Disease-recapitulating animal models are valuable tools in preclinical development for the study of compounds. In the case of fibrotic pulmonary diseases such as idiopathic pulmonary fibrosis (IPF), the bleomycin model of lung injury in the mouse is widely used. To evaluate bleomycin-induced changes in the lung, we employed a quantitative, multimodal approach. Using in vivo microcomputed tomography (μCT), we demonstrated radiographic changes associated with disease progression in aeration levels of the lung parenchyma. There exists an unmet need for a quantitative, high-resolution imaging probe to detect pulmonary fibrosis, particularly that can differentiate between inflammatory and fibrotic components of the disease. Matrix remodeling and overexpression of extracellular matrix (ECM) proteins such as collagen and fibronectin are hallmarks of organ fibrosis. A splice variant of fibronectin containing extra domain A (FnEDA) is of particular interest in fibrosis due to its high level of expression in diseased tissue, which is confirmed here using immunohistochemistry (IHC) in mouse and human lungs. An antibody against FnEDA was evaluated for use as an imaging tool, particularly by using in vivo single-photon emission computed tomography (SPECT) and ex vivo near-infrared (NIR) fluorescence imaging. These data were further corroborated with histological tissue staining and fibrosis quantitation based on a Modified Ashcroft (MA) score and a digital image analysis of whole slide lung tissue sections. The fusion of these different approaches represents a robust integrated workflow combining anatomical and molecular imaging technologies to enable the visualization and quantitation of disease activity and treatment response with an inhibitor of the TGFβ signaling pathway.

In this work, the impact of physicochemical modifications on pharmacokinetics and in vivo targeting of a small molecule fibroblast activation protein inhibitor (FAPI) imaging ligand in a murine model of rheumatoid arthritis was evaluated. While similar ligands have been well-reported in oncology for molecular imaging and radiotherapy, there are limited reports of FAPI derivatives in targeted applications in immunology. As inflammation may increase both specific and non-specific delivery of targeted agents in general, we sought to identify the optimal targeted molecular imaging probe characteristics for efficient cell surface engagement. A series of FAPI derivatives were synthesized and their physicochemical properties modified via conjugation of fluorescent dyes and/or an albumin-binding small molecule. The impact of these modifications on cell surface binding affinity was assessed using an overexpressing cell line. Additionally, a thorough mechanistic characterization of fibroblast activation protein (FAP) cell surface internalization was evaluated in both overexpressing and endogenously expressing cells. Lastly, the pharmacokinetics and in vivo uptake in inflamed arthritic paws were characterized via near-infrared (NIR) imaging. All targeted molecular imaging agents tested maintained strong nanomolar binding affinity to cell surface FAP independent of chemical modification. The murine fibroblast-like synoviocytes expressed lower absolute cell-surface FAP compared to a transfected line, and the net internalization half-life measured for the transfected cells via flow cytometry was 7.2 h. The unmodified FAPI ligand exhibited the poorest in vivo targeting, likely resulting from its large apparent volume of distribution (62.7 ml) and rapid systemic clearance (t1/2 = 0.5 h). Conjugation of a charged, hydrophilic AF647 fluorophore decreased systemic clearance (t1/2 = 2.1 h) and demonstrated a 2-fold improvement in blocking FAPI-800CW engagement of FAP in vivo when compared to blocking of FAPI-800CW with FAPI with up to 2.8-fold improvements noted for the equivalent albumin binding construct comparison.

A painful, chronic condition, Rheumatoid Arthritis, is marked by bone erosion and soft tissue swelling at the joint. As treatments are investigated in pre-clinical models, characterizing disease progression is integral to assessing treatment efficacy. Here, in vivo and ex vivo micro-computed tomography (µCT) are used in parallel with traditional caliper score measurement to quantify physiological changes in the tarsal region in a murine, collagen-induced arthritis model. In vivo imaging methods, which are validated here through comparison to ex vivo and caliper methods, afford longitudinal analysis of both bone and soft tissue through a single image acquisition. This method removes the subjectivity of swelling quantification which is inherently associated with traditional caliper measurements. Histopathology offers an additional assessment of bone erosion and inflammation by providing a microscopic characterization of disease activity. In comparison to untreated animals, daily prednisolone (glucocorticoid) treatment is shown to restore bone volume, as reflected through in vivo and ex vivo µCT images, as well as histopathology. Prednisolone-associated reduction in inflammation is shown through in vivo µCT soft tissue volume measurements, paw caliper measurements, and histopathology. The findings reported here provide a comprehensive validation of in vivo µCT with a sensitivity that enables characterization of pre-clinical disease assessment in response to treatment in a murine, collagen-induced arthritis model.

Objectives To assess the ability of monoclonal antibodies (mAbs) specific for fibronectin extra-domain A (FnEDA) to target diseased tissues of mouse collagen induced arthritis (mCIA) models. To explore the parameters of the targeting exhibited by anti-FnEDA mAbs including timing and location. Methods Targeting capabilities of anti-FnEDA mAbs were demonstrated by biodistribution study where i.v. injected antibodies were detected by conjugated near-infrared (NIR) fluorophore, 125 I label and immunohistochemistry (IHC) of the injected antibody. Location of FnEDA expression in both mCIA and human RA tissue were mapped by IHC. Quantification of anti-FnEDA mAbs targeted to disease tissue was measured by whole-body autoradiography (WBA). Timing of the targeting was interrogated with fluorescent and confocal microscopy using anti-FnEDA mAbs labeled with different fluorophores and injected at different times. Results Anti-FnEDA mAbs show specific targeting to diseased paws of mCIA animal. The targeting was focused on inflamed synovium which is consistent with FnEDA expression profile in both mCIA and human RA tissues. Anti-FnEDA mAbs accumulated in diseased tissue at pharmacologically relevant concentrations, the targeting was sustained for up to 14 days and FnEDA was able to support targeting of multiple doses of anti-FnEDA mAbs given 5 days apart. Conclusion FnEDA is specifically upregulated in the inflamed tissues of mCIA. Antibodies specific for FnEDA can be useful as molecular delivery vehicles for disease specific targeting of payloads to inflamed joint tissue.

Purpose To image colon-expressed alternatively spliced D domain of tenascin C in preclinical colitis models using near infrared (NIR)-labeled targeted molecular imaging agents. Procedures. A human IgG1 with nanomolar binding affinity specific to the alternatively spliced D domain of tenascin C was generated. Immunohistochemistry identified disease-specific expression of this extracellular matrix protein in the colon of mice given dextran sulfate sodium in the drinking water. The antibody reagent was labeled with the NIR fluorophore IRDye 800CW via amine chemistry and intravenously dosed to evaluate in vivo targeting specificity. Increasing doses of imaging agent were given to estimate the saturating dose. Results The NIR-labeled proteins successfully targeted colonic lesions in a murine model of colitis. Co-administration of a molar excess competing unlabeled dose reduced normalized uptake in diseased colon by > 70%. Near infrared ex vivo images of colon resected from diseased animals showed saturation at doses exceeding 1 nmol and was confirmed with additional quantitative ex vivo biodistribution. Cellular-level specificity and protein stability were assessed via microscopy. Conclusions Our imaging data suggest the alternatively spliced D domain of tenascin C is a promising target for delivery-based applications in inflammatory bowel diseases.

Intestinal permeability and neutrophil activity are closely linked to inflammatory bowel disease (IBD) pathophysiology. Here we discuss two techniques for assessing permeability and neutrophil activity in mouse IBD models using near infrared (NIR) detection. To address the limitation of visible light readouts—namely high background—IRDye 800CW was used to enable rapid, non-terminal measurements of intestinal permeability. The increased sensitivity of NIR readouts for colon permeability is shown using dextran sulfate sodium (DSS) and anti-CD40 murine colitis models in response to interleukin-22 immunoglobulin Fc (IL22Fc) fusion protein and anti-p40 monoclonal antibody treatments, respectively. In addition to enhanced permeability, elevated levels of neutrophil elastase (NE) have been reported in inflamed colonic mucosal tissue. Activatable NIR fluorescent probes have been extensively used for disease activity evaluation in oncologic animal models, and we demonstrate their translatability using a NE-activatable reagent to evaluate inflammation in DSS mice. Confocal laser endomicroscopy (CLE) and tissue imaging allow visualization of spatial NE activity throughout diseased colon as well as changes in disease severity from IL22Fc treatment. Our findings with the 800CW dye and the NE probe highlight the ease of their implementation in preclinical IBD research.

While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte-associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4-binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1-/- mice that received naive donor CD4+ T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2-bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.

Background: Intestinal permeability is increased in many gut disorders, but is difficult to measure in humans.Currently, intestinal permeability is assessed in people using surrogate measures of barrier function, such as concentrations of circulating bacterial lipopolysaccharide (LPS), indicators of exposure to gut bacteria such as antibodies to LPS, LPS binding protein, and α-1-acid glycoprotein, and fecal α-1-antitrypsin.Many studies have used dual sugar absorption tests to probe gut permeability, which measure the ratios in urine of a disaccharide (generally lactulose) to a monosaccharide (generally rhamnose or lactulose) following oral ingestion.If barrier function is impaired, the ratios of the heavier to the lighter sugar are elevated.This theoretically sound test is compromised by specimen handling and assay challenges, and reliance on an arbitrarily time point for assessment.Pyrazine-derived fluorophores MB-402 (MW=422, peak excitation l=500 nm, peak emission=620 nm) and MB-301 (MW=198, peak excitation l=405 nm, peak emission=540 nm) detect small bowel injury in rats challenged with indomethacin (Sci Rep 2017;7:10888).Following oral ingestion, these fluorophores can be measured through the skin using transcutaneous sensor technology, as is now employed in clinical studies to measure glomerular filtration rates, and provide more robust tracings and dynamic range in a dose response model.Methods.We tested in our rat model MB-404 (MW = 492, peak excitation=500 nm, peak emission=620 nm).MB-404 contains eight hydroxyl groups (vs.four in MB-402), retains MB-402's acid-resistance, and is soluble in water.We gavaged MB-404 ( 92mg/kg) in combination with MB-301 (16 mg/kg).Results.Urinary MB-404:MB-301 ratios recapitulate (Fig 1) MB-402:MB-301 urine ratios.Simultaneous continuous transcutaneous monitoring detects each fluorophore in the extracellular space of the rats over eight hours resemble the cumulative urinary clearance of the fluorophores, demonstrating the feasibility of measuring tracer uptake from the gut without taking possession of urine or other fluids.Transcutaneous monitoring and urinary concentrations also enabled us to test if the ratios of MB-404 and MB-301 were superior to the uptake and clearances of individual tracers.The transcutaneous assessment measurement of extracellular MB-404 by itself (Fig 2) discriminated injured vs. control rats better than did ratios of the two fluorophores.Conclusion.MB-404's greater solubility offers advantages over MB-402 in dual fluorophore tests of gut permeability.Most importantly, MB-404 uptake from the gut following oral ingestion offers greater sensitivity as a single tracer than when used in tandem with MB-301.The ability to test gut permeability with a single tracer will facilitate upcoming trials of fluorophore tracers in people with intestinal enteropathy.

During drug development, tissue samples serve as indicators of disease activity and pharmacodynamic responses. Reliable non-invasive measures of drug target engagement will facilitate identification of promising new treatments. Here we develop and validate a novel bi-transgenic mouse model of myotonic dystrophy type 1 (DM1) in which expression of either DsRed or GFP is determined by alternative splicing of an upstream minigene that is mis-regulated in DM1. Using a novel in vivo fluorescence spectroscopy system, we show that quantitation of the DsRed/GFP ratio provides an accurate estimation of splicing outcomes in muscle tissue of live mice that nearly doubles throughput over conventional fluorescence imaging techniques. Serial in vivo spectroscopy measurements in mice treated with a C16 fatty acid ligand conjugated antisense (LICA) oligonucleotide reveal a dose-dependent therapeutic response within seven days, confirm a several-week duration of action, and demonstrate a two-fold greater target engagement as compared to the unconjugated parent oligonucleotide.

Because of the heterogeneous nature of articular cartilage tissue, penetration of potential therapeutic molecules for osteoarthritis (OA) through the articular surface (AS) is complex, with many factors that affect transport of these solutes within the tissue. Therefore, the goal of this study is to investigate how the size of antibody (Ab) variants, as well as application of cyclic mechanical loading, affects solute transport within healthy cartilage tissue. Penetration of fluorescently tagged solutes was quantified using confocal microscopy. For all the solutes tested, fluorescence curves were obtained through the articular surface. On average, diffusivities for the solutes of sizes 200 kDa, 150 kDa, 50 kDa, and 25 kDa were 3.3, 3.4, 5.1, and 6.0 μm2/s from 0 to 100 μm from the articular surface. Diffusivities went up to a maximum of 16.5, 18.5, 20.5, and 23.4 μm2/s for the 200 kDa, 150 kDa, 50 kDa, and 25 kDa molecules, respectively, from 225 to 325 μm from the surface. Overall, the effect of loading was very significant, with maximal transport enhancement for each solute ranging from 2.2 to 3.4-fold near 275 μm. Ultimately, solutes of this size do not diffuse uniformly nor are convected uniformly, through the depth of the cartilage tissue. This research potentially holds great clinical significance to discover ways of further optimizing transport into cartilage and leads to effective antibody-based treatments for OA.

Lymphangioleiomyomatosis (LAM) is a rare disease in women. Patients with LAM develop metastatic smooth-muscle cell adenomas within the lungs, resulting in reduced pulmonary function. LAM cells contain mutations in tuberous sclerosis genes (TSC1 or TSC2), leading to up-regulation of mTORC1 activity and elevated proliferation. The origin of LAM cells remains unknown; however, inactivation of Tsc2 gene in the mouse uterus resulted in myometrial tumors exhibiting LAM features, and approximately 50% of animals developed metastatic myometrial lung tumors. This suggests that LAM tumors might originate from the uterine myometrium, possibly explaining the overwhelming prevalence of LAM in female. Here, we demonstrate that mouse Tsc2-null myometrial tumors exhibit nearly all the features of LAM, including mTORC1/S6K activation, as well as expression of melanocytic markers and matrix metalloproteinases (MMPs). Estrogen ablation reduces S6K signaling and results in Tsc2-null myometrial tumor regression. Thus, even without TSC2, estradiol is required to maintain tumors and mTORC1/S6K signaling. Additionally, we find that MMP-2 and -9, as well as neutrophil elastase (NE), are overexpressed in Tsc2-null myometrial tumors in an estrogen-dependent fashion. In vivo fluorescent imaging using MMP- or NE-sensitive optical biomarkers confirms that protease activity is specific to myometrial tumors. Similar to LAM cells, uterine Tsc2-null myometrial cells also overexpress melanocytic markers in an estrogen-dependent fashion. Finally, we identify glycoprotein NMB (GPNMB) as a melanocytic marker up-regulated in Tsc2-null mouse uteri and human LAM samples. Our data highlight the potential importance of estradiol in LAM cells, suggesting that anti-estrogen therapy may be a treatment modality. Furthermore, proteases and GPNMB might be useful LAM biomarkers.

This paper presents an algorithm for accurately solving the full two-dimensional governing equations, along with the interface conditions that govern laminar/laminar annular/stratified internal condensing flows. The simulation approach - which can be generalized to adiabatic and evaporating flows, a 3-D level-set technique, and so on - uses a sharp-interface model, separate liquid and vapor domain computational solutions with interface conditions embedded as boundary conditions, and a moving grid technique to locate the dynamic wavy interface (in amplitude and phase) by a method of characteristics solution of the interface tracking equation. The moving grid is spatially fixed for a defined number of instants, but changes when the current marker instant advances in time.

This paper highlights: (i) numerical methods developed to solve annular/stratified internal condensing flow problems, and (ii) the assessed effects of transverse gravity and surface tension on shear driven (horizontal channels) and gravity driven (inclined channels) internal condensing flows. A comparative study of the flow physics is presented with the help of steady and unsteady computational results obtained from the numerical solutions of the full two-dimensional governing equations for annular internal condensing flows. These simulations directly apply to recently-demonstrated innovative condenser operations which make the flow regime annular over the entire length of the condenser. The simulation algorithm is based on an active integration of our own codes developed on MATLAB with the standard single-phase CFD simulation codes available on COMSOL. The approach allows for an accurate wave simulation technique for the highly sensitive shear driven annular condensing flows.This simulation approach employs a sharp-interface model and uses a moving grid technique to accurately locate the dynamic interface by the solution of the interface tracking equation (employing the method of characteristics) along with the rest of the governing equations. The 4th order time-step accuracy in the method of characteristics has enabled, for the first time, the ability to track time-varying interface locations associated with wave phenomena and accurate satisfaction of all the interface conditions - including the more difficult to satisfy interfacial mass-flux equalities.A combination of steady and unsteady simulation results are also used to identify the effects of transverse gravity, axial gravity, and surface tension on the growth of waves. The results presented bring out the differences within different types of shear driven flows and differences between shear driven and gravity driven flows.The unsteady wave simulation capability has been used here to do the stability analysis for annular shear-driven steady flows. In stability analysis, an assessment of the dynamic response of the steady solutions to arbitrary instantaneous initial disturbance are obtained. The results mark the location beyond which the annular regime transitions to a non-annular regime (experimentally known to be a plug-slug regimes).The computational prediction of heat-flux values agree with the experimentally measured values (at measurement locations) obtained from relevant runs of our in-house experiments. Also, a comparison between the computationally predicted and experimentally measured values regarding the length of the annular regime is possible, and will be presented elsewhere.

Studies investigating the interaction of Candida with a single host cell type such as phagocytes or epithelial cells are typically performed in vitro. The advantages of in vitro studies include the ability to compare multiple conditions in one experiment, the ability to perform thorough biochemical and immunologic analyses, the relatively short duration of experiments, lower experimental costs, and the ability to reduce the use of laboratory animals. The major disadvantage of in vitro experiments is that it is not possible to fully replicate the myriad of cellular and extracellular signals provided by the host environment. Several animal models of candidiasis are regularly used, including models of oropharyngeal, vaginal, and disseminated candidiasis. Information about the interaction of specific host cell types with Candida organisms in vivo can sometimes be obtained through conventional histology or immunohistochemistry, but these analyses require sacrifice of the animal and thus are often limited to a single time point during the course of infection. Several fluorescent proteins have recently been codon optimized for expression in yeast. The authors suspect that any strain of Candida that has relatively bright fluorescence could be used in this model. For imaging, an inverted confocal microscope with an enclosed stage incubator chamber and generic stage insert is used. Recently, a stage insert was constructed allowing the use of an Olympus FV1000 laser scanning confocal microscope at the confocal microscopy core facility. Images were successfully obtained using both 10x, 0.4to 0.45-numerical-aperture and 20x, 0.75-NA dry objective lenses.

We demonstrate the use of an enzyme-activatable fluorogenic probe, Neutrophil Elastase 680 FAST (NE680), for in vivo imaging of neutrophil elastase (NE) activity in tumors subjected to photodynamic therapy (PDT). NE protease activity was assayed in SCC VII and EMT6 tumors established in C3H and BALB/c mice, respectively. Four nanomoles of NE680 was injected intravenously immediately following PDT irradiation. 5 h following administration of NE680, whole-mouse fluorescence imaging was performed. At this time point, levels of NE680 fluorescence were at least threefold greater in irradiated versus unirradiated SCC VII and EMT6 tumors sensitized with Photofrin. To compare possible photosensitizer-specific differences in therapy-induced elastase activity, EMT6 tumors were also subjected to 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-PDT. NE levels measured in HPPH-PDT-treated tumors were twofold higher than in unirradiated controls. Ex vivo labeling of host cells using fluorophore-conjugated antibodies and confocal imaging were used to visualize Gr1+ cells in Photofrin-PDT-treated EMT6 tumors. These data were compared with recently reported analysis of Gr1+ cell accumulation in EMT6 tumors subjected to HPPH-PDT. The population density of infiltrating Gr1+ cells in treated versus unirradiated drug-only control tumors suggests that the differential in NE680 fold enhancement observed in Photofrin versus HPPH treatment may be attributed to the significantly increased inflammatory response induced by Photofrin-PDT. The in vivo imaging of NE680, which is a fluorescent reporter of NE extracellular release caused by neutrophil activation, demonstrates that PDT results in increased NE levels in treated tumors, and the accumulation of the cleaved probe tracks qualitatively with the intratumor Gr1+ cell population.

The reported experimental results involve fully condensing flows of pure FC-72 vapor on a horizontal condensing surface (316 stainless steel) which is the bottom surface (wall) of a rectangular cross-section duct of 2 mm height, 15 mm width, and 1 m length. The sides and top of the duct are made of clear plastic. The experimental system in which this condenser is used is able to control steady-in-the-mean (termed quasi-steady) mass flow rate, exit pressure, and wall cooling conditions. It has been found that, with the condenser mean (time averaged) inlet mass flow rate, exit pressure, and wall cooling condition held fixed at steady values, there is a very strong sensitivity to high amplitude pressure fluctuations and flow rate pulsations at the condenser inlet. This sensitivity often significantly alters the condenser mean inlet pressure, pressure drop, local heat transfer rates (>200% increase at certain locations), and the condensing flow morphology. These effects are representative of fluctuations/pulsations that are typically encountered in applications but are either not accounted for or are not detected. The effects of imposed fluctuations/pulsations, as opposed to cases involving negligible imposed fluctuations/pulsations, are dependent on the amplitude and the frequency content of the imposed fluctuations and this is discussed in a separate paper. A significant upstream annular regime portion of the reported shear/pressure driven fully condensing flows operate under conditions where the laboratory’s transverse gravity effects are negligible and, therefore, the identified sensitivity phenomenon is highly relevant to zero- or micro-gravity conditions. The micro-gravity relevance of this sensitivity for the annular regime phenomenon is currently also being demonstrated with the help of computations and simulations.

We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH)-mediated photodynamic therapy (PDT) of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV) administration of 1 μmol kg-1 HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1+/CD11b+ leukocytes and major histocompatibility complex class II (MHC-II)+ cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1+ cells in response to therapy. The maximum accumulation of Gr1+ cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1+ cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1+ cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.