Competitive inhibition enzyme immunoassays for the measurement of human IgG, IgA and IgM

Competitive-inhibition enzyme immunoassays for the measurement of human IgG, IgA and IgM are described. These assays can be readily performed with commercial antisera and a recently developed method for purifying human IgA and IgM with high yield. The assays described are specific, with undetectable (<0.5%) cross-reactivity between the immunoglobulin classes in all systems, except with purified IgM, which cross-reacted to 1.9% with the IgG enzyme immunoassay.Minimal detectable concentrations of 2.5±0.8 ng/ml for IgG 4.2±0.9 ng/ml for IgA and 7.2±1.4 ng/ml for IgM were recorded, indicating that these assays are particularly sensitive. There is little within-assay variation (mean coefficient of variation = 3.9%), although the between-assay variation was substantially greater (mean coefficient of variation = 23.5%). These assay systems appear to be particularly suited to the measurement of immunoglobulin production by lymphocytes in culture. In such studies the assay must be specific, sensitive and be capable of discriminating between levels of immunoglobulin produced in response to various experimental treatments.     

Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA)

Competitive-inhibition enzyme immunoassays for the measurement of human IgG, IgA and IgM are described. These assays can be readily performed with commercial antisera and a recently developed method for purifying human IgA and IgM with high yield. The assays described are specific, with undetectable (<0.5%) cross-reactivity between the immunoglobulin classes in all systems, except with purified IgM, which cross-reacted to 1.9% with the IgG enzyme immunoassay.

Minimal detectable concentrations of 2.5±0.8 ng/ml for IgG 4.2±0.9 ng/ml for IgA and 7.2±1.4 ng/ml for IgM were recorded, indicating that these assays are particularly sensitive. There is little within-assay variation (mean coefficient of variation = 3.9%), although the between-assay variation was substantially greater (mean coefficient of variation = 23.5%). These assay systems appear to be particularly suited to the measurement of immunoglobulin production by lymphocytes in culture. In such studies the assay must be specific, sensitive and be capable of discriminating between levels of immunoglobulin produced in response to various experimental treatments.     

Cryopreservation of human embryos and oocytes

The success rate of human embryo cryopreservatlon depends ontechnical and embryonic parameters. First of all, the cryoprotectantcan affect embryo survival as we found by comparing two freeze-thawprocedures using propanediol (PROH) (1.5 mol) alone or withsucrose (0.1 mol). Embryo survival was significantly enhancedwith sucrose (62 versus 32%). Embryo quality is another majorparameter involved in the success of freezing; the rates ofpositive survival were found to be 67% for morphologically normalembryos versus 49% for embryos with fragments (P < 0.001).The efficiency of embryo cryopreservatlon in an IVF programmecould be estimated in 1986: a woman with extra embryos, storedafter transfer of 3–4 fresh embryos (16% of all cydes),can expect a 22% pregnancy rate per transfer of fresh embryosand a 32% pregnancy rate per collection after transfer of thestored eggs. A comparative study of the cryopreservability ofimmature or mature oocytes was performed in humans. Human oocyteshave a low survival rate (36%) whatever the cryopreservationprotocol or the initial maturation stage. Immature human oocytescould survive freezing and thawing, mature and be fertilizedin vitro, but with a very low efficiency.     

Are clinical and biological IVF parameters correlated with chromosomal disorders in early Life: a multicentric study

A multicentric study was carried out to analyse in a large series:(i) the chromosomal status of unfertilized oocytes, (ii) errorsat fertilization and (iii) the chromosomal complement of cleavedembryos. Parameters such as type of sterility, maternal age,stimulation treatment, doses of gonadotrophins administeredand oocyte preincubation time before insemination were studiedin relation to the incidence of chromosome abnormalities. Twenty-sixper cent of the unfertilized oocytes and 29.2% of the embryoshad chromosome anomalies. Maternal age significantly increasedthe rate of aneuploidy in oocytes: 38% in patients over 35 years(versus 24% in younger patients). Fertilization-related abnormalitieswere significant, i.e. 1.6% parthenogenesis and 6.4% polyploidy.Unexplained infertility was correlated with an increase in therate of parthenogenesis (4.2%) when compared with tubal infertility(1.2%). Triploidy was found to be correlated with three parameters.A lower rate of triploidy was observed in the group of couplesreferred because of male sterility (1.9% versus 6.3% for tuba1sterility), in HMG-treated patients (2.4% versus 7% with analoguesof LHRH/HMG) and with a short 2-h preincubation time beforeinsemination (3% versus 7.2% for > 2 h). A general modelfor natural selection against embryos carrying a chromosomeimbalance was proposed.     

Human prolactin release induced by follicle stimulating hormone, luteinizing hormone and human chorionic gonadotrophin

Plasma prolactin levels rise in stimulated cycles. To clarifythe effects of gonadotrophin on the lactotrophs, three studieswere performed. First, plasma concentrations of prolactin duringclomiphene citrate (CC)-human menopausal gonadotrophin (HMG)-humanchononic gonadotrophin (HCG) treatment of women enrolled forin-vitro fertilization (IYF) were compared with those duringHMG-HCG administration while under pituitary suppression witha gonadotrophin releasing hormone (GnRH) analogue (buserelin).Women suppressed with buserelin had higher basal levels of PRLin plasma (14.4 ± 4.3 nglml versus 6.9 ± 1.4 ng/ml,P<0.001). Only buserelin-suppressed women showed a significantrise in plasma prolactin before HCG administration, while bothpatient groups had marked prolactin peaks after HCG injection.This peak was higher in the buserelin group (71.9 ± 50.7ng/ml versus 52.6 ± 29.7 ng/ml). The second study showedthat plasma levels of prolactin of 6 post- menopausal womenwere significantly increased 48 h after an injection of 5000IU HCG, i.m. (24.9 ± 17.4 ng/ml versus 12.4 ±6.2 ng/ml P<0.05). Third, plasma prolactin was studied in5 women over 30 days after surgical castration. An upward trendwas observed similar to that of endogenous gonadotrophin, withthe change in prolactin values closely correlating with thechange in concentrations of follicule stimulating hormone (P<0.005).All these findings suggest that human gonadotrophins stimulatelactotrophs.     

医用生物力学参数的数字化与数字医学

目的了解医用生物力学研究中,各种科技参数进行数字化处理的必要性、可行性和迫切性;阐明国内外数字人和数字医学研究的发展过程;展望数字医学今后的发展前景。方法收集了数字人和数字医学各阶段发展资料;分析了这一领域的科研工作优点和存在的问题。结果整理了国内外数字人研究发展简要资料,提出了数字医学今后发展的主要方向和应重视的事项。结论数字化可以提高医用生物力学科研的质量和效率,数字医学有重大发展前景。     

Prevalence and genotype distribution of human papillomavirus in women living with HIV/AIDS in an area of Northeast Brazil

Women infected by human immunodeficiency virus (HIV) are more likely to manifest oncogenic viral infections including human papillomavirus (HPV). It was investigated the HPV prevalence, genotype distribution and HPV relationship with cervical lesions among women living with HIV in Sergipe state, Northeast Brazil. A prevalence survey was conducted including 270 HIV-infected women who attended the reference center for HIV in Sergipe from August 2014 to November 2017. Cervical samples were processed by the polymerase chain reaction for HPV-DNA detection. Among the 270 HIV-infected women, 190 (70.4%) were between 26 and 49 years old and 159 (55.6%) were coinfected with HPV. Among the coinfected women, 24 viral types were identified; 113 (72%) subjects had high-risk HPV types, and the most prevalent was HPV 16 (53/35.3%). Positive HPV status was statistically associated with having 0 to 8 years of schooling compared with ≥9 years of schooling; and have been diagnosed with HIV infection less than 5 years ago compared with more than 10 years. Cytological abnormalities were found in 13.4% (31/231) of women, most with high-grade squamous intraepithelial lesions (16/51.6%). However, of women who had no cytological lesions or malignancy (200/86.6%), almost half were HPV DNA-positive (99/49.5%). In conclusion, the prevalence of HPV among women living with HIV in Sergipe was high. There was a high frequency of high-risk HPV infection, and a wide diversity of genotypes were detected, with HPV 16 being the most frequent.     

Description of novel class I alleles encountered during routine registry typing in 2007

Twenty-six novel human leukocyte antigen (HLA) class I alleles are described: 3 HLA-A alleles, 19 HLA-B alleles and 4 HLA-C alleles. Only one of the novel alleles (HLA-B*0753) was found in multiple individuals and likely is not uncommon in the population. Nineteen (∼70%) of the 26 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Four of these single nucleotide variants are silent substitutions, and one creates a null allele. The remaining novel alleles differ from their most similar allele by two to six nucleotide substitutions. Some of the new alleles encode novel codons and unique amino acid changes at polymorphic positions in the HLA-B lows (codons 30, 67 and 72), while HLA-Cw*0347 encodes an amino acid change at a position not previously reported to be polymorphic for this locus.     

Isolation and culturing of hepatocytes from human livers

Summary Using a modified collagenase digestion procedure, we were successful in the isolation of viable hepatocytes from human liver surgical wastes, unused transplantable livers, and diseased livers from transplant recipients. The modified procedures for isolation involved collagenase perfusion of a sample of limited size (<50 g) with only one cut surface, perfusion via only one blood vessel, the use of an appropriate amount of collagenase, as well as manual massaging of the liver sample during critical stages of the collagenase perfusion. Using this modified procedures, we had close to 100% success rate in the isolation of viable hepatocytes from the human liver samples.     

The interactions between human dendritic cells and microbes; possible clinical applications of dendritic cells

The dendritic cells comprise several subsets that induce and regulate the immune responses against foreign and self-antigens, and that can therefore function as initiators of protective immunity and inducers of central or peripheral tolerance. The different subpopulations of dendritic cells interact with and also influence other cell populations of the immune system, such as T and B lymphocytes and natural killer cells. The factors that determine the given dendritic cell functions depend on the state of maturation and the local microenvironment. The interactions between dendritic cells and microorganisms are rather complex, but progress in the past few years has shed light on several aspects of these interactions. This review lays emphasis on the interactions between human dendritic cells, important components of the intima of arterial specimens at areas predisposed to atherosclerotic lesions, and Chlamydia pneumoniae and cytomegalovirus, the human pathogens most strongly implicated in the development of atherosclerosis. In addition, several examples of the potential clinical applications of dendritic cells are described.Received 13 January 2004; accepted by A. Falus 22 March 2004     

Comparison between human serum and Albuminar-20 (TM) supplement for in-vitro fertilization

Patient or fetal cord serum is commonly used as a protein supplement to culture media used in in-vitro fertilization (IVF). To eliminate the variability and possible hazards related to the use of human serum, a well-defined protein supplement, Albuminar-20 (Armour Pharmaceutical Cy) was evaluated as a substitute for serum. Prior to its application in the human, Earle's culture media supplemented with 0.5% (w/v) bovine serum albumin, 8% (v/v) decomplemented patient serum or 2.25% (v/v) Albuminar-20 were compared in a mouse bioassay. For the three different conditions, the percentages of blastocysts formed after 120 h in-vitro culture were respectively 91.2, 85.2 and 87.8% (NS). In the human IVF, a controlled comparison was performed from October to December 1988, between Earle's medium supplemented with patients' serum or Albuminar-20. When oocytes and spermatozoa were cultured in these two media, the fertilization rates were similar, 58.9% in human serum versus 59.4% in Albuminar-20. After further culture, the morphological quality of the cleaved embryos was better in the embryos cultured in Albuminar-20. The higher pregnancy rate in Albuminar-20 was correlated with the better morphological appearance of the embryos and their more advanced cleavage stage at the time of transfer. Therefore, Albuminar-20 can be considered as a suitable protein supplement in human IVF.     

Characterization of 104 novel alleles at the HLA-A, -B, and -DRB1 loci from National Marrow Donor Program volunteer donors

One hundred and four novel human leukocyte antigen (HLA) alleles are described from volunteer donors of the National Marrow Donor Program: 37 HLA-A alleles, 37 HLA-B alleles, and 30 HLA-DRB1 alleles. Seventeen (∼16%) of the novel alleles were found in multiple individuals and likely are relatively common in the population. Seventy-two (∼69%) of the 104 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Nine of these single nucleotide variants are silent substitutions and three create null alleles. The remaining novel alleles differ from their most similar allele by two to seven nucleotide substitutions. Some of the novel alleles encode amino acid changes at positions not previously reported to be polymorphic, such as codons 6 and 11 in HLA-A alleles and codons 5, 105, and 141 in HLA-B alleles. Interestingly, one of the novel HLA-DRB1 alleles (*1471) has a change that is not the typical glycine/valine dimorphism at codon 86, which plays a key role in peptide binding to DR molecules. This is only the second DRB1 allele described that encodes an amino acid other than glycine or valine at this position.     

Immunoglobulin VH gene expression in human B cell lines and tumors: biased VH gene expression in chronic lymphocytic leukemia

We have studied frequencies of VH gene utilization in a panel of monoclonal Epstein-Barr virus (EBV)-transformed B cell lines derived from human adult and fetal tissues as well as in monoclonal B cells obtained from fresh chronic lymphocytic leukemia (CLL) samples. The results show that IgM-secreting EBV cell lines from both fetal and adult tissues utilize VH genes from particular families roughly in proportion to estimated family size, suggesting that the repertoire of sigM-positive B cells in both fetal and adult organs is 'normalized' with respect to the V(H) gene family. In contrast, we find a highly biased pattern of VH gene expression in CLLs. The significance of these findings is discussed in the context of mechanisms that could be involved in normal B cell repertoire development and in the process of malignant transformation of precursors of CLL.     

Comparison of the seroprevalence of human metapneumovirus and human respiratory syncytial virus

Human metapneumovirus (hMPV) is a virus that induces human respiratory syncytial virus (hRSV)-like illnesses, ranging from upper respiratory tract infection to severe bronchiolitis and pneumonia. The 100 serum samples from children aged 1 month to 5 years were tested for the presence of hMPV and hRSV antibodies using an indirect immunofluorescence assay and a neutralizing-antibody assay, respectively. The seroprevalence of hMPV was significantly lower than that of hRSV in children over 4-months-old (43% vs. 60%, P < 0.025), and the difference was particularly notable between the ages of 4 months and 1 year (11% vs. 48%, P = 0.006). The results suggest that primary infection with hMPV occurs somewhat later than that with hRSV.     

Replication of human syncytium-forming virus in human cells: effect of certain biological factors and selective chemicals

The growth characteristics of the human syncytium-forming virus (HSFV) were examined in several human cell lines of normal and malignant origins and composing of either fibroblastic or epithelial-like cells. Virus production occurred only in the fibroblastic diploid cell lines: HEF (human embryonic cells, Flow #5,000) and HFDL #645 (human fetal diploid lung), but not in the epithelial-like heteroploid cell lines: RA (a continuous line of human amnion), #999 (human bone marrow), and KB (carcinoma of the nasopharynx). While the single-cycle growth pattern of the virus in HEF and HFDL #645 cell lines were essentially similar, the virus yield per cell was greater in the HFDL #645 cells. Furthermore, the physiological state of the cell had a marked effect on virus production. Subconfluent actively growing HFDL #645 cells produced higher yields of virus than density-inhibited confluent HFDL #645 cell cultures. The replication of HSFV was inhibited by actinomycin D at concentrations that did not interfere with poliovirus replication (0.001 to 0.01 microgram/ml). Pretreatment and posttreatments of infected cell cultures with either the polycation polybrene (hexadimethrine bromide) or the synthetic glucocorticosteroid dexamethasone did not enhance HSFV production.     

Chromosome analysis of human oocytes and embryos: does delayed fertilization increase chromosome imbalance?

Thirty per cent of a sample of 120 unfertilized human oocytescarried chromosome abnormalities highly correlated with maternalage (38% in patients >35, as compared with 24% in youngerpatients). Fertilized eggs, when observed 17 h after insemination,showed in 1.6% a single pronudeus suggesting parthenogeitelicactivation. In 92% of the cases two pronuclei were observedand the rate of chromosome anomalies depended on the morphologicalaspect of the embryos. Triploidy was also encountered in 6.4%of the eggs leading to an overall rate of chromosome aberrationsreaching 29.2%. Delayed fertilization drastically increasedthe rate of chromosome anomalIes (87%) as well as the rate ofmosaicism: 30% versus 10.6% in timely fertilized eggs. The highrate of chromosome disorders in early life after in-vitro fertilization(IVF) raises the ethical question of the opportunity of carryingout a genetic control of normality in human embryos at the preimplantationstage.     

Monoclonal Antibodies Against Human Chorionic Gonadotropin (hCG): I. Production,Specificity, and Intramolecular Binding Sites*

ABSTRACT: Thirty-nine monoclonal antibody (MCA) producing hybridoma cell lines derived from fusions of mouse myeloma cells with spleen cells from mice immunized with human chorionic gonadotropin (hCG) have been established. Their products have been tested in radioimmunoassays using ¹²⁵I-labeled hCG, luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), the alpha (α) and beta (β) subunits of hCG and LH, and the C-terminal peptide 109–145 (CTP) of CG. All MCA were, in addition, tested in indirect immunofluorescence (IIF) on paraffin sections of human pituitary glands. According to the intramolecular localization of the determinants recognized, three main groups of MCA can be distinguished: 1) MCA directed against epitopes on the β-chain (α-MCA), 2) MCA directed against (β-chain determinants (β-MCA), and 3) MCA reacting with a conformational determinant only present on the native hormone and not on either subunit (con-formational-MCA). All α-MCA cross-react with human LH, FSH, and TSH. The β-MCA do not react with FSH or TSH, but do react to a varying degree with LH. The conformational-MCA show no binding of labeled FSH or TSH and very little or no cross-reactivity with LH.     

Search for human herpesvirus 6 and human cytomegalovirus in bronchoalveolar lavage from patients with human immunodeficiency virus-1 and respiratory disorders

Virus isolation and viral DNA detection by the polymerase chain reaction were used to investigate the presence of human herpesvirus 6 (HHV-6) and human cytomegalovirus (HCMV) in bronchoalveolar lavage from 34 human immunodeficiency virus-1 (HIV-1)-infected patients with respiratory disorders. The aim was to assess the presence of reactivated HHV-6 in lung tissues for a subsequent evaluation of the frequency of virus involvement in respiratory clinical manifestations in the course of HIV-1 infection. Bronchoalveolar lavage samples were tested for the presence of HCMV, as a routine investigation within a protocol monitoring opportunistic infections in symptomatic HIV-1 patients. Whereas HCMV DNA was detected by the polymerase chain reaction in 12 bronchoalveolar lavage specimens, 10 of which were also positive for virus isolation, all samples were negative for HHV-6 by both virological procedures. The HHV-6 DNA finding in bronchoalveolar lavage from an HIV-1-seronegative patient with renal carcinoma, investigated accidentally together with the bronchoalveolar lavage specimens from HIV-1 seropositive patients, stressed the HHV-6 polymerase chain reaction-negative results in the bronchoalveolar lavage samples under study. It is concluded that the lung may be a target organ for HCMV infection in HIV-1-seropositive patients affected by respiratory symptoms but that this does not seem to be the case for HHV-6. © 1996 Wiley-Liss, Inc.     

Identification of the Complement Regulatory Protein CD59 in Human Colostrum and Milk

PROBLEM: Complement lytic activity has been demonstrated, and a potential for its activation is present in human colostrum and milk. This necessitates the presence of regulatory mechanisms protecting epithelial cells in the oropharynx and the gastrointestinal tract of the infant, the milk cellular elements, and bacteria colonizing the oropharynx and the gastrointestinal tract. Lactoferrin and C1 inhibitor have been attributed such a role. However, it is likely that additional protection against the cytolytic activity of the membrane attack complex is required. This has lead us to investigate the presence of the complement regulatory protein CD59 in human colostrum and milk, and to further characterize the source of secretion. METHOD: Samples of human colostrum and milk were obtained from volunteers at different stages of lactation, and separated into fat, skim milk, and milk cellular elements by centrifugation. Normal human mammary gland tissues were obtained from patients undergoing biopsy for benign conditions. SDS-PAGE and Western blotting, and an immuno dot-blot assay were used to identify CD59 in human milk. Immunohistochemistry was performed on all tissue samples and cytospins of the milk cellular elements, using monoclonal antibodies to CD59. RESULTS: CD59 was present in cell-free colostrum and milk as a 19–25 kDa glycoprotein. No variation in CD59 levels was detected between colostrum and milk. CD59 was present in great amounts in the cytoplasm and was highly expressed on the surface membrane on mammary gland acinar and ductal epithelial cells, while the milk cellular elements contained CD59 mainly in their cytoplasm. CONCLUSION: The complement regulatory protein CD59 present in cell-free human colostrum and milk may exert its effects both in the mammary gland and in the oropharynx and gastrointestinal tract of the infant. The lobuloalveolar epithelial cells in the mammary gland are the likely source of secretion.     

A haplotype HLA-A*3201 – B*0702 – Cw*07 with a new HLA-C allelic variant closely related to HLA-Cw*0702 found in a Caucasian patient suffering from leukaemia

The novel allele human leucocyte antigen (HLA)-Cw*0746 differs from HLA-Cw*070101 by three nucleotide exchanges at codons 45 and 66 in exon 2 and at codon 99 in exon 3.