bluuebunny/crossref_metadata_2025_split huggingface dataset:
TRAF3 regulates Pim2 and c-Myc-mediated B cell survival
Authors: Whillock Amy Louise , Mambetsariev Nurbek , Lin Wai W. , Stunz Laura L. , Bishop Gail A. 
Published: January 2023

Abstract
Tumor necrosis factor receptor associated factor 3 (TRAF3) is an adaptor protein that plays an important role in B cell homeostasis. TRAF3 deletions or inactivating mutations are common in human B cell malignancies. In human and mouse B cells, TRAF3 negatively regulates signaling of several receptors important for B cell homeostasis (including BAFFR, CD40, and IL-6R). B cell specific TRAF3-deficient (B-Traf3−/−) mice have enlarged spleens and lymph nodes and an increased frequency of B cell tumors as they age. B cells from B-Traf3−/− mice have markedly increased survival and autoantibody production. The mechanisms by which TRAF3 regulates B cell survival are not yet well defined. We found that TRAF3-deficient mouse B cells and human multiple myeloma cell lines with low expression of TRAF3 had increased RNA and protein expression of the pro-survival kinase Pim2. Additionally, TRAF3-deficient mouse B cells had increased phosphorylation of Pim2 targets BAD, p70S6K, and 4E-BP1. TRAF3-deficient B cells showed increased survival relative to TRAF3-sufficient B cells after treatment with the Pim inhibitors SGI-1776 and TP-3654. Pim2 can promote survival by phosphorylating and stabilizing the oncogenic transcription factor c-Myc. Loss of TRAF3 led to transcription-independent c-Myc protein elevation that was dependent on Pim2. K48 polyubiquitination of c-Myc was decreased in the absence of TRAF3, consistent with increased c-myc protein stability. TRAF3 deficiency correlated with B cell resistance to apoptosis mediated by the c-Myc transcription inhibitor JQ1. Our results show that TRAF3 promotes B cell survival by regulating expression of and interaction between Pim2 and c-Myc.
The pro-bacterial effect of Type 1 Interferon signaling requires its own negative regulator USP18
Authors: Shaabani Namir , Honke Nadine , Prinz Marco , Knobeloch Klaus-peter , Zhang Dong-Er , Teijaro John R. 
Published: January 2023

Abstract
Elevated Type 1 interferon (IFN-I) production following bacterial and viral infections has been causally linked with promoting host susceptibility to primary and secondary bacterial infections, some which can result in severe morbidity and Mortality. Using a Listeria monocytogenes (L.m.) model system, we found that among the many Interferon-induced genes (ISGs), USP18 is a central ISG that increases susceptibility to primary and secondary L.m. infection downstream of IFN-I signaling. Knocking out Usp18 specifically in DCs enhanced bacterial control, despite an elevated IFN-I gene signature compared to WT mice. Moreover, Usp18 pro-bacterial effect hinges on the ability of Usp18 to inhibit TNF-α signaling. We extended our findings to demonstrate that Usp18 expression also regulates IFN-I sensitive Staphylococcus aureus infection, suggesting that Usp18 may regulate many IFN-I-sensitive bacterial infections. In summary, we identify a single downstream ISG responsible for promoting primary and secondary bacterial infection – a significant conceptual advance. Targeting host USP18 will have the distinct advantage of exerting less selective pressure on bacterial populations and thus likely attenuate the rate at which resistance develops.
Circulating bacterial DNA is associated with disease severity in patients with S. aureus bacteremia
Authors: Gutierrez Johnny , Guimaraes Alessander , Peck Melicent C. , Barhanu Aklile , Xu Min , Cao Yi , Kim Janice , Yan Donghong , Chang Joanna K. , Dinoso Jason , Koss Catherine , Clemenzi-Allen Angelo , Chambers Henry F. , Rosenberger Carrie , Baruch Amos 
Published: January 2023

Abstract
Staphylococcus aureus bacteremia is a leading cause of morbidity and mortality and yet tools to assess severity of infection and response to treatment are still lacking. This study aimed to establish a link between bacterial DNA quantified in blood and clinical severity in bacteremic patients. We developed a workflow coupled with a highly sensitive quantitative PCR method to quantify S. aureus bacterial DNA. This assay was used to measure whole blood S. aureus DNA (SA-DNA) and plasma-derived S. aureus cell-free DNA (SA-cfDNA) in a set of longitudinal samples from 45 patients with confirmed S. aureus bacteremia. SA-DNA was detectable at baseline and declined following treatment with antibiotics. Notably, baseline SA-DNA correlated with disease severity and levels were sustained in cases of complicated infections versus with non-complicated infections. In addition, plasma-derived S. aureus cell-free DNA could be readily detected in bacteremic patients and showed concordance with whole blood SA-DNA. High levels of SA-DNA and SA-cfDNA were associated with metrics of clinical severity, including longer durations of treatment, elevated white blood cell count, and positive blood cultures. Taken together, these data suggest that circulating bacterial DNA is more reflective of whole body bacterial load than blood culture and may aid in monitoring clearance of tissue reservoirs of infection during antibiotic therapy.
Comparative genomic analysis of Escherichia coli strains resistant to grazing by the social amoeba Dictyostelium discoideum
Authors: Snyder Michelle , Hazen Tracy , Michalski Jane , Rasko David 
Published: January 2023

Abstract
Diarrheagenic Escherichia coli remain a significant health challenge, particularly in developing countries. Challenges in the use of small animal models for enteric infections highlight the need for development of alternative model systems. The social amoeba, Dictyostelium discoideum, which phagocytizes bacteria for nutritional purposes, serves as a host model for several pathogenic bacteria, including E. coli. Previous studies characterized interactions of D. discoideum with commensal and extraintestinal E. coli. In the current study, we extend this characterization to analyze the grazing interactions of D. discoideum with 35 representative diarrheagenic, extraintestinal and commensal E. coli strains. We found that E. coli strains sensitive and resistant to D. discoideum grazing are present in each pathotype. Whole genome phylogenomic analysis revealed that grazing resistance to D. discoideum is distributed among the phylotypes, although resistant strains are enriched in the B2 phylogenomic group. Comparative genomic analysis identified genes that were more prevalent among strains resistant to D. discoideum grazing. While not providing direct insight into conserved mechanisms of resistance, these identified genes do provide avenues for further investigation. Phenotypic analysis revealed that E. coli strains resistant to D. discoideum grazing also exhibited increased resistance to the reducing agent, β-ME. These data extend the characterization of interactions between D. discoideum and E. coli, and through comparative genomics identify candidate E. coli genes that may be associated with resistance to grazing by the model phagocyte D. discoideum, and by extension with resistance to phagocytic immune cells.
NK cells inhibit anti-M.bovis BCG T cell responses by lysing BCG-infected macrophages and aggravating pulmonary inflammation
Authors: Fang Min , Wang Dongfang , Gu Xiuling , Liu Xiaoman , Wei Songtao , Wang Bin 
Published: January 2023

Abstract
Tuberculosis remains a threat to public health. The major problem for curing this disease is latent infection, of which the underlying mechanisms are still not fully understood. Previous studies indicate that NK cells do not play a role in inhibiting the growth of Mycobacterium tuberculosis in the lung, and recent studies have revealed that NK cells regulate the adaptive immunity during mycobacterial infection. By using a novel mouse model of direct lung infection with M.bovis BCG, we found that the presence of NK cells postponed the priming and activation of T cells after BCG infection. In addition, depletion of NK cells before infection alleviated pulmonary pathology. Further studies showed that NK cells lysed BCG-infected macrophages in an NKG2D dependent manner. Thus, NK cells did not play a direct role in control BCG, but by lysing BCG-infected macrophages, NK cells aggravated the pulmonary inflammation and impaired anti-BCG T cell immunity. Our results may have important implications for the design of immune therapy to treat tuberculosis.
TGF-β1 regulates epidermal LCs in a Smad-independent pathway
Authors: HUANG LINTING , Li Gui-Hua , Zhang Xilin , Yin Congcong , Zhou Li , Mi Qing-sheng 
Published: January 2023

Abstract
Transforming growth factor-β1 (TGF-β1) is a key factor for Langerhans cell (LC) homeostasis and maintaining in the epidermis, but the underlying mechanisms in TGF-β signaling pathways remain elusive. Canonically, binding of TGF-β to TGF-βR activates Smad2 or Smad3, which subsequently heterodimerizes with Smad4 and then translocates into the nucleus acting as transcriptional regulators of TGF-β1 target genes. In order to investigate if Smad-dependent pathways are required for epidermal LC maintenance, we first crossed CSF1R-Cre and hLangerin-Cre transgenic mice with Smad2fl/fl and Smad4fl/fl, mice to generate the mutant mice with conditional Smad2 and Smad4 deletion at birth (CSF1Rcre) or 3 days after birth (hlangerinCre). Surprisingly, deletion of individual Smad2 or Smad4 in LCs did not significantly affect their ratios in skin and their expression of MHC-II, CD80 and CD86 in both models. The phagocytic capacity of LCs from KO mice was unaltered in vivo and in vitro compared to WT LCs. Furthermore, upon in vitro stimulation, MHC-II, CD80 and CD86 were comparably unregulated on LCs between KO and WT mice. We further tested LC number and maturation in conventional Smad3KO mice as well as Smad2/4 double KO mice induced by CSF1Rcre, the number and maturation of LCs are comparable between mutant and WT mice. Overall, our data suggest that conventional TGF-β/Smad2/3/4 signaling pathways may not be required for maintaining LC homeostasis and immature stage in the epidermis and LC maturation upon stimulation. The underlying mechanisms that Smad2/3/4-independent TGF-β pathways involved in LC maintenance and function remain to be further determined.
Oropharyngeal (OPG) vaccination activates head and neck lymphoid tissues required for immune protection against mucosal Brucella infection
Authors: Clapp Beata , Yang Xinghong , Hoffman Carol , Pascual David W. 
Published: January 2023

Abstract
Human brucellosis remains a global health problem with no human vaccines existing. Most human infections occur mucosally with eventual systemic brucellae dissemination. Current interventions primarily focus on the systemic disease, rather than considering neutralizing the infection at its source. A number of studies show that the OPG mucosa is the principal site of brucellae uptake following oral or inhalational exposures. We have devised an OPG infection method using an attenuated Brucella mutant by allowing the mice to drink (ad bibitum) from a pipette tip. BALB/c mice were fed once daily for 3 days with 108 CFUs. Individual head and neck lymph nodes (HNLNs), and spleens were examined at 2 weeks post-infection for bacterial colonization and proinflamatory cytokine responses. Elevated colonization was observed in submandibular LNs (SmLNs) and deep cervical LNs (CLNs). Modest colonization was detected in the parotid LNs (PrLNs) and spleens. Splenic and individual HNLN lymphocytes were analyzed for IFN-g, TNF-a, granzyme B, and perforin production by flow cytometry. SmLN, CLN, and PrLN IFN-g-producing CD8+ T cells significantly exceeded IFN-g-producing CD4+ T cells by 2.5-, 2.5-, and 1.5-fold, respectively. Increased numbers of CD4+ and CD8+ T cells expressing TNF-a, granzyme B, and perforin were also observed. All activated lymphocytes were predominantly of effector-memory phenotype, CD44+CD62LlowCCR7low. These studies demonstrate that ad bibitum vaccination against brucellosis results in colonization and activation of the HNLNs, mimicking natural human infection, enabling future inquiry into the immune mechanisms responsible for protection to mucosal Brucella infections. Work supported by NIH AI-125546.
Large-scale use of knockout validation to confirm antibody specificity
Authors: Zhong Zhen , Sassi Monica , Heaton Sam , Koch Sofia , De Block Gert , Conlon Daniel , Lochead Julia , Dreja Hanna , Munro Melanie , Solache Alejandra , Hamilton Bruce 
Published: January 2023

Abstract
Antibodies are among the most common tools in basic science and clinical research. However, an increasing number of studies have shown that not all antibodies are specific, leading to a growing issue of experimental irreproducibility. This is in part due to the cross-reactivity between antibodies and off-target proteins, as well as the variability between different antibody batches. These unspecific antibodies result in wasted time and resources and compromise the advancement of science.
In response to this, Abcam launched a knockout (KO) validation program in 2015 as one of its antibody quality control protocols. This initiative uses KO cells lines made possible through a partnership with Horizon Discovery. Target genes are mutated via CRISPR-Cas9 within a haploid cell line, which results in a frameshift and a complete loss of gene expression. These KO cell lines function as true negative controls that can be used at a large scale to confirm antibody specificity to the protein of interest.
We have KO-validated hundreds of antibodies, including several that are relevant to immunology and immuno-oncology research. Here we present the data related to key targets validated in western blot, flow cytometry and immunocytochemistry. The antibodies have been tested in KO and wild-type cells to confirm their specificity. By providing researchers with reliable and specific antibodies that work first time, we hope to minimize wasted resources and improve antibody reproducibility.
Magnetic enrichment of tumor infiltrating T cells reduces experimental time and prevents biased functional characterization
Authors: Evaristo Cesar , Siemer Ramona , Agorku David , Brauner Janina , Hardt Olaf , Dose Christian , Richter Anne 
Published: January 2023

Abstract
Tumor-Infiltrating (TI) T cells play an important role in anti-tumor immunity, highlighted by multiple immunotherapy strategies approved by the FDA. However, the benefit of current immunotherapies is still limited, such that additional research is needed to improve efficacy. Preclinical tumor models are essential to advance immunotherapy research. However, tumor composition is highly variable and T cell numbers can be very limited. Even flow cytometry can be very time consuming. Importantly, TI T cells are embedded in a cellular environment where antigen is abundant and surrounding cells express immunomodulatory molecules, such that unbiased cell-intrinsic functional characterization is impossible. Pre-enrichment of TI T cells is highly desirable to reduce hands-on time, and generate high quality and reliable data.
We developed new CD4, CD8 and Pan T cell specific enrichment reagents for magnetic cell sorting from mouse tumors. Single cell suspension was obtained by automated dissociation. T cell isolation was done either manually, or using semi or full automation. TI T cell frequencies ranged from 0.2–9%, were isolated to purities above 80% with yields ranging from 60–95%. Pan T cell isolation maintained the original CD4:CD8 ratio. CD69 was not upregulated after 18h culture, demonstrating that isolation did not result in T cell activation. Importantly, functional analysis of bulk vs. purified cells revealed significant influence of tumor and stromal environment in cytokine expression by TI T cells, which was not due to the isolation protocol.
Our data, demonstrates that magnetic enrichment reduces time of analysis, increases quality of analysis and is essential for the functional characterization of TI T cells.
Cross platform validation of a unique method for deep genomic and proteomic analysis of rare immune cell populations
Authors: Lopez Peter , Gregory Michael , Miller Kelley , Fuhrman Kit , Ray Karina , Hinerfeld Douglas 
Published: January 2023

Abstract
Advances in flow and mass cytometry expand the number of cell parameters that can be interrogated improving our understanding of the myriad immune cell types. These technologies, however, are limited in the types of analytes profiled in a single sample. In addition to efforts to understand the immune system for the treatment of autoimmune diseases, the immune system is increasingly a target for cancer therapeutics. These efforts require deep characterization to understand rare cell populations, requiring next-generation methods that expand on flow cytometry.
3D Flow™ Analysis integrates flow cytometry cell sorting and the nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay* to characterize sorted immune cell populations with direct digital counting of 770 RNA and 30 proteins. To determine if the method is agnostic to the cell sorting technology used, 3D Flow analysis was run with the Becton Dickenson FACSARIA IIu, Sony SH800z, and Beckman Coulter MoFloXDP. Stimulated and unstimulated PBMC were co-stained with flow antibodies for CD3, CD8, and CD4 plus 30 DNA-labeled antibodies for NanoString readout. 5,000 CD3+/CD4+ and CD3+/CD8+ cells were sorted on each platform and immediately lysed to release cellular RNA and DNA tags from NanoString antibodies. RNA and DNA tags were directly hybridized to NanoString barcodes and run on the nCounter system.
Expression of 30 proteins and 770 RNA across cell populations correlated between the three sorting platforms, showing 3D Flow is agnostic to the sorting technology used. Importantly, high-plex RNA data was obtained without additional molecular biology methods, such as RNA purification or library construction, simplifying incorporation into a flow cytometry laboratory.
Rapid Specificity Determination and FACS Selection of Plasma Cells for Single Cell Antibody Cloning
Authors: McBride Kevin M. , Mu Yunxiang , Zelazowska Monika Anna , Dong Qiwen , Krug Laurie , Plummer Joshua 
Published: January 2023

Abstract
Plasma cells (PC) are terminally differentiated antibody secreting cells that are the major source of serum immunoglobulins (Ig). Identifying antigen specific PCs is an important goal for both immunologic studies and monoclonal antibody production. However, the lack of Ig surface expression has impeded direct identification of PCs expressing antigen specific antibodies. We devised a simple method termed antigen specificity of plasma cell determination (ASPCeD), to isolate live PCs producing antigen specific antibodies from total spleen or bone marrow populations using one-step flow cytometry. To demonstrate the utility of this technique we immunized mice against a viral protein immunogen, and combined ASPCeD with established single cell immunoglobulin cloning. We rapidly produced sequence diverse, recombinant monoclonal antibodies, highly specific for the viral protein target and infected cells. Furthermore, we have produced post-translational modification specific antibodies using this technique. Thus, ASPCeD is a novel method to identify antigen specific PCs and an improved means for high throughput recombinant antibody production over existing methodology.
The Benzathine Penicillin G (BPG) reformulation preferences study – the importance of cultural awareness and appropriate governance concerning Rheumatic Fever related research in New Zealand
Authors: Sika-Paotonu Dianne , Tiatia Ramona , Sung Yun K. , Thornley Craig , Betty Bryan , Wineera-Parai Ranei , Eddie Barbara , Baker Michael , Maloney Margaret , Sanchez-Felix Manuel , Spector Jonathan , Carapetis Jonathan 
Published: January 2023

Abstract
Acute Rheumatic Fever (ARF) is the autoimmune condition caused by untreated group A streptococcal (GAS) infection of the throat and possibly skin. Repeated or severe untreated attacks of ARF can cause permanent heart damage known as rheumatic heart disease (RHD). ARF/RHD rates in the Pacific (Samoa, Tonga and Fiji) have been reported as being 5 times higher than that found in NZ. Pacific Peoples living in NZ currently comprise ~ 7% of the NZ population so prioritization of ARF/RHD prevention, treatment and management efforts within the Pacific region and NZ is warranted due to high incidence rates and migration between countries. As a GAS vaccine is not yet available, the most effective recommended preventative measure for ARF requires painful monthly injections of Benzathine Penicillin G (BPG) be given intramuscularly for 10 years. Known as secondary prophylaxis, the purpose is to prevent GAS infections that can lead to ARF and cause RHD. A reformulated BPG is desperately needed. To support the generation of a more appropriate BPG reformulation that encourages treatment adherence, this work explored the BPG reformulation preferences of predominantly Māori and Pacific children/teens in New Zealand currently receiving monthly BPG intramuscular injections, their families and healthcare providers. The appropriate governance, cultural leadership and guidance essential for this project work to progress safely by the research team was provided by Pasifika and Māori health researchers, leaders, representatives, scientists and academics with careful considerations integrated into the development of the software applications and into the research work.
A bead-based multiplex immunoassay for cancer autoantibody biomarker discovery
Authors: Pepin D , Godeny Michael , Lie Wen-Rong 
Published: January 2023

Abstract
Accurate, reliable and affordable blood tests are needed for early detection of cancer. Here we show data for the development and validation of a sensitive Luminex® bead-based multiplex immunoassay for detecting cancer autoantibodies in multiple cancer types. The cancer autoantibody biomarkers in this new multiplex panel include: CTAG1B/NY-ESO-1, Cyclin B/CCNB1, ENO1, Galectin-1, Galectin-3, Her2, HIF1α, HSP60, IMP2/IGF2BP2/p62, IMP3/IGF2BP3/KOC, MUC1, p16-INK4a, p53, SOX2, and Survivin/BIRC5. This multiplex immunoassay is sensitive and robust. In a 96-well plate, a mixture of recombinant protein-immobilized magnetic bead sets was incubated with serum samples for 2 hours with gentle shaking; after washing, the beads were incubated with detection antibody phycoerythrin (PE)-labeled anti-human IgG for 1.5 hours. After the final wash, the beads were resuspended in sheath fluid and the median fluorescent intensity (MFI) data of 50 beads per bead set were analyzed on a Luminex® reader. The performance of this panel was demonstrated using a set of serum samples from 78 cancer cases and 18 healthy controls. Elevated cancer autoantibodies were detected in serum from patients with various cancer types, as compared to healthy controls. In conclusion, this cancer autoantibody multiplex immunoassay has potential application as a non-invasive tool for studying early detection, diagnosis or monitoring of cancer.