Substance P promotes epidural fibrosis via induction of type 2 macrophages

In response to spinal surgery,neurons secrete a large amount of substance P into the epidural area.Substance P is involved in macrophage differentiation and fibrotic disease.However,the specific roles and mechanisms of substance P in epidural fibrosis remain unclear.In this study,we established a mouse model of L1–L3 laminectomy and found that dorsal root ganglion neurons and the macrophages infiltrating into the wound area released sphingolipids.In vitro experiments revealed that type 1 macrophages secreted substance P,which promoted differentiation of type 1 macrophages towards a type 2 phenotype.High-throughput mRNA-seq analysis revealed that the sphingolipid metabolic pathway may be involved in the regulation of type 2 macrophages by substance P.Specifically,sphingomyelin synthase 2,a component of the sphingolipid metabolic pathway,promoted M2 differentiation in substance P-treated macrophages,while treating the macrophages with LY93,a sphingomyelin synthase 2 inhibitor,suppressed M2 differentiation.In addition,substance P promoted the formation of neutrophil extracellular traps,which further boosted M2 differentiation.Blocking substance P with the neurokinin receptor 1 inhibitor RP67580 decreased the number of M2 macrophages in the wound area after spinal surgery and alleviated epidural fibrosis,as evidenced by decreased fibronectin,α-smooth muscle actin,and collagen I in the scar tissue.These results demonstrated that substance P promotes M2 macrophage differentiation in epidural fibrosis via sphingomyelin synthase 2 and neutrophil extracellular traps.These findings provide a novel strategy for the treatment of epidural fibrosis.

A SARS-CoV-2 neutralizing antibody discovery by single cell sequencing and molecular modeling

Although vaccines have been developed,mutations of SARS-CoV-2,especially the dominant B.1.617.2(delta)and B.1.529(omicron)strains with more than 30 mutations on their spike protein,have caused a significant decline in prophylaxis,calling for the need for drug improvement.Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms.The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments,providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies.A total of 128 sequences were obtained after sequencing 196 memory B cells,and 42 sequences were left after merging extremely similar ones and discarding incomplete ones,followed by homology modeling of the antibody variable region.Thirteen candidate sequences were expressed,of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants.The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.

Characterization and potential diagnostic application of monoclonal antibodies specific to rabies virus

Objective：Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem.It is necessary to develop standard rabies virus diagnostic tools,especially for diagnosing the strains prevalent in China.Methods：Monoclonal antibodies（MAbs）specific to rabies virus were produced and characterized by enzyme linked immunosorbent assay（ELISA）,isotyping,affinity assay,immunofluorescence assay（IFA）,and immunocytochemistry.The MAb,whose affinity was higher for antigen,was used to establish an antigen captureELISA（AC-ELISA）detection system and test the efficiency by using clinical samples.Results：The heavy chain subclasses of two MAbs were all determined to be IgG2a.The 3C7 MAb showed stronger reactivity with rabies virus protein than the 2C5 MAb in an ELISA analysis,whereas the 3C7 MAb showed the highest affinity for antigen.IFA and immunocytochemistry results also indicated that the two MAbs could recognize rabies virus protein in its native form in cell samples.Data obtained using clinical samples showed that rabies virus could be detected by AC-ELISA detection system using the 3C7 MAb.Conclusion：It was potentially useful for the further development of highly sensitive,easily handled,and relatively rapid detection kits/tools for rabies surveillance in those areas where rabies is endemic,especially in China.

Using SELDI-TOF-MS technology for screening serum markers of hepatic carcinoma in rats

Objective： To identify potential serum markers of hepatic carcinoma in rats through Surface-Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry（SELDI-TOF-MS） Technology. Methods： A rat model of hepatic carcinoma was established. The serum samples of hepatic carcinoma and normal rats were analyzed via SELDI-TOF-MS Technology. The changes of the serum protein fingerprint patterns were observed between the experimental group of hepatic carcinoma and the controls. The analysis was conducted by statistical software-Biomarker Wizard. Results： Fifty-six protein peaks in the serums were found. Within m/z 0-20 000, the protein peaks of rrdz 1158, 8 835 and 15 302 of hepatic carcinoma serums were obviously higher in the rat models compared with those in the controls（P 〈 0.01）. Conclusion： Three peaks were considered as potential biomarkers according to the serum protein fingerprint patterns of the hepatic carcinoma group and the control group.

Generation of monoclonal antibodies against n-3 fatty acid desaturase

Dear Editor： Omega-3 polyunsaturated fatty acids （n-3 PUFAs） are essential fatty acids for normal cellular functions and have been used for prevention and treatment of many diseases, including coronary heart disease, dia- betes, and cancers. n-3 PUFAs and n-6 PUFAs have been shown to decrease and increase the severity of several human diseases, respectively. Unfortunately,

Pre-evaluation of humoral immune response of Bactrian camels by the quantification of Th2 cytokines using real-time PCR

With the increasing immunological studies on camels due to the advantage of their single-chain antibodies for humanizations,it is demanding to develop an easy-to-handle evaluation method of their humoral immune response before proceeding with immunization of foreign antigens that may be toxic to camels.In this study,we quantitatively determined the expression levels of T-helper 2(Th2) cytokines in peripheral blood lymphocytes obtained from Bactrian camels by real-time PCR.The recorded kinetic profiles resulting from the immunization of ovalbumin(OVA) indicated that after immunization,Th2 cytokines including interleukin(IL) families such as IL-4,IL-10,and IL-13 in the camels were up-regulated by a factor of 1.78,3.15,and 1.22,respectively,which was validated by traditional enzyme-linked immunosorbent assay(ELISA) methods.Unlike ELISA which requires specific enzyme-labeled antibodies,this established method based on the minimal amount of blood samples holds an advantage in the preliminary evaluation of camel humoral immune response with desirable precision,which is meaningful for biomedical explorations of camel-derived antibodies.

Improving antibody affinity through in vitro mutagenesis in complementarity determining regions

High-affinity antibodies are widely used in diagnostics and for the treatment of human diseases.However,most antibodies are isolated from semi-synthetic libraries by phage display and do not possess in vivo affinity maturation,which is triggered by antigen immunization.It is therefore necessary to engineer the affinity of these antibodies by way of in vitro assaying.In this study,we optimized the affinity of two human monoclonal antibodies which were isolated by phage display in a previous related study.For the 42A1 antibody,which targets the liver cancer antigen glypican-3,the variant T57H in the second complementarity-determining region of the heavy chain(CDR-H2)exhibited a 2.6-fold improvement in affinity,as well as enhanced cell-binding activity.For the I4A3 antibody to severe acute respiratory syndrome coronavirus 2,beneficial single mutations in CDR-H2 and CDR-H3 were randomly combined to select the best synergistic mutations.Among these,the mutation S53P-S98T improved binding affinity(about 3.7 fold)and the neutralizing activity(about 12 fold)compared to the parent antibody.Taken together,single mutations of key residues in antibody CDRs were enough to increase binding affinity with improved antibody functions.The mutagenic combination of key residues in different CDRs creates additive enhancements.Therefore,this study provides a safe and effective in vitro strategy for optimizing antibody affinity.

Galectin-14 promotes hepatocellular carcinoma tumor growth via enhancing heparan sulfate proteoglycan modification

Hepatocellular carcinoma(HCC)is a highly heterogeneous malignancy and lacks effective treatment.Bulk-sequencing of different gene transcripts by comparing HCC tissues and adjacent normal tissues provides some clues for investigating the mechanisms or identifying potential targets for tumor progression.However,genes that are exclusively expressed in a subpopulation of HCC may not be enriched or detected through such a screening.In the current study,we performed a single cell-clone-based screening and identified galectin-14 as an essential molecule in the regulation of tumor growth.The aberrant expression of galectin-14 was significantly associated with a poor overall survival of liver cancer patients with database analysis.Knocking down galectin-14 inhibited the proliferation of tumor growth,whereas overexpressing galectin-14 promoted tumor growth in vivo.Non-targeted metabolomics analysis indicated that knocking down galectin-14 decreased glycometabolism;specifically that glycoside synthesis was significantly changed.Further study found that galectin-14 promoted the expression of cell surface heparan sulfate proteoglycans(HSPGs)that functioned as co-receptors,thereby increasing the responsiveness of HCC cells to growth factors,such as epidermal growth factor and transforming growth factor-alpha.In conclusion,the current study identifies a novel HCC-specific molecule galectin-14,which increases the expression of cell surface HSPGs and the uptake of growth factors to promote HCC cell proliferation.

T cells expressing a LMP1-specific chimeric antigen receptor mediate antitumor effects against LMP1-positive nasopharyngeal carcinoma cells in vitro and in vivo

T cells modified with chimeric antigen receptor are an attractive strategy to treat Epstein-Barr virus（EBV） associated malignancies.The EBV latent membrane protein 1（LMP1） is a 66-KD integral membrane protein encoded by EBV that consists of transmembrane-spanning loops.Previously,we have identified a functional signal chain variable fragment（scFv） that specifically recognizes LMP1 through phage library screening.Here,we constructed a LMP1 specific chimeric antigen receptor containing anti-LMP1 scFv,the CD28 signalling domain,and the CD3ζchain（HELA/CAR）.We tested its functional ability to target LMP1 positive nasopharyngeal carcinoma cells.HELA/CAR cells were efficiently generated using lentivirus vector encoding the LMP1-specific chimeric antigen receptor to infect activated human CD3＋ T cells.The HELA/CAR T cells displayed LMP1 specific cytolytic action and produced IFN-γ and IL-2 in response to nasopharyngeal carcinoma cells overexpressing LMP1.To demonstrate in vivo anti-tumor activity,we tested the HELA/CAR T cells in a xenograft model using an LMP1 overexpressing tumor.Intratumoral injection of anti-LMP1 HELA/CAR-T cells significantly reduced tumor growth in vivo.These results show that targeting LMP1 using HELA/CAR cells could represent an alternative therapeutic approach for patients with EBV-positive cancers.

c-Met-targeted chimeric antigen receptor T cells inhibit hepatocellular carcinoma cells in vitro and in vivo

c-Met is a hepatocyte growth factor receptor overexpressed in many tumors such as hepatocellular carcinoma(HCC).Therefore,c-Met may serve as a promising target for HCC immunotherapy.Modifying T cells to express c-Met-specific chimeric antigen receptor(CAR)is an attractive strategy in treating c-Met-positive HCC.This study aimed to systematically evaluate the inhibitory effects of 2^(nd)-and 3^(rd)-generation c-Met CAR-T cells on hepatocellular carcinoma(HCC)cells.Here,2^(nd)-and 3^(rd)-generation c-Met CARs containing an anti-c-Met singlechain variable fragment(scFv)as well as the CD28 signaling domain and CD3ζ(c-Met-28-3ζ),the CD137 signaling domain and CD3ζ(c-Met-137-3ζ),or the CD28 and CD137 signaling domains and CD3ζ(c-Met-28-137-3ζ)were constructed,and their abilities to target c-Met-positive HCC cells were evaluated in vitro and in vivo.All c-Met CARs were stably expressed on T cell membrane,and c-Met CAR-T cells aggregated around c-Met-positive HCC cells and specifically killed them in vitro.c-Met-28-137-3ζCAR-T cells secreted more interferon-gamma(IFN-γ)and interleukin 2(IL-2)than c-Met-28-3ζCAR-T cells and c-Met-137-3ζCAR-T cells.Compared with c-Met low-expressed cells,c-Met CAR-T cells secreted more cytokines when co-cultured with c-Met high-expressed cells.Moreover,c-Met-28-137-3ζCAR-T cells eradicated HCC more effectively in xenograft tumor models compared with the control groups.This study suggests that 3^(rd)-generation c-Met CAR-T cells are more effective in inhibiting c-Met-positive HCC cells than 2^(nd)-generation c-Met CAR-T cells,thereby providing a promising therapeutic intervention for c-Met-positive HCC.

Nanobody-based immunosensing methods for safeguarding public health

Immunosensing methods are biosensing techniques based on specific recognition of an antigen-antibody immunocomplex,which have become commonly used in safeguarding public health.Taking advantage of antibody-related biotechnological advances,the utilization of an antigen-binding fragment of a heavy-chain-only antibody termed as'nanobody'holds significant biomedical potential.Compared with the conventional full-length antibody,a single-domain nanobody retaining cognate antigen specificity possesses remarkable physicochemical stability and structural adaptability,which enables a flexible and efficient molecular design of the immunosensing strategy.This minireview aims to summarize the recent progress in immunosensing methods using nanobody targeting tumor markers,environmental pollutants,and foodborne microbes.

Screening and identification of human Zn T8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library

Zinc transporter 8 （ZnT8） is a major autoantigen and a predictive marker in type 1 diabetes （T1D）. To investigate ZnT8-specific antibodies, a phage display library from TID was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment （scFv） phage display library consists of approximately 1 ~ l0s clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Topl0F＇ and then purified by affin- ity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.

Histone methyltransferase Nsd2 ensures maternal-fetal immune tolerance by promoting regulatory T-cell recruitment

Regulatory T cells(Tregs)are fundamentally important for maintaining systemic immune homeostasis and are also required for immune tolerance at the maternal-fetal interface during pregnancy.Recent studies have suggested that epigenetic regulation is critically involved in Treg development and function.However,the role of H3K36me has not yet been investigated.Here,we found that the H3K36me2 methyltransferase Nsd2 was highly expressed in Tregs.Although loss of Nsd2 did not impair systemic Treg development or function,the level of Tregs at the maternal-fetal interface was significantly decreased in pregnant Nsd2 conditional knockout mice.Consequently,maternal-fetal immune tolerance was disrupted in the absence of Nsd2 in Tregs,and the pregnant mice showed severe fetal loss.Mechanistically,Nsd2 was found to upregulate CXCR4 expression via H3K36me2 modification to promote Treg cell recruitment into the decidua and suppress the anti-fetal immune response.Overall,our data identified Nsd2 as a critical epigenetic regulator of Treg recruitment for maternal-fetal tolerance.

Schiff-base silver nanocomplexes formation on natural biopolymer coated mesoporous silica contributed to the improved curative effect on infectious microbes

Infectious microbes that spread easily in healthcare facilities remain as the severe threat for the public health,especially among immunocompromised populations.Given the intricate problem of dramatic increase in resistance to common biocides,the development of safe and efficient biocide formulated agents to alleviate drug resistance is highly demanding.In this study,Schiff-base ligands were successfully formed on natural biopolymer of epsilon-poly-L-lysine(ε-PL)decorated aldehyde functionalized mesoporous silica SBA-15(CHO-SBA-15)for the selective coordination of silver ions,which was affirmed by various physicochemical methods.Besides the identified broad-spectrum antibacterial activities,the as-prepared Schiff-base silver nanocomplex(CHO-SBA-15/ε-PL/Ag,CLA-1)exhibited an improved inhibitory effect on infectious pathogen growth typified by Escherichia coli and Staphylococcus aureus in comparison with two control silver complexes without Schiff-base conjugates,SBA-15/ε-PL/Ag and CHO-SBA-15/Ag,respectively.In addition,CLA-1 remarkably inhibited the growth of Mycobacterium tuberculosis due to the excellent antimicrobial activity of silver species.Significantly,CLA-1 kills Candida albicans cells,inhibits biofilm formation,and eliminates preformed biofilms,with no development of resistance during continuous serial passaging.The antifungal activity is connected to disruption of bacterial cell membranes and increased levels of intracellular reactive oxygen species.In mouse models of multidrug-resistant C.albicans infection,CLA-1 exhibited efficient in vivo fungicidal efficacy superior to two antifungal drugs,amphotericin B and fluconazole.Moreover,CLA-1 treatment induces negligible toxicity against normal tissues with safety.Therefore,this study reveals the pivotal role of the molecular design of Schiff-base silver nanocomplex formation on biopolymer surface-functionalized silica mesopores as a green and efficient nanoplatform to tackle infectious microbes.