Studies on Lectin Mediated Agglutination Reaction on Red Blood Cell Surface Antigens Using Hot and Cold Water Plant Extracts

Lectin has various physiological roles in cell agglutination, based on their carbohydrate-binding properties, plant lectins are widely used for the detection, segregation, and characterization of glycoconjugates. Rhesus (Rh) factor is a protein that is inherited and found on the surface of red blood cells. If the surface protein is present, the RBC is Rh positive; otherwise, it is Rh-negative in nature. In this paper, we use agglutination reactions to investigate the effect of different cold and hot water extracted plants on RBC antigens as an alternative to commercial monoclonal antibodies. Extensive research on the sequence homology and 3-D structure of various plant lectins suggests that they have been conserved throughout evolution and may play important physiological roles that are still unknown.

Clinical Activity of Anti-HER2-Antibody Drug Conjugates in HER2-Mutated Metastatic Cancer Patients

Background/Objective: Antibody drug conjugates (ADCs) deliver potent cytotoxic therapy in a highly targeted manner by binding cancer-specific cell surface antigens. HER2 is a receptor tyrosine kinase that is commonly amplified in breast and gastric cancers, but more recently has been shown to harbor gain-of-function activating mutations in several cancer types. Recent observations have suggested that HER2-ADCs may be viable therapies for patients that harbor these activating mutations. This research retrospectively explores the efficacy of HER2-ADCs in metastatic cancer patients in a real-world setting. Methods: Patient information was gathered from the Precision Genomics program at IU Health, narrowed for specific mutation criteria (HER2 pathogenic mutation), cross referenced with OncoKB to legitimize oncogenicity, and selected for patients prescribed HER2-ADCs. For these patients, pre-treatment and mid-treatment CT-scans were analyzed following RECIST protocols. The primary outcomes were overall response rate (ORR), clinical benefit rate (CBR), and progression free survival (PFS). Secondary outcomes were to tabulate the frequency and clinical characteristics of HER2-mutated tumors. Results: Out of 517 patients with somatic HER2 mutations, 60 patients had a pathogenic HER2 mutation. Of these 60 patients, the most common tumor types were 26.67% Breast and 11.67% Bladder/Urothelial. 11 of 60 patients were prescribed a HER2-ADC (Trastuzumab Emtansine = 10, Trastuzumab Deruxtecan = 1). 8 of 11 patients were evaluable for response with RECIST criteria with 1 patient having a partial response, 4 patients having stable disease and 3 patients having progressive disease. ORR=13%, CBR=63%, Median PFS = 2.77 months (95% CI: 2.15-3.39 months). Conclusion/Implications: To our knowledge, this is the first report of HER2-ADCs demonstrating clinical activity in HER2-mutated cancers across tumor types. Further clinical trials are ongoing that will validate these initial findings.

A GDPase/UDPase Bifunctional Enzyme From Candida Albicans: Purification and Biochemical Characterization

The most frequently isolated human fungal pathogen is Candida albicans which is responsible for about 50% of all Candida infections. In healthy individuals, this organism resides as a part of the normal microbiota in equilibrium with the host. However, under certain conditions, particularly in immunocompromised patients, this opportunistic pathogen adheres to host cells causing serious systemic infections. Thus, much effort has been dedicated to the study of its physiology with emphasis on factors associated to pathogenicity. A representative analysis deals with the mechanisms of glycoprotein assembly as many cell surface antigens and other macromolecules that modulate the immune system fall within this chemical category. In this regard, studies of the terminal protein glycosylation stage which occurs in Golgi vesicles has led to the identification of nucleotidases that convert glycosyltransferase-generated dinucleotides into the corresponding mononucleotides, thus playing a double function: their activity prevent inhibition of further glycosyl transfer by the accumulation of dinucleotides and the resulting mononucleotides are exchanged by specific membrane transporters for equimolecular amounts of sugar donors from the cytosol. Here, using a simple protocol for protein separation we isolated a bifunctional nucleotidase from C. albicans active on GDP and UDP that was characterized in terms of its molecular mass, response to bivalent ions and other factors, substrate specificity and affinity. Results are discussed in terms of the similarities and differences of this nucleotidase with similar counterparts from other organisms thus contributing to the knowledge of a bifunctional diphosphatase not described before in C. albicans.

The Impact of Inflammation on the Immune Responses to Transplantation: Tolerance or Rejection?

Transplantation (Tx) remains the optimal therapy for end-stage disease (ESD) of various solid organs. Although alloimmune events remain the leading cause of long-term allograft loss, many patients develop innate and adaptive immune responses leading to graft tolerance. The focus of this review is to provide an overview of selected aspects of the effects of inflammation on this delicate balance following solid organ transplantation. Initially, we discuss the inflammatory mediators detectable in an ESD patient. Then, the specific inflammatory mediators found post-Tx are elucidated. We examine the reciprocal relationship between donor-derived passenger leukocytes (PLs) and those of the recipient, with additional emphasis on extracellular vesicles, specifically exosomes, and we examine their role in determining the balance between tolerance and rejection. The concept of recipient antigen-presenting cell “cross-dressing” by donor exosomes is detailed. Immunological consequences of the changes undergone by cell surface antigens, including HLA molecules in donor and host immune cells activated by proinflammatory cytokines, are examined. Inflammation-mediated donor endothelial cell (EC) activation is discussed along with the effect of donor-recipient EC chimerism. Finally, as an example of a specific inflammatory mediator, a detailed analysis is provided on the dynamic role of Interleukin-6 (IL-6) and its receptor post-Tx, especially given the potential for therapeutic interdiction of this axis with monoclonal antibodies. We aim to provide a holistic as well as a reductionist perspective of the inflammation-impacted immune events that precede and follow Tx. The objective is to differentiate tolerogenic inflammation from that enhancing rejection, for potential therapeutic modifications. (Words 247).

Anti-Inflammatory Effects of Novel Glycyrrhiza Variety Wongam In Vivo and In Vitro

Licorice is the common name of Glycyrrhiza species, which is an important plant for edible and medicinal purposes; however, Glycyrrhiza resources have become limited because of desertification, depletion of natural resources, and environmental restrictions. For this reason, a novel Glycyrrhiza variety named Wongam, a hybrid of G. glabra and G. uralensis, was developed by the Korea Rural Development Administration. To elucidate the antiallergic inflammatory effects of Wongam, we investigated its effects using a compound-48/80-induced anaphylaxis in vivo model and PMA/A23187-stimulated HMC-1 cells and immunoglobulin E (IgE)/DNP-stimulated RBL-2H3 cells in in vitro models. Wongam treatment reduced mortality and serum IgE levels and downregulated proinflammatory cytokines and chemokines in a compound-48/80-induced anaphylaxis mouse model. Wongam decreased histamine release and the expression of proinflammatory cytokines in HMC-1 and RBL-2H3 cells. Wongam treatment downregulated the expression of chemokines, T helper 2 cytokines, and cell surface antigens in PMA/A23187-stimulated HMC-1 cells. We confirmed that these effects were associated with the inhibition of the MAPK and NF-κB signaling pathways by Wongam. The present study suggests that Wongam ameliorates mast-cell-mediated allergic inflammatory responses by reducing mast cell activation and may serve as an effective agent for the prevention and treatment of allergic inflammatory responses.

Arsenite exposure inhibits the erythroid differentiation of human hematopoietic progenitor CD34+ cells and causes decreased levels of hemoglobin

Arsenic exposure poses numerous threats to human health. Our previous work in mice has shown that arsenic causes anemia by inhibiting erythropoiesis. However, the impacts of arsenic exposure on human erythropoiesis remain largely unclear. We report here that low-dose arsenic exposure inhibits the erythroid differentiation of human hematopoietic progenitor cells (HPCs). The impacts of arsenic (in the form of arsenite; As3+) on red blood cell (RBC) development was evaluated using a long-term culture of normal human bone marrow CD34+-HPCs stimulated in vitro to undergo erythropoiesis. Over the time course studied, we analyzed the expression of the cell surface antigens CD34, CD71 and CD235a, which are markers commonly used to monitor the progression of HPCs through the stages of erythropoiesis. Simultaneously, we measured hemoglobin content, which is an important criterion used clinically for diagnosing anemia. As compared to control, low-dose As3+ exposure (100 nM and 500 nM) inhibited the expansion of CD34+-HPCs over the time course investigated; decreased the number of committed erythroid progenitors (BFU-E and CFU-E) and erythroblast differentiation in the subsequent stages; and caused a reduction of hemoglobin content. These findings demonstrate that low-dose arsenic exposure impairs human erythropoiesis, likely by combined effects on various stages of RBC formation.

EPCO-14. MULTIFACETED TRANSCRIPTOMIC AND PROTEOMIC ANALYSES IDENTIFIED PUTATIVE ALTERNATIVE SPLICING-DERIVED CELL SURFACE ANTIGENS IN GLIOMA

BACKGROUND To develop effective immunotherapy for gliomas, it is crucial to expand the repertoire of targetable antigens. Recent studies have suggested that alternative splicing (AS), or its deriving tumor-specific junctions (“neojunctions”), could generate cryptic amino acid sequences that can be a source of neoantigens. In this study, we investigated neojunctions based on multifaceted transcriptomic and proteomic analyses, seeking the potential cell surface antigens that may be targeted by CAR. METHODS For screening, we analyzed bulk RNA-sequencing data of TCGA-GBM/LGG with high tumor purity (n = 429) and GTEx normal tissues (n = 9,166). Cohorts of spatially mapped intratumoral samples and longitudinally collected tumors were used to determine clonality and stability of the candidate neojunctions. Nanopore long-read amplicon sequencing was deployed to confirm the full-length transcript sequence. Their protein-level expression was explored by analyzing the Clinical Proteomic Tumor Analysis Consortium (CPTAC)-GBM proteomics dataset. RESULTS In the screening analysis comparing TCGA and GTEx datasets, we identified 218 neojunctions with adequate expression, prevalence, and tumor-specificity. Of these, 12 were predicted to be cell-surface antigens. Eight of the 12, such as BCAN, DLL3, and PTPRZ1, were also observed in multiple cases of another validation dataset. In the analysis of tumors with spatially mapped intratumoral samples, 7 of the 12 were recurrently detected in no less than 50% of the samples in multiple cases. In addition, 5 of the 12 were found to be conserved in primary and recurrent pairs of tumors in multiple cases. Full-length transcript sequencing corroborated our predictions based on short reads, and also demonstrated more complex AS patterns. Finally, CPTAC-GBM proteomics analysis identified one cryptic peptide that substantiated the corresponding transcriptome-based prediction. CONCLUSION: We identified neojunctions with the potential to generate cell-surface antigens. These multifaceted transcriptomic and proteomic analyses provide the rationale to pursue the development of immunotherapy targeting neojunction-derived antigens.

132 HLA-independent T cell receptors effectively target low abundance antigens

BackgroundChimeric antigen receptors (CARs) engage antigen independently of HLA and enable sustained T cell proliferation when they are endowed with both activating and costimulatory functions. While remission rates have been noticeably elevated in numerous clinical trials targeting CD19, CD22 or BCMA, relapses are common. One of the several underlying relapse mechanisms is antigen escape, which refers to a relapsing tumor that is either negative for the targeted antigen or expresses the latter at a low level. Failure to eliminate antigen-low tumors raises questions about the sensitivity of CARs and the minimum antigen density that is required for effective tumor eradication. Unlike CARs, TCRs engage antigen in an HLA-dependent manner, and they do so with high sensitivity. We hypothesized that a TCR/CD3 complex containing the same heavy and light immunoglobulin chains as a CAR will display increased sensitivity to the target antigen.MethodsWe edited the TRAC locus in human primary T cells to establish a novel antigen receptor structure, termed HLA-independent TCR or HIT receptor, by incorporating into the TCR/CD3 complex the same heavy and light chains as those of a corresponding CAR. We assessed their antigen sensitivity against a panel of cell lines expressing different antigen levels, analyzing their cytotoxicity, cytokine secretion, signaling response and degranulation activity. HIT and CAR T cells were further evaluated for their anti-tumor response using established ALL and AML mouse models.ResultsCD19-TRAC-HIT and CD19-TRAC-CAR T cells lysed wild-type NALM6 (~27,000 CD19 molecules) and NALM6 variants with 100-fold less CD19. As CD19 levels decreased further, CAR T cells no longer killed their target, in contrast to HIT T cells. HIT T cells showed increased expression of IFN-gamma, IL-2 and TNF-alpha upon exposure to NALM6 cells expressing ~20 CD19 molecules per cell, compared to CAR T cells. This increased sensitivity of HIT receptors correlated to their greater signaling response, upon exposure to the low-antigen-density NALM6. Phospho-proteomic analyses further confirmed this increased response of HIT T cells to low antigen levels. Altogether, these results confirm that HIT receptors endow T cells with greater antigen sensitivity than canonical CARs. We further showed that HIT T cells have higher in vivo anti-tumor activity compared to CAR T cells in mice bearing low-antigen-density ALL or AML.ConclusionsHIT receptors consistently afford high antigen sensitivity and mediate tumor recognition beyond what current CARs can provide. HIT receptors open new prospects for targeting cell surface antigens of low abundance.Ethics ApprovalEight- to 12-week-old NOD/SCID/IL-2Rgamma-null (NSG) male mice (Jackson Laboratory) were used under a protocol approved by the MSKCC Institutional Animal Care and Use Committee.

Diagnostic, Prognostic, Predictive, and Monitoring Role of Neutrophil CD11b and Monocyte CD14 in Neonatal Sepsis

Background. Sepsis is a critical medical condition that requires additional diagnostic considerations. Recently, focus has shifted to the diagnosis of sepsis using new markers to overcome the limitations of traditional laboratory diagnostic modalities. Neutrophil CD11b (nCD11b) and monocyteCD14 (mCD14) cell surface antigens have been shown to be useful in such diagnostic consideration. Aim. To investigate the diagnostic, monitoring, prognostic, and predictive roles of nCD11b and mCD14 as sepsis biomarkers in comparison to each other and to traditional laboratory sepsis parameters in order to select the best fit for routine daily use in neonatal intensive care units (NICUs). Subject. The study included 188 neonates from Ain Shams University Hospitals’ NICUs, who were divided into two groups: the control group ( n = 100 ) and the sepsis group ( n = 88 ). Highly sensitive CRP (hs-CRP), complete blood count (CBC), blood culture, and nCD11b and mCD14 evaluations were all part of the laboratory sepsis evaluation (done by flow cytometry technology). Positive blood culture results (BACT/ALERT system) confirmed the sepsis diagnosis. Twenty-four enrolled sepsis neonates were subjected to follow-up assessments, and they were divided into two groups based on clinical improvement: improved sepsis and sepsis without improvement. In order to predict performance evaluation, the subjected neonates were reclassified according to their outcome into survivors’ and nonsurvivors’ group. Results. Sepsis patients had a significant increase in mCD14 MFI values when compared to controls. With sensitivity 75.4 percent, specificity 71.9 percent, efficacy 73.3 percent, and AUC 0.703, mCD14 MFI at cutoff 9.36 could distinguish the presence of septicemia. Significant increases in both mCD14 MFI and nCD11b MFI ( P = 0.001 ) were observed in the severe sepsis/septic shock group compared to the nonsevere sepsis group. The combined measurement of CD14 MFI at cutoff 9.97 and CD14 percent at cutoff 44.7 percent yielded the best predictive performance. Conclusion. Sepsis patients had a significant increase in mCD14 MFI comparable to the controls. mCD14 MFI demonstrated better diagnostic, prognostic, and predictive results than nCD11b. hs-CRP outperformed mCD14 and nCD11b in terms of diagnostic efficacy and AUC. In the monitoring of sepsis patients, both mCD14 and nCD11b produced unsatisfactory results. Currently, the routine use of mCD14 or nCD11b as sepsis biomarkers in neonatal ICUs is not justified.