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Histologic overexpression of the estrogen receptor α (ER) is a well-established prognostic marker in breast cancer. Noninvasive imaging techniques that could detect ER overexpression would be useful in a variety of settings where patients' biopsies are problematic to obtain. This study focused on developing, by in vivo MRI, strategies to measure the level of ER expression in an orthotopic mouse model of human breast cancer. Specifically, novel ER-targeted contrast agents based on pyridine-tetra-acetate-Gd(III) chelate (PTA-Gd) conjugated to 17β-estradiol (EPTA-Gd) or to tamoxifen (TPTA-Gd) were examined in ER-positive or ER-negative tumors. Detection of specific interactions of EPTA-Gd with ER were documented that could differentiate ER-positive and ER-negative tumors. In vivo competition experiments confirmed that the enhanced detection capability of EPTA-Gd was based specifically on ER targeting. In contrast, PTA-Gd acted as an extracellular probe that enhanced ER detection similarly in either tumor type, confirming a similar vascular perfusion efficiency in ER-positive and ER-negative tumors in the model. Finally, TPTA-Gd accumulated selectively in muscle and could not preferentially identify ER-positive tumors. Together, these results define a novel MRI probe that can permit selective noninvasive imaging of ER-positive tumors in vivo. Cancer Res; 71(24); 7387–97. ©2011 AACR.

In vivo mapping of the transcapillary fluxes in tumors can help predict the efficacy of delivery of blood-borne anticancer drugs. These fluxes are primarily affected by the vascular permeability and the pressure gradients across the blood vessels’ walls. We describe herein high-resolution dynamic contrast-enhanced magnetic resonance imaging of the influx and outflux transcapillary transfer rates in vivo in invasive MDA-MB-231 tumors orthotopically inoculated in severe combined immunodeficient mice. The tumors were noted for rapid growth, impaired drainage of fluid, and subsequent formation of cysts. Consequently, the time evolution of the contrast enhancement, induced by i.v. injection of Gadolinium diethylene-triamine-penta-acetate, exhibited two distinct patterns: transcapillary transfer in the cellular regions and simple diffusion in the cyst fluid. Both processes were analyzed at pixel resolution applying to each a physiological model and a corresponding algorithm. In the cellular region, the influx and outflux transcapillary transfer rates decreased during tumor growth; however, an increased disparity between the transfer constants was observed, with the outflux rate exceeding the influx rate. This quantitative spatial and temporal mapping of this disparity can provide a means to assess the physiological barriers to tracer delivery. It is hypothesized that both the increased disparity in transcapillary transfer rates and impaired fluid drainage in these tumors could arise from the development of interstitial hypertension.

Supplementary Video 3 from Real-time Imaging of Lymphogenic Metastasis in Orthotopic Human Breast Cancer

Supplementary Figures from High-Resolution Magnetic Resonance Imaging of Disparities in the Transcapillary Transfer Rates in Orthotopically Inoculated Invasive Breast Tumors

Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFP) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis. (Cancer Res 2006; 66(16): 8037-41)

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Supplementary Video 2 from Real-time Imaging of Lymphogenic Metastasis in Orthotopic Human Breast Cancer

Supplementary Video 1 from Real-time Imaging of Lymphogenic Metastasis in Orthotopic Human Breast Cancer

We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity, two of its chemical moieties (tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of IL-1β, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug candidate for the treatment of ARDS in COVID-19 patients.

Conventional treatments of peripheral vascular disease and coronary artery disease have partial success but are still limited. Methods to deliver angiogenic factors into ischemic areas using gene, protein and cell therapies are faced with difficult issues such a delivery, effective concentration and duration of action. Tissue engineering offers the possibility of creating a functional self-contained three-dimensional (3D) unit that works as a coordinated biological pump that can secrete a whole range of angiogenic factors. We report a tissue engineering approach using decellularized micro-fragments and mesenchymal stem cells (MSCs) to create a vascular inducing device (VID). Proteomic analysis of the decellularized micro-fragments and of the VIDs reveals a large number of extracellular-matrix (ECM) proteins. Moreover, the VIDs were found to transcribe and secrete a whole repertoire of angiogenic factors in a sustained manner. Furthermore, preliminary results of implantation VIDs into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice indicate formation of vascular network at the site within a week. We propose that those VIDs could serve as a safe, localized, simple and powerful method for the treatment of certain types of vascular diseases.

The partial success of tumor immunotherapy induced by checkpoint blockade, which is not antigen-specific, suggests that the immune system of some patients contain antigen receptors able to specifically identify tumor cells. Here we focused on T-cell receptor (TCR) repertoires associated with spontaneous breast cancer. We studied the alpha and beta chain CDR3 domains of TCR repertoires of CD4 T cells using deep sequencing of cell populations in mice and applied the results to published TCR sequence data obtained from human patients. We screened peripheral blood T cells obtained monthly from individual mice spontaneously developing breast tumors by 5 months. We then looked at identical TCR sequences in published human studies; we used TCGA data from tumors and healthy tissues of 1,256 breast cancer resections and from 4 focused studies including sequences from tumors, lymph nodes, blood and healthy tissues, and from single cell dataset of 3 breast cancer subjects. We now report that mice spontaneously developing breast cancer manifest shared, Public CDR3 regions in both their alpha and beta and that a significant number of women with early breast cancer manifest identical CDR3 sequences. These findings suggest that the development of breast cancer is associated, across species, with biomarker, exclusive TCR repertoires., Author summary More than 1,100 cancer treatments are currently in clinical testing. Of these, 295 are immune-oncology medicines and vaccines. For Checkpoint Immunotherapy alone, 52,539 patients were enrolled in 2017. Of these treatments, 108 are for breast cancer, the leading cancer diagnosed in women. Although checkpoint immunotherapy has yet to be proven beneficial in most types of breast cancer, current observations of which specific T cell go into the tumor, and how these cells are connected to the response to treatment, have lead to a new understating of the role of the immune system. Yet, and in spite of this intense research into the role of T cells in tumors, very little is known about how clones of T cells behave during tumor formation. The paper we present here studies this clonal behavior over time. We study it during mammary tumor development in mice. We see that a set of T cell clones, which comes from a diversity of different nucleotide sequences, stands out in its behavior compared with other T cell clones. Importantly, we show that these mouse T cell clones have a strong connection to human breast cancer. The specific identification of a subset of T cell clones, together with the behavior of these clones over time, is critical to our understanding of the effect of modifications to the immune system, such as the modifications made by immunotherapy treatments, and to our understanding of how to better control the behavior of the immune system.

Leukocyte transendothelial migration is one of the most important step in launching an inflammatory immune response and chronic inflammation can lead to devastating diseases. Leukocyte migration inhibitors are considered as promising and potentially effective therapeutic agents to treat inflammatory and auto-immune disorders. In this study, based on previous trioxotetrahydropyrimidin based integrin inhibitors that suboptimally blocked leukocyte adhesion, twelve molecules with a modified scaffold were designed, synthesized, and tested in vitro for their capacity to block the transendothelial migration of immune cells. One of the molecules, namely, methyl 4-((2-(tert-butyl)-6-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) methyl) phenoxy) methyl) benzoate, (compound 12), completely blocked leukocyte transendothelial migration, without any toxic effects on immune or endothelial cells (IC50 = 2.4 µM). In vivo, compound 12 exhibited significant therapeutic effects in inflammatory bowel disease (IBD)/Crohn’s disease, multiple sclerosis, fatty liver disease, and rheumatoid arthritis models. A detailed acute and chronic toxicity profile of the lead compound in vivo did not reveal any toxic effects. Such a type of molecule might therefore provide a unique starting point for designing a novel class of leukocyte transmigration blocking agents with broad therapeutic applications in inflammatory and auto-immune pathologies.

Cancer immunotherapy by checkpoint blockade proves that an effective immune response to a tumor can be induced clinically. However, little is known about the evolution of tumor-associated T-cell receptor (TCR) repertoires without intervention. Here we studied TCR repertoire evolution in mice spontaneously developing mammary tumors; we sequenced peripheral blood alpha and beta TCRs of CD4+CD62L+CD44− T cells monthly for 8 months in 10 FVB/NJ mice transgenic at the Erbb2 locus, all developing tumors; 5 FVB/NJ mice without the transgene were age-matched controls. Sequences were either private (restricted to one mouse) or public (shared among mice); public sequences were either exclusive to the tumor group or inclusive among different groups. We now report that 1), public AA sequences were each encoded by many different nucleotide sequences (NT) recombinations (convergent recombination; CR); 2) mice developing tumors evolved tumor-exclusive public sequences, derived initially from private or from inclusive public sequences; and 3) tumor-exclusive public sequences in mice were also present among published public TCR sequences from human breast cancer patients. These cross-species tumor-exclusive TCR sequences manifested high CR; but the AA sequences shared by mice and humans did not share NT sequences. Thus, tumor-exclusive TCR AA sequences across species are selected from different NT recombination events. The roles of tumor-exclusive TCR repertoires in advancing or inhibiting tumor development and the effects of tumor immunotherapy on these T cells remain to be seen.

Aberrant inflammation appears to be a pathogenic factor in autoimmune diseases and other noxious inflammatory conditions in which the inflammatory process is misapplied, exaggerated, recurrent or chronic. The protein molecules involved in pathogenic inflammation—disease-associated proteins (DAP)—which include chemokines, cytokines, and growth factors and their receptors, appear normal; their networks of interaction are at fault. Here we demonstrate a new approach to network regulation of inflammation based on peptide sequence motifs shared by the second extra-cellular loop (ECL2) of different chemokine receptors; previously known chemokine receptor binding sites have not involved the ECL2 loop. These motifs of 9 amino acids, which we detected by sequence alignment, manifest very low E-values compared with slightly modified sequence variations, indicating that they were not likely to have evolved by chance. To test whether this shared sequence network (SSN) might serve a regulatory function, we synthesized...

Organophosphate (OP) based pesticides are highly toxic compounds that are still widely used in agriculture around the world. According to World Health Organization (WHO) data, it is estimated that between 250,000 and 370,000 deaths occur yearly around the globe as a result of acute intoxications by pesticides. Currently available antidotal drug treatments of severe OP intoxications are symptomatic, do not reduce the level of intoxicating OP in the body and have limited ability to prevent long-term brain damage. Pesticide poisonings present a special therapeutic challenge since in many cases, such as with parathion, their toxicity stems from their metabolites that inhibit the essential enzyme acetylcholinesterase. Our goal is to develop a new treatment strategy for parathion intoxication by combining a catalytic bioscavenger that rapidly degrades the intoxicating parathion-metabolite (paraoxon) in the blood, with a glutamate bioscavenger that reduces the elevated concentration of extracellular glutamate in the brain following OP intoxication. We report on the development of a novel catalytic bioscavenger by directed evolution of serum paraoxonase 1 (PON1) that effectively detoxifies paraoxon in-vivo. We also report preliminary results regarding the utilization of this PON1 variant together with a recombinant human enzyme glutamate oxaloacetate transaminase 1 (rGOT1), suggesting that a dual PON-GOT treatment may increase survival and recovery from parathion and paraoxon intoxications.

The estrogen receptor (ER) is a major prognostic biomarker of breast cancer, currently determined in surgical specimens by immunohistochemistry. Two new ER-targeted probes, pyridine-tetra-acetate-Gd chelate (PTA-Gd) conjugated either to 17β-estradiol (EPTA-Gd) or to tamoxifen (TPTA-Gd), were explored as contrast agents for molecular imaging of ER. In solution, both probes exhibited a micromolar ER binding affinity, fast water exchange rate (∼107 s−1), and water proton-relaxivity of 4.7–6.8 mM−1 s−1. In human breast cancer cells, both probes acted as estrogen agonists and enhanced the water protons T1 relaxation rate and relaxivity in ER-positive as compared to ER-negative cells, with EPTA-Gd showing a higher ER-specific relaxivity than TPTA-Gd. In studies of breast cancer tumors in vivo, EPTA-Gd induced the highest enhancement in ER-positive tumors as compared to ER-negative tumors and muscle tissue, enabling in vivo detection of ER. TPTA-Gd demonstrated the highest enhancement in muscle tissue indicating nonspecific interaction of this agent with muscle components. The extracellular contrast agents, PTA-Gd and GdDTPA, showed no difference in the perfusion capacity of ER-positive and -negative tumors confirming the specific interaction of EPTA-Gd with ER. These findings lay a basis for the molecular imaging of the ER using EPTA-Gd as a template for further developments. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.

Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by ∼10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) ∼ 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.

Talin1 is a key integrin coactivator. We investigated the roles of this cytoskeletal adaptor and its target integrins in B-cell lymphogenesis, differentiation, migration, and function. Using CD19 Cre-mediated depletion of talin1 selectively in B cells, we found that talin1 was not required for B-cell generation in the bone marrow or for the entry of immature B cells to the white pulp of the spleen. Loss of talin1 also did not affect B-cell maturation into follicular B cells but compromised differentiation of marginal zone B cells. Nevertheless, serum IgM and IgG levels remained normal. Ex vivo analysis of talin1-deficient spleen B cells indicated a necessary role for talin1 in LFA-1 and VLA-4 activation stimulated by canonical agonists, but not in B-cell chemotaxis. Consequently, talin1 null B splenocytes could not enter lymph nodes nor return to the bone marrow. Talin1 deficiency in B cells was also impaired in the humoral response to a T cell-dependent antigen. Collectively, these results indicate that talin1 is not required for follicular B-cell maturation in the spleen or homeostatic humoral immunity but is critical for integrin-dependent B lymphocyte emigration to lymph nodes and optimal immunity against T-dependent antigens.

Although mesenchymal stromal cells (MSCs) exhibit marked immunoregulatory activity through multiple mechanisms, their potential to completely evade rejection upon transplantation into allogeneic recipients is controversial. To directly address this controversy, the survival of luciferase-labeled MSCs (Luc+ MSCs) was evaluated by imaging in allogeneic recipients. This analysis showed that although MSCs exhibited longer survival compared to fibroblasts (Fib), their survival was significantly shorter compared to that exhibited in syngeneic or in immune-deficient Balb-Nude or non-obese diabetic severe combined immunodeficiency (NOD-SCID) recipients. Graft rejection in re-challenge experiments infusing Luc+ Fib into mice, which had previously rejected Luc+ MSCs, indicated potential induction of immune memory by the MSCs. This was further analyzed in T-cell antigen receptor (TCR) transgeneic mice in which either CD4 TEA mice or CD8 T cells (2C mice) bear a TCR transgene against a specific MHC I or MHC II, respectively. Thus, following a re-challenge with MSCs expressing the cognate MHC haplotype, an enhanced percentage of 2C CD8+ or TEA CD4+ T cells exhibited a memory phenotype (CD122+, CD44+, and CD62Llow). Collectively, these results demonstrate that MSCs are not intrinsically immune-privileged, and under allogeneic settings, these cells induce rejection, which is followed by an immune memory. Considering that the use of allogeneic or even a third party (“off the shelf”) MSCs is commonly advocated for a variety of clinical applications, our results strongly suggest that long-term survival of allogeneic MSCs likely represents a major challenge. Disclosure of potential conflicts of interest is found at the end of this article.

Intravenous immunoglobulin (IVIG) administration has been beneficially used for the treatment of a variety of autoimmune diseases including myasthenia gravis (MG). We have demonstrated that IVIG administration in experimental autoimmune MG (EAMG) results in suppression of disease that is accompanied by decreased Th1 cell and B cell proliferation. Chromatography of pooled human immunoglobulins (IVIGs) on immobilized IgG, isolated from rats with EAMG, results in a complete depletion of the suppressive activity of the IVIG. Moreover, the eluate from this EAMG-specific antibody column retains the immunosuppressive activity of IVIG. This study supports the notion that the therapeutic effect of IVIGs is mediated by an antigen-specific anti-immunoglobulin (anti-idiotypic) activity that is essential for its suppressive activity.

The mononuclear phagocyte (MP) system is a body-wide macrophage (MΦ) and dendritic cell (DC) network, which contributes to tissue homeostasis, inflammation, and immune defense. The in vivo origins of MPs remain poorly understood. Here, we use an adoptive precursor cell transfer strategy into MP-depleted mice to establish the in vivo differentiation sequence from a recently identified MΦ/DC-restricted bone marrow (BM) precursor (MDP) via BM and blood intermediates to peripheral MΦs and DCs. We show that MDPs are in vivo precursors of BM and blood monocytes. Interestingly, grafted Gr1high “inflammatory” blood monocytes shuttle back to the BM in the absence of inflammation, convert into Gr1low monocytes, and contribute further to MP generation. The grafted monocytes give rise to DCs in the intestinal lamina propria and lung, but not to conventional CD11chigh DCs in the spleen, which develop during homeostasis from MDPs without a monocytic intermediate.

Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFP) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis. (Cancer Res 2006; 66(16): 8037-41)

Tumor response to blood borne drugs is critically dependent on the efficiency of vascular delivery and transcapillary transfer. However, increased tumor interstitial fluid pressure (IFP) forms a barrier to transcapillary transfer, leading to resistance to drug delivery. We present here a new, noninvasive method which estimates IFP and its spatial distribution in vivo using contrast-enhanced magnetic resonance imaging (MRI). This method was tested in ectopic human non–small-cell lung cancer which exhibited a high IFP of ∼28 mm Hg and, for comparison, in orthotopic MCF7 human breast tumors which exhibited a lower IFP of ∼14 mm Hg, both implanted in nude mice. The MRI protocol consisted of slow infusion of the contrast agent [gadolinium-diethylenetriaminepentaacetic acid (GdDTPA)] into the blood for ∼2 hours, sequential acquisition of images before and during the infusion, and measurements of T1 relaxation rates before infusion and after blood and tumor GdDTPA concentration reached a steady state. Image analysis yielded parametric images of steady-state tissue GdDTPA concentration with high values of this concentration outside the tumor boundaries, ∼1 mmol/L, declining in the tumor periphery to ∼0.5 mmol/L, and then steeply decreasing to low or null values. The distribution of steady-state tissue GdDTPA concentration reflected the distribution of IFP, showing an increase from the rim inward, with a high IFP plateau inside the tumor. The changes outside the borders of the tumors with high IFP were indicative of convective transport through the interstitium. This work presents a noninvasive method for assessing the spatial distribution of tumor IFP and mapping barriers to drug delivery and transport. (Cancer Res 2006; 66(8): 4159-66)

The clinical detection of evolving acute tubular necrosis (ATN) and differentiating it from other causes of renal failure are currently limited. The maintenance of the corticomedullary sodium gradient, an indicator of normal kidney function, is presumably lost early in the course of ATN. Herein, sodium magnetic resonance imaging (23Na MRI) was applied to study the early alteration in renal sodium distribution in rat kidneys 6 h after the induction of ATN. Three-dimensional gradient echo sodium images were recorded at 4.7 T with high spatial resolution. ATN was produced by the administration of radiologic contrast medium, combined with inhibition of nitric oxide and prostaglandin synthesis. The sodium images revealed that the sham-controlled kidney exhibited a linear increase in sodium concentration along the corticomedullary axis of 30+/-2 mmol/l/mm, resulting in an inner medulla to cortex sodium ratio of 4.3+/-0.3 (n=5). In the ATN kidney, however, the cortico-outer medullary sodium gradient was reduced by 21% (P0.01, n=7) and the inner medulla to cortex sodium ratio was decreased by 40% (P0.001, n=7). Small, though significant, increments in plasma creatinine at this time inversely correlated with the decline in the corticomedullary sodium gradient. Histological findings demonstrated outer medullary ATN involving 4% of medullary thick ascending limbs. Hence, 23Na MRI non-invasively quantified changes in the corticomedullary sodium gradient in the ATN kidney when morphologic tubular injury was still focal and very limited. MRI detection of corticomedullary sodium gradient abnormalities may serve to identify evolving ATN at its early phases.

Renal function is highly correlated with the sodium concentration gradient along the corticomedullary axis. The application of 3D high-resolution sodium magnetic resonance imaging (MRI) provided a means to quantify in vivo the spatial and temporal changes in renal tissue sodium concentration under normal and diuretic conditions. A detailed, pixel-by-pixel analysis of the intact rat kidney sodium MR images yielded a quantitative measure of the corticomedullary sodium gradient before and at early and later times after the administration of two distinct diuretic agents, furosemide and mannitol. Furosemide, a loop diuretic, induced a fivefold reduction in the cortical-outer medullary sodium gradient, whereas mannitol, an osmotic diuretic, did not affect this gradient. Both diuretics induced a 50% decrease in the sodium concentration of the inner medulla; however, mannitol exerted its effect twice as fast as furosemide with a 2.5-min exponential decay constant. These specific changes were attributed to the different mechanism of action and site of activity of each diuretic agent. Thus, high-resolution (23)Na MRI offers a unique, noninvasive tool for functional imaging of the kidney physiology.

In vivo mapping of the transcapillary fluxes in tumors can help predict the efficacy of delivery of blood-borne anticancer drugs. These fluxes are primarily affected by the vascular permeability and the pressure gradients across the blood vessels’ walls. We describe herein high-resolution dynamic contrast-enhanced magnetic resonance imaging of the influx and outflux transcapillary transfer rates in vivo in invasive MDA-MB-231 tumors orthotopically inoculated in severe combined immunodeficient mice. The tumors were noted for rapid growth, impaired drainage of fluid, and subsequent formation of cysts. Consequently, the time evolution of the contrast enhancement, induced by i.v. injection of Gadolinium diethylene-triamine-penta-acetate, exhibited two distinct patterns: transcapillary transfer in the cellular regions and simple diffusion in the cyst fluid. Both processes were analyzed at pixel resolution applying to each a physiological model and a corresponding algorithm. In the cellular region, the influx and outflux transcapillary transfer rates decreased during tumor growth; however, an increased disparity between the transfer constants was observed, with the outflux rate exceeding the influx rate. This quantitative spatial and temporal mapping of this disparity can provide a means to assess the physiological barriers to tracer delivery. It is hypothesized that both the increased disparity in transcapillary transfer rates and impaired fluid drainage in these tumors could arise from the development of interstitial hypertension.

Diffusion MRI studies revealed specific morphological and physiological properties of MCF7 tumors implanted in the mammary gland of immunodeficient mice. These tumors mimic the histological and pathophysiological properties of human breast cancer in patients. The experiments were conducted by (1) applying varying diffusion gradient strengths, G d , from 0 to 20 G/cm and a short diffusion time (t d = 16 ms) in order to minimize the effect of restriction and exchange of water between the intra- and extracellular compartments, and (2) applying a strong constant gradient and diffusion times up to 96 ms, revealing water restriction and exchange. The normalized signal intensity was plotted against the diffusion weighting factor b, taking into account interaction with the imaging gradients. The curves were analyzed by applying a bi-exponential decay function assuming two exchanging water compartments, with fast and slow diffusion coefficients. The amplitudes and decay constants of the two exponents, a fast and a slow one, were related to the fraction and apparent diffusion coefficients of the extra- and intracellular water, respectively, considering contributions of restriction and exchange. During tumor progression the distribution of the diffusion parameters for the same experimental protocol varied and became less homogeneous. This was predominantly due to variations in the cellularity and increased necrosis. Upon treatment of the tumors with a new anti-estrogenic drug, tamoxifen methiodide, the changes in the diffusion parameters indicated increased cell swelling. Hence, this cytostatic response to treatment was detected before actual cell death was apparent. The potential capacity of diffusion MRI is of high clinical relevance and may help improve the noninvasive diagnosis and followup of treatment of this devastating disease.

Imaging of the vasculature and its functioning over the entire lesion may significantly aid in cancer diagnosis, assessment of prognosis, and therapeutic evaluation. In the current study we present a dynamic three-dimensional deuterium magnetic resonance imaging method that determines the intravascular volume fraction and water perfusion rate at a resolution of 2 mm(2)/pixel. The method was tested and utilized to characterize the vasculature of orthotopic MCF7 human breast cancer tumors in CD1-NU athymic mice. A new algorithm based on Patlak's kinetic model was developed to analyze the dynamic images acquired during and after termination of infusion with deuterated water. The resulting parametric maps spanned a wide range from 0.4 to 35.2% for the intravascular volume fraction and from 4 x 10(-6) to 3.9 x 10(-3) min(-1) for the perfusion rate and exhibited high intratumoral and intertumoral heterogeneity at both parameters. The intravascular volume fraction did not correlate with the corresponding perfusion rate, demonstrating the irregular outgrowth of tumor neovascularization. Averaging the data or analyzing at spatially degraded resolution completely masked the presence of both "hot spots" and hypoxic loci, highlighting the critical importance of high spatial resolution. The method is applicable to other types of tumors and animal models and may be extended to humans.

Prion diseases are caused by abnormal folding of the prion protein. The paradigm is that the prion protein is not immunogenic because the immune system must be tolerant to such a self protein. In an attempt to identify immunogenic prion peptides, we immunized Lewis rats with peptides that fitted the MHC class II RT1.B 1 motif. Both humoral and cellular immunity to the prion peptides were obtained without any harmful effects to young animals. However, when 8-month-old rats were immunized, a sixth (6/36) of the rats developed severe skin inflammation with concomitant hair loss. These findings suggest that immunity to self-prion peptides can be readily induced in Lewis rats and that this immune response may have pathogenic consequences in older rats. © 2001 Academic Press

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats either by active immunization with myelin basic protein (MBP) or by adoptive transfer using anti-MBP specific CD4+T cells. Treatment with human polyclonal immunoglobulins (IgG) effectively suppressed active EAE. Time-dependent experiments demonstrated that the effect of IgG was manifested only when treatment was given immediately after immunization; administration from day 7 after disease induction did not suppress the disease. In the adoptive transfer model of EAE, IgG had no effect in vivo. However, pretreatment in vitro of the antigen-specific T-cells with IgG inhibited their ability to mediate adoptive EAE, as it did in active EAE. Similarly, in vitro IgG pretreatment of the antigen-specific T-cells suppressed the proliferative response to MBP. Fluorescent Activated Cell Sorter (FACS) analysis demonstrated the binding of IgG to activated T-cell lines that was inhibited by soluble Fc molecules. The differential effects of IgG on active EAE and on the adoptive transfer of EAE suggest that IgG in vivo can suppress disease by acting during the early phase of the immune response which involves naive T cells. The inhibition of T-cell proliferation and adoptive transfer of EAE by incubation of T cells in vitro appears to require higher concentrations of IgG than those obtained in vivo.

Dexanabinol (HU-211) is a synthetic non-psychotropic cannabinoid which suppresses TNF-alpha production in the brain and peripheral blood. The effects of dexanabinol in rat experimental autoimmune encephalomyelitis (EAE) were studied using different doses, modes of administration and time regimes. Dexanabinol, 5 mg/kg i.v. given once after disease onset (day 10), significantly reduced maximal EAE score. Increasing the dose or treatment duration resulted in further suppression of EAE. Drug administration at earlier phases during disease induction was not effective. Histological studies supported the clinical findings demonstrating reduction in the inflammatory response in the brain and spinal cord in animals treated with dexanabinol. The results suggest that dexanabinol may provide an alternative mode of treatment for acute exacerbations of multiple sclerosis (MS).

The differential capacity of iron oxide microspheres and of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) to serve as contrast agents that can map the microcirculation of MCF7 human breast cancer implanted in nude mice has been examined by high resolution MRI. Modulation of signal intensity in T2*-weighted, gradient-echo images after iron oxide administration and the temporal signal enhancement after Gd-DTPA administration were monitored and analyzed at a spatial resolution of 98 x 98 x 500 microm and 195 x 390 x 1,000 microm, respectively. The pathophysiologic features revealed in the contrast-enhanced images were analyzed in reference to those obtained from the corresponding high resolution T2-weighted, spin-echo images and from histologic sections stained with hematoxylin and eosin and with an endothelial cell marker. The results showed that iron oxide microspheres can aid in the characterization of gross histopathologic features and in the assessment of the distribution of the microvasculature, whereas Gd-DTPA estimates the permeability of the microvessels to this agent and determines the cellularity (cell volume fraction) in the vicinity of the vessels.

Purpose:The role of intravenous immunoglobulin (IVIG) in the prevention of severe ovarian hyperstimulation syndrome (OHSS) was evaluated.

Transplantation of T cell–depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders and can mediate immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. Here, we address this barrier using donor-derived central memory CD8+ T cells (Tcms), directed against third-party antigens. Our results show that fully allogeneic or (hostXdonor)F1-Tcm, support donor chimerism (> 6 months) in sublethally irradiated (5.5Gy) mice, without GVHD symptoms. Chimerism under yet lower irradiation (4.5Gy) was achieved by combining Tcm with short-term administration of low-dose Rapamycin. Importantly, this chimerism resulted in successful donor skin acceptance, whereas third-party skin was rejected. Tracking of host anti-donor T cells (HADTCs), that mediate TDBMT rejection, in a novel bioluminescence-imaging model revealed that Tcms both induce accumulation and eradicate HADTCs in the LNs,concomitant with their elimination from other organs, including the BM. Further analysis with 2-photon microcopy revealed that Tcms form conjugates with HADTCs, resulting in decelerated and confined movement of HADTCs within the LNs in an antigen-specific manner. Thus, anti–third-party Tcms support TDBMT engraftment under reduced-conditioning through lymph-node sequestration and deletion of HADTCs, offering a novel and potentially safe approach for attaining stable hematopoietic chimerism.

Dynamic gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced MRI was followed during growth and regression of MCF7 human breast tumors implanted in nude mice in the presence of estrogen and tamoxifen, respectively. Gradient-echo and spin-echo sequences were applied at a temporal resolution of 12 and 100 seconds, respectively, and a spatial resolution of 195 x 390 x 1000 microns. Maps of initial rates of contrast enhancement demonstrated stimulation of local growth of permeable microcapillaries at regions bordering necrotic areas, resulting from tamoxifen treatment. This localized angiogenic stimulation was confirmed by immunohistochemical staining of endothelial cells. After 1 week of tamoxifen treatment, the fraction of tumor pixels exhibiting rapid initial rate of contrast enhancement increased significantly from .28 +/- .05 to .46 +/- .06. In parallel, the fraction of tumor area showing contrast enhancement 3 minutes after Gd-DTPA injection also increased significantly, from .42 +/- .06 to .58 +/- .06. On the basis of these changes, it was possible to assess the response to tamoxifen therapy at an early stage.

The activation of T cells by antigens or mitogens leads to the secretion of cytokines and enzymes that shape the inflammatory response. Among these molecular mediators of inflammation is a heparanase enzyme that degrades the heparan sulfate scaffold of the extracellular matrix (ECM). Activated T cells use heparanase to penetrate the ECM and gain access to the tissues. We now report that among the breakdown products of the ECM generated by heparanase is a trisulfated disaccharide that can inhibit delayed-type hypersensitivity (DTH) in mice. This inhibition of T-cell mediated inflammation in vivo was associated with an inhibitory effect of the disaccharide on the production of biologically active tumor necrosis factor alpha (TNF-alpha) by activated T cells in vitro; the trisulfated disaccharide did not affect T-cell viability or responsiveness generally. Both the in vivo and in vitro effects of the disaccharide manifested a bell-shaped dose-response curve. The inhibitory effects of the trisulfated disaccharide were lost if the sulfate groups were removed. Thus, the disaccharide, which may be a natural product of inflammation, can regulate the functional nature of the response by the T cell to activation. Such a feedback control mechanism could enable the T cell to assess the extent of tissue degradation and adjust its behavior accordingly.

Multiple sclerosis (MS) is a central nervous system demyelinating disease of implicated autoimmune aetiology. The effect was evaluated of intravenous gammaglobulin (IVIg), a successful therapy in various autoimmune diseases, in relapsing-remitting MS patients treated for three years. IVIg treatment significantly reduced the number and severity of acute exacerbations and resulted in a lesser neurological disability. There were no significant short or long-term adverse effects to IVIg treatment. To clarify the putative therapeutic effects of IVIg, this treatment was examined in the animal model of experimental autoimmune encephalomyelitis (EAE) in the rat. IVIg suppressed active EAE in relation to disease severity and duration, despite the presence of T-cell reactivity to specific antigens, while the treatment had no effect on passive EAE induced by adoptive transfer of myelin basic protein specific CD4 + T-cells. It is concluded that IVIg treatment may be a promising treatment in relapsing-remitting MS as it can alter the natural course of the disease.

It has been reported previously that intravenous administration of normal human immunoglobulins (IVIg) to human patients can suppress the clinical signs of certain autoimmune diseases. However, the mechanism(s) by which normal Ig interferes with the various disorders and the scheduling of treatment have been poorly delineated. To study these questions, we examined IVIg treatment of two experimentally induced T cell autoimmune diseases in rats: experimental autoimmune encephalomyelitis (EAE) and adjuvant arthritis (AA). We now report that IVIg treatment (0.4 g/kg) inhibited the active induction of both EAE and AA, and that this treatment did not affect the acquisition of resistance to reinduction of EAE. The importance of the site of administration and schedule of treatment were studied in the AA model. Ig was effective when given intravenously, but not when administrated subcutaneously or intraperitoneally. IVIg treatment was effective when given daily from immunization to outbreak of disease; but it was also effective when given once at the time of immunization or once 2 wk after induction of AA, just at the clinical outbreak of disease. Administration of IVIg between immunization and outbreak of AA was less effective. Prevention of disease by IVIg occurred despite the presence of T cell reactivity to the specific antigens in the disease. In fact, IVIg administrated to naive rats activated T cell reactivity to some self-antigens. Nevertheless, IVIg treatment led to decreased production of the inflammatory cytokine TNF alpha. Thus, IVIg treatment may exert its therapeutic power not by inhibiting T cell recognition of self-antigens, but by inhibiting the biological consequences of T cell recognition.

Histologic overexpression of the estrogen receptor α (ER) is a well-established prognostic marker in breast cancer. Noninvasive imaging techniques that could detect ER overexpression would be useful in a variety of settings where patients' biopsies are problematic to obtain. This study focused on developing, by in vivo MRI, strategies to measure the level of ER expression in an orthotopic mouse model of human breast cancer. Specifically, novel ER-targeted contrast agents based on pyridine-tetra-acetate-Gd(III) chelate (PTA-Gd) conjugated to 17β-estradiol (EPTA-Gd) or to tamoxifen (TPTA-Gd) were examined in ER-positive or ER-negative tumors. Detection of specific interactions of EPTA-Gd with ER were documented that could differentiate ER-positive and ER-negative tumors. In vivo competition experiments confirmed that the enhanced detection capability of EPTA-Gd was based specifically on ER targeting. In contrast, PTA-Gd acted as an extracellular probe that enhanced ER detection similarly in either tumor type, confirming a similar vascular perfusion efficiency in ER-positive and ER-negative tumors in the model. Finally, TPTA-Gd accumulated selectively in muscle and could not preferentially identify ER-positive tumors. Together, these results define a novel MRI probe that can permit selective noninvasive imaging of ER-positive tumors in vivo. Cancer Res; 71(24); 7387–97. ©2011 AACR.

Endogenous tissue inhibitors of metalloproteinases (TIMPs) have key roles in regulating physiological and pathological cellular processes. Imitating the inhibitory molecular mechanisms of TIMPs while increasing selectivity has been a challenging but desired approach for antibody-based therapy. TIMPs use hybrid protein-protein interactions to form an energetic bond with the catalytic metal ion, as well as with enzyme surface residues. We used an innovative immunization strategy that exploits aspects of molecular mimicry to produce inhibitory antibodies that show TIMP-like binding mechanisms toward the activated forms of gelatinases (matrix metalloproteinases 2 and 9). Specifically, we immunized mice with a synthetic molecule that mimics the conserved structure of the metalloenzyme catalytic zinc-histidine complex residing within the enzyme active site. This immunization procedure yielded selective function-blocking monoclonal antibodies directed against the catalytic zinc-protein complex and enzyme surface conformational epitopes of endogenous gelatinases. The therapeutic potential of these antibodies has been demonstrated with relevant mouse models of inflammatory bowel disease. Here we propose a general experimental strategy for generating inhibitory antibodies that effectively target the in vivo activity of dysregulated metalloproteinases by mimicking the mechanism employed by TIMPs.

Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine stromal cell derived factor-1 (SDF-1) to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4(+) human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, whereas β2-adrenergic antagonist inhibited both steady state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady-state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.

Background: Chemokines regulate the pathways that restrict homing of specific subsets of immune cells, and thereby fine tune the immune response at specific lymphoid and peripheral tissues. CCL2 is a chemokine that induces migration of monocytes, memory T cells, and dendritic cells. Previously, we demonstrated that pM levels of CCL2 dramatically inhibit migration of T cells. The aim was to test whether subphysiological doses of CCL2 can ameliorate murine colitis and inflammation-induced colorectal cancer. Methods: TNBS (2,4,6 trinitrobenzene sulfonic acid) colitis and dextran sodium sulfate (DSS) colitis were induced in Balb/c and C57BL/6 mice, respectively. Mice were treated daily with intraperitoneal CCL2 injections. Disease activity was assessed clinically, histologically, and by measuring inflammatory cytokine levels. In addition, an inflammatory cancer model was induced by azoxymethane-DSS (AOM-DSS) in Balb/c mice. Mice were treated daily with CCL2 for 11 weeks and then assessed for number of tumors in the colons. Results: Daily administration of CCL2 (60–120 ng) significantly decreased the development of TNBS- and DSS-induced colitis. In a DSS-AOM model, CCL2-treated mice developed significantly fewer tumors (P < 0.005) at 11 weeks. Chronic inflammation in the CCL2-treated mice was significantly less pronounced as compared to phosphate-buffered saline-treated mice. Conclusions: Administration of pM levels of CCL2 significantly inhibits migration of T cells in amelioration of TNBS and DSS colitis and inhibits development of colorectal cancer in an AOM-DSS colitis model in mice. Thus, pM levels of CCL2 may be clinically beneficial as an antiinflammatory agent in IBD. (Inflamm Bowel Dis 2010)

The effects of 17 beta-estradiol versus tamoxifen on the growth and metabolism of MCF7 human breast cancer cells, in culture and in tumors implanted in nude mice, were studied by 31P and 13C nuclear magnetic resonance spectroscopy and by proton magnetic resonance imaging. In culture, the content of the phosphate metabolites including nucleoside triphosphates (NTP), phosphomonoesters, phosphodiesters and inorganic phosphate (Pi) were not affected by tamoxifen treatment. However, in the presence of estrogen the rate of glucose consumption and lactate production via glycolysis (270 and 280 fmol/cell.h, respectively) were twice that of tamoxifen treated cells. Estrogen rescue of tamoxifen treated cells indicated that glycolysis induction occurs at the early stages of the hormonal response. The in vivo studies included recording of proton images that provided an accurate measure of tumor size and distribution of tumor cells, necrotic regions and stromal tissue. Tamoxifen caused enhanced necrosis extending from the center of the tumor during the first two days of treatment (12 h to 6 days). This was followed by growth of reparative tissue along with tumor regression. Tamoxifen also modified the content of the phosphate metabolites, increasing markedly (P less than 0.0002) the ratio of NTP to Pi from 0.41 before treatment to 1.75 9-19 days after treatment. This change was attributed to the enhanced growth of repair tissue. The results provide new information regarding the response of human breast cancer to hormonal treatment and suggest a mechanism for the induction of tumor regression by tamoxifen.

The role of c-Myc in estrogen regulation of vascular endothelial growth factor (VEGF) and of the vasculature function has been investigated in breast cancer cells and tumors. The studies were performed on MCF7 wild-type cells and MCF7-35im clone, stably transfected with an inducible c-Myc gene. In vitro and ex vivo methods for investigating molecular events were integrated with in vivo magnetic resonance imaging of the vascular function. The results showed that the c-Myc upregulation by estrogen is necessary for the transient induction of VEGF transcription; however, overexpression of c-Myc alone is not sufficient for this induction. Furthermore, both c-Myc and the activated estrogen receptor α (ERα) were shown to co-bind the VEGF promoter in close proximity, indicating a novel mechanism for estrogen regulation of VEGF. Studies of long-term estrogen treatment and overexpression of c-Myc alone demonstrated regulation of stable VEGF expression levels in vitro and in vivo, maintaining steady vascular permeability in tumors. However, withdrawal of estrogen from the tumors resulted in increased VEGF and elevated vascular permeability, presumably due to hypoxic conditions that were found to dominate VEGF overexpression in cultured cells. This work revealed a cooperative role for ERα and c-Myc in estrogen regulation of VEGF and the ability of c-Myc to partially mimic estrogen regulation of angiogenesis. It also illuminated the differences in estrogen regulation of VEGF during transient and long-term sustained treatments and under different microenvironmental conditions, providing a complementary picture of the in vitro and in vivo results.

Our studies focus on the pathways that restrict homing of specific subsets of immune cells, and thereby fine-tune the immune response at specific lymphoid and peripheral tissues. Here, we report that CCL2 (at picomolar [pM] levels) renders both murine and human T cells defective in their ability to develop CCR7-triggered activation of LFA-1– and LFA-1–mediated adhesion strengthening to endothelial ICAM-1 both in vitro and in vivo. CCL2 also attenuated lymphocyte chemotaxis toward lymph node chemokines. Consequently, low-dose CCL2 inhibited lymphocyte homing to peripheral lymph nodes but did not affect lymphocyte trafficking through the spleen. Impaired homing of lymphocytes to peripheral lymph nodes resulted in attenuated progression of both asthma and adjuvant arthritis. Thus, pM levels of circulating CCL2 can exert global suppressive effects on T-cell trafficking and differentiation within peripheral lymph nodes, and may be clinically beneficial as an anti-inflammatory agent.

Intravenous immunoglobulin (IVIG) administration has been beneficially used for the treatment of a variety of autoimmune diseases including myasthenia gravis (MG). We have demonstrated that IVIG administration in experimental autoimmune MG (EAMG) results in suppression of disease that is accompanied by decreased Th1 cell and B cell proliferation. Chromatography of pooled human immunoglobulins (IVIG) on immobilized IgG, isolated from rats with EAMG or from MG patients, results in a depletion of the suppressive activity of the IVIG. Moreover, reconstitution of the activity-depleted IVIG with the eluted minute IVIG fractions that had been adsorbed onto the EAMG- or MG-specific columns recovers the depleted immunosuppressive activity. This study supports the notion that the therapeutic effect of IVIG is mediated by an antigen-specific anti-immunoglobulin (anti-idiotypic) activity that is essential for its suppressive activity.

Previously, we reported that a peptide, p458, from the sequence of the mammalian 60-kDa heat shock protein (hsp60) molecule can serve as a carrier in conjugate vaccines with capsular polysaccharide (CPS) molecules of various bacteria. These conjugate vaccines were effective injected in PBS without added adjuvants. We now report that p458 conjugated to pneumococcal CPS type 4 (PS4) manifests innate adjuvant effects: it stimulated mouse macrophages to secrete IL-12 and induced the late appearance of PS4 on the macrophage surface in a TLR4-dependent manner; PS4 alone or conjugated to other carriers did not stimulate macrophages in vitro. The injection of macrophages manifesting PS4 on the surface into mice induced long-term resistance to lethal Streptococcus pneumoniae challenge. The TLR4 ligand LPS could also induce the late appearance on the surface of unconjugated PS4 and resistance to challenge in injected mice. Resistance was not induced by macrophages containing only internalized PS4 or by pulsed macrophages that had been lysed. Glutaraldehyde-fixed macrophages pulsed with PS4 did induce resistance to lethal challenge. Moreover, bone marrow-derived dendritic cells activated by LPS and pulsed with unconjugated CPS were also effective in inducing resistance to lethal challenge. Resistance induced by the PS4-pulsed bone marrow-derived dendritic cell was specific for pneumococcal CPS serotypes (type 3 or type 4) and was associated with the induction of CPS-specific IgG and IgM Abs.