bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microbubbles: A high-throughput platform for the sustained culture, characterization, and isolation of single cells based on morphology, cytokine production, and antibody secretion.
Authors: Kobie James , DeLouise Lisa , Scheinman Andrew 
Published: January 2023

Abstract
Analysis of lymphocytes in bulk masks the heterogeneity of cellular processes. Through advancements in single cell technologies finer resolution and the true complexities of cell populations can be revealed. A major limitation of single cell technologies, especially those that enable high-throughput screening is the poor survival and proliferation of individual cells when binned separately in vitro. We have developed a novel platform technology, termed microbubbles which are scalable arrays of individual nanoliter size wells of unique spherical architecture and gas exchange properties that enable single cells to rapidly condition their microenvironment and enable long-term culture. Additionally, morphological heterogeneity is maintained in the microbubbles, unlike in conventional tissue culture wells. Microbubble arrays are compatible with conventional fluorescent microscopes and morphology and cell surface protein expression can be easily monitored longitudinally. Secreted cellular products such as antibodies and cytokines accumulate and are readily detected from cells in real-time using fluorescent probes. Arrays with 100,000 microbubbles, contained within a footprint of a standard microscope slide, can be screened using custom software in brightfield and three fluorescent channels within 10 minutes, and rare cells of interest identified based on morphological and/or functional properties are easily recovered for downstream analysis. Applications of microbubbles developed thus far include identification and isolation of rare antibody secreting cells based on antibody-specificity for monoclonal antibody development, monitoring T cell cytokine production, and characterizing cancer stem cells.
Poly Lactic-co-glycolic acid nanoparticles encapsulation enhance the effectiveness of curcumin for rescuing trafficking-defective gp91phox in chronic granulomatous disease leukocytes.
Authors: Yen Chia Liang , Liao Chia Li , Shieh Dar Bin , Wu Ping Ching , Yeh Chen Sheng , Chang Chuan Fa , Jiang Si Tse , Shieh Chi Chang 
Published: January 2023

Abstract
Mutations in leukocyte NADPH oxidase genes lead to defective reactive oxygen species (ROS) production and cause chronic granulomatous disease (CGD) in humans. Previously we have shown that the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump inhibitor, thapsigargin, rescues the endoplasmic reticulum (ER) retention of the mutant protein in a CGD patient with a gp91phox (H338Y) mutation. Curcumin, a natural constituent of Curcuma longa, is also a SERCA pump inhibitor. However, the clinical effectiveness of curcumin is not satisfactory due to its poor in vivo bioavailability and potential cytotoxicity. In this study, we investigated the effects of organelle-targeting nanoparticles on the delivery of curcumin to leukocytes for the treatment of CGD. First, we investigated the cytotoxicity of curcumin with poly (lactic-co-glycolic acid) (PLGA) nanoparticles encapsulation. Next, we investigated the therapeutic potential of PLGA encapsulated curcumin in mice expressing gp91phox (H338Y) mutant protein. Our results showed that (1) ER-targeting peptide decoration enhanced the localization of PLGA encapsulated curcumin in the ER region; (2) PLGA encapsulated curcumin induced ER calcium release, but the following extracellular calcium entry and ER stress-associated cell death was inhibited; (3) The in vivo test showed that PLGA encapsulated curcumin with ER-targeting peptide decoration enhanced leukocytes gp91phox (H338Y) mutant protein processing and the ROS production. This study showed the therapeutic potential of PLGA encapsulated curcumin for diseases with protein-trafficking defective mutations. Efficient organelle-targeting may promote the efficiency of nanoparticle-based curcumin delivery system.
Functional stability of ACD anticoagulated blood samples stored for 72 hours at room temperature.
Authors: Kunz Hawley E , Quiriarte Heather , Nelman-Gonzalez Mayra , Chew Darilyn , Crucian Brian 
Published: January 2023

Abstract
Characterization of the immune system in remote locations is challenging, requiring technical expertise and specific analytical equipment. Immune functional assessments require live cells, and the window of viability following blood donation is limited. To test the ability of ACD blood collection tubes to expand this window of viability, cellular function was analyzed over 4 consecutive days, beginning on the date of blood collection. T-cell activation, characterized by CD69 and CD25 expression, was measured following a 24-hour stimulation with soluble anti-CD3 and anti-CD28 antibodies (CD3/CD28) or with staphylococcus enterotoxins A and B (SEA+SEB). Mitogen stimulated cytokine profiles were determined following a 48-hour stimulation with CD3/CD28, phytohemagglutinin and ionomycin (PMA+I), or lipopolysaccharide (LPS). T-cell activation following stimulation with SEA+SEB was not altered by the processing delay; however, following stimulation with CD3/CD28, T-cell activation increased over the 72 hour period. The processing delay did not affect cytokine production following CD3/CD28 stimulation. Following stimulation with LPS and PMA+I, results were varied. While some cytokine responses were unaltered following the processing delay, others were impaired. These findings confirm the general viability of whole blood stored at room temperature (68 to 72°F) for 48 to 72 hours in ACD tubes and suggest many phenotypic and functional assays are feasible on blood samples aged as described. Investigators using this protocol should confirm their specific culture technique and measured output. This protocol may assist studies of subjects in remote locations with no immediate access to laboratory instrumentation.
Application of super-resolution microscopy to the study of B and T lymphocyte mitochondria morphology and metabolism
Authors: Roesler Alexander Scott , Miozzo Pietro , Akkaya Billur , Smelkinson Margery , Brzostowski Joseph , Kabat Juraj , Traba Javier , Dorward David W , Pierce Susan K , Akkaya Munir 
Published: January 2023

Abstract
High-resolution microscopy techniques have advanced our understanding of lymphocyte biology. An emerging focus in the field of immunometabolism on activation-induced metabolic reprogramming and its effects on immune cell function will require in-depth analyses of mitochondria. However, even with newer imaging technologies, obtaining super-resolution images of lymphocyte mitochondria remains challenging due to their small cytoplasmic sizes. Here, we compared standard confocal laser microscopy with three commonly used super-resolution methods to analyze activation-induced mitochondrial changes in B and T cells. Naïve B cells were stimulated in vitro via Toll-like receptor 9 and/or the B cell receptor, and naïve T cells were differentiated in vitro into activated or T regulatory phenotypes. The cells were imaged using standard laser confocal microscopy, AiryScan, stimulated emission depletion (STED) microscopy, and computerized tomography electron microscopy (TEM). Mitochondrial morphology was best resolved by staining both the mitochondrial membrane and matrix simultaneously. In preliminary analyses, we observed activation-induced changes in the distribution of lymphocyte mitochondria. Of the imaging methods used, only TEM allowed for 3D reconstructions of the mitochondria’s internal structure, which revealed swelling of the inner matrix and significant loss of cristae in B cells, but not in T cells. In conclusion, the super-resolution light microscopy techniques we developed appear to be powerful tools for immunometabolism studies of lymphocyte mitochondria, having demonstrated clear visualization even at the molecular level, while TEM answered key questions about intra-mitochondrial morphology.
Dynamic characterization of the phagocytic process utilizing an automated microfluidic platform
Authors: Chen Cindy , Yeh Victor , Booth Ross , Helton James , cappione amedeo 
Published: January 2023

Abstract
Phagocytosis is a complex pathway that involves removal of pathogens, foreign particles, or cell debris through engulfment by unique cell types. Such innate responses provide the first line of immune defense with evidence showing their critical involvement in prevention of chronic inflammation or autoimmune disease, tissue remodeling and removal of apoptotic tumor cells following therapeutic intervention. Traditional tools evaluate cell behavior in bulk context thus masking inherent cellular heterogeneity or employ end-point assays thus losing temporal process dynamics. Moreover, non-adherent immune cells are mobile complicating single cell analysis. Characterizing functional changes with single cell resolution requires precise spatial cell positioning, controlled micro-environment and high-throughput multiplexed measurement. We developed a novel microfluidic approach to trap single non-adherent immune cells in a microfluidic platform capable of perfusion-based media supply with gas and temperature control. The device is comprised of a controller system and optically transparent microfluidic plates, each with four culture chambers. Each chamber [dimension: 3mm × 3mm × 40μm (L × W × H)] contains a trap array tailored to the dimensions of the cells of interest. Each unit can be addressed by programmable manipulation of up to six reagents, enabling uninterrupted real-time live cell imaging assays in response to changing conditions. To demonstrate utility, the platform was used in conjunction with fluorescent markers to monitor the phagocytic process in real-time via, including temporal changes in morphology and the rate of phagosome formation/inhibition in the presence of different stimuli and concentration.
Treatment for severe combined immunodeficiency (SCID) in the era of newborn screening (NBS)
Authors: Wahlstrom Justin , Nguyen Alan , Dvorak Christopher C. , Puck Jennifer M , Cowan Morton J 
Published: January 2023

Abstract
SCID is associated with excellent survival when treated with allogeneic hematopoietic cell transplantation (HCT), gene therapy (GT), or enzyme replacement therapy (ERT) within 3–4 months of birth or prior to infection. NBS for SCID results in the diagnosis by 2–3 weeks of age. This retrospective analysis describes therapy and immune reconstitution of 28 infants diagnosed by NBS in California since 2010 and treated at the University of California, San Francisco at a median of 56 (24–217) days of age. HCT with alkylating chemotherapy (N=6), serotherapy alone (N=6), or no conditioning (N=12) was used in 24 infants, and ERT followed by definitive GT at University of California, Los Angeles, was used in 4 ADA-deficient infants. Overall survival at &gt;2 years was 93% with 1 death from HHV6 and 1 from CMV. The median time to CD4 T cell recovery (&gt;200 T cells/uL) was 73 (10–96) days for ERT patients prior to GT, and 138 days (14–220) for HCT patients. Time from first appearance of donor chimerism to CD4 recovery was evaluable for 14 HCT survivors who lacked pre-HCT maternal chimerism or whose donors were not their mothers. After donor chimerism appeared in peripheral blood at a median of 21.5 (14–169) days, appearance of T cells took 32 (0–84) more days. Second transplant or stem cell boost was required for 10 of 24 patients at a median of 77 (35–382) days following first HCT; for patients eligible for analysis (N=15), need for 2nd therapy was significantly higher in those without detectable donor chimerism by 28 days following therapy (0% vs 83%, p=0.04). NBS for SCID allowed rapid institution of anti-infective and definitive treatment to optimize outcomes, and early detection of donor chimerism was associated with successful T cell recovery.
STAT6 and T-bet double deficient mouse serving as allograft recipient is a new viable animal model to study lung transplantation
Authors: Chen Rongjuan , Chen Qirui , Li Hui , Ochando Jordi , Li Liwu , Ding Yaozhong , Xu Jiangnan 
Published: January 2023

Abstract
The long-term success of lung transplantation is mainly limited by the development of obliterative bronchiolitis (OB) in which IL-17 plays a critical role. However, murine orthotopic allogeneic lung transplant model in wild type recipients develops IFNγ-dominant acute allograft rejection. Our previous research demonstrated that additional STAT6 deficiency to T-bet deficiency resulted in Th17-dominant immune responses, and also restored CD4 T cells to produce IFNγ which is essential to immunosuppression and immunological tolerance. In this study, murine orthotopic lung transplants were performed in C57BL/6 wild type or T-bet/STAT6 double knout-out (DKO) mice using BALB/c donors. Vigorous allograft rejection was observed in T-bet/STAT6 DKO recipients with much higher levels of IL-17 but comparable levels of IFNγ expression to that in wild type recipients. Analysis of the graft-infiltrating cells displayed a significantly decreased CD4:CD8 ratio in T-bet/STAT6 DKO recipients compared to wild type recipients, but not as extremely as that observed in T-bet KO recipients, demonstrating more balanced CD4 and CD8 responses in DKO recipients. Our results indicate T-bet and STAT6 double deficient recipients of lung allografts develop obliterative airway inflammation that is mediated by both CD4 and CD8 responses, more closely resembling OB pathogenesis, and provide a new viable model to study lung transplant rejection.
Silencing of lncRNA NEAT1 induces tolerogenic dendritic cells and immune tolerance in heart transplantation
Authors: Wu Jian , Li Shuang , Zheng Yang , Zhang Maomao , Zhang Hanlu , Sun Yong , Yu Bo 
Published: January 2023

Abstract

Objective
Increasing research have focused on the potential therapeutic value of tolerogenic DC (tol-DC) in clinical organ transplantation. However, the role of long non-coding RNA (lncRNA) in tol-DC remains to be elucidated. Our report aims to illuminate the relationship between lncRNA NEAT1 and DC function in inmune tolerance of heart transplantation.

Methods and Results
We identified lncRNA NEAT1 was upregulated in cardiac allografts of mice by microarray. Applying Flow Cytometry, we found that LPS-dependent maturation of DC was inhibited after siNEAT1 characterizing the low expression of costimulatory molecular CD80, CD86 and MHCII. A mixed lymphocytere action (MLR) was performed to dectect the function of DC in simulating T cells. CD4+CD25+FoxP3+T cells were significantly increased in siNEAT1-DC, however, the proliferation of T cells was suppressed. Bioinformatic databases and dual-luciferase reporter gene assay confirmed that Neat1 can regulate mircoRNA let-7i. In addition, BALB/c recipients transfused with siNEAT1-DC had significantly prolonged allograft survival. Histological examination of cardiac allografts showd slight cell infiltration in recipients transfused with siNEAT1-DC.

Conclusion
LncRNA NEAT1 can regulate DC maturation and function, and transfusion of LPS-induced DC transfected with siNEAT1 prolonged allograft survival, besides, we identified that NEAT1 can regulate mircoRNA let-7i. This study enriched the network of interaction between lncRNA and mircoRNA and may provide a new therapeutic target in heart transplantation.
KEAP1 gene editing using CRISPR/Cas9 for therapeutic NRF2 activation in human T cells
Authors: Noel Sanjeev , Lee Sul A , Sadasivam Mohanraj , Hamad Abdel Rahim A , Rabb Hamid 
Published: January 2023

Abstract

Background
T lymphocytes mediate ischemia reperfusion injury (IRI) during organ transplantation. We recently demonstrated that augmentation of NRF2 dependent antioxidant response in T lymphocytes by deleting KEAP1 significantly protects from IR induced kidney injury. In order to develop clinically translatable T cell specific antioxidant therapy, we used CRISPR technology to delete KEAP1, the inhibitor of NRF2, in human Jurkat T cells and primary T cells.

Methods
We targeted KEAP1 exon 2 using site specific guide RNA. Briefly, 5×105 cells were electroporated 1.5μM of Cas9 protein and 1.8μM crRNA:tracrRNA complex. Control cells were electroporated without cas9:guide RNA complex. Cells were harvested 72h after electroporation and assessed for cell number and viability, KEAP1 editing and qPCR based analysis of NRF2 target genes.

Results
Electroporation of cas9:guide RNA complex had no adverse effect on cell expansion and viability (≥ 95%) in either Jurkat or primary T cells. Furthermore, genomic cleavage analysis showed editing of KEAP1 in upto 75% Jurkat cells. qPCR based studies in Jurkat cells showed significant (p≤0.05) increases in NRF2 targets NQO1 (~10 fold), HO-1 (~13 fold) and GCLM (~2.6 fold) mRNA following KEAP1 deletion, compared to control cells. Similarly, CRISPR mediated KEAP1 editing in primary T cells resulted in significant increase (p≤0.05) in NQO1 (~24 fold), HO-1 (~48 fold), GCLM (~6 fold) and GCLC (~3.5 fold) compared to control cells.

Conclusions
These data show that KEAP1 editing is feasible using CRISPR technology to augment NRF2 regulated antioxidant response in primary human T cells. This approach could improve immune cell based therapy for IRI during transplantation and help treat allograft rejection.
Hyperlipidemia alters Treg function through effects on Akt
Authors: Hyde Michael , Bagley Jessamyn , Iacomini John 
Published: January 2023

Abstract
Hyperlipidemia is a common health condition in the transplant patient population. This condition causes end-stage heart disease in approximately 40% of all patients requiring a heart transplant, developing in 50% of heart transplant recipients after the first year and 95% of patients within 5 years of transplant. We have recently shown that hyperlipidemia promotes an aggressive rejection response that results in accelerated rejection of allogeneic heart transplants and resistance to tolerance induction. These results appear to be related to effects of hyperlipidemia on regulatory T cells (Tregs), including activation of Akt. Here we examined how activation of Akt might alter Treg function. Our data indicates that hyperlipidemia down-regulated regulators of Akt, allowing for increased Akt phosphorylation. Interestingly, hyperlipidemia preferentially affects the activation through phosphorylation of specific Akt isoforms and isoform specific targets. Akt isoforms activated because of hyperlipidemia in Tregs are not activated in CD4 effectors, suggesting a specific role in Tregs. Our data lead us to conclude that hyperlipidemia alters Treg function by altering specific Akt isoforms. These data may lead to new approaches that can be used to down or up-regulate Treg function.
Biomaterial scaffolds for localized IL-33 release to modulate the local immune environment and enhance extrahepatic allogeneic islet transplant
Authors: Liu Jeffrey M , Luo Xunrong , Zhang Xiaomin , Shea Lonnie D 
Published: January 2023

Abstract
Allogeneic islet transplantation represents a promising therapeutic option to restore endogenous insulin production in Type-1 diabetes patients. Clinically, new methods of regulating the immune response against the islet allograft without using general immunosuppression are required. We have previously demonstrated the viability of transplanting syngeneic islets on porous poly-lactide-co-glycolide (PLG) scaffolds within the epididymal fat pad to restore long-term euglycemia. The use of a biomaterial scaffold also provides an opportunity to incorporate novel immunomodulatory factors that can modify the local immune response to transplanted cells. Due to the prevalence of ST2+ regulatory T cells and M2 macrophages linked to promotion of an anti-inflammatory phenotype within adipose tissue, we hypothesized scaffold-mediated IL-33 release could promote a tolerogenic environment to extend islet allograft survival. We found incubation of an IL-33 loaded scaffold in vitro with naïve T cells undergoing anti-CD3 TCR stimulation stimulated release of IL-13, demonstrating maintenance of IL-33 bioactivity. We found that IL-33 loaded scaffolds implanted into the epididymal fat pad expanded the CD4+ Foxp3+ population within the graft site, most strikingly amongst CD4+ Foxp3+ ST2+ Tregs. Using an allogeneic murine islet transplant model, preliminary data showed diabetic C57BL/6 recipients receiving 250 Balb/c islets on IL-33 scaffolds had extended graft survival time compared to BSA-loaded control scaffolds. Collectively, these results indicate the potential for IL-33 to modulate the local immune response to support graft function, and support ongoing studies to investigate mechanisms to extend graft survival.
A Comparison of the Antigenic Properties of Different Strains of Bacillus Typhosus
Authors: Hooker Sanford B. 
Published: January 2023

Summary and Conclusions
By means of the alexin-fixation reaction it has been found possible to demonstrate consistent antigenic differences among some strains of B. typhosus. In accordance with these differences a number of strains have been tentatively allocated in three fairly well-defined groups. Group I is made up of strains of rather recent isolation; no strain in this group has been under artificial cultivation for more than two years. Older strains, isolated from three to fifteen years ago, compose Group II. The third group is made up of those strains that fall neither in Groups I nor II, and that cross-fix irregularly with each other and with the members of the other groups. Group III strains are all more than three years old.
By means of agglutinin-absorption experiments, findings have been obtained that, in general, harmonize strikingly with the results of cross-fixation. This confirmatory evidence, secured by a widely different serologic method, tends strongly to prove that these antigenic differences among typhoid strains, as demonstrated by alexin-fixation, represent an actual condition and are not fortuitous.
It is considered that the evidence of antigenic differences thus far discovered among different strains of B. typhosus is sufficiently valid to warrant the presentation of these data, and sufficiently encouraging to justify extension of this work, especially with regard to the comparative protection afforded by sera obtained by immunization with strains of different character.
The facts that a serum immune to any recently isolated strain cross-fixes with all other strains, old or young, while sera immune to older strains do not so cross-fix, would lead to the seemingly justified assumption that only do the young strains contain all of the antigenic complexes of typhoid bacillary protein. The logical conclusion, then, would be that a young strain ought to afford the most efficient protection when used for prophylactic immunization against typhoid fever. However, the results of the work on agglutinin-absorption, if correctly interpreted, do not warrant the assumption that a univalent vaccine is sufficient. Therefore, although the grouping of strains here given is but tentative, it seems advisable, for the present, to recommend the use of a balanced polyvalent typhoid vaccine, for immunizing and therapeutic purposes, compounded in accordance with these groups. The experimental work here set forth distinctly does not uphold the prevailing practice of employing a single old strain for prophylactic immunization against typhoid fever.
It is a pleasure here to acknowledge my gratitude to Dr. F. P. Gay for the helpful interest he has taken in this work.