bluuebunny/crossref_metadata_2025_split huggingface dataset:
Performance Evaluation of an Enhanced-Sensitivity Interferon-gamma ELISpot Assay Kit (132.5)
Authors: Moskowitz Keith A , Lathey Janet L , Jeffrey Lisa , Branch Rodshawn , Hickman Scott , Moshkoff Dmitry , Manak Mark 
Published: January 2023

Abstract
Human Interferon-gamma (IFN-y) ELISpot assays are designed for the detection of IFN-y secreting cells at the single cell level. We have developed and validated an ELISpot assay with enhanced sensitivity (SC) and compared its performance relative to a common commercial kit (CK). Three manufactured lots of ELISpot kits (SC1-3) and one CK were analyzed in cellular titration experiments using PHA stimulation of cryopreserved PBMC from four subjects. The minimum quantity of cells required to elicit a single spot-forming cell (SFC) per well was 25,000 for CK compared to 7,000 for SC. In many cases, &gt; 50,000 cells were required to measure a response using the CK. Slopes of log/log plots were 2.3 for CK versus 1.7 for SC, with no overlap at the 95% CI level. At the upper detection limit (200,000 cells/well), the CK averaged 108±46 SFC/well, while SC 1-3 measured 264±79, 377±120 and 231 ±95 SFC/well, respectively, demonstrating a broader linear range. Differences in sensitivity and range are possibly due to spot size, SC produced spots 9 x 10-3 mm2 (95% CI 7-11) while CK averaged 19 x 10-3 mm2 (95% CI 17-21), P &lt; 0.001. The same PBMC were also analyzed using CEF (CMV, EBV and Influenza A – specific peptide pools) and CMV peptide stimulants and media controls. For CEF, the mean values for SC 1-3 were 100.0, 90.0, and 86.6 SFC/well, vs. 77.7 for CK. The CMV responses were 104.3, 80.2 and 74.4 SFC/well for SC 1-3 vs. 77.1 for CK. PBMC added with no stimulus, gave backgrounds of &lt;2 SFC/well in all cases. The average within run coefficients of variation (CV) were similar- 10.2, 14.1 and 18.9 for SC 1-3 and 13.8 for CK when stimulated using CEF and 20.8 to 26.4 with CMV. No significant differences were observed for means or variability between CK and SC 1-3. In summary, SeraCare has developed and validated an IFN-y ELISpot kit having an enhanced dynamic range and sensitivity.
Using Immunology as a Model to Teach Cell Communication. Including: A Practical ELISA for High School Biology (34.1)
Authors: Brokaw Ann , Cobb Brian 
Published: January 2023

Abstract
This educational unit is developed to illuminate the inherent connections between cell communication and immunology through analysis of cell signaling in the context of an introduction to vertebrate immune responses. The students will gain knowledge of the two interrelated fields through a variety of lectures, written assignments, and a computer lab simulation, culminating in a final laboratory experiment entitled, “Determination of Antibody Concentrations by Modified ELISA”. Both the educational unit and the laboratory exercise are developed for a typical advanced high school AP Biology or second year biology course setting, budget, and equipment. Students will complete a macro-scale ELISA (Enzyme-linked Immunosorbent Assay) to provide hands-on experience with one of the most fundamental immunological assays used in modern research laboratories. Upon conclusion of this curriculum, students will have developed a deeper understanding of not only immunology and cell communication pathways, but also an appreciation for common laboratory tools in the fields of immunology and medicine.
CSI Ann Arbor: A Middle School WebQuest Look at PCR Technology (34.2)
Authors: Lundy Philip Alan , Lukacs Nicholas W. 
Published: January 2023

Abstract
A WebQuest is a journey of understanding using information found on the Internet. Students are placed in a situation and guided through their quest, finishing with an individual or group project. At the Middle School level students are being introduced to the concepts of genetics and DNA. This curriculum is designed as an extension to a student’s understanding of DNA and an introduction to the polymerase chain reaction (PCR), an essential technology used in immunological research. Every piece of DNA contains areas called introns that seem to provide no genetic information. In 1984, Dr. Alex Jeffreys of the University of Leicester found that some of these areas contain repeated sequences, ranging from 20 to over 100 base pairs. These repeating sections of DNA are called Variable Number Tandem Repeats or VNTRs. In forensics, several different VNTRs from the same individual are amplified using PCR to provide a DNA fingerprint that is unique to that individual.
For this WebQuest, students take on the role of an undergraduate student paying his or her way through college as a midnight watchman in a Crime Scene Investigation lab. Someone in the lab is constantly stealing the watchman’s chewing gum, (a middle school crime of the highest level), and he/she decides to take matters into their own hands to find the guilty party. The student begins his/her web research with a review of basic DNA information. The student continues on with an introduction to PCR technology and demonstrations of real world examples. Students collect this information and record their results in their lab notebook. Following this information search, they use their acquired knowledge to collect evidence and run a DNA fingerprint to determine the guilty party. Finally the students present their findings to their classmates via PowerPoint, video, or poster.
Our Body’s Response to Infectious Diseases: A Middle School Unit on Infectous Diseases and the Immune System (34.3)
Authors: Paul Debra L. , Budd Ralph C. 
Published: January 2023

Abstract
This unit, written for grades 5 – 8, gives students an introduction to infectious diseases and the immune system. Students will discover the source of the cholera epidemic that broke out in London in 1854, learn how a pathogen can enter the body and how infectious disease spread. They will conduct experiments involving the washing of their hands and about how the skin acts as a barrier, particpate in an activity showing how antigens and antibodies fit together, and develop skits that show each of the body’s lines of defense. Guest speakers from the medical center will talk with the students about infectious disease and and how stress affects the immune system. The unit will culminate with a visit to a DNA Analysis Facility, Microscopy Imagining Department and Flow Cytometry Department.
The Autoimmune Lymphoproliferative Syndrome (ALPS) Associated with a Novel Mutation in Exon 6 of the FAS (TNFRSF6) Gene (35.14)
Authors: Schaffer Frederick M , Frankel Bruce M , Spicer Eleanor K , Cavalier MaryEllen 
Published: January 2023

Abstract
ALPS is the result of dysregulated lymphocyte FAS-mediated apoptosis and is characterized by lymphadenopathy, autoimmune phenomena, the expansion of a CD3+/CD4−CD8− T cell population, hypergammaglobulinemia, and an increased risk for lymphoma. We present the laboratory and clinical studies of a 6 y/o female with a history of adenopathy, severe neutropenia, microcytic anemia, and recurrent systemic infections. She has hypergammaglobulinemia (IgG = 2020 mg/dL; IgM = 413; IgA = 695 mg/dL), an elevated RF, an elevated CD3+/CD4−CD8− lymphocyte fraction, normal in vitro lymphocyte proliferation and normal titers to tetanus, diphtheria and pneumococcal antigens. Bone marrow aspirate and biopsy were unremarkable. We suspected a diagnosis of ALPS; therefore, exons 1–9 of the FAS (TNFRSF6) gene were PCR-amplified from genomic DNA. Bidirectional sequencing demonstrated a novel heterozygous mutation, I184V, an A&gt;G change resulting in a substitution of the normal isoleucine codon (ATT) with a mutant valine codon (GTT) in exon 6. This is a previously unpublished mutation in association with ALPS. The presence of the I184V mutation was confirmed by restriction fragment analysis using the Bsr1 endonuclease. This A&gt;G mutation appears to create a splice acceptor site that could strongly compete with the actual acceptor site located 5′ to exon 6. This mutation further causes an in-frame 15 amino acid (AA) deletion (which includes a cysteine) which most likely effects the folding of the resultant protein. Studies to investigate and assess the novel splice adaptor site and the effects of the 15 AA deletion on FAS expression and on in vitro apoptosis are currently in progress.
Porcine MHC Class II Haplotype Divergence (35.15)
Authors: Luetkemeier Erin , Schook Larry B 
Published: January 2023

Abstract
The swine major histocompatibility complex (MHC) located in SSC7, is a gene dense region with a role in antigen presentation and controlling immune responsiveness. Although highly polymorphic, serological approaches to identify and define alleles have been disappointing. To determine the extent of MHC polymorphisms of class II genes, DNA from divergent world breeds of unrelated pigs was analyzed for SNPs. Primers targeting exon 2 of DQB and DRB were shown to be locus specific and amplified both loci for each animal. Expression of exon 2 PCR amplicons for both genes was verified through sequence analysis revealing 30 haplotypes comprised of over 20 different alleles among the 9 breeds. Located within the two genes were a total of 24 polymorphic sites, 22 of these were located within the antigen recognition site (DRB had two in the alpha helix) where 17 of those were nonsynonymous substitutions (7 synonymous). The breeds analyzed were selected to represent geographic areas in order to determine if haplotype divergence correlated with location of origin. The first group was identified as “Asian” and included Meishan, Erhualian and Lanyu breeds. The second group represented the “British Isles” (Hampshire, Yorkshire and Berkshire breeds) and the third group was identified as “Continental” (Duroc, Large White, and Landrace breeds). Fewer breed specific alleles were observed in breeds selected for lower fat and longer carcasses. SNPs in DRB appear to be under positive Darwinian selection with a dN/dS value of 13 as opposed to DQB with a value of 1 (no selective pressure or purifying selection) To confirm expression of the PCR products and eliminate the possibility of pseudogenes, RT-PCR with primers from intron-exon boundaries were utilized and verified the absence of pseudogene amplification and revealed highest RNA expression in DRB especially in heart, bone marrow, lung, small intestine and lymph node.
NK cell inhibition receptor genes and HLA –DRB1*1201 in Spontaneous Clearance of Hepatitis Virus Infection. (35.16)
Authors: Romero Viviana A , Zuñiga Joaquin , Azocar Jose , Clavijo Olga P , Chung Raymond T , Amos Christopher I , Yunis Edmond J 
Published: January 2023

Abstract
Host genetic factors play an important role in the evolution of hepatitis C virus (HCV) infection. The aim of this study was to analyze the MHC-KIR combinations associated with the susceptibility and clinical outcome of HCV infection in drug users with Puerto Rican American ethnic background. A total of 160 HCV infected drug users (121 with chronic viremia (CV) and 39 with spontaneous clearance (SC)). Frequencies of KIR inhibitory and activating receptors and MHC class I ligands in HCV infected patients were analyzed using DNA methods. We also used a novel approach to rule out genetic stratification. We report a significant association between HLA-C1/C1 and KIR2DL3/KIR2DL3 with SC was detected confirming previous studies in Caucasian and African Americans. In addition we found MHC class II associations with clinical outcome different to those reported in Caucasians. Our results support the existence of interactions between HLA-C1 and KIR2DL3 genes in HCV viral outcome consistent with loss of inhibition mediated by HLA-C1 and inhibitory functions of KIR2DL3 together with the presence of HLA-DRB1*1201 consistent with negative epistasis.
Transcriptional regulation of L-selectin (35.19)
Authors: Dang Xitong , Raffler Nikolai A , Ott Kenneth R , Francis Joshua , Mirmira Raghavendra G , Ley Klaus 
Published: January 2023

Abstract
L-selectin mediates tethering and rolling of lymphocytes in high endothelial venules. To study the transcriptional regulation of L-selectin, we cloned 5′-flanking sequences of the murine L-selectin gene 1084 bp upstream of the translational start site. Analysis of the 5′-flanking sequence shows no TATA box, but an Initiator-like element present around position −104. Multiple transcription initiation sites were mapped between −119 and −15 by 5′RACE. Transfection of serially truncated 5′-flanking regions linked to a reporter gene in Jurkat and EL-4 cells identified that nucleotides −2 to −284 contained the elements to drive high level gene expression. Transfection of the constructs into COS 7 cells, which lacks L-seletin, showed negligible luciferase activity. Within this region we identified several cis-elements, including IRF-2, Sp1, Ets-1, MZF-1. Site-directed mutagenesis of the putative binding sites showed that both Sp1 and Ets binding sites were positive regulatory elements. EMSA and CHIP confirmed the specific binding of Sp1, MZF-1, and Ets1. These results provide a molecular basis for further investigation of the involvement of L-selectin in regulating lymphocyte tethering and rolling.
Regulation of MHC Class II Transcription by the 19S Proteasomal ATPase S8 (35.25)
Authors: Bhat Kavita P , Turner Jonathan D , Greer Susanna F 
Published: January 2023

Abstract
The 26S proteasome is a multi-subunit complex involved in the turnover of polyubiquitinated proteins. Increasing evidence in yeast shows that individual ATPase subunits of the 19S proteasome lid play non-proteolytic roles in transcription regulation and histone modification. To determine if 19S ATPases play similar roles in mammalian transcription, we studied the role of the 19S ATPase S8 in the transcriptional regulation of Major Histocompatibility Complex (MHC) class II genes. Following IFN-γ stimulation, S8 associated in vivo with both the MHC class II master regulator, the Class II transactivator (CIITA), and the MHC proximal promoter. In the absence of S8, both MHC II promoter activity and MHC gene expression were decreased. Although CIITA expression remained constant in the absence of S8, CIITA association at the MHC class II promoter was drastically reduced and this reduction was not affected by proteasome inhibition. Finally, although S8 rapidly associated with the MHC class II proximal promoter following IFN-γ stimulation, the 26S proteasome demonstrated a substantial delay in association. Together, these data demonstrate that S8 plays a crucial but non-proteolytic role in the transcriptional regulation of MHC class II genes and illustrate involvement of 19S proteasome in mammalian transcription.
This work is supported by grants from the NMSS, The Georgia Cancer Coalition and GSU (to S.F. Greer).
Monocyte Polarization is Partly Mediated through Epigenetic Changes which Modify Gene Expression (35.31)
Authors: Sullivan Kathleen E , Garrett Stacey 
Published: January 2023

Abstract
Monocytes and macrophages respond to multiple changes in their milieu by altering their gene expression. Monocytes treated with γIFN alter their morphology and have an enhanced capacity for the production of inflammatory mediators. In contrast, exposure of monocytes to IL-4 dampens the inflammatory response. We utilized gene expression arrays to characterize both the direct effects of γIFN, αIFN, and IL-4 polarization as well as to define the effect of pretreatment with these cytokines on subsequent responses to a common pro-inflammatory stimulus, immune complexes. Each cytokine pretreatment COMPLETELY altered subsequent responses to immune complexes. For example, only 10% of the chemokines detected as upregulated after immune complexes in the absence of cytokine pretreatment were seen when any of the cytokines were utilized as pretreatment. γIFN induced a set of changes which were not completely reversed after a washout and rest period nor after re-treatment with IL-4. This suggested that γIFN induced a type of change in the cell which semi-permanently altered the expression of some genes. To explore the mechanism of this aspect of polarization, chromatin immunoprecipitation assays were performed. These assays demonstrated that γIFN induces increased H4 acetylation at the TNFα promoter. Together, these data suggest that polarization can be long lasting and may act in part by altering the epigenome.
Ets-1 is a negative regulator of Th17 differentiation (35.34)
Authors: Moisan Jacques , Grenningloh Roland , Bettelli Estelle , Oukka Mohamed , Ho I-Cheng 
Published: January 2023

Abstract
IL-17 is a proinflammatory cytokine that plays a role in the clearance of extracellular bacteria and contributes to the pathology of many autoimmune and allergic conditions. IL-17 is produced mainly by T lymphocytes and a newly characterized subset of T helper (Th) cells producing IL-17 has recently been described and termed Th17. Although the role of Th17 cells in the pathology of autoimmune diseases is well established, the transcription factors regulating the differentiation of this subset remain poorly characterized. We report here that the Ets-1 transcription factor is a negative regulator of Th17 differentiation. Naïve CD4 T cells from Ets-1 knockout (KO) mice expressed increased levels of IL-17 and other Th17-specific genes, including IL-17F, IL-22, and IL-23 receptor, following differentiation to the Th17 pathway with TGFβ and IL-6. The increased IL-17 production in Ets-1 KO Th17 cells appeared to be cell intrinsic and was probably caused by altered expression of RORγT, a transcription factor that is essential for the differentiation of Th17 cells. Ets-1 KO mice displayed increased mucus production in their lungs that was associated with increased expression of IL-17, IL-17F, but not IFNγ. Overall, our data demonstrate a role for Ets-1 in inhibiting the differentiation of Th17 cells and broadens the knowledge of the transcriptional program regulating Th17 cell lineage commitment.
The work was supported by a Senior Investigator Award from the Crohn’s and Colitis Foundation of America and a RO3 grant from NIH.
Genetic control of variegated KIR gene expression: polymorphisms of the bi-directional KIR3DL1 promoter are associated with distinct frequencies of gene expression (35.37)
Authors: Anderson Stephen K , Pascal Veronique , Martin Maureen P , Carrington Mary , Li Hongchuan 
Published: January 2023

Abstract
The recent discovery of the bi-directional nature of the KIR gene promoters suggests that the relative strength of competing sense and antisense promoter elements might affect the probability of gene expression. Analysis of a panel of donors has revealed the presence of several functionally relevant promoter polymorphisms, clustered mainly in the inhibitory KIR family members, especially the KIR3DL1 alleles. The percentage of peripheral blood NK cells expressing either KIR3DL1 or KIR3DS1 was evaluated in individuals heterozygous at this locus. The KIR3DL1*001 and KIR3DS1 alleles were found to be expressed on a significantly higher percentage of NK cells than all other KIR3DL1 alleles. High frequency of expression was associated with a specific promoter polymorphism at a Sp1 transcription factor-binding site, and this change increased the relative promoter activity in the sense orientation. The KIR2DS4 promoter region is nearly identical to the KIR3DL1 promoter, and several polymorphisms are present, however none of the KIR2DS4 polymorphisms affect promoter activity in vitro and no association was observed between promoter polymorphisms and frequency of expression.
This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Funded by NCI Contract N01-CO-12400
Association of genetic variations in inflammatory and fibrogenic genes with progressive massive fibrosis in coal miners (35.51)
Authors: Yucesoy Berran , Johnson Victor J , Fluharty Kara , Kashon Michael L , Slaven James , Kissling Grace , Germolec Dori , Vallyathan Val , Luster Michael I 
Published: January 2023

Abstract
Progressive massive fibrosis (PMF) is a chronic interstitial lung disease with a complex etiology. Cytokines, growth factors and cell-surface adhesion molecules play crucial roles in the pathogenesis of pulmonary fibrosis by mediating inflammation, fibroblast proliferation, angiogenesis and collagen synthesis. We examined the influence of functional single nucleotide polymorphisms (SNP) in interleukin-1 and 6, tumor necrosis factor-α, transforming growth factor-β, vascular endothelial growth factor, epithelial growth factor and intercellular adhesion molecule-1 genes on the development of PMF. A total of 648 ex-coal miners from the National Coal Workers Autopsy Study were examined in a case-control study. Genotype analysis was performed on genomic DNA using 5′ nuclease PCR assay. There were no significant differences in the frequencies of individual SNPs between PMF and control groups. However, gene-gene interactions were found between TNFα −308/−238, VEGF+405 / ICAM-1+241 and VEGF+405 / ICAM-1+241 / IL-6+174 (p&lt;0.05) variants. These data suggest that genetic variant of cytokine and fibrogenic genes may play role in individual susceptibility to PMF initiated presumably by coal dust.
Supported in part by the NIEHS-IAG (Y1-ES-0001).
The findings have not been formally disseminated by the NIOSH and should not be construed to represent any agency determination or policy.&lt;
Anergy is a Survival Advantage for the Maintenance of Memory CD4+ T Cells (36.10)
Authors: Dalai Sarat Kumar , Sadegh-Nasseri Scheherazade 
Published: January 2023

Abstract
The hallmarks of specific T cell immunity include proliferative expansion, and acquisition of effector function. The outcome is resolution of the infection and the generation of memory T cells (Tmem). Although majority of Tmem cells die following clearance of infection, a fraction survives through lifetime and is protective on recall. It is not clear how Tmem decline with the fall of antigenic load and survive so long in the absence of antigen. Using CpG mixed with antigen to mimic an infection, we have shown that CD4+ Tmem cells undergo anergy upon encountering suboptimal doses of antigen, a condition that associates with the fall of antigenic load expected post resolution of an infection. We demonstrate that follicular B (B2) cells, and not DCs, induce anergy in CD4+ Tmem by upregulating CTLA-4 expression. These findings suggest that B cells bearing high affinity antigen receptor regulate responsiveness of Tmem by capturing the lowest levels of antigen likely present after the clearance of an infection, and signal Tmem to undergo anergy. We also show that the anergized cells are activated by stimulatory signals generated by innate immunity at the onset of a subsequent infection. Our study suggest that anergy is a mechanism to regulate long-term survival of Tmem in the absence of antigen by preventing unnecessary proliferations that lead to death.
Supported by R01GM53549 and R01AI063764 grants to SS-N.