Effect of IgG dimer concentration on the binding ability of IgG Fc fragment in IVIG to THP-1 cell surface receptors / 中国输血杂志

【Objective】 To investigate the effect of immunoglobulin G (IgG) dimer concentration of intravenous immunoglobulin (IVIG) on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Methods】 Firstly, protein purification and high performance liquid chromatography (HPLC) were used to prepare different concentrations of IgG dimers. After that, IgG dimer was added to IVIG to prepare IVIG containing different concentrations of IgG dimer. Finally, based on the method established in our laboratory, we analyzed the effect of IgG dimer concentration in IVIG on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Results】 When the concentration of IgG dimer in IVIG was 1.11%-10.30%, its binding ability to Fc receptors on the surface of THP-1 cell was 97.67%-135.33%, and this binding ability was positively correlated with the concentration of IgG dimer. When the IgG dimer concentration exceeded 13.22%, the binding ability had no correlation with the IgG dimer concentration. 【Conclusion】 A certain concentration of IgG dimer can promote the binding ability of the IgG Fc fragment in IVIG to receptors on the surface of THP-1 cells, which needs further verification from animal experiments and clinical data.

Diversity of IgG autoantibodies of 4 Chinese intravenous immunoglobulins / 中国输血杂志

【Objective】 To develop methods to display the IgG autoantibody repertoire of intravenous immunoglobulin (IVIG) products, analyze the different types of antibodies and study the diversity of IgG autoantibody in 4 IVIG preparations from different Chinese manufacturers. 【Methods】 Two-dimensional gel electrophoresis and immunoblotting with human umbilical vein endothelial cell (HUVEC) proteins were used to demonstrate the IgG autoantibody repertoire and the human protein microarray with bioinformatics analysis was employed to profile the immune reactive autoantigens of the 4 IVIG preparations. 【Results】 The methods to showcase the autoantibody repertoire and study the antibody diversity of IVIG were successfully established. High-quality repertoires of IVIG autoantibodies and biological information about self-proteins that can be recognized were obtained. There was a significant difference in the recognition of the quantity and variety of the self-antigens by different IVIG products. The number of antibodies against HUVEC proteins in four products ranged from 241-386. The number of proteins recognized on the human protein chip ranged from 292-435, with 172 human self-proteins recognized by all four products. 【Conclusion】 Demonstration of antibody repertoire and protein chip technology can be used to analyze IVIG products′ IgG autoantibody repertoire. All four preparations tested in this study exhibited a broad spectrum of antibodies against HUVEC proteins and human proteome microarray, each product had its unique antibody repertoire characteristics.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses of these vaccines and the development of new variant-derived ones1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells (MBCs)5-9. It remains unclear, however, whether the additional doses induce germinal centre (GC) reactions where reengaged B cells can further mature and whether variant-derived vaccines can elicit responses to novel epitopes specific to such variants. Here, we show that boosting with the original SARS- CoV-2 spike vaccine (mRNA-1273) or a B.1.351/B.1.617.2 (Beta/Delta) bivalent vaccine (mRNA-1273.213) induces robust spike-specific GC B cell responses in humans. The GC response persisted for at least eight weeks, leading to significantly more mutated antigen-specific MBC and bone marrow plasma cell compartments. Interrogation of MBC-derived spike-binding monoclonal antibodies (mAbs) isolated from individuals boosted with either mRNA-1273, mRNA-1273.213, or a monovalent Omicron BA.1-based vaccine (mRNA-1273.529) revealed a striking imprinting effect by the primary vaccination series, with all mAbs (n=769) recognizing the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted approach, we isolated mAbs that recognized the spike protein of the SARS-CoV-2 Omicron (BA.1) but not the original SARS-CoV-2 spike from the mRNA-1273.529 boosted individuals. The latter mAbs were less mutated and recognized novel epitopes within the spike protein, suggesting a naive B cell origin. Thus, SARS-CoV-2 boosting in humans induce robust GC B cell responses, and immunization with an antigenically distant spike can overcome the antigenic imprinting by the primary vaccination series.

Germinal centre-driven maturation of B cell response to SARS-CoV-2 vaccination

Germinal centres (GC) are lymphoid structures where vaccine-responding B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells (BMPCs)1-4. Induction of the latter is a hallmark of durable immunity after vaccination5. SARS-CoV-2 mRNA vaccination induces a robust GC response in humans6-8, but the maturation dynamics of GC B cells and propagation of their progeny throughout the B cell diaspora have not been elucidated. Here we show that anti-SARS-CoV-2 spike (S)-binding GC B cells were detectable in draining lymph nodes for at least six months in 10 out of 15 individuals who had received two doses of BNT162b2, a SARS-CoV-2 mRNA vaccine. Six months after vaccination, circulating S-binding MBCs were detected in all participants (n=42) and S-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of single-cell RNA sequencing of responding blood and lymph node B cells from eight participants and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1540 S-specific B cell clones. SHM accumulated along the B cell differentiation trajectory, with early blood plasmablasts showing the lowest frequencies, followed by MBCs and lymph node plasma cells whose SHM largely overlapped with GC B cells. By three months after vaccination, the frequency of SHM within GC B cells had doubled. Strikingly, S+ BMPCs detected six months after vaccination accumulated the highest level of SHM, corresponding with significantly enhanced anti-S polyclonal antibody avidity in blood at that time point. This study documents the induction of affinity-matured BMPCs after two doses of SARS-CoV-2 mRNA vaccination in humans, providing a foundation for the sustained high efficacy observed with these vaccines.

SARS-CoV-2 mRNA vaccination elicits robust and persistent T follicular helper cell response in humans

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we show that frequency of TFH correlates with that of S-binding germinal center B cells. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLADPB1* 04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

The plasmablast response to SARS-CoV-2 mRNA vaccination is dominated by non-neutralizing antibodies that target both the NTD and the RBD

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast derived mAbs from subjects who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, that the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to seasonal human coronaviruses OC43 and HKU1 spike proteins. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine induced RBD binding antibodies may provide substantial protection against viral variants carrying E484K.

A public vaccine-induced human antibody protects against SARS-CoV-2 and emerging variants

The emergence of antigenically distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility is a public health threat. Some of these variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies, which principally target the receptor binding domain (RBD) on the virus spike glycoprotein. Here, we describe 2C08, a SARS-CoV-2 mRNA vaccine-induced germinal center B cell-derived human monoclonal antibody that binds to the receptor binding motif within the RBD. 2C08 broadly neutralizes SARS-CoV-2 variants with remarkable potency and reduces lung inflammation, viral load, and morbidity in hamsters challenged with either an ancestral SARS-CoV-2 strain or a recent variant of concern. Clonal analysis identified 2C08-like public clonotypes among B cell clones responding to SARS-CoV-2 infection or vaccination in at least 20 out of 78 individuals. Thus, 2C08-like antibodies can be readily induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern. One Sentence SummaryProtection against SARS-CoV-2 variants by a potently neutralizing vaccine-induced human monoclonal antibody.

Increased complement activation is a distinctive feature of severe SARS-CoV-2 infection.

Complement activation has been implicated in the pathogenesis of severe SARS-CoV-2 infection. However, it remains to be determined whether increased complement activation is a broad indicator of critical illness (and thus, no different in COVID-19). It is also unclear which pathways are contributing to complement activation in COVID-19, and, if complement activation is associated with certain features of severe SARS-CoV-2 infection, such as endothelial injury and hypercoagulability. To address these questions, we investigated complement activation in the plasma from patients with COVID-19 prospectively enrolled at two tertiary care centers. We compared our patients to two non-COVID cohorts: (a) patients hospitalized with influenza, and (b) patients admitted to the intensive care unit (ICU) with acute respiratory failure requiring invasive mechanical ventilation (IMV). We demonstrate that circulating markers of complement activation (i.e., sC5b-9) are elevated in patients with COVID-19 compared to those with influenza and to patients with non-COVID-19 respiratory failure. Further, the results facilitate distinguishing those who are at higher risk of worse outcomes such as requiring ICU admission, or IMV. Moreover, the results indicate enhanced activation of the alternative complement pathway is most prevalent in patients with severe COVID-19 and is associated with markers of endothelial injury (i.e., Ang2) as well as hypercoagulability (i.e., thrombomodulin and von Willebrand factor). Our findings identify complement activation to be a distinctive feature of COVID-19, and provide specific targets that may be utilized for risk prognostication, drug discovery and personalized clinical trials. SUMMMARYComplement has been implicated in COVID-19. However, whether this is distinctive of COVID-19 remains unanswered. Ma et al report increased complement activation in COVID-19 compared to influenza and non-COVID respiratory failure, and demonstrate alternative pathway activation as a key marker of multiorgan failure and death.

The research status of using of physical restraint and restraint minimization act / 中国实用护理杂志

Physical restraint is a conventional nursing practice at home and abroad. AS the use of physical restraint in the hospital, the risk has been increasingly emerging. Many scholars have already realized the necessity of Restraint Minimization Act and the voices of reducing the using of physical restraint run high constantly. Therefore, this paper would review the status quo of the using of physical restraint, advantages and disadvantages, ethical issue, influencing factors and restraint minimization act domestic and international, so as to provide help and support for further research.

The clinical significance of combined heart lung ultrasound on severe left heart failure with pulmonary hypertension / 中华医学超声杂志（电子版）

Objective To investigate the clinical significance of combined heart and lung ultrasound in patients with severe left heart failure and pulmonary hypertension. Methods From March 2016 to June 2017, 75 patients with grade Ⅲ and Ⅳ heart failure and dyspnea were enrolled in Qingdao Municipal Hospital Affiliated to Qingdao University. Thirty-three patients had normal pulmonary artery pressure (normal pulmonary arterial pressure group), 25 patients had mild pulmonary hypertension (mild pulmonary hypertension group), and 17 patients had moderate to severe pulmonary hypertension (moderate to severe pulmonary hypertension group). The patient′s plasma B-type natriuretic peptide (BNP) was measured. Left ventricular diameter (LVD), right ventricular diameter (RVD), and left ventricular ejection fraction (LVEF) were measured by echocardiography. The patient′s lungs were observed by lung ultrasonography, and its number was recorded. One-way analysis of variance was used to compare the differences of LVD, RVD, and LVEF in three groups of patients with severe left heart failure. Further comparison between groups was performed using LSD-t test. Kruskal-wallis H test was used to compare the plasma BNP concentration and B-line number in three groups of patients with severe left heart failure. The Mann-Whitney U test was used to further compare the groups. The receiver operating characteristic (ROC) curve of pulmonary hypertension diagnosed by plasma BNP concentration and B line number in patients with severe left heart failure were drwan. Results The concentrations of BNP in patients with normal pulmonary arterial pressure, mild pulmonary hypertension, and moderate to severe pulmonary hypertension were 890 (614, 1516), 1460 (1245, 1950), and 2660 (1670, 3279) ng/L, respectively. The number of B line was 12 (9, 16), 17 (14, 18), 26 (20, 28), and the RVD was (22.1±1.7), (24.9±2.0), (26.3±2.8) mm, respectively. The number of B-line and RVD in the moderate-severe pulmonary hypertension group were both lager than those in the mild pulmonary hypertension group, and the number of B-line and RVD in the mild pulmonary hypertension group were both lager than those in the normal pulmonary artery pressure group. There was significant difference between any two groups (BNP concentration: U=210.500, P < 0.05; U=47.000, 73.000, both P < 0.001;B line number:U=189.000,P < 0.05;U=38.5000,64.000,both P < 0.001;RVD:t=0.553, 0.623, both P<0.001; t=0.656, P<0.05). There was no significant difference in LVD and LVEF between the three groups of patients. The ROC curve showed that the optimal threshold for the diagnosis of pulmonary hypertension in patients with severe left heart failure with BNP concentration was 1225 ng/L. The sensitivity was 85.7%,the specificity was 69.7%,the area under the curve was 0.814,and the 95% CI was 0.717 to 0.911. The optimal threshold for diagnosis of pulmonary hypertension in patients with severe left heart failure was B line number 14, the sensitivity was 88.1%, specificity was 66.7%, the area under the curve was 0.836, and 95%CI was 0.747 to 0.925.Conclusion Patients with severe left heart failure at different pulmonary artery pressure levels have different B-line findings, and the number of B-line increases with the severity of pulmonary hypertension, which warrants further study and application.

Acoustic radiation force impulse elastography in differential diagnosis of breast masses / 中华超声影像学杂志

ObjectiveTo explore the application value of acoustic radiation force impulse(ARFI) in the differential diagnosis of breast lesions.Methods Eighty six patients with 107 lesions were performed ARFI elastography.The stiffness of the masses in virtual touch tissue image(VTI) were scored.The pathological diagnosis as the gold standard,drawed the ROC curve to find the cut off point of area ratio to predict breast cancer.Shear wave velocity(SWV) within the breast masses were measured using virtual touch tissue quantification(VTQ) technique.ResultsThe elastography hardness score higher than grade 4 was mainly distributed in the malignancies compared to benign lesions( P ＜0.01).1.53 as the cut-off point of area ratio,sensitivity and specificity of diagnosing breast cancer were 95.7 ％,92.5 ％.SWV of malignant group higher than benign group,the difference was statistically significant (P ＜ 0.001 ).Conclusions Hardness score in VTI imaging,area ratio of benign and malignant lesions,SWV within the breast masses can help the differential diagnosis of breast lesions.

A novel approach for identifying the heme-binding proteins from mouse tissues / 基因组蛋白质组与生物信息学报·英文版

Heme is a key cofactor in aerobic life, both in eukaryotes and prokaryotes. Because of the high reactivity of ferrous protoporphyrin IX, the reactions of heme in cells are often carried out through heme-protein complexes. Traditionally studies of heme-binding proteins have been approached on a case by case basis, thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues. The procedure described here is aimed at profiling heme-binding proteins in mouse tissues sequentially by 1) purification of heme-binding proteins by heme-agarose, an affinity chromatographic resin; 2) isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis; 3) identification of heme-binding proteins by mass spectrometry. In five mouse tissues, over 600 protein spots were visualized on 2-DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF, in which most proteins belong to heme related. This methodology makes it possible to globally characterize the heme-binding proteins in a biological system.

The epitope study on the SARS-CoV nucleocapsid protein / 基因组蛋白质组与生物信息学报·英文版

The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS.