Induction of antigen-specific isotype switching by in vitro immunization of human naive B lymphocytes

The use of in vitro immunization technology for the generation of human antigen-specific antibodies has essentially resulted in low affinity IgM antibodies, resembling an in vivo primary immune response. We now describe a detailed reproducible protocol for a two-step in vitro immunization, which yields isotype switched, antigen-specific human antibodies. The immunizing antigen was a 30aa synthetic peptide, containing both a B (15aa V3 peptide of the HIV-1) and a T helper cell epitope (15aa peptide from tetanus toxin). The immunization protocol includes: (i) a selection procedure of donors with a memory T cell response against tetatus toxoid; (ii) immunization of mature naive peripheral B lymphocytes in two distinct phases, involving a primary and a secondary step. None of the donors which were examined after primary 7immunization showed at any time an IgG anti-V3 specific antibody response, while all the donors showed an IgM response. After the secondary immunization step, anti-V3 antibodies of both IgM and IgG isotypes were detected. The switch frequency event was high among the tested donors (5/8).     

血小板活化因子、癌胚抗原、糖类抗原724在结直肠癌中的表达及与临床病理特征的关系

目的:探讨血小板活化因子(PAF)、癌胚抗原(CEA)、糖类抗原724(CA-724)在结直肠癌(CRC)患者术前血清中的阳性表达率及与临床病理特征的关系、发病的独立危险因素。方法:选取2019年12月至2021年10月于包头医学院第一附属医院就诊的CRC患者75例为CRC组,另选取同时期在本院体检的健康志愿者75例为对照组。采用酶联免疫吸附(ELISA)法分别检测CRC患者术前及对照组的晨空腹外周静脉血PAF含量;回顾性分析上述入组患者术前血清CEA、CA-724含量,进行统计分析。结果:(1)CRC组PAF、CEA、CA-724单独检测和联合检测的阳性率分别为25.33%、42.67%、30.67%、74.67%,均高于对照组,且联合检测的阳性率均高于单独检测指标的阳性率,组间差异均具有统计学意义(P<0.05);(2)单因素方差分析结果显示,血清CEA表达水平的影响因素为患者的淋巴结转移(P<0.05);血清CA-724表达水平的影响因素为患者的年龄(P<0.05);(3)二元Logistic回归分析结果显示,PAF表达在临床病理特征之间的差异无统计学意义(P&...     

A quantitative analysis of the distribution of Purkinje cell axonal collaterals in different zones of the cat's cerebellum: an intracellular HRP study

Summary Purkinje cells in zones a and b of the vermis and zone c in the intermediate cortex of the anterior lobe were intracellularly injected with horseradish peroxidase and the distribution patterns of the varicosities present on their axonal collaterals were quantified and compared at the light microscopic level. The data derived from this study reveal that each individual axonal arbor had a unique pattern of distribution. However, certain principles of distribution could be applied to the collaterals regardless of the cortical location of the parent cell, including the following: 1) the total number of varicosities derived from the axonal collaterals of individual Purkinje cells is relatively uniform; 2) the collateral plexi have a greater extent in the sagittal plane as compared to the transverse plane; 3) the majority of varicosities remain within 200–300 m of the parent cell in both the sagittal and transverse planes; 4) there is a heterogenous distribution of varicosities within the area traversed by the axonal branches; and 5) the majority of varicosities are located within the Purkinje cell layer. Although there were similarities in the pattern of distribution for collaterals, there were also variations which distinguished the plexi in the three zones. The collaterals of zone a cells tended to be the most confined in both the sagittal and transverse planes. In contrast, several cells in zone b and c had branches that extended for relatively long distances in the sagittal plane. In zone b the collaterals have an asymmetric distribution around the cell of origin in the transverse plane. In zones a and c there is a tendency for a more symmetric pattern of distribution in this plane. The similarities in the number and laminar distribution of varicosities, as well as the predominantly sagittal orientation of the collaterals in all zones of the cerebellar cortex suggest that the collaterals subserve a common function throughout the cerebellar cortex. However, variations indicate that there may be subtle differences in the way recurrent collaterals process information in the cerebellar cortex that may be related to the functional heterogeneity or the location of the targets of the collaterals in the three zones analyzed.     

Distribution of 2-naphthol sulphotransferase and its endogenous substrate adenosine 3′-phosphate 5′-phosphosulphate in human tissues

The activity of sulphotransferase towards 2-naphthol and the concentration of its endogenous substrate, adenosine 3'-phosphate 5'-phosphosulphate (PAPS), have been measured in five specimens of human liver, lung, and kidney, and the mucosa from the ileum and the ascending, descending and sigmoid colon. The activity of 2-naphthol sulphotransferase (mean nmol.min-1.mg-1 protein) was 1.82 (liver); 0.034 (kidney); 0.19 (lung); 0.64 (ileum); 0.47 (ascending colon); 0.50 (descending colon); 0.40 (sigmoid colon). The concentration of PAPS (mean nmol.g-1 wet tissue) was 22.6 (liver); 4.8 (kidney); 4.3 (lung); 12.8 (ileum); 8.1 (ascending colon); 7.5 (descending colon); 6.2 (sigmoid colon). The concentration of PAPS and the activity of 2-naphthol sulphotransferase were higher in the liver than in the extrahepatic tissues. There was significant difference between ileum and ascending colon, both the activity of sulphotransferase and the concentration of PAPS being higher in the former. 2-Naphthol sulphotransferase activity and the concentration of PAPS have consistent distribution patterns. Differences between the tissues studied were more marked for sulphotransferase than for its endogenous substrate.     

The molecular basis of classic Ehlers-Danlos syndrome: a comprehensive study of biochemical and molecular findings in 48 unrelated patients

Classic Ehlers-Danlos syndrome (EDS) is characterized by fragile and hyperextensible skin, atrophic scarring, and joint hypermobility. Mutations in the COL5A1 and the COL5A2 gene encoding the alpha1(V) and the alpha2(V) chains, respectively, of type V collagen have been shown to cause the disorder, but it is unknown what proportion of classic EDS patients carries a mutation in these genes. We studied fibroblast cultures from 48 patients with classic EDS by SDS-PAGE for the presence of type V collagen defects. An abnormal collagen pattern was detected in only 2 out of 48 cell lines, making this a poor method for routine diagnostic evaluation. A total of 42 out of 48 (88%) patients were heterozygous for an expressed polymorphic variant in COL5A1. cDNA from 18 (43%) of them expressed only one COL5A1 allele. In 37 patients, the COL5A1/A2 genes were then analyzed by SSCP and conformation sensitive gel electrophoresis (CSGE). A total of 26 patients that were mutation-negative after SSCP/CSGE screening were reanalyzed by dHPLC. In addition, 11 other patients were analyzed by dHPLC only. In total, 17 mutations leading to a premature stop codon and five structural mutations were identified in the COL5A1 and the COL5A2 genes. In three patients with a positive COL5A1 null-allele test, no causal mutation was found. Overall, in 25 out of 48 patients (52%) with classic EDS, an abnormality in type V collagen was confirmed. Variability in severity of the phenotype was observed, but no significant genotype-phenotype correlations emerged. The relatively low mutation detection rate suggests that other genes are involved in classic EDS. We excluded the COL1A1, COL1A2, and DCN gene as major candidate genes for classic EDS, since no causal mutation in these genes was found in a number of patients who tested negative for COL5A1 and COL5A2.     

1519例胎儿心电图的临床价值和相关因素分析

目的对1519例胎儿全部实行常规胎儿心电图(FECG)监测工作,探讨胎儿心电图应用情况,并对异常FECG结合临床,进行临床原因分析.方法采用随机湘样方法,应用杭州产FECG-D型胎儿心电图分析仪进行监测.结果发现采用横导联方式进行监测检出率为53.5%:在孕22w进行监测检出率为45.8%;异常FECG检出率为7.64%.结论对1519例FECG的监测发现采用横导联方式并在孕22w～26w期间进行监测检出率最高、波形效果好.并对监测发现的导致FECG异常改变的临床原因与胎儿宫内窘迫的关系进行浅要分析.     

Candidate genes associated with ageing and life expectancy in the Jerusalem longitudinal study

In an exploratory study, 11 common polymorphisms were examined for contributing to longevity including: apolipoprotein E (apoE), methylenetetrahydrofolate reductase (MTHFR), cathepsin D (CAD), superoxide dismutase 2 (SOD2), angiotensinogen (AGT) and insulin-like growth factor 2 (IGF2), Leiden factor 7, p53 oncogene, dopamine D4 receptor (DRD4) and the serotonin transporter (SERT). Genotype and allele frequencies of these genes were compared in 224 older (75 years) Jewish Jerusalem residents of Ashkenazi ethnicity to a group of 441 younger subjects (22 years). Nominally significant results provide suggestive evidence in the Ashkenazi group that apoE, MHTFR, SOD2, IGF2 ApaI, and factor VII are risk factors for a single outcome, survival to 75. Overall, the more genetically homogenous Ashkenazi ethnic group showed evidence for association in five genes examined suggesting that future studies in this population would gainfully focus on this ethnic group.     

Virion-bound ICAM-1 and activated LFA-1: a combination of factors conferring resistance to neutralization by sera from human immunodeficiency virus type 1-infected individuals independently of the disease status and phase

The role of the supplementary interaction between virion-bound host ICAM-1 and LFA-1 on target cells in sensitivity to neutralization of human immunodeficiency virus type 1 (HIV-1) is poorly studied. Serum samples from four long-term nonprogressors (LTNPs) and sequential sera from one progressor were used to assess neutralization sensitivity of isogenic ICAM-1-negative and ICAM-1-bearing HIV-1(NL4-3), a prototype of T-cell-line-adapted viruses. We found that virus neutralization sensitivity to the studied sera is not modified by the additional interaction between virally embedded ICAM-1 and LFA-1 under an inactive state. However, expression on the target cell surface of an activated LFA-1 form renders ICAM-1-bearing virus particles, but not viruses devoid of ICAM-1, more refractory to neutralization by sera from three out of four LTNPs and all sequential sera from the person who has experienced a progression of the HIV-1-associated disease. Although no conclusive correlation could be drawn between virus susceptibility to neutralization and the disease status or stages of HIV-1 infection, these findings demonstrate that other nonspecific virus-cell interactions mediated by virion-anchored host proteins and their normal cognate ligands on target cells represent factors that can affect the mechanism of HIV-1 neutralization.     

A Novel Mutation in the GATA1 Gene Associated with Acute Megakaryoblastic Leukemia in a Korean Down Syndrome Patient

Although acquired mutations in the GATA1 gene have been reported for Down syndrome-related acute megakaryoblastic leukemia (DS-AMKL) in Caucasians, this is the first report of a Korean Down syndrome patient with AMKL carrying a novel mutation of the GATA1 gene. A 3-yr-old Korean girl with Down syndrome was admitted to our hospital complaining of pallor and fever. The findings of a peripheral blood smear and bone marrow study were compatible with the presence of AMKL. A chromosome study showed 48,XX,-7,+21c,+21,+r[3]/47,XX,+21c[17]. Following GATA1 gene mutation analysis, a novel mutation, c.145dupG (p.Ala49GlyfsX18), was identified in the N-terminal activation domain of the GATA1 gene. This mutation caused a premature termination at codon 67 and expression of an abnormal GATA-1 protein with a defective N-terminal activation domain, and the absence of full-length GATA-1 protein. This case demonstrates that a leukemogenic mechanism for DS-AMKL is contributed by a unique collaboration between overexpressed genes from trisomy 21 and an acquired GATA1 mutation previously seen in Caucasians and now in a Korean patient.