Flow cytometric characterisation of cells of differing densities isolated from human term placentae and enrichment of villous trophoblast cells

Manoussaka, Maria; Jackson, David J.; Lock, Rebecca; Sooranna, Suren R.; Kumpel, Belinda M.

doi:10.1016/j.placenta.2004.06.008

Cited by 24 publications

(19 citation statements)

References 26 publications

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“…As shown in Fig. 6A, the prepared cell populations were almost exclusively positive for cytokeratin-7 and negative for vimentin, showing that almost all cells were primary CTB, whereas only minor proportions accounted for mesenchymal cells (57,58,75). Consistent with the potency to neutralize HCMV entry and cell-to-cell spread, the neutralization potency of all PC NAb measured on CTB was significantly higher than that of the NAb targeting gH or CMV-HIG ( Fig.…”

Section: ) (B)supporting

confidence: 58%

“…The CTB were isolated on a discontinuous gradient of Percoll (GE Healthcare, Uppsala, Sweden; 50, 45, 35, and 30%) by centrifugation. Cells migrating to the 35%/45% Percoll interface were recovered and immunopurified by negative selection with simultaneous treatment with anti-CD9 (to exclude EnC, FB, platelets, smooth muscle, extravillous trophoblast cells, B cells, and monocytes) and anti-CD45RA (to exclude leukocytes) antibodies and magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) (56,57). The purity of the CTB population was assessed by cytokeratin-7 staining and was on average Ͼ97%.…”

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confidence: 99%

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Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection

Chiuppesi

Wussow

Johnson

et al. 2015

J Virol

127

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Human cytomegalovirus (HCMV) elicits neutralizing antibodies (NAb) of various potencies and cell type specificities to prevent HCMV entry into fibroblasts (FB) and epithelial/endothelial cells (EpC/EnC). NAb targeting the major essential envelope glycoprotein complexes gB and gH/gL inhibit both FB and EpC/EnC entry. In contrast to FB infection, HCMV entry into EpC/EnC is additionally blocked by extremely potent NAb to conformational epitopes of the gH/gL/UL128/130/131A pentamer complex (PC). We recently developed a vaccine concept based on coexpression of all five PC subunits by a single modified vaccinia virus Ankara (MVA) vector, termed MVA-PC. Vaccination of mice and rhesus macaques with MVA-PC resulted in a high titer and sustained NAb that blocked EpC/EnC infection and lower-titer NAb that inhibited FB entry. However, antibody function responsible for the neutralizing activity induced by the MVA-PC vaccine is uncharacterized. Here, we demonstrate that MVA-PC elicits NAb with cell type-specific neutralization potency and antigen recognition pattern similar to human NAb targeting conformational and linear epitopes of the UL128/130/ 131A subunits or gH. In addition, we show that the vaccine-derived PC-specific NAb are significantly more potent than the anti-gH NAb to prevent HCMV spread in EpC and infection of human placental cytotrophoblasts, cell types thought to be of critical importance for HCMV transmission to the fetus. These findings further validate MVA-PC as a clinical vaccine candidate to elicit NAb that resembles those induced during HCMV infection and provide valuable insights into the potency of PC-specific NAb to interfere with HCMV cell-associated spread and infection of key placental cells. IMPORTANCEAs a consequence of the leading role of human cytomegalovirus (HCMV) in causing permanent birth defects, developing a vaccine against HCMV has been assigned a major public health priority. We have recently introduced a vaccine strategy based on a widely used, safe, and well-characterized poxvirus vector platform to elicit potent and durable neutralizing antibody (NAb) responses targeting the HCMV envelope pentamer complex (PC), which has been suggested as a critical component for a vaccine to prevent congenital HCMV infection. With this work, we confirm that the NAb elicited by the vaccine vector have properties that are similar to those of human NAb isolated from individuals chronically infected with HCMV. In addition, we show that PCspecific NAb have potent ability to prevent infection of key placental cells that HCMV utilizes to cross the fetal-maternal interface, suggesting that NAb targeting the PC may be essential to prevent HCMV vertical transmission. Human cytomegalovirus (HCMV) is the most common infectious cause of permanent births defects worldwide, often resulting in auditory and cognitive abnormalities and in rare cases even in multiorgan failure and death (1-4). Congenital HCMV infection occurs in 0.05 to 1% of all pregnancies, and 10 to 25% of congenitally infected newborns d...

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Section: ) (B)supporting

confidence: 58%

mentioning

confidence: 99%

Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection

Chiuppesi

Wussow

Johnson

et al. 2015

J Virol

127

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“…Following published methods (40)(41)(42)(43)(44), trophoblast from gestational age matched normal and GDM placentas were isolated ( Fig. 1) and purified (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The trophoblast cells from the placentas were isolated and then purified by following the published methods (40)(41)(42)(43)(44). Placentas were placed in sterile trays, maternal side facing up.…”

Section: Methodsmentioning

confidence: 99%

GDM-associated insulin deficiency hinders the dissociation of SERT from ERp44 and down-regulates placental 5-HT uptake

Li¹,

Hadden²,

Singh³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Serotonin (5-HT) transporter (SERT) regulates the level of 5-HT in placenta. Initially, we found that in gestational diabetes mellitus (GDM), whereas free plasma 5-HT levels were elevated, the 5-HT uptake rates of trophoblast were significantly down-regulated, due to impairment in the translocation of SERT molecules to the cell surface. We sought to determine the factors mediating the down-regulation of SERT in GDM trophoblast. We previously reported that an endoplasmic reticulum chaperone, ERp44, binds to Cys200 and Cys209 residues of SERT to build a disulfide bond. Following this posttranslational modification, before trafficking to the plasma membrane, SERT must be dissociated from ERp44; and this process is facilitated by insulin signaling and reversed by the insulin receptor blocker AGL2263. However, the GDM-associated defect in insulin signaling hampers the dissociation of ERp44 from SERT. Furthermore, whereas ERp44 constitutively occupies Cys200/ Cys209 residues, one of the SERT glycosylation sites, Asp208 located between the two Cys residues, cannot undergo proper glycosylation, which plays an important role in the uptake efficiency of SERT. Herein, we show that the decrease in 5-HT uptake rates of GDM trophoblast is the consequence of defective insulin signaling, which entraps SERT with ERp44 and impairs its glycosylation. In this regard, restoring the normal expression of SERT on the trophoblast surface may represent a novel approach to alleviating some GDM-associated complications.serotonin | ERp44 | insulin | serotonin transporter | gestational diabetes mellitus

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“…The digested cells were put onto a 10-70% discontinuous percoll gradient (GE Healthcare Bio-sciences AB, Uppsala, Sweden) in a 50 ml centrifuge tube and centrifuged at 1500 × g for 15 min. The purified cytotrophoblast cells were carefully collected in the region corresponding to a density of 1.035-1.064 g/l [30], and the percoll removing step was repeated. The cell pellet was suspended in 5 ml of medium and filterd with 30 µm MACS ® Pre-Separation Filters (Miltenyi Biotec K.K, Tokyo, Japan).…”

Section: Isolation and Culture Of Human Trophoblast Cellsmentioning

confidence: 99%

Expression and Role of SNAT3 in the Placenta

et al. 2009

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The number of characters in the abstract; 1079 characters without spaces 2 The number of characters in the entire manuscripts; 28922 characters without spaces The number of illustrations; 8The number of references; 40 3 AbstractGlutamine is the most versatile amino acid and its plasma concentration is the highest of all amino acid. Many transporters are therefore involved in glutamine uptake or efflux. Glutamine is actively released from the placenta into fetal circulation. In this study, we examined the alteration of transporters that transport glutamine into fetal circulation as gestation progresses. High expression levels of system A and y + L were found in the rat placenta in the late period of pregnancy and the expression levels of these transporters increased as gestation progressed (p<0.05). On the other hand, the expression of SNAT3, the system N transporter, was detected in the early period of pregnancy and its expression level decreased as gestation progressed (p<0.05). SNAT3 was also found to be expressed in isolated human primary cytotrophoblast cells and its expression level was decreased by their differentiation into syncytiotrophoblast cells (p<0.05). Since this regulation is closely related to glutamine synthetase expression, SNAT3 may play a key role in providing glutamine corresponding to glutamine synthetase function in the early period of gestation. This is the first report on the expression of SNAT3 in the placenta in the early stage of pregnancy.

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Flow cytometric characterisation of cells of differing densities isolated from human term placentae and enrichment of villous trophoblast cells

Cited by 24 publications

References 26 publications

Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection

Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection

GDM-associated insulin deficiency hinders the dissociation of SERT from ERp44 and down-regulates placental 5-HT uptake

Expression and Role of SNAT3 in the Placenta

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