Transcriptome Analysis Reveals the Induction of Apoptosis-Related Genes by a Monoclonal Antibody against a New Epitope of CD99 on T-Acute Lymphoblastic Leukemia.

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.

Neutralizing antibody and T-cell responses against SARS-CoV-2 variants by heterologous CoronaVac/ChAdOx-1 vaccination in elderly subjects with chronic obstructive pulmonary disease.

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).

The ligation of CD4 molecules, expressed on monocytes by an anti-CD4 monoclonal antibody, inhibits T cell activation and monocyte mobility.

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.

Levels and durability of neutralizing antibodies against SARS-CoV-2 Omicron and other variants after ChAdOx-1 or BNT162b2 booster in CoronaVac-primed elderly individuals.

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.

Cannabinoid Receptor 1 Agonist ACEA and Cannabinoid Receptor 2 Agonist GW833972A Attenuates Cell-Mediated Immunity by Different Biological Mechanisms.

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.

Humoral and cellular immune responses against SARS-CoV-2 variants of concern induced by heterologous CoronaVac/ChAdOx-1 versus homologous ChAdOx-1 vaccination in the elderly.

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.

Chimeric single-chain variable fragment-human immunoglobulin G crystallizable fragment antibody against GD2 for neuroblastoma targeted immunotherapy.

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.

Neutralizing Antibody and T-Cell Responses against SARS-CoV-2 Wild-Type and Variants of Concern in Chronic Obstructive Pulmonary Disease Subjects after ChAdOx-1/ChAdOx-1 Homologous Vaccination: A Preliminary Study.

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.

Development of IgY-Based Indirect Competitive ELISA for the Detection of Fluoroquinolone Residues in Chicken and Pork Samples.

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 (IC50) 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.

Optimization of culture conditions for stable expression of recombinant fc-fused human extracellular CD99 in HEK293T cells.

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.

Correlation Analysis of Anti-SARS-CoV-2 RBD IgG and Neutralizing Antibody against SARS-CoV-2 Omicron Variants after Vaccination.

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.

Neutralizing antibody and T cell responses against SARS-CoV-2 variants of concern following ChAdOx-1 or BNT162b2 boosting in the elderly previously immunized with CoronaVac vaccine.

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.

An IgM monoclonal antibody against domain 1 of CD147 induces non-canonical RIPK-independent necroptosis in a cell type specific manner in hepatocellular carcinoma cells.

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.

Differential CD147 Functional Epitopes on Distinct Leukocyte Subsets.

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.

Differentiation and activation of human CD4 T cells is associated with a gradual loss of myelin and lymphocyte protein.

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.

The presence of membrane bound CD99 ligands on leukocyte surface.

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.

Impact of the detection of ζ-globin chains and hemoglobin Bart's using immunochromatographic strip tests for α0-thalassemia (--SEA) differential diagnosis.

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.

Anti-human CD63 monoclonal antibody COS3A upregulates monocyte-induced IL-10 excretion leading to diminution of CD3-mediated T cell response.

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.

Immunostick Test for Detecting ζ-Globin Chains and Screening of the Southeast Asian α-Thalassemia 1 Deletion.

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.

Sandwich antibody-based biosensor system for identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria.

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.