Expression Of Differentiation Antigens Research Articles

IMMU-07. THE IMMUNE LANDSCAPE OF PEDIATRIC BRAIN TUMORS: A TRANSCRIPTOMIC ANALYSIS

Abstract Despite standard-of-care therapy, pediatric brain tumors are now the leading cause of cancer-related death in children, highlighting an urgent need for the development of new treatments. Immunotherapy is actively being evaluated as a therapeutic approach to pediatric brain tumors, however, more research is needed to reveal additional antigenic targets that exist in these malignancies. In this study, our team performed a transcriptomic analysis of pediatric brain tumor RNA sequencing data within the Children’s Brain Tumor Network (CBTN) to identify intra and extracellular antigens that are highly expressed across major tumor types. Interestingly, our team observed that most tumors had high gene expression of a group of antigens that were unique to that specific tumor type. Specifically, our analysis revealed that diffuse midline gliomas (DMGs) highly expressed the extracellular antigens CA9, CTCFL, and ST8SIA1, whereas atypical rhabdoid tumors (ATRTs) highly expressed MUC1 and TEK. Other tumors that displayed high gene expression for a variety of antigenic targets included H3-wildtype high-grade gliomas (HGGs), and ependymomas (EPNs). Strikingly, we observed minimal difference in antigen expression in primary tumors when compared to patient-matched recurrences. Beyond antigen expression, we evaluated the expression of antigen processing machinery (APM) which includes major histocompatibility complexes (MHCs). Our findings revealed that DMGs had the lowest level of APM expression, however, these tumors uniquely expressed high levels of the immune checkpoints ADORA2A, CD276, and KLRC1. Contrarily, ATRTs showed a high expression of immune checkpoints CD274 (PD-L1), PVR, and CD80. Other tumors included in our analysis had unique expression patterns of antigen, as well as APM and immune checkpoints. Our findings illustrate that pediatric brain tumors have distinct expression patterns of antigens, APM, and immune checkpoints that are specific to tumor type, emphasizing diverse, rather than general, immunotherapeutic approaches must be considered for childhood brain tumors.

Enhanced Immunohistochemistry Interpretation with a Machine Learning-Based Expert System.

In recent decades, machine-learning (ML) technologies have advanced the management of high-dimensional and complex cancer data by developing reliable and user-friendly automated diagnostic tools for clinical applications. Immunohistochemistry (IHC) is an essential staining method that enables the identification of cellular origins by analyzing the expression of specific antigens within tissue samples. The aim of this study was to identify a model that could predict histopathological diagnoses based on specific immunohistochemical markers. The XGBoost learning model was applied, where the input variable (target variable) was the histopathological diagnosis and the predictors (independent variables influencing the target variable) were the immunohistochemical markers. Our study demonstrated a precision rate of 85.97% within the dataset, indicating a high level of performance and suggesting that the model is generally reliable in producing accurate predictions. This study demonstrated the feasibility and clinical efficacy of utilizing the probabilistic decision tree algorithm to differentiate tumor diagnoses according to immunohistochemistry profiles.

Thy-1 restricts steatosis and liver fibrosis in steatotic liver disease.

Steatotic liver disease (SLD) is generally considered to represent a hepatic manifestation of metabolic syndrome and includes a disease spectrum comprising isolated steatosis, metabolic dysfunction-associated steatohepatitis, liver fibrosis and ultimately cirrhosis. A better understanding of the detailed underlying pathogenic mechanisms of this transition is crucial for the design of new and efficient therapeutic interventions. Thymocyte differentiation antigen (Thy-1, also known as CD90) expression on fibroblasts controls central functions relevant to fibrogenesis, including proliferation, apoptosis, cytokine responsiveness, and myofibroblast differentiation. The impact of Thy-1 on the development of SLD and progression to fibrosis was investigated in high-fat diet (HFD)-induced SLD wild-type and Thy-1-deficient mice. In addition, the serum soluble Thy-1 (sThy-1) concentration was analysed in patients with metabolic dysfunction-associated SLD stratified according to steatosis, inflammation, or liver fibrosis using noninvasive markers. We demonstrated that Thy-1 attenuates the development of fatty liver and the expression of profibrogenic genes in the livers of HFD-induced SLD mice. Mechanistically, Thy-1 directly inhibits the profibrotic activation of nonparenchymal liver cells. In addition, Thy-1 prevents palmitic acid-mediated amplification of the inflammatory response of myeloid cells, which might indirectly contribute to the pronounced development of liver fibrosis in Thy-1-deficient mice. Serum analysis of patients with metabolically associated steatotic liver disease syndrome revealed that sThy-1 expression is correlated with liver fibrosis status, as assessed by liver stiffness, the Fib4 score, and the NAFLD fibrosis score. Our data strongly suggest that Thy-1 may function as a fibrosis-protective factor in mouse and human SLD.

Electrospun photothermal tellurium nanosheet-loaded polycaprolactone/gelatin nanofibers to promote wound healing

Low-cost and biodegradable photothermal wound dressings with remarkable therapeutic effects are highly desirable for next-generation wound healing. Herein, we report an efficient photothermal wound dressing mat made of tellurium nanosheet (TeNS)-loaded electrospun polycaprolactone/gelatin (PCL/GEL) nanofibers. The TeNS-loaded PCL/GEL nanofibrous architectures showed antibacterial efficacy against Escherichia coli and Staphylococcus aureus of 87.68% and 94.57%, respectively. Under near-infrared (NIR) light illumination, they can facilitate cell proliferation as revealed by in vitro scratch assay. The results from in vivo skin wounds combined with tissue staining experiments further showed that the TeNS-loaded dressing could substantially promote wound healing under photothermal conditions. Using immunohistochemical analysis, we found that the TeNS-loaded PCL/GEL nanofibers [Formula: see text] NIR group have a high expression of specific antigens in epidermal growth factor (EGF) ([Formula: see text]) and endothelial cell adhesion molecule-31 (CD31, [Formula: see text]), verifying that the nanofibrous mat can stimulate EGF generation and microvessel proliferation. Furthermore, the PCL/GEL/Te[Formula: see text]NIR group has the lowest expression in endothelial cell adhesion molecule-68 (CD68, [Formula: see text]), suggesting that the nanofibrous mats have a high anti-inflammatory efficiency. Our work sheds light on the development of novel nonanti-inflammatory wound dressings via photothermal sterilization and the promotion of cell growth using two-dimensional (2D) nanosheets.

Abstract LB170: AND-gate dual-targeting EGFR and PD-L1 with a split prodrug converter enzyme for cancer treatment

Abstract Conventional cancer chemotherapies, while effective, often encounter "on-target, off-tissue" cytotoxicity linked to target prevalence. Although antibody-drug conjugates (ADCs) offer improved targeting, they face limitations in drug payload capacity. Therefore, a potent chemotherapy without off-tissue effects or payload limitations is in crucial need. To address the demand, we developed an AND-gate-based dual-targeting approach that minimizes off-tissue cytotoxicity with tune-able payload, exploiting a pair of split human simplex virus thymidine kinase (HSV-TK), which converts the prodrug ganciclovir (GCV) into a cytotoxic form with high specificity. We first successfully split a pair of HSV-TK (Split TK) to exhibit the possibility of an AND-gate drug activation system. HSV-TK was split into N-fragment (nTK) and C-fragment (cTK) with a 20 amino acid overlap, transduced into HEK293T cells, and treated with ganciclovir for 72 hours. Cells expressing one TK half showed IC50 >1000 µM, while co-expression of cTK and nTK reduced IC50 to ~3 µM, which is comparable to full-length TK. Successful splitting of HSV-TK demonstrated the feasibility of dual-targeting. We then validated this AND-gate targeting in cancer treatment. Split TK[HH1] was assessed in treatment of triple-negative breast cancer (TNBC) cells. TNBC lacks the overexpression of common breast cancer targets, which poses a significant challenge in treatment strategies. We designed a specific split TK pair to treat MDA-MD-231 TNBC, with MCF-7 cells as a control. MDA-MB-231 has upregulated epidermal growth factor receptor (EGFR) and programmed death-ligand 1(PD-L1) expression, while MCF-7 is high in EGFR but low in PD-L1. To enable a split TK pairs that selectively reconstituted HSV-TK activity in MDA-MB-231 cell, we choose epidermal growth factor (EGF) as the targeting ligand for EGFR, and PD-1 extracellular domain for PD-L1. We fused nTK to EGF (nTK-EGF), and the extracellular domain of programmed cell death protein 1 (PD-1) to cTK (PD-1-cTK). Fusion proteins were expressed in SHuffle E. coli, purified, and introduced into MDA-MB-231 and MCF-7 cells, together with 100 µM GCV. After 7 days of treatment, individual nTK-EGF and PD-1-cTK treatments with GCV showed minimal cytotoxicity. However, combined treatment of nTK-EGF and PD-1-cTK significantly decreased cell viability in MDA-MB-231 by ~20%, while MCF-7 remained healthy. In summary, we have successfully constructed and validated an AND-gate-based dual-targeting approach that minimizes off-tissue cytotoxicity with tune-able payload for cancer treatment. This versatile AND-gate dual-targeting system has the great potential of a paradigm-shifting strategy of cancer treatment, particularly for the cancers that cannot be treated by targeting a single antigen. The distinct cytotoxicity based on differential antigen expression introduces a novel cancer therapy category to overcome the targeting difficulties of triple-negative breast cancer (TNBC) and is potentially generalizable to other cancers. Citation Format: Mengrou Lu, Yunhui Xing, Keng-Jung Lee, Xi Ren, Justin R. Pritchard, Boyle C. Cheng, Hongzhang He, Siyang Zheng. AND-gate dual-targeting EGFR and PD-L1 with a split prodrug converter enzyme for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB170.

Turning Trash Into Treasure: A Simple Protocol for Human Mesenchymal Stromal Cell Isolation From Used Bone Marrow Collection Kits.

The therapeutic potential of mesenchymal stromal cells (MSCs) has been extensively investigated in both preclinical and clinical settings. Recent years have witnessed the emergence of numerous isolation protocols and culture techniques, ranging from the selection of subpopulations to preserve stemness to preconditioning strategies aimed at enhancing therapeutic efficacy, tailored to the specific tissue source. In this protocol, we present a straightforward and cost-effective method for isolating human MSCs (hMSCs) from discarded bone marrow collection kits (comprising bag and filter systems) originally intended for removing impurities and unwanted cellular debris from the collected bone marrow aspirate, ensuring the purity of the stem cell population during stem cell transplantation. Utilizing basic laboratory equipment, we demonstrate the isolation of hMSCs, highlighting the expression of specific surface antigens, and multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages in vitro. This sustainable and resource-efficient approach not only contributes to reducing medical waste but also holds promise for advancing regenerative medicine applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human mesenchymal stromal cells from bone marrow collection kits Basic Protocol 2: Culture of human mesenchymal stromal cells Basic Protocol 3: Characterization of human mesenchymal stromal cells with flow cytometry analysis Basic Protocol 4: Characterization of human mesenchymal stromal cells with multilineage differentiation under in vitro conditions.

Development of a Protocol for Obtaining a Homologous Cell Product of Animal Origin Intended for Preclinical Studies of Antitumor Vaccine CaTeVac.

When developing a program of preclinical studies of human cell-based drugs intended for adoptive immunotherapy of cancer patients, the biological effect should be substantiated by data describing their immunological action. Administration and study of human autologous dendritic cell vaccine to immunocompetent animals are not adequate in terms of immunological compatibility. It is possible to use immunocompromised, knockout, or transgenic animals or to obtain a homologous cellular product, namely, a preparation based on animal cells using a technology similar to obtaining the original preparation for clinical practice in humans. Within the framework of this study, we have developed a protocol for obtaining a homologous cell product based on animal dendritic cells (mice, rats) according to a similar technology for obtaining human vaccine dendritic cells, and demonstrated the comparability of morphological characteristics and expression of differentiation antigens of dendritic cells (CD11c, CD80, CD86, and CD83) of animals (mice) and humans.

Multiomics Characterization of Normal CD34+ Hematopoietic Progenitor Cells (HPC) and Blasts in the Bone Marrow (BM) and Peripheral Blood (PB) of Patients with Acute Myeloid Leukemia (AML)

BACKGROUNG: Multiparameter flow cytometry (MFC) is traditionally used to asses MRD in AML. However, its sensitivity and specificity lag behind other methods and this precludes a broader application in routine practice. It could be hypothesized the emerging multiomics such as single-cell RNA and antibody sequencing could uncover new differences from normal and leukemia associated immunophenotypes (DfN/LAIP). In addition, the identification of DfN/LAIP may be easier in PB when compared to the BM due to the scarcity of CD34+ HPC in the former sample. AIM: Uncover new DfN/LAIP for improved MFC discrimination between normal CD34+ HPC and blasts in the BM and PB of AML patients. METHODS: A total of 47 samples were analyzed in this study. Namely, paired BM and PB from 5 young healthy adults (20-23yo) and 5 PB samples from older healthy adults (47-88yo); paired BM and PB of 11 newly diagnosed AML (44-79yo) patients, and paired BM and PB collected in 5 of the 11 AML patients at the time of MRD assessment. We used fluorescence activated cell sorting (FACS) to isolate CD34+ HPC from the BM and PB of healthy adults as well as blasts from AML patients. scCITE-Seq with a panel of 600 genes and 50 oligo-conjugated antibodies, selected according to their role in stem cell identification, lineage differentiation, clonal hematopoiesis and aberrant expression in myeloid neoplasms was performed. After quality control filtering, a total of 111,334 cells were analyzed and differential expression of genes and antigens was considered if an adjusted P-value <.05 and a log 2FoldChange (FC) >|1| were observed. RESULTS: We first analyzed the phenotype of paired BM and PB 32,637 CD34+ HPC from younger healthy adults. There were 82 deregulated genes, 50 overexpressed in BM and 32 in PB. CLEC12A, CEBPA and HBB were amongst the top overexpressed genes in BM, whereas CRHBP, ADGRG6 and MEIS1 showed the highest upregulation in PB. These results suggest partial transcriptional overlap between BM and PB CD34+ HPC, with the former showing reduced differentiation. There were no differences in antigen expression. Next, we analyzed the potential effect of aging in the phenotype of 34,102 CD34+ HPC from the PB of young and older healthy adults. There were 34 genes differentially expressed including BCORL1, CBLB, HRAS and KDM6A. These findings might reflect an upregulation of genes associated with clonal hematopoiesis. Furthermore, there were 13 antigens differentially expressed including CD9, CD10, CD33, CD38, CD62L and CD371. The comparison of 56,387 blasts in paired BM and PB samples from newly diagnosed AML patients showed near overlap with only HBB being differentially expressed. There were no differences in antigen expression. We then investigated the extent of phenotypic differences between normal CD34+ HPC vs blasts in the BM and PB of healthy adult's vs AML patients. There were 224 and 15 differentially expressed genes between BM cell types, whereas in PB the numbers were 182 and 11, respectively. The markers that contributed the most in the discrimination between CD34+ HPC vs blasts in PB were WT1, ADM, HMOX1, DDIT3, HOPX, CD371, CD45RA, CD133 and CD47. The overlap between DfN/LAIP identified in PB and BM was 31%. Four of the five patients monitored after treatment were MRD positive in BM using MFC. Of note, BM and PB CD34+ HPC isolated from the five AML patients (including one MRD negative in BM) clustered separately of normal BM and PB CD34+ HPC from healthy adults. There was near overlap between BM and PB cells from AML patients. The markers that contributed the most in the discrimination between PB CD34+ HPCfrom healthy adult's vs resistant cells from AML patients were CXCL16, LILRA5, MME, CD274, CTSL, MINPP1 and AHSP. CONCLUSIONS: Normal CD34+ HPC in PB showed partial overlap with the BM counterpart, with a few phenotypic differences probably related to less differentiation and the effects of aging and clonal hematopoiesis. The phenotype of BM and PB blasts from newly diagnosed AML patients nearly overlapped, which indicates that DfN/LAIP may be equally applicable in BM and PB. Accordingly, the markers included in the scRNA/Ab-seq panel effectively discriminated CD34+ HPC from blasts regardless of the sample of origin. Furthermore, we showed phenotypic divergence between PB CD34+ HPC of healthy adults' vs AML patients after treatment. Taken together, this study identified new DfN/LAIP markers for improved assessment of MRD using MFC in the PB of AML patients.

Immunohistochemical Evaluation of the Expression of Specific Membrane Antigens in Patients with Pancreatic Ductal Adenocarcinoma.

(1) Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. The lack of validated disease biomarkers makes timely diagnosis challenging in most cases. Cell membrane and surface proteins play a crucial role in several routes of oncogenesis. The aim of this study was to evaluate the expression of six membrane antigens on PDAC (CA 19-9, mucin 1 and 4 (MUC1, MUC4), mesothelin (MSLN), Annexin A10 (ANXA10), Glypican-1 (GPC-1)) and their correlation with oncologic outcomes. (2) Methods: Immunohistochemical staining for CA 19.9, MUC1, MUC4, MSLN, ANXA10, and GPC-1 of surgical samples of 50 consecutive patients with PDAC was performed. Antigen expression for tumor, ductal, and acinar tissues was classified according to the histo-score (H-score) by two pathologists. (3) Results: Recurrence rate was 47% and 18 patients (36%) deceased (median follow-up 21.5 months). Immunostaining for CA 19-9 and MUC1 showed a significantly higher expression in the neoplastic tissue compared to non-tumor ductal and acinar tissues (p < 0.001). MUC4, MSLN, ANXA10, and GPC-1 were selectively expressed in the neoplastic tissue (p < 0.001). A CA 19-9 H-score value >270 was independently associated with a worse overall survival (p = 0.05) and disease-free survival (p = 0.05). (4) Conclusions: CA 19-9 and MUC1 are highly expressed in PDAC cells. The histological expression of CA 19-9 may predict prognosis. MUC4, MSLN, ANXA10, and GPC-1 are selectively expressed by neoplastic tissue and may represent a potential histological biomarker of disease.

Unveiling the Etiopathogenic Spectrum of Hypophysitis: A Narrative Review.

Hypophysitis, a rare inflammatory disorder of the pituitary gland, has seen an uptick in reported cases in recent years. Our objective is to summarize the most recent research on the etiopathogenesis, molecular mechanisms, and genetics of both primary and secondary hypophysitis. Primary autoimmune hypophysitis (PAH): During the acute phase of the disease, the pituitary gland in enlarged due to the infiltration of T and B lymphocytes. The chronic phase is characterized by progressive and irreversible pituitary atrophy. APA may play a role in the management, diagnosis, and prognosis of PAH. Specific autoantibodies such as anti-GH, anti-PIT-1, and anti-T-PIT have been found in patients with hypophysitis and hypopituitarism. A recent study suggested that a mechanism of escaping clonal deletion and mounting an immune response against self antigens can explain the unusual nature of the immune response observed in PAH patients. A cytokine array shows the presence of gamma-interferon and interleukin-17. Patients carrying mutations in the PIT1 or PROP1 genes may present PAH. Individuals carrying the HLA DQ8 haplotype are four times more likely to develop PAH. Immune checkpoint inhibitors induce hypophysitis (IIHs): IIHs is an increasingly frequent toxicity of in patients on treatment with inhibitors targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death-1 (PD-1). ICIs inhibit the CTLA-4 pathway, leading to overactivation of T lymphocytes. The binding of PD-1/PD-L1 suppresses the activity of T cells, promotes the conversion of T-helpers into T-regulatory cells, and activates pro-survival signaling pathways in cancer cells. Cytokines play a crucial role in IIHs. B-cell infiltration has been observed in IIHs, suggesting that antibody-mediated pituitary injury may contribute. Genetic polymorphisms of CTLA-4 and PD-1 genes can increase the risk of IIHs. HLA alleles may also be involved in the onset of IIHs; this HLA association presents a possible alternative mechanistic hypothesis. IIHs may also be linked to a paraneoplastic syndrome triggered by ectopic expression of pituitary specific antigens. SARS-CoV-2-related hypophysitis: Recently, the literature has reported occurrences of hypophysitis associated with the SARS-CoV-2 virus; long COVID-19 may also present as infundibulo-neuro-hypophysitis. The virus enters the central nervous system because of its distinct interaction with angiotensin-converting enzyme receptors via spike proteins binding the capillary endothelium, and it directly damages the pituitary cells. The effect of SARS-CoV-2 can occur indirectly through inflammation and the release of cytokines. The exact mechanism remains ambiguous. The available data on endocrine complications associated with the SARS-CoV-2 vaccine are scant. Nonetheless, isolated cases of hypophysitis have been documented. Treatment of hypophysitis: Glucocorticoids are the cornerstone in managing primary hypophysitis, given their targeted action on inflammation. A better understanding of the etiopathogenesis and molecular mechanism of hypophysitis can lead to more effective and personalized treatment strategies.

Enhancement of immune surveillance in breast cancer by targeting hypoxic tumor endothelium: Can it be an immunological switch point?

Breast cancer ranks second among the causes of cancer-related deaths in women. In spite of the recent advances achieved in the diagnosis and treatment of breast cancer, further study is required to overcome the risk of cancer resistance to treatment and thereby improve the prognosis of individuals with advanced-stage breast cancer. The existence of a hypoxic microenvironment is a well-known event in the development of mutagenesis and rapid proliferation of cancer cells. Tumor cells, purposefully cause local hypoxia in order to induce angiogenesis and growth factors that promote tumor growth and metastatic characteristics, while healthy tissue surrounding the tumor suffers damage or mutate. It has been found that these settings with low oxygen levels cause immunosuppression and a lack of immune surveillance by reducing the activation and recruitment of tumor infiltrating leukocytes (TILs). The immune system is further suppressed by hypoxic tumor endothelium through a variety of ways, which creates an immunosuppressive milieu in the tumor microenvironment. Non responsiveness of tumor endothelium to inflammatory signals or endothelial anergy exclude effector T cells from the tumor milieu. Expression of endothelial specific antigens and immunoinhibitory molecules like Programmed death ligand 1,2 (PDL-1, 2) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3) by tumor endothelium adds fuel to the fire by inhibiting T lymphocytes while promoting regulatory T cells. The hypoxic microenvironment in turn recruits Myeloid Derived Suppressor Cells (MDSCs), Tumor Associated Macrophages (TAMs) and T regulatory cells (Treg). The structure and function of newly generated blood vessels within tumors, on the other hand, are aberrant, lacking the specific organization of normal tissue vasculature. Vascular normalisation may work for a variety of tumour types and show to be an advantageous complement to immunotherapy for improving tumour access. By enhancing immune response in the hypoxic tumor microenvironment, via immune-herbal therapeutic and immune-nutraceuticals based approaches that leverage immunological evasion of tumor, will be briefly reviewed in this article. Whether these tactics may be the game changer for emerging immunological switch point to attenuate the breast cancer growth and prevent metastatic cell division, is the key concern of the current study.

Study on the Relationship between Integrin 2A and Drug Resistance in Chronic Myeloid Leukemia

To explore the expression pattern and clinical significance of Integral membrane protein 2A（ITM2A） in drug resistant patients with chronic myeloid leukemia (CML). The expression of ITM2A in CML was evaluated by qRT-PCR, Western blot and immunocytochemistry. In order to understand the possible biological effects of ITM2A, apoptosis, cell cycle and myeloid differentiation antigen expression of CML cells were detected by flow cytometry after over-expression of ITM2A. The nuderlying molecular mechanism of its biological effect was explored. The expression of ITM2A in bone marrow of CML resistant patients was significantly lower than that of sensitive patients and healthy donors（P<0.05）. The CML resistant strain cell K562R was successfully constructed in vitro. The expression of ITM2A in the resistant strain was significantly lower than that in the sensitive strain(P<0.05). Overexpression of ITM2A in K562R cells increased the sensitivity of K562R cells to imatinib and blocked the cell cycle in G2 phase(P<0.05), but did not affect myeloid differentiation. Mechanistically, up-regulation of ITM2A reduced phosphorylation in ERK signaling (P<0.05). The expression of ITM2A was low in patients with drug resistance of CML, and the low expression of ITM2A may be the key factor of imatinib resistance in CML.

IgE-mediated Anisakis allergy in children.

Anisakids are nematodes responsible for different clinical patterns in humans. The well-known human-infecting Anisakis species include members of the Anisakis simplex (AS) complex. Humans usually contract anisakiasis through ingestion of raw or undercooked seafood containing Anisakis larvae. Once Anisakis has been ingested, patients may develop disease driven directly by Anisakis larvae and/or by allergic reaction due to this nematode. The capability of inducing allergic reactions depends on the expression of specific antigens by nematodes and host factors. This study aims to resume actual knowledge about AS and Anisakiasis with regard to epidemiology, pathophysiology, clinical presentation, diagnosis, and treatment. Particular attention is paid to Anisakis allergens and their cross-reactivity on available diagnostic methods, and defining a diagnostic pathway for Anisakis allergy. Because only a few data are available in the literature about pediatric population, we focus on this group of patients specifically.

Differential antigen expression between human apocrine sweat glands and eccrine sweat glands

Bromhidrosis has a great negative impact on personal occupation and social psychology. It is not yet clear whether bromhidrosis is caused by apocrine sweat glands or the co-action of apocrine sweat glands and eccrine sweat glands. To distinguish between apocrine sweat glands and eccrine sweat glands, specific antigen markers for apocrine sweat glands and eccrine sweat glands must be found first. In the study, we detected the expression of K7, K18, K19, Na+-K+-2Cl- cotransporter 1 (NKCC1), carbonic anhydrase II (CAII), Forkhead transcription factor a1 (Foxa1), homeobox transcription factor engrailed homeobox1 (En1), gross cystic disease fluid protein-15 (GCDFP-15), mucin-1 (MUC-1), cluster of differentiation 15 (CD15) and apolipoprotein (APOD) in eccrine sweat glands and apocrine sweat glands by immunofluorescence staining. The results showed that K7, K18, K19, Foxa1, GCDFP-15 and MUC-1 were expressed in both apocrine and eccrine sweat glands, CD15 and APOD were only expressed in apocrine sweat glands, and CAII, NKCC1 and En1 were only expressed in eccrine sweat glands. We conclude that CD15 and APOD can serve as specific markers for apocrine sweat glands, while CAII, NKCC1 and En1 can serve as specific markers for eccrine sweat glands to differentiate the two sweat glands.

Systematic Evaluation of AML-Associated Antigens Identifies Novel Anti-U5 snRNP200 Therapeutic Antibodies for the Treatment of AML

Numerous antibody-based immunotherapies are in development for AML including antibodies targeting CD47, CD70, CD123, CD33, and TIM3. However, systematic evaluation of the distribution of AML-associated antigens in combination with Fc receptors and the immune microenvironment required for antibody-mediated therapeutic responses have been limited. Additionally, efforts to maximize antibody Fc receptor engagement on immune effector cells in AML patients via Fc region engineering have not been explored. Here we applied a custom 36-color flow cytometry panel tailored for AML patient bone marrow along with proteogenomic assessment via CITE-seq to simultaneously assess known and novel antibody target expression on AML cells, immune microenvironment, and Fc receptor expression across immune subsets. Application to samples from 7 age-matched healthy donors and 51 newly diagnosed AML patients allowed for unbiased identification of the malignant blast population via UMAP projection. Quantification of differential antigen expression on blasts and leukemic stem cells versus normal hematopoietic stem and progenitor cell (HSPC) subsets, identified that out of 9 AML putative antibody targets (CD123, TIM3, CD33, CD47, CD90, CD38, CD25, CD70, U5 snRNP200), only U5 snRNP200 is expressed on blasts but not normal HSPCs (Fig. A). Characterization of the distribution of Fc receptors revealed that AML patients exhibit reduced cell-surface activating Fc receptors (CD16, CD32A) on NK and non-classical monocytes with concomitant increases in inhibitory (CD32B) Fc receptors on monocytes and B-cells. Moreover, AML patients had fewer classical monocytes and type 2 conventional dendritic (cDC2) cells than normal age-match control marrow. Given the unique cell surface expression of U5 snRNP200 on AML blasts compared with normal HSPCs, we deeply interrogated the distribution of this marker throughout normal and malignant hematopoiesis. While U5 snRNP200 expression was not seen on normal HSPCs, U5 snRNP200 was present on mature B-cells and a subset of NK cells in normal adult human and mouse hematopoietic cells. U5 snRNP200 is an RNA helicase which is an essential conserved component of the spliceosome. Its known function is limited to the nucleus and it is therefore unexpected that it would be expressed on the cell surface. To rigorously confirm expression of U5 snRNP200 on the cell membrane, we knocked in a HaloTag at the N-terminus of the locus encoding U5 snRNP200 within K562 cells and traced expression of tagged endogenous U5 snRNP200 protein across cellular compartments. This clearly revealed cell surface localization of the full-length protein on the surface of AML cells. We next conducted genome-wide CRISPR screens in two human AML cell lines to identify regulators of surface U5 snRNP200 expression. In both cell lines, the top gene required for cell surface U5 snRNP200 expression was CD32A. CRISPR knockout of CD32A in U937 cells reduced U5 snRNP200 surface expression, while restoration of CD32A rescued U5 snRNP200 surface expression. AML-associated expression of U5 snRNP200 was present on a number of syngeneic immunocompetent mouse models of AML including MLL-AF9, RN2 (MLL-AF9 + NRAS G12D), transgenic EVI1 rearranged/SF3B1K700E mutant, and Tet2/Zrsr2 double knockout mice. To test the therapeutic potential of targeting cell surface U5 snRNP200, we engineered several anti-murine U5 snRNP200 antibody variants featuring modified Fc regions to assess the possible roles of engagement of Fc receptors on immune cell subsets to enhance AML cell killing. In both the RN2 and EVI1/SF3B1 mutant models, anti-U5 snRNP200 antibodies featuring the Fc region modified to engage activating Fc receptors and induce antibody-dependent cellular cytotoxicity (IgG2a) provided superior survival benefit compared to IgG1 or IgG2b U5 snRNP200 antibodies or a mutant IgG1 antibody (IgG1 D265A) which does not bind Fc receptors (Fig. B). These data provide a high-density roadmap of the distribution of known and novel AML-associated antigens together with the Fc receptor distribution and immune microenvironment in AML. The results motivated discovery of a novel antibody-therapeutic targeting aberrant cell-surface U5 snRNP200 and research into mechanisms for cell surface trafficking of U5 snRNP200. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nucleic acid vaccination strategies for ovarian cancer.

High grade serous carcinoma (HGSC) is one of the most lethal ovarian cancers that is characterised by asymptomatic tumour growth, insufficient knowledge of malignant cell origin and sub-optimal detection. HGSC has been recently shown to originate in the fallopian tube and not in the ovaries. Conventional treatments such as chemotherapy and surgery depend upon the stage of the disease and have resulted in higher rates of relapse. Hence, there is a need for alternative treatments. Differential antigen expression levels have been utilised for early detection of the cancer and could be employed in vaccination strategies using nucleic acids. In this review the different vaccination strategies in Ovarian cancer are discussed and reviewed. Nucleic acid vaccination strategies have been proven to produce a higher CD8+ CTL response alongside CD4+ T-cell response when compared to other vaccination strategies and thus provide a good arena for antitumour immune therapy. DNA and mRNA need to be delivered into the intracellular matrix. To overcome ineffective naked delivery of the nucleic acid cargo, a suitable delivery system is required. This review also considers the suitability of cell penetrating peptides as a tool for nucleic acid vaccine delivery in ovarian cancer.

Immunohistochemical evaluation of the expression of specific membrane antigens in patients with pancreatic ductal adenocarcinoma undergoing surgery

Immunohistochemical evaluation of the expression of specific membrane antigens in patients with pancreatic ductal adenocarcinoma undergoing surgery

An update on the use of cryoablation and immunotherapy for breast cancer.

The use of cryoablation, a minimally-invasive image-guided technique to target and kill cancer cells, continues to gain traction within the medical field and with patients. This includes the use of cryoablation for the treatment of small breast cancers and focal sites of metastatic disease. In comparison to open surgical approaches, length of hospital stay and recovery time are decreased with the use of cryoablation. Research studies have also found that cryoablation may actually enhance tumor susceptibility to immunotherapy agents. Immunotherapy enhances a person's own immune system to identify and attack cancer cells. It is proposed that after cryoablation there is increased expression of tumor specific antigens which the body can recognize as foreign invaders and with the combination of immunotherapy, result in an even more robust and efficient attack on the cancer cells. In this review we aim to highlight some of the recent advances in cryoablation which support the potential for cryoablation to induce these tumor-specific immune responses and thus supporting the use of combining cryoablation and immunotherapy for the treatment of breast cancer.

Enhanced radiation-induced immunogenic cell death activates chimeric antigen receptor T cells by targeting CD39 against glioblastoma

Chimeric antigen receptor (CAR)-T cells directed to solid tumors have been less effective, due in part to the low or lost expression of specific tumor antigens. Herein, we developed a different strategy to enhance CAR-T cell persistence and efficacy by producing a multispecific CAR-T or vaccine based on immunogenic cell death (ICD). We demonstrated that ionizing radiation activates STAT1-IRF1-CD39 axis to upregulate CD39 expression to form an immunosuppressive tumor microenvironment (TME) to enhance radioresistance. CD39 blockade accumulates extracellular ATP, which activates NLRP3 inflammasome in dendritic cells via P2X7 receptor, thereby promoting radiation-induced ICD. Multispecific CAR-T cells in vitro prepared by elevated ICD suppress the growth of xenografts in nude mice. Radiation and CD39 inhibition-induced ICD of glioma stem cells as a vaccine enhance CAR-T expansion in peripheral blood, multifunctionality in the TME, and antitumor effect in a glioma model. The multispecificity of CAR-T cells, targeting CAR and tumor antigens, vastly enhances the function of conventional CAR-T cells, stimulates a native immune response, and overcomes obstacles of specific antigen loss or low expression of target cells in antitumor therapy.

Complex genetic and histopathological study of 15 patient-derived xenografts of aggressive lymphomas

AbstractNon-Hodgkin lymphomas (NHL) represent the most common hematologic malignancies. Patient-derived xenografts (PDXs) are used for various aspects of translational research including preclinical in vivo validation of experimental treatment approaches. While it was repeatedly demonstrated that PDXs keep majority of somatic mutations with the primary lymphoma samples, from which they were derived, the composition of PDX tumor microenvironment (TME) has not been extensively studied. We carried out a comparative genetic and histopathological study of 15 PDX models derived from patients with various types of NHL including diffuse large B-cell lymphoma (DLBCL; n = 7), Burkitt lymphoma (BL; n = 1), mantle cell lymphoma (MCL; n = 2), and peripheral T-cell lymphomas (PTCL; n = 5). Whole exome sequencing (WES) of the PDXs and primary lymphoma cells was implemented in 13 out of 15 cases with available DNA samples. Standard immunohistochemistry (IHC) was used to analyze the composition of PDX TME. WES data confirmed that PDXs maintained the genetic heterogeneity with the original primary lymphoma cells. In contrast, IHC analysis revealed the following recurrently observed alterations in the composition of PDX tumors: more blastoid lymphoma cell morphology, increased proliferation rate, lack of non-malignant cellular components including T cells and (human or murine) macrophages, and significantly lower intratumoral microvessel density and microvessel area composed of murine vessels. In addition, PDX tumors derived from T-NHL displayed additional differences compared to the primary lymphoma samples including markedly lower desmoplasia (i.e., the extent of both reticular and collagen fibrosis), loss of expression of cytotoxic granules (i.e., perforin, TIA, granzyme B), or loss of expression of T-cell specific antigens (i.e., CD3, CD4, CD8). Our data suggest that despite keeping the same genetic profiles, PDX models of aggressive NHL do not recapitulate the microenvironmental heterogeneity of the original lymphomas. These findings have implications on the relevance of PDX models in the context of preclinical research.