Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.     

GICA和CLIA联合检测SARS-CoV-2特异性抗体的检测策略研究

评价胶体金免疫层析法(GICA)与化学发光法(CLIA)联合检测对降低新型冠状病毒(SARS-CoV-2)特异性抗体假阳性的效果。收集2020年1月22日至2020年3月5日就诊于川北医学院附属医院及南充市中心医院的19例SARS-CoV-2确诊患者不同时段的血清33份,55例非SARS-CoV-2、其他病原体感染及自身免疫性疾病患者的血清55份,采用GICA和CLIA分别对血清SARS-CoV-2 IgM、IgG进行检测,并对结果进行分析。GCIA检测SARS-CoV-2 IgM、IgG的敏感性分别为100.0%、94.74%,与CLIA(92.86%和100.0%)比较没有差异(P>0.05);GCIA检测SARS-CoV-2 IgM、IgG的特异性分别为70.91%、74.55%,明显低于CLIA的特异性(98.18%和89.09%)(P<0.01);两种方法检测SARS-CoV-2 IgM、IgG结果具有一致性(P<0.001),Kappa值分别为0.434,0.406;ROC曲线分析发现,GCIA检测SARS-CoV-2 IgM、IgG的AUC分别为0.855、0.846,明显低于CLIA(0.955和0.945)(P<0.05)。两种方法联合检测SARS-CoV-2 IgM、IgG的敏感性分别为92.86%、94.74%,特异性分别为100.0%、100.0%;ROC曲线分析显示,联合检测SARS-CoV-2 IgM、IgG的AUC分别为0.964、0.974,高于两种方法的单独检测。GICA和CLIA联合检测能有效提高SARS-CoV-2 IgM和IgG的检测特异性,值得临床推广应用。     

Degradation of yolk platelets in the early amphibian embryo is regulated by fusion with late endosomes

The eggs of many animal species contain a large store of yolk platelets, lipid droplets and glycogen granules; these are consumed during early embryogenesis. However, the mechanisms by which degradation of these stored materials occurs during early embryogenesis are not clearly understood. The mechanisms underlying yolk degradation in amphibian (newt) embryos were investigated. Electron microscopy using an anion marker, cationic ferritin, revealed that yolk platelets were degraded after fusion with late endosomes containing primary lysosomes. Electron microscopy and the results of experiments using a number of reagents with selective effects on intracellular transport suggested that yolk degradation activity in early amphibian embryos may be regulated at the point of fusion between late endosomes and yolk platelets.     

Calculation of NMR relaxation, covolume, and scattering-related properties of bead models using the SOLPRO computer program

The hydrodynamic properties of macromolecules and bioparticles, represented by bead models, can be calculated using methods implemented in the computer routine HYDRO. Recently, a new computer routine, SOLPRO, has been presented for the calculation of various SOLution PROperties. These include (1) time-dependent electro-optic and spectroscopic properties related to rotational diffusion, (2) non-dynamic properties like scattering curves, and (3) dimensionless quantities that combine two or more solution properties in a form which depends on the shape of the macromolecule but not on its size. In the present work we describe the inclusion of more of those types of properties in a new version of SOLPRO. Particularly, we describe the calculation of relaxation rates in nuclear magnetic resonance (NMR). For dipolar coupling, given the direction of the dipole the program calculates values of the spectral density, from which the NMR relaxation times can be obtained. We also consider scattering-related properties, namely the distribution of distances for the bead model, which is directly related to the angular dependence of scattered intensity, and the particle's longest distance. We have devised and programmed a procedure to calculate the covolume of the bead model, related to the second virial coefficient and, in general, to the concentration dependence of solution properties. Various shape-dependent dimensionless quantities involving the covolume are calculated. In this paper we also discuss some aspects, namely bead overlapping and hydration, that are not explicitely included in SOLPRO, but should be considered by the user. Received: 25 May 1998 / Revised version: 30 July 1998 / Accepted: 30 July 1998     

Tetraspanins connect several types of Ig proteins: IgM is a novel component of the tetraspanin web on B-lymphoid cells

The tetraspanins form a family of about 30 molecules mainly expressed on the cell surface. They have been reported to be involved in many physiological or pathological processes, such as fertilization, immune response, development of the nervous system, and metastasis, as well as in infectious diseases (HCV, malaria, etc.). The tetraspanins may play a role as organizers of multimolecular complexes on the cell surface associating numerous proteins, the tetraspanin web. To better define the composition of the tetraspanin web, its characterization has been recently performed using mass spectrometry and proteomics. We report the proteomic analysis of tetraspanin complexes on B-lymphoid cells. Immunoprecipitation experiments were performed using mAbs directed against the tetraspanin CD9, and associated molecules were identified by MALDI-TOF (matrix assisted laser desorption ionization time of flight) mass spectrometry. This led to the identification of IgM as a novel component of the complexes. Thus, tetraspanins may connect several types of proteins with Ig domains, including HLA-DR, EWI-2, and IgM, that may play a role in immune responses.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Detection of specific antibodies in cord blood, infant and maternal saliva and breast milk to staphylococcal toxins implicated in sudden infant death syndrome (SIDS)

The common bacterial toxins hypothesis of sudden infant death syndrome (SIDS) is that nasopharyngeal bacterial toxins can trigger events leading to death in infants with absent/low levels of antibody that can neutralise the toxins. The aim of this study was to investigate nasopharyngeal carriage of Staphylococcus aureus and determine levels of immunity in the first year of life to toxic shock syndrome toxin (TSST-1) and staphylococcal enterotoxin C (SEC). Both toxins have been implicated in SIDS cases. Seventy-three mothers and their infants (39 males and 34 females) were enrolled onto the study. The infants had birth dates spread evenly throughout the year. In infants, S. aureus carriage decreased significantly with age (P<0.001). Between 40% and 50% of infants were colonised with S. aureus in the first three months of life and 49% of the isolates produced one or both of the staphylococcal toxins. There was a significant correlation between nasopharyngeal carriage of S. aureus in mothers and infants in the three months following the birth (P<0.001). Carriage of S. aureus in infants and their mothers was not significantly associated with levels of antibody to TSST-1 or SEC in cord blood, adult saliva or breast milk. Infants colonised by S. aureus had higher levels of salivary IgA to TSST-1 than infants who were culture negative. Analysis of cord blood samples by a quantitative ELISA detected IgG bound to TSST-1 and SEC in 95.5% and 91.8% of cases respectively. There was a marked variation in levels of maternal IgG to both TSST-1 and SEC among cord blood samples. Maternal age, birth weight, and seasonality significantly affected the levels of IgG binding to TSST-1 or SEC. Analysis of infant saliva samples detected IgA to TSST-1 and SEC in the first month after birth; 11% of samples tested positive for salivary IgA to TSST-1 and 5% for salivary IgA to SEC. By the age of two months these proportions had increased to 36% and 33% respectively. More infants who used a dummy tested positive for salivary IgA to TSST-1 compared to infants who did not use a dummy. Levels of IgA to TSST-1 and SEC detected in the breast-milk samples varied greatly among mothers. There was a trend for infants receiving breast milk with low levels of antibody to TSST-1 or SEC to have higher levels of salivary antibody to the toxins. In conclusion, passive immunity to toxins implicated in SIDS cases varies greatly among infants. Infants are able to mount an active mucosal immune response to TSST-1 and SEC in the first month of life.     

Helicobacter pylori in familial clusters based on antibody profile

Studies have shown a high prevalence of Helicobacter pylori infection in close communities and that intrafamilial spread during early childhood may be a route of transmission. A total of 72 household members from 21 families were enrolled in this study. Sera from individuals showed 50/72 (69.4%) seropositive for IgG against H. pylori by ELISA. Western blots showed diversity in the protein profiles with molecular masses ranging from approximately 8 to 130 kDa. Cohen's kappa statistical analysis of the blot patterns showed that nine families demonstrated similar profiles (100%), while 4 other families showed varying similarities (17-50%). The results support the hypothesis of intrafamilial transmission of H. pylori. Furthermore, serological studies can be used as an effective approach to determine the familial status in relation to H. pylori infection.     

Production and characterization of monoclonal immunoglobulin A antibodies directed against Salmonella H:g,m flagellar antigen

Hybridomas were generated after intragastral immunization of BALB/c mice with live Salmonella suberu and subsequent fusion between isolated spleen lymphoblasts and myeloma cells. Three monoclonal antibodies (MAbs) of immunoglobulin A (IgA) isotype were selected and characterized. All of them were found to recognize the H:g epitope in enzyme-linked immunosorbent assay and immunoblotting but did not react with all H:g-expressing strains in slide agglutination test. All MAbs strongly agglutinated Salmonella enteritidis type strain and a large number of S. enteritidis clinical isolates. They were not bactericidal in the presence of complement. All hybridoma clones produced secretory IgA forms, which were found in the gastrointestinal tract of mice bearing hybridoma as a subcutaneous 'backpack' tumor or after intravenous application of purified MAbs. The IgA MAbs stability demonstrated in different tests together with their antigen specificity and strong agglutination ability make them a useful diagnostic tool for serotyping of Salmonella strains.