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Considering the limited information on the impact of PM 2.5 content on ocular health, a follow-up study was conducted on 50 healthy adults. Samples were collected twice, once before the PM 2.5 exposure season and again after exposure. Daily PM 2.5 concentration data was gathered from Thung Satok monitoring station. All subjects completed the self-structured ocular symptom questionnaire. The concentrations of 1-OHP were determined using HPLC-FLD. Logistic regression analysis investigated the relationship between PM 2.5 toxicity and ocular symptoms. The findings revealed that daily PM 2.5 concentrations surpassed the WHO-recommended range by around threefold. Exposure to PM 2.5 significantly raised the likelihood of ocular redness (adjusted OR: 12.39, 95% CI), watering (adjusted OR: 2.56, 95% CI), and dryness (adjusted OR: 5.06, 95% CI). Additionally, these symptoms had an exposure-response relationship with increasing 1-OHP levels. Ocular symptoms worsened in frequency and severity during the high PM 2.5 season, showing a strong link to elevated PM 2.5 levels. Lymphocyte counts were also positively correlated with redness, watering, and dryness during high PM 2.5 exposure. In conclusion, our study shows that subjects exposed to higher PM 2.5 levels presented more significant ocular surface alterations., (© 2024. The Author(s).)

The ongoing adjustment and fluctuation of energy, climate, and economic policies leads to potential policy uncertainty. The potential uncertainty arising from these policies has a wide range of effects on renewable energy technology, leading to changes in investment decisions or consumption patterns. Hence, given the worldwide shift towards sustainable technologies, it is critical to assess how these policy uncertainties will affect renewable power. Given this backdrop, this study delves into the impact of energy policy uncertainty, climate policy uncertainty, economic policy uncertainty, and government effectiveness on renewable energy in the United States. The cross-quantilogram (CQ) methodology is employed by using monthly data from 2002 to 2022. The outcomes reveal that energy policy uncertainty effectively promotes renewable energy in the short run, whereas it serves as a barrier to the adoption of renewables in the long run. Economic policy uncertainty reduces renewable energy consumption in the short run, but it increases in the long run. Moreover, climate policy uncertainty accelerates the transition to low-carbon energy sources. Finally, government effectiveness is a significant supportive element of the energy transition towards renewables., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The explicit identification of CD8 + T cell subpopulation is important for deciphering the role of CD8 + T cells for protecting our body against invading pathogens and cancer. Our generated monoclonal antibody (mAb), named FE-1H10, recognized two novel subpopulations of peripheral blood CD8 + T cells, FE-1H10 + and FE-1H10 - CD8 + T cells. The molecule recognized by mAb FE-1H10 (FE-1H10 molecules) had a higher distribution on effector memory CD8 + T cell subsets. The functions of FE-1H10 - and FE-1H10 + CD8 + T cells were investigated. T cell proliferation assays revealed that FE-1H10 - CD8 + T cells exhibited a higher proliferation rate than FE-1H10 + CD8 + T cells, whereas FE-1H10 + CD8 + T cells produced higher levels of IFN-γ and TNF-α than FE-1H10 - CD8 + T cells. In T cell cytotoxicity assays, FE-1H10 + CD8 + T cells were able to kill target cells better than FE-1H10 - CD8 + T cells. RNA-sequencing analysis confirmed that these subpopulations were distinct: FE-1H10 + CD8 + T cells have higher expression of genes involved in effector functions (IFNG, TNF, GZMB, PRF1, GNLY, FASL, CX3CR1) while FE-1H10 - CD8 + T cells have greater expression of genes related to memory CD8 + T cell populations (CCR7, SELL, TCF7, CD40LG). The results suggested that mAb FE-1H10 identifies two novel distinctive CD8 + T cell subpopulations. The FE-1H10 + CD8 + T cells carried a superior functionality in response to tumour cells. The uncover of these novel CD8 + T cell subpopulations may be the basis knowledge of an optional immunotherapy for the selection of potential CD8 + T cells in cancer treatment., (© 2024 John Wiley & Sons Ltd.)

The diagnosis of mycobacterial infections, including both the Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), poses a significant global medical challenge. This study proposes a novel approach using immunochromatographic (IC) strip tests for the simultaneous detection of MTBC and NTM. Traditional methods for identifying mycobacteria, such as culture techniques, are hindered by delays in distinguishing between MTBC and NTM, which can affect patient care and disease control. Molecular methods, while sensitive, are resource-intensive and unable to differentiate between live and dead bacteria. In this research, we developed unique monoclonal antibodies (mAbs) against Ag85B, a mycobacterial secretory protein, and successfully implemented IC strip tests named 8B and 9B. These strips demonstrated high concordance rates with conventional methods for detecting MTBC, with positivity rates of 93.9% and 85.9%, respectively. For NTM detection, the IC strip tests achieved a 63.2% detection rate compared to culture methods, considering variations in growth rates among different NTM species. Furthermore, this study highlights a significant finding regarding the potential of MPT64 and Ag85B proteins as markers for MTBC detection. In conclusion, our breakthrough method enables rapid and accurate detection of both MTBC and NTM bacteria within the BACTEC MGIT system. This approach represents a valuable tool in clinical settings for distinguishing between MTBC and NTM infections, thereby enhancing the management and control of mycobacterial diseases. KEY POINTS: • Panel of mAbs for differentiating MTB versus NTM • IC strips for diagnosing MTBC and NTM after the BACTEC MGIT • Combined detection of MTP64 and Ag85B enhances diagnostic accuracy., (© 2024. The Author(s).)

CD99 was demonstrated to be a potential target for antibody therapy on T-acute lymphoblastic leukemia (T-ALL). The ligation of CD99 by certain monoclonal antibodies (mAbs) induced T-ALL apoptosis. However, the molecular basis contributing to the apoptosis of T-ALL upon anti-CD99 mAb engagement remains elusive. In this study, using our generated anti-CD99 mAb clone MT99/3 (mAb MT99/3), mAb MT99/3 engagement strongly induced apoptosis of T-ALL cell lines, but not in non-malignant peripheral blood cells. By transcriptome analysis, upon mAb MT99/3 ligation, 13 apoptosis-related genes, including FOS, TNF, FASLG, BCL2A1, JUNB, SOCS1, IL27RA, PTPN6, PDGFA, NR4A1, SGK1, LPAR5 and LTB, were significantly upregulated. The epitope of CD99 recognized by mAb MT99/3 was then identified as the VDGENDDPRPP at residues 60-70 of CD99, which has never been reported. To the best of our knowledge, this is the first transcriptome data conducted in T-ALL with anti-CD99 mAb engagement. These findings provide new insights into CD99 implicated in the apoptosis of T-ALL. The identification of a new epitope and apoptosis-related genes that relate to the induction of apoptosis by mAb MT99/3 may serve as a new therapeutic target for T-ALL. The anti-CD99 mAb clone MT99/3 might be a candidate for further development of a therapeutic antibody for T-ALL therapy.

Energy transition to greener systems has been a focal point in climate policy agendas across countries as the negative environmental impacts of fossil fuel technologies have become more evident Displacing fossil fuels with clean energy alternatives in this regard is essential for meeting global climate objectives. In this context, the study analyzes the role of disaggregated renewable energy sources on fossil fuel displacement in 36 Organisation for Economic Cooperation and Development (OECD) countries in the period 2000-2020. The findings demonstrate a discernible trend in the displacement of fossil fuels by various forms of renewable energy sources. It is found that to effectively displace 1% of fossil fuels, it is necessary to achieve an average increase of 1.15% in renewable generation capacity. In addition, a one-to-one displacement of fossil fuels occurs with hydropower, demonstrating its higher level of competitiveness and effectiveness in displacing fossil fuels. Moreover, there is a partial displacement of fossil fuels by solar and wind power. These findings suggest that renewable energy sources are progressively advancing towards effectively displacing fossil fuels., (© 2024. The Author(s).)

Background: Data on humoral and cellular immune responses against SARS-CoV-2 after receiving heterologous CoronaVac/ChAdOx-1 (CoVac/ChAd) vaccination in subjects with chronic obstructive pulmonary disease (COPD) are still limited. Therefore, we determined the neutralizing antibody (NAb) and T-cell responses against SARS-CoV-2 wild type (WT) and variants of concern (VOCs) in COPD patients., Methods: The levels of NAb as well as specific CD4 and CD8 T-cell responses against SARS-CoV-2 WT and VOCs were determined in COPD patients before and after vaccination., Results: Four weeks after vaccinations, the median levels of % inhibition of NAb against SARS-CoV-2 WT, Alpha, Beta, and Delta variants were significantly higher compared to pre-vaccination. The induction of NAb against Omicron was very low compared to other variants. At four weeks after vaccination, in comparison to pre-vaccination, the increasing trend of TNF-α-, IFN-γ-, IL-4-, IL-17-, IL-10-, and FasL-producing CD4 T-cells upon stimulation with WT spike peptides were demonstrated. No difference in T-cell responses to spike peptides of Alpha, Beta, and Delta variants and their WT homologs was observed., Conclusion: Heterologous CoVac/ChAd vaccine induced the production of NAb against SARS-CoV-2 WT, Alpha, Beta, and Delta variants, but low for Omicron in COPD patients. Induction of CD4 T-cell subset responses was slightly observed by this vaccine regimen., Clinical Trials Registry: This study was approved by the Clinical Trials Registry (Study ID: TCTR20210822002)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: CD4, a leukocyte surface glycoprotein, is mainly expressed on CD4+ T cells, but is also expressed on monocytes. The difference in the expression level and structure of CD4 on T cells and monocytes predicts the different functions of this molecule in both cell types. Although the function of CD4 on T cells is well characterized, little is known about that expressed on primary monocytes., Objective: In this study, we investigated the immunoregulation function of CD4 on peripheral blood monocytes., Methods: Methods: CD4 molecule on monocyte was ligated by anti-CD4 monoclonal antibody (mAb), MT4/3. The effect of mAb MT4/3 on T cell proliferation, cytokine production, the expression of monocyte costimulatory molecules, monocyte migration, and macrophage differentiation were investigated. Moreover, the molecular weight of CD4 on peripheral blood monocyte was carried out by Western immunoblotting., Results: We demonstrated that mAb MT4/3 inhibited anti-CD3 induced T cell proliferation, cytokine production, and the expression of monocyte costimulatory molecules. The ligation of only CD4 on monocytes was sufficient to inhibit T cell activation. Moreover, mAb MT4/3 could inhibit monocyte migration in a transwell migration assay, but not affect the differentiation of monocytes to macrophages. Using purified primary monocytes, the molecular weight of CD4 expressed on monocytes was identified as 55 kDa., Conclusions: The CD4 molecule expressed on monocytes might play an important role in the regulation of immune responses in both innate and adaptive immunity. Understanding the novel role of CD4 on monocytes in immunoregulation is valuable in the development of new therapeutic approaches.

The outbreak of the SARS-CoV-2 Omicron variant raised the need for vaccine boosting. We evaluated the efficiency of the third booster vaccine, ChAdOx-1 or BNT162b2, in causing a neutralizing antibody (NAb) response and its durability against the Omicron and other variants in elderly individuals previously vaccinated with 2-dose CoronaVac inactivated vaccine. After receiving 2-dose CoronaVac, only 2.2% of subjects had NAbs against the Omicron variant above the cut-off value. Four weeks after boosting, the number of subjects who had NAb levels above the cut-off values in the ChAdOx-1 and BNT162b2 vaccine boosting groups increased to 41.7% and 54.5%, respectively. However, after 12 and 24 weeks of boosting with any vaccines, NAb levels against the Omicron variant dramatically waned. Twenty-four weeks after boosting, only 2% had high levels of NAbs against the Omicron variant. Compared to other variants, the Omicron variant was less responsive to boosting vaccines. The waning rate of NAb levels for the Omicron variant was much faster than that observed in the Alpha, Beta and Delta variants. To combat the Omicron variant, the fourth booster dose is, therefore, recommended for elderly individuals., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) are components in the endocannabinoid system that play significant roles in regulating immune responses. There are many agonists for the cannabinoid receptors; however, their effects on T cell regulation have not been elucidated. In the present study, we determined the effects of the CB1 selective agonist ACEA and the CB2 selective agonist GW833972A on T cell responses. It was found that both agonists impaired anti-CD3 monoclonal antibody induced T cell proliferation. However, ACEA and GW833972A agonists down-regulated the expression of activation markers on CD4 + and CD8 + T cells and co-stimulatory molecules on B cells and monocytes in different manners. Moreover, only GW833972A suppressed the cytotoxic activities of CD8 + T cells without interfering in the cytotoxic activities of CD4 + T cells and NK cells. In addition, the CB2 agonist, but not CB1 agonist, caused the reduction of Th1 cytokine production. Our results demonstrated that the CB1 agonist ACEA and CB2 agonist GW833972A attenuated cell-mediated immunity in different mechanisms. These agonists may be able to be used as therapeutic agents for inducing T cell hypofunction in inflammatory and autoimmune diseases.

Background: The concept of heterologous vaccination against SARS-CoV-2 infection has been adopted in Thailand with limited data on the induction of humoral and cellular immunity, particularly the CoronaVac/ChAdOx-1 (CoVac/ChAd) regimen in the elderly., Objective: In this study, the immune responses of the elderly induced by heterologous CoVac/ChAd and homologous ChAdOx-1 (ChAd/ChAd) vaccinations were demonstrated., Methods: A prospective observational study involving healthy participants aged ≥ 60 years who received heterologous CoVac/ChAd or homologous ChAd/ChAd vaccination was conducted. Surrogate neutralizing antibody (NAb) and T-cell responses against the SARS-CoV-2 wild type (WT) and variants of concern were determined at pre and post vaccinations., Results: At 4 and 12 weeks after heterologous or homologous vaccination, the NAb levels against WT, Alpha, Beta, and Delta variants between each group were not significantly different, except for significant lower NAb against the Beta variant in heterologous group at 12 weeks after vaccination. The NAb against the Omicron at 4 weeks post-vaccination were below the cutoff level for antibody detection in both groups. However, higher spike-specific CD4 T cell producing IFN-γ and TNF-α in the heterologous than the homologous vaccination were observed. Insignificant difference of cellular immune responses to spike-peptides of Alpha, Beta, and Delta variants and their WT homologues was demonstrated., Conclusions: In the elderly, heterologous CoVac/ChAd vaccination could induce NAb response against the WT and non-Omicron variants not different from the homologous ChAd/ChAd vaccination. Both regimens could not give adequate NAb of the Omicron strain. The heterologous vaccination, however, induced higher spike-specific Th1 cell response.

Aim: The present study aims to generate chimeric mouse single-chain variable fragment (scFv) and immunoglobulin G1 (IgG1) crystallizable fragment (Fc) antibody against disialoganglioside (GD2) for the treatment of neuroblastoma (NB). The generated scFv-IgG Fc antibody, lacking first constant domain of heavy chain (CH1), is of a smaller size than the natural antibody and has anti-tumor activity., Methods: Vector for scFv-IgG Fc antibody was constructed and scFv-IgG Fc antibody was expressed in human embryonic kidney 293T (HEK293T) cell line. Purification of scFv-IgG Fc antibody from the culture supernatant of transfected HEK293T cells was performed by Protein G affinity chromatography. The structure and binding activity of scFv-IgG Fc antibody were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting (WB), and immunofluorescence techniques. Anti-tumor activities by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were determined., Results: Using plasmid fusion-human IgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2), a plasmid vector encoding chimeric mouse scFv and hIgG1 Fc antibody against GD2 was successfully constructed. This vector was transfected into human HEK293T cells to produce scFv-IgG Fc antibody. The transfected HEK293T cells could produce chimeric scFv-IgG Fc antibody against GD2, which lacks the IgG heavy chain CH1 domain but carries CH2 and CH3 domains. The chimeric antibodies could be purified from the culture supernatant of the transfected HEK293T culture in the presence of zeocin drug. The produced GD2 scFv-IgG Fc antibodies, which are smaller in size than the intact antibody, could trigger the killing of GD2 expressed NB cell line SH-SY5Y by ADCC and ADCP mechanisms., Conclusions: The results indicate that chimeric scFv-hIgG Fc antibody, lacking heavy chain CH1 domain, could mediate antibody induced anti-tumor activities. The small size of this type of chimeric antibody may be employed as anti-GD2 antibody for NB therapy., Competing Interests: The authors declare that they have no conflicts of interest., (© The Author(s) 2023.)

Data on immunogenicity of adenovirus-vectored vaccine in chronic obstructive pulmonary disease (COPD) patients is limited. Therefore, we aimed to determine the humoral and cellular immune responses after homologous ChAdOx-1 vaccination in subjects with COPD. COPD subjects and age- and sex-matched healthy elderly receiving ChAdOx-1 homologous vaccination were included. The levels of neutralizing antibodies (NAb) and specific CD4 and CD8 T-cell responses against SARS-CoV-2 wild-type (WT) and variants of concern (VOCs: Alpha, Beta, Delta, and Omicron) were measured. Eight COPD patients were matched with eight control participants. After vaccination for 4 and 12 weeks, % inhibition of NAb against Alpha, Beta, and Delta in both groups were comparable and significantly higher than baseline. The percentage inhibition of NAb at the 12th week was significantly dropped from the 4th week in each group. The NAb against the Omicron variant, however, were much lower than Alpha, Beta, Delta variants. The increasing trend in the number of CD4 T-cells producing TNF-α, IFN-γ, IL-10, and FasL upon stimulation with spike peptides of WT and VOCs was observed in COPD patients compared to the healthy group. These responses were not observed in CD8 T-cells. Homologous ChAdOx-1 vaccination could induce comparable NAb against the SARS-CoV-2 WT, Alpha, Beta, Delta, and Omicron variants between COPD and healthy elderly. The CD4 T-cell responses did not differ between COPD patients and healthy control.

CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T-cell activation, cell adhesion, and cell migration. We have also demonstrated that CD99 and its ligands regulate proinflammatory cytokines in NK cells, monocytes and activated T cells. These data suggest CD99 as a potential therapeutic target in cancer. However, the molecular mechanisms by which CD99 and CD99 counter receptors participate in such processes are unclear. High-quality CD99 recombinant proteins produced in large amounts are essential for biological studies and clinical research. In this study, we optimized the various culture conditions for increasing amounts of recombinant protein production with good biological activity. Intracellular immunofluorescence staining was performed to identify the highly expressing CD99HIgG cells. We further investigated the culture conditions for recombinant protein production. A double antibody sandwich enzyme-linked immunosorbent assay was employed to determine the level of secreted CD99HIgG proteins in the culture supernatant of various culture conditions. Later, affinity chromatography using protein G was used to purify CD99HIgG proteins from the culture supernatant of three proper culture conditions. According to our previous report, which utilized Western blotting, the purified CD99HIgG obtained from all tested culture conditions is composed of the CD99 extracellular part fused with the human IgG Fc part in dimer form. For biological activity, the obtained CD99HIgG proteins showed the ability to ligate with the CD99 counter receptor, resulting in the induction of cytokine production., Competing Interests: Declaration of competing interest The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Fluoroquinolones (FQs) are among the antibiotics whose widespread use in farm-raised animals results in potentially harmful residues in the end products. Additionally, most Thai farmers use antibiotics. Amoxicillin and enrofloxacin were commonly used by pig farms, and hens were given enrofloxacin to prevent immunization side effects. Moreover, antibiotic overuse has harmed food safety in the long term, and the use of low-dose antibiotics causes bacterial resistance. Herein, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to make a fast, easy, sensitive, and cost-effective method for monitoring FQs residues. After immunizing hens with mixed multi-hapten ciprofloxacin-bovine serum albumin (CPFX-BSA) with norfloxacin-bovine serum albumin (NFX-BSA), the IgY antibody purified from egg yolk was used for the detection of FQs residues in chicken and pork samples. The efficiency of the IgY antibody showed excellent sensitivity, with 50% inhibitory concentration (IC 50 ) of enrofloxacin at 0.05 µg/mL, far below the MRLs defined by the EU for muscle samples, which was not to exceed 100 µg/kg. The recovery range for chicken muscle samples spiked with ENFX concentrations of 1.00-0.01 µg/mL was 86.65-112.71%, similar to pork samples, which were 84.24-117.22.2%. This method has a lot of potential for analyzing fluoroquinolones in complex samples quickly, easily, and at a low cost on-site. The IgY-based ic ELISA was developed to detect ciprofloxacin (CPFX), norfloxacin (NFX), and enrofloxacin (ENFX) residues; it confirms that IgY could be a promising choice for the detection of antibiotic residues in food samples.

Background: Enoyl-CoA hydratase short-chain 1 (ECHS1) is a key mitochondrial enzyme that is involved in valine catabolism and fatty acid beta-oxidation. Mutations in the ECHS1 gene lead to enzymatic deficiency, resulting in the accumulation of certain intermediates from the valine catabolism pathway. This disrupts the pyruvate dehydrogenase complex and the mitochondrial respiratory chain, with consequent cellular damage. Patients present with a variable age of onset and a wide spectrum of clinical features. The Leigh syndrome phenotype is the most frequently reported form of the disease. Herein, we report a case of a male with ECHS1 deficiency who was diagnosed at 8 years of age. He presented severe dystonia, hyperlordosis, moderate to severe kyphoscoliosis, great difficulty in walking, and severe dysarthria. A valine-restricted and total fat-restricted diet was considered as a therapeutic option after the genetic diagnosis. An available formula that restricted branched-chain amino acids and especially restricted valine was used. We also restricted animal protein intake and provided a low-fat diet that was particularly low in dairy fat., Results: This protein- and fat-restricted diet was initiated with adequate tolerance and adherence. After three years, the patient noticed an improvement in dystonia, especially in walking. He currently requires minimal support to walk or stand. Therefore, he has enhanced his autonomy to go to school or establish a career for himself. His quality of life and motivation for treatment have greatly increased., Conclusions: There is still a substantial lack of knowledge about this rare disorder, especially knowledge about future effective treatments. However, early diagnosis and treatment with a valine- and fat-restricted diet, particularly dairy fat-restricted diet, appeared to limit disease progression in this patient with ECHS1 deficiency., (© 2022. The Author(s).)

CD147/Basigin/EMMPRIN is overexpressed in several cancerous tissues and it has been shown to induce matrix metalloproteinases (MMPs) whose expression is associated with cancer metastasis. Thus, targeting CD147 with monoclonal antibodies (mAbs) potentially has therapeutic applications in cancer immunotherapy. Here, we report the use of anti-CD147 mAbs targeting domain 1 of CD147, namely M6-1D4 (IgM), M6-1F3 (IgM), M6-2F9 (IgM) and M6-1E9 (IgG2a), against several human cancer cell lines. Strikingly, IgM but not IgG mAbs against CD147, especially clone M6-1D4, induced acute cellular swelling, and this phenomenon appeared to be specifically found with hepatocellular carcinoma (HCC) cells. Furthermore, molecular investigation upon treating HepG2 cells with M6-1D4 showed unfolded protein response (UPR) activation, autophagosome accumulation, and cell cycle arrest, but without classic apoptosis related features. More interestingly, prolonged M6-1D4 treatment (24 h) resulted in irreversible oncosis leading to necroptosis. Furthermore, treatment with a mixed lineage kinase domain-like psuedokinase (MLKL) inhibitor and partial knockout of MLKL resulted in reduced sensitivity to necroptosis in M6-1D4-treated HepG2 cells. Surprisingly however, the observed necroptotic signaling axis appeared to be non-canonical as it was independent of receptor-interacting serine/threonine-protein kinase (RIPK) phosphorylation. In addition, no cytotoxic effect on human dermal fibroblast (HDF) was observed after incubation with M6-1D4. Taken together, this study provides clues to target CD147 in HCC using mAbs, as well as sheds new light on a novel strategy to kill cancerous cells by the induction of necroptosis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Various vaccines have been developed to control the COVID-19 pandemic, but the available vaccines were developed using ancestral SARS-CoV-2 wild-type (WT) strains. Commercial anti-SARS-CoV-2 receptor binding domain (RBD) antibody assays have been established and employed for validation of vaccine efficacy. However, these assays were developed before the SARS-CoV-2 variants of concern (VOCs) emerged. It is unclear whether anti-RBD IgG levels can predict immunity against VOCs. In this study, we determined the correlations between the levels of anti-RBD IgG and neutralizing antibodies (NAbs) against SARS-CoV-2 variants in vaccinated subjects. After vaccination, 100% of subjects showed an anti-RBD IgG response, whereas 82, 79, 30, 75, and 2% showed NAb responses against WT, Alpha, Beta, Delta, and Omicron variants, respectively. A high correlation was observed between anti-RBD IgG and NAbs against WT, Alpha, Beta, and Delta, but not so for the Omicron NAbs. Among subjects with high levels of anti-RBD IgG, 93, 93, 71, 93, and 0% of them had NAbs against WT, Alpha, Beta, Delta, and Omicron variants, respectively. These results indicate that anti-RBD IgG levels cannot be used as a predictor for the presence of NAbs against the globally dominant SARS-CoV-2 Omicron variant.

Background: The existence of SARS-CoV-2 variants of concern (VOCs) in association with evidence of breakthrough infections despite vaccination resulted in the need for vaccine boosting. In elderly individuals, information on the immunogenicity of booster vaccinations is limited. In countries where the CoronaVac inactivated vaccine is the primary vaccine, the appropriate boosting regimen is not clear. Immunologic studies of the effects of booster vaccination against VOCs, particularly Delta and Omicron, following CoronaVac in elderly individuals are helpful for policy makers. In this study, we determined the immune responses against VOCs following ChAdOx-1 or BNT162b2 boosting in elderly individuals previously immunized with CoronaVac., Results: Before boosting, the median % inhibition of neutralizing antibodies (NAbs) against the wild-type (WT), Alpha, Beta, Delta and Omicron variants in the ChAdOx-1 and BNT162b2 groups was 52.8% vs. 53.4, 36.6% vs. 39.9, 5.2% vs. 13.7, 34.3% vs. 44.9, and 20.8% vs. 18.8%, respectively. After boosting with ChAdOx-1 or BNT162b2, the % inhibition of NAbs were increased to 97.3% vs. 97.4, 94.3% vs. 97.3%, 79.9 vs. 93.7, 95.5% vs. 97.5, and 26.9% vs. 31.9% for WT, Alpha, Beta, Delta and Omicron variants, respectively. Boosting with BNT162b2 induced significantly higher NAb levels than boosting with ChAdOx-1 against the Alpha, Beta and Delta variants but not the WT and Omicron variants. NAb levels against Omicron variant were not significantly different before and after boosting with ChAdOx-1 or BNT162b2. To evaluate T-cell responses, S peptides of the WT, Alpha, Beta and Delta variants were used to stimulate T cells. Upon stimulation, the expression of IL-17A in CD8 T cells was higher in the BNT162b2 group than in the ChAdOx-1 boosting group. However, IFN-γ production in CD4 and CD8 T cells did not significantly differ under all vaccination regimens. The expression of FasL in CD4 T cells, but not CD8 T cells, was higher in the BNT162b2-boosted group., Conclusion: Boosting with either ChAdOx-1 or BNT162b2 in CoronaVac-primed healthy elderly individuals induced high NAb production against all examined VOCs except Omicron. BNT162b2 stimulated higher NAb and some T-cell responses than ChAdOx-1. Vaccine boosting is, therefore, recommended for elderly individuals previously immunized with CoronaVac., (© 2022. The Author(s).)

CD147, a member of the immunoglobulin (Ig) superfamily, is widely expressed in several cell types. CD147 molecules have multiple cellular functions, such as migration, adhesion, invasion, energy metabolism and T cell activation. In particular, recent studies have demonstrated the potential application of CD147 as an effective therapeutic target for cancer, as well as autoimmune and inflammatory diseases. In this study, we elucidated the functional epitopes on CD147 extracellular domains in T cell regulation using specific monoclonal antibodies (mAbs). Upon T cell activation, the anti-CD147 domain 1 mAbs M6-1E9 and M6-1D4 and the anti-CD147 domain 2 mAb MEM-M6/6 significantly reduced surface expression of CD69 and CD25 and T cell proliferation. To investigate whether functional epitopes of CD147 are differentially expressed on distinct leukocyte subsets, PBMCs, monocyte-depleted PBMCs and purified T cells were activated in the presence of anti-CD147 mAbs. The mAb M6-1E9 inhibited T cell functions via activation of CD147 on monocytes with obligatory cell-cell contact. Engagement of the CD147 epitope by the M6-1E9 mAb downregulated CD80 and CD86 expression on monocytes and IL-2, TNF-α, IFN-γ and IL-17 production in T cells. In contrast, the mAb M6-1D4 inhibited T cell function via activation of CD147 on T cells by downregulating IL-2, TNF-α and IFN-γ. Herein, we demonstrated that certain epitopes of CD147, expressed on both monocytes and T cells, are involved in the regulation of T cell activation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pata, Surinkaew, Takheaw, Laopajon, Chuensirikulchai and Kasinrerk.)

Upon generation of monoclonal antibodies to the T cell antigen receptor/CD3 (TCR/CD3) complex, we isolated mAb MT3, whose reactivity correlates inversely with the production of IFN-γ by human peripheral blood T lymphocytes. Using eukaryotic expression cloning, we identified the MT3 antigen as myelin-and-lymphocyte (MAL) protein. Flow cytometry analysis demonstrates high surface expression of MAL on all naïve CD4 + T cells whereas MAL expression is diminished on central memory- and almost lost on effector memory T cells. MAL - T cells proliferate strongly in response to stimulation with CD3/CD28 antibodies, corroborating that MAL + T cells are naïve and MAL - T cells memory subtypes. Further, resting MAL - T cells harbor a larger pool of Ser59- and Tyr394- double phosphorylated lymphocyte-specific kinase (Lck), which is rapidly increased upon in vitro restimulation. Previously, lack of MAL was reported to prevent transport of Lck, the key protein tyrosine kinase of TCR/CD3 signaling to the cell membrane, and to result in strongly impaired human T cell activation. Here, we show that knocking out MAL did not significantly affect Lck membrane localization and immune synapse recruitment, or transcriptional T cell activation. Collectively, our results indicate that loss of MAL is associated with activation-induced differentiation of human T cells but not with impaired membrane localization of Lck or TCR signaling capacity., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Objective: CD99, a leukocyte surface molecule, reportedly plays an important role in several cellular processes. However, the role of CD99 in T cell regulation remains unclear, as the CD99 ligand associated with T-cell regulation has not yet been identified. Our previous study showed that recombinant CD99 bound to CD99 ligands was expressed on monocytes, NK cells and dendritic cells. This interaction regulates the expression of IL-6 and TNF-α in CD3 + T cells following T cell activation. In the present study, we confirmed the presence of CD99 ligands in immune cells., Results: A recombinant CD99-human IgG fusion protein, CD99HIgG, was produced and used to search for CD99 ligand expression in various hematopoietic cell lines. Among several cell lines, THP-1 monocytic cell line showed strong positive reaction for CD99HIgG, and CD99 and CD99 ligand complexes were pulled-down using a DTSSP cross-linker. The study demonstrated the presence of the membrane bound CD99 ligand, and CD99 ligand candidates were identified via LC-MS/MS. These results may be useful to further identify the CD99 ligands, and to fully comprehend the role of CD99 in immunoregulation.

α0-Thalassemia is an inherited hematological disorder caused by the deletion of α-globin genes. The Southeast Asian deletion (--SEA) is the most common type of α0-thalassemia observed in Southeast Asian countries. Regarding WHO health policy, an effective α0-thalassemia screening strategy is needed to control new severe α-thalassemia cases. In this study, a monoclonal antibody panel was used to develop immunochromatographic (IC) strip tests for detecting the Hb Bart's and ζ-globin chain. Among 195 samples, all α0-thalassemia traits (78 α0-thalassemia (--SEA) and 4 α0-thalassemia (--THAI)) had low MCV or MCH values. The sensitivity, specificity, PPV and NPV of the IC strip tests for ζ-globin and Hb Bart's for screening α0-thalassemia (--SEA) within the low MCV or MCH samples were 100%, 65.2%, 90.7%, 100% and 96.2%, 47.8%, 86.6%, 78.6%, respectively. All 4 α0-thalassemia (--THAI) traits were negative for ζ-globin chains but positive for Hb Bart's using the IC strip tests. These results led to a α0-thalassemia screening being proposed in which blood samples are first evaluated by MCV, MCH and Hb typing. Samples with high MCV and MCH values are excluded for the presence of the α0-thalassemia gene. Samples with low MCV or MCH values are assayed using the developed IC strip tests, where only samples testing positive are further assayed for α0-thalassemia by PCR. Patients with Hb H, EA Bart's or EF Bart's diseases do not need to use this IC strip assay. Thus, in this study, a simple and cost effective α0-thalassemia point of care test was developed., Competing Interests: All authors have read the journal’s policy on disclosure of potential conflicts of interest and declare that WK, CT and SF hold the patent application number 20080233659 entitled “Process of screening for alpha-thalassemia carrier using immunochromatographic strip test”. The assignee is the National Science and technology Development Agency (NSTDA), publication date 25 September 2008. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Human CD63 has been reported to play a role either as an inhibitor or as a co-stimulator of T- cell responses, although the mechanism of this is unclear. In this study, an anti-human CD63 monoclonal antibody (mAb) COS3A was used to monitor the role of CD63 in T-cell activation. MAb COS3A could inhibit CD3-mediated T-cell proliferation and CD25 expression in peripheral blood mononuclear cells (PBMCs), used as a study model, but the suppressive effect was not observed when purified T-cells were used instead of PBMCs. The inhibitory phenomenon was associated with downregulation of IL-2 and IFN-γ by T-cells, but upregulation of IL-10 by monocytes. Neutralizing IL-10 with anti-IL-10 mAb improved the T-cell response, indicating the role of IL-10 in T-cell suppression. In this study, monocytes were demonstrated to play a role in impeding T-cell activation by the anti-CD63 mAb COS3A. This is the first evidence that anti-CD63 mAb induces IL-10 secretion by monocytes, which later play a role in T-cell hypo-responsiveness., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Couples who carry α-thalassemia-1 deletion are at 25% risk of having a fetus with hemoglobin Bart's hydrops fetalis. Southeast Asian deletion (--(SEA)) is the most common type of α-thalassemia 1 among Southeast Asian populations. Thus, identification of the (--(SEA)) α-thalassemia 1 carrier is necessary for controlling severe α-thalassemia in Southeast Asian countries., Results: Using our generated anti ζ-globin chain monoclonal antibodies (mAbs) clones PL2 and PL3, a simple immunostick test for detecting ζ-globin chain presence in whole blood lysates was developed. The procedure of the developed immunostick test was as follows. The immunostick paddles were coated with 50 μg/mL of mAb PL2 as capture mAb, or other control antibodies. The coated immunostick was dipped into cocktail containing tested hemolysate at dilution of 1:500, 0.25 μg/mL biotin-labeled mAb PL3 and horseradish peroxidase-conjugated streptavidin at dilution of 1:1000. The immunostick was then dipped in precipitating substrate and the presence of ζ-globin chain in the tested sample was observed by the naked eye. Upon validation of the developed immunostick test with various types of thalassemia and normal subjects, 100% sensitivity and 82% specificity for detection of the (--(SEA)) α-thalassemia-1 carriers were achieved. The mAb pre-coated immunostick can be stored at room temperature for at least 20 weeks., Conclusion: In this study, a novel simple immunostick test for the screening of (--(SEA)) α-thalassemia 1 carriers was presented. The developed immunostick test, within a single test, contains both positive and negative internal procedural controls., Competing Interests: Competing InterestsThe authors declare that they have no competing interest.

Natural killer (NK) cells are innate lymphoid cells having potent cytolytic function that provide host defense against microbial infections and tumors. Using our generated monoclonal antibody (mAb), named FE-1H10, new NK cell sub-populations in peripheral blood were identified. The molecules recognized by mAb FE-1H10 were expressed on a sub-population of CD3-CD56dim NK cells. The epitope recognized by mAb FE-1H10 was demonstrated to be N-glycan and proven to be different from CD57. Upon K562 stimulation, the CD56dimFE-1H10+ NK cell sub-population exhibited significantly lower cytolytic function with low ability to degranulate and release cytolytic granules compared to the CD56dimFE-1H10- NK cell sub-population. Moreover, the CD56dimFE-1H10+ NK cells produced less IFN-γ and TNF-α than the CD56dimFE-1H10- NK cells. We demonstrated here that mAb FE-1H10 could identify two sub-populations of circulating CD56dim NK cells with different functions. Our discovery of new sub-populations of NK cells improves our understanding of NK cell biology and may lead to the development of new approaches for NK cell therapy., (© The Japanese Society for Immunology. 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

CD99 has been reported to be involved in T cell regulation. CD99 ligand involvement in the regulation of T cell activation has been postulated. In this study, recombinant CD99 proteins were produced and used as a tool for determining the role of CD99 and its ligand interaction. Recombinant CD99 proteins induced the upregulation of IL-6 and TNF-α expression, but not IFN-γ, in anti-CD3 monoclonal antibody activated T cells. The cytokine alteration was not observed in unstimulated T cells indicating the cytokine upregulation required the signal from T cell activation. The upregulation of IL-6 and TNF-α was, in addition, observed in CD3- mononuclear cell population including monocytes and NK cells. The recombinant CD99 proteins, however, did not affect either CD25, CD69 or MHC class II expression or T cell proliferation, upon T cell activation. The CD99 ligands were demonstrated to be expressed on monocytes, NK cells and dendritic cells, but not on B and T cells. Our results indicated the presence of CD99 ligands on leukocyte surface. Interaction between CD99 and its ligands involves the regulation of cytokine production., Competing Interests: The authors have declared that no competing interests exist.

CD99, a leukocyte surface glycoprotein, has been implicated in many cellular processes including cell adhesion, cell migration and T cell activation. Our previous study demonstrated the anti-CD99 monoclonal antibody (mAb) clone MT99/3 inhibited T cell activation; however, the mechanism is unclear. In this study, we demonstrated that CD99 expressed on monocytes played a role in the inhibition of T cell activation. Anti-CD99 mAb MT99/3 downregulated the expression of costimulatory molecule CD86, but upregulated IL-6, IL-10 and TNF-α production by monocytes. The inhibitory effect of mAb MT99/3 required cell to cell contact between monocytes and lymphocytes. The soluble mediators produced by monocytes alone were insufficient to induce hypo-function of T lymphocytes. In summary, we demonstrated that ligation of CD99 on monocytes by anti-CD99 mAb MT99/3 could mediate T cell hypo-responsiveness. These findings provide the first evidence of the role of CD99 on monocytes that contributes to T cell activation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mycobacterial infection, leading to pulmonary disease, remains a world health problem. Clinical symptoms of pulmonary disease caused by Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) are very similar. A rapid method for the differentiation of MTBC and NTM infection is essential for appropriate therapy. In this study, we aim to establish an antibody-based biosensor system for the identification of MTBC and NTM infection. Monoclonal antibodies (mAbs) specific for Ag85B proteins of mycobacteria were generated and characterized. The generated anti-Ag85B mAb clones AM85B-5 and AM85B-8 reacted to Ag85B of Mycobacterium spp.; in contrast, clone AM85B-9 specifically reacted to Ag85B of MTBC. By employing the produced mAbs, single and sandwich antibody-based biosensors using bio-layer interferometry were established for determination of Ag85B proteins. The sandwich antibody-based biosensor system was demonstrated to be suitable for detection of Ag85B protein and identification of MTBC and NTM. Using anti-Ag85B mAbs AM85B-8 and AM85B-9 as immobilized antibodies on sensor chips and using mAb AM85B-5 as secondary antibody, the established sandwich antibody-based biosensor could discriminate MTBC and NTM. The developed biosensor system can be used for culture confirmation of mycobacteria and speciation to MTBC and NTM.

Background: Soluble CD147 (sCD147) is the shed form of membrane-bound CD147, which is involved in the regulation of cellular functions. The presence of sCD147 in body fluids is associated with several diseases., Objective: In this study, we aimed to establish antibody (Ab) biosensors for the simultaneous differential detection of the general and truncated forms of sCD147., Method: By combining biolayer interferometry technology (BLItz) and different anti-CD147 monoclonal antibodies (mAbs) specific to different extracellular domains of the CD147 molecule, Ab-based biosensors were established to rapidly measure and characterize sCD147 isoforms., Results: Two types of Ab-biosensors, desginated the single Ab-biosensor and double Ab-biosensor, were established for the measurment and characterization of sCD147 isoforms. For the single Ab-biosensor system, monoclonal antibodies specific for CD147 domain 1 (D1) or domain 2 (D2) were immobilized on the sensor tips and used for the quantification of sCD147 using a BLItz optical interferometric biosensor. For the double Ab-biosensor system, following the single Ab-biosensor step, secondary anti-CD147 mAbs specific for each domain of the CD147 molecule were added and monitored by a BLItz biosensor. By combining the results obtained from the single Ab- and double Ab-biosensors, sCD147 isoforms including the general form (D1 linked to D2) and the truncated forms (sCD147 containing D1 or D2) could be determined., Conclusions: This method may be a beneficial tool for the determination of sCD147 isoforms for disease diagnosis and prognosis as well as for the definition of the cellular mechanisms of the immune system.

T cells play a crucial role in orchestrating body immune responses. T cell hyperfunction, however, leads to inflammation and induction of autoimmune diseases. Understanding of T cell regulation mechanisms and successful modulation of T cell responses is beneficial in treatment of disease associated to T cell hyperresponsiveness. Our previous study indicated that monoclonal antibody (mAb) P-3E10, a mAb to Na, K ATPase β3 subunit, inhibited anti-CD3-induced PBMC proliferation. In the current study, we further investigated the mechanism of mAb P-3E10 in the induction of T cell hypofunction. We demonstrated that mAb P-3E10 decreased T cell proliferation and Th1, Th2 and Th17 cytokine production. Monocytes were the cells playing a key role in mediation of mAb P-3E10 induced T cell hypofunction. The inhibition of T cell activation by mAb P-3E10 required cell contact between monocytes and T cells. The mAb P-3E10 induced the down-expression level of MHC class II and CD86 and increased IL-6, IL-10 and TNF-α production of monocytes. We concluded that ligation of the Na, K ATPase β3 subunit on monocytes by mAb P-3E10 arbitrated T cell hypofunction. This mAb might be a promising novel immunotherapeutic antibody for the treatment of hyperresponsive T cell associated diseases., Competing Interests: The authors have declared that no competing interests exist.

Hemoglobin F (HbF) in blood lysate can be accurately measured by various methods, including immunoassay. In this study, we have produced polyclonal antibody (pAb) against HbF and established a modified sandwich-type ELISA for HbF quantification in blood lysates. The modified sandwich ELISA utilized anti-γ-globin monoclonal antibody clones Thal N/B as the capture antibody (Ab) coated on solid-phase, fluorescein isothiocyanate (FITC)-labeled pAb as the detecting Ab, and HPR-labeled anti-FITC Ab as the signal-generating Ab. By using an optimized blood lysate dilution, the HbF could be measured with no interference from hemoglobin Bart's (Hb Bart's) and hemoglobin Portland (Hb Portland 1) presented in α-thalassemia carriers. HbF levels measured by the modified sandwich ELISA were comparable to those quantified by the standard cation-exchange high performance liquid chromatography. We suggested that this modified sandwich ELISA was able to accurately measure HbF levels even in α-thalassemia carriers containing Hb Bart's and Hb Portland 1 and be an alternative method for HbF measurement.

Overexpression of a disintegrin and metalloproteinase 9 (ADAM9) has been shown in various types of cancer. Some studies have reported inconclusive findings regarding chromosomal aberrations in the ADAM9 -containing region and ADAM9 expression in oral cancer. Therefore, in this study, ADAM9 protein expression was determined and compared between oral squamous cell carcinoma (OSCC) and normal oral tissues, and between oral cancer cell lines and human oral keratinocytes (HOKs). In total, 34 OSCC and 10 healthy paraffin-embedded tissue sections were probed with an anti-ADAM9 antibody, and the immunohistochemical score was determined by multiplying the percentage of positively stained cells with the intensity score. Four different oral cancer and eight independent HOK cell lines were cultured, and the expression of membrane ADAM9 and active ADAM9 at 84 kDa in these cell lines was assayed by flow cytometry and western blot hybridization, respectively. The results showed that the median immunohistochemical score of ADAM9 expression in OSCC tissues was significantly greater than that in normal tissues (P<0.001). Furthermore, among OSCC cases, intense staining of ADAM9 expression was detected in well-differentiated and in moderately-differentiated OSCC; ADAM9 expression was also correlated with an increased degree of cell differentiation ( r =0.557; P=0.001). Expression of membrane ADAM9 was present in 3/4 cancer cell lines. Expression of active ADAM9 varied among all the tested cell lines, but significantly higher ADAM9 expression was present in certain cancer cell lines than those in HOKs (P<0.05). In summary, ADAM9 expression is enhanced in OSCC and oral cancer cell lines, suggesting its role in the pathogenesis of oral cancer. Similar to the overexpression of ADAM9 in well-differentiated prostate cancer, high degrees of ADAM9 expression have also been observed in well-differentiated OSCC.

Background: Human trefoil factor (TFF) peptides consist of three members: TFF1, TFF2 and TFF3. TFF3 is the most abundant TFF peptide in saliva. TFF3 homodimer was suggested to be involved in apoptosis inhibition and malignancy. Determination of TFF3 homodimer expression profiles in saliva may lead to new information about oral biology and diseases. The objective of this study was to generate monoclonal antibodies (mAbs) against TFF3 and apply the produced mAbs for the establishment of ELISA for quantification of dimeric TFF3 in saliva., Results: With our modified hybridoma technique, three hybridoma clones producing anti-TFF3 mAbs having IgG isotype were generated. The mAbs were specific for TFF3 with no cross-reactivity to other TFFs. Using the generated mAbs, a modified-sandwich ELISA with high sensitivity for the quantification of dimeric TFF3 in saliva was developed. Using this ELISA, the amount of dimeric TFF3 in saliva could be measured., Conclusions: A modified-sandwich ELISA for the quantification of TFF3 dimeric form was established. The established ELISA will be a valuable tool for facilitating the investigation of the physiological roles and the diagnostic values of TFF3 in oral diseases. The concept of this modified-sandwich ELISA may be applied for the determination of other homodimeric peptides of interest.

Enamel-renal-gingival syndrome (ERGS; OMIM #204690), a rare autosomal recessive disorder caused by mutations in FAM20A, is characterized by nephrocalcinosis, nephrolithiasis, amelogenesis imperfecta, hypoplastic type, gingival fibromatosis and other dental abnormalities, including hypodontia and unerupted teeth with large dental follicles. We report three patients and their families with findings suggestive of ERGS. Mutation analysis of FAM20A was performed in all patients and their family members. Patients with homozygous frameshift and compound heterozygous mutations in FAM20A had typical clinical findings along with periodontitis. The other had a novel homozygous missense mutation in exon 10, mild gingival fibromatosis and renal calcifications. The periodontitis in our patients may be a syndrome component, and similar findings in previous reports suggest more than coincidence. Fam20a is an allosteric activator that increases Fam20c kinase activity. It is hypothesized that lack of FAM20A activation of FAM20C in our patients with FAM20A mutations might have caused amelogenesis imperfecta, abnormal bone remodeling and periodontitis. Nephrocalcinosis appears not to be a consistent finding of the syndrome and the missense mutation may correlate with mild gingival fibromatosis. Here we report three patients with homozygous or compound heterozygous mutations in FAM20A and findings that extend the phenotypic spectrum of this disorder, showing that protein truncation is associated with greater clinical severity.

In this study, we describe the application of a molecular biology technique for the production of mouse polyclonal antibodies (pAbs) specific to human cell surface molecules. Production of the pAb specific to the human CD99 surface molecule was used as the study model. The retroviral expression system was employed to generate human CD99 expressing mouse myeloma cells. After cell sorting and single cell cloning, a myeloma clone which stably expressed high levels of human CD99 on its surface was established. The human CD99 expressing mouse myeloma cells were then used as the immunogen for immunization of BALB/c mice. As endogenous proteins of mouse myeloma cells possess self-non-immunogenicity for BALB/c mice, after immunization, only the expressed human CD99 molecules induce antibody response. After three immunizations, high titers of mouse anti-CD99 pAbs were successfully produced. The produced pAb specifically reacted to both recombinant human CD99 and native CD99 molecules expressed on human blood cells. The established technology is simple and valuable for the production of pAbs specific to human CD99 membrane proteins which can be used for characterization of the CD99 molecule., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background and Objectives: The expression of A Disintegrin and Metalloproteinase 8 (ADAM8) is associated with several inflammatory diseases. Elevated ADAM8 levels have been shown in gingival crevicular fluid of patients with chronic periodontitis. The objective of this study was to investigate ADAM8 expression in chronic periodontitis tissues compared with that in normal tissues. ADAM8 expression and its inductive mechanism were examined in human gingival epithelial cells (HGECs) and human gingival fibroblasts., Material and Methods: Total RNA and protein were extracted from gingival biopsies of 33 patients with chronic periodontitis and those of 23 healthy volunteers. ADAM8 mRNA and protein expression was analyzed by real-time polymerase chain reaction, immunoblotting and immunohistochemistry. ADAM8 expression in control and stimulated cells in the presence or absence of specific inhibitors for mitogen-activated protein kinase pathways was assayed by real-time polymerase chain reaction, immunoblotting, flow cytometry and immunofluorescence., Results: ADAM8 mRNA and protein expression in chronic periodontitis tissues was significantly greater than that in normal tissues (p < 0.01). Significantly increased ADAM8 expression was detected in the gingival epithelium of chronic periodontitis tissues (p < 0.001). ADAM8 mRNA expression in HGECs, but not in human gingival fibroblasts, was significantly induced by stimulation with Fusobacterium nucleatum (p < 0.05), partially via the p44/42 mitogen-activated protein kinase pathway. ADAM8 expression in the cell lysates and on the surface of HGECs was induced by stimulation with F. nucleatum., Conclusion: ADAM8 expression is increased in inflamed chronic periodontitis tissues and localized within gingival epithelium, consistent with an upregulation of ADAM8 expression in F. nucleatum-stimulated HGECs, suggesting a possible role of ADAM8 in innate immunity of periodontal tissue., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To quantitatively measure the increased expression of Akt2 and its phosphorylated form (p-Akt) in oral cancer cell lines and investigate the post-translational mechanism for Akt2 and p-Akt overexpression., Methods: Three oral cancer cell lines and three cell lines of primary human oral keratinocytes (HOKs) were cultured and the degrees of Akt2 and p-Akt expression was evaluated by immunoblot analysis and flow cytometry. Each cell line was incubated with cycloheximide, an inhibitor of new protein synthesis, for various times to quantitatively determine the remaining expression levels of Akt2 and p-Akt by flow cytometry. The localization of Akt2 and p-Akt was assessed by immunofluorescence., Results: The levels of Akt2 and p-Akt proteins were significantly higher in cancer cell lines than those in HOKs (P < 0.05). When the new protein synthesis was blocked by cycloheximide treatment, the degradation rate of Akt2 and p-Akt in oral cancer cells was significantly lower than that in HOKs (P < 0.05). Both Akt2 and p-Akt were more intensely stained in the cytoplasm of cancer cells, whereas HOKs expressed Akt2 and p-Akt only minimally., Conclusion: Both Akt2 and p-Akt were overexpressed in oral cancer cells, which may be partly explained by a reduced rate of protein degradation in order to maintain high cytosolic levels., (© 2015 Wiley Publishing Asia Pty Ltd.)

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) accelerates tumor invasion and metastasis via activation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) expression. The authors were interested in whether the scFv-M6-1B9 intrabody against EMMPRIN that retains EMMPRIN in endoplasmic reticulum could be a potential tool to suppress cervical cancer invasion through inhibition of uPA. The chimeric adenoviral vector Ad5/F35-scFv-M6-1B9 was transferred into human cervical carcinoma HeLa cells to produce the scFv-M6-1B9 intrabody against EMMPRIN. Cell surface expression of EMMPRIN, the membrane-bound uPA, the enzymatic activity of secreted uPA, and the invasion ability were analyzed. The scFv-M6-1B9 intrabody successfully diminished the cell surface expression of EMMPRIN and the membrane-bound uPA on HeLa cells. uPA activity from tissue culture media of EMMPRIN-downregulated HeLa cells was decreased. The invasion ability of HeLa cells harboring scFv-M6-1B9 intrabody was also suppressed. These results suggested that the scFv-M6-1B9 intrabody might represent a potential approach for invasive cervical cancer treatment. The application of scFv-M6-1B9 intrabody in animal experiments and preclinical studies would be investigated further.

Background: Monoclonal antibodies (mAbs) have become essential tools in life science research and in medicine, because of their extreme specificity. Several mAbs against leukocyte surface molecules have been generated in our laboratory. From these, mAb COS3A was selected for biochemical and functional analysis., Objective: To analyze the properties and function of the mAb COS3A., Method: Cellular distribution was analyzed by immunofluorescence staining and flow cytometry. Biochemical characterization of the molecular target of COS3A was approached by immunoprecipitation, Western blotting and amino acid sequencing using LC-MS. N-glycosidase F treatment of COS3A-precipitated protein and culture of U937 cells in the presence of tunicamycin before cell lysate preparation were used to study the glycosylation state of proteins. Phagocytosis was examined by flow cytometry., Results: MAb COS3A bound specifically to a molecule expressed on the surface of various human hematopoietic cells and cell lines but not on erythrocytes. The antigen had a molecular weight of 30-70 kDa, which was reduced to 25 kDa by elimination of N-linked glycan. LC-MS data and immunoprecipitation indicated that mAb COS3A bound specifically to the CD63 molecule. Remarkably, functional analysis demonstrated that mAb COS3A dramatically reduced granulocyte phagocytosis., Conclusions: The mAb COS3A recognized the CD63 molecule and strongly diminished granulocyte phagocytosis of E. coli, suggesting that CD63 may play a crucial role in the initial step of phagocytosis. MAb COS3A is, therefore, suitable for both biochemical and functional studies of CD63, and may be used for further study of the mechanism of phagocytosis and also in therapeutic approaches.

A silicon nitride Ion Sensitive Field Effect Transistor (ISFET) based immunosensor was developed as a low-cost and label-free electrical detection for the detection of antigen 85 complex B (Ag85B). The sensing membrane of the ISFET was modified with 3-aminopropyltriethoxysilane (APTES) followed by glutaraldehyde (GA), yielding an aldehyde-terminated surface. This group is available for immobilization of a monoclonal antibody against a recombinant Ag85B protein (anti-Ag85B antibody). The optimal concentration for anti-Ag85B antibody immobilization onto the modified ISFET was 100 μg ml -1 . This optimal condition provided the maximal binding capability and minimal non-specific background signal. The binding event between the recombinant Ag85B antigen and anti-Ag85B antibody on the ISFET surface is presented by monitoring the gate potential change at a constant drain current. The dose response for the recombinant Ag85B protein showed a linear response between 0.12 and 1 μg ml -1 without significant interference from other recombinant proteins. The analytical imprecision (CV%) and accuracy of this Ag85B protein biosensor were 9.73-10.99% and 95.29%, respectively. In addition, an irrelevant antibody and other recombinant proteins were employed as a negative control to demonstrate the non-specific interaction of the antigen and antibody. The success of this immunosensor system for Ag85B protein detection facilitates the construction of a promising device which can shorten the turnaround time for the diagnosis of tuberculosis compared to a standard culture method. Furthermore, this device could also be applied for real-time growth monitoring of Mycobacterium tuberculosis in a mycobacterial culture system.

The relationship between P-gp and CD147 in the regulation of MDR in leukemic cells has not been reported. This study aimed to investigate the correlation between CD147 and P-gp in the regulation of drug resistance in the K562/ADR leukemic cell line. The results showed that drug-resistant K562/ADR cells expressed significantly higher P-gp and CD147 levels than drug-free K562/ADR cells. To determine the regulatory effect of CD147 on P-gp expression, anti-CD147 antibody MEM-M6/6 significantly decreased P-gp and CD147 mRNA and protein levels. This is the first report to show that CD147 mediates MDR in leukemia through the regulation of P-gp expression.

Aims: To exploit immunomagnetic separation combined with PCR with confronting two-pair primers (IMS-PCR-CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens., Methods and Results: Monoclonal antibody (mAb) against the mycobacterial antigen, 85B (Ag85B), was coupled with magnetic particles for specific immunomagnetic separation (IMS) of Mycobacterium spp. Immunofluorescence assay indicated the capability of mAb to bind to Ag85B in both the recombinant and the native form. The IMS combined with PCR-CTPP targeting the mycobacterial lep B gene was further implemented using 133 sputum samples with acid-fast bacilli grading from negative to 3+. The results showed the sensitivity and specificity of IMS-PCR-CTPP vs gold standard culture method were 89·9 and 88·6% respectively., Conclusions: The IMS-PCR-CTPP method shortens the time for tuberculosis (TB) diagnosis from months to a day. This method is also suitable for investigation of MTC and epidemiological study of Myco. bovis in sputum specimens., Significance and Impact of the Study: This study is the first report emphasizing the combination of IMS and PCR-CTPP for the detection of MTC and simultaneous identification of Myco. bovis from sputum. It could be used for TB diagnosis in resource-limited countries with high TB burden., (© 2016 The Society for Applied Microbiology.)

Autoantibodies against interferon-gamma (IFN-γ) can cause immunodeficiency and are associated with various opportunistic infections. In the present study, we investigated other cellular immune parameters for a better understanding of the immunodeficiency condition in the patients. The numbers of WBC, monocytes and NK cells were increased in patients with anti-IFN-γ autoantibodies (AAbs). Upon TCR activation, T cell proliferation and IL-2 receptor of the patients remained intact. Nonetheless, the Th1 cytokine (IFN-γ and TNF-α) production was up-regulated. The production of Th2 (IL-4) and Th17 (IL-17) cytokines was unchanged. We suggest that, in addition to the presence of anti-IFN-γ autoantibodies, alterations in the cellular immune functions may also contribute to this immunodeficiency.

Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A2, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart's hydrops fetalis in which no α-globin chain is produced.

The current available assays cannot differentiate the stages of phagocytosis. We, therefore, established methods for concurrent detection of antigen attachment and engulfment by phagocyte using latex beads coated with lipopolysaccharide, rabbit IgG, and carboxyfluorescein diacetate succinimidyl ester. The generated beads were incubated with whole blood at 37°C for 1 hr and stained with PE-Cy5.5 anti-rabbit IgG antibody. By flow cytometry, attachment and phagocytic processes could be detected, simultaneously. The established method is a valuable tool for diagnosis of phagocytic disorder and study of molecules involved in phagocytosis.

This study was carried out to understand the effect of the recombinant multivalent extracellular matrix metalloproteinase inducer (EMMPRIN) extracellular domain, designated as rmEMMPRINex, on the apoptotic cell death of human leukemia U937 cells. Expression of monocarboxylate transporter 1 (MCT1) and caspase-9 in U937 treated with rmEMMPRINex was investigated in this study. Levels of membrane MCT1 and intracellular procaspase-9 were decreased in rmEMMPRINex-treated cells in comparison to controls. However, the expression of activated caspase-9 was undetectable. rmEMMPRINex also induced DNA fragmentation and apoptosis in U937 cells. Taken together, we concluded that interaction of rmEMMPRINex with U937 cells leads to inhibition of MCT1 membrane expression, intracellular activation of procaspase-9, followed by DNA fragmentation and apoptosis. This may contribute to the conceptual development of novel cancer drugs in the future.

Background: Several molecules are known to be involved in T-cell activation via the TCR/CD3 complex and while the mechanisms of late T cell signaling have been well characterized, the very early events are still not fully understood., Objective: The aim of this study was to identify yet unknown molecules associated with the TCR/CD3 complex., Results: To identify new molecules associated with the TCR/CD3 complex, a monoclonal antibody termed MT3 was produced by immunoprecipitated beads immunization. Colocalization of the MT3 mAb recognizing molecules with the TCR/CD3 complexes was verified by confocal microscopic analysis. The surface antigen recognized by MT3 antibody was expressed on a subpopulation of CD3+ T cells, and on both CD4+ and CD8+ lymphocytes. The antigen was also expressed on na?ve CD4+ T cells and on a subset of memory CD4+ T cells. In contrast, in the CD8 population, the majority of MT3+ cells were found in the na?ve population. The MT3 mAb recognizing molecules were also expressed on red blood cells but only in particular subjects. Similar to peripheral blood leukocytes, MT3 mAb recognizing molecules are exclusively expressed on T cell lines., Conclusions: Based on the cellular distribution patterns and confocal microscopic analysis, the MT3 mAb recognizing molecule that we investigated is proposed to be a TCR/CD3 associated molecule and might be involved in the antigen recognition of T cells.

Na,K ATPase plays an important role in the regulation of Na(+) and K(+) ions that are required for normal resting membrane potential and various cellular functions. Na,K ATPase is composed of two subunits, α and β subunits. Engagement of the β subunit by an agonistic monoclonal antibody (mAb) P-3E10 inhibited T cell activation and induced the G0/G1 cell cycle arrest. In addition, mAb P-3E10 decreased CD25 expression. The mAb P-3E10, however, did not inhibit the proliferation of cell lines and the phagocytosis activity of phagocytes, and did not interfere with the Na,K ATPase activity. These results indicate that mAb P-3E10 reacts to the β subunit and, as a consequence, brings about the regulation of the T cell activation without disturbing the Na,K pump activity. By sequential immunoprecipitation, we demonstrated the expression of the β3 subunit free form apart from the α subunit. In this study, we propose that the β3 subunits of Na,K ATPase are expressed separately from the α subunit, and play a role in regulation of the immune response., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Couples in which both partners carry the α-thalassemia-1 trait have a 25% risk of hemoglobin Bart's hydrops fetalis in each pregnancy. Identification of α-thalassemia-1 trait is, therefore, necessary in order to control this severe form of α-thalassemia. We have generated monoclonal antibodies specific to the ζ-globin chain without cross reaction with other globin chains. A simple and sensitive ELISA was developed by using poly-l-lysine to increase the protein binding to the ELISA plate. The developed poly-l-lysine pre-coated ELISA has a very high sensitivity (100%) and specificity (97%) for detection of carriers of α-thalassemia-1 with Southeast Asian-type deletion.