Production Of Specific Antibodies Research Articles

Immunostimulatory and immunoadjuvant capacities of soluble Rhamnan-type Ulva oligosaccharides

Algae polysaccharides were discovered to act as pathogen-associated molecular patterns (PAMPs) to trigger immune responses, while bioactivities of oligosaccharides have not been thoroughly explored. Ulva oligosaccharides from Ulva prolifera were obtained using PL25 family ulvan lyase and evaluated for cell viability and phagocytic activity. Ulva oligosaccharides with 542 Da (OUP), consisting of tetrasaccharide (∆UA-RhaS-Xyl-RhaS) and disaccharides (∆UA-RhaS), exhibited the most pronounced phagocytic activities. Besides, OUP could effectively stimulate the expressions of proinflammatory cytokines and chemokines like TNF-α, IL-1β, IL-6, IFN-γ and CXCL1 in primary macrophages and subsequently promote the proliferation and activation of T and B lymphocytes in vitro. Transcriptome analysis also gave rise to the systemic proinflammatory responses triggered by OUP in macrophages, such as activation of multiple PRRs-related signaling pathways and downstream JAK-STAT and NF-κB signaling. Furthermore, in an in vivo mouse OVA immunization model, OUP upregulated the specific anti-OVA IgG antibody production in vivo, pointing to its immunoadjuvant capacity. OUP increases cell proliferation and secretion of IL-4 and IFN-γ of splenocytes in response to OVA stimulation ex vivo, thereby enhancing both Th1 and Th2 immune responses. Collectively, all this data suggests that OUP could function as the immune stimulator to enhance host immune response to infections.

Homozygosity in any HLA locus is a risk factor for specific antibody production: the taboo concept 2.0.

In a cooperative study of the University Hospital Leipzig, University of Leipzig, and the Charité Berlin on kidney transplant patients, we analysed the occurrence of HLA-specific antibodies with respect to the HLA setup of the patients. We aimed at the definition of specific HLA antigens towards which the patients produced these antibodies. Patients were typed for the relevant HLA determinants using mainly the next-generation technology. Antibody screening was performed by the state-of-the-art multiplex-based technology using microspheres coupled with the respective HLA alleles of HLA class I and II determinants. Patients homozygous for HLA-A*02, HLA-A*03, HLA-A*24, HLA-B*07, HLA-B*18, HLA-B*35, HLA-B*44, HLA-C*03, HLA-C*04, and HLA-C*07 in the class I group and HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*07, HLA-DRB1*15, HLA-DQA1*01, HLA-DQA1*05, HLA-DQB1*02, HLA-DQB1*03(7), HLA-DQB1*06, HLA-DPA1*01, and HLA-DPB1*04 in the class II group were found to have a significant higher antibody production compared to the heterozygous ones. In general, all HLA determinants are affected. Remarkably, HLA-A*24 homozygous patients can produce antibodies towards all HLA-A determinants, while HLA-B*18 homozygous ones make antibodies towards all HLA-B and selected HLA-A and C antigens, and are associated with an elevation of HLA-DRB1, parts of DQB1 and DPB1 alleles. Homozygosity for the HLA class II HLA-DRB1*01, and HLA-DRB1*15 seems to increase the risk for antibody responses against most of the HLA class I antigens (HLA-A, HLA-B, and HLA-C) in contrast to HLA-DQB1*03(7) where a lower risk towards few HLA-A and HLA-B alleles is found. The widely observed differential antibody response is therefore to be accounted to the patient's HLA type. Homozygous patients are at risk of producing HLA-specific antibodies hampering the outcome of transplantation. Including this information on the allocation procedure might reduce antibody-mediated immune reactivity and prevent graft loss in a patient at risk, increasing the life span of the transplanted organ.

Sar1A overexpression in Chinese hamster ovary cells and its effects on antibody productivity and secretion

Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles.

Analysis of the Immunogenicity of African Swine Fever F317L Protein and Screening of T Cell Epitopes.

The African swine fever virus (ASFV) encodes numerous proteins characterized by complex immune escape mechanisms. At present, the structure and function of these proteins, including the F317L protein, have yet to be fully elucidated. In this study, we examined the immunogenicity of the F317L protein. Mice were subcutaneously immunized with the F317L protein using initial and subsequent booster doses, and, at the 28th day post-treatment, we assessed the humoral and cellular immune responses of mice. The F317L protein stimulated production of specific antibodies and activated humoral immune responses. In addition, F317L stimulated the production of large amounts of IFN-γ by splenic lymphocytes, thereby activating cellular immune responses. Using informatics technology, we predicted and synthesized 29 F317L protein T cell epitopes, which were screened using IFN-γ ELISpot. Among these, the F25 (246SRRSLVNPWT255) peptide was identified as having a stronger stimulatory effect than the full-length protein. Collectively, our findings revealed that the ASFV F317L protein can stimulate both strong humoral and cellular immunity in mice, and that the F25 (246SRRSLVNPWT255) peptide may be a potential active T cell epitope. These findings will provide a reference for further in-depth studies of the F317L protein and screening of antigenic epitopes.

Differential protective impact of peptide vaccine formulae targeting the lung- and liver-stage of challenge Schistosoma mansoni infection in mice

The study aimed to elicit protective immune responses against murine schistosomiasis mansoni at the parasite lung- and liver stage. Two peptides showing amino acid sequence similarity to gut cysteine peptidases, which induce strong memory immune effectors in the liver, were combined with a peptide based on S. mansoni thioredoxin peroxidase (TPX), a prominent lung-stage schistosomula excretory-secretory product, and alum as adjuvant. Only one of the 2 cysteine peptidases-based peptides in a multiple antigenic peptide construct (MAP-3 and MAP-4) appeared to adjuvant protective immune responses induced by the TPX peptide in a MAP form. Production of TPX MAP-specific IgG1 serum antibodies, and increase in lung interleukin-1 (IL-1), uric acid, and reactive oxygen species (ROS) content were associated with significant (P < 0.05) 50 % reduction in recovery of lung-stage larvae. Increase in lung triglycerides and cholesterol levels appeared to provide the surviving worms with nutrients necessary for a stout double lipid bilayer barrier at the parasite-host interface. Surviving worms-released products elicited memory responses to the MAP-3 immunogen, including production of specific IgG1 antibodies and increase in liver IL-33 and ROS. Reduction in challenge worm burden recorded 45 days post infection did not exceed 48 % associated with no differences in parasite egg counts in the host liver and small intestine compared to unimmunized adjuvant control mice. Alum adjuvant assisted the second peptide, MAP-4, in production of IgG1, IgG2a, IgG2b and IgA specific antibodies and increase in liver ROS, but with no protective potential, raising doubt about the necessity of adjuvant addition. Accordingly, different vaccine formulas containing TPX MAP and 1, 2 or 3 cysteine peptidases-derived peptides with or without alum were used to immunize parallel groups of mice. Compared to unimmunized control mice, significant (P < 0.05 to < 0.005) 22 to 54 % reduction in worm burden was recorded in the different groups associated with insignificant changes in parasite egg output. The results together indicated that a schistosomiasis vaccine able to entirely prevent disease and halt its transmission still remains elusive.

RORγt inverse agonist TF-S14 inhibits Th17 cytokines and prolongs skin allograft survival in sensitized mice

Chronic antibody mediated rejection (AMR) is the major cause of solid organ graft rejection. Th17 contributes to AMR through the secretion of IL17A, IL21 and IL22. These cytokines promote neutrophilic infiltration, B cell proliferation and donor specific antibodies (DSAs) production. In the current study we investigated the role of Th17 in transplant sensitization. Additionally, we investigated the therapeutic potential of novel inverse agonists of the retinoic acid receptor-related orphan receptor gamma t (RORγt) in the treatment of skin allograft rejection in sensitized mice. Our results show that RORγt inverse agonists reduce cytokine production in human Th17 cells in vitro. In mice, we demonstrate that the RORγt inverse agonist TF-S14 reduces Th17 signature cytokines in vitro and in vivo and leads to blocking neutrophilic infiltration to skin allografts, inhibition of the B-cell differentiation, and the reduction of de novo IgG3 DSAs production. Finally, we show that TF-S14 prolongs the survival of a total mismatch grafts in sensitized mice. In conclusion, RORγt inverse agonists offer a therapeutic intervention through a novel mechanism to treat rejection in highly sensitized patients.

In silico and in vivo Investigations of the Immunoreactivity of Klebsiella pneumoniae OmpA Protein as a Vaccine Candidate.

The growing threat of antibiotic resistance and Klebsiella pneumoniae infection in healthcare settings highlights the urgent need for innovative solutions, such as vaccines, to address these challenges. This study sought to assess the potential of using K. pneumoniae OmpA as a vaccine candidate through both in silico and in vivo analyses. The study examined the OmpA protein sequence for subcellular localization, antigenicity, allergenicity, similarity to the human proteome, physicochemical properties, B-cell epitopes, MHC binding sites, tertiary structure predictions, molecular docking, and immune response simulations. The ompA gene was cloned into the pET-28a (+) vector, expressed, purified and confirmed using Western blotting analysis. IgG levels in the serum of the immunized mice were measured using ELISA with dilutions ranging from 1:100 to 1:6400, targeting rOmpA and K. pneumoniae ATCC 13883. The sensitivity and specificity of the ELISA method were also assessed. The bioinformatics analysis identified rOmpA as a promising vaccine candidate. The immunized group demonstrated significant production of specific total IgG antibodies against rOmpA and K. pneumoniae ATCC1 13883, as compared to the control group (p < 0.0001). The titers of antibodies produced in response to bacterial exposure did not show any significant difference when compared to the anti-rOmpA antibodies (p > 0.05). The ELISA test sensitivity was 1:3200, and the antibodies in the serum could accurately recognize K. pneumoniae cells. This study is a significant advancement in the development of a potential vaccine against K. pneumoniae that relies on OmpA. Nevertheless, additional experimental analyses are required.

SUPPRESSOR CELLS (MDSC) REGULATE THE T-CELL RESPONSE IN OLD MICE IMMUNIZED WITH THE WHOLE-VIRION SARS-CoV-2 VACCINE

Myeloid-derived suppressor cells (MDSCs) are an immunosuppressive population involved in pathological processes associated with chronic inflammation. In recent years, the role of these cells in the regulation of a number of non-pathological processes, in particular, in the development of vaccine-induced immune response, has been actively discussed. This work was aimed at investigating the role of MDSC in the development of an immune response in old mice immunized with an inactivated whole virion SARS-CoV-2. Immunization of old animals induced an increase in the relative content of B cells and the production of specific antibodies to S1 and N proteins of SARS-CoV-2 in titers comparable to those of young animals. At the same time, elevated MDSC levels in aged animals negatively correlated with the level of CD8+ T cells. Splenic MDSCs in old animals were characterized by increased production of reactive oxygen species compared to young animals. In vitro experiments showed that MDSC depletion increased antigen-specific production of IFNγ by CD4+ T cells. The data obtained suggest a negative role for MDSC in regulating the T-cell vaccine-induced response to inactivated SARS-CoV-2 and encourage further studies in this direction, which may be a key to developing approaches to increase the efficacy of vaccines against SARS-CoV-2 in at-risk groups.

Sexual Dimorphism in the Physiopathology and Immune Response during Acute Toxocara canis Infection.

Toxocara canis (T. canis) is a helminth parasite of zoonotic and veterinary health significance that causes the disease known as Toxocariasis. This disease has been associated with conditions of poverty, especially in tropical climate zones throughout the world. Although it rarely causes important clinical manifestations, T. canis can lead to blindness, meningoencephalitis, or other nervous manifestations in humans. Moreover, some studies show its importance in the development of tumor growth, which have been associated with the parasite's ability to modulate the host's immune response. While different studies have evaluated the immune response during this disease, currently, there are no studies where the infection is analyzed from the perspective of sexual dimorphism. To evaluate sex differences in susceptibility, we analyzed lesions and parasite loads in lung and liver at 7 days post-infection. In addition, immune cell subpopulations were analyzed in spleen, mesenteric and peripheral lymph nodes. Finally, the production of cytokines and specific antibodies were determined in the serum. Statical analyses were performed using a Two-way ANOVA and a post-hoc Bonferroni multiple comparison test. Female rats had a higher number of larvae in the liver, while male rats had them in the lungs. The percentages of immune cells were evaluated, and in most cases, no significant differences were observed. Regarding the cytokines production, infection can generate a decrease in Th1 such as IL-1β in both sexes and IL-6 only in females. In the case of Th2, IL-4 increases only in infected males and IL-5 increases in males while decreasing in females due to the effect of infection. IL-10 also decreases in both sexes as a consequence of the infection, and TGF-β only in females. Finally, the infection generates the production of antibodies against the parasite, however, their quantity is lower in females. This study demonstrates that T. canis infection is dimorphic and affects females more than males. This is due to a polarization of the inadequate immune response, which is reflected as a higher parasite load in this sex.

Abstract 2760: Novel GPCR recombinant monoclonal antibodies for cancer biology research

Abstract Recombinant technology is becoming the preferred production mechanism for creation of antibody reagents used in academic and clinical cancer biology research. Recombinant antibodies (rAbs) are superior to standard polyclonal and hybridoma-generated monoclonal reagents as they offer reproducible performance and consistent, scalable supply. A major focus of antibody manufacturers is to leverage rAb production strategies to generate antibodies against challenging targets for which there are few, if any, reliable antibodies. G-protein coupled receptors (GPCRs) are encoded by more than 820 human genes and represent the largest membrane protein superfamily in the genome. These seven-pass transmembrane domain receptors are involved in a multitude of physiological and pathophysiological processes related to various chronic diseases, including an array of malignancies. Perhaps 30-40% of all therapeutics are directed against GPCR activity, and ~350 of the non-olfactory GPCRs are thought to be potential candidates for drug development. However, for a slew of technical reasons, the development and validation of high-quality antibodies against GPCRs has proven difficult. In an effort to facilitate further research into GPCRs, GeneTex is combining its recombinant antibody production platform with enhanced validation techniques to make specific monoclonal antibodies against GPCRs with direct relevance to cancer and other diseases. GeneTex’s rAb production protocol employs a multi-parameter fluorescence-activated single cell sorting (FACS)-based methodology to identify and select antigen-specific IgG+ memory B cells from an immunized rabbit (Starkie et al., 2016). The heavy and light chain variable region genes from single cells are amplified, cloned, and co-expressed in mammalian cells to generate a monoclonal IgG. Application-specific testing is performed in conjunction with knockout/knockdown (KO/KD), differential expression comparison in cells and tissues, cell fraction enrichment (i.e., membrane extracts versus whole cell extracts), and other methodologies. Antibodies directed against extracellular GPCR domains are validated for specificity using GPCR arrays (CDI Labs, Baltimore, MD). This workflow has resulted in the production of specific antibodies for a number of GPCRs studied by cancer biologists. These include novel recombinant monoclonal antibodies to the dopamine D2 receptor (DRD2), retinoic acid-induced protein 3 (RAI3), CXC motif chemokine receptor 2 (CXCR2), CXCR4, and CXCR7. All of these antibodies were validated for at least two applications, while the specificity of three (i.e., RAI3, CXCR4, and CXCR7) is supported by KO/KD data. GPCR array data argues for the specificity of the other two antibodies (i.e., DRD2 and CXCR2). This same production/validation approach will be applied as GeneTex targets the remaining GPCRs. Citation Format: Alexander Ball, Yung Lin Hsieh, Chun Kai Huang, Sega Huang, Shang Ru Wu, Yue Yun Chang, Jiming Liu, Po Shin Peng, Chia Yi Lin. Novel GPCR recombinant monoclonal antibodies for cancer biology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2760.

Role of Skewed X-Chromosome Inactivation in Common Variable Immunodeficiency.

The term common variable immunodeficiency (CVID) encompasses a clinically diverse group of disorders, mainly characterized by hypogammaglobulinemia, insufficient specific antibody production, and recurrent infections. The genetics of CVID is complex, and monogenic defects account for only a portion of cases, typically <30%. Other proposed mechanisms include digenic, oligogenic, or polygenic inheritance and epigenetic dysregulation. In this study, we aimed to assess the role of skewed X-chromosome inactivation (XCI) in CVID. Within our cohort of 131 genetically analyzed CVID patients, we selected female patients with rare variants in CVID-associated genes located on the X-chromosome. Four patients harboring heterozygous variants in BTK (n = 2), CD40LG (n = 1), and IKBKG (n = 1) were included in the study. We assessed XCI status using the HUMARA assay and an NGS-based method to quantify the expression of the 2 alleles in mRNA. Three of the 4 patients (75%) exhibited skewed XCI, and the mutated allele was predominantly expressed in all cases. Patient 1 harbored a hypomorphic variant in BTK (p.Tyr418His), patient 3 had a pathogenic variant in CD40LG (c.288+1G>A), and patient 4 had a hypomorphic variant in IKBKG (p.Glu57Lys) and a heterozygous splice variant in TNFRSF13B (TACI) (c.61+2T>A). Overall, the analysis of our cohort suggests that CVID in a small proportion of females (1.6% in our cohort) is caused by skewed XCI and highly penetrant gene variants on the X-chromosome. Additionally, skewed XCI may contribute to polygenic effects (3.3% in our cohort). These results indicate that skewed XCI may represent another piece in the complex puzzle of CVID genetics.

Specific antibody production using recombinant proteins to elucidate seed transmission and nuclear localization of Coguvirus citrulli and Coguvirus henanense in radicles of watermelon crop

Watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2, both belonging to the genus Coguvirus (family Phenuiviridae), have been identified in watermelon plants in Brazil. To study tissue tropism and the potential for seed transmission of these viruses, we initially planned to produce specific antibodies. However, difficulties in isolating and propagating the virus in host plants hindered the purified virus preparations. To overcome this problem, the nucleocapsid (N) proteins of WCLaV-1 and −2 were produced using the pepper ringspot virus vector. The N protein genes and the vector backbone were prepared by (RT-)PCR and ligated by Gibson assembly. The constructs were agro-infiltrated in Nicotiana benthamiana plants. The expressed N proteins were purified and used for polyclonal antibody production. The specificity of both antibodies was confirmed by antigen-coating ELISA, tissue-blot immunobinding assay and Western blot. By antigen-coating ELISA demonstrated that WCLaV-1 showed 93.1% of seed-transmission, while WCLaV-2 showed only 17.8%. The N protein of WCLaV-1 was detected in the cytoplasm of the seed tissues. It was also found in the nuclei of the radicle, as confirmed by confocal microscopy. We concluded that the antibodies exhibited both a high titer and sufficient specificity for use in ELISA-based diagnostics and for subcellular localization study.

Construction of and evaluation of the immune response to two recombinant pseudorabies viruses expressing the B119L and EP364R proteins of African swine fever virus.

African swine fever (ASF) is an infectious disease caused by ASF virus (ASFV), which is characterized by high infectivity, rapid onset of disease, and a high mortality rate. Outbreaks of ASFV have caused great economic losses to the global pig industry, and there is a need to develop safe and effective vaccines. In this study, two recombinant pseudorabies virus (PRV) strains, rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L, expressing the EP364R and B119L protein, respectively, of ASFV, were constructed by homologous recombination technology. Western blotting and immunofluorescence analysis showed that these foreign proteins were expressed in cells infected with the recombinant strains. The strains showed good genetic stability and proliferative characteristics for 20 passages in BHK-21 cells. Both of these strains were immunogenic in mice, inducing the production of specific antibodies against the expressed ASFV proteins while providing protection against lethal challenge with PRV. Thus, the recombinant strains rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L could be used as candidate vaccines for both ASFV and PRV. In addition, our study identifies two potential target genes for the development of safe and efficient ASFV vaccines, provides a reference for the construction of bivalent ASFV and PRV vaccines, and demonstrates the feasibility of developing a live ASFV vector vaccine.

Plant-derived galactolipids enhance specific antibody production and induce class-switch as vaccine adjuvant

Various plant-derived compounds can activate immune responses against bacterial infections, and this property contributes to them being developed as effective and safe adjuvants for vaccines. This study evaluated the potential adjuvant effects of a galactolipid-enriched fraction generated from the medicinal plant Crassocephalum rabens (designated CRA). Heat shock protein 60 of periodontal disease pathogen Actinobacillus actinomycetemcomitans (AaHSP60) was taken as an antigen and mixed with CRA. The AaHSP60/CRA mixture was then injected intraperitoneally into the BALB/c mice. Titers and affinity of specific antibodies were measured by ELISA. Cytokine profiles in mouse serum or culture media of AaHSP60/CRA-treated splenocytes were analyzed by cytokine multiplex assay and ELISA kits. B cell differentiation and macrophage activation were determined by phenotyping. CRA dramatically enhanced specific antibody titers and induced Ig class switch, as shown by increases in the IgG2a, IgG2b, and IgG3 proportions of total Ig in mouse serum. Furthermore, CRA-induced anti-AaHSP60 antibodies had cross-reactivity to other bacterial HSP60s. Cell-based and animal results demonstrated that CRA induced the release of IL-21 and B cell activating factor (BAFF), which stimulated B cell differentiation. CRA enhanced cell proliferation, uptake ability, and antigen presentation in mouse phagocytes. CRA served as a vaccine adjuvant that enhance mouse immunity against pathogenic antigens. CRA strengthened the activation and capabilities of phagocytes and B cells. Therefore, CRA may be a promising adjuvant for bacterial vaccines including periodontal disease.

Understanding hypergammaglobulinemia in experimental or natural visceral leishmaniasis.

Nonspecific hypergammaglobulinemia (HGG) occurs in symptomatic human visceral leishmaniasis (VL) caused by L. L. infantum. This study assessed this finding in experimental infection in hamsters and natural infection in dogs. The serum concentration of proteins, albumin and globulins was determined through the biuret and bromocresol green reaction, where the HGG was better expressed through the albumin/globulin (A/G) ratio. HGG was associated with a higher concentration of specific anti-glycan antibodies (BSA-G)/promastigote soluble extract (PSE) and the presence of circulating immune complexes (IC) by dissociative enzyme-linked immunoassay (ELISA). The study found monovalent IC in 37.9% (PSE) and 50% (BSA-G) of sera from infected hamsters, with increased frequency as the disease progressed. HGG was found in >60% of the samples in dogs with VL, associated with higher levels of specific immunoglobulin (Ig)A and IgM, but not IgG, determined using the PSE and BSA-G ELISA. HGG was associated with the presence of monovalent IC in 58.9% (PSE) and 63.4% (BSA-G) positive dog samples. HGG may result not only from the nonspecific activation of B cells, with greater production of specific and nonspecific antibodies, but also due to lower IgG excretion due to the presence of soluble monovalent IC. HGG correlates to the progression of VL and may be a marker for manifested disease.

Comparative analysis of tropomyosin allergenicity in three different species of molluscs: insights into the role of amino acid composition in IgE epitopes.

Limited research has been conducted on the differences in allergenicity among Alectryonella plicatula tropomyosin (ATM), Haliotis discus hannai tropomyosin (HTM), and Mimachlamys nobilis tropomyosin (MTM) in molluscs. Our study aimed to comprehensively analyze and compare their immunoreactivity, sensitization, and allergenicity while simultaneously elucidating the underlying molecular mechanisms involved. We assessed the immune binding activity of TM utilizing 86 sera from allergic patients and evaluated sensitization and allergenicity through two different types of mouse models. The dot-blot and basophil activation test assays revealed strong immunoreactivity for HTM, ATM, and MTM, with HTM exhibiting significantly lower levels compared to ATM. In the BALB/c mouse sensitization model, all TM groups stimulated the production of specific antibodies, elicited IgE-mediated immediate hypersensitivity responses, and caused an imbalance in the IL-4/IFN-γ ratio. Similarly, in the BALB/c mouse model of food allergy, all TM variants induced IgE-mediated type I hypersensitivity responses, leading to the development of food allergies characterized by clinical symptoms and an imbalance in the IL-4/IFN-γ ratio. The stimulation ability of sensitization and the severity of food allergies consistently ranked as ATM > MTM > HTM. Through an in-depth analysis of non-polar amino acid frequency and polar hydrogen bonds, HTM exhibited higher frequencies of non-polar amino acids in its amino acid sequence and IgE epitopes, in comparison with ATM and MTM. Furthermore, HTM demonstrated a lower number of polar hydrogen bonds in IgE epitopes. Overall, HTM exhibited the lowest allergenic potential in both allergic patients and mouse models, likely due to its lower polarity in the amino acid sequence and IgE epitopes.

Extracellular DNA level as an indicator of the activity of inflammatory reactions in patients with rheumatoid arthritis and asthma

BACKGROUND: We performed a clinical and laboratory examination of patients with rheumatoid arthritis and bronchial asthma. Nosologies were selected to examine extracellular DNA changes in the blood during immunopathological processes based on generally accepted views on the role of helper balance in disease pathogenesis. The degree of disease activity in rheumatoid arthritis primarily depends on the severity of inflammatory changes in the joints; therefore, it is closely associated with a shift in the balance of helpers toward the Th1. According to today’s concepts, the pathogenesis of asthma is determined by the intensive production of specific antibodies, which are controlled by activated Th2 lymphocytes. AIM: To study extracellular DNA changes in the blood of patients during immunopathological processes depending on the Th1/Th2 balance ratio and compare these data with the parameters of standard laboratory tests and indicators of neutrophil activities and their ability to netosis. METHODS: Extracellular DNA concentration was determined using the Quant-ITTM PicoGreen fluorescent reagent for double-stranded DNA. Neutrophils isolated in a double-density gradient of ficoll–urografin were cultured with or without the addition of phorbol myristate acetate followed by Sytox Green. RESULTS: The studied immunopathological conditions had significantly decreased extracellular DNA levels in the plasma of patients with asthma, which sharply contrasted with its increase in patients with arthritis. Laboratory parameters confirmed the presence of local inflammation in patients with asthma without obvious symptoms of a systemic inflammatory response, while in patients with arthritis, inflammatory changes in the joints were accompanied by an increased C-reactive protein level, indicating a systemic reaction of the body in response to inflammatory stimuli. CONCLUSIONS: Thus, the state of the Th1/Th2 balance is assumed as one of the significant regulators that determined the extracellular DNA concentration in the blood. According to the proposed hypothesis, the shift in this ratio toward the predominance of Th1 cells should promote the development of inflammatory processes in the body and increase the amount of extracellular DNA, while a shift in the balance of helpers toward the dominance of Th2 lymphocytes should suppress these processes and consequently decrease the extracellular DNA in the blood.

Promising directions in the development of new vaccines against papillomavirus infection

Papillomavirus infection is one of the most common viral infections worldwide. Highly oncogenic types of human papillomavirus (HPV) are the root cause of cancer of the cervix, vulva, vagina, genitals in men, anal cancer, as well as cancer of the oropharyngeal region. Currently, three HPV vaccines have been registered and successfully used in Russia: divalent (HPV types 16, 18) and quadrivalent (HPV types 6, 11, 16, 18). In the countries of America and Europe, a nine-valent vaccine is available, which contains nine types of HPV (6, 11, 16, 18, 31, 33, 45, 52, 58). These vaccines are preventive, have high immunogenicity and induce the production of specific antibodies. The action of preventive vaccines is aimed at creating specific humoral immunity to the capsid proteins of the virus, which leads to the prevention of HPV infection. Nevertheless, there are no specific medicines on the pharmaceutical market aimed at treating an already established pathology.&#x0D; Currently, active development of HPV vaccines with both preventive and therapeutic effects is underway. Candidates of therapeutic HPV vaccines undergo various stages of research. Experimental vaccination systems on animal models provide encouraging results. If preventive and therapeutic HPV vaccines prove to be effective in patients as well as in animal models, then pillomavirus infection and related malignant diseases can be controlled by vaccination.

Comparison of cow's milk allergy models highlighted higher humoral and Th2 immune responses in BALB/c than C3H/HeNCrl mice

Cow's milk allergy (CMA) is common in early childhood and the incidence is increasing. However, its mechanisms of action are still not fully understood due to the range of different clinical symptoms. So far, the development of different mouse models has been the best choice to study the molecular mechanisms triggering allergy. However, the selection of suitable strains for the establishment of animal models truly representative of associated human pathologies is still a challenge. Hence, we focused on both C3H/HeNCrl and BALB/c mice to characterize their susceptibility to CMA. After intraperitoneal sensitization, BALB/c and C3H/HeNCrl strains were challenged with β-lactoglobulin (BLG), and compared in allergic symptoms and active immune response, which assessed by specific antibody production and cytokine release. At first, both groups exhibited anaphylaxis, showed specific BLG-related IgE, Th2 response and seemed both suitable for the development of CMA models. However, a detailed analysis revealed that BALB/c had both stronger humoral and Th2 immune responses, producing more antibodies (IgE and IgG/IgG1/IgG2a), and releasing higher levels of Th2-associated cytokines (IL-4, IL-5, IL-13) compared to C3H/HeNCrl mice. Therefore, BALB/c strain would represent a preferential choice in the establishment of CMA models. This study highlights the subtle differences and major outcomes in the selection of mouse strains for the development of suitable food allergy models.

Targeting kinase ITK treats autoimmune arthritis via orchestrating T cell differentiation and function

IL-2 inducible T cell kinase (ITK) is critical in T helper subset differentiation and its inhibition has been suggested for the treatment of T cell-mediated inflammatory diseases. T follicular helper (Tfh), Th17 and regulatory T cells (Treg) also play important roles in the development of rheumatoid arthritis (RA), while the role of ITK in the development of RA and the intricate balance between effector T and regulatory T cells remains unclear. Here, we found that CD4+ T cells from RA patients presented with an elevated ITK activation. ITK inhibitor alleviated existing collagen-induced arthritis (CIA) and reduced antigen specific antibody production. Blocking ITK kinase activity interferes Tfh cell generation. Moreover, ITK inhibitor effectively rebalances Th17 and Treg cells by regulating Foxo1 translocation. Furthermore, we identified dihydroartemisinin (DHA) as a potential ITK inhibitor, which could inhibit PLC-γ1 phosphorylation and the progression of CIA by rebalancing Th17 and Treg cells. Out data imply that ITK activation is upregulated in RA patients, and therefore blocking ITK signal may provide an effective strategy to treat RA patients and highlight the role of ITK on the Tfh induction and RA progression.