Activity of synthetic enzymes of tetrahydrobiopterin in the human placenta

Tetrahydrobiopterin (BH4) is an essential co-factor for nitric oxide (NO) synthase (NOS) and regulates the production of NO, or endothelium-derived relaxation factor. Although NOS is highly expressed in the placenta and NO plays a critical role in the regulation of feto-placental circulation, the mechanism maintaining the level of BH4 is not known. To investigate the de novo synthesis of BH4 in the human placenta, the activity of guanosine triphosphate cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR) in the chorionic tissue during the first, second and third trimester of pregnancy was analyzed. GTPCH activity was the lowest of the three enzymes and became negligible after the second trimester. There was no significant change in PTPS activity throughout pregnancy. Although SR activity decreased significantly after the second trimester, the levels remained abundant throughout pregnancy. These results showed that GTPCH is a rate-limiting enzyme and the total activity of the de novo synthesis of BH4 is negligible in the mature placenta after the second trimester when fetal growth is accelerated. The present study suggests that the level of BH4 in the placenta depends principally on the system other than de novo synthesis. The salvage pathway is considered the most potent system, which is formed by the transfer of the substrates from the fetus and their enzymatic conversion to BH4 in the placenta.     

CD44 variant 6 in endometrioid carcinoma of the uterus: its expression in the adenocarcinoma component is an independent prognostic marker

The expression of variant isoforms of CD44 (CD44v) correlates with the metastatic potential of various carcinomas. In endometrial cancer, however, the significance of CD44v-expression as a prognostic indicator has not been fully investigated, nor has it been compared with that of p53, estrogen receptor or Ki67. Surgical material consisted of 14 atypical endometrial hyperplasias (AEH) and 163 endometrial carcinomas (EC). Expression of CD44s, v3 and v6 in carcinoma tissue, and other prognostic markers were immunohistochemically evaluated. The expression in the squamous differentiation was strictly excluded for the evaluation of immunohistochemistry, because the significance was different from that in the adenocarcinoma component. CD44s was frequently expressed in AEH and EC. On the other hand, CD44v3- and v6-positivities were rare or nonexistent in AEH, but were observed in 8 and 35% of EC, respectively. CD44v3-expression correlated significantly with histologic grade and lymph node metastasis. However, there was no correlation between CD44v6 expression and any clinicopathologic factor, nor were other prognostic markers expressed. Univariate analysis revealed that each CD44 was a prognostic determinant in the patients with EC. However, employing multivariate analysis, there were only three independent factors: p53 overexpression, CD44v6 expression and myometrial invasion. CD44v6 expression in the adenocarcinoma component may directly affect the behavior of carcinoma and the prognosis of patients with EC.     

No difference in serum leptin concentrations between urban-dwelling Austronesians and Non-Austronesians in Papua New Guinea.

Pacific Islands populations can be broadly divided into Austronesians (AN) and Non-Austronesians (NAN); obesity and type 2 diabetes are prevalent in the former, although leptin levels in both groups have seldom been investigated. Thirty-seven (20 male and 17 female) adult pairs, matched by age and percent body fat, from AN-speaking Balopa and NAN-speaking Huli, all of whom migrated to settle in Port Moresby, the capital of Papua New Guinea, were selected for comparison of their serum leptin concentrations. The Balopa did not differ significantly from the Huli in age (30.5 +/- 9.7 and 30.0 +/- 8.7 years for males, 33.7 +/- 8.9 and 34.1 +/- 7.5 years for females, respectively) or percent body fat (19.4 +/- 5.6 and 18.8 +/- 4.6 for males, 34.1 +/- 6.2 and 33.3 +/- 5.0 for females), although the BMI of females was lower in the Balopa (26.4 +/- 4.9) than in the Huli (29.7 +/- 4.7) (P = 0.02). In both ethnic groups, females had markedly higher leptin concentrations than males, but there was no significant inter-group difference in males (3.5 +/- 2.6 and 3.1 +/- 4.7 ng/ml, P = 0.14) or females (22.7 +/- 12.9 and 19.7 +/- 11.9 ng/ml, P = 0.40), after controlling for lifestyle factors and serum lipids. Multiple regression analysis revealed that significant predictors of leptin concentration were % body fat (beta = 0.58), sex (male, 0; female, 1; beta = 0.27), and smoker status (non-smoker, 0; smoker, 1; beta = -0.15) (R(2) = 0.80), implying that the leptin concentration was primarily determined by lifestyle-derived body fatness. In conclusion, the NAN populations do not endogenously differ in leptin status from the AN populations, who have been recognized as a typical group with a "thrifty" genotype.     

IDO expression on decidual and peripheral blood dendritic cells and monocytes/macrophages after treatment with CTLA-4 or interferon-gamma increase in normal pregnancy but decrease in spontaneous abortion

Recent data demonstrated that CD4+CD25+ regulatory T (Treg) cells and an enzyme called indoleamine 2,3-dioxygenase (IDO) mediate maternal tolerance to the fetus. Interestingly, Treg cells express the CTLA-4 molecule on their surface, and B7 (CD80/86) ligation by CTLA-4 enhanced IDO activity of dendritic cells (DCs) and monocytes by the induction of interferon gamma (IFN-gamma) production. In this study, we studied the IDO expression on peripheral blood monocytes and decidual monocytes or DCs after treatment with CTLA-4/Fc fusion protein or IFN-gamma using flow cytometry. IDO expressions on both peripheral blood DC and decidual DC and monocytes were up-regulated during normal pregnancy. On the other hand, both IDO expression on DC and monocytes after IFN-gamma treatment or CTLA-4 treatment were decreased in spontaneous abortion cases. The expression of CD86 on peripheral blood and decidual monocytes and DC in spontaneous abortion cases was lower compared with those in normal pregnancy subjects. Also, IFN-gamma production by decidual and peripheral blood mononuclear cells after CTLA-4/Fc treatment in spontaneous abortion cases was significantly lower than those in normal pregnancy subjects. These data suggest that CTLA-4 on Treg cells up-regulates IDO expression on decidual and peripheral blood DC and monocytes by the induction of IFN-gamma production.     

Generation of functional and mature dendritic cells from cord blood and bone marrow CD34+ cells by two-step culture combined with calcium ionophore treatment

The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34+ hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34+ progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34+ hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7--11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-alpha. By the two-step culture, total nucleated cells were increased 208+/-66 (+/-SD, n=13), or 94+/-29 (n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34+ cells at the time of starting culture. Out of the total nucleated cells, 23 +/-10.4% of cells in CB cell culture and 25 +/-5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a+ cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34+ cells using SCF, GM-CSF and TNF-alpha. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a+ DC generated from CD34+ cells, and the phenotypes and functions of these CD1a+ DC could be enhanced efficiently by treatment with a calcium ionophore agent.     

Mutational screening of APP gene in patients with early-onset Alzheimer disease utilizing mismatched PCR-RFLP

To elucidate the frequency of mutations of the β/A4 amyloid protein precursor (APP) gene in early-onset Alzheimer disease, we designed a mismatched PCR-RFLP that can identify all kinds of missense mutations at codon 717 in addition to the seven kinds of known mutations at exon 17. When we screened mutations at exon 17 utilizing this method and the double missense mutations at exon 16 of the APP gene by PCR-RFLP, no cases revealed mutations of the APP gene among 13 familial and 54 sporadic cases, except one family (OS-1) that had previously been reported and used as a positive control of APP717(Val → Ile). Our results support the hypothesis that mutations in the APP gene are not major causes in early-onset Alzheimer disease.     

Expression of connexin 32 gap junction protein in the kidneys during fetal development of the hamster (Mesocricetus auratus)

The expression of gap junction protein was examined immunohistochemically using affinity-purified antibody against rat liver gap junction protein, connexin 32 (Cx32), in the kidneys of fetal (gestation days 13–16) and adult Syrian golden hamsters. Phalloidin histochemical staining, PNA- and RCA I-lectin stainings, NCAM immunostaining, and alkaline phosphatase and Na⁺-K⁺-ATPase enzyme-histochemical staining were performed in combination with Cx32 immunostaining. The kidney sections were observed with a confocal scanning laser microscope. By gestation day 13, Cx32 immunoreactivity was observed in the differentiating tubules. The Cx32 staining was localized on the lateral cell membrane of the cells lining the developing proximal tubules, while the S-shaped bodies, developing distal tubules, and collecting tubules showed no positive immunostaining. As the kidney developed, the density of Cx32 immunoreactivity increased. As the gap junction provides pathways for cell-cell communication, the development of Cx32 expression may imply that this structure plays an important role in renal tubule development. Confocal scanning laser microscopy provided a clear image of the fluorescence-labeled cell structures, free from out-of-focus blur. Using the same sections, stereoscopic images were easily reconstructed from serial optical sections, and were helpful in understanding the spatial distribution of Cx32 expression in the developing fetal proximal tubules.     

Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the method's sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase; however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.     

Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo

Notch receptors and their ligands contribute to many developmental systems, but it is not apparent how they function after birth, as their null mutants develop severe defects during embryogenesis. Here we used the Cre-loxP system to delete the Delta-like 1 gene (Dll1) after birth and demonstrated the complete disappearance of splenic marginal zone B cells in Dll1-null mice. In contrast, T cell development was unaffected. These results demonstrated that Dll1 was dispensable as a ligand for Notch1 at the branch point of T cell-B cell development but was essential for the generation of marginal zone B cells. Thus, Notch signaling is essential for lymphocyte development in vivo, but there is a redundancy of Notch-Notch ligand signaling that can drive T cell development within the thymus.     

Ultrastructural changes of bile duct epithelium in primary biliary cirrhosis in relation to progression of bile duct loss

Summary Using wedge liver biopsies from patients with primary biliary cirrhosis (PBC), ultrastructural features of the intrahepatic bile ducts in livers with slight or no bile duct loss were compared with those in livers with advanced bile duct loss and in extrahepatic cholestasis (EHC).Most changes in the biliary epithelium in PBC were similar to those in EHC. Microvillous loss and bleb formation, mitochondrial damage and increase in endoplasmic reticulum and ribosomes were found in PBC irrespective of the degree of bile duct loss, and also in EHC. These changes were present almost equally at any level of the biliary tree, and are presumed to represent a variety of non-specific lesions of biliary epithelial cells. As the loss of bile ducts in PBC progressed, cytoskeletal filaments and cytophagosomes increased in number and basement membranes were more thickened and reduplicated. These changes were more or less conspicuous in smaller branches of the biliary tree, and were also prominent in EHC. They might be causally related to the bile flow disturbance in the liver. Lateral intercellular spaces were irregularly dilated and contained osmiophilic membranous and/or granular material, similar to that found in duct lumena, within and without the basement membrane, and in the cytoplasm of periductal macrophages. Furthermore, pinocytotic vesicles were increased in the biliary cytoplasm facing periphery. These findings suggest possible alteration of the permeability of biliary epithelial cells, probably in the direction from the lumena to the periductal tissue. Such changes were found in PBC livers with virtual absence of bile duct loss, and the significance of this phenomenon is discussed.