Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP. Two coding substitutions were found in comparison with the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429]PLAP, into wild-type Chinese hamster ovary cells. We determined the k(cat) and K(m), of the PLAP S, F, and D allozymes using the non-physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples.     

Stress and immune mediators in miscarriage.

BACKGROUND: Stress is thought to be abortogenic and psycho-neuro-immunological pathways have been suggested to be involved in triggering miscarriages. From experiments in pregnant mice exposed to stress some insights into the underlying mechanisms have been gained, delineating immunological imbalances as a cause of pregnancy failure. In order to test the validity of the conclusions drawn from murine experiments and the role of stress in human pregnancy loss, the following study was performed. METHODS: We used an established perceived stress questionnaire and measured the stress score of women with a confirmed diagnosis of first trimester spontaneous abortion (n = 94). Decidual tissue was investigated by immunohistochemistry and in-situ hybridization to detect the presence and distribution of immunocompetent decidual cells [CD56(+) natural killer (NK) cells, CD8(+)and CD3(+) T cells, tryptase(+) mast cells (MCT(+)) and tumour necrosis factor (TNF)-alpha(+) cells]. The patient cohort was divided into women experiencing low or high levels of stress. RESULTS: In the decidua of women with high stress scores we observed significantly higher numbers of MCT(+), CD8(+) T cells and TNF-alpha(+) cells per mm(2) tissue (P < or = 0.05). No significant differences between individuals with lower or higher stress scores could be observed with respect to decidual CD56(+) NK and CD3(+) T cells. CONCLUSIONS: Using a questionnaire to score perceived stress in humans may be a valid approach to assess non-biased stress scores. Stress-triggered abortion in humans, identified by a questionnaire, can be linked to immunological imbalances.     

用高分辨G带和人工细菌染色体荧光原位杂交技术分析中国儿童孤独症患者的染色体改变

目的 检测中国儿童孤独症患者的特征性染色体改变。方法 应用高分辨G带和人工细菌染色体(bacterial artificial chromosome，BAC)荧光原位杂交(flourescence in situ hybridization，FISH)分析68例中国儿童孤独症患者的染色体改变。结果 用G带分析观察到有染色体改变的4例患者，分别为1例t(4；6)(q23-24；p21)、1例21p 和2例9号染色体臂间倒位。BAC FISH进一步证实易位病例，而且更精确[t(4；6)(q25-26；p21-1)]；涉及7号、15号、2号染色体的BAC FISH均未观察到文献中报道的染色体改变；而9号染色体的臂间倒位和21p 因无BAC克隆而无法证实。结论 用G带和BAC FISH发现少数中国孤独症患者有染色体改变，但远没有文献中报道的10％～48％那么高。BAC FISH有助于精确地确定染色体易位断裂点。     

Possible susceptibility of the HLA-DPB1*0402 and HLA-DPB1*04 alleles to unexplained recurrent abortion: analysis by means of polymerase chain reaction-restricted fragment length polymorphism method

PROBLEM: To clarify whether HLA-DP antigens are associated with patient population of unexplained recurrent abortion. METHOD OF STUDY: The frequency of HLA-DPB1 alleles in patients with unexplained recurrent abortion, and the compatibility of HLA-DPB1 alleles between patient couples, were studied using a polymerase chain reaction (PCR)-restricted fragment length polymorphism (RFLP) method. Thirty patients who had a history of unexplained primary recurrent abortion, and their husbands, were typed for HLA-DPB1 genotype. Two hundred and ninety-nine base pair fragments from the second exon of HLA-DPB1 genes were selectively amplified using the PCR-primers. After amplification, the DNAs were digested with restriction endonucleases, and subjected to electrophoresis in a 12% polyacrilamide gel to determine HLA-DPB1 genotype. RESULTS: The frequency of HLA-DPB1*0402 and DPB1*04 alleles in the patient group (n = 30) was significantly increased, as compared to that in the normal fertile women (n = 30). The frequency of HLA-DPB1*04 allele in the patient group was significantly increased, as compared to that in the general population (n = 112). No significant compatibility of HLA-DPB1 alleles could be observed between patient couples and normal fertile couples. CONCLUSION: These findings suggest a possible new class II association with patient population of unexplained recurrent abortion.     

Adenosine Deaminase and Human Reproduction: A Comparative Study of Fertile Women and Women with Recurrent Spontaneous Abortion

PROBLEM: We have investigated the possible role of adenosine deaminase (ADA) genetic polymorphism in human fertility through a comparative study of couples with recurrent spontaneous abortion (RSA) and healthy puerperae. METHOD OF STUDY: Adenosine deaminase phenotype has been determined in 209 women with repeated episodes of unexplained spontaneous abortion (RSA) and their husbands, as well as in 115 healthy pregnant women from the population of Rome. An independent sample of 286 puerperae along with their newborn infants in the population of Penne was also studied. RESULTS: The proportion of carriers of ADA*2 allele, which is associated with the lowest enzymatic activity, is lower among women with RSA than among healthy pregnant women from the same population of Rome. Preliminary observations suggest a protective effect of ADA*2 against the development of autoantibodies in RSA. Such an effect seems to be mediated by an interaction with ABO blood groups. In the population of Penne the proportion of women carrying ADA*2 allele is higher among those who have had two or more previously born children than among women with only one or no children. CONCLUSIONS: The data suggest that women carrying the ADA*2 allele are better protected against the spontaneous loss of embryos and have a higher fertility rate.     

Suppressed Cell-Mediated Immunity and Monocyte and Natural Killer Cell Activity Following Allogeneic Immunization of Women with Spontaneous Recurrent Abortion

Spontaneous recurrent abortion (SRA) has been treated by means of immunization with paternal or third-party white blood cells, yet the immunological basis for SRA and for the role of immunization protocols in pregnancy outcome remains controversial. To elucidate this question, nine women with SRA were immunized with paternal mononuclear cells and studied before and 2 weeks after immunization. Seven women who became pregnant gave birth to live newborns. Secretion of the T helper 1 cytokines IL-2 and interferon- by patients' mononuclear cells decreased, while production of IL-10 increased. The levels of natural killer and lymphokine-activated killer cell-mediated cytotoxicity were markedly decreased. Monocyte functions such as secretion of IL-l, tumor necrosis factor a, IL-6, and cytotoxic activity decreased concurrently with elevations in IL-10 and transforming growth factor secretion. Production of IL-12, a pivotal regulatory cytokine, decreased. Furthermore, B7/1 expression on patients' mononuclear cells was downregulated. This resulted in a decrease in monocyte costimulatory activity of purified T cells with soluble anti-CD3, paralleled by a decline in allogeneic proliferative responses. These results suggest that the improved pregnancy success rate in women with SRA following immunization may be partly related to suppression of cell-mediated immunity and monocyte and natural killer cell activity.     

Psychosocial factors regulating natural-killer cell activity in recurrent spontaneous abortions

PROBLEM: The preconceptional natural-killer cell (NK) activity predicts subsequent miscarriage among women with unexplained recurrent spontaneous abortion (RSA). Psycho-neuro-immuno-endocrine network has recently been proposed as a mechanism for abortions. We therefore examined which psychosocial factors influenced the NK activity among women with RSA. METHOD OF STUDY: We measured the preconceptional NK activity of 61 women with a history two consecutive unexplained first-trimester miscarriages and no live births. We also administered semi-structured interviews and a battery of self-report questionnaires to assess their social support, personality, self-esteem and psychiatric symptoms. RESULTS: The preconceptional NK activity was negatively correlated with the women's neuroticism personality trait (r= -0.32, P = 0.01) and current depressive symptoms (r = -0.26, P= 0.05), and positively correlated with their self-esteem (r = 0.34, P = 0.01). CONCLUSIONS: In addition to several substances such as transforming-growth-factor beta and granulocyte-macrophase colony-stimulating factor, we found that low neuroticism, low depression scale score and high self-esteem contributed to high NK activity among women with RSA.     

Partial duplication 16q: report of two affected siblings resulting from a maternal translocation and literature review

Two siblings with a partial duplication 16q, born to a woman with a balanced translocation (6;16), are described. The first infant died at 8 weeks of age; the second died at 4 months. Fifteen other cases of duplications involving 16q have been reported, all of them derived from a balanced parental translocation. The most frequent physical findings have included dysmorphic facies characterized by high forehead, prominent nose, antimongoloid slant, malformed ears, and micrognathia, as well as flexion contractures of the joints, deformity of the feet, and genital hypoplasia in the male. Anorectal, intestinal and cardiac malformations were less frequent findings. Most of the affected infants died at ages ranging from 8 days to 6 months. The few with longer survival (up to 6 years) had a shorter, more distal segment duplication of chromosome 16. Although intrauterine growth retardation and microcephaly were not always present at birth, most of the infants had postnatal growth failure. The phenotypic and clinical findings of the two infants in this report are compared with those of previously reported cases, from which there appears to be correlation of the length of the 16q duplication with clinical phenotype and survivals.     

肱骨远端的解剖观测与肱骨假体的设计

目的:为临床肱骨远端假体的设计与安放提供相关数据。方法:测量120块(男35对、女25对)成人干燥肱骨远端相关结构,对所测得结果用SPSS软件处理。结果:(1)肱骨远端前倾角和肱骨滑车外旋角,男女分别为(35.78±5.12)°、(36.33±5.06)°和(5.35±1.13)°、(5.55±1.22)°。(2)内上髁至外上髁最大宽度、滑车最内侧缘至小头最外侧缘宽度、肱骨滑车和肱骨小头宽度男女分别为(58.92±4.03)、(55.75±3.85);(45.34±3.15)、(42.18±3.01);(24.82±1.74)、(21.78±1.51)和(17.69±1.23)、(16.39±1.21)mm。(3)肱骨滑车和肱骨小头最大矢径男女分别为(23.35±2.06)、(23.13±2.15)和(19.47±1.38)、(19.13±2.19)mm。男女之间和左右之间各测量数据无显著性差异(P>0.05)。个体间有较大差异。结论:肱骨远端各项目的测量值,可供设计与安放肱骨假体提供解剖参数。     

Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts

BACKGROUND: Manual puncture of the trophectoderm of human blastocysts with a needle before vitrification increases their survival rate, but the embryos take a long time to re-expand. This study examined whether causing human blastocysts to collapse by manual pipetting before vitrification would allow more rapid re-expansion and improve pregnancy rates. METHODS: After embryo transfer in IVF cycles, surplus embryos that developed to the expanded blastocyst stage were placed in cryoprotectant and then artificially shrunk by mechanical pipetting with a fine hand-drawn glass pipette slightly smaller in diameter than the blastocyst. The shrunken embryos were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. The blastocysts were thawed by warming and then dilution in 1 mol/l sucrose. RESULTS: Of 49 expanded vitrified blastocysts, 48 (98%) re-expanded within 3 h after warming. Following transfer (48 blastocysts in 28 cycles), 14 women (50%) became clinically pregnant, and the implantation rate was 33% (16/48). Eight healthy babies have been born in six deliveries, and the other eight pregnancies are ongoing. To date, there have been no spontaneous abortions. CONCLUSIONS: The results suggest that artificial shrinkage with pipetting is a simple and effective technique to assist successful cryopreservation of expanded blastocysts by vitrification.