Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle Antibodies (SNAbs) for Selective Depletion of Target Cells.

Monoclonal antibodies (mAb) have had a transformative impact on treating cancers and immune disorders. However, their use is limited by high development time and monetary cost, manufacturing complexities, suboptimal pharmacokinetics, and availability of disease-specific targets. To address some of these challenges, we developed an entirely synthetic, multivalent, Janus nanotherapeutic platform, called Synthetic Nanoparticle Antibodies (SNAbs). SNAbs, with phage-display-identified cell-targeting ligands on one "face" and Fc-mimicking ligands on the opposite "face", were synthesized using a custom, multistep, solid-phase chemistry method. SNAbs efficiently targeted and depleted myeloid-derived immune-suppressor cells (MDSCs) from mouse-tumor and rat-trauma models, ex vivo. Systemic injection of MDSC-targeting SNAbs efficiently depleted circulating MDSCs in a mouse triple-negative breast cancer model, enabling enhanced T cell and Natural Killer cell infiltration into tumors. Our results demonstrate that SNAbs are a versatile and effective functional alternative to mAbs, with advantages of a plug-and-play, cell-free manufacturing process, and high-throughput screening (HTS)-enabled library of potential targeting ligands.

Reconstitution of Low-Density Lipoproteins with Fatty Acids for the Targeted Delivery of Drugs into Cancer Cells.

Low-density lipoproteins (LDLs) are a class of nanocarriers for the targeted delivery of therapeutics into aberrant cells that overexpress the LDL receptor. A facile procedure is used for reconstituting the hydrophobic core of LDLs with a binary fatty acid mixture. Facilitated by the tumor targeting capability of the apolipoprotein, the reconstituted, drug-loaded LDLs can effectively target cancer cells that overexpress the LDL receptor while showing minor adverse impact on normal fibroblasts. According to a hypothesized mechanism, the reconstituted LDLs can also enable metabolism-triggered drug release while preventing the payloads from lysosomal degradation. This study demonstrates that LDLs reconstructed with fatty acids hold great promise to serve as effective and versatile nanocarriers for targeted cancer therapy.

IL-2-armored peptide-major histocompatibility class I bispecific antibodies redirect antiviral effector memory CD8+ T cells to induce potent anti-cancer cytotoxic activity with limited cytokine release.

T cell engagers (TCEs) are becoming an integral class of biological therapeutic owing to their highly potent ability to eradicate cancer cells. Nevertheless, the widespread utility of classical CD3-targeted TCEs has been limited by narrow therapeutic index (TI) linked to systemic CD4+ T cell activation and aberrant cytokine release. One attractive approach to circumvent the systemic activation of pan CD3+ T cells and reduce the risk of cytokine release syndrome is to redirect specific subsets of T cells. A promising strategy is the use of peptide-major histocompatibility class I bispecific antibodies (pMHC-IgGs), which have emerged as an intriguing modality of TCE, based on their ability to selectively redirect highly reactive viral-specific effector memory cytotoxic CD8+ T cells to eliminate cancer cells. However, the relatively low frequency of these effector memory cells in human peripheral blood mononuclear cells (PBMCs) may hamper their redirection as effector cells for clinical applications. To mitigate this potential limitation, we report here the generation of a pMHC-IgG derivative known as guided-pMHC-staging (GPS) carrying a covalent fusion of a monovalent interleukin-2 (IL-2) mutein (H16A, F42A). Using an anti-epidermal growth factor receptor (EGFR) arm as a proof-of-concept, tumor-associated antigen paired with a single-chain HLA-A *02:01/CMVpp65 pMHC fusion moiety, we demonstrate in vitro that the IL-2-armored GPS modality robustly expands CMVpp65-specific CD8+ effector memory T cells and induces potent cytotoxic activity against target cancer cells. Similar to GPS, IL-2-armored GPS molecules induce modulated T cell activation and reduced cytokine release profile compared to an analogous CD3-targeted TCE. In vivo we show that IL-2-armored GPS, but not the corresponding GPS, effectively expands grafted CMVpp65 CD8+ T cells from unstimulated human PBMCs in an NSG mouse model. Lastly, we demonstrate that the IL-2-armored GPS modality exhibits a favorable developability profile and monoclonal antibody-like pharmacokinetic properties in human neonatal Fc receptor transgenic mice. Overall, IL-2-armored GPS represents an attractive approach for treating cancer with the potential for inducing vaccine-like antiviral T cell expansion, immune cell redirection as a TCE, and significantly widened TI due to reduced cytokine release.

Enhanced immune responses to vaccine antigens in the corneal stroma.

To examine the widely accepted dogma that the eye is an immune-privileged organ that can suppress antigen immunogenicity, we explored systemic immune responses to a model vaccine antigen (tetanus toxoid) delivered to six compartments of the rodent eye (ocular surface, corneal stroma, anterior chamber, subconjunctival space, suprachoroidal space, vitreous body). We discovered that antigens delivered to corneal stroma induced enhanced, rather than suppressed, antigen-specific immune responses, which were 18- to 30-fold greater than conventional intramuscular injection and comparable to intramuscular vaccination with alum adjuvant. Systemic immune responses to antigen delivered to the other ocular compartments were much weaker. The enhanced systemic immune responses after intrastromal injection were related to a sequence of events involving the formation of an antigen "depot" in the avascular stroma, infiltration of antigen-presenting cells, up-regulation of MHC class II and costimulatory molecules CD80/CD86, and induction of lymphangiogenesis in the corneal stroma facilitating sustained presentation of antigen to the lymphatic system. These enhanced immune responses in corneal stroma suggest new approaches to medical interventions for ocular immune diseases and vaccination methods.

Robust production of monovalent bispecific IgG antibodies through novel electrostatic steering mutations at the CH1-Cλ interface.

Bispecific antibodies represent an increasingly large fraction of biologics in therapeutic development due to their expanded scope in functional capabilities. Asymmetric monovalent bispecific IgGs (bsIgGs) have the additional advantage of maintaining a native antibody-like structure, which can provide favorable pharmacology and pharmacokinetic profiles. The production of correctly assembled asymmetric monovalent bsIgGs, however, is a complex engineering endeavor due to the propensity for non-cognate heavy and light chains to mis-pair. Previously, we introduced the DuetMab platform as a general solution for the production of bsIgGs, which utilizes an engineered interchain disulfide bond in one of the CH1-CL domains to promote orthogonal chain pairing between heavy and light chains. While highly effective in promoting cognate heavy and light chain pairing, residual chain mispairing could be detected for specific combinations of Fv pairs. Here, we present enhancements to the DuetMab design that improve chain pairing and production through the introduction of novel electrostatic steering mutations at the CH1-CL interface with lambda light chains (CH1-Cλ). These mutations work together with previously established charge-pair mutations at the CH1-CL interface with kappa light chains (CH1-Cκ) and Fab disulfide engineering to promote cognate heavy and light chain pairing and enable the reliable production of bsIgGs. Importantly, these enhanced DuetMabs do not require engineering of the variable domains and are robust when applied to a panel of bsIgGs with diverse Fv sequences. We present a comprehensive biochemical, biophysical, and functional characterization of the resulting DuetMabs to demonstrate compatibility with industrial production benchmarks. Overall, this enhanced DuetMab platform substantially streamlines process development of these disruptive biotherapeutics.

Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine.

Despite recent success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability, remain. Although mRNA, pDNA, and viral-vector based vaccines are being administered, no protein subunit-based SARS-CoV-2 vaccine is approved. Molecular adjuvants targeting pathogen-recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulate various PRRs, including toll-like receptors (TLRs) and retinoic-acid-inducible gene I-like receptors (RIG-I). We hypothesized that targeting the same PRRs using adjuvants on nanoparticles along with a stabilized spike (S) protein antigen could provide broad and efficient immune responses. Formulations targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer-nanoparticles (NPs) were combined with the S1 subunit of S protein and assessed in vitro with isogeneic mixed lymphocyte reactions (iso-MLRs). For in vivo studies, the adjuvanted nanoparticles were combined with stabilized S protein and assessed using intranasal and intramuscular prime-boost vaccination models in mice. Combination NP-adjuvants targeting both TLR and RIG-I (MPLA+PUUC, CpG+PUUC, or R848+PUUC) differentially increased proinflammatory cytokine secretion (IL-1ß, IL-12p70, IL-27, IFN-ß) by APCs cultured in vitro, and induced differential T cell proliferation. When delivered intranasally, MPLA+PUUC NPs enhanced local CD4+CD44+ activated memory T cell responses while MPLA NPs increased anti-S-protein-specific IgG and IgA in the lung. Following intramuscular delivery, PUUC-carrying NPs induced strong humoral immune responses, characterized by increases in anti-S-protein IgG and neutralizing antibody titers and germinal center B cell populations (GL7+ and BCL6+ B cells). MPLA+PUUC NPs further boosted S-protein-neutralizing antibody titers and T follicular helper cell populations in draining lymph nodes. These results suggest that SARS-CoV-2-mimicking adjuvants and subunit vaccines could lead to robust and unique route-specific adaptive immune responses and may provide additional tools against the pandemic.

Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine.

Despite success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability remain. Molecular adjuvants targeting pattern recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulates various PRRs, including toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors. We hypothesized that targeting PRRs using molecular adjuvants on nanoparticles (NPs) along with a stabilized spike protein antigen could stimulate broad and efficient immune responses. Adjuvants targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer NPs were combined with the S1 subunit of spike protein and assessed in vitro with isogeneic mixed lymphocyte reactions (isoMLRs). For in vivo studies, the adjuvant-NPs were combined with stabilized spike protein or spike-conjugated NPs and assessed using a two-dose intranasal or intramuscular vaccination model in mice. Combination adjuvant-NPs simultaneously targeting TLR and RIG-I receptors (MPLA+PUUC, CpG+PUUC, and R848+PUUC) differentially induced T cell proliferation and increased proinflammatory cytokine secretion by APCs in vitro. When delivered intranasally, MPLA+PUUC NPs enhanced CD4+CD44+ activated memory T cell responses against spike protein in the lungs while MPLA NPs increased anti-spike IgA in the bronchoalveolar (BAL) fluid and IgG in the blood. Following intramuscular delivery, PUUC NPs induced strong humoral immune responses, characterized by increases in anti-spike IgG in the blood and germinal center B cell populations (GL7+ and BCL6+ B cells) in the draining lymph nodes (dLNs). MPLA+PUUC NPs further boosted spike protein-neutralizing antibody titers and T follicular helper cell populations in the dLNs. These results suggest that protein subunit vaccines with particle-delivered molecular adjuvants targeting TLR4 and RIG-I could lead to robust and unique route-specific adaptive immune responses against SARS-CoV-2.

Synthesis of hydrophilic superparamagnetic magnetite nanoparticles via thermal decomposition of Fe(acac), in 80 vol% TREG + 20 vol% TREM.

In this paper, we report single step synthesis of hydrophilic superparamagnetic magnetite nanoparticles by thermolysis of Fe(acac)3 and their characterization of the properties relevant to biomedical applications like hyperthermia and magnetic resonance imaging (MRI). Size and morphology of the particles were determined by Transmission electron microscopy (TEM) while phase purity and structure of the particles were identified by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Magnetic properties were evaluated using vibrating sample magnetometer (VSM) and superconducting quantum interference device (SQUID) measurements. The as prepared nanoparticles were found to be superparamagnetic with the blocking temperature of 136 K and were easily suspendable in water. Cytotoxicity studies on human cervical (SiHa), mouse melanoma (B16F10) and mouse primary fibroblast cells demonstrated that up to a dose of 0.1 mg/ml, the magnetite nanoparticles were nontoxic to the cells. To evaluate the feasibility of their uses in hyperthermia and MRI applications, specific absorption rate (SAR) and spin-spin relaxation time (T2) were measured respectively. SAR has been calculated to be above 80 Watt/g for samples with the iron concentration of 5-20 mg/ml at 10 kA/m AC magnetic field and 425 kHz frequency. r2 relaxivity value was measured as 358.4 mM(-1)S(-1) which is almost double as compared to that of the Resovist, a commercially available MRI contrast agent. Thus the as-prepared magnetite nanoparticles may be used for hyperthermia and MRI applications due to their promising SAR and r2 values.

Single-cell RNA-seq of out-of-thaw mesenchymal stromal cells shows tissue-of-origin differences and inter-donor cell-cycle variations.

BACKGROUND: Human Mesenchymal stromal cells (hMSCs) from various tissue sources are widely investigated in clinical trials. These MSCs are often administered to patients immediately after thawing the cryopreserved product (out-of-thaw), yet little is known about the single-cell transcriptomic landscape and tissue-specific differences of out-of-thaw human MSCs. METHODS: 13 hMSC samples derived from 10 "healthy" donors were used to assess donor variability and tissue-of-origin differences in single-cell gene expression profiles. hMSCs derived and expanded from the bone marrow (BM) or cord tissue (CT) underwent controlled-rate freezing for 24 h. Cells were then transferred to the vapor phase of liquid nitrogen for cryopreservation. hMSCs cryopreserved for at least one week, were characterized immediately after thawing using a droplet-based single-cell RNA sequencing method. Data analysis was performed with SC3 and SEURAT pipelines followed by gene ontology analysis. RESULTS: scRNA-seq analysis of the hMSCs revealed two major clusters of donor profiles, which differ in immune-signaling, cell surface properties, abundance of cell-cycle related transcripts, and metabolic pathways of interest. Within-sample transcriptomic heterogeneity is low. We identified numerous differentially expressed genes (DEGs) that are associated with various cellular functions, such as cytokine signaling, cell proliferation, cell adhesion, cholesterol/steroid biosynthesis, and regulation of apoptosis. Gene-set enrichment analyses indicated different functional pathways in BM vs. CT hMSCs. In addition, MSC-batches showed significant variations in cell cycle status, suggesting different proliferative vs. immunomodulatory potential. Several potential transcript-markers for tissue source differences were identified for further investigation in future studies. In functional assays, both BM and CT MSCs suppressed macrophage TNFα secretion upon interferon stimulation. However, differences between donors, tissue-of-origin, and cell cycle are evident in both TNF suppression and cytokine secretion. CONCLUSIONS: This study shows that donor differences in hMSC transcriptome are minor relative to the intrinsic differences in tissue-of-origin. hMSCs with different transcriptomic profiles showed potential differences in functional characteristics. These findings contribute to our understanding of tissue origin-based differences in out-of-thaw therapeutic hMSC products and assist in the identification of cells with immune-regulatory or survival potential from a heterogeneous MSC population. Our results form the basis of future studies in correlating single-cell transcriptomic markers with immunomodulatory functions.

Development of systemic immune dysregulation in a rat trauma model of biomaterial-associated infection.

Orthopedic biomaterial-associated infections remain a major clinical challenge, with Staphylococcus aureus being the most common pathogen. S. aureus biofilm formation enhances immune evasion and antibiotic resistance, resulting in a local, indolent infection that can persist long-term without symptoms before eventual hardware failure, bone non-union, or sepsis. Immune modulation is an emerging strategy to combat host immune evasion by S. aureus. However, most immune modulation strategies are focused on local immune responses at the site of infection, with little emphasis on understanding the infection-induced and orthopedic-related systemic immune responses of the host, and their role in local infection clearance and tissue regeneration. This study utilized a rat bone defect model to investigate how implant-associated infection affects the systemic immune response. Long-term systemic immune dysregulation was observed with a significant systemic decrease in T cells and a concomitant increase in immunosuppressive myeloid-derived suppressor cells (MDSCs) compared to non-infected controls. Further, the control group exhibited a regulated and coordinated systemic cytokine response, which was absent in the infection group. Multivariate analysis revealed high levels of MDSCs to be most correlated with the infection group, while high levels of T cells were most correlated with the control group. Locally, the infection group had attenuated macrophage infiltration and increased levels of MDSCs in the local soft tissue compared to non-infected controls. These data reveal the widespread impacts of an orthopedic infection on both the local and the systemic immune responses, uncovering promising targets for diagnostics and immunotherapies that could optimize treatment strategies and ultimately improve patient outcomes.

TLR7 and RIG-I dual-adjuvant loaded nanoparticles drive broadened and synergistic responses in dendritic cells in vitro and generate unique cellular immune responses in influenza vaccination.

Although the existing flu vaccines elicit strong antigen-specific antibody responses, they fail to provide effective, long term protection - partly due to the absence of robust cellular memory immunity. We hypothesized that co-administration of combination adjuvants, mirroring the flu-virus related innate signaling pathways, could elicit strong cellular immunity. Here, we show that the small molecule adjuvant R848 and the RNA adjuvant PUUC, targeting endosomal TLR7s and cytoplasmic RLRs respectively, when delivered together in polymer nanoparticles (NP), elicits a broadened immune responses in mouse bone marrow-derived dendritic cells (mBMDCs) and a synergistic response in both mouse and human plasmacytoid dendritic cells (pDCs). In mBMDCs, NP-R848-PUUC induced both NF-κB and interferon signaling. Interferon responses to co-delivered R848 and PUUC were additive in human peripheral blood mononuclear cells (PBMCs) and synergistic in human FLT3-differentiated mBMDCs and CAL-1 pDCs. Vaccination with NPs loaded with H1N1 Flu antigen, R848, and PUUC increased percentage of CD8+ T-cells in the lungs, percentage of antigen-specific CD4-T-cells in the spleen, and enhanced overall cytokine-secreting T cell percentages upon antigen restimulation. Also, in the spleen, T lymphopenia, especially after in vitro restimulation with dual adjuvants, was observed, indicating highly antigen-reactive T cells. Our results demonstrate that simultaneous engagement of TLR7 and RIG-I pathways using particulate carriers is a potential approach to improve cellular immunity in flu vaccination.

Polymeric Pathogen-Like Particles-Based Combination Adjuvants Elicit Potent Mucosal T Cell Immunity to Influenza A Virus.

Eliciting durable and protective T cell-mediated immunity in the respiratory mucosa remains a significant challenge. Polylactic-co-glycolic acid (PLGA)-based cationic pathogen-like particles (PLPs) loaded with TLR agonists mimic biophysical properties of microbes and hence, simulate pathogen-pattern recognition receptor interactions to safely and effectively stimulate innate immune responses. We generated micro particle PLPs loaded with TLR4 (glucopyranosyl lipid adjuvant, GLA) or TLR9 (CpG) agonists, and formulated them with and without a mucosal delivery enhancing carbomer-based nanoemulsion adjuvant (ADJ). These adjuvants delivered intranasally to mice elicited high numbers of influenza nucleoprotein (NP)-specific CD8+ and CD4+ effector and tissue-resident memory T cells (TRMs) in lungs and airways. PLPs delivering TLR4 versus TLR9 agonists drove phenotypically and functionally distinct populations of effector and memory T cells. While PLPs loaded with CpG or GLA provided immunity, combining the adjuvanticity of PLP-GLA and ADJ markedly enhanced the development of airway and lung TRMs and CD4 and CD8 T cell-dependent immunity to influenza virus. Further, balanced CD8 (Tc1/Tc17) and CD4 (Th1/Th17) recall responses were linked to effective influenza virus control. These studies provide mechanistic insights into vaccine-induced pulmonary T cell immunity and pave the way for the development of a universal influenza and SARS-CoV-2 vaccines.

Impaired bone healing following treatment of established nonunion correlates with serum cytokine expression.

Delayed union and nonunion are a significant concern in long bone fractures and spinal fusions. Treatment of nonunion often entails multiple revision surgeries that further increase the financial, physical, and emotional burden on patients. The optimal treatment strategy for nonunions remains unclear in many cases, and the risk of complications after revision procedures remains high. This is in part due to our limited understanding of the biological mechanisms that inhibit proper bone healing and lead to nonunion. And yet, few preclinical models directly investigate how healing is impacted after establishment of nonunion, with most instead primarily focusing on treatment immediately after a fresh bone injury. Here, we utilized a critical size femoral defect model in rats where treatment was delayed 8 weeks post-injury, at which time nonunion was established. In this study, acute and delayed treatments with bone morphogenetic protein-2 (BMP-2) were assessed. We found that delayed treatment resulted in decreased bone formation and reduced mechanical strength compared to acute treatment, even when BMP-2 dose was increased by 2.5 times the acute treatment dose. Interestingly, serum cytokine analysis at 12 weeks post-treatment revealed signs of chronic immune dysregulation after delayed treatment. In particular, non-responders (rats that did not exhibit defect bridging) demonstrated higher overall expression of inflammatory cytokines, including TNFα and IL-1ß, compared to responders. These findings suggest that re-establishing long-term immune homeostasis may be critical for successful bone healing, particularly after nonunion. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:299-307, 2019.

Modification of primary amines to higher order amines reduces in vivo hematological and immunotoxicity of cationic nanocarriers through TLR4 and complement pathways.

For decades, cationic polymer nanoparticles have been investigated for nucleic acid delivery. Despite promising in vitro transfection results, most formulations have failed to translate into the clinic due to significant in vivo toxicity - especially when delivered intravenously. To address this significant problem, we investigated the detailed mechanisms that govern the complex in vivo systemic toxicity response to common polymeric nanoparticles. We determined that the toxicity response is material dependent. For branched polyethylenimine (bPEI) nanoparticles - toxicity is a function of multiple pathophysiological responses - triggering of innate immune sensors, induction of hepatic toxicity, and significant alteration of hematological properties. In contrast, for chitosan-based nanoparticles - systemic toxicity is primarily driven through innate immune activation. We further identified that modification of primary amines to secondary and tertiary amines using the small molecule imidazole-acetic-acid (IAA) ameliorates in vivo toxicity from both nanocarriers by different, material-specific mechanisms related to Toll-like receptor 4 activation (for bPEI) and complement activation driven neutrophil infiltration (for chitosan), respectively. Our results provide a detailed roadmap for evaluating in vivo toxicity of nanocarriers and identifies potential opportunities to reduce toxicity for eventual clinical translation.

Extended delivery of vaccines to the skin improves immune responses.

Vaccines prevent 2-3 million childhood deaths annually; however, low vaccine efficacy and the resulting need for booster doses create gaps in immunization coverage. In this translational study, we explore the benefits of extended release of licensed vaccine antigens into skin to increase immune responses after a single dose in order to design improved vaccine delivery systems. By administering daily intradermal injections of inactivated polio vaccine according to six different delivery profiles, zeroth-order release over 28 days resulted in neutralizing antibody titers equivalent to two bolus vaccinations administered one month apart. Vaccinations following this profile also improved immune responses to tetanus toxoid and subunit influenza vaccine but not a live-attenuated viral vaccine, measles vaccine. Finally, using subunit influenza vaccine, we demonstrated that daily vaccination by microneedle patch induced a potent, balanced humoral immunity with an increased memory response compared to bolus vaccination. We conclude that extended presentation of antigen in skin via intradermal injection or microneedle patch can enhance immune responses and reduce the number of vaccine doses, thereby enabling increased vaccination efficacy.

3D in vitro microvascular model-based lymphoma model.

Diffuse large B-cell lymphoma (DLBCL) is a particularly aggressive cancer, impacting the lives of approximately 20,000 people annually in the United States. Elucidating cellular interactions that occur within the microenvironment of DLBCL tumors is crucial to the successful development of therapeutic strategies for this condition. As the in vivo microenvironment of DLBCL is quite complex and variable, in vitro platforms that can sufficiently recapitulate these multifaceted cellular interactions without introducing the complexities of in vivo systems are vital for understanding the pathophysiology of this disease. In this chapter, we present a method for fabrication and development of an in vitro DLBCL-on-chip model in which a fully vascularized, perfusable, microfluidic traverses a DLBCL tumor cell-laden hydrogel that successfully recapitulates hallmark attributes and cellular interaction that occur within the DLBCL tumor microenvironment. As this microfluidic approach makes use of common laboratory items and does not require traditional photolithography to fabricate, this system represents a vital tool that can unlock previously inaccessible research areas of the DLBCL tumor microenvironment to researchers across numerous fields.

A synthetic stroma-free germinal center niche for efficient generation of humoral immunity ex vivo.

B cells play a major role in the adaptive immune response by producing antigen-specific antibodies against pathogens and imparting immunological memory. Following infection or vaccination, antibody-secreting B cells and memory B cells are generated in specialized regions of lymph nodes and spleens, called germinal centers. Here, we report a fully synthetic ex-vivo system that recapitulates the generation of antigen-specific germinal-center (GC) like B cells using material-surface driven polyvalent signaling. This synthetic germinal center (sGC) reaction was effectively induced using biomaterial-based artificial "follicular T helper cells (TFH)" that provided both natural CD40-CD40L ligation as well as crosslinking of CD40 and by mimicking artificial "follicular dendritic cells (FDC)" to provide efficient, polyvalent antigen presentation. The artificial sGC reaction resulted in efficient B cell expansion, immunoglobulin (Ig) class switching, and expression of germinal center phenotypes. Antigen presentation during sGC reaction selectively enhanced the antigen-specific B cell population and induced somatic hyper-mutations for potential affinity maturation. The resulting B cell population consisted primarily of GC-like B cells (centrocytes) as well as some plasma-like B cells expressing CD138. With concurrent cell sorting, we successfully created highly enriched populations of antigen-specific B cells. Adoptive transfer of these GC-like B cells into non-irradiated isogeneic or non-lethally irradiated congenic recipient mice showed successful engraftment and survival of the donor cells for the 4 week test period. We show that this material-surface driven sGC reaction can be successfully applied to not only splenic B cells but also B cells isolated from more therapeutically relevant sources such as peripheral blood mononuclear cells (PBMCs), thus making our current work an exciting prospect in the new era of personalized medicine and custom-immunotherapy.

Comparative evaluation of heating ability and biocompatibility of different ferrite-based magnetic fluids for hyperthermia application.

In this study, lauric acid-coated, superparamagnetic, nanoparticle-based magnetic fluids of different ferrites (Fe(3)O(4), MnFe(2)O(4), and CoFe(2)O(4)) were prepared and compared in terms of heating ability and biocompatibility to evaluate the feasibility of use in hyperthermia treatment of cancer. All the magnetic fluids prepared had particles of average sizes 9-11 nm. Heating ability of these magnetic fluids was evaluated by calorimetric measurement of specific absorption rate (SAR) at 300 kHz frequency and 15 kA/m field. Fe(3)O(4) and MnFe(2)O(4) showed higher SAR (120 and 97 W/g of ferrite, respectively) than CoFe(2)O(4) (37 W/g of ferrite). In vitro study on BHK 21 cell lines showed dose-dependent cell viability for all the magnetic fluids. Threshold-biocompatible ferrite concentration for all the magnetic fluids was 0.1 mg/mL. Above 0.2 mg/mL, CoFe(2)O(4) was more toxic than the other magnetic fluids. On intravenous injection of different doses (50, 200, and 400 mg/kg body weight) of magnetic fluids in mice, no significant changes in hematological and biochemical parameters were observed for Fe(3)O(4) and MnFe(2)O(4). With CoFe(2)O(4), an increase in SGPT levels at a dose rate of 400 mg/kg body weight was observed, indicating its mild hepatotoxic effect. However, histology of different vital organs showed no pathological changes for all the three magnetic fluids. Further, long term in vivo evaluation of biocompatibility of the lauric acid-coated ferrites is warranted. This study shows that lauric acid-coated, superparamagnetic Fe(3)O(4) and MnFe(2)O(4) may be used for hyperthermia treatment and are to be preferred over CoFe(2)O(4).

Can the intestine perform some functions of the kidney?

The majority of patients in countries like India and Pakistan with end-stage renal disease (ESRD) die without renal replacement therapy due to lack of adequate resources. The use of the intestinal mucosa as a semipermeable membrane for removal of urea and creatinine from the body has been previously studied using various types of intestinal lavage for gut dialysis. This study was undertaken in an animal model to assess the applicability, cost of therapy, and acceptability of the method for potential application in humans. Renal failure was induced in six dogs by bilateral ureteric ligation along with six healthy controls. Dialysis fluid was introduced per rectum as an enema, which was repeatedly administered. Clearances of serum creatinine and urea were assessed. Mean recovery of creatinine and urea in dialysate in the present study was around 8.925 mmol/l and around 207.74 micromol//l, respectively. The mean clearances of serum creatinine and urea were, respectively, 0.0683 and 0.0633 ml/sec. Enteral dialysis was effective and, considering its minimal cost (monthly cost will be around US$35-40) vis a vis available methods, it holds promise for the treatment of patients with ESRD. The creation of an appendicostomy for repeated introduction of antegrade enemas would be a consideration.

Combinatorial Delivery of Dual and Triple TLR Agonists via Polymeric Pathogen-like Particles Synergistically Enhances Innate and Adaptive Immune Responses.

Despite decades of research very few vaccine-adjuvants have received FDA approval. Two fundamental challenges plague clinical translation of vaccine-adjuvants: reducing acute toxicities that result from systemic diffusion of many soluble adjuvants, and delivering multiple adjuvants at the same time to mimic the synergistic immune-stimulation of pathogens, while being safe. In order to address these barriers, we evaluated combinations of four clinically relevant immune-agonists, specifically Toll-like receptor (TLR) ligands, using biodegradable, polymer microparticles. We tested them alone and in combinations of 2 or 3, for a total of 10 unique conditions. We evaluated primary bone-marrow-derived Dendritic Cell phenotypes and functionality, and identified several synergistic combinations. We picked a dual and a triple adjuvant combination, TLR4/TLR9 and TLR4/TLR7/TLR9, for further evaluation and found that both combinations promoted antigen cross-presentation in vitro. Studies in mice using the model antigen Ovalbumin, showed that both combinations enhanced lymph node germinal center and T follicular helper cell responses. The triple adjuvant combination showed increased antigen-specific antibody titer with an overall balanced Th1/Th2 response, while the dual combination promoted Th1-polarized IgG responses. Our results show how polymeric particulate-carriers can be adopted to safely deliver combinatorial adjuvants and selectively synergize specific types of immune responses for vaccine applications.