Enantioselective Synthesis of Chiral Organosilicon Compounds by Organocatalytic Asymmetric Conjugate Addition of Boronic Acids to ß-Silyl-α,ß-Unsaturated Ketones.

Herein, we report (R)-3,3'-(3,5-(CF3)2-C6H3)2-BINOL-catalyzed enantioselective conjugate addition of organic boronic acids to ß-silyl-α,ß-unsaturated ketones, furnishing moderate to excellent yields of the corresponding ß-silyl carbonyl compounds with stereogenic centers in excellent enantioselectivities (up to 98% ee). Moreover, the catalytic system features mild reaction conditions, high efficiency, broad substrate scope, and easy scale-up.

Guidelines on the treatment with integrated traditional Chinese medicine and western medicine for severe coronavirus disease 2019.

Severe Coronavirus Disease 2019 (COVID-19) is characterized by numerous complications, complex disease, and high mortality, making its treatment a top priority in the treatment of COVID-19. Integrated traditional Chinese medicine (TCM) and western medicine played an important role in the prevention, treatment, and rehabilitation of COVID-19 during the epidemic. However, currently there are no evidence-based guidelines for the integrated treatment of severe COVID-19 with TCM and western medicine. Therefore, it is important to develop an evidence-based guideline on the treatment of severe COVID-19 with integrated TCM and western medicine, in order to provide clinical guidance and decision basis for healthcare professionals, public health personnel, and scientific researchers involved in the diagnosis, treatment, and care of COVID-19 patients. We developed and completed the guideline by referring to the standardization process of the "WHO handbook for guideline development", the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system, and the Reporting Items for Practice Guidelines in Healthcare (RIGHT).

Polysaccharide from Phellinus Igniarius activates TLR4-mediated signaling pathways in macrophages and shows immune adjuvant activity in mice.

Polysaccharide from Phellinus igniarius (PPI) is known for its immune-regulating effect with low toxicity. Toll like receptor 4 (TLR4) is important in both innate and adaptive immune responses and considered to be a promising target for new immune adjuvants. In this study, PPI was investigated for its effect on activating TLR4 in RAW264.7 and peritoneal macrophages. The adjuvant potential of PPI was evaluated in OVA-immunized mice. The results showed PPI treatment significantly increased the secretion and the mRNA expression of both MyD88 dependent and TRIF dependent cytokines. IRAK-1, a key molecule on the downstream of MyD88, was polyubiquitinated while IRF-3, another key molecule on the downstream of TRIF, was phosphorylated obviously after the treatment of PPI. The phosphorylation of molecules involved in both NF-κB pathway and MAPK pathway were significantly up-regulated after PPI treatment. In addition, the effects of PPI on the macrophages almost completely disappeared after treating the cells with the TLR4 antagonist TAK-242. Further in vivo results showed PPI significantly increased the serum OVA-specific antibody and the OVA-specific spleen cell proliferation. Taken together, PPI can specifically stimulate TLR4 and activate both MyD88 and TRIF pathways. PPI has immune adjuvant activity and may become a new potential immune adjuvant.

Development of genetically engineered iNKT cells expressing TCRs specific for the M. tuberculosis 38-kDa antigen.

INTRODUCTION: The invariant natural killer T (iNKT) cell has been shown to play a central role in early stages immune responses against Mycobacterium tuberculosis (Mtb) infection, which become nonresponsive (anergic) and fails to control the growth of Mtb in patients with active tuberculosis. Enhancement of iNKT cell responses to Mtb antigens can help to resist infection. STUDY DESIGN AND METHODS: In the present study, an Mtb 38-kDa antigen-specific T cell receptor (TCR) was isolated from human CD8(+) T cells stimulated by 38-kDa antigen in vitro, and then transduced into primary iNKT cells by retrovirus vector. RESULTS: The TCR gene-modified iNKT cells are endowed with new features to behave as a conventional MHC class I restricted CD8(+) T lymphocyte by displaying specific antigen recognition and anti-Mtb antigen activity in vitro. At the same time, the engineered iNKT cells retaining its original capacity to be stimulated proliferation by non-protein antigens α-Gal-Cer. CONCLUSIONS: This work is the first attempt to engineer iNKT cells by exogenous TCR genes and demonstrated that iNKT cell, as well as CD4(+) and CD8(+) T cells, can be genetically engineered to confer them a defined and alternative specificity, which provides new insights into TCR gene therapy for tuberculosis patients, especially those infected with drug-resistant Mtb.

Limited T cell receptor repertoire diversity in tuberculosis patients correlates with clinical severity.

BACKGROUND: The importance of CD4⁺ and CD8⁺ T cells in protection against tuberculosis (TB) is well known, however, the association between changes to the T cell repertoire and disease presentation has never been analyzed. Characterization of T-cells in TB patients in previous study only analyzed the TCR ß chain and omitted analysis of the Vα family even though α chain also contribute to antigen recognition. Furthermore, limited information is available regarding the heterogeneity compartment and overall function of the T cells in TB patients as well as the common TCR structural features of Mtb antigen specific T cells among the vast numbers of TB patients. METHODOLOGY/PRINCIPAL FINDINGS: CDR3 spectratypes of CD4⁺ and CD8⁺ T cells were analyzed from 86 patients with TB exhibiting differing degrees of disease severity, and CDR3 spectratype complexity scoring system was used to characterize TCR repertoire diversity. TB patients with history of other chronic disease and other bacterial or viral infections were excluded for the study to decrease the likely contribution of TCRs specific to non-TB antigens as far as possible. Each patient was age-matched with a healthy donor group to control for age variability. Results showed that healthy controls had a normally diversified TCR repertoire while TB patients represented with restricted TCR repertoire. Patients with mild disease had the highest diversity of TCR repertoire while severely infected patients had the lowest, which suggest TCR repertoire diversity inversely correlates with disease severity. In addition, TB patients showed preferred usage of certain TCR types and have a bias in the usage of variable (V) and joining (J) gene segments and N nucleotide insertions. CONCLUSIONS/SIGNIFICANCE: Results from this study promote a better knowledge about the public characteristics of T cells among TB patients and provides new insight into the TCR repertoire associated with clinic presentation in TB patients.

HGF-transgenic MSCs can improve the effects of tissue self-repair in a rabbit model of traumatic osteonecrosis of the femoral head.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is generally characterized as an irreversible disease and tends to cause permanent disability. Therefore, understanding the pathogenesis and molecular mechanisms of ONFH and developing effective therapeutic methods is critical for slowing the progress of the disease. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an experimental rabbit model of early stage traumatic ONFH was established, validated, and used for an evaluation of therapy. Computed tomography (CT) and magnetic resonance (MR) imaging confirmed that this model represents clinical Association Research Circulation Osseous (ARCO) phase I or II ONFH, which was also confirmed by the presence of significant tissue damage in osseous tissue and vasculature. Pathological examination detected obvious self-repair of bone tissue up to 2 weeks after trauma, as indicated by revascularization (marked by CD105) and expression of collagen type I (Col I), osteocalcin, and proliferating cell nuclear antigen. Transplantation of hepatocyte growth factor (HGF)-transgenic mesenchymal stem cells (MSCs) 1 week after trauma promoted recovery from ONFH, as evidenced by a reversed pattern of Col I expression compared with animals receiving no therapeutic treatment, as well as increased expression of vascular endothelial growth factor. CONCLUSIONS/SIGNIFICANCE: These results indicate that the transplantation of HGF-transgenic MSCs is a promising method for the treatment for ONFH and suggest that appropriate interference therapy during the tissue self-repair stage contributes to the positive outcomes. This study also provides a model for the further study of the ONFH etiology and therapeutic interventions.

Development of genetically engineered CD4+ and CD8+ T cells expressing TCRs specific for a M. tuberculosis 38-kDa antigen.

Cell-mediated immunity is critical to the clearance of Mycobacterium tuberculosis due to the primarily intracellular niche of this pathogen. Adoptive transfer of M. tuberculosis-specific effector T cells has been shown to confer immunity to M. tuberculosis-infected recipients resulting in M. tuberculosis clearance. However, it is difficult to generate sufficient numbers of M. tuberculosis antigen-specific T cells in a short time. Recent studies have developed T cell receptor (TCR) gene-modified T cells that allow for the rapid generation of large numbers of antigen-specific T cells. Many TCRs that target various tumor and viral antigens have now been isolated and shown to have functional activity. Nevertheless, TCRs specific for intracellular bacterial antigens (including M. tuberculosis antigens) have yet to be isolated and their functionality confirmed. We isolated M. tuberculosis 38-kDa antigen-specific HLA class I and class II-restricted TCRs and modified the TCR gene C regions by substituting nine amino acids with their murine TCR homologs (minimal murinization). Results showed that both wild-type and minimal murinized TCR genes were successfully cloned into retroviral vectors and transduced into primary CD4(+) and CD8(+) T cells and displayed anti-M. tuberculosis activity. As expected, minimal murinized TCRs displayed higher cell surface expression levels and stronger anti-M. tuberculosis activity than wild-type TCRs. To the best of our knowledge, this is the first report describing TCRs targeting M. tuberculosis antigens and this investigation provides the basis for future TCR gene-based immunotherapies that can be designed for the treatment of immunocompromised M. tuberculosis-infected patients.

Cancer of the gastrointestinal tract results in a restricted T-cell repertoire dependent upon tumor differentiation.

We investigated the influence of tumor tissue differentiation on the diversity of TCR repertoire. CDR3 spectratypes of CD4(+) and CD8(+) T cell subsets were analyzed from 27 patients with gastrointestinal tract tumors exhibiting varying degrees of differentiation. A CDR3 spectratype complexity scoring system was used to quantify the diversity of TCR repertoire. Each patient was matched with an age-matched healthy group to control for age variability. Results show that the complexity scores (TCR repertoire diversity) have a significant correlation with the degree of tumor differentiation, which provides useful information for understanding immune response in cancer patients.

Normalization of T cell receptor repertoire diversity in patients with advanced colorectal cancer who responded to chemotherapy.

To investigate the correlation between normalization of T cell receptor (TCR) repertoire and remission of advanced colorectal cancer. Forty-one patients were randomly assigned to receive either folinic acid/fluorouracil/irinotecan alone (n = 20) or folinic acid/fluorouracil/irinotecan in combination with recombinant human endostatin (n = 21). Efficacy and toxicity were evaluated, and changes in TCR repertoire diversity were assessed by detecting the spectratypes of TCR complementarity-determining region three before and after several cycles of therapy. A scoring system was used to quantify changes in the TCR repertoire over time. The results demonstrated that the TCR repertoire exhibited a higher degree of normalization among patients undergoing remission relative to patients experiencing tumor progression. The results of the current study showed a positive correlation between TCR repertoire normalization and remission of colorectal cancer, suggesting that dynamic monitoring of TCR repertoire diversity may have potential prognostic value in the clinical setting.

[Differentiation of 5-azacytidine-induced bone marrow stromal stem cells transduced with hepatocyte growth factor gene].

OBJECTIVE: To examine the differentiation of 5-azacytidine-induced bone marrow stromal stem cells (BMSCs) into cardiomyocyte-like cells and hepatocyte growth factor (HGF) gene expression after HGF gene transfection. METHODS: 5-azacytidine was used to induce beagle dog BMSCs to differentiate into cardiomyocyte-like cells. Morphological observation and immunohistochemistry were performed to detect the expression of the markers of cardiomyocyte-like cells including beta-myosin heavy chain (beta-MHC) and alpha-sarcomeric actin. The cells were then transfected with Ad-HGF, and the mRNA and protein expressions of HGF gene were detected by RT-PCR and ELISA, respectively. RESULTS: After 4 weeks of induction with 5-azacytidine, the BMSCs differentiated into cardiomyocyte-like cells. The expressions of HGF at the mRNA and protein levels were confirmed in the cells after transfection with Ad-HGF. The peak HGF protein secretion was 10(3) ng/ml at 48 h after transfection. CONCLUSION: Ad-HGF can efficiently transfect BMSCs induced with 5-azacytidine, and this result provides basic experimental evidence for biotherapy of ischemic heart disease using BMSCs.

[Effect of vascular endothelial growth factor and tumor necrosis factor receptor for treatment of avascular necrosis of the femoral head in rabbits].

OBJECTIVE: To evaluate the therapeutic effect of vascular endothelial growth factor (VEGF) and tumor necrosis factor receptor (TNFR) on avascular necrosis of the femoral head in rabbits. METHODS: Avascular necrosis of the femoral head was induced in 26 New Zealand white rabbits by injections of horse serum and prednisolone. The rabbits were then divided into VEGF/TNFR treatment group, VEGF treatment group, and untreated model group, with another 4 normal rabbits as the normal control group. In the two treatment groups, the therapeutic agents were injected percutaneously into the femoral head. Enzyme-linked immunosorbent assay was performed to determine the concentration of TNF-alpha in rabbit serum followed by pathological examination of the changes in the bone tissues, bone marrow hematopoietic tissue and the blood vessels in the femoral head. RESULTS: Compared with the model group, the rabbits with both VEGF and TNFR treatment showed decreased serum concentration of TNF-alpha with obvious new vessel formation, decreased empty bone lacunae in the femoral head and hematopoietic tissue proliferation in the bone marrow cavity. CONCLUSION: Percutaneous injection of VEGF and TNFR into the femoral head can significantly enhance bone tissue angiogenesis and ameliorate osteonecrosis in rabbits with experimental femoral head necrosis.

[Cloning of Rcet3, a novel gene related to family 2 cystatins].

OBJECTIVE: To clone the full-length Rcet3 gene, a novel gene related to family 2 cystatins, from mouse testis or other tissues. METHODS: Rcet3 gene was cloned using digital differential display (DDD) and RT-PCR was performed for cloning the full-length Rcet3 gene from adult mouse testis cDNA library with sequence analysis. RESULTS: Rcet3 cDNA was 610 bp in length, consisting of 4 exons to encode a protein with 140 amino acid residues. The encoded protein contained a potential signal peptide and a cystatin domain, but lacked critical consensus site important for cysteine protease inhibition. These characteristics could be seen in the Cres subgroup related to the family 2 cystatins. Rcet3 was specifically expressed in adult mouse testis, epididymis and the cerebrum, but at higher levels in the testis than in the epididymis and cerebrum. CONCLUSION: Rcet3 may be a new member of Cres subgroup of family 2 cystatins.

A modified peptide stimulation method for efficient amplification of cytomegalovirus (CMV)-specific CTLs.

CMV-specific immunity is essential for control of human cytomegalovirus (HCMV) infection. Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the ex vivo production of cells for adoptive transfer is labor intensive and expensive. We report here a modified peptide stimulation method using CMV-specific epitope peptides to stimulate PBMCs for generation of CMV-specific CTLs. This method permits efficient amplification of CMV-specific CTLs and provides a large number of cells for FACS analysis from a single blood sample. Significantly, it achieves high frequencies of tetramer staining of CD8+ T cells allowing the data of different individuals to be easily compared and sequentially evaluated. Thus, this approach expands and selects HLA-restricted CMV-pp65-reactive T-cell lines of high specificity for potential adoptive immunotherapy.

[CDR3 spectratyping and clonal expansion of peripheral blood T cells in a patient with beta-mediterranean anaemia following peripheral blood stem cell transplantation].

OBJECTIVE: To study the CDR3 spectratyping and clonal expansion of T cells in the peripheral blood mononuclear cells (PBMCs) of patients with beta-mediterranean anemia patient undergoing allogeneic peripheral blood stem cell (PBSC) transplantation. METHODS: The total RNA was isolated from the PBMCs of a nine-year-old boy with beta-mediterranean anemia before and after PBSC transplantation, and at the time of occurrence of graft-versus-host disease (GVHD). The CDR3 length was analyzed using immunoscope technique, and the characteristics of the T cell receptor (TCR) on the T cells with clonal expansion were examined by gene sequencing. RESULTS: The 24 TCR BV CDR3 repertoire showed Gaussian distribution in the PBMCs isolated before the transplantation, and some of the TCR BV family CDR3 showed skewing in the PBMCs isolated 23 days after transplantation and at the onset of GVHD (28 days after transplantation), suggesting the clonal expansion of the donor PBSCs. CONCLUSION: The host PBMCs show muti-clonal expansion 23 days after PBSC transplantation, and in the stage of GVHD, some of the TCR BV family T cells show significant monoclonal expansion. Analysis of TCR CDR3 spectratyping and the molecular characteristics of specific TCR may help evaluate the immune reconstitution following the transplantation and indicate specific treatment for potential GVHD.

[Recombinant type 1 adeno-associated virus-mediated transfection of ECV304 cells with enhanced green fluorescent protein gene in vitro].

OBJECTIVE: To assess the feasibility of recombinant type 1 adeno-associated virus (rAAV1) as a vector for gene therapy of corneal neovascularization. METHODS: The rAAV1 vector carrying enhanced green fluorescence protein (EGFP) gene (rAAV1-EGFP) was transfected into ECV304 cells at different multiplicities of infection (MOI=5 x 10(3), 5 x 10(4), 5 x 10(5)). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage determined by flow cytometry. MTT assay was used to assess the proliferation of the transfected cells. RESULTS: The cells with rAAV1-EGFP transfection at MOI of 5 x 10(5) began to exhibit GFP expression 2 days after transfection, and the fluorescence intensity increased with the MOI used for transfection. GFP expression reached the maximum on day 7, at the point of which the transduction efficiency of rAAV1-EGFP in ECV304 cells was 45.90%, 58.56% and 68.31% corresponding to MOIs of 5 x 10(3), 5 x 10(4), and 5 x 10(5), respectively. MTT assay did not reveal significant difference in the absorbance between the transfected cells and the control cells at 72 and 96 h after transfection. CONCLUSION: arAAV1-EGFP gene can be stably and efficiently expressed in ECV304 cells without causing cell growth inhibition, suggesting the potential of rAAV1 as a safe and efficient vector for gene therapy of corneal neovascularization.

Comparison of four methods for the purification and refolding of human interleukin-2-mouse granulocyte/macrophage colony-stimulating factor fusion protein.

The combination of IL-2 (interleukin-2) and GM-CSF (granulocyte/macrophage colony-stimulating factor) has been broadly studied in antitumour immune therapy, but its efficacy is uncertain. To better exert the activities of the two cytokines and study them in a mouse model, we have constructed a bifunctional protein, hIL-2-mGM-CSF (human IL-2-mouse GM-CSF), fused to a C-terminal tag of six histidine residues (His(6)). The fusion protein was expressed in Escherichia coli as IBs (inclusion bodies). After extracting and clarifying the IBs, four methods of protein purification and refolding were compared in order to optimize the preparation technique. Of these methods, the best result was obtained with a four-step process consisting of (1) purification with denaturing affinity chromatography, (2) followed by fully denaturing the protein with system conversion, (3) then refolding by isovolumetric ultrafiltration and (4) finally, purification by anion-exchange chromatography. The purity of the hIL-2-mGM-CSF was approx. 95%, yielding approx. 20 mg of protein/l of culture. The fusion protein retained the natural activities of IL-2 and GM-CSF, with specific activities of 8.7 x 10(6) and 1.1 x 10(7) i.u./mg respectively. Flow-cytometric analysis indicated that hIL-2-mGM-CSF could specifically bind to the corresponding receptor-positive cells. The present study provides important preliminary information for studying the antitumour activity of hIL-2-mGM-CSF in vivo, which will facilitate future clinical research into the use of hIL-2/hGM-CSF in immune therapy.

Expression, purification, and refolding of recombinant fusion protein hIL-2/mGM-CSF.

OBJECTIVE: To study the activities of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (hIL-2/mGM-CSF). METHODS: SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli (E. coli) BL21 (DE3) in inclusion body (IB) form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. RESULTS: The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein /L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4 x 10(6) IU/mg and 7.1 x 10(6) IU/mg, respectively. CONCLUSION: This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hIL-2/mGM-CSF used in immune therapy.

[Complementarity-determining region 3 analysis of T cell receptor beta chain variable region in peripheral blood mononuclear cells cultured with interleukin-2].

OBJECTIVE: To analyze the drift of the complementarity-determining region 3 (CDR3) of T cell receptor beta (TCRbeta) chain variable region in T cells of healthy volunteers cultured with interleukin-2 (IL-2). METHODS: T cells were isolated from the peripheral blood and cultured in vitro in the presence of IL-2. The non-specific killing effect of the cells was analyzed by LDH releasing assay, and the distribution of TCRbeta chain CDR3 in healthy volunteers by immunoscope spectratyping method to evaluate the clonality of the T cells. RESULTS: The results showed Gaussian distribution of TCR Vbeta gene CDR3 in healthy volunteers. The T cell cultured with IL-2, however, displayed some anomalous and oligoclonal expansion in different TCR Vbeta families without killing effect against nasophargngal carcinoma cell line CNE2. CONCLUSION: IL-2 may affect TCRbeta chain CDR3 distribution in T cells cultured in vitro.