cross-reactivity. In addition, time-consuming staining steps were shortened by using polymer-based secondary antibodies. Taking advantage of the nature of the chromogen 3-amino-9-ethylcarbazole (AEC), which is used as a horseradish peroxidase substrate for antibody detection, the AEC-derived signals in the first color development were easily eliminated by alcohol treatment. Therefore, the signals from the first staining did not interfere with those from the subsequent second staining, which used the chromogen 3,3'-diaminobenzidine. The co-localization of antigens within the same cell could be confirmed using this method, because cell images of the individual dye staining steps could be obtained and developed. The images from each step could be expressed in pseudo-colors in a dark field by using a computer. As a result, merged images could be constructed that resembled the images acquired by the fluorescent immunolabeling technique. The resolution of this method enabled analysis of the coexpression of two antigens in the same cell in the same section. The authors have named this staining technique the elucidation of the coexpression of two antigens in a cell using antibodies from the same species (ECSS).

Endothelial cell activation and proliferation in ovarian tumors: Two distinct steps as potential markers for antiangiogenic therapy response

Ovarian cancer is the second leading cause of cancer-related death in women worldwide. Since most patients are diagnosed in advanced disease stages, the starting point, early steps and molecular mechanisms of ovarian cancer angiogenesis are still incompletely characterized. Most immunohistochemical studies for assessment of microvessel density (MVD) in ovarian tumors are based on CD31, CD34 and CD105 immunostaining of tumor blood vessels. Yet, the proliferative status of tumor blood vessel endothelial cells has not as yet been used in the assessment of tumor blood vessels. The present study investigated the Ki67 proliferative index of tumor blood vessel endothelial cells highlighted with the CD34 panendothelial marker and the CD105 endothelial marker in ovarian cancers by antigen co-localization with a doublestaining immunohistochemical method. Lack of co-localization of CD105 and Ki67 in all types of ovarian tumors together with the presence of CD34+/Ki67-positive endothelial cells suggest that endothelial cell activation and proliferation are distinct steps in ovarian tumors. Differences in the proliferation index were observed in endothelial cells from blood vessels of the tumor core and those of the tumor peripheral zones. Potential specific targeting of activated and proliferating tumor blood vessels may provide clues for improving antiangiogenic therapy efficiency.

A practical calibration procedure for fluorescence colocalization at the single organelle level

We have determined practical requirements for antigen colocalization on subcellular structures. The calibration standards used were 175-nm fluorescent microspheres and microtubules (approximately 255 nm) in cultured astrocytes. The colocalization problem became apparent with detection of anti-alpha-tubulin labelling in three colour channels, when images were not identical. Complete superimposition could only be achieved by shifting the single colour image stacks relative to one another in three dimension, and images in the xy plane. The errors could be traced in chromatic aberration (100 nm each between blue and green, and green and red), and to a lateral pixel shift (approximately 150 nm). For such colocalization, it was essential to apply image projection on the camera chip at high magnification (200 x), focus steps (100 nm) smaller than required by the Nyquist criterion and very narrow band filter sets, in addition to high-aperture achromat lenses. Several steps in the deconvolution settings and in 3D reconstruction had to be standardized. As a result, the 175-nm microspheres, which displayed roughly symmetric diffraction patterns above and below the object plane, were reconstructed as spherical objects. Several neighbouring microtubules could be resolved with a limit < 200 nm. Also, three-colour colocalization on a single tubule was validated. Applying this setup, it was possible to colocalize several antigens in astrocytes, at the level of organelles, presumptive exocytosis vesicles. We colocalized glutamate and 14-3-3 protein as well as synaptophysin and 14-3-3 protein, which may be involved in early steps of exocytosis. The practical parameters validate colocalization on subcellular structures at a resolution limit below conventional light microscopy.

In vivo Colocalization of Antigen and CpG within Dendritic Cells Is Associated with the Efficacy of Cancer Immunotherapy

Immunostimulatory cytidyl guanosyl (CpG) motifs are of great interest as cancer vaccine adjuvants. They act as potent inducers of Th1 responses, including the activation of cytotoxic CD8(+) T lymphocytes (CTL). Whereas animal models have provided clear evidence that CpG enhances antitumor immunity, clinical trials in humans have thus far been less successful. Applying cryosurgery as an instant in situ tumor destruction technique, we now show that timing of CpG administration crucially affects colocalization of antigen and CpG within EEA-1(+) and LAMP-1(+) compartments within dendritic cells in vivo. Moreover, antigen/CpG colocalization is directly correlated with antigen cross-presentation, the presence of CTL, and protective antitumor immunity. Thus, failure or success of CpG as a vaccine adjuvant may depend on colocalization of antigen/CpG inside DCs and hence on the timing of CpG administration. These data might aid in the design of future immunotherapeutic strategies for cancer patients.