Objective To construct a library of human-derived anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) single-chain variable fragments (scFv) and screen for broad-spectrum neutralizing antibodies to identify candidate molecules for the development of diagnostic and therapeutic agents. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of patients who had recovered from novel coronavirus infection. Total RNA was extracted from these PBMCs and reverse transcribed into cDNA, which was used as a template for constructing a human anti-SARS-CoV-2 scFv library. Phage display technology was used to screen for scFv antibodies specific to the SARS-CoV-2 S protein. Full-length IgG antibodies were synthesized through sequence analysis and human IgG expression, and their binding capacity and neutralizing activity against SARS-CoV-2 were evaluated. Results A human-derived scFv antibody library against SARS-CoV-2 with a capacity of 1.56×10⁷ CFU was successfully constructed. Two specific scFv antibodies were screened from this library and expressed as full-length IgG antibodies (IgG-A10 and IgG-G6). IgG-A10 exhibited strong neutralizing activity against both the original SARS-CoV-2 strain (WT) and the XBB subvariant of the Omicron variant. However, the neutralizing activity of this antibody against the JN.1 sub lineage of the Omicron BA.2.86 variant was moderate. Conclusion This study has successfully constructed a human anti-SARS-CoV-2 scFv antibody library from the peripheral blood of recovered patients, and screened and expressed anti-SARS-CoV-2 IgG antibodies with neutralizing activity, laying a foundation for the prevention, diagnosis, and treatment of SARS-CoV-2 infection.
Humans ; Single-Chain Antibodies/genetics* ; SARS-CoV-2/immunology* ; COVID-19/immunology* ; Immunoglobulin G/genetics* ; Antibodies, Viral/genetics* ; Peptide Library ; Spike Glycoprotein, Coronavirus/immunology* ; Antibodies, Neutralizing/immunology* ; Leukocytes, Mononuclear/immunology* ; Broadly Neutralizing Antibodies/immunology*

Objective To clarify the mechanism that HIV infection mediates mitochondrial damage of CD4⁺ T lymphocytes (CD4⁺ T cells) through mitogen-activated protein kinase (MAPK) pathway. Methods From October 1st, 2022 to March 31st, 2023, 47 HIV-infected people who received antiretroviral therapy (ART) for 4 years were recruited, including 22 immune non-responders (INR) and 25 responders (IR); and 26 sex and age-matched control participants (HC) who were negative for HCV, HBV, and HIV infections. The immune parameters were analyzed by flow cytometry. Finally, peripheral blood mononuclear cells (PBMCs) from HC or HIV patients were treated with MAPK pathway inhibitor SB203580, and the changes of mitochondrial function of CD4⁺ T cells were observed. Results Compared with HC group, the proportion of CD4⁺ T cells in PBMCs in INR group and IR group was significantly lower, and the proportion of CD4⁺ T cells in PBMCs in INR group was significantly lower than that in IR group. In addition, the proportion of naive (CD45RA⁺CD27⁺)T cells in PBMCs in INR group was significantly lower than that in HC group and IR group. Compared with HC group and IR group, the proportions of CD4⁺PD-1⁺, CD4⁺Av⁺ and CD4⁺MO⁺ in PBMCs in INR group and the proportions of CD45RA⁺CD27⁺PD-1⁺, CD45RA⁺CD27⁺Av⁺, CD45RA⁺CD27⁺MO⁺ in CD4⁺ T cell subsets increased significant. Compared with HC-con group, the basal respiration, maximal respiration and adenosine triphosphate(ATP) production of CD4⁺ T cells in HIV-con group decreased significantly, and JC-1 (green/red) in CD4⁺ T cells increased significantly. Compared with HIV-con group, the basal respiration, maximal respiration, ATP production and respiratory potential of CD4⁺ T cells in HIV-SB203580 group increased significantly, and the JC-1 (green/red) in CD4⁺ T cells decreased significantly. Conclusion Abnormal activation of the MAPK signaling pathway is observed in HIV patients receiving ART treatment, especially in CD4⁺ T cells of INR patients, which may lead to impaired mitochondrial function and abnormal CD4⁺ T cell homeostasis.
Humans ; HIV Infections/immunology* ; Male ; Mitochondria/drug effects* ; Female ; CD4-Positive T-Lymphocytes/metabolism* ; Adult ; Middle Aged ; MAP Kinase Signaling System/drug effects* ; Pyridines/pharmacology* ; Imidazoles/pharmacology* ; Leukocytes, Mononuclear/immunology*

OBJECTIVE: The aim of this study is to investigate the effects of oral cadmium (Cd) ingestion on the pulmonary immune response. METHODS: Determination of Cd content in lungs and histopathological evaluation of the tissue was performed in rats following 30-day oral Cd administration (5 and 50 mg/L). Antioxidant enzyme defense (superoxide dismutase and catalase), cell infiltration, and production of tumor necrosis factor (TNF) and interferon (IFN)-γ, as well as the activity of myeloperoxidase (MPO), nitric oxide (NO), and various cytokines [interleukin (IL)-1β, IL-6, IL-10, and IL-17] were investigated. RESULTS: Cd caused tissue damage and cell infiltration in the lungs, and this damage was more pronounced at higher doses. Cd deposition resulted in lung inflammation characterized by a dose-dependent IL-1β increase in lung homogenates, increased TNF levels at both doses, and IL-6 stimulation at low doses with inhibition observed at higher doses. Cd exerted differential effects on lung leukocytes isolated by enzyme digestion, and these effects were characterized by a lack of change in the production of reactive oxygen and nitrogen species, an inhibition of IL-1β and TNF, and stimulation of MPO and IFN-γ. The higher capacity of Cd-exposed lung cells to respond to the opportunistic pathogen Staphylococcus epidermidis was demonstrated in vitro. CONCLUSION The potential of ingested Cd to exert both proinflammatory and immunosuppressive effects on pulmonary tissue inflammation and immune reactivity highlights the complex immunomodulatory actions of this metal.
Administration, Oral ; Animals ; Cadmium ; administration & dosage ; toxicity ; Leukocytes ; metabolism ; Lung ; drug effects ; immunology ; pathology ; Male ; Rats ; Staphylococcus epidermidis ; Toxicity Tests, Subchronic

OBJECTIVE: To study the correlation of IL-37 with T lymphocytes subsets and NK cells in ITP patients, and to explore its possible mechanisms involved in the pathogenesis of ITP. METHODS: Forty-five patients with newly diagnosed ITP(newly diagnosed group), 32 patients of complete remission (remission group) and 22 healthy persons(control group) were selected. The serum level of IL-37 in 3 groups was determined by enzyme linked immunosorbent assay (ELISA). The mRNA expression of IL-37, IL-17 and IL-18 in peripheral blood mononuclear cells（PBMNC） in 3 groups was measured by real-time fluorescence quantitative polymerase chain reaction (PCR). The number of IL-18RαCD4 T cells and Tim-3NK cells in the peripheral blood in 3 groups was detected by flow cytometry (FCM). RESULTS: The serum level of IL-37 in the peripheral blood of ITP patients in the newly diagnosed group was significantly higher than that in the control group and the remission group(P＜0.01) . The expression level of IL-37 in PBMNC of the ITP patients in newly diagnosed group was higher than that in the control group and the remission group(P＜0. 05). The expression level of IL-17 and IL-18 in PBMNC of the ITP patients in newly diagnosed group was higher than that in the control group and the remission group(P＜0. 01); the expression of IL-18Rα in CD4 T cells in newly diagnosed group was significantly higher than that in both the control and the remission group(P＜0.01).The expression of Tim-3 in NK cells in ITP patients was significantly lower than that in the control group (P＜0. 01). In ITP patients, the serum IL-37 level and IL-18RαCD4T cells ratio both negatively correlated with Plt count (r=-0.58, r=-0.48) moreo-ver the serum IL-37 level also negatively correlated with amount of CD4 T cells and NK cells (r=-0.29, r=-0.28), but positively correlated with amount of CD8 T cells (r=0.329). CONCLUSION The IL-37 and its receptors may play an immunoregulatory role in CD4 T cells and NK cells, the IL-37 may be a therapeutic target for ITP patients.
CD8-Positive T-Lymphocytes ; Flow Cytometry ; Humans ; Interleukin-1 ; immunology ; Killer Cells, Natural ; Leukocytes, Mononuclear ; Purpura, Thrombocytopenic, Idiopathic ; T-Lymphocyte Subsets

Astragalus membranaceus may be a potential therapy for childhood asthma but its driving mechanism remains elusive. The main components of A. membranaceus were identified by HPLC. The children with asthma remission were divided into two combination group (control group, the combination of budesonide and terbutaline) and A. membranaceus group (treatment group, the combination of budesonide, terbutaline and A. membranaceus). The therapeutic results were compared between two groups after 3-month therapy. Porcine peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by using density gradient centrifugation on percoll. The levels of FoxP3, EGF-β, IL-17 and IL-23 from PBMCs and serum IgE were measured. The relative percentage of Treg/Th17 cells was determined using flow cytometry. The main components of A. membranaceus were calycosin-7-O-glucoside, isoquercitrin, ononin, calycosin, quercetin, genistein, kaempferol, isorhamnetin and formononetin, all of which may contribute to asthma therapy. Lung function was significantly improved in the treatment group when compared with a control group (P < 0.05). The efficacy in preventing the occurrence of childhood asthma was higher in the treatment group than the control group (P < 0.05). The levels of IgE, IL-17 and IL-23 were reduced significantly in the treatment group when compared with the control group, while the levels of FoxP3 and TGF-β were increased in the treatment group when compared with the control group (P < 0.05). A. membranaceus increased the percentage of Treg cells and reduced the percentage of Th17 cells. A. membranaceus is potential natural product for improving the therapeutic efficacy of combination therapy of budesonide and terbutaline for the children with asthma remission by modulating the balance of Treg/Th17 cells.
Animals ; Asthma ; drug therapy ; immunology ; Astragalus propinquus ; chemistry ; Budesonide ; administration & dosage ; Cells, Cultured ; Child ; Child, Preschool ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Humans ; Immunologic Factors ; administration & dosage ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lung ; drug effects ; physiology ; Male ; Swine ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Terbutaline ; administration & dosage ; Th17 Cells ; cytology ; drug effects ; Treatment Outcome

BackgroundDevelopment of innovative immunotherapy is imperative to improve the poor survival of the nasopharyngeal carcinoma (NPC) patients. In this study, we evaluated the T cell response to melanoma-associated antigen (MAGE)-A1, MAGE-A3, or synovial sarcoma X-2 (SSX-2) in the peripheral blood of treatment-naive NPC patients. The relationship of responses among the three proteins and the human leukocyte antigen (HLA)-A types were analyzed to provide evidence of designing novel therapy.

MethodsSixty-one NPC patients admitted into the Tumor Hospital affiliated to the Xinjiang Medical University between March 2015 and July 2016 were enrolled. Mononuclear cells were isolated from the peripheral blood before any treatment. HLA-A alleles were typed with Sanger sequence-based typing technique. The T cell response to the MAGE-A1, MAGE-A3, or SSX-2 was evaluated with the Enzyme-Linked ImmunoSpot assay. Mann-Whitney U-test was used to compare the T cell responses from different groups. Spearman's rank correlation was used to analyze the relationship of T cell responses.

ResultsHLA-A*02:01, A*02:07, and A*24:02 were the three most frequent alleles (18.9%, 12.3%, and 11.5%, respectively) among the 22 detected alleles. 31.1%, 19.7%, and 16.4% of the patients displayed MAGE-A1, MAGE-A3, or SSX-2-specific T cell response, respectively. The magnitudes of response to the three proteins were 32.5, 38.0, and 28.7 SFC/10 peripheral blood mononuclear cells, respectively. The T cell response against the three proteins correlated with each other to different extent. The percentage of A*02:01 and A*24:02 carriers were significantly higher in patients responding to any of the three proteins compared to the nonresponders.

ConclusionMAGE-A1, MAGE-A3, or SSX-2-specific T cell responses were detectable in a subgroup of NPC patients, the frequency and magnitude of which were correlated.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Alleles ; Antigens, Neoplasm ; immunology ; metabolism ; Carcinoma ; immunology ; metabolism ; Female ; HLA-A Antigens ; metabolism ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Middle Aged ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; immunology ; metabolism ; Neoplasm Proteins ; metabolism ; Sarcoma, Synovial ; immunology ; metabolism ; Young Adult

OBJECTIVE: To identify differentially expressed genes in peripheral blood mononuclear cells between patients with continuous mild-to-moderate asthma and healthy controls using mRNA microarray in order to explore the underlying signaling pathways and clarify the roles of CD4+ T cells in the pathogenesis of asthma. METHODS: Global transcriptomic profiles of the CD4+ T cells were defined by using Agilent Sure Print G3 Human GE 8×60K microarray. Enrichment pathways were analyzed with Ingenuity Pathway Analysis (IPA) software. RESULTS: Compared with controls, 805 genes were up-regulated, 192 were down-regulated in asthma patients. Among these, the expression of 38 annotated genes have varied by 4 times or more. Expression of CD300A was inversely proportional to the absolute value of eosinophils (r=-0.89, P=0.02) as well as the proportion of eosinophils (r=-0.94, P=0.004), while CSF1R was inversely proportional to PD20 (r=-0.83, P=0.04) and AQLQ (r=-0.88, P=0.02) by correlation analysis. CONCLUSION Numerous pathophysiological pathways may be involved in the pathogenesis of asthma. Above findings have provided a basis for the delineation the pathogenesis of asthma.
Antigens, CD ; genetics ; Asthma ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; Case-Control Studies ; Eosinophils ; Gene Expression Profiling ; Humans ; Leukocytes, Mononuclear ; Oligonucleotide Array Sequence Analysis ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Receptors, Immunologic ; genetics ; Transcriptome

Recent studies have shown that diet can affect the body's immunity. Roughage of dairy cows consists of a variety of plant materials which make different contributions to health. This study investigated the effect of different roughages on the immunity of dairy cows. Serum, peripheral blood mononuclear cells (PBMCs), and milk samples were collected from 20 multiparous mid-lactation cows fed mixed forage (MF)- or corn straw (CS)-based diets. Expression profile analysis was used to detect the differentially expressed genes (DEGs) from PBMCs. The results showed that milk protein in the MF group increased to 3.22 g/100 ml, while that of the CS group milk was 2.96 g/100 ml; by RNA sequencing, it was found that 1615 genes were differentially expressed between the CS group and the MF group among the 24 027 analyzed probes. Gene ontology (GO) and pathway analysis of DEGs suggested that these genes (especially genes coding cytokines, chemokine and its receptors) are involved in the immune response. Results were confirmed at the protein level via detecting the levels of interleukin-2 (IL-2), IL-6, IL-10, IL-12, leptin (LEP), interferon-γ (IFN-γ), transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) in peripheral blood by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay analysis. Our data supported the conclusions that the protein content in milk of the MF group was higher than that of the CS group, the CS-based diets induced more release of cytokines than the MF-based diets in dairy cows' PBMCs, and milk protein content may be affected by cytokines.
Animals ; Cattle/immunology* ; Cytokines/physiology* ; Diet ; Female ; Gene Ontology ; Leukocytes, Mononuclear/immunology* ; Milk/chemistry* ; Transforming Growth Factor beta/physiology* ; Zea mays

Leukocyte differentiation antigens (LDAs) play important roles in the immune system, by serving as surface markers and participating in multiple biological activities, such as recognizing pathogens, mediating membrane signals, interacting with other cells or systems, and regulating cell differentiation and activation. Data mining is a powerful tool used to identify novel LDAs from whole genome. LRRC25 (leucine rich repeat-containing 25) was predicted to have a role in the function of myeloid cells by a large-scale "omics" data analysis. Further experimental validation showed that LRRC25 is highly expressed in primary myeloid cells, such as granulocytes and monocytes, and lowly/intermediately expressed in B cells, but not in T cells and almost all NK cells. It was down-regulated in multiple acute myeloid leukemia (AML) cell lines and bone marrow cells of AML patients and up-regulated after all-trans retinoic acid (ATRA)-mediated granulocytic differentiation in AML cell lines and acute promyelocytic leukemia (APL; AML-M3, FAB classification) cells. Localization analysis showed that LRRC25 is a type I transmembrane molecule. Although ectopic LRRC25 did not promote spontaneous differentiation of NB4 cells, knockdown of LRRC25 by siRNA or shRNA and knockout of LRRC25 by the CRISPR-Cas9 system attenuated ATRA-induced terminal granulocytic differentiation, and restoration of LRRC25 in knockout cells could rescue ATRA-induced granulocytic differentiation. Therefore, LRRC25, a potential leukocyte differentiation antigen, is a key regulator of ATRA-induced granulocytic differentiation.
Antigens, Differentiation ; immunology ; metabolism ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Granulocytes ; cytology ; drug effects ; immunology ; metabolism ; Humans ; Leukocytes ; cytology ; drug effects ; immunology ; metabolism ; Membrane Proteins ; antagonists & inhibitors ; immunology ; metabolism ; RNA, Small Interfering ; pharmacology ; Tretinoin ; pharmacology

OBJECTIVE: The aim of this study was to investigate the effect of saliva of patients with chronic periodontitis (CPD) on the differentiation, activation, and secretion of osteoclast-maturing mediators of macrophages. METHODS: A total of 40 saliva samples were collected from healthy donors (n=20) and severe periodontitis patients (n=20). Peripheral blood mononuclear cells (PBMCs) and THP-1 monocyte line cells were challenged with 15% saliva for 5 days. The phenotype, surface marker, and phagocytosis of macrophages were analyzed by flow cytometry and microscopy. Osteoclast-maturing mediators were assayed by using enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: When PBMCs were treated with CPD saliva for 5 days, 61.25%±11.33% of cells were transformed into large granular cells; 86.78%±13.69% of large granular cells were identified as CD14⁺⁺CD16⁺ macrophages. When THP-1 cells were treated with CPD saliva, most cells attached to the bottom of cell culture plates, thereby exhibiting macrophage morphology and releasing additional osteoclast-maturing mediators. Furthermore, the phagocytosis of THP-1 cells considerably increased in the presence of CPD saliva (66.35%±9.67%) compared with medium control (33.33%±7.52%), or healthy saliva (40.71%±3.52%). CONCLUSIONS Saliva from patients with CPD can induce macrophage differentiation, activate phagocytose microorganisms, and secrete osteoclast-maturing mediators.
Cell Differentiation ; Humans ; Leukocytes, Mononuclear ; Macrophages ; Monocytes ; Periodontitis ; immunology ; Saliva