Biomedical subjects
Publications and source records attributed to G C Douglas.
This study reports the characterization of chloroplast DNA (cpDNA) variation of Fraxinus excelsior at five loci and the successful development of primer pairs for the amplification of three of these containing mononucleotide microsatellites. We detected high levels of haplotype variation among provenances of Fraxinus around Europe and within Ireland.
Infection of human placental syncytiotrophoblast cells with HIV requires direct contact with infected leukocytes. In vitro investigations into mechanisms regulating placental HIV transmission and into the development of therapeutic interventions have been hampered by difficulties inherent in quantitating HIV levels in cocultures of infected lymphocytes and adherent multinucleated syncytiotrophoblast cells. Here, we have used fluorescence in situ hybridization (FISH) for the direct detection of HIV-1 RNA within syncytiotrophoblast cells combined with laser scanning cytometry (LSC) to quantitate HIV levels exclusively in the syncytiotrophoblast cells. HIV-1-infected lymphocytic MOLT-4 cells were cocultured with primary human syncytiotrophoblast cells. Lymphocytic cells were identified with an anti-vimentin antibody and Cy5. HIV RNA was localized by in situ hybridization, using a digoxigenin-labeled riboprobe detected by Oregon Green, and nuclei were stained with 7-aminoactinomycin D. The three-color cocultures were analyzed by LSC to remove unwanted cell populations and quantitate HIV expression levels. The total HIV RNA level (green fluorescence integral) in each colony was normalized for cell size by dividing by the total DNA content (red fluorescence integral). The nuclear-normalized fluorescence integral was 2.3 times higher in infected cocultures than in uninfected cultures. When cocultures were incubated with 10 microM AZT, the green/red fluorescence integral value was significantly lower than that of cocultures incubated in the absence of AZT, corresponding to a 78% reduction in fluorescence. Laser scanning cytometry can be used to quantitate cell-mediated HIV infection in syncytiotrophoblast cells and should allow drug assessment studies and studies aimed at understanding the mechanism of virus entry into trophoblast cells to be carried out.
Despite their potential importance in placental HIV infection and placental immune function, nothing is known about the expression of chemokine receptors by human syncytiotrophoblast cells. Immunocytochemical analysis revealed that primary cultures of term syncytiotrophoblast cells express CCR1, CCR3, CXCR4, and CCR6. Immunohistochemical examination of cryosections of term placental villous tissue confirmed the expression of CCR3, CXCR4, and CCR6 by trophoblast cells. The primary syncytiotrophoblast cultures showed no reactivity with antibodies against CCR5. In the villous tissue sections, CCR5 was detected in stromal cells and blood vessel walls but was not found in trophoblast cells. RT-PCR analysis of RNA extracted from cultured syncytiotrophoblast cells confirmed that the cells express message for CCR1, CCR3, CXCR4, CCR6 and CCR10. No transcripts corresponding to CCR2b, CCR5, or CCR8 were detected. Other experiments showed that exposure of syncytiotrophoblast cells to soluble SDF-1alpha elicited a calcium mobilization response, consistent with the expression of functional CXCR4. Thus, human syncytiotrophoblast cells express CXCR4, a known co-receptor for TCL-tropic HIV-1 isolates but do not express CCR5, the major co-receptor for M-tropic isolates. In addition to implications for the maternal-fetal transmission of HIV, the expression of chemokine receptors by syncytiotrophoblast cells could be important in other aspects of placental immune function.
Knowledge of chemokine receptor expression by human syncytiotrophoblast has important implications for our understanding of maternal-fetal HIV transmission as well as for understanding the regulation of placental growth and development. This review discusses what is known about chemokine receptor expression by trophoblast and other placental cells. In addition, new data are presented showing that CXCR4 is expressed on the syncytiotrophoblast surface. In other new studies, leukocyte-mediated HIV-1Lai(an X4 strain) infection of syncytiotrophoblast cultures was reduced when stromal derived factor-1alpha was added to the cocultures, consistent with a role for CXCR4. The available information on chemokine receptor expression by trophoblast is discussed in terms of the apparent selective transmission of R5 strains. Studies in other systems indicate that caution must be used in predicting chemokine receptor usage by different HIV isolates, particularly when the route of infection is cellmediated.
The objective of this work was to develop an in vitro system that would extend the usefulness of the macaque as a model for studying trophoblast invasion and spiral artery modification. We sought to determine whether trophoblast cells isolated from early gestation macaque placentas expressed vitronectin receptors and tested the idea that these receptors play a role in trophoblast migration and adhesion. Cytotrophoblast cells were isolated from 40-100 day macaque placentas, cultured, and characterized by immunofluorescence microscopy and flow cytometry. The cells expressed alphaV, beta3, and beta1 integrins on their surfaces. Immunohistochemical analysis of early gestation placentas and decidua basalis confirmed that intravascular trophoblast cells express alphaVbeta3/beta5. Using migration chambers we found that the trophoblast cells migrated towards vitronectin but not towards bovine serum albumin. This specific migration was blocked by preincubating the trophoblast cells with anti-vitronectin receptor (alphaVbeta3/beta5) antibodies. In other experiments, macaque trophoblast cells adhered to myometrial endothelial cells in a time-dependent manner and adhesion was significantly blocked by antibodies against alphaVbeta3/beta5 integrin. The results suggest that vitronectin receptors expressed by macaque trophoblast cells play a role in the migratory activity of these cells and may also be important in mediating attachment to endothelium.
During placental development in higher primates trophoblast cells invade maternal blood vessels and migrate along the luminal surface of endothelium. In the present study, the adherence of human cytotrophoblast cells to endothelial cells has been characterized to test the hypothesis that vitronectin receptors (alpha(v) integrins) play a role in intra-luminal trophoblast migration. Adherence was measured using a quantitative fluorescence-based assay and was found to increase in a time-dependent fashion up to about 2 h after which it leveled off. Adhesion was detectable at 4 degrees C but was greatly reduced compared to that seen at 37 degrees C. Adhesion was partially blocked by antibodies against alpha(v)beta3/beta5 integrin, beta1 integrin and by antibodies against P-selectin. Antibodies against beta3 integrin subunits had no effect. Adhesion was reduced by galactose-6-phosphate and fructose-6-phosphate. Flow cytometric analysis revealed alpha(v) integrin on the surface of cytotrophoblast and endothelial cells. Beta1 integrin was detected on the surface of endothelial cells and on cytokine-stimulated cytotrophoblast cells. Beta3 and beta5 integrins were not detected on the surface of either cell type, although beta3 was detected using permeabilized endothelial cells. These results raise the possibility that alpha(v) integrins expressed by both cytotrophoblast cells and endothelial cells, and P-selectin expressed by endothelial cells, may be important in facilitating trophoblast adhesion and migration along the uterine microvasculature.
We have investigated the binding and internalization of alpha2-macroglobulin and serum albumin by human placental syncytiotrophoblast cells in vitro. The time course (obtained at 4 degrees C) of alpha2-macroglobulin binding indicated that an equilibrium was reached after 4 h. The binding of 125I-labelled alpha2-macroglobulin to syncytiotrophoblast cells was competitively reduced in the presence of excess unlabelled alpha2-macroglobulin. When the concentration-dependence of binding was examined over a wide concentration range, non-linear regression analysis yielded a Kd of 6.4 nM. In the case of albumin, binding was weak and ligand dissociated from the cell surface during aqueous washing making it impractical to analyze the binding reaction. In other experiments, syncytiotrophoblast cells were incubated with 125I-labelled alpha2-macroglobulin at 37 degrees C. Under these conditions, trypsin-resistant cell-associated radioactivity increased with time consistent with ligand internalization. 125I-labelled-ligand was internalized with a t1/2 of about 5 min. After a lag period some radioactivity was released back into the incubation medium. When measured at times up to 210 min, this was found to consist of mostly TCA-precipitable material that had been lost from the cell surface. However, when the incubation was extended to 24 h, almost 15% of the initial cell-associated radioactivity was released to the extracellular medium as TCA-soluble material, consistent with a slow rate of ligand degradation. The specific binding of 65Zn-labelled alpha2M was similar to that of the 125I-labelled ligand and trypsin-resistance measurements provided evidence of alpha2M-mediated 65Zn uptake. These results support a role for syncytiotrophoblast in the metabolism of alpha2-macroglobulin during pregnancy and are also consistent with a role for alpha2-macroglobulin in the maternal-fetal transport of zinc.
Integrin-mediated adhesive interactions are viewed in the context of HIV transmission to susceptible cells and fertilization. The ability of a low-molecular-weight non-peptide integrin modulator to inhibit HIV infection (virus-to-cell and cell-to-cell) and sperm-egg fusion is demonstrated. It is concluded that integrin-modulating substances offer significant promise as female-controlled means for preventing sexual transmission of HIV (whatever entity acts as HIV vector in semen and other penile secretions) and as female-controlled contraceptives.
Therapeutic interventions and public education are reducing pediatric AIDS cases in developed countries, but the number of HIV-infected women and children is still a major global concern. The finding that human sperm-associated HIV can be transmitted to oocytes following in vitro fertilization provides a novel viewpoint from which to consider not only the problem of HIV transmission to children but also transmission to women. In the present paper we will first discuss some recent findings that offer new perspectives on the role of the placenta, and particularly the trophoblast, in maternal-fetal transmission of HIV. Results will be presented showing that cell-mediated infection of syncytiotrophoblast cells requires direct contact between infected lymphocytes and trophoblast. We will also discuss possible routes of transmission of HIV to both mothers and their offspring in the light of data providing evidence of gametic infection. These hypothetical routes include trophoblast-mediated infection of maternal uterine cells during implantation and trophoblast-mediated infection of maternal blood cells during pregnancy. Clearly, more studies are needed in order to assess the significance and relative contribution of these routes in the transmission of HIV.
Dimethyl sulfoxide (DMSO) exerts a number of biological effects, the most frequently cited being induction of cell differentiation. The compound also increases invasiveness and metastatic potential. In contrast to the many reports of DMSO-induced cell differentiation, we report here that DMSO inhibits the morphological differentiation of human cytotrophoblast cells to syncytiotrophoblast, as revealed by immunofluorescence staining for desmosomal protein and nuclei. Cytotrophoblast cells treated with DMSO under differentiation-inducing conditions remained mononucleated with intense desmosomals staining. The effect was dose dependent, with a maximal effect seen at 1.5% DMSO. Concentrations of < or = 0.5% had no effect and concentrations > 2% were cytotoxic. In addition to these morphological changes, DMSO inhibited secretion of human chorionic gonadotropin in a dose-dependent manner. At a concentration of 1.5%, DMSO inhibited secretion by 70%. If cytotrophoblast cells were cultured in the presence of DMSO and then switched to DMSO-free medium, they proceeded to differentiate normally. While the precise mechanism of action remains unknown, judicious use of DMSO may be a useful tool for studying and manipulating the differentiation of human trophoblast cells in vitro. The findings also indicate that care should be used in interpreting results obtained using DMSO as a carrier in drug and inhibitor studies.
Cells with fibroblast-like features were isolated from the villous tissue of normal term human placentas. Immunocytochemical characterization of the cells showed that they were vimentin-positive but negative for factor-VIII, CD14 and CD4. Thus, the cells are mesenchymal and are not endothelial cells, macrophages or trophoblast. These cells were exposed to nine different cell-free virus isolates, including seven isolates of human immunodeficiency virus type 1 (HIV-1), one HIV-2 isolate and one simian immunodeficiency virus isolate (SIVmac251). The susceptibility of the cells to infection was evaluated by immunocytochemical and virological techniques. No evidence of infection could be found using immunofluorescence microscopy or by p24 antigen capture and reverse transcriptase assays. However, virus rescue experiments using 11 different target cell types provided evidence that the placental fibroblasts were susceptible to infection with HIV-1Lai, HIV-1IIIB, HIV-2CBL-20, and SIVmac251, yet were resistant to infection by all other isolates. The infected fibroblasts exhibited neither cytopathic effects nor released virus into the culture medium. For each infected fibroblast population, some, but not all, indicator target cell lines or human peripheral blood mononuclear cells were able to rescue the respective virus. Based on these observations, we conclude that placental fibroblasts can be infected with HIV during transplacental transmission and could act as virus reservoirs, capable of infecting other fetal cells.
Transferrin receptor expression has been examined in primary cultures of morphologically differentiated placental syncytiotrophoblast cells. More than 90% of the cells were multinucleated. Incubation of syncytiotrophoblast for 4 days in the presence of iron salts had no effect on receptor expression assessed by measuring the binding of 125I-labelled transferrin. However, incubation of cells in the presence of human diferric transferrin (10-100 microM) led to a 50% decrease in surface and intracellular receptor expression. This down-regulation was not accompanied by a significant decrease in receptor synthesis. In contrast to syncytiotrophoblast, expression of intracellular transferrin receptors in non-differentiated cytotrophoblast cells decreased when cells were cultured with iron salts; this was accompanied by decreased receptor synthesis. Addition of diferric transferrin to cytotrophoblast cells led to a 50% reduction in surface and intracellular receptor expression, similar to that seen in the syncytiotrophoblast. This reduction was accompanied by a decrease in receptor synthesis. In contrast to that of most cell types, the expression and distribution of trophoblast transferrin receptors were not altered by insulin, epidermal growth factor or hydrocortisone. These characteristics of syncytiotrophoblast transferrin receptor expression may assist in ensuring a supply of iron to the fetus regardless of the maternal iron status.
We previously showed that a significant number of failing pregnancies are associated with production of human chorionic gonadotropin (hCG) having relatively low bioactivity. The present study was designed to compare the secretion of intact, immunoreactive hCG to the secretion of bioactive hCG during trophoblast differentiation, and to test the hypothesis that the lower bioactive: immunoreactive hCG ratios in failing pregnancies are related to reduced or impaired trophoblast differentiation. Cytotrophoblast cells were isolated from term placentas and cultured under conditions that induced or did not induce syncytiotrophoblast formation. Culture media were collected at regular intervals up to 72 h and levels of immunoreactive and bioactive hCG were measured. The differentiation of cytotrophoblast cells to multinucleated syncytiotrophoblast was monitored by immunocytochemistry and electron microscopy. During the 72 h culture period, concentrations of immunoreactive and bioactive hCG increased in both differentiating and non-differentiating cells. However, the concentrations of immunoreactive and bioactive hCG were higher under culture conditions that promoted trophoblast differentiation. Furthermore, the ratio of bioactive hCG to immunoreactive hCG was higher in differentiating cultures. When differentiation was inhibited by dimethyl sulfoxide, the secretion of bioactive hCG was reduced and the bioactive: immunoreactive hCG ratio did not change. These findings are consistent with the idea that production of bioactive hCG accompanies syncytiotrophoblast formation.
We have previously shown that lymphocytic cells adhere to cultured syncytiotrophoblast and that this may be important in the lymphocyte-mediated infection of trophoblast with the human immunodeficiency virus (HIV). During the course of studies aimed at investigating the role of cell surface carbohydrates in adhesion, it was discovered that a contaminant of commercial fucose-1-phosphate, dicyclohexylamine, inhibited MOLT-trophoblast adhesion. Dicyclohexylamine and the related compounds, cyclohexylamine and hexylamine, inhibited adhesion in a dose-responsive manner with half-maximal inhibition seen at about 4 mM. While the pressor effects of cyclohexylamine, the principal metabolite of cyclamate, are well known, this is the first report of an effect of this and related compounds on cell adhesion activity. The inhibitory effect was reversible and, at concentrations less than 25 mM, did not result in loss of cell viability. Several possible mechanisms of action of cyclohexylamine were examined in an attempt to explain the effect on adhesion. No evidence was found to suggest that the effects of cyclohexylamine were due to inhibition of polyamine synthesis, increase in intracellular Ca2+ concentration or to a lysosomotropic effect. The concentrations of cyclohexylamine used are within the range of plasma concentrations attainable in humans, raising the possibility that the in vitro effects described here may also occur in vivo. The results also suggest that caution should be used in the interpretation of results obtained from experiments where cell adhesion is blocked using exogenous monosaccharides that are in the form of dicyclohexylammonium salts. Appropriate controls must be included or, if possible, sodium, potassium or barium salts should be chosen.
BACKGROUND: The success of kidney transplant surgery and ureteral reconstruction requires the preservation of the ureteral blood supply. Because of its potential vulnerability to surgical trauma during transplant and reconstructive surgery, the ureteral vasculature merits a full anatomical description. METHODS: The microvascular anatomy of the ureter was studied in male New Zealand white rabbits by light microscopy and transmission electron microscopy and scanning electron microscopy of vascular corrosion casts and alkali digested tissue. RESULTS: The rabbit ureter is supplied predominantly by a branch of the renal artery proximally (cranial ureteral artery) and by a branch of the vesicular artery distally (caudal ureteral artery). Minor vascular continuities are also present between the capillary beds of the ureter and those of the renal pelvis cranially and the bladder wall caudally. There are no external vascular connections to the middle ureter with the exception of a single, small vein which drains into the inferior vena cava. A single group of longitudinal arteries and veins runs the full length of the ureter within the adventitia. Branches of these longitudinal vessels pass tangentially through the muscularis to supply a vascular complex within the lamina propria. This complex in turn supports a rich, mucosal capillary plexus located at the junction between the transitional epithelium and the lamina propria. In the fixed ureter the capillary plexus lies in grooves formed by displacement of the basal layers of the overlying transitional epithelium. The capillaries are continuous or fenestrated, are often invested with pericytes, and are distributed uniformly around the entire circumference of the ureter. CONCLUSIONS: The ureteral vasculature exhibits several unique features related to its function in urine conduction and its ability to accommodate expansion and contraction. The combination of techniques used provides a clear three-dimensional view of this vasculature. Our findings also confirm that, because of its limited blood supply, the ureter may be very susceptible to injury during renal transplantation or other abdominal surgery.
We previously demonstrated that syncytiotrophoblast (ST) cells from term human placentas could be infected when cocultured with HIV-infected lymphocytic cells. Here, we have used fluorescence microscopy and transmission electron microscopy to examine the kinetics of this infection process. Molt-4 clone 8 cells infected with HIV-1Lai or filtered supernatant from these cultures were incubated with ST cells for different times. In cell-associated infection, immunofluorescence microscopy revealed that some ST colonies were positive for HIV core proteins (p24,p55) after 1 hr. The number of positive colonies and the intensity of the ST-associated fluorescence increased with time. Transmission electron microscopy showed viral particles with HIV morphology associated with the ST cell surface at 1 hr. Immature virions with budding morphology were observed at 2 hr. In cell-free infection, positive p24,p55 staining was first detected in a few ST colonies at 4 hr. The number of positive colonies increased with time. At 24 hr, the fluorescence pattern and intensity resembled that seen with cell-mediated infection at 4 hr. Transmission electron microscopy revealed an increasing number of viral particles associated with the ST cell plasma membrane with respect to time, and budding virions first appeared at 8 hr. These results demonstrate that HIV infection of placental ST cells proceeds very rapidly in culture and that, furthermore, cell-associated infection of ST is much more efficient than the infection with cell-free virus.
Lactoferrin, an iron-binding glycoprotein that is abundant in milk of some species, has been suggested to play a key role in the absorption of iron in human infants. This hypothesis is based on the dominant role of lactoferrin as an iron-binding component in human milk and on the occurrence of lactoferrin receptors in brush-border membranes in infants' intestines. The piglet may be a useful model to evaluate the biological activity of lactoferrin because we have previously found the presence of a lactoferrin receptor in brush-border membranes from suckling piglets. In this study, viable enterocytes were isolated from 6- to 20-day-old suckling piglets. Binding studies were performed at 4 degrees C using 125I-labeled porcine lactoferrin. Scatchard analysis of equilibrium binding data showed an apparent binding constant (Kd) of 2 x 10(-6) M (SD = 0.6 x 10(-6)). This affinity is in close agreement with previous results obtained using isolated brush-border membrane vesicles. Bovine lactoferrin inhibited the binding of porcine lactoferrin. Porcine transferrin, however, did not affect porcine lactoferrin binding significantly. Thus, lactoferrin binding is highly specific. When enterocytes were incubated with 125I-labeled lactoferrin at 37 degrees C, the amount of cell-associated radioactivity exceeded the surface binding capacity of the cells by almost fivefold. This finding agrees with the continuous binding and subsequent internalization of 125I-labeled lactoferrin. The isolated piglet enterocyte seems to provide a useful model for further studies of the mechanism of receptor-mediated absorption of lactoferrin.
Explore the source record for details and available documents.