Activated Tim-3/Galectin-9 participated in the development of multiple myeloma by negatively regulating CD4 T cells.

The interaction between Tim-3 on T cells and its ligand Galectin-9 negatively regulates the cellular immune response. However, the regulation of Tim-3/Galectin-9 on CD4 T cell subsets in multiple myeloma (MM) remains unclear. The aim of this study was to investigate the relationship between the regulation of CD4 T cell subsets by the Tim-3/Galectin-9 pathway and clinical prognostic indicators in MM. Tim-3/Galectin-9 were detected by flow cytometry, PCR and ELISA in 60 MM patients and 40 healthy controls, and its correlation with clinical prognostic parameters was analyzed. The expressions of Tim-3 on CD4 T cells, Galectin-9 mRNA in PBMC and level of Galectin-9 protein in serum were significantly elevated in MM patients, especially those with poor prognostic indicators. In MM patients, Tim-3 was highly expressed on the surfaces of Th1, Th2, and Th17 cells, but lowly expressed on Treg. Moreover, level of cytokine IFN-γ in serum was negatively correlated with Tim-3+Th1 cell and Galectin-9mRNA, Galectin-9 protein level. In addition, cell culture experiments showed that the anti-tumor effect and the ability to secrete IFN-γ were restored by blocking the Tim-3/Galectin-9 pathway. In MM patients, Tim-3/Galectin-9 is elevated and associated with disease progression, by inhibiting the cytotoxic function of Th1, and also promoting Th2 and Th17 to be involved in immune escape of MM. Therefore, Tim-3/Galectin-9 may serve as a new immunotherapeutic target for MM patients.

[Expression and Clinical Significance of Helper T Cells 9 and Its Sytokines Interleukin 9 in Chronic Lymphocytic Leukemia].

OBJECTIVE: To investigate the expression and clinical significance of T helper cell 9 (Th9) and its cytokine interleukin 9(IL-9) in peripheral blood of patients with chronic lymphocytic leukemia(CLL). METHODS: A total of 43 newly diagnosed patients with chronic lymphocytic leukemia in the First Affiliated Hospital of Xinjiang Medical University from June 2021 to June 2022 were selected as the case group. The patients were divided into Binet A group (13 cases), Binet B group (20 cases) and Binet C group (10 cases) by Binet staging system, and 20 healthy volunteers who underwent physical examinationin in our hospital in the same period served as control group. The proportion of Th9 cells in peripheral blood was detected by flow cytometry, the expression level of Th9 specific transcription factors PU.1 and IRF4 was detected by Western blot, and the expression level of serum cytokine IL-9 was detected by ELISA. The proportion of Th9, the expression of PU.1, IRF4 and IL-9 in each group were compared, and the correlation between the proportion of Th9, IL-9 and clinicopathological indexes of CLL patients was analyzed. RESULTS: The proportion of Th9, the expression of PU.1, IRF4 and IL-9 in CLL group were significantly higher than those in control group (P<0.05), the proportion of Th9 and the expression of IL-9 in Binet B and C group were higher than those in Binet A group (P<0.05), but there was no significant difference in the proportion of Th9 cells between Binet B group and C group (P>0.05). The expression of IL-9 in Binet C group was significantly higher than that in Binet B group (P<0.05) . The proportion of Th9 cells and IL-9 were highly expression in patients with ß2 microglobulin abnormality, IGHV unmutation, P53 abnormality and hepatosplenic lymph node enlargement(P<0.05), but not related to age and sex (P>0.05). The results of Spearman correlation analysis showed that the proportion of Th9 in patients with CLL was negatively correlated with the lymphocytic account and lymphocyte proportion(rs=-0.32，rs=-0.34). The proportion of Th9 and IL-9 were positively correlated with Binet stage, Rai stage and CLL-IPI Scoring (rs=0.79，rs=0.54，rs=0.58； rs=0.72，rs=0.63，rs=0.45), but not with WBC, CD4+ T cells and CD8+T cells (P>0.05). The proportion of Th9 was positively correlated with IL-9 (rs=0.53). CONCLUSION: Th9 cells and IL-9 are abnormally highly expressed in CLL, which is related to the poor prognosis of CLL.

Salvianolic acid B from Salvia miltiorrhiza bunge: A potential antitumor agent.

Salvia miltiorrhiza Bunge (Lamiaceae) is a perennial herb widely found in China since ancient times with a high economic and medicinal value. Salvianolic acid B (Sal-B) is an important natural product derived from Salvia miltiorrhiza and this review summarizes the anticancer activity of Sal-B. Sal-B inhibits tumor growth and metastasis by targeting multiple cell signaling pathways. This review aims to review experimental studies to describe the possible anticancer mechanisms of Sal-B and confirm its potential as a therapeutic drug.

Glyceroglycolipids in marine algae: A review of their pharmacological activity.

Glyceroglycolipids are major metabolites of marine algae and have a wide range of applications in medicine, cosmetics, and chemistry research fields. They are located on the cell surface membranes. Together with glycoproteins and glycosaminoglycans, known as the glycocalyx, they play critical roles in multiple cellular functions and signal transduction and have several biological properties such as anti-oxidant and anti-inflammatory properties, anti-viral activity, and anti-tumor immunity. This article focused on the sources and pharmacological effects of glyceroglycolipids, which are naturally present in various marine algae, including planktonic algae and benthic algae, with the aim to highlight the promising potential of glyceroglycolipids in clinical treatment.

Subvisible Particle Analysis of 17 Monoclonal Antibodies Approved in China Using Flow Imaging and Light Obscuration.

In the study, subvisible particles in 205 samples from 17 commercial mAb drug products approved in China were analyzed using light obscuration (LO) and flow imaging microscopy (FIM) methods. For each method, a total 633 tests (runs) were performed. In the tests, samples in state of lyophilized powder or syringe package had significantly higher particle concentrations. It was confirmed by analyzing the 205 drug product samples that FIM particle counts are generally higher than LO counts. The cause of the higher counts of FIM method than LO counts was examined by looking into the contribution of proteinaceous, translucent particles in the samples. The data of the study showed that the number of proteinaceous, translucent particles was a factor in the elevated counts of FIM method compared to LO method.

ZmCCA1a on Chromosome 10 of Maize Delays Flowering of Arabidopsis thaliana.

Maize (Zea mays) is a major cereal crop that originated at low latitudes, and thus photoperiod sensitivity is an important barrier to the use of tropical/subtropical germplasm in temperate regions. However, studies of the mechanisms underlying circadian regulation in maize are at an early stage. In this study we cloned ZmCCA1a on chromosome 10 of maize by map-based cloning. The gene is homologous to the Myb transcription factor genes AtCCA1/AtLHY in Arabidopsis thaliana; the deduced Myb domain of ZmCCA1a showed high similarity with that of AtCCA1/AtLHY and ZmCCA1b. Transiently or constitutively expressed ZmCCA1a-YFPs were localized to nuclei of Arabidopsis mesophyll protoplasts, agroinfiltrated tobacco leaves, and leaf and root cells of transgenic seedlings of Arabidopsis thaliana. Unlike AtCCA1/AtLHY, ZmCCA1a did not form homodimers nor interact with ZmCCA1b. Transcripts of ZmCCA1a showed circadian rhythm with peak expression around sunrise in maize inbred lines CML288 (photoperiod sensitive) and Huangzao 4 (HZ4; photoperiod insensitive). Under short days, transcription of ZmCCA1a in CML288 and HZ4 was repressed compared with that under long days, whereas the effect of photoperiod on ZmCCA1a expression was moderate in HZ4. In ZmCCA1a-overexpressing A. thaliana (ZmCCA1a-ox) lines, the circadian rhythm was disrupted under constant light and flowering was delayed under long days, but the hypocotyl length was not affected. In addition, expression of endogenous AtCCA1/AtLHY and the downstream genes AtGI, AtCO, and AtFt was repressed in ZmCCA1a-ox seedlings. The present results suggest that the function of ZmCCA1a is similar, at least in part, to that of AtCCA1/AtLHY and ZmCCA1b, implying that ZmCCA1a is likely to be an important component of the circadian clock pathway in maize.

A robust and stable reporter gene bioassay for anti-IgE antibodies.

Immunoglobin E (IgE)-related allergy constitutes a high proportion in allergic diseases. The production of specific IgE is key to evoking serial cascades and pathological reactions. Thus, targeting IgE is a different therapeutic approach from symptomatic treatments. Monoclonal antibodies (mAbs) against IgE were developed and a humanized antibody, omalizumab, was approved by five countries. It could inhibit the binding of IgE with epsilon receptor I of crystallizable fragment (FcεRI), thus preventing anaphylactic reactions. However, no bioactivity assay, which is the critical quality attribute and should thoroughly reflect the clinical mechanism, has been established to date. In commercial lot release, only the enzyme-linked immunosorbent assay (ELISA) method was applied, which only reflects the binding of omalizumab to IgE but not the subsequent reaction. In scientific research works, human FcεRI-transfected RBL-2H3 cells were used to indicate degranulation based on the detection of ß-hexosaminidase. Nevertheless, this method needs much work to stabilize the response and, hence, is not suitable for routine usage in commercial production and control of antibodies. To evaluate the bioactivity of anti-IgE antibodies including omalizumab using a simple assay that reflects the following mechanism of actions (MOA) after binding, we established an RBL-2H3 cell line transfected with both the α subunit of human FcεRI and nuclear factor-activated T cell (NFAT) response elements, the latter is conjugated with a luciferase gene, which could shed luminescence when substrates exist. The method was proven to possess good specificity, accuracy, linearity, and precision and may be utilized as a supplement to anti-IgE antibody bioactivity assays in terms of development, lot release, stability, and comparability studies. Graphical abstract The mechanism sketch of reporter gene assay for bioactivity determination of anti-IgE antibodies by RBL-2H3/FcεRIα/NFAT-Luc cells (left) and representative curves generated by the reporter gene assay (right).

Analytical Similarity of a Proposed Biosimilar BVZ-BC to Bevacizumab.

BVZ-BC (bevacizumab-biosimilar candidate) is a proposed biosimilar to bevacizumab. Bevacizumab binds to vascular endothelial growth factor (VEGF) type A and prevents the interaction of VEGF with its receptors on the surface of endothelial cells, neutralizing angiogenesis required for the growth, persistence, and metastases of solid tumors. An analytical comparison of BVZ-BC and bevacizumab was performed using state-of-the-art analytical techniques, including biochemical and biophysical characterization, biological activity, and immunological properties. Multiple attributes of the molecules were evaluated, including amino acid sequence, disulfide structure, glycan profiles, free thiol content, isoelectric point, protein content, subvisible particles, higher-order structure such as near- and far-ultraviolet circular dichroism and differential scanning calorimetry, product purity and product-related impurities, and process-related impurities. Biological activity assessment employed orthogonal assays such as the VEGF cell-based bioassay and the VEGF enzyme-linked immunosorbent assay, and Fc functional assays to interrogate all expected biological activities. An accumulation of 18 batches of bevacizumab (sourced in China, manufactured in Europe, Roche) and 10 batches of BVZ-BC representing unique drug product lots from each individual drug substance lot were utilized in this study. The analytical similarity between BVZ-BC and bevacizumab was assessed and demonstrated the similarities of all of the quality attributes between BVZ-BC and bevacizumab.

Role of ADAM10 and ADAM17 in CD16b shedding mediated by different stimulators.

OBJECTIVE: To investigate the main proteinases responsible for CD16b shedding under different stimulators. METHODS: HEK293 cell line stably expressing CD16b was constructed by lentivirus system. The cell line was then overexpressed with a disintegrin and metalloproteinase 10 (ADAM10) or ADAM17, suppressed with short hairpin RNA of ADAM10 or ADAM17, and reconstituted with ADAM10 or ADAM17, respectively. After each treatment, the cell line was stimulated with ionomycin or phorbol 12-myristate- 13-acetate (PMA) for 12 hours. The soluble CD16b released from cell membrane was detected by immunoprecipition and immunoblot. Quantitation was then implemented to compare the amount of soluble CD16b in cell supernatant after stimulation. RESULTS: HEK293 cell line stably expressing CD16b was successfully established. When CD16b expressing cell line was overexpressed with ADAM10, shedding of CD16b was increased after stimulation with ionomycin but not PMA; when the cell line overexpressed with ADAM17, shedding of CD16b was increased after stimulation with PMA but not ionomycin. Similarly, when ADAM10 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with ionomycin; when ADAM17 was suppressed by short hairpin RNA, CD16b shedding was decreased after stimulation with PMA. The shedding of CD16b was increased again when CD16b expressing cell line was reconstituted with ADAM10 and stimulated by ionomycin or reconstituted with ADAM17 and stimulated by PMA. CONCLUSIONS: Both ADAM10 and ADAM17 could shed CD16b, but they possess differed preferences. ADAM10 is the main sheddase under stimulation of ionomycin, while ADAM17 is the main sheddase under stimulation of PMA.

Ectodomain shedding of Fcalpha receptor is mediated by ADAM10 and ADAM17.

FcalphaR (CD89) plays important roles in immunoglobulin A (IgA)-mediated immune responses. Soluble forms of FcalphaR (sFcalphaR) are found in the culture supernatants of FcalphaR-expressing cells, in human serum and in the serum of FcalphaR transgenic mice, and have been suggested to be produced through a proteolytic process. However, little is known about the mechanism involved in the proteolytic release of sFcalphaR. In this study, we investigated the shedding mechanism of FcalphaR and determined the nature of the proteinase involved in FcalphaR shedding. In chemical inhibitor assays, shedding of FcalphaR was dramatically inhibited by EDTA, EGTA and a broad-spectrum metalloproteinase inhibitor, GM6001, suggesting that a metalloproteinase was responsible for FcalphaR shedding. Overexpression of dominant-negative mutants of ADAM (a disintegrin and metalloproteinase) 10 and ADAM17 markedly inhibited the production of sFcalphaR. Finally, knockdown of both endogenous ADAM10 and endogenous ADAM17 inhibited FcalphaR shedding, demonstrating that ADAM10 and ADAM17 were involved in the shedding of FcalphaR. The characterization of ADAM10 and ADAM17 as sFcalphaR-releasing enzymes provides a novel insight into the molecular mechanism of sFcalphaR production and will help in further elucidation of the physiological and pathological roles of sFcalphaR.