The T-locus - inspiration and distraction?

The T/t locus was a major focus of study by mouse geneticists during the 20th century. In the 70s, as the study of cell surface antigens controlling transplantation antigens was taking off, several laboratories hypothesized that alleles of this locus would control cell surface antigens important for embryonic development. One such antigen, the embryonal carcinoma F9 antigen was said to be an example. Other antigens were described on sperm and embryos that were said to be controlled by alleles at the T/t complex. These findings were later found to be false. The history of the findings and their refutation is described.

Germ cell tumors, cell surface markers, and the early search for human pluripotent stem cells.

Many strands of research by different groups, starting from teratocarcinomas in the laboratory mouse, later moving the corresponding human tumors, contributed to the isolation and description of human pluripotent stem cells (PSCs). In this review, I highlight the contributions from my own research, particularly at the Wistar Institute during the 1980s, when with my colleagues we characterized one of the first clonal lines of pluripotent human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, and identified key features including cell surface antigen markers that have since found a place in the study and exploitation of human PSC. Much of this research depended upon close teamwork with colleagues, many in other laboratories, who contributed different expertise and experience. It was also often driven by circumstance and chance rather than pursuit of a grand design.

Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer.

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting.

Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues.

BACKGROUND AIMS: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. METHODS: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. RESULTS: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. CONCLUSIONS: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.

A CD138-independent strategy to detect minimal residual disease and circulating tumour cells in multiple myeloma.

CD138 (also termed SDC1) has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug-resistant residual and circulating MM cells were shown to have lower expression of this marker. In this study, we have shown that residual MM cells following bortezomib treatment are hypoxic. This combination of drug exposure and hypoxia down-regulates their CD138 expression, thereby making this marker unsuitable for detecting residual or other hypoxic MM cells, such as circulating tumour cells, in MM. Hence, we developed an alternative biomarker set which detects myeloma cells independent of their hypoxic and CD138 expression status in vitro, in vivo and in primary MM patients. The new markers were able to identify a clonal CD138-negative population as minimal residual disease in the bone marrow and peripheral blood of MM patients. Further investigation to characterize the role of this population as a prognostic marker in MM is warranted.

Candidate Biomarkers for Targeting in Type 1 Diabetes; A Bioinformatic Analysis of Pancreatic Cell Surface Antigens.

OBJECTIVE: Type 1 diabetes (T1Ds) is an autoimmune disease in which the immune system invades and destroys insulin-producing cells. Nevertheless, at the time of diagnosis, about 30-40% of pancreatic beta cells are healthy and capable of producing insulin. Bi-specific antibodies, chimeric antigen receptor regulatory T cells (CAR-Treg cells), and labeled antibodies could be a new emerging option for the treatment or diagnosis of type I diabetic patients. The aim of the study is to choose appropriate cell surface antigens in the pancreas tissue for generating an antibody for type I diabetic patients. MATERIALS AND METHODS: In this bioinformatics study, we extracted pancreas-specific proteins from two large databases; the Human Protein Atlas (HPA) and Genotype-Tissue Expression (GTEx) Portal. Pancreatic-enriched genes were chosen and narrowed down by Protter software for the investigation of accessible extracellular domains. The immunohistochemistry (IHC) data of the protein atlas database were used to evaluate the protein expression of selected antigens. We explored the function of candidate antigens by using the GeneCards database to evaluate the potential dysfunction or activation/hyperactivation of antigens after antibody binding. RESULTS: The results showed 429 genes are highly expressed in the pancreas tissue. Also, eighteen genes encoded plasma membrane proteins that have high expression in the microarray (GEO) dataset. Our results introduced four structural proteins, including NPHS1, KIRREL2, GP2, and CUZD1, among all seventeen candidate proteins. CONCLUSION: The presented antigens can potentially be used to produce specific pancreatic antibodies that guide CARTreg, bi-specific, or labeling molecules to the pancreas for treatment, detection, or other molecular targeted therapy scopes for type I diabetes.

Evolution of developmental and comparative immunology in poultry: The regulators and the regulated.

Avian has a unique immune system that evolved in response to environmental pressures in all aspects of innate and adaptive immune responses, including localized and circulating lymphocytes, diversity of immunoglobulin repertoire, and various cytokines and chemokines. All of these attributes make birds an indispensable vertebrate model for studying the fundamental immunological concepts and comparative immunology. However, research on the immune system in birds lags far behind that of humans, mice, and other agricultural animal species, and limited immune tools have hindered the adequate application of birds as disease models for mammalian systems. An in-depth understanding of the avian immune system relies on the detailed studies of various regulated and regulatory mediators, such as cell surface antigens, cytokines, and chemokines. Here, we review current knowledge centered on the roles of avian cell surface antigens, cytokines, chemokines, and beyond. Moreover, we provide an update on recent progress in this rapidly developing field of study with respect to the availability of immune reagents that will facilitate the study of regulatory and regulated components of poultry immunity. The new information on avian immunity and available immune tools will benefit avian researchers and evolutionary biologists in conducting fundamental and applied research.

The study of neural antibodies in neurology: A practical summary.

The field of Autoimmune Neurology is expanding rapidly, with new neural antibodies being identified each year. However, these disorders remain rare. Deciding when to test for these antibodies, when and what samples are to be obtained, how to handle and study them correctly, and how to interpret test results, is complex. In this article we review current diagnostic techniques and provide a comprehensive explanation on the study of these patients, in an effort to help with correct diagnosis minimizing false positive and false negative results. We also propose routine storage of samples and referral of certain cases to specialized research laboratories.

Detection of Homologous Blood Transfusion in Sport Doping by Flow Cytofluorimetry: State of the Art and New Approaches to Reduce the Risk of False-Negative Results.

This article presents the results of a study aimed to give new suggestions and strategies for improving the implementation of the flow cytofluorimetry-based method for the detection of homologous blood transfusions in doping control. The method is based on the recognition of the phenotypic mismatch between minority blood group antigens possessed by the donor and the recipient. Two strategies have been followed to reduce the risk of false-negative results: (i) the monitoring of a broader range of erythrocytes surface antigens; and (ii) the application of different surface erythrocyte staining protocols, tailored on the different antigens and the type of antigenic mismatch that had to be detected (whether it is the donor or the recipient who expresses or not the antigen to be detected). Special attention has also been focused on the time factor, to avoid prolonged sample storage, since hemolysis may have a significant impact on the reliability and quality of the results. Our experimental evidence suggests that the risk of false-negative results can be minimized by (i) the expansion of the antigen panel, with the inclusion of four additional targets; (ii) a more accurate selection of the gating area of the red blood cells; (iii) the choice of a better fluorochrome (alexa fluor 488) to be conjugated to the secondary antibody; and (iv) the implementation of different staining protocols depending on the nature of the double population to be detected (donor expressing vs. recipient non-expressing and vice versa). The combination of the above approaches allowed a significant reduction of false-negative results, assessed on samples simulating a homologous blood transfusion between two compatible subjects.

Comparative assessment of the cell-surface antigens and gene expression profiles of the gingival tissue biomarkers in subjects with fixed functional and removable functional orthodontic appliances.

Background: This study aimed to examine the cellular components of the gingiva during orthodontic treatment with fixed and removable appliances. The cellular and molecular cues of pathologies of the gingival tissue associated with the use of different orthodontic appliances could be studied. Materials and methods: Tissue samples of gingiva were received from healthy patients undergoing gingivectomy for aesthetic purpose and from patients with fixed and removable functional orthodontic appliances undergoing gingivectomy for gingival overgrowth. The collected samples were stored in a sterile container with phosphate-buffered saline and to carry out further processes it was transported to the laboratory. Results: Cells positive for ECAD and NCAD were found to be increased in fixed appliances where as CD90 and CD105 positive cells showed no significant difference in all the three groups. CD24 and CD146 positive cells were increased significantly in removable and fixed than normal whereas CD133 positive cells were decreased in removable and fixed than normal. CD44 positive cells showed no noticeable change in all three groups. The gene expression levels of KRT5, SOX2, NANOG, and CXCL5 were found to be significantly increased in removable and fixed appliance groups. However, KRT8, CXCL10, and TIMP1 were increased only in fixed appliance group but CXCL10 showed decreased expression in removable appliance group. KRT6A, MYC, and MMP9 were decreased in fixed appliance group whereas MYC and MMP9 were increased in removable appliance group. KRT6A, KRT8, and TIMP1 showed no significant difference in removable appliance group. Conclusion: This study demonstrated essential roles of various genes, showing their contribution in regulating cell proliferation and migration in both the removable and fixed functional appliances.

A Novel Methodology for Detecting Variations in Cell Surface Antigens Using Cell-Tearing by Optical Tweezers.

The quantitative analysis of cell surface antigens has attracted increasing attention due to the antigenic variation recognition that can facilitate early diagnoses. This paper presents a novel methodology based on the optical "cell-tearing" and the especially proposed "dilution regulations" to detect variations in cell surface antigens. The cell attaches to the corresponding antibody-coated slide surface. Then, the cell-binding firmness between a single cell and the functionalized surface is assayed by optically tearing using gradually reduced laser powers incorporated with serial antibody dilutions. Groups B and B3 of red blood cells (RBCs) were selected as the experiment subject. The results indicate that a higher dilution called for lower power to tear off the cell binding. According to the proposed relative-quantitative analysis theory, antigenic variation can be intuitively estimated by comparing the maximum allowable dilution folds. The estimation result shows good consistency with the finding in the literature. This study suggests a novel methodology for examining the variation in cell surface antigens, expected to be widely capable with potential sensor applications not only in biochemistry and biophysics, but also in the micro-/nano- engineering field.

What's a Biofilm?-How the Choice of the Biofilm Model Impacts the Protein Inventory of Clostridioides difficile.

The anaerobic pathogen Clostridioides difficile is perfectly equipped to survive and persist inside the mammalian intestine. When facing unfavorable conditions C. difficile is able to form highly resistant endospores. Likewise, biofilms are currently discussed as form of persistence. Here a comprehensive proteomics approach was applied to investigate the molecular processes of C. difficile strain 630Δerm underlying biofilm formation. The comparison of the proteome from two different forms of biofilm-like growth, namely aggregate biofilms and colonies on agar plates, revealed major differences in the formation of cell surface proteins, as well as enzymes of its energy and stress metabolism. For instance, while the obtained data suggest that aggregate biofilm cells express both flagella, type IV pili and enzymes required for biosynthesis of cell-surface polysaccharides, the S-layer protein SlpA and most cell wall proteins (CWPs) encoded adjacent to SlpA were detected in significantly lower amounts in aggregate biofilm cells than in colony biofilms. Moreover, the obtained data suggested that aggregate biofilm cells are rather actively growing cells while colony biofilm cells most likely severely suffer from a lack of reductive equivalents what requires induction of the Wood-Ljungdahl pathway and C. difficile's V-type ATPase to maintain cell homeostasis. In agreement with this, aggregate biofilm cells, in contrast to colony biofilm cells, neither induced toxin nor spore production. Finally, the data revealed that the sigma factor SigL/RpoN and its dependent regulators are noticeably induced in aggregate biofilms suggesting an important role of SigL/RpoN in aggregate biofilm formation.

Identification of Cell Surface Targets for CAR T Cell Immunotherapy.

Immunotherapy has become a prominent approach for the treatment of cancer. Targeted killing of malignant cells by adoptive transfer of chimeric antigen receptor (CAR) T cells is a promising immunotherapy technique in oncology. However, the identification of cell surface antigens unique to tumor cells against which CAR T cells can be engineered has historically been challenging and not well documented in solid tumors. Here, we describe a generalized method to construct a cell subtype-specific surface antigen profile (i.e., surfaceome) from cell lines and identify high-confidence antigens as effective targets for CAR T cell therapy by integrating transcriptomics and cell surface proteomics. This method is widely applicable to all therapies utilizing CAR T cells, such as cancer, as well as infectious and autoimmune diseases.

[Recent progress in autoimmune encephalitis and its related disorders].

After the discovery of a series of autoantibodies against neuronal cell surface antigens (NSAs) of the CNS in the past 10 years, the concept of encephalitis has changed dramatically. Accordingly, a practical, syndrome-based diagnostic approach to autoimmune encephalitis was proposed in 2016. These autoantibodies have also been identified in a subset of overlapping encephalitis and demyelinating syndrome, epilepsy, first episode psychosis, movement disorders, post-herpes simplex encephalitis, progressive dementia, postpartum psychosis, stiff-person spectrum disorders, or non-REM/REM sleep behavior disorder. Although not all neuronal antibody tests are available in Japan, we have entered a new era that we have to make a correct diagnosis and start appropriate immunotherapy based on initial neurological assessment and conventional tests, without being constrained by conventional fixed ideas or normal-appearing brain MRIs while waiting for neuronal antibody test results. Although many issues need to be resolved in Japan in terms of diagnosis and treatment in autoimmune encephalitis, this review focusses on recent progress in autoimmune encephalitis and its related disorders closely related to clinical practice, including Hashimoto encephalopathy and new-onset refractory status epilepticus (NORSE).

Systematic screening of 96 Schistosoma mansoni cell-surface and secreted antigens does not identify any strongly protective vaccine candidates in a mouse model of infection.

Background: Schistosomiasis is a major parasitic disease affecting people living in tropical and sup-tropical areas. Transmission of the parasite has been reported in 78 countries, causing significant morbidity and around 200,000 deaths per year in endemic regions. The disease is currently managed by the mass-administration of praziquantel to populations at risk of infection; however, the reliance on a single drug raises the prospect of parasite resistance to the only treatment widely available. The development of an effective vaccine would be a more powerful method of control, but none currently exists and the identification of new immunogens that can elicit protective immune responses therefore remains a priority. Because of the complex nature of the parasite life cycle, identification of new vaccine candidates has mostly relied on the use of animal models and on a limited set of recombinant proteins. Methods: In this study, we have established an infrastructure for testing a large number of vaccine candidates in mice and used it to screen 96 cell-surface and secreted recombinant proteins from Schistosoma mansoni. This approach, using standardised immunisation and percutaneous infection protocols, allowed us to compare an extensive set of antigens in a systematic manner. Results: Although some vaccine candidates were associated with a statistically significant reduction in the number of eggs in the initial screens, these observations could not be repeated in subsequent challenges and none of the proteins studied were associated with a strongly protective effect against infection. Conclusions: Although no antigens individually induced reproducible and strongly protective effects using our vaccination regime, we have established the experimental infrastructures to facilitate large-scale systematic subunit vaccine testing for schistosomiasis in a murine infection model.

Identification of dentinogenic cell-specific surface antigens in odontoblast-like cells derived from adult dental pulp.

BACKGROUND: Odontoblast is a unique progenitor that plays a role in dentin formation. So far, the dentinogenic differentiation of dental pulp stem cells and the role of surface molecules of odontoblasts in dentinogenesis are not well known yet. In this study, we obtained odontoblast-like cells from human dental pulp cells and screened odontoblast-specific cell surface antigens by decoy immunization. METHODS: Through decoy immunization with intact odontoblast-like cells derived from human dental pulp cells, we constructed 12 monoclonal antibodies (mAbs) of IgG type, and their binding affinities for cell surface of odontoblast-like cells were analyzed by flow cytometry. Immunoprecipitation, mass spectrometry, and immunohistochemistry were performed to demonstrate odontoblast-specific antigens. Odontoblasts were sorted by these mAbs using magnetic-activated cell sorting system, and their mineralization efficiency was increased after sorting. RESULTS: We constructed 12 mAbs of IgG type, which had a strong binding affinity for cell surface antigens of odontoblast-like cells. In human adult tooth, these mAbs accumulated in the odontoblastic layer between dentin and pulp and in the perivascular region adjacent to the blood vessels in the pulp core. Cell surface expression of the antigenic molecules was increased during odontogenic cytodifferentiation and decreased gradually as dentinogenic maturation progressed. Proteomic analysis showed that two representative antigenic molecules, OD40 and OD46, had the potential to be components for cell adhesion and extracellular matrix structures. CONCLUSION: These results suggest that mAbs will be useful for detecting and separating odontoblasts from the primary pulp cells and other lineage cells and will provide information on the structures of extracellular matrix and microenvironment that appears during the dentinogenic differentiation.

Prospective Isolation of Poised iPSC Intermediates Reveals Principles of Cellular Reprogramming.

Cellular reprogramming converts differentiated cells into induced pluripotent stem cells (iPSCs). However, this process is typically very inefficient, complicating mechanistic studies. We identified and molecularly characterized rare, early intermediates poised to reprogram with up to 95% efficiency, without perturbing additional genes or pathways, during iPSC generation from mouse embryonic fibroblasts. Analysis of these cells uncovered transcription factors (e.g., Tfap2c and Bex2) that are important for reprogramming but dispensable for pluripotency maintenance. Additionally, we observed striking patterns of chromatin hyperaccessibility at pluripotency loci, which preceded gene expression in poised intermediates. Finally, inspection of these hyperaccessible regions revealed an early wave of DNA demethylation that is uncoupled from de novo methylation of somatic regions late in reprogramming. Our study underscores the importance of investigating rare intermediates poised to produce iPSCs, provides insights into reprogramming mechanisms, and offers a valuable resource for the dissection of transcriptional and epigenetic dynamics intrinsic to cell fate change.

Detection and Characterization of Autoantibodies to Neuronal Cell-Surface Antigens in the Central Nervous System.

Autoimmune encephalitis (AIE) is a group of disorders in which autoantibodies directed at antigens located on the plasma membrane of neurons induce severe neurological symptoms. In contrast to classical paraneoplastic disorders, AIE patients respond well to immunotherapy. The detection of neuronal surface autoantibodies in patients' serum or CSF therefore has serious consequences for the patients' treatment and follow-up and requires the availability of sensitive and specific diagnostic tests. This mini-review provides a guideline for both diagnostic and research laboratories that work on the detection of known surface autoantibodies and/or the identification of novel surface antigens. We discuss the strengths and pitfalls of different techniques for anti-neuronal antibody detection: (1) Immunohistochemistry (IHC) and immunofluorescence on rat/primate brain sections; (2) Immunocytochemistry (ICC) of living cultured hippocampal neurons; and (3) Cell Based Assay (CBA). In addition, we discuss the use of immunoprecipitation and mass spectrometry analysis for the detection of novel neuronal surface antigens, which is a crucial step in further disease classification and the development of novel CBAs.