Inflammation plays a central role in the pathogenesis of acute lung injury (ALI) during both the acute pneumonitis stage and progression into the chronic fibroproliferative phase, leading to pulmonary fibrosis. Currently, there is an unmet clinical and research need for noninvasive ways to monitor lung inflammation through targeting of immunoregulatory pathways contributing to ALI pathogenesis. In this study, we evaluated the role of targeted imaging of very late antigen-4 (VLA-4), as a key integrin mediating the adhesion and recruitment of immune cells to inflamed tissues, in quantifying lung inflammation in a mouse model of lipopolysaccharide-induced ALI. Methods: ALI was induced by a single intratracheal administration of lipopolysaccharide (10, 20, or 40 μg per mouse) in C57BL/6J mice. Control mice were intratracheally instilled with sterile phosphate-buffered saline. VLA-4-targeted PET/CT was performed 24 h after intravenous injection of a 64 Cu-labeled high-affinity peptidomimetic ligand referred to as 64 Cu-LLP2A, which is conjugated with the chelator (1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) and a polyethylene glycol 4 linker, at day 2 after the induction of ALI. Ex vivo biodistribution of 64 Cu-LLP2A was determined by γ-counting of harvested organs. The severity of lung inflammation was assessed histologically and by measuring the expression of inflammatory markers in the lung tissue lysates using reverse transcription quantitative polymerase chain reaction. Results: Intratracheal lipopolysaccharide instillation led to an acute inflammatory response in the lungs, characterized by increased expression of multiple inflammatory markers and infiltration of myeloid cells, along with a significant and specific increase in 64 Cu-LLP2A uptake, predominantly in a peribronchial distribution. There was a strong correlation between the lipopolysaccharide dose and 64 Cu-LLP2A uptake, as quantified by in vivo PET (R 5 0.69, P , 0.01). Expression levels of both subunits of VLA-4, that is, integrins α 4 and β 1 , significantly correlated with the expression of multiple inflammatory markers, including tumor necrosis factor-α, interleukin-1β, and nitric oxide synthase-2, highlighting the potential of VLA-4 as a surrogate marker of acute lung inflammation. Notably, in vivo 64 Cu-LLP2A uptake significantly correlated with the expression of multiple inflammatory markers and VLA-4. Conclusion: Our study demonstrates the feasibility of molecular imaging of VLA-4, as a mechanistically relevant target in ALI, and the accuracy of VLA-4-targeted PET in quantification of ongoing lung inflammation in a murine model.
The complement system is an integral component of the humoral immune system, and describes a cascade of interacting proteins responsible for the opsonization and lysis of foreign pathogens, in addition to the recruitment of immune cells. However, complement activation is also implicated in the progression and complication of immune dysfunctions such as sepsis. Microparticle (MP) biomaterials capable of tuning the local magnitude of serum complement activation could improve complement-mediated cytotoxicity to serum-resistant bacteria or calm an overactive immune response during sepsis. We demonstrate that model Fc-functionalized microparticles can be designed to either enhance or diminish the local cytotoxic effect of complement activation in human serum. The particles were formed with either the antibody Fc domains oriented outward from the particle surface or randomly adsorbed in a non-oriented fashion. In the oriented Fc form, complement products were directly sequestered to the particle surface, including C5a, a potent anaphylatoxin that, when elevated, is associated with poor sepsis prognosis. The oriented particle also lowered the cytotoxicity of serum and thus decreased the antibiotic effect when compared to serum alone. Conversely, the non-oriented microparticles were found to sequester similar levels of C5a, but much lower levels of iC3b and TCC on the microparticle surface, thereby increasing the amount of the soluble terminal complement complex. In addition, the non-oriented microparticles extend the distance over which TCC forms and enhance serum cytotoxicity to bacteria. Together, these two types of complement-modulating particles provide the first biomaterial that can functionally modify the range of complement activation at sites distant from the particle surface. Thus, biomaterials that exploit Fc presentation provide new possibilities to functionally modulate complement activation to achieve a desired clinical result.
Standard dead-end sample filtration is used to improve sample purity, but is limited as particle build-up fouls the filter, leading to reduced recovery. The fouling layer can be periodically cleared with backflush algorithms applied through a customized fluidic actuator using variable duty cycles, significantly improving particulate recovery percentage. We show a Pulse Width Modulation (PWM) process can periodically backflush the filter membrane to repeatedly interrupt cake formation and reintegrate the fouling layer into the sample, improving net permeate flux per unit volume of sample by partially restoring filter flux capacity. PWM flow for 2.19 um (targeted) and 7.32 um (untargeted) polystyrene microbeads produced 18-fold higher permeate concentration, higher recovery up to 68.5%, and an 8-fold enrichment increase, compared to a uniform flow. As the duty cycle approaches 50%, the recovery percentage monotonically increases after a critical threshold. Further, we developed and validated a mathematical model to determine that fast, small-volume backflush pulses near 50% duty cycle yield higher recovery by decreasing fouling associated with the cake layer. Optimized PWM flow was then used to purify custom particles for immune activation, achieving 3-fold higher recovery percentage and providing a new route to improve purification yields for diagnostic and cellular applications. Dead-end filtration using patterned microsieves, fiber meshwork, and membranes of various materials is a standard technique to isolate desired particles of various sizes and is often used in clinical and laboratory settings for therapeutic and diagnostic applications 1-4. Both biological and physical suspensions can be filtered to yield high purity and enrichment at a high throughput. Dead-end filters are especially susceptible to fouling, however, which leads to lower recovery percentage and yield as a direct result 2,5,6. Because a decreasing yield negatively impacts therapeutic quality 7-9 , clinical and industrial therapeutic manufacturing will frequently change or increase the surface area of the dead-end filter 10 , or switch to crossflow filtration modalities 11,12 , which further decreases the throughput and increases processing time. Membrane fouling is caused by pore blocking followed by cake layer formation, resulting in an exponential decay with time in the flux of permeating particulate 13-19. Membrane fouling also affects crossflow systems and, in this case, numerous studies were conducted to disrupt cake formation and reintegrate particulates into the bulk flow feed stream 11,12,20-25. For example, crossflow filtration can disrupt caking by implementing an oscillatory flow with a sinusoidal flow velocity or a pulsatile flow, consisting of a steady flow with oscillations superimposed 20. These studies examined the effects of numerous waveforms, including variations of sinusoids, saw tooth, and square waves 12,20,23. Oscillatory and pulsatile techniques showed improved clearance of the crossflow membranes, leading to an incre...
Immune checkpoint inhibitors (ICIs) have revolutionized cancer care, but many patients with poorly immunogenic tumors fail to benefit. Preclinical studies have shown that external beam radiotherapy (EBRT) can synergize with ICI to prompt remarkable tumor regression and even eradication. However, EBRT is poorly suited to widely disseminated disease. Targeted radiopharmaceutical therapy (TRT) selectively delivers radiation to both the primary tumor and the metastatic sites, and promising results achieved with this approach have led to regulatory approval of certain agents (e.g., 177 Lu-PSMA-617/Pluvicto for metastatic prostate cancer). To further improve therapeutic outcomes, combining TRT and ICI is a burgeoning research area, both preclinically and in clinical trials. Here we introduce basic TRT radiobiology and survey emerging and clinically translated TRT agents that have been combined with ICI.
Granulomas form during tuberculosis (TB) and restrain bacterial dissemination but are also sites of mycobacterial replication and persistence. The immunometabolic state of cells in granulomas has immunologic and diagnostic relevance and PET-CT with the glucose analog FDG demonstrates that granuloma glucose uptake is dynamic and heterogenous within a host. Basic details on glucose uptake, including the cells responsible for FDG PET signal, have not been resolved and filling these gaps will improve interpretation of PET data in TB. Our objective was to identify relationships between glucose (FDG) uptake and granuloma composition and to identify factors that drive this process in M. tuberculosis-infected cynomolgus macaques. We used glucose transporter 1 (GLUT1) to identity cells that may be using glucose as an energy source in granulomas, and compared these data with the cell’s microenvironment, and the granuloma’s bacteria load and FDG PET data to determine how these factors influence GLUT1 expression. We found that GLUT1 was strongly expressed by myeloid cell subsets in specific granuloma microenvironments and this pattern was conserved in granulomas from different organs. We also identified macrophage subsets and T cells that may be important contributors to a granuloma’s potential glucose (FDG) uptake when their GLUT1 expression and population sizes were considered. We also correlated granuloma bacteria loads and hypoxia with GLUT1 expression, suggesting that bacterial antigens and hypoxic conditions drive a granuloma’s glucose uptake. Taken together, our data suggest that granuloma glycolysis and FDG uptake are driven, in part, by cell subset-specific responses to a granuloma’s microbial and microenvironmental milieu. This work was supported by NIH grants AI134183 and AI118195.
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