Differential expression of the immediate-early 2 and 3 proteins in developing mouse brains infected with murine cytomegalovirus.

Murine cytomegalovirus (MCMV) immediate-early (IE) 2 protein has been reported to be dispensable for growth and latency in mice. Therefore, its role in viral pathogenesis and tissue tropism is not known. Here we prepared specific antibodies to the IE2 and IE3 proteins by using fusion proteins expressed in Escherichia coli as antigens. Immunostaining of MCMV-infected cultured fibroblasts revealed IE2 protein to be expressed diffusely in the nucleoplasm similar to the IE1 protein. In contrast, expression of the IE3 protein, 88 kDa, exhibited a punctate pattern in the nucleus in the early phase of infection then diminished. In the brain of neonatal mice infected with MCMV, both IE2 and IE3 proteins were detected immunohistochemically in the cells of the ventricular walls early in infection. When the infection was prolonged, the IE2 protein was expressed in neurons of the cortex and hippocampus, while the IE3 protein was preferentially expressed in glial cells in the early phase of infection, and its levels declined during the infection. These results suggest that the IE2 protein may play a role in persistent infection in neurons, whereas the IE3 protein, expressed preferentially in glial cells, may play the main role in acute infection.

3-D fractal tumor growth of epithelial ovarian cancer.

PURPOSE: A fractal is a shape made of parts similar to the whole. Our objective was to determine whether surface growth patterns in malignant epithelial ovarian tumors are 3-D fractal, and if the mean fractal dimension differs according to histologic types. METHODS: After the images of photographs of 139 resected malignant epithelial ovarian tumors were digitized, the fractal dimensions of surface of solid portions were measured using 3-D fractal analysis software. RESULTS: The mean fractal dimensions of the surface of a solid area of tumor in serous, mucinous, endometrioid, and clear cell adenocarcinoma were 2.320, 2.224, 2.229, and 2.298, respectively. Those of serous and mucinous cystadenoma of low malignant potential (LMP) were 2.398 and 2.282, respectively. These values were significantly greater than the topological dimension of a surface (= 2). The mean fractal dimensions of a solid area of tumor inside the cyst for serous, mucinous, endometrioid, and clear cell carcinoma were 2.347, 2.223, 2.228, and 2.310, respectively. The values for serous and mucinous cystadenoma of LMP were 2.398 and 2.282, respectively. CONCLUSION: This study shows that the surface of a solid area of malignant epithelial ovarian tumors has a 3-D fractal structure, and the mean fractal dimension may differ according to histologic types.

Preoperative diagnosis of colouterine fistula secondary to diverticulitis by sonohysterography with contrast medium.

Colouterine fistulae secondary to sigmoid diverticulitis are unusual. Methods for diagnosis remain to be established. We report a case with a colouterine fistula in which sonohysterography detected the flow of ultrasound contrast medium between the uterine cavity and the sigmoid colon through the posterior uterine wall, thus confirming the diagnosis. The diagnosis was further substantiated by a charcoal challenge test. The patient underwent en bloc resection of the uterus, Fallopian tubes, ovaries and sigmoid colon, the organs involved with diverticulitis. This is the first report to describe a colouterine fistula successfully diagnosed by sonohysterography using ultrasound contrast medium.

A primary cell culture system for human cytotrophoblasts of proximal cytotrophoblast cell columns enabling in vitro acquisition of the extra-villous phenotype.

Cytotrophoblast (CT) differentiation into the extra-villous phenotype is a crucial process in initiating their invasion into the decidua and thereby developing the placenta. However, how CTs differentiate into extra-villous CTs (EVCTs) is not fully elucidated. To address this, a suitable culture model for CTs has been long-sought. But this has been hampered by annoying problems such as; cell aggregation, in vitro syncytialization, low plating efficiency, etc. The aim of this study is to develop a culture system in which CTs differentiate into EVCTs. CTs were isolated from the first trimester placenta using density gradient separation and immuno-depletion using anti-CD9 antibody to remove contaminating fibroblasts and EVCTs. The resultant isolated CTs were found to have the character similar to poorly differentiated CTs comprising proximal cytotrophoblastic cell columns as confirmed by immunocytochemical and flowcytometric analyses. When cultured on type 4 collagen-coated plates in culture media containing low calcium concentration, CTs neither aggregated nor syncytialized, remaining mononuclear and monolayer state. Interestingly, cultured CTs gradually upregulated integrin alpha1, CD9, and human leukocyte antigen (HLA)-G; the known markers specific for EVCTs invading into the decidua diffusely. Hence, the CT culture system provides a sophisticated experimental model in which highly purified CTs acquire the extra-villous phenotype without syncytialization.

Sonographic assessment of the cervix before, during and after a uterine contraction is effective in predicting the course of labor.

OBJECTIVE: To investigate whether the degree of change in cervical length during a uterine contraction is predictive of subsequent progression of labor. METHODS: The subjects were 73 uncomplicated parturient women at term. We observed the cervix before, during and after a uterine contraction by transvaginal ultrasound in the first stage of labor and determined the degree of cervical shortening during the contraction relative to the cervical length before contraction. We related the degree of cervical shortening to labor patterns at the time of the ultrasound examination, which were retrospectively determined by reviewing the partogram. RESULTS: The cervix was shortened in length by about 50% on average during a uterine contraction in the normal course of labor. The degree of cervical shortening was significantly greater in the normal latent and active phases than it was in the prolonged latent phase, protracted active phase and false labor, whereas there were no differences between the former two phases nor between the latter three phases. Nulliparous and parous women exhibited almost the same degree of shortening in the normal latent and active phases. CONCLUSIONS: Real-time ultrasound observation of the cervix during uterine contraction could help differentiate inefficient uterine contractions from normal ones and thus predict the subsequent course of labor.

Assessment of para-aortic lymph nodes by intraoperative sonography in gynecological malignancies: a preliminary report.

OBJECTIVE: To investigate the usefulness of intraoperative sonography for the assessment of para-aortic lymph nodes in gynecological malignancies. METHODS: The assessment of para-aortic lymph node swelling by computed tomography (CT), palpation during surgery and intraoperative sonography was performed in 43 women with a gynecological malignancy. The sensitivity, specificity and predictive values of each technique for assessment of node metastasis were examined in the 33 women who underwent para-aortic lymphadenectomy. For each of the three assessment techniques, the number of para-aortic lymphadenectomies that would have been performed and the number of women who would have had missed metastasis were evaluated assuming a para-aortic lymphadenectomy would be performed only when swollen nodes were detected. RESULTS: Para-aortic nodes were assessed to be swollen by CT, palpation, and intraoperative sonography, respectively, in one, six and 10 of 43 women in total, and in one, six and nine of the 33 women who underwent para-aortic lymphadenectomy. The sensitivity and negative predictive value of intraoperative sonography were 100%, while the specificity and positive predictive value of CT were 100%. If para-aortic lymphadenectomy had been performed only when swollen nodes were detected by intraoperative sonography, the number performed would have decreased from 33 to nine without missing lymph-node metastasis. CONCLUSION: Intraoperative sonography has potential for the assessment of para-aortic lymph nodes in gynecological malignancies.

Myocardial hypertrophy of the recipient twins in twin-to-twin transfusion syndrome and cerebral palsy.

OBJECTIVES: This study was performed to determine risk factors for cerebral palsy (CP) in monochorionic twins, especially with twin-to-twin transfusion syndrome (TTTS). METHODS: In 33 pathologically confirmed monochorionic pregnancies, we analyzed the incidence of cardiovascular and neurological complications. RESULTS: Seventeen cases were diagnosed as TTTS. Myocardial hypertrophy was detected in seven recipient twins of TTTS, but not found in the donor twins of TTTS or in non-TTTS. CP developed in six out of 29 in TTTS and one out of 32 in non-TTTS. In TTTS, all the cases with CP were seen in the recipient twins. The development of CP was significantly associated with cardiovascular complications such as myocardial hypertrophy and hydropic changes. CONCLUSIONS: Myocardial hypertrophy found in the recipient twins in TTTS seems to be a risk for developing CP later on, which may have implications in understanding the pathogenesis of CP.

Sonographic determination of fetal size from 20 weeks of gestation onward correlates with birth weight.

OBJECTIVE: To evaluate the clinical efficacy of ultrasonographic measurement of fetal weight, estimated fetal weight (EFW) was analyzed in relation to birth weight. METHOD: Data were collected retrospectively from the medical records of 548 term pregnancies unassociated with fetal malformations. The subjects underwent sonographic examinations from 20-23 weeks onward at 4 weeks intervals for the estimation of EFW. Subjects were divided into 6 subgroups based on birth weight deviation (BWD) (A: - 3.0 < or = BWD < -2.0; B: -2.0 < or = BWD < -1.5; C: -1.5 < or = BWD < -1.0; D: - 1.0 < or = BWD < -0.5; E: -0.5 < or = BWD < + 0.5 and F: + 0.5 < or = BWD). Distribution and changes in EFW deviation (EFWD) with gestational age were analyzed related to BWD. RESULT: A difference in fetal growth based on EFWD was noted in small for gestational age (SGA) cases compared to non-SGA cases, and the difference was observed as early as 20-23 weeks. EFWD was significantly correlated with BWD from 20-23 weeks to term. CONCLUSION<==: Sonographic determination of fetal growth from 20 weeks of gestation onward correlated with birth weight deviation, and this emphasized the clinical value of evaluating fetal growth during the latter half of pregnancy.

Polymorphism of human leukocyte antigen-E gene in the Japanese population with or without recurrent abortion.

PROBLEM: The aim of this study was to investigate the gene frequencies and shared alleles of human leukocyte antigen (HLA)-E gene in Japanese couples with or without recurrent abortion. METHOD OF STUDY: Polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis was carried out to detect polymorphism in exon 3 of the HLA-E gene in 30 Japanese couples with recurrent abortion and 38 normal Japanese couples with proven fertility. RESULTS: No point mutation was detected in exon 3 of HLA-E in both recurrent aborters and normal controls. HLA-EG and HLA-ER alleles were detected with frequencies of 66.7% and 33.3% in couples with recurrent abortion and 69.2% and 30.8 in normal couples, respectively. The gene frequency of HLA-EG was higher than that of HLA-ER, which is contrary to that found in Caucasian, African-American and Hispanic people but similar to Chinese people. The frequency of each allele was not significantly different between recurrent aborters and normal controls. The number of shared alleles between each couple with recurrent abortion is not significantly different from that with normal controls. CONCLUSION: Allele frequencies of HLA-E were suggested to be different in Asian people from those in other ethnic people. In light of no specific distribution pattern in recurrent aborters, HLA-E polymorphism does not seem to play a role in the pathogenesis of recurrent abortion.

Human leukocyte antigen-G-expressing cells differently modulate the release of cytokines from mononuclear cells present in the decidua versus peripheral blood.

PROBLEM: To better understand the role of human leukocyte antigen (HLA)-G in regulating the T helper (Th)1/Th2 cytokine balance, one of key conditions in determining the fate of pregnancy, we asked whether the presence of HLA-G protein altered the release of cytokines from both decidual mononuclear cells and peripheral blood mononuclear cells (PBMCs). METHOD OF STUDY: The amounts of cytokines released from decidual mononuclear cells and PBMCs were compared in the presence or absence of HLA-G-expressing cells. RESULTS: When cocultured with HLA-G-expressing cells, the amounts of tumor necrosis factor-alpha and interferon-gamma released from decidual mononuclear cells and PBMCs were decreased, while the amounts of interleukin (IL)-4 from PBMCs was increased, with IL-4 release from decidual mononuclear cells being unchanged. CONCLUSIONS: Upon contact with HLA-G, decidual mononuclear cells, and PBMCs as well, modulate their ability to release cytokines in a way that may shift the Th1/Th2 balance towards relative Th2 dominance, suggesting a role for HLA-G in maintaining pregnancy.

Soluble HLA-G influences the release of cytokines from allogeneic peripheral blood mononuclear cells in culture.

Exquisitely regulated cytokine balance during early pregnancy is thought to be necessary for promoting survival of the fetal allograft. Our previous studies have demonstrated that membrane-bound human leukocyte antigen (mHLA-G) expressed on trophoblasts is one of the key factors in regulating cytokine balance by shifting the Th1/Th2 balance toward Th2 polarization, a favourable milieu for the maintenance of pregnancy. Given that trophoblasts secrete soluble HLA-G (sHLA-G), we examined its biological roles in comparison with mHLA-G. We cultured peripheral blood mononuclear cells (PBMC) with either the HLA-A and -B-deficient B lymphoblast cell line (721.221 cells) or the same cell line transfected with mHLA-G (721.221-G1 cells), in the presence or absence of recombinant sHLA-G. Cytokine concentrations in the culture media were determined by enzyme-linked immunosorbent assay. In contrast to mHLA-G protein, sHLA-G stimulated the release of tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma, whereas it reduced the release of interleukin (IL)-3, regardless of the presence of the presence of a stimulatory effect of the mHLA-G-expressing cells. Although mHLA-G reduced the release of IL-4, sHLA-G did not have any effect. Conversely, sHLA-G stimulated the release of IL-10 whereas mHLA-G was without effect. These results suggest that sHLA-G regulates the release of cytokines from PBMC chiefly by counterbalancing mHLA-G, and thereby may play a role in maintaining pregnancy.

Generation of periventricular leukomalacia by repeated umbilical cord occlusion in near-term fetal sheep and its possible pathogenetical mechanisms.

Periventricular leukomalacia (PVL) is a major cause of cerebral palsy. However, pathogenetic mechanisms of PVL have not been fully understood. Although it has been postulated that umbilical cord compression is related to the development of PVL, no animal experiments clearly demonstrated an association of umbilical cord occlusion with 'periventricular' white matter lesions. The purpose of this study is to determine whether umbilical cord occlusions could produce periventricular white matter lesions in fetal sheep and to examine how changes in fetal cardiovascular and metabolic variables are related to the induction of brain damage. Fourteen near-term fetal sheep underwent umbilical cord occlusion (3-min total cord occlusions 5 times at 5-min intervals). Dissections performed 24 h after cord occlusion revealed that periventricular white matter lesions were produced in 7 out of 14 sheep fetuses. According to the pattern of brain damage, we classified the fetal sheep into three groups: 5 fetuses with dominant lesions in the periventricular white matter (group I), 4 fetuses with brain lesions in the cerebral cortex and thalamus (group II) and 5 fetuses with no or minimal brain lesions (group III). Group I showed higher blood pressure and higher plasma lipid peroxide levels before cord occlusion compared to the other groups, while group II showed systemic hypotension during cord occlusion. No significant differences in changes in pH, PaCO2, PaO2 and heart rate were found between the three groups. It is speculated that PVL might be produced by an association of preexisting chronic circulatory instability with an acute episode of severe repetitive cord occlusion.

A movable gestational sac in association with a myometrial defect.

An ultrasound examination in a woman at 9 weeks' gestation demonstrated a gestational sac with a live fetus in the left uterine horn which was surrounded by an extremely thin outer lining. An interstitial pregnancy was suspected and laparoscopy was performed. During laparoscopy the purple bulge in the left horn gradually reduced in size and eventually disappeared following the external pressure by the tip of the forceps. We believe this pressure caused contraction of the superficial myometrial fibers resulting in a shift in the location of the gestational sac to the central area of the uterus. The pregnancy continued and on Cesarean section at 34 weeks' gestation, a membranous protrusion of the uterine wall in the left horn was noticed. A myometrial defect caused by an intrauterine intervention in the patient's previous pregnancy was suspected to be the cause of this "movable gestational sac" phenomenon.

Hyperemesis gravidarum associated with thyrotoxicosis and a past history of an eating disorder.

We present a case of severe hyperemesis gravidarum (HG) associated with thyrotoxicosis in a woman with a past history of an eating disorder. She had developed persistent HG from early pregnancy until about at the end of the second trimester with a body loss of 14 kg. Total parenteral nutrition was effective in alleviateing HG. It is suggested that even a past history of an eating disorder could be at risk of developing HG.

A fetus with Prader-Willi syndrome showing normal diurnal rhythm and abnormal ultradian rhythm on heart rate monitoring.

Clinical features of Prader-Willi syndrome in neonates are marked hypotonia with the absence of crying and feeding difficulty so that prenatal diagnosis of Prader-Willi syndrome is strongly hoped in order to provide appropriate medical and psychological care for neonates and their families. However, the clinical picture of Prader-Willi syndrome in utero has not been well described. We report a pregnancy associated with Prader-Willi syndrome manifesting polyhydramnios, large biparietal diameter of the fetus and characteristic fetal heart rate pattern: prolonged inactive periods and diurnal variation of the incidence of heart rate accelerations. These findings may offer a clue to the prenatal diagnosis of Prader-Willi syndrome, although molecular cytogenetics is mandatory for the definite diagnosis.

Genomic structure of mouse and human genes for DNA-PKcs interacting protein (KIP).

DNA-dependent protein kinase (DNA-PK) is a nuclear protein serine/threonine kinase in a wide variety of vertebrate species and it has a role in the DNA repair and recombination process of lymphoid development. DNA-PK is composed of a large catalytic subunit (DNA-PKcs) and DNA-binding protein, Ku. Recently, the mouse and human DNA-PKcs interacting proteins (Kip/KIP) have been reported. In this report, we have determined the complete genomic structure of mouse and human Kip/KIP genes. The total length of mouse Kip gene and human KIP gene are approximately 5.7 kb and 3.6 kb in genomic DNAs, respectively. Both of genes consist of 7 exons.

Polymorphisms of RhD(Va) and a new RhD(Va)-like variant found in Japanese individuals.

BACKGROUND AND OBJECTIVES: Red cell type RhD(Va) lacks epD1 and 5 and is encoded by hybrid RHD-CE(5)-D alleles. We analyzed RhD(Va) and RhD(Va)-like samples in Japanese blood donors. MATERIALS AND METHODS: Ten RhD(Va) samples lacked epD1 and 5 and 3 RhD(Va)-like variants also lacked, epD2 and a part of 6/7. We identified the full-length nucleotide sequences of the complementary DNA (cDNA) synthesized from 4 samples: 3 of type D(Va) and the 4th a D(Va)-like variant. RESULTS: Although their sequences differed from each other, all the substitutions were exclusively in exon 5. Three D(Va) samples had hybrid RHD-CE(5)-D alleles, but the D(Va)-like variant had a unique nucleotide substitution with a single amino acid change, E233K. Exon 5 of the genomic DNA from all 13 samples was analyzed by sequencing. No other sequences were identified. CONCLUSION: All RhD(Va) and RhD(Va)-like variants had the substitution for E233. E233 seems to be a determinant of epD1 and 5. A new category of RhD variant, DYO, was identified.

Immunotherapy prevents recurrent abortion without influencing natural killer receptor status.

PROBLEM: We assessed the expression of natural killer (NK) receptors in recurrent aborters before and after immunotherapy using their husbands' peripheral blood mononuclear cells (PBMCs). METHOD OF STUDY: Using stored PBMCs from recurrent aborters before and after the immunotherapy, the expression of NK receptors, CD158a, CD158b, CD159 and CD94, were analyzed using monoclonal antibodies for respective receptors. The diversity of killer activatory receptors (KARs) and killer inhibitory receptors (KIRs) was also examined using reverse transcriptase-polymerase chain reaction (RT-PCR)-single strand conformation polymorphism (SSCP) method. RESULTS: In recurrent aborters, no apparent changes in NK receptor expression and the balance between KARs and KIRs were found before and after the immunotherapy. CONCLUSION: The allo-human leukocyte antigen (HLA)-stimulation caused by the immunotherapy for recurrent aborters did not affect the expression of NK receptors and the ratio of KARs to KIRs regardless of the outcome of subsequent pregnancies, suggesting that recurrent aborters may benefit from the immunotherapy through mechanisms unrelated to alteration in NK receptor status.