bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessment of Immunogenicity of a Peptibody Based Protein Therapeutic Using a Surface Plasmon Resonance based Biosensor Immunoassay using a Biacore 3000 (132.7)
Authors: Hokom Martha M. , Jawa Vibha , Mytych Daniel , Swanson Steve 
Published: January 2023

Abstract
Administration of therapeutic biologic products is often accompanied by an unwanted immune response that requires monitoring. We used a BIAcore based biosensor immunoassay that can detect antibodies against a peptibody protein-based therapeutic and its peptide domain.. The development and validation of the multi-flow cell surface plasmon resonance based technology is described in this presentation. The biosensor immunoassay measures the interaction of serum antibodies to immobilized ligands in real time. This binding interaction is further confirmed to be the result of an antibody by the use of species-specific secondary antibodies (anti-F(ab)’2) to detect the bound antibodies. The binding is further characterized by a competition step using the soluble drug. The fully validated assay has a limit of detection of 50ng/mL and 200 ng/mL for the peptibody and the peptide respectively, and a limit of quantification of 400 and 800 ng/mL respectively.
In summary, we developed and validated a sensitive and robust biosensor immunoassay for the detection of antibodies against a peptibody based drug that meets the requirements necessary to support the clinical development of this therapeutic.
PERIPHERAL BLOOD MONONUCLEAR CELL GENE ARRAY PROFILES IN PATIENTS WITH OVERACTIVE BLADDER (132.8)
Authors: Bluth Martin H , Bluth Mark J , Johns Christopher , Khan Sohail , Lin Yin Yao , Zenilman Michael E , Cheung Wellman 
Published: January 2023

Abstract
Introduction: Overactive bladder (OAB) confirmation of disease often requires invasive approaches. We have previously demonstrated that peripheral blood mononuclear cells (PBMC) can provide a reporter function in solid organ retroperitoneal disease. Here we investigated the utility of using PBMC as a marker for patients with OAB.
Methods: 21 patients were assessed for OAB and structural integrity. Patients with a history of recent surgery or infection were excluded. Whole blood was obtained from patients and appropriately matched controls (n=6) and PBMC were separated by density centrifugation. RNA was isolated from PBMC and converted to aRNA and subjected to microarray analysis (human U133A 2.0).
Results: Microarray analysis revealed that 16 genes were differentially regulated (8 upregulated and 8 downregulated) in all patients with OAB compared with controls. Sex based analysis demonstrated 74 genes differentially regulated in males (25 upregulated and 49 downregulated), and 30 in females (13 upregulated and 17 downregulated). Of these, PDGF, MFAP3L, a microfibrillar-associated protein, and TPM1 (tropomyosin) were downregulated in all sets analyzed.
Conclusions: Microarray analysis revealed many genes which were differentially regulated in PBMC from OAB patients, including regulatory/structural genes; PDGF, MFAP and TPM1 may be important in regulating structural integrity of bladder and supporting tissues. These data suggest that blood derived leukocytes can provide a reporter function for patients with solid organ disease and may serve as a diagnostic marker and/or monitor response to therapy.
A Rapid Automated Method for the Sequential Isolation of CD19, CD3 and CD33 cells from One Tube of Whole Blood. (132.9)
Authors: Fairhurst Maureen A , Nauer Melany , Thomas Terry E. 
Published: January 2023

Abstract
Chimerism analysis is typically performed on small blood samples. Analysis of purified cell subsets requires techniques which can isolate &gt;1 cell type from an entire starting sample rather than a divided cell suspension. High cell recovery is essential. Ficoll steps often result in cell loss of 50% while certain lysis and wash steps can affect granulocyte content. We describe a method of sequential selections, starting with 4–5 mL of human whole blood and using a fully automated pipetting robot (RoboSep®).
CD19 and CD3 and CD33 positive cell fractions were isolated using immunomagnetic, column-free positive and negative selection (EasySep®). Briefly, cells were first labeled with antibody targeting CD19 positive cells. These were then coupled to magnetic nanoparticles and the sample was placed in a magnet. The supernatant with unlabeled cells was removed to a new tube, leaving isolated CD19 positive cells in the magnet. The supernatant was next labeled with anti-CD3 antibody, magnetic nanoparticles, placed in a magnet and the supernatant was removed to a new tube leaving isolated CD3 positive cells. Finally, a cocktail of antibodies was used to label and remove the remaining unwanted cells (CD33 negative) from the supernatant. The resultant CD33 positive cells were collected in the supernatant fraction leaving CD33 negative cells. Assessment by flow cytometry yielded average purities over 90% for all cell types.
T and B Cell Pathways: An Introduction to the Immune System (34.4)
Authors: Paul Mark R. , Budd Ralph C. 
Published: January 2023

Abstract
This presentation will summarize a two-week curriculum designed for an introductory High School Biology Class. This unit meets Vermont Standards for the Human Body (7.14, bbb), The Problem Solving Process (2.2), and Relationships (3.10). This unit also meets Grade Expectations for Human Health and Disease and the Immune system.
Students first learned about different kinds of diseases and the structure and function of viruses and bacteria. Next they read an article and answered questions on Typhoid Mary. They were then given two weeks to solve a case study involving Lyme disease, after which they learned more about Lyme disease and performed an ELISA lab for Lyme.
Students received background information from a textbook and a power point presentation. They also roll played how the immune system responds to a first infection of Borrelia burgdorferi. For a culminating activity students built functional models that simulated either humoral or cell mediated immunity. The basic premise of designing these models was the Mouse Trap Game or Rube Goldberg design using whatever materials they could rustle up. We also used RASMOL so that the student s could view a 3-D model of the Lyme antibody and antigen.
IgH intronic enhancer, Eμ, and associated matrix attachment regions are required for efficient somatic hypermutation (35.1)
Authors: Li Fubin , Eckhardt Laurel A 
Published: January 2023

Abstract
Somatic hypermutation is a critical mechanism for generating antibody with high affinity. Although the enzyme critical to this process, AID, has been identified, the mechanism that largely limits its activity to the Ig loci is not known. Prior studies have shown that IgH gene transcription levels are positively correlated with somatic hypermutation frequency. The intronic enhancer, Eμ, both stimulates IgH transcription and is essential for promoting efficient V region gene assembly in the IgH locus. It consists of both a core region (cEμ) and two flanking matrix attachment regions (MARs). A recent study reported normal levels of somatic hypermutation in the B cells of cEμ-deficient mice. In the present study, we tested whether deletion of both cEμ and MARs had an effect on somatic hypermutation. We found that the mutation frequency for the Eμ-deficient allele was reduced to approximately 1/3 of wild-type levels. This dramatic decrease in mutation frequency suggests that cEμ, together with its associated MARs, plays an important role in somatic hypermutation within the IgH locus.
Supported by NIH grant AI030653 to L.A.E. Research infrastructure and instrumentation at Hunter College is supported by NIH RCMI grant RR-03037.
Ataxia-Telangiectasia, Associated with a Rare 1339C&gt;T ATM mutation, presenting in infancy with Pneumocystis carinii pneumonia, and an elevated serum IgM. (35.11)
Authors: Schaffer Frederick M , Virella-Lowell Isabel , Gatti Richard A , Gossage David L 
Published: January 2023

Abstract
Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder manifested by progressive immunodeficiency, progressive neurologic dysfunction, and an elevated cancer predisposition. Characteristic symptoms are rarely encountered during infancy.
We present the laboratory and clinical studies of a patient with respiratory distress at age 3 months due to pneumocystis pneumonia. Laboratory studies demonstrated an elevated serum IgM (840 mg/dL), recurrent neutropenia, lymphopenia (CD3, CD4, CD8, and CD19), diminished in vitro lymphocyte proliferation, a negative HIV PCR, and normal ADA and PNP levels. Flow cytometry demonstrated normal lymphocyte expression of CD40 and the CD40 ligand. Sequencing, after PCR-amplification of genomic DNA, demonstrated no apparent mutation consistent with type 2 hyper IgM syndrome. Based on a significantly elevated serum alpha-fetoprotein and early neurologic deficits, a diagnosis of A-T was suspected. Subsequently, her cells were found to be radiosensitive, based on a Colony Survival Assay. Western blot analysis of nuclear lysates of patient cells failed to detect ATM protein. Molecular analysis of the ATM gene identified a rare homozygous 1339C&gt;T mutation (previously reported in a Druze A-T family: Hum Mutat 11:69–75, 1998). Notably, our A-T case (AT219LA) demonstrates early and atypical findings of an opportunistic infection and elevated serum IgM. The patient is currently thriving on IVIG and the prophylactic use of Septra.
Amino acid residues in Rag1 crucial for DNA hairpin formation (35.12)
Authors: Lu Catherine Pei-ju , Sandoval Hector , Rice Phoebe A , Roth David B 
Published: January 2023

Abstract
The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, utilizing a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acids to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265, and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (Y, K, [E], F, R, K) may share functional similarities with the YREK motif in IS4 family transposases.
Modifications of CD40 regulate defective CD40 signals in B cells from a patient with non-X-linked hyper IgM syndrome (HIGM) (35.13)
Authors: Lu Kristina T. , Covey Lori R. 
Published: January 2023

Abstract
Our previous studies have demonstrated a CD40 signaling defect in B cells from a patient with non-X-linked HIGM (pt1). To elucidate the cause for impaired CD40 signals, we utilized a mini-EBV transformed cell line with inducible latent membrane protein 1 (LMP1) expression (LCLtet). The LMP1 signaling pathway appears to be intact, however CD40 activation was unable to promote cell division and transcriptional synthesis of cMyc in pt1-LCLtet cells. We examined mediators of the CD40 signaling pathway and found impaired localization of CD40 to microdomains, thereby impeding TNF receptor-associated factor recruitment and degradation after 2h of CD40 activation. However, prolonged CD40 ligation was sufficient to induce proper CD40 distribution, suggesting that the pt1 B cells possess a higher CD40 activation threshold that requires a greater magnitude of extensive and sustained CD40 signals to achieve signal transmission. Furthermore, pt1 B cells have a unique CD40 expression profile, indicative of an isoform with post-translational modifications. Specifically, CD40 on pt1 B cells is constitutively hyperphosphorylated and not differentially regulated by LMP1 or CD40 signals. These data collectively suggest that modulation of post-translational modifications, which mediate CD40 function and activity, may provide further insight into the cause of defective CD40 signals in this unique form of HIGM. (AI37081)
Unambiguous HLA Typing for Studies of Human Immunity (35.18)
Authors: Cate Steve , Roselius Kassi , Hildebrand William 
Published: January 2023

Abstract
The Major Histocompatibility Complex (MHC) is recognized as the most polymorphic region of the human genome. Our HLA typing laboratory at the University of Oklahoma Health Sciences Center has performed more than 30,000 HLA typings in the past 10 years. We have systematically overcome various capacity and cost constraints to provide precision, DNA sequence-based, HLA typing data for studies of transplantation, vaccine development, and autoimmunity. A confounding variable in the HLA typing field is data heterogeneity; different HLA laboratories provide different levels of resolution when reporting an HLA type for autoimmune, transplant, and vaccine studies. For example, HLA-A*02, A*0201, or A*0201/09/43N/66/75/83N/89/97 might all be reported for a like sample. Such data heterogeneity hinders data integration and comparison. On the basis of our experience with HLA typing and with functional assays such as the ELISpot and tetramer staining, we propose a system of HLA typing that encompasses known functional regions of the HLA molecule. The DNA sequencing of the appropriate exons at various HLA loci, along with the resolution of known null substitutions, will provide cost efficient HLA typing data that can be integrated among different laboratories. Improved HLA data uniformity will ultimately strengthen studies of human immune responses and disease.
“Allelic” exclusion despite 15 IgH loci in nurse shark (35.2)
Authors: Lee Victor , Malecek Karolina , Hsu Ellen 
Published: January 2023

Abstract
Nurse sharks have an alternative IgH organization consisting of 15 independent miniloci (VH-D1-D2-JH-Cμ). Rearrangement takes place primarily within the locus, and we found there is no strictly regulated order of the three recombination events that occur among the four gene segments. In each B cell only a few IgH rearrange. Aside from the selected functional VDDJ, out-of-frame and intermediate configurations with one or two recombination events are present; these all are transcribed and can become targets of hypermutation. The data are consistent with a stochastic initiation of rearrangement in B cells. In contrast, in thymocytes, multiple IgH each undergo one or two recombination events but are otherwise inert. We speculate there are different levels of activation, one occurring at many IgH in precursor lymphocytes that renders the gene segments accessible to recombinase, and a second B-cell specific factor that, in limiting amounts, leads to efficient V(D)J rearrangement and transcription.
The transcription factor GATA-3 regulates IL-4 responses in B cells (35.23)
Authors: Aud Dee , Pai Sung-Yun , Ho I-Cheng , Peng Stanford L. 
Published: January 2023

Abstract
The GATA-3 transcription factor plays a central role in T helper type 2 development, but its role in B cells has not been well described, even though GATA-3 is present in naïve B cells. To study the role of GATA-3 in B cells, mice with a floxed Gata3 allele (Gata3fl/fl) mice were crossed to CD19-Cre transgenic mice, allowing B cell-specific deletion of GATA-3. B cell development was grossly normal in Gata3fl/fl CD19-Cre, but surprisingly, Gata3fl/fl CD19-Cre B cells showed a decreased ability to proliferate (in an IL-4 dose dependent manner) but an increased ability to produce IgG1 and IgE in response to anti-CD40 and/or IL-4 in vitro. These findings may reflect an interaction between GATA-3 and type 2 Ig-regulating transcription factors such as NF-kB or STAT6, because GATA-3 can modulate the activities of these factors in vitro. These findings demonstrate a critical yet surprising role for GATA-3 in the regulation of IL-4-mediated responses in B cells.
Identification and characterization of a conserved negative regulatory element in the human DAP10 gene (35.27)
Authors: BURGESS STEVEN , MARUSINA ALINA , BORREGO FRANCISCO , COLIGAN JOHN 
Published: January 2023

Abstract
The NKG2D receptor is an activation receptor expressed on all NK cells and the majority of CD8+ T cells that can bind stress-inducible ligands such as MICA/B. Human NKG2D signals through its association with DAP10, which can couple to the PI3-K signaling pathway upon NKG2D ligation leading to cellular activation. Inappropriate activation of NK and/or T cells by NKG2D has been implicated in a variety of human diseases and thus the determination of the factors that regulate NKG2D/DAP10 expression is important. We have embarked on a study in which to understand the molecular mechanisms governing transcription of the DAP10 gene. We have identified a highly conserved intron between exons III and IV within DAP10. By cloning gene fragments of DAP10 into pGLbasic for luciferase reporter assays, we have shown that intron III contains a negative regulatory element capable of completely abolishing the promoter activity of DAP10. Progressive deletions of the conserved intron III allowed us to localize the negative regulatory element to a region about 80 base pairs in size that contains a putative GATA-3 binding site. The putative silencer within intron III appears to be both orientation and location dependent. Using ChIP assays, we have also shown that intron III contains high levels of acetylated chromatin in resting NK cells. Further studies are underway to further characterize the negative regulatory element contained within the conserved intron of the DAP10 gene.
c-maf, NFAT2 and NFkB p65 specifically cooperate to augment HIV-1 transcription in IL-4 producing CD4 T cells (35.28)
Authors: Zhang Mingce , Cron Randy Q. 
Published: January 2023

Abstract
HIV-1, the cause of AIDS, preferentially replicates in IL-4 producing cells, and this coincides with a Th2 switch linked with disease progression. Others suggested differential chemokine receptor expression accounts for increased infectivity of Th2 cells. However, we confirm HIV-1 expression is increased in IL-4 producing CD4 T cells but is irrespective of viral tropism (T-tropic, M-tropic) for different chemokine receptors. Instead, we note increased HIV-1 long terminal repeat (LTR) transcription in IL-4 producing primary CD4 T cells. Using in vitro footprinting, we identified a binding site for the Th2 restricted transcription factor, c-maf (required for IL-4), located adjacent and proximal to the dual NFkB/NFAT sites in the proximal LTR. We show c-maf from activated primary CD4 T cells binds the HIV-1 LTR in vitro (EMSA) and in vivo (ChIP of virally infected cells). Interestingly, c-maf binds in conjunction with NFAT2, but not NFAT1, and with NFkB p65, but not p50. Over-expression of c-maf, NFAT2, and NFkB p65 synergistically augment HIV-1 transcription and p24/gag expression. Conversely, c-maf specific siRNA introduced into IL-4 producing primary CD4 T cells inhibits HIV-1 transcription. Thus, we identified a host factor, c-maf, present in IL-4 producing CD4 T cells that contributes to increased HIV-1 transcription in Th2 cells via binding to the LTR in cooperation with specific NFAT and NFkB family members.
Regulation of epsilon germline transcription and switch region mutations by IgH locus 3′ enhancers in transgenic mice (35.3)
Authors: Severinson Eva , Laurencikiene Jurga 
Published: January 2023

Abstract
Germline (GL) transcription is regulated by specific promoters and Immunoglobulin heavy chain (IgH) 3′ locus enhancers and is necessary for Ig class switch recombination (CSR). We have generated different transgenic lines containing the GL epsilon promoter, switch (S)_psilon region and constant (C)_psilon region with or without the DNase I-sensitive regions (HS)3A-HS1,2 or HS3B-HS4 3′ IgH enhancer pairs. The enhancerless construct was expressed in B cells activated by interleukin (IL)-4 and cluster of differentiation (CD)40, thus resembling regulation of the endogenous gene. Both enhancer-containing transgenes efficiently increased expression in B cells and were strongly up-regulated by stimuli. In addition, S epsilon regions of the transgene containing HS3B-HS4 were mutated in activated, sorted B cells. Such mutations are known to precede CSR and are dependent on activation induced cytidine deaminase (AID). Our findings show that all elements necessary for recruitment of the recombination machinery are present in the transgene containing HS3 and HS4. These enhancers probably provide something more specific than mere increased accessibility of switch regions. We propose that transcription factors binding the enhancers help to target the recombination machinery to the switch regions.
This work was supported by grants from the Swedish Research Council, the Swedish Institute and the Karolinska Institutet
New Sequence of Strategies to Define Gene Transcriptional Territories in Lymphocytes: The Perforin Example (35.32)
Authors: Lichtenheld Mathias Georg , Zúñiga-Pflücker Juan Carlos , Rao Anjana , Pipkin Matthew E. 
Published: January 2023

Abstract
Distal regulatory regions control transcription by promoter interactions that are limited to a territory to avoid non-target genes. Few loci are defined with the scrutiny needed to fully understand how a territory is established, maintained and fenced off. Here we approach the human perforin gene (PRF1). First, we transferred an epigenetically silenced PRF1 within its chromosome into a rodent CTL cell line. Afterward PRF1 expression was turned on equivalently to the endogenous mouse gene without perturbing bordering genes. Ultra long-range DHS assays showed epigenetic remodeling during chromosome transfer and delineated a 150 kb territory with 16 DHS sites for PRF1. When a corresponding BAC was introduced into the CTL, transgene expression proceeded with locus control region activity dependent on 4 distal DHS sites that also protected PRF1 from transcriptional perturbations by bordering genes. Finally, differentiation of transgenic ES cells showed the area contained all regulatory domains required for developmentally appropriate PRF1 expression during in vitro lymphopoiesis. Thus, we defined a new sequence of strategies that reliably characterizes autonomous transcriptional territories in lymphocytes.
Common Transcriptional Control Complexes of Murine CD21 and CD23 Suggests Stage Specific Repression (35.35)
Authors: Debnath Irina , Roundy Kirstin , Weis Janis , Weis John 
Published: January 2023

Abstract
The expression of CD21 and CD23 is coincident in differentiating B cells during the T1 to T2 splenic transition stage. To define the factors regulating the expression of CD21 and CD23, we have used Chromatin Immunoprecipitation to map candidate transcription factors. Total naïve splenocytes showed constitutive binding of Oct1, NFATc3, YY1, NFkB-p52, PU.1, Pax5, E2A and RBP-Jk; to CD21 sequences (promoter and first intron) and NFkB-p52, Pax5, NFATc3, E2A and RBP-Jk to CD23 promoter sequences. Analysis of naïve T and B cell subsets showed constitutive binding of YY1, NFkB-p52, Pax5, Oct1 and NFATc3 proteins to CD21 sequences and NFkB-p52, Pax5, NFATc3 to CD23 promoter in B cells, but no specific binding of NFATc3 or Pax5 in T cell populations. Transcripts and protein for various NFAT species are present in splenic B and T cell (NFATc1, c2, c3, c4, NFAT5), but only a subset of NFAT proteins were defined to form constitutive complexes with the CD21 and CD23 gene sequences of resting T and B cells. The shared transcriptional control elements of CD21 and CD23 did not suggest an obvious means of alternative transcriptional regulation of these genes in marginal zone (MZ) B cells where CD21 cell surface expression and transcription are high and that of CD23 is absent or very low. These data suggest a model where MZ cells are derived directly from T1 splenic precursors.
NIH-AI-24158 to JHW and NIH-AI-32223 to JJW