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Cells Jun 2024holds significant therapeutic potential; however, its nonspecific invasiveness results in off-target effects. The purpose of this study is to evaluate whether...
holds significant therapeutic potential; however, its nonspecific invasiveness results in off-target effects. The purpose of this study is to evaluate whether specificity can be improved by surface display of scFv directed against dendritic cells' endocytic receptor, DEC205, and immune checkpoint PD-L1. Anti-DEC205 scFv was anchored to the surface either directly via glycosylphosphatidylinositol (GPI) or by fusion with the SAG1 protein. Both constructs were successfully expressed, but the binding results suggested that the anti-DEC-SAG1 scFv had more reliable functionality towards recombinant DEC protein and DEC205-expressing MutuDC cells. Two anti-PD-L1 scFv constructs were developed that differed in the localization of the HA tag. Both constructs were adequately expressed, but the localization of the HA tag determined the functionality by binding to PD-L1 protein. Co-incubation of displaying anti-PD-L1 scFv with tumor cells expressing/displaying different levels of PD-L1 showed strong binding depending on the level of available biomarker. Neutralization assays confirmed that binding was due to the specific interaction between anti-PD-L1 scFv and its ligand. A mixed-cell assay showed that expressing anti-PD-L1 scFv predominately targets the PD-L1-positive cells, with negligible off-target binding. The recombinant RH-PD-L1-C strain showed increased killing ability on PD-L1+ tumor cell lines compared to the parental strain. Moreover, a co-culture assay of target tumor cells and effector CD8+ T cells showed that our model could inhibit PD1/PD-L1 interaction and potentiate T-cell immune response. These findings highlight surface display of antibody fragments as a promising strategy of targeting replicative strains while minimizing nonspecific binding.
Topics: Toxoplasma; Single-Chain Antibodies; Humans; B7-H1 Antigen; Cell Line, Tumor; Animals; Dendritic Cells
PubMed: 38891106
DOI: 10.3390/cells13110975 -
Foods (Basel, Switzerland) May 2024Chlorphenamine maleate is a prohibited additive found in herbal teas and health foods. Excessive intake of this substance can result in adverse health effects. In this...
Chlorphenamine maleate is a prohibited additive found in herbal teas and health foods. Excessive intake of this substance can result in adverse health effects. In this study, two novel haptens, PEM and bepotastine (PB1), mimicking chlorphenamine maleate structure were designed and synthesized based on molecular simulation for developing two corresponding polyclonal antibodies (PEM-Ab and PB1-Ab), respectively. Afterward, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed to quickly and accurately detect chlorphenamine maleate in herbal teas using PB1-Ab, which has a high sensitivity and specificity. For chlorphenamine maleate, the half-maximal inhibitory concentration (IC) and limit of detection (LOD) of PB1-Ab under ideal circumstances were found to be 1.18 µg/L and 0.07 µg/L, respectively. Besides, an environmentally friendly sample pre-treatment strategy was employed that allowed easy and effective elimination of complex matrices. The ic-ELISA method observed the average recovery rate from 87.7% to 94.0% with the variance coefficient (CV) ranging from 2.2% to 9.4%. Additionally, the identification of 25 commercially available herbal teas using liquid chromatography-tandem mass spectrometry (LC-MS/MS) further confirmed the validity of our detection. The results of the two methods are consistent. Overall, the proposed ic-ELISA could be an ultrasensitive and reliable method for chlorphenamine maleate adulterated in foods or exposure to the environment.
PubMed: 38890838
DOI: 10.3390/foods13111609 -
MAbs 2024Integrins are cell surface receptors that mediate the interactions of cells with their surroundings and play essential roles in cell adhesion, migration, and...
Integrins are cell surface receptors that mediate the interactions of cells with their surroundings and play essential roles in cell adhesion, migration, and homeostasis. Eight of the 24 integrins bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, comprising the RGD-binding integrin subfamily. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands to fulfill specific functions in cellular processes. Antibodies against individual RGD-binding integrins are desirable for investigating their specific functions, and were selected here from a synthetic yeast-displayed Fab library. We discovered 11 antibodies that exhibit high specificity and affinity toward their target integrins, i.e. αVβ3, αVβ5, αVβ6, αVβ8, and α5β1. Of these, six are function-blocking antibodies and contain a ligand-mimetic R(G/L/T)D motif in their CDR3 sequences. We report antibody-binding specificity, kinetics, and binding affinity for purified integrin ectodomains, as well as intact integrins on the cell surface. We further used these antibodies to reveal binding preferences of the αV subunit for its 5 β-subunit partners: β6 = β8 > β3 > β1 = β5.
Topics: Humans; Saccharomyces cerevisiae; Integrin beta Chains; Integrin alphaV; Integrins; Peptide Library; Cell Surface Display Techniques; Protein Binding; Antibody Specificity
PubMed: 38889315
DOI: 10.1080/19420862.2024.2365891 -
RMD Open Jun 2024The objective of this study is to evaluate whether anti-neutrophil cytoplasmic antibody (ANCA) seropositivity and antigen specificity at diagnosis have predictive...
OBJECTIVES
The objective of this study is to evaluate whether anti-neutrophil cytoplasmic antibody (ANCA) seropositivity and antigen specificity at diagnosis have predictive utility in paediatric-onset small vessel vasculitis.
METHODS
Children and adolescents with small vessel vasculitis (n=406) stratified according to the absence (n=41) or presence of ANCA for myeloperoxidase (MPO) (n=129) and proteinase-3 (PR3) (n=236) were compared for overall and kidney-specific disease activity at diagnosis and outcomes between 1 and 2 years using retrospective clinical data from the ARChiVe/Paediatric Vasculitis Initiative registry to fit generalised linear models.
RESULTS
Overall disease activity at diagnosis was higher in PR3-ANCA and MPO-ANCA-seropositive individuals compared with ANCA-negative vasculitis. By 1 year, there were no significant differences, based on ANCA positivity or specificity, in the likelihood of achieving inactive disease (~68%), experiencing improvement (≥87%) or acquiring damage (~58%). Similarly, and in contrast to adult-onset ANCA-associated vasculitis, there were no significant differences in the likelihood of having a relapse (~11%) between 1 and 2 years after diagnosis. Relative to PR3-ANCA, MPO-ANCA seropositivity was associated with a higher likelihood of kidney involvement (OR 2.4, 95% CI 1.3 to 4.7, p=0.008) and severe kidney dysfunction (Kidney Disease Improving Global Outcomes (KDIGO) stages 4-5; OR 6.04, 95% CI 2.77 to 13.57, p<0.001) at onset. Nonetheless, MPO-ANCA seropositive individuals were more likely to demonstrate improvement in kidney function (improved KDIGO category) within 1 year of diagnosis than PR3-ANCA seropositive individuals with similarly severe kidney disease at onset (p<0.001).
CONCLUSIONS
The results of this study suggest important paediatric-specific differences in the predictive value of ANCA compared with adult patients that should be considered when making treatment decisions in this population.
Topics: Humans; Antibodies, Antineutrophil Cytoplasmic; Male; Female; Child; Adolescent; Peroxidase; Myeloblastin; Retrospective Studies; Kidney Diseases; Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis; Biomarkers; Child, Preschool; Prognosis; Predictive Value of Tests
PubMed: 38886004
DOI: 10.1136/rmdopen-2024-004315 -
JCI Insight Jun 2024Antibody-mediated depletion studies have demonstrated that CD8+ T cells are required for effective immune control of SIV. However, this approach is confounded by several...
Antibody-mediated depletion studies have demonstrated that CD8+ T cells are required for effective immune control of SIV. However, this approach is confounded by several factors, including reactive CD4+ T cell proliferation, and further provides no specificity information. We circumvented these limitations by selectively depleting CD8+ T cells specific for the Gag epitope CTPYDINQM (CM9) via the administration of immunotoxin-conjugated tetrameric complexes of CM9/Mamu-A*01. Immunotoxin administration effectively depleted circulating but not tissuelocalized CM9-specific CD8+ T cells, akin to the bulk depletion pattern observed with antibodies directed against CD8. However, we found no evidence to indicate that circulating CM9-specific CD8+ T cells suppressed viral replication in Mamu-A*01+ rhesus macaques during acute or chronic progressive infection with a pathogenic strain of SIV. This observation extended to macaques with established infection during and after continuous antiretroviral therapy. In contrast, natural controller macaques experienced dramatic increases in plasma viremia after immunotoxin administration, highlighting the importance of CD8+ T cell-mediated immunity against CM9. Collectively, these data showed that CM9-specific CD8+ T cells were necessary but not sufficient for robust immune control of SIV in a nonhuman primate model and, more generally, validated an approach that could inform the design of next-generation vaccines against HIV-1.
PubMed: 38885329
DOI: 10.1172/jci.insight.174168 -
Molecular Therapy. Methods & Clinical... Jun 2024Over 4% of the global population is estimated to live with autoimmune disease, necessitating immunosuppressive treatment that is often chronic, not curative, and carries...
Over 4% of the global population is estimated to live with autoimmune disease, necessitating immunosuppressive treatment that is often chronic, not curative, and carries associated risks. B cells have emerged as key players in disease pathogenesis, as evidenced by partial responsiveness to B cell depletion by antibody-based therapies. However, these treatments often have transient effects due to incomplete depletion of tissue-resident B cells. Chimeric antigen receptor (CAR) T cells targeting B cells have demonstrated efficacy in refractory systemic lupus erythematosus. To this end, we developed an anti-CD19 CAR T cell product candidate, CABA-201, containing a clinically evaluated fully human CD19 binder (IC78) with a 4-1BB costimulatory domain and CD3 zeta stimulation domain for treatment refractory autoimmune disease. Here, we demonstrate specific cytotoxic activity of CABA-201 against CD19 Nalm6 cells with no off-target effects on primary human cells. Novel examination of CABA-201 generated from primary T cells from multiple patients with autoimmune disease displayed robust CAR surface expression and effective elimination of the intended target autologous CD19 B cells . Together, these findings support the tolerability and activity of CABA-201 for clinical development in patients with autoimmune disease.
PubMed: 38883975
DOI: 10.1016/j.omtm.2024.101267 -
Journal of Pregnancy 2024There is insufficient evidence to assess the risk of the production of clinically important alloimmune irregular red blood cell (RBC) antibodies in first-time pregnant... (Comparative Study)
Comparative Study
Comparison of the Detection Rate and Specificity of Irregular Red Blood Cell Antibodies Between First-Time Pregnant Women and Women With a History of Multiple Pregnancies Among 18,010 Chinese Women.
There is insufficient evidence to assess the risk of the production of clinically important alloimmune irregular red blood cell (RBC) antibodies in first-time pregnant women. Using the microcolumn gel antiglobulin method, 18,010 Chinese women with a history of pregnancy and pregnant women were screened for irregular RBC antibodies, and for those with positive test results, antibody specificity was determined. The detection rate and specificity of irregular RBC antibodies in women with a history of multiple pregnancies (two or more) and first-time pregnant women were determined. In addition to 25 patients who passively acquired anti-D antibodies via an intravenous anti-D immunoglobulin injection, irregular RBC antibodies were detected in 121 (0.67%) of the 18,010 women. Irregular RBC antibodies were detected in 93 (0.71%) of the 13,027 women with a history of multiple pregnancies, and antibody specificity was distributed mainly in the Rh, MNSs, Lewis, and Kidd blood group systems; irregular RBC antibodies were detected in 28 (0.56%) of the 4983 first-time pregnant women, and the antibody specificity was distributed mainly in the MNSs, Rh, and Lewis blood group systems. The difference in the percentage of patients with irregular RBC antibodies between the two groups was insignificant ( = 1.248, > 0.05). Of the 121 women with irregular RBC antibodies, nine had anti-Mur antibodies, and one had anti-Di antibodies; these antibodies are clinically important but easily missed because the antigenic profile of the reagent RBCs that are commonly used in antibody screens does not include the antigens that are recognized by these antibodies. Irregular RBC antibody detection is clinically important for both pregnant women with a history of multiple pregnancies and first-time pregnant women. Mur and Di should be included in the antigenic profile of reagent RBCs that are used for performing antibody screens in the Chinese population.
Topics: Humans; Female; Pregnancy; Erythrocytes; China; Adult; Pregnancy, Multiple; Isoantibodies; Rho(D) Immune Globulin; Sensitivity and Specificity; Antibody Specificity; MNSs Blood-Group System; Asian People; Kidd Blood-Group System; East Asian People
PubMed: 38883212
DOI: 10.1155/2024/5539776 -
Frontiers in Cellular and Infection... 2024As a contagious and chronic disease in the livestock industry, Paratuberculosis is a significant threat to dairy herds' genetic and economic resources. Due to intensive...
INTRODUCTION
As a contagious and chronic disease in the livestock industry, Paratuberculosis is a significant threat to dairy herds' genetic and economic resources. Due to intensive breeding and high production of dairy cattle, the incidence and prevalence are higher. Developing non-destructive diagnostic methods for the early detection and identification of healthy animals is paramount for breeding programs. Conventional methods are almost entirely destructive, have low accuracy, lack precision, and are time-consuming. Near-infrared spectroscopy (NIRS) and aquaphotomics can detect changes in biofluids and thus have the potential to diagnose disease. This study aimed to investigate the diagnostic ability of NIRS and aquaphotomics for Paratuberculosis in dairy cattle.
METHODS
Blood plasma from dairy cattle was collected in the NIR range (1,300 nm to 1,600 nm) 60 days before and 100 days to 200 days after calving in two groups, positive and negative, using the same consecutive enzyme-linked immunosorbent assay test results three times as a reference test.
RESULTS
NIRS and aquaphotomics methods invite 100% accuracy, sensitivity, and specificity to detect Paratuberculosis using data mining by unsupervised method, Principal Component Analysis, and supervised methods: Soft Independent Modeling of Class Analogiest, Linear Discriminant Analysis, Quadratic Discriminant Analysis, Partial Least Square-Discriminant Analysis, and Support Vector Machine models.
DISCUSSION
The current study found that monitoring blood plasma with NIR spectra provides an opportunity to analyze antibody levels indirectly via changes in water spectral patterns caused by complex physiological changes, such as the amount of antibodies related to Paratuberculosis by aquagram.
Topics: Animals; Cattle; Paratuberculosis; Spectroscopy, Near-Infrared; Cattle Diseases; Sensitivity and Specificity; Mycobacterium avium subsp. paratuberculosis; Female; Dairying; Enzyme-Linked Immunosorbent Assay
PubMed: 38873096
DOI: 10.3389/fcimb.2024.1374560 -
Journal of Immunological Methods Jun 2024When the membrane protein CD40 ligand (CD40L) on activated T cells binds the receptor CD40 on B-cells, it provides a co-stimulatory signal for B cell activation....
When the membrane protein CD40 ligand (CD40L) on activated T cells binds the receptor CD40 on B-cells, it provides a co-stimulatory signal for B cell activation. Dysregulation of the CD40L:CD40 axis is associated with inflammatory and autoimmune diseases. The presence of soluble CD40L (sCD40L) in plasma is implicated in several diseases, from cardiovascular and autoimmune diseases to different types of cancer, and sCD40L has been suggested as a valuable marker of disease. If sCD40L is to be used as a biomarker, being able to precisely measure and quantify the levels of sCD40L in human blood samples is of utmost importance. We demonstrate the development of a sandwich-type time-resolved immunofluorometric assay for quantification of sCD40L in plasma or serum samples. For this, we generate 29 monoclonal anti-CD40L antibodies, and from these, we select the optimal combination of capture antibody and detection antibody. A number of variables were tested: the influence of the type of sample (comparing 3 different blood collection tubes for serum sampling and 4 different types of tubes for plasma sampling), the influence of freeze-thaw cycles, the influence of sampling time during night and day, and the influence of centrifugation of the samples. We found a very similar level of sCD40L in paired EDTA plasma and serum samples. Out of 100 healthy blood donor samples 61 had a level of sCD40L below the detection level of the assay, whereas the remaining 39 samples had ranging levels of sCD40L from 1.14 to 33.14 ng/mL. In summary, we present a time-resolved immunofluorometric assay based on paired monoclonal antibodies, ensuring high specificity, sensitivity, and homogeneity. The Eu-based assay additionally provides consistent assay readouts due to the extended decay time not seen in standard enzyme-linked immunosorbent assays. The assay paves the way for specific and consistent quantification of sCD40L in human plasma and serum samples.
PubMed: 38871279
DOI: 10.1016/j.jim.2024.113710 -
Pathology, Research and Practice Jun 2024Expression and function of TRPC3 and TRPC6 in the pancreas is a controversial topic. Investigation in human tissue is seldom. We aimed to provide here a detailed...
BACKGROUND
Expression and function of TRPC3 and TRPC6 in the pancreas is a controversial topic. Investigation in human tissue is seldom. We aimed to provide here a detailed description of the distribution of TRPC3 and TRPC6 in the human exocrine and endocrine pancreas.
METHODS
We collected healthy samples from cadavers (n = 4) and visceral surgery (n = 4) to investigate the respective expression profiles using immunohistochemical tracing with knockout-validated antibodies.
RESULTS
TRPC3- and TRPC6-proteins were detected in different pancreatic structures including acinar cells, as well as epithelial ductal cells from intercalate, intralobular, and interlobular ducts. Respective connective tissue layers appeared unstained. Endocrine islets of Langerhans were clearly and homogenously immunolabeled by the anti-TRPC3 and anti-TRPC6 antibodies. Insular α, β, γ, and δ cells were conclusively stained, although no secure differentiation of cell types was performed.
CONCLUSIONS
Due to aforementioned antibody specificity verification, protein expression in the immunolabeled localizations can be accepted. Our study in human tissue supports previous investigations especially with respect to acinar and insular α and β cells, while other localizations are here reported for the first time to express TRPC3 and TRPC6, ultimately warranting further research.
PubMed: 38870712
DOI: 10.1016/j.prp.2024.155403