Akt and SHP-1 are DC-intrinsic checkpoints for tumor immunity.
2016; 1 (18)
VACCINES. A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells.
Science (New York, N.Y.)
2015; 348 (6241): aaa8205
BM-derived DC (BMDC) are powerful antigen-presenting cells. When loaded with immune complexes (IC), consisting of tumor antigens bound to antitumor antibody, BMDC induce powerful antitumor immunity in mice. However, attempts to employ this strategy clinically with either tumor-associated DC (TADC) or monocyte-derived DC (MoDC) have been disappointing. To investigate the basis for this phenomenon, we compared the response of BMDC, TADC, and MoDC to tumor IgG-IC. Our findings revealed, in both mice and humans, that upon exposure to IgG-IC, BMDC internalized the IC, increased costimulatory molecule expression, and stimulated autologous T cells. In contrast, TADC and, surprisingly, MoDC remained inert upon contact with IC due to dysfunctional signaling following engagement of Fcγ receptors. Such dysfunction is associated with elevated levels of the Src homology region 2 domain-containing phosphatase-1 (SHP-1) and phosphatases regulating Akt activation. Indeed, concomitant inhibition of both SHP-1 and phosphatases that regulate Akt activation conferred upon TADC and MoDC the capacity to take up and process IC and induce antitumor immunity in vivo. This work identifies the molecular checkpoints that govern activation of MoDC and TADC and their capacity to elicit T cell immunity.
View details for PubMedID 27812544
View details for PubMedCentralID PMC5085602
Distribution of alkaline phosphatase, osteopontin, RANK ligand and osteoprotegerin in calcified human carotid atheroma.
The protein journal
2015; 34 (5): 315–28
Genital Chlamydia trachomatis (Ct) infection induces protective immunity that depends on interferon-γ-producing CD4 T cells. By contrast, we report that mucosal exposure to ultraviolet light (UV)-inactivated Ct (UV-Ct) generated regulatory T cells that exacerbated subsequent Ct infection. We show that mucosal immunization with UV-Ct complexed with charge-switching synthetic adjuvant particles (cSAPs) elicited long-lived protection in conventional and humanized mice. UV-Ct-cSAP targeted immunogenic uterine CD11b(+)CD103(-) dendritic cells (DCs), whereas UV-Ct accumulated in tolerogenic CD11b(-)CD103(+) DCs. Regardless of vaccination route, UV-Ct-cSAP induced systemic memory T cells, but only mucosal vaccination induced effector T cells that rapidly seeded uterine mucosa with resident memory T cells (T(RM) cells). Optimal Ct clearance required both T(RM) seeding and subsequent infection-induced recruitment of circulating memory T cells. Thus, UV-Ct-cSAP vaccination generated two synergistic memory T cell subsets with distinct migratory properties.
View details for DOI 10.1126/science.aaa8205
View details for PubMedID 26089520
View details for PubMedCentralID PMC4605428
Adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release.
2014; 32 (24): 2882–95
Ectopic vascular calcification is a significant component of atherosclerotic disease. Osteopontin (OPN), Osteoprotegerin (OPG), Receptor Activator of NFκB Ligand (RANKL), and alkaline phosphatase (ALP) are each thought to play central roles in the calcification or demineralization of atherosclerotic lesions. Abnormalities in the balance of these proteins may lead to perturbations in bone remodeling and arterial calcification. The purpose of this study was to measure the distribution of these proteins in human carotid lesions and to elucidate possible mechanism(s) whereby they control the deposition or depletion of arterial calcification. Thirty-three patients who had undergone carotid endarterectomy (CEA) within the previous 18 months and 11 control patients were enrolled. CEA specimens were analyzed by EBCT for calcification content in terms of Agatston (AGAT) and Volume scores. CEA specimens were then cut into 5 mm segments which were homogenized and extracted. Extracts were analyzed for tissue levels of calcium, phosphorus, ALP, OPN, RANKL, and OPG. Fasting blood samples were analyzed for the same components. In CEA tissue segments, the calcification levels (CHA AGAT) were inversely associated with the levels of OPG (r = -0.432/-0.579, p < 0.05) and positively associated with the levels of RANKL (r = 0.332/0.415, p < 0.05). In turn, the tissue levels of OPG were associated with homologous serum levels of OPG (r = 0.820/0.389, p < 0.001), and the tissue levels of RANKL were associated with the serum levels of homologous RANKL (r = 0.739/0.666, p < 0.0001). This study suggests that serum levels of OPG and RANKL may be useful biomarkers for estimating the degree of calcification in carotid atherosclerotic lesions.
View details for DOI 10.1007/s10930-015-9620-3
View details for PubMedID 26307009
Engineered nanomedicine for myeloma and bone microenvironment targeting.
Proceedings of the National Academy of Sciences of the United States of America
2014; 111 (28): 10287–92
Augmentation of immunogenicity can be achieved by particulate delivery of an antigen and by its co-administration with an adjuvant. However, many adjuvants initiate strong systemic inflammatory reactions in vivo, leading to potential adverse events and safety concerns. We have developed a synthetic vaccine particle (SVP) technology that enables co-encapsulation of antigen with potent adjuvants. We demonstrate that co-delivery of an antigen with a TLR7/8 or TLR9 agonist in synthetic polymer nanoparticles results in a strong augmentation of humoral and cellular immune responses with minimal systemic production of inflammatory cytokines. In contrast, antigen encapsulated into nanoparticles and admixed with free TLR7/8 agonist leads to lower immunogenicity and rapid induction of high levels of inflammatory cytokines in the serum (e.g., TNF-a and IL-6 levels are 50- to 200-fold higher upon injection of free resiquimod (R848) than of nanoparticle-encapsulated R848). Conversely, local immune stimulation as evidenced by cellular infiltration of draining lymph nodes and by intranodal cytokine production was more pronounced and persisted longer when SVP-encapsulated TLR agonists were used. The strong local immune activation achieved using a modular self-assembling nanoparticle platform markedly enhanced immunogenicity and was equally effective whether antigen and adjuvant were co-encapsulated in a single nanoparticle formulation or co-delivered in two separate nanoparticles. Moreover, particle encapsulation enabled the utilization of CpG oligonucleotides with the natural phosphodiester backbone, which are otherwise rapidly hydrolyzed by nucleases in vivo. The use of SVP may enable clinical use of potent TLR agonists as vaccine adjuvants for indications where cellular immunity or robust humoral responses are required.
View details for DOI 10.1016/j.vaccine.2014.02.027
View details for PubMedID 24593999
View details for PubMedCentralID PMC4059049
A vector-free microfluidic platform for intracellular delivery
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (6): 2082-2087
Bone is a favorable microenvironment for tumor growth and a frequent destination for metastatic cancer cells. Targeting cancers within the bone marrow remains a crucial oncologic challenge due to issues of drug availability and microenvironment-induced resistance. Herein, we engineered bone-homing polymeric nanoparticles (NPs) for spatiotemporally controlled delivery of therapeutics to bone, which diminish off-target effects and increase local drug concentrations. The NPs consist of poly(D,L-lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and bisphosphonate (or alendronate, a targeting ligand). The engineered NPs were formulated by blending varying ratios of the synthesized polymers: PLGA-b-PEG and alendronate-conjugated polymer PLGA-b-PEG-Ald, which ensured long circulation and targeting capabilities, respectively. The bone-binding ability of Ald-PEG-PLGA NPs was investigated by hydroxyapatite binding assays and ex vivo imaging of adherence to bone fragments. In vivo biodistribution of fluorescently labeled NPs showed higher retention, accumulation, and bone homing of targeted Ald-PEG-PLGA NPs, compared with nontargeted PEG-PLGA NPs. A library of bortezomib-loaded NPs (bone-targeted Ald-Bort-NPs and nontargeted Bort-NPs) were developed and screened for optimal physiochemical properties, drug loading, and release profiles. Ald-Bort-NPs were tested for efficacy in mouse models of multiple myeloma (MM). Results demonstrated significantly enhanced survival and decreased tumor burden in mice pretreated with Ald-Bort-NPs versus Ald-Empty-NPs (no drug) or the free drug. We also observed that bortezomib, as a pretreatment regimen, modified the bone microenvironment and enhanced bone strength and volume. Our findings suggest that NP-based anticancer therapies with bone-targeting specificity comprise a clinically relevant method of drug delivery that can inhibit tumor progression in MM.
View details for DOI 10.1073/pnas.1401337111
View details for PubMedID 24982170
View details for PubMedCentralID PMC4104924
Non-invasive assessment of failure torque in rat bones with simulated lytic lesions using computed tomography based structural rigidity analysis
JOURNAL OF BIOMECHANICS
2011; 44 (3): 552-556
Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.
View details for DOI 10.1073/pnas.1218705110
View details for Web of Science ID 000315209800033
View details for PubMedID 23341631
View details for PubMedCentralID PMC3568376
Single-Step Assembly of Homogenous Lipid - Polymeric and Lipid - Quantum Dot Nanoparticles Enabled by Microfluidic Rapid Mixing
2010; 4 (3): 1671-1679
This study applies CT-based structural rigidity analysis (CTRA) to assess failure torque of rat femurs with simulated lytic defects at different locations (proximal and distal femur) and diameters (25% and 50% of the cross-section at the site), and compared the results to those obtained from mechanical testing. Moreover, it aims to compare the correlation coefficients between CTRA-based failure torque and DXA-based aBMD versus actual failure torque. Twenty rats were randomly assigned to four equal groups of different simulated lesions based on size and location. Femurs from each animal underwent micro-computed tomography to assess three-dimensional micro-structural data, torsional rigidity using structural rigidity analysis and dual energy X-ray absorptiometry to assess bone mineral density. Following imaging, all specimens were subjected to torsion. Failure torque predicted from CT-derived structural rigidity measurements was better correlated with mechanically derived failure torque [R(2)=0.85] than was aBMD from DXA [R(2)=0.32]. In summary, the results of this study suggest that computed tomography based structural rigidity analysis can be used to accurately and quantitatively measure the mechanical failure torque of bones with osteolytic lesions in an experimental rat model. Structural rigidity analysis can provide more accurate predictions on maximal torque to mechanical failure than dual energy X-ray absorptiometry based on bone mineral density.
View details for DOI 10.1016/j.jbiomech.2010.09.022
View details for Web of Science ID 000287551000030
View details for PubMedID 20926079
View details for PubMedCentralID PMC4405884
Quantitative Segmentation of Principal Carotid Atherosclerotic Lesion Components by Feature Space Analysis Based on Multicontrast MRI at 1.5 T
IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING
2009; 56 (2): 352-360
A key challenge in the synthesis of multicomponent nanoparticles (NPs) for therapy or diagnosis is obtaining reproducible monodisperse NPs with a minimum number of preparation steps. Here we report the use of microfluidic rapid mixing using hydrodynamic flow focusing in combination with passive mixing structures to realize the self-assembly of monodisperse lipid-polymer and lipid-quantum dot (QD) NPs in a single mixing step. These NPs are composed of a polymeric core for drug encapsulation or a QD core for imaging purposes, a hydrophilic polymeric shell, and a lipid monolayer at the interface of the core and the shell. In contrast to slow mixing of lipid and polymeric solutions, rapid mixing directly results in formation of homogeneous NPs with relatively narrow size distribution that obviates the need for subsequent thermal or mechanical agitation for homogenization. We identify rapid mixing conditions that result in formation of homogeneous NPs and show that self-assembly of polymeric core occurs independent of the lipid component, which only provides stability against aggregation over time and in the presence of high salt concentrations. Physicochemical properties of the NPs including size (35-180 nm) and zeta potential (-10 to +20 mV in PBS) are controlled by simply varying the composition and concentration of precursors. This method for preparation of hybrid NPs in a single mixing step may be useful for combinatorial synthesis of NPs with different properties for imaging and drug delivery applications.
View details for DOI 10.1021/nn901433u
View details for Web of Science ID 000275858200052
View details for PubMedID 20166699
View details for PubMedCentralID PMC2923464
HER-2-Targeted Nanoparticle-Affibody Bioconjugates for Cancer Therapy
2008; 3 (12): 1839-1843
Microfluidic platform for controlled synthesis of polymeric nanoparticles
2008; 8 (9): 2906-2912
The purpose of this paper is to evaluate the capability of feature space analysis (FSA) for quantifying the relative volumes of principal components (thrombus, calcification, fibrous, normal intima, and lipid) of atherosclerotic plaque tissue in multicontrast magnetic resonance images (mc-MRI) acquired in a setup resembling clinical conditions ex vivo. Utilizing endogenous contrast, proton density, T1-weighted, and T2-weighted images were acquired for 13 carotid endarterectomy (CEA) tissues under near-clinical conditions (human 1.5 T GE Excite scanner with sequence parameters comparable to an in vivo acquisition). An FSA algorithm was utilized to segment and quantify the principal components of atherosclerotic plaques. Pilot in vivo mc-MRI images were analyzed in the same way as the ex vivo images for exploring the possible adaptation of this technique to in vivo imaging. Relative abundance of principal plaque components in CEA tissues as determined by mc-MRI/FSA were compared to those measured by histology. Mean differences +/- standard deviations were 5.8 +/- 4.1% for thrombus, 1.5 +/-1.4 % for calcification, 4.0 +/-2.8% for fibrous, 8.2 +/- 10% for normal intima, and 2.4 +/- 2.2% for lipid. Reasonable quantitative agreement between the classification results obtained with FSA and histological data was obtained for near-clinical imaging conditions. Combination of mc-MRI and FSA may have an application for determining atherosclerotic lesion composition and monitoring treatment in vivo.
View details for DOI 10.1109/TBME.2008.2003100
View details for Web of Science ID 000265372700019
View details for PubMedID 19272944
Co-delivery of hydrophobic and hydrophilic drugs from nanoparticle-aptamer bioconjugates
2007; 2 (9): 1268-1271
Quantification of carotid atherosclerotic plaque components using feature space analysis and magnetic resonance imaging.
Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
2006; 1: 3102-3105
A central challenge in the development of drug-encapsulated polymeric nanoparticles is the inability to control the mixing processes required for their synthesis resulting in variable nanoparticle physicochemical properties. Nanoparticles may be developed by mixing and nanoprecipitation of polymers and drugs dissolved in organic solvents with nonsolvents. We used rapid and tunable mixing through hydrodynamic flow focusing in microfluidic channels to control nanoprecipitation of poly(lactic- co-glycolic acid)- b-poly(ethylene glycol) diblock copolymers as a model polymeric biomaterial for drug delivery. We demonstrate that by varying (1) flow rates, (2) polymer composition, and (3) polymer concentration we can optimize the size, improve polydispersity, and control drug loading and release of the resulting nanoparticles. This work suggests that microfluidics may find applications for the development and optimization of polymeric nanoparticles in the newly emerging field of nanomedicine.
View details for DOI 10.1021/nl801736q
View details for Web of Science ID 000259140200053
View details for PubMedID 18656990
Atherosclerosis is one of the main causes of cardiovascular disease, accounting for more than one third of all deaths in the United States, there is a growing need to develop non-invasive techniques to assess the severity of atherosclerotic plaque burden. Recent research has suggested that not the size of the atherosclerotic plaque but rather its composition is indicative for plaque rupture as the underlying event of stroke and acute coronary syndrome. With its excellent soft-tissue contrast, magnetic resonance imaging (MRI) is a favored modality for examining plaque composition. In an ex-vivo study, aimed to show the feasibility of quantifying the components of carotid atherosclerotic plaques in-vivo, we acquired multi-contrast MRI images of 13 freshly excised endarterectomy tissues with commercially available MRI sequences and a human surface coil. Feature space analysis (FSA) was utilized in four representative tissues to determine the total relative abundance of calcific, lipidic, fibrotic, thrombotic and normal components as well as in consecutive 2 mm sections across the carotid bifurcation in each tissue. Excellent qualitative agreement between the FSA results and the results obtained from histological methods was observed. This study demonstrates the feasibility of combining MRI with FSA to quantify carotid atherosclerotic plaques in-vivo.
View details for PubMedID 17945756