Engineered CD147-Deficient THP-1 Impairs Monocytic Myeloid-Derived Suppressor Cell Differentiation but Maintains Antibody-Dependent Cellular Phagocytosis Function for Jurkat T-ALL Cells with Humanized Anti-CD147 Antibody.

CD147 is upregulated in cancers, including aggressive T-ALL. Traditional treatments for T-ALL often entail severe side effects and the risk of relapse, highlighting the need for more efficacious therapies. ADCP contributes to the antitumor response by enhancing the ability of phagocytic cells to engulf cancer cells upon antibody binding. We aimed to engineer CD147KO THP-1 cells and evaluated their differentiation properties compared to the wild type. A humanized anti-CD147 antibody, HuM6-1B9, was also constructed for investing the phagocytic function of CD147KO THP-1 cells mediated by HuM6-1B9 in the phagocytosis of Jurkat T cells. The CD147KO THP-1 was generated by CRISPR/Cas9 and maintained polarization profiles. HuM6-1B9 was produced in CHO-K1 cells and effectively bound to CD147 with high binding affinity (KD: 2.05 ± 0.30 × 10-9 M). Additionally, HuM6-1B9 enhanced the phagocytosis of Jurkat T cells by CD147KO THP-1-derived LPS-activated macrophages (M-LPS), without self-ADCP. The formation of THP-1-derived mMDSC was limited in CD147KO THP-1 cells, highlighting the significant impact of CD147 deletion. Maintaining expression markers and phagocytic function in CD147KO THP-1 macrophages supports future engineering and the application of induced pluripotent stem cell-derived macrophages. The combination of HuM6-1B9 and CD147KO monocyte-derived macrophages holds promise as an alternative strategy for T-ALL.

Dimeric Ankyrin with Inverted Module Promotes Bifunctional Property in Capturing Capsid to Impede HIV-1 Replication.

Several anti-HIV scaffolds have been proposed as complementary treatments to highly active antiretroviral therapy. AnkGAG1D4, a designed ankyrin repeat protein, formerly demonstrated anti-HIV-1 replication by interfering with HIV-1 Gag polymerization. However, the improvement of the effectiveness was considered. Recently, the dimeric molecules of AnkGAG1D4 were accomplished in enhancing the binding activity against HIV-1 capsid (CAp24). In this study, the interaction of CAp24 against the dimer conformations was elucidated to elaborate the bifunctional property. The accessibility of the ankyrin binding domains was inspected by bio-layer interferometry. By inverting the second module of dimeric ankyrin (AnkGAG1D4NC-CN), the CAp24 interaction KD was significantly reduced. This reflects the capability of AnkGAG1D4NC-CN in simultaneously capturing CAp24. On the contrary, the binding activity of dimeric AnkGAG1D4NC-NC was indistinguishable from the monomeric AnkGAG1D4. The bifunctional property of AnkGAG1D4NC-CN was subsequently confirmed in the secondary reaction with additional p17p24. This data correlates with the MD simulation, which suggested the flexibility of the AnkGAG1D4NC-CN structure. The CAp24 capturing capacity was influenced by the distance of the AnkGAG1D4 binding domains to introduce the avidity mode of AnkGAG1D4NC-CN. Consequently, AnkGAG1D4NC-CN showed superior potency in interfering with HIV-1 NL4-3 WT and HIV-1 NL4-3 MIRCAI201V replication than AnkGAG1D4NC-NC and an affinity improved AnkGAG1D4-S45Y.

Development of an immunochromatographic strip test for rapid detection of a potent neutralizing anti-interferon gamma antibody in adult-onset immunodeficiency.

BACKGROUND: Adult-onset immunodeficiency (AOID) is associated with the presence of anti-interferon gamma autoantibodies (anti-IFN-γ autoAbs), which neutralizes IFN-γ signaling and leads to susceptibility to intracellular opportunistic infections. However, measuring neutralizing autoAbs is not practical for general clinical laboratories. OBJECTIVE: This study aims to develop a competitive immunochromatographic (IC) strip test for detecting autoAbs that recognize the same epitope as a potent neutralizing antibody. METHODS: The competitive IC strip test detecting autoAbs that recognize the same epitope as mouse anti-IFN-γ monoclonal antibody, clone B27 (B27 mAb) was fabricated and used to determine B27 epitope recognizing autoAb (B27 AAb) in AOID plasma. The competitive ELISA was used as a comparative method. AR patients. RESULTS: The efficacy of the IC strip test was compared with competitive ELISA and found a percent positive agreement of 91.30%, a percent negative agreement of 79.31%, and a percent overall agreement of 84.62%. CONCLUSIONS: The results from the competitive IC strip test were consistent with those from competitive ELISA, indicating that the generated IC strip could detect B27 AAb in AOID plasma.

Updating on Roles of HIV Intrinsic Factors: A Review of Their Antiviral Mechanisms and Emerging Functions.

BACKGROUND: Host restriction factors are cellular proteins that inhibit specific steps of the viral life cycle. Since the 1970s, several new factors have been identified, including human immunodeficiency virus-1 (HIV-1) replication restriction. Evidence accumulated in the last decade has substantially broadened our understanding of the molecular mechanisms utilized to abrogate the HIV-1 life cycle. SUMMARY: In this review, we focus on the interaction between host restriction factors participating in the early phase of HIV-1 infection, particularly CA-targeting proteins. Host factors involved in the late phase of the replication cycle, such as viral assembly and egress factors, are also described. Additionally, current reports on well-known antiviral intrinsic factors, as well as other viral restriction factors with their emerging roles, are included. CONCLUSION: A comprehensive understanding of the interactions between viruses and hosts is expected to provide insight into the design of novel HIV-1 therapeutic interventions.

Biological properties of reverse ankyrin engineered for dimer construction to enhance HIV-1 capsid interaction.

BACKGROUND: Assembly and budding in the late-stage of human immunodeficiency virus type 1 (HIV-1) production rely on Gag protein polymerization at the inner leaflet of the plasma membrane. We previously generated a monomeric ankyrin repeat protein (Ank1D4) that specifically interacts with capsid protein (CAp24) of HIV-1, however this protein had modest binding affinity. OBJECTIVE: This study aimed to improve the avidity of Ank1D4 by generating two Ank1D4 dimers: (Ank1D4NC-NC) and its inverted form (Ank1D4NC-CN), with each domain connected by a flexible (G4S)4 linker peptide. METHODS: Binding properties of monomeric and dimeric Ank1D4 was performed by capture enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA was used to examine bifunctional module of dimeric Ank1D4. Ank1D4NC-NC and Ank1D4NC-CN were evaluated using bio-layer interferometry (BLI), compared to monomeric Ank1D4. RESULTS: Similar binding surfaces were observed in both dimers which was comparable with monomeric Ank1D4. The interaction of Ank1D4NC-CN with CAp24 was significantly greater than that of Ank1D4NC-NC and Ank1D4 by capture ELISA. Ank1D4NC-CN also exhibited bifunctionality using a sandwich ELISA. The KD of Ank1D4NC-CN, Ank1D4NC-NC and monomeric Ank1D4 was 3.5 nM, 53.7 nM, and 126.2 nM, respectively using bio-layer interferometry analysis. CONCLUSIONS: This study provides a strategy for increasing Ank1D4 avidity through the construction of novel inverted dimers with a flexible linker. Ank1D4NC-CN may provide an alternative treatment strategy for inhibiting HIV-1 replication.

Suppression of µ1 subunit of the adaptor protein complex 2 reduces dengue virus release.

Dengue virus (DENV) requires clathrin-mediated endocytosis for its entry into the cells where the adaptor protein complex (AP) is vital for the clathrin-coated vesicle formation. The role of AP-2 was previously examined in the early stages of DENV infection; however, the role of AP-2 in the late stage of DENV infection was not determined. The µ1 subunit of AP-2 (AP2M1) is one of the most important cytoplasmic carrier domains in clathrin-mediated endocytosis and the phosphorylation of this subunit by the kinase enzyme, AP-2 associated protein kinase 1 (AAK1), stimulates clathrin and supports the cell surface receptor incorporation. In the present study, we primarily aimed to investigate the role of AP2M1 by gene silencing approach as well as using naked DENV RNA transfection into AP2M1 knockdown cells. Secondarily, an inhibitor of AAK1, sunitinib was used to investigate whether AAK1 could influence the virus production in DENV-infected Huh7 cells. The knockdown of AP2M1 in the DENV-infected Huh7 cells displayed a reduction in the viral titer at 24 h post-infection. Furthermore, experiments were conducted to bypass the DENV internalization using a naked DENV RNA transfection into the AP2M1 knockdown cells. Higher intracellular DENV RNA, DENV E protein, and intracellular virion were observed, whereas the extracellular virion production was comparably less than that of control. Treatment with sunitinib in DENV-infected Huh7 cells was able to reduce extracellular virion production and was consistent with all four serotypes of DENV. Therefore, our findings demonstrate the role of AP2M1 in the exocytosis step of DENV replication leading to infectious DENV production and the efficacy of sunitinib in suppressing virus production during the infection with different serotypes of DENV.

Impairment of a membrane-targeting protein translated from a downstream gene of a "self-cleaving" T2A peptide conjunction.

The requirement for reliable bicistronic or multicistronic vectors in gene delivery systems is at the forefront of bio/biomedical technology. A method that provides an efficient co-expression of multiple heterologous proteins would be valuable for many applications, especially in medical science for treating various types of disease. In this study, we designed and constructed a bicistronic expression vector using a self-cleaving 2A peptide derived from a virus of the insect Thosea asigna (T2A). This exhibited the most efficient cleavage of the 2A sequence. Two versions of the T2A-based vector were constructed by switching the DNA sequences encoding the proteins of interest, the N-myristoylated protein and the nuclear-homing protein, upstream and downstream of the 2A linker, respectively. Our results showed that similar levels of mRNA expression were found and 100% of cleavage efficiency of T2A was observed. Nevertheless, we also reported the cleared evidence that the N-myristoylated protein cannot be placed downstream of the 2A sequence. Since the protein product fails to translocate to the plasma membrane due to altered myristoylation process, the gene position of the T2A-based vector is meaningful for the subcellular localization of the N-myristoylated protein. Therefore, the observation was marked as a precaution for using the 2A peptide. To adopt the 2A peptide technology for generating the bicistronic or multicistronic expression, the vector design should be carefully considered for the transgene position, signal sequences, and post-translational modifications of each individual protein.

Integration of Image Pattern Recognition and Photon Sensor for Analyzing Cytokine Gene Expression Using πCode MicroDisc.

Current quantitative gene expression detection in genomic and transcriptomic research heavily relies on quantitative real-time PCR (qPCR). While existing multiplex gene detection techniques offer simultaneous analysis of multiple targets, we present an alternative assay capable of detecting gene expression simultaneously within a single well. This highly sensitive method utilizes πCode MicroDiscs, featuring unique identification patterns and fluorescent detection. Our study compared this multiplex πCode platform with a qPCR platform for profiling cytokine gene expression. The πCode MicroDisc assay successfully demonstrated the expression of polymerization markers for M1- and M2-like macrophages generated from THP-1-derived macrophages in a qualitative assay. Additionally, our findings suggest a pattern agreement between the πCode assay and the qPCR assay, indicating the potential of the πCode technology for comparative gene expression analysis. Regarding the inherent sensitivity and linearity, the developed πCode assay primarily provides qualitative gene expression to discriminate the polarization of macrophages. This remarkable capability presents substantial advantages for researchers, rendering the technology highly suitable for high-throughput applications in clinical diagnosis and disease monitoring.

Evaluating the ability of different chaperones in improving soluble expression of a triple-mutated human interferon gamma in Escherichia coli.

Human interferon gamma (hIFN-γ) plays a pivotal role as a soluble cytokine with diverse functions in both innate and adaptive immunity. In a previous investigation, we pinpointed three critical amino acid residues, i.e., threonine (T) 27, phenylalanine (F) 29, and leucine (L) 30, on the IFN-γ structure, which are integral to the epitope recognized by anti-IFN-γ autoantibodies. It is crucial to impede the interaction between this epitope and autoantibodies for effective therapy in adult-onset immunodeficiency (AOID). However, the challenge arises from the diminished solubility of the T27AF29L30A mutant in Escherichia coli BL21(DE3). This study delves into a targeted strategy aimed at improving the soluble expression of IFN-γ T27AF29AL30A. This is achieved through the utilization of five chaperone plasmids: pG-KJE8, pKJE7, pGro7, pG-Tf2, and pTf16. These plasmids, encoding cytoplasmic chaperones, are co-expressed with the IFN-γ mutant in E. coli BL21(DE3), and we meticulously analyze the proteins in cell lysate and inclusion bodies using SDS-PAGE and Western blotting. Our findings reveal the remarkable efficacy of pG-KJE8, which houses cytoplasmic chaperones DnaK-DnaJ-GrpE and GroEL-GroES, in significantly enhancing the solubility of IFN-γ T27AF29AL30A. Importantly, this co-expression not only addresses solubility concerns but also preserves the functional dimerized structure, as confirmed by sandwich ELISA. This promising outcome signifies a significant step forward in developing biologic strategies for AOID.

Role of cathepsin B in dengue virus-mediated apoptosis.

Dengue virus (DENV) infection is one of the most important mosquito-borne viral diseases, which is endemic in the tropical and sub-tropical regions. Patients with dengue hemorrhagic fever (DHF) generally present hemorrhagic tendencies, plasma leakage, thrombocytopenia, and hemoconcentration. Hepatic dysfunction is also a crucial feature of DENV infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show cellular apoptosis, which apparently relate to the pathogenesis. Cathepsins, which are cysteine proteases inside the lysosome, were previously reported to be up-regulated in patients with DHF. However, their functions during DENV infection have not been thoroughly investigated. We show for the first time that DENV induces lysosomal membrane permeabilization. The resulting cytosolic cathepsin B and S contributed to apoptosis via caspase activation. The activity of caspase 3 was significantly reduced in DENV-infected HepG2 cells treatedwith cathepsin B or S inhibitors. Treatment with cathepsin B inhibitor also reduced the activity of caspase 9, suggesting that cathepsin B activates both caspase-9 and caspase-3. Reduced cathepsin B expression, effected by RNA interference, mimicked pharmacological inhibition of the enzyme and confirmed the contribution of cathepsin B to apoptotic events induced by DENV in HepG2 cells.

Compound A, a dissociated glucocorticoid receptor modulator, reduces dengue virus-induced cytokine secretion and dengue virus production.

Dengue Virus (DENV) infection is an important mosquito-borne viral disease and its clinical symptoms range from a predominantly febrile disease, dengue fever (DF), to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Increased levels of cytokines - the so-called 'cytokine storm', contribute to the pathogenesis of DHF/DSS. In this study, we compared the expression of cytokine genes between mock-infected and DENV-infected HepG2 cells using a real-time PCR array and revealed several up-regulated chemokines and cytokines, including CXCL10 and TNF-α. Compound A (CpdA), a plant-derived phenyl aziridine precursor containing anti-inflammatory action and acting as a dissociated nonsteroidal glucocorticoid receptor modulator, was selected as a candidate agent to modulate secretion of DENV-induced cytokines. CpdA is not a glucocorticoid but has an anti-inflammatory effect with no metabolic side effects as steroidal ligands. CpdA significantly reduced DENV-induced CXCL10 and TNF-α secretion and decreased leukocyte migration indicating for the first time the therapeutic potential of CpdA in decreasing massive immune activation during DENV infection.

Interaction of dengue virus nonstructural protein 5 with Daxx modulates RANTES production.

Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called 'cytokine storm', is observed in the patients with DHF/DSS. A complex interaction between DENV proteins and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production.

Determination of a distinguished interferon gamma epitope recognized by monoclonal antibody relating to autoantibody associated immunodeficiency.

Anti-interferon gamma autoantibodies (anti-IFN-γ autoAbs) neutralize the IFN-γ-mediated functions, contributing to immunodeficiency. A particular autoAb in patient serum had been previously demonstrated to recognize the same determinant on IFN-γ as the neutralizing anti-IFN-γ monoclonal antibody clone B27 (B27 mAb). This study explored the epitope recognized by B27 mAb. The specific peptide sequence recognized by B27 mAb, TDFLRMMLQEER, was retrieved from a phage display random peptide library. Sequence alignment and homology modeling demonstrated that the queried phage peptide sequence and structure were similar to amino acids at position 27-40 (TLFLGILKNWKEES) of the human IFN-γ. This determinant resides in the contact surface of IFN-γ and interferon gamma receptor 1. To elucidate the crucial amino acids, mutations were introduced by substituting T27 and T27F29L30 with alanine or deleting the amino acid residues T27-L33. The binding of B27 mAb to IFN-γ T27A using western blotting was lesser than that to wild-type. The interaction with triple mutant and T27-L33 deletion mutant using western blotting and sandwich ELISA was abolished. The finding demonstrated that T27, F29, and L30 are critical residues in the B27 antigenic determinant. Identification of the functional domain of IFN-γ decrypted the relevance of neutralizing autoAb in adult-onset immunodeficiency.

Immunoreactivity of humanized single-chain variable fragment against its functional epitope on domain 1 of CD147.

Domain 1 of CD147 participates in matrix metalloproteinase (MMP) production and is a candidate for targeted therapy to prevent cancer invasion and metastasis. A functional mouse anti-CD147 monoclonal antibody, M6-1B9, was found to recognize domain 1 of CD147, and its respective mouse single-chain variable fragment (ScFvM61B9) was subsequently generated. The EDLGS epitope candidate for M6-1B9 was identified using the phage display peptide technique in this study. For future clinical applications, humanized ScFv specific to domain 1 of CD147 (HuScFvM61B9) was partially adopted from the hypervariable sequences of parental mouse ScFvM61B9 and grafted onto suitable human immunoglobulin frameworks. Molecular modelling and simulation were performed in silico to generate the conformational structure of HuScFvM61B9. These results elucidated the amino acid residues that contributed to the interactions between CDRs and the epitope motif. The expressed HuScFvM61B9 specifically interacted with CD147 at the same epitope as the original mAb, M6-1B9, and retained immunoreactivity against CD147 in SupT1 cells. The reactivity of HuScFvM61B9 was confirmed using CD147 knockout Jurkat cells. In addition, the inhibitory effect of HuScFvM61B9 on OKT3-induced T-cell proliferation as M6-1B9 mAb was preserved. As domain 1 is responsible for cancer invasion and metastasis, HuScFvM61B9 would be a candidate for cancer targeted therapy in the future.

Dynamic of anti-spike receptor binding domain (RBD) levels and short-term adverse events following a heterologous booster dose of BNT162b2 after two doses of CoronaVac in Thai health care workers.

BACKGROUND: CoronaVac was administered as the primary COVID-19 vaccine for Thai health care workers (HCWs) in early 2021 in response to the epidemic of new variants. This study aimed to evaluate the dynamic of humoral immune response as well as the short-term side effects resulting from the booster dose of BNT162b2 following completion of a CoronaVac double-dose in Thai HCWs. METHODS: This study was conducted at a teaching hospital in Northern Thailand during August and September 2021. The participants were 50 HCWs who were vaccinated with 2 doses of CoronaVac and were scheduled to receive a booster dose of BNT162b2. Anti-SARS-CoV-2 IgG antibodies levels and short-term side effects were assessed. The anti-RBD level was determined using Architect SARS-CoV-2 IgG II Quant (Abbott). RESULT: Of the 50 participants, 37 were female. The median age was 33.0 years old. The average time between the second CoronaVac shot and the BNT162b2 booster shot was 81.7 days (SD = 25.0). The median anti-SARS-CoV-2 IgG antibody level on booster vaccination date, as well as day 14, and day 28 after the booster were 335.5 AU/ml, 31,613.5 AU/ml, and 20,311.9 AU/ml, respectively. Fourteen days after the booster, 94% of participants had anti-SARS-CoV-2 IgG antibody levels higher than 50.0 AU/ml. Being female, higher log anti-SARS-CoV-2 IgG antibodies prior to booster vaccination, and longer interval between the second shot and the booster shot were found to be significantly associated with higher levels of anti-SARS-CoV-2 IgG antibodies at both day 14 and day 28 after the booster. There were no reports of serious adverse events. CONCLUSION: A booster dose of BNT162B2 promoted a high level of anti-SARS-CoV-2 IgG antibodies among HCWs who received 2 doses of CoronaVac. The time between the second CoronaVac shot and the booster shot should be at least three months. There were no severe adverse effects observed.

Role of CD137 signaling in dengue virus-mediated apoptosis.

Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.

Performance of Affinity-Improved DARPin Targeting HIV Capsid Domain in Interference of Viral Progeny Production.

Previously, a designed ankyrin repeat protein, AnkGAG1D4, was generated for intracellular targeting of the HIV-1 capsid domain. The efficiency was satisfactory in interfering with the HIV assembly process. Consequently, improved AnkGAG1D4 binding affinity was introduced by substituting tyrosine (Y) for serine (S) at position 45. However, the intracellular anti-HIV-1 activity of AnkGAG1D4-S45Y has not yet been validated. In this study, the performance of AnkGAG1D4 and AnkGAG1D4-S45Y in inhibiting wild-type HIV-1 and HIV-1 maturation inhibitor-resistant replication in SupT1 cells was evaluated. HIV-1 p24 and viral load assays were used to verify the biological activity of AnkGAG1D4 and AnkGAG1D4-S45Y as assembly inhibitors. In addition, retardation of syncytium formation in infected SupT1 cells was observed. Of note, the defense mechanism of both ankyrins did not induce the mutation of target amino acids in the capsid domain. The present data show that the potency of AnkGAG1D4-S45Y was superior to AnkGAG1D4 in interrupting either HIV-1 wild-type or the HIV maturation inhibitor-resistant strain.

Latticed Gold Nanoparticle Conjugation via Monomeric Streptavidin in Lateral Flow Assay for Detection of Autoantibody to Interferon-Gamma.

Adult-onset immunodeficiency syndrome (AOID) patients with autoantibodies (autoAbs) against interferon-gamma (IFN-γ) generally suffer from recurrent and recalcitrant disseminated non-tuberculous mycobacterial diseases. Since the early stages of AOID do not present specific symptoms, diagnosis and treatment of the condition are not practical. A simplified diagnostic method for differentiating AOID from other immunodeficiencies, such as HIV infection, was created. Anti-IFN-γ is generally identified using enzyme-linked immunosorbent assay (ELISA), which involves an instrument and a cumbersome process. Recombinant IFN-γ indirectly conjugated to colloidal gold was used in the modified immunochromatographic (IC) strips. The biotinylated-IFN-γ was incorporated with colloidal-gold-labeled 6HIS-maltose binding protein-monomeric streptavidin (6HISMBP-mSA) and absorbed at the conjugate pad. The efficacy of the IC strip upon applying an anti-IFN-γ autoAb cut-off ELISA titer of 2500, the sensitivity and specificity were 84% and 90.24%, respectively. When a cut-off ELISA titer of 500 was applied, the sensitivity and specificity were 73.52% and 100%, respectively.

Sirtuin Family Members Selectively Regulate Autophagy in Osteosarcoma and Mesothelioma Cells in Response to Cellular Stress.

The class III NAD+ dependent deacetylases-sirtuins (SIRTs) link transcriptional regulation to DNA damage response and reactive oxygen species generation thereby modulating a wide range of cellular signaling pathways. Here, the contribution of SIRT1, SIRT3, and SIRT5 in the regulation of cellular fate through autophagy was investigated under diverse types of stress. The effects of sirtuins' silencing on cell survival and autophagy was followed in human osteosarcoma and mesothelioma cells exposed to DNA damage and oxidative stress. Our results suggest that the mitochondrial sirtuins SIRT3 and 5 are pro-proliferative under certain cellular stress conditions and this effect correlates with their role as positive regulators of autophagy. SIRT1 has more complex role which is cell type specific and can affect autophagy in both positive and negative ways. The mitochondrial sirtuins (SIRT3 and SIRT5) affect both early and late stages of autophagy, whereas SIRT1 acts mostly at later stages of the autophagic process. Investigation of potential crosstalk between SIRT1, SIRT3, and SIRT5 revealed several feedback loops and a significant role of SIRT5 in regulating SIRT3 and SIRT1. Results presented here support the notion that sirtuin family members play important as well as differential roles in the regulation of autophagy in osteosarcoma vs. mesothelioma cells exposed to DNA damage and oxidative stress, and this can be exploited in increasing the response of cancer cells to chemotherapy.

Neutralizing Activity of Anti-interferon-γ Autoantibodies in Adult-Onset Immunodeficiency Is Associated With Their Binding Domains.

Adult-onset immunodeficiency (AOID) with anti-interferon-γ (IFN-γ) autoantibodies (autoAbs) is an emerging immunodeficiency syndrome in Asian countries. The presence of neutralizing anti-IFN-γ autoAbs are significantly associated with severe disseminated opportunistic infections. However, the characteristics of the neutralizing antibodies in patients are poorly defined. To better understand the properties of the anti-IFN-γ autoAbs in patients with opportunistic infections, a simplified competitive-binding ELISA was developed. The domains recognized by anti-IFN-γ autoAbs were assessed based on their competition with commercial neutralizing mouse anti-IFN-γ monoclonal antibodies (mAbs). First, the binding affinity and neutralizing capacity of these mAbs (clones B27, B133.5, and MD-1) were characterized. Kinetic analysis and epitope binning using bio-layer interferometry showed the comparable binding affinity of these mAbs to full-length IFN-γ and to the adjacent binding region. These mAbs did not recognize the synthetic 20-mer peptides and inhibited IFN-γ-mediated functions differently. In a competitive-binding ELISA, the anti-IFN-γ autoAbs in AOID serum blocked B27, B133.5, and MD-1 mAb binding. This evidence suggested that the autoAbs that competed with neutralizing mouse anti-IFN-γ mAbs recognized a discontinuous epitope of homodimeric IFN-γ as these mAbs. The patient autoAbs that recognized the B27 epitope exhibited strong neutralizing activity that was determined by the functional analysis. Our results demonstrated the heterogeneity of the autoAbs against IFN-γ in AOID patients and the different patterns among individuals. These data expand upon the fundamental knowledge of neutralizing anti-IFN-γ autoAbs in AOID patients.