Rapin Osathanondh

Induction of Tenascin-C in Cardiac Myocytes by Mechanical Deformation ROLE OF REACTIVE OXYGEN SPECIES

Mechanical overload may change cardiac structure through angiotensin II-dependent and angiotensin II-independent mechanisms. We investigated the effects of mechanical strain on the gene expression of tenascin-C, a prominent extracellular molecule in actively remodeling tissues, in neonatal rat cardiac myocytes. Mechanical strain induced tenascin-C mRNA (3.9 ± 0.5-fold, p < 0.01, n = 13) and tenascin-C protein in an amplitude-dependent manner but did not induce secreted protein acidic and rich in cysteine nor fibronectin. RNase protection assay demonstrated that mechanical strain induced all three alternatively spliced isoforms of tenascin-C. An angiotensin II receptor type 1 antagonist inhibited mechanical induction of brain natriuretic peptide but not tenascin-C. Antioxidants such as N-acetyl-l-cysteine, catalase, and 1,2-dihydroxy-benzene-3,5-disulfonate significantly inhibited induction of tenascin-C. Truncated tenascin-C promoter-reporter assays using dominant negative mutants of IκBα and IκB kinase β and electrophoretic mobility shift assays indicated that mechanical strain increases tenascin-C gene transcription by activating nuclear factor-κB through reactive oxygen species. Our findings demonstrate that mechanical strain induces tenascin-C in cardiac myocytes through a nuclear factor-κB-dependent and angiotensin II-independent mechanism. These data also suggest that reactive oxygen species may participate in mechanically induced left ventricular remodeling.

Comparison of the ontogeny of protein gene product 9.5, chromogranin A and proliferating cell nuclear antigen in developing human lung

Pulmonary neuroendocrine cell products, especially bombesin‐like peptides, are important modulators of fetal lung growth, morphogenesis and maturation. In the present study, we describe the ontogeny of protein gene product 9.5 (PGP 9.5) in 28 midtrimester human fetal lungs, in comparison to chromogranin A (CGA), a marker of differentiated neuroendocrine cells, and proliferating cell nuclear antigen (PCNA), which is expressed by actively dividing cells. PGP 9.5 immunostaining colocalized with CGA in many cells, although the peak abundance of PGP 9.5 preceded that of CGA by 4 to 6 weeks. In addition, a novel staining pattern was noted for PGP 9.5: diffuse cytoplasmic staining of undifferentiated epithelial cells, which was demonstrated by all of the airways before 15 weeks gestation. After gestational week 15, only the smallest airways demonstrated this pattern. PCNA immunostaining demonstrated age‐dependent regional variation. All samples had approximately 25% epithelial cells immunopositive for PCNA. Between 11 and 14 weeks gestation over 50% of the mesenchymal cells were PCNA positive. This mesenchymal staining decreased after 14 weeks, and was rare by week 19. There was no definite correlation between the immunostaining for PGP 9.5 and that for PCNA, although PGP 9.5 positive cells were usually PCNA negative. These observations suggest that other growth factors produced by non‐neuroendocrine epithelial cells also participate in lung development. Microsc. Res. Tech. 37:62–68, 1997.

The fetal thyroid: normal and abnormal sonographic measurements.

The thyroid of 31 fetuses at low risk for perinatal thyroid disease were evaluated sonographically. The transverse width and circumference of the fetal thyroid was measured prospectively to provide normative values for each gestational age. In addition, the thyroid of 23 fetuses at risk for thyroid disease were examined sonographically and compared to the control group. At birth, 18 of the neonates had no evidence of thyroid dysfunction, whereas 5 newborns had goiters and abnormal thyroid function. The fetal thyroid measurements for these 5 neonates were above the upper limit of the 95% confidence interval compared to the control group. The other 18 fetuses in the group at risk for thyroid disease but without evidence of thyroid dysfunction at birth had fetal thyroid measurement within the normal range.

Divergent responses by human and mouse thyroids to human chorionic gonadotropin in vitro

hCG is a known stimulator of mouse thyroid in vivo. Studies were therefore performed to ascertain whether the thyroid-stimulating activity of hCG in the mouse could also be demonstrated by the in vitro techniques that had failed to show any activity of hCG in the human thyroid. When labeled with 125I and incubated at 22 degrees C in 20 mM Tris-0.5% bovine serum albumin (Tris-BSA), pH 7.45, with increasing concentrations (70-300 micrograms protein/ml) of a mouse thyroid fraction, a purified hCG preparation [( 125I]hCG) showed 5-12% specific binding. In contrast, its binding to a human thyroid particulate fraction, over the same range of protein concentrations, did not exceed 1%. When similar studies were performed at 37 degrees C in 10 mM Tris-50 mM NaCl-0.5% BSA, pH 7.45, [125I]hCG showed no detectable binding either to the human or the mouse thyroid fractions. At concentrations ranging from 1 to 20 mIU/ml (0.9-18 X 10(-9) M), bTSH stimulated cAMP release from human thyroid slices into the medium in a dose-dependent manner. In contrast, hCG concentrations from 10(3) to 10(4) IU/ml (2-20 X 10(-6) M) were without effect on cAMP release. bTSH, at concentrations of 4.5 and 9.0 mIU/ml (4 and 8 X 10(-9)M), stimulated cAMP release from the mouse thyroid, producing in the medium approximately 11- and 28-fold increases in cAMP concentration. hCG also stimulated cAMP release from the mouse thyroid, the increases being approximately 2.3- and 1.8-fold, in the presence of 2270 and 4540 IU/ml (4.5 and 9.0 X 10(-6) M), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Levels of urinary beta‐core fragment, total oestriol, and the ratio of the two in second‐trimester screening for Down syndrome

Levels of beta‐core fragment and total oestriol in second‐trimester maternal urine samples were measured in 32 Down syndrome pregnancies and 206 control pregnancies. Beta‐core fragment and total oestriol values were corrected for the urinary creatinine level and expressed as multiples of the control medians (MOM). In addition, the ratio of the beta‐core fragment level to the total oestriol level, without creatinine correction, was calculated, and expressed as MOM values. The median beta‐core fragment, total oestriol, and ratio levels in Down syndrome cases were 5·42, 0·64, and 9·32 MOM, respectively. In the Down syndrome pregnancies, 66 per cent of the beta‐core fragment levels were above the 95th centile of control levels, while 22 per cent of the total oestriol levels were below the fifth centile of control levels. In combination with maternal age, measurement of beta‐core fragment and total oestriol levels in Down syndrome pregnancy resulted in an 80 per cent detection rate at a 5 per cent false‐positive rate. Use of the ratio resulted in a univariate detection rate of 72 per cent. In combination with maternal age, the ratio resulted in a detection rate of 81 per cent at a 5 per cent false‐positive rate. Based on this unmatched study, the measurement of a ratio of beta‐core fragment to total oestriol levels, without the need for creatinine correction, may be useful in screening for fetal Down syndrome in second‐trimester urine.

Expression of messenger ribonucleic acid for epidermal growth factor receptor and its ligands, epidermal growth factor and transforming growth factor-α, in human first- and second-trimester fetal ovary and uterus

OBJECTIVES The epidermal growth factor receptor, a 170 kd polypeptide with tyrosine kinase activity, is used as the common receptor by two homologous polypeptide growth factors, epidermal growth factor and transforming growth factor-alpha. The activation of the epidermal growth factor receptor results in effects including deoxyribonucleic acid synthesis and cellular differentiation. Epidermal growth factor, transforming growth factor-alpha, and the epidermal growth factor receptor are reported to be associated with adult reproduction. However, the pattern of gene expression for the epidermal growth factor receptor and its ligands in human fetal reproductive tissues has not been detailed previously. STUDY DESIGN We studied the expression of messenger ribonucleic acid encoding three polypeptides, epidermal growth factor receptor, epidermal growth factor, and transforming growth factor-alpha, in 10-, 15-, 19-, and 22-week human fetal ovaries and uteri. Ribonucleic acid was prepared from the fetal tissues and made into complementary deoxyribonucleic acid by reverse transcription. The complementary deoxyribonucleic acid was amplified by polymerase chain reaction, using primers specific for the human epidermal growth factor receptor, epidermal growth factor, and transforming growth factor-alpha. RESULTS We found that epidermal growth factor receptor messenger ribonucleic acid was present in all stages of ovarian and uterine tissues studied. In addition, both epidermal growth factor and transforming growth factor-alpha messenger ribonucleic acid was found in all four stages of ovarian development. Epidermal growth factor messenger ribonucleic acid was detected in all four stages of fetal uterine development. CONCLUSIONS This is the first demonstration, to our knowledge, of epidermal growth factor receptor, epidermal growth factor, and transforming growth factor-alpha messenger ribonucleic acid expression in human first- and second-trimester uterus and ovary.

mRNAs for insulin-like growth factor-II (IGF-II) and variant IGF-II are co-expressed in human fetal ovary and uterus

Insulin-like growth factor-II (IGF-II) is postulated to have autocrine and/or paracrine functions in developing fetal tissues, but has never been reported in human fetal reproductive organs. The forms of IGF-II found in normal human serum include a 67 amino acid form and a variant form resulting from alternate splicing of the mRNA such that Ser-29 is replaced by four other amino acid residues. We studied the expression of mRNA encoding IGF-II in human fetal ovaries and uteruses of 10, 15, 19 and 22 weeks of gestation. By reverse transcription followed by polymerase chain reaction (PCR), we identified the co-expression of two mRNAs encoding IGF-II in all developmental stages of fetal ovaries and uteruses tested. One of the PCR amplified fragments was 9 nucleotides larger than the other. The PCR amplified ovarian and uterine DNA fragments were mapped by digestion with the restriction endonucleases AvaII and PvuII and both the IGF-II fragment and the larger IGF-II fragment produced the anticipated DNA patterns by gel electrophoresis. The PCR amplified DNA fragments were cloned and sequenced to confirm that the expressed mRNAs encoded IGF-II and variant IGF-II. We conclude that IGF-II and variant IGF-II mRNA co-expression occurs in the human fetal female genital tract and that the two forms of the growth factors may have physiologic roles in reproductive tract development.

Phenotypic characteristics of lymphoid populations of middle gestation human fetal liver, spleen and thymus

Mononuclear cells isolated from liver, spleen and thymus of fetuses between 18 and 24 weeks gestational age were stained for a number of lymphoid cell markers by indirect immunofluorescence and analyzed by flow cytometry. Studies were carried out on freshly isolated mononuclear cell preparations and on cultured cells after selective expansion in interleukin 2 (IL2). Many mononuclear cells in fresh isolates of liver and spleen could not be identified with antibodies to mature T- and B-cell markers. An average of 3% of isolated liver cells and 34% of isolated spleen cells stained positively for CD3, and 19% of liver cells and 37% of spleen cells stained positively for CD20. Lymphoid cells of the fetal thymus were an average 67% CD3+, 76% CD4+, 84% CD8+, and showed greater CD45RO staining (93%) than mononuclear cells of other tissues. Propagation of liver and spleen cell populations in culture favored CD3 phenotypes and CD8 phenotypes. Propagated T cell populations of liver and spleen were primarily TCR alpha/beta+ (81% in liver, 85% in spleen), suggesting a selective advantage in IL2 expansion of alpha/beta T cells over gamma/delta T cells. Propagated gamma/delta T cells of liver and spleen were predominantly TCR gamma/delta 2+. Whereas propagated cells of liver and spleen consisted of approximately 10% gamma/delta+ cells, thymus-derived cells expanded in culture were only an average of 2% TCR gamma/delta+, demonstrating a rarity of IL2-responsive gamma/delta T cells in middle gestation fetal thymus.

Sonographic findings in otocephaly (synotia).

Otocephaly (synotia) is a rare congenital malformation characterized by a hypoplastic or absent mandible, close-set temporal bones, and abnormal position and development of the auricles. Cyclopia, cranial meningocele or encephalocele, and other abnormalities may be present. 16 High-resolution real-time imaging equipment has allowed detection of a variety of neural tube and facial malformations. To our knowledge, the ultrasound findings in otocephaly have not been previously described. \Ve report the use of ultrasonography in the antenatal diagnosis of otocephaly. Pathologic correlation is provided.

Phenotypic characteristics of lymphocyte populations isolated from middle gestation human placenta

In order to characterize the phenotypic composition of populations of lymphoid cells in maternal and fetal tissues during the period of middle gestation, mononuclear cells were isolated from maternal peripheral blood, fetal spleen, fetal thymus and placenta of 18-24 week pregnancies. Peripheral blood and placental isolates were stained for a number of lymphoid cell markers by indirect immunofluorescence and analyzed by flow cytometry. Studies were performed on both freshly isolated mononuclear cell preparations and in vitro cultured cells after selective expansion in interleukin 2 (IL2). Fresh placental mononuclear cell isolates were an average 20% CD3+; their CD4/CD8 ratios varied among individuals. An average of 68% of the lymphocytes isolated from maternal peripheral blood were CD3+. Placental and maternal peripheral blood isolates had comparable percentages of CD16+ and CD20+ cells, while CD56+ cells were present at significantly greater numbers in the lymphocyte compartment of placenta (17%) than in peripheral blood (3%; P < 0.01). Lymphocyte isolates were expanded by culture with IL2 and PHA and stained to determine if propagated lymphocyte populations are representative of initial isolates. Expansion of all lymphocyte isolates favored CD3 phenotypes and CD8 phenotypes. Compared to expanded placenta-derived populations, expanded peripheral blood lymphocytes were similar with regard to percentages of all phenotypes except gamma/delta T cells which represented more of placental lymphocytes (10%) than peripheral lymphocytes (5%; P < 0.01). Surface HLA typing determined propagated placenta-derived lymphocytes to be of maternal and not fetal origin. In vitro propagation of placental mononuclear cell isolates may therefore provide populations of maternal CD3+ lymphocytes for assessment of function and specificity.