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Biomedical subjects

P M Starkey

Publications and source records attributed to P M Starkey.

At least 19 recordsLinked to original sources
PubMed1992

Isolation and characterisation of a subpopulation of human chorionic cytotrophoblast using a monoclonal anti-trophoblast antibody (NDOG2) in flow cytometry.

Human cytotrophoblast cells, isolated from term amniochorion by enzymic digestion and Percoll gradient centrifugation, were characterised by flow cytometry. A panel of 12 anti-trophoblast monoclonal antibodies was screened for labelling of these cells in flow cytometry and the results compared with immunoperoxidase labelling of cytospin preparations and tissue sections. All 12 antibodies were positive for trophoblast on tissue sections, 11/12 were positive on cytospins but only two (NDOG2 and GB25) gave consistent results in flow cytometry. Two-colour labelling with NDOG2 and W6/32, an antibody to HLA-A, -B, -C, demonstrated that 88% of the NDOG2-positive cells also express Class I major histocompatibility complex (MHC) antigens. The NDOG2-positive cytotrophoblast subpopulation was isolated by flow cytometry in sufficient purity (greater than 95%) and yield (3.1 x 10(6)) for use in functional studies in vitro.

Antibodies, Monoclonal↗
PubMed1992

Tumor necrosis factor-alpha selectively stimulates prostaglandin F2 alpha production by macrophages in human term decidua.

OBJECTIVE: Our objective was to investigate the effect of tumor necrosis factor-alpha on prostaglandin production by human term decidual cell subtypes in vitro. STUDY DESIGN: We measured the effect of tumor necrosis factor-alpha on prostaglandins F2 alpha, E2, D2, F metabolite, and E metabolite production by decidual cells (n = 4) with radioimmunoassay. We used flow cytometry after labeling with an antibody to histocompatibility antigen DR, L243, which is specific for macrophages in this tissue, to prepare pure populations of decidual macrophages (n = 3). Differences in prostaglandin output were analyzed by Wilcoxon and Kruskal-Wallis tests. RESULTS: Tumor necrosis factor-alpha stimulated prostaglandin F2 alpha output by unfractionated decidual cells, without altering the output of any other prostaglandins. Tumor necrosis factor-alpha (10 nmol/L) increased prostaglandin F2 alpha production from decidual macrophages (HLA-DR-positive cells) by about threefold: from a median of 727 (range 423 to 1226) to a median of 1974 (range of 1550 to 2201), fmol/10(6) cells per 18 hours, as compared with a 1.4-fold increase from nonmacrophages from a median of 247 fmol/10(6) cells per 18 hours (range 125 to 611) to a median of 340 fmol/10(6) cells per 18 hours (range 201 to 505). CONCLUSION: Stimulation of decidual prostaglandin F2 alpha production by tumor necrosis factor-alpha may be important in the etiology of spontaneous labor at term or preterm labor associated with infection.

Adult↗
PubMed1992

Isolation and purification of human endometrial stromal and glandular cells using immunomagnetic microspheres.

Isolation of pure preparations of the different cell populations of human endometrium is a prerequisite for studies of in-vitro function. Sieving of dispersed endometrial cells, followed by adsorption onto immunomagnetic microspheres coated with antibody to Thy-1 was used to separate glandular and stromal cells. The purity of these cell populations was checked with antibodies to cytokeratin and Thy-1. The stromal cells were 98% pure and 90% viable, gland cells were 82% pure with 76% viability. The purified cells were able to proliferate in vitro as shown by thymidine incorporation.

Antigens, Surface↗
PubMed1992

Production of granulocyte colony-stimulating factor at the materno-foetal interface in human pregnancy.

A bioassay specific for human granulocyte colony-stimulating factor (G-CSF) was developed and used to measure G-CSF production in human pregnancy tissues. G-CSF was secreted by both foetal chorionic villous and maternal decidual tissues taken in the first trimester and at term. The level of G-CSF production by placental tissue was 6750 (1250-10,000) units of bioactivity per g of tissue in 48 hr in the first trimester and 104 (83-190) U/g at term. Bioactive G-CSF was also secreted by decidual tissue, more in the first trimester than at term. ELISA immunoassays measured 75 (10-820) ng/g/48 hr of G-CSF antigen from first trimester placenta, 15 (10-50) ng/g from first trimester decidua and less than 2 ng/g from term placenta. RNA isolated from decidual and chorionic villous tissue or from cells purified by flow cytometry, contained G-CSF mRNA in both tissues. In decidua, mRNA for G-CSF was confined to the macrophages, and cytotrophoblast from term amniochorion contained no detectable G-CSF mRNA. No G-CSF, measured as bioactivity or as mRNA, was detectable in choriocarcinoma cell lines.

Biological Assay↗
PubMed1992

Prostaglandin D2 production by term human decidua: cellular origins defined using flow cytometry.

OBJECTIVE: To measure prostaglandin (PG) D2 output by human decidua at term and to identify the cell population(s) responsible for its production. METHODS: Decidual cell suspensions were prepared enzymatically and the cells were labeled with either of two monoclonal antibodies: F10/89/4, which recognizes the leukocyte common antigen (CD45), or L243, which labels HLA-DR and is specific for macrophages in this tissue. Cells were sorted on a Coulter EPICS 541 flow cytometer. Prostaglandin levels were measured by radioimmunoassay. RESULTS: Human decidua is an important intrauterine source of PGD2 at term. Twenty-nine percent (median) of decidual cells were CD45-positive and 12% were HLA-DR-positive; sorted cell populations were 95% pure. Prostaglandin D2 output (fmol/10(6) cells per 3 hours) by bone marrow-derived (CD45-positive) cells was significantly higher than that by non-bone marrow-derived cells: median 63 (range 35-67) versus 20 (13-21), respectively; HLA-DR-positive cells (macrophages) had the highest PGD2 production rate (186, range 97-288 fmol/10(6) cells per 3 hours). Under basal conditions, PGD2 production by unsorted term decidual cells was not influenced by labor. CONCLUSION: Bone marrow-derived cells (macrophages) are the major source of decidual PGD2 at term. Further studies are required to investigate the possible role of PG production by human decidual macrophages in the mechanism of term and/or preterm labor.

Cell Separation↗
PubMed1991

Variation during the menstrual cycle of immune cell populations in human endometrium.

Morphometric analysis and immunohistology of tissue sections have been used to assess variation, during the normal menstrual cycle, of the bone marrow-derived cell populations in human endometrium. Levels of T cells and macrophages were found to be relatively constant throughout the cycle. In contrast, numbers of large granular lymphocytes, identified as being CD56-positive, were generally low between days 10 and 19, but increased sharply in the latter part of the luteal phase, decreasing again after menstruation. This LGL population is known to be abundant in first trimester pregnancy decidua, and is presumed to play a role in early pregnancy success.

Antigens, CD↗
PubMed1991

Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua.

Large granular lymphocytes (LGL) are the most abundant cell type in first trimester human pregnancy decidua. We have shown previously that CD56-positive decidual LGL have cytotoxic activity against the natural killer (NK) target K562, and that this cytotoxicity is augmented by pretreatment with interleukin-2 (IL-2). We now report that flow cytometrically purified populations of CD56-positive decidual LGL have no cytotoxic activity against either the BeWo choriocarcinoma cell line or freshly isolated term trophoblast. Incubation of unfractionated decidual cells with IL-2 induced cytotoxicity against BeWo, but term trophoblast remained resistant to lysis. Both BeWo and trophoblast showed much lower binding frequencies to decidual or peripheral blood cells than K56 targets, and excess trophoblast did not inhibit cytotoxic activity against K562. This suggests that the resistance of trophoblast to lysis by either decidual or peripheral blood LGL is due to the lack of accessible NK target structures on the surface of trophoblast.

Choriocarcinoma↗
PubMed1991

Expression of intermediate filament in endometrial glands changes with the onset of pregnancy and in endometriosis.

Appropriate endometrial differentiation is believed to be a prerequisite for pregnancy success. This study investigates the expression of two intermediate filament proteins, cytokeratin and vimentin, in human endometrium and first trimester decidua and in ectopic endometrium from women with endometriosis. Stromal elements, including vascular endothelial cells, were consistently vimentin-positive and cytokeratin-negative. Surface and glandular epithelial cells of human endometrium co-expressed vimentin and cytokeratin during all stages of the menstrual cycle, but failed to express vimentin after the onset of pregnancy. This suggests that intermediate filaments, and especially vimentin, may have a role to play in the proliferation and/or differentiation of the endometrial glands during decidualization. Ectopic endometrium showed a staining pattern similar to normal endometrium.

Decidua↗
PubMed1991

Identification by flow cytometry of the prostaglandin-producing cell populations of term human decidua.

Human term decidua produces prostaglandins (PGs) which have been implicated in the initiation of human parturition. Using flow cytometry to isolate pure cell populations, we have investigated the cell types responsible for decidual PG production. Cell dispersions were prepared enzymatically from decidua vera isolated from term placentae, and were incubated in Dulbecco's Modified Eagle's Medium containing 0.25% bovine serum albumin at 37 degrees C. PGF2 alpha and PGE2 output were measured by radioimmunoassay of the conditioned medium. Production of PGF2 alpha (fmol/10(6) cells per 3 h) exceeded that of PGE2 at 273 (108-322) versus 97 (38-127) respectively (median (range]. The decidual cell dispersions were then incubated with monoclonal antibodies (anti-CD45 which labels the leukocyte common antigen or anti-human leukocyte antigen class II (HLA-DR) which is specific for macrophages in this tissue) and sorted by flow cytometry. The resultant antibody-positive and -negative cell populations were incubated and PG production was measured. Controls showed that antibody labelling and sorting did not alter PG production. PGF2 alpha and PGE2 output by bone marrow-derived (CD45-positive) cell populations exceeded that of non-bone marrow-derived (CD45-negative) cells. Furthermore, we were able to demonstrate that the HLA-DR-positive macrophage population had the highest PGF2 alpha and PGE2 production rates in human term decidua in vitro.

Antibodies, Monoclonal↗
PubMed1991

Expression on cells of early human pregnancy decidua, of the p75, IL-2 and p145, IL-4 receptor proteins.

Immunohistological studies of human first trimester pregnancy decidua demonstrated the presence of the p75 interleukin-2 receptor (IL-2R) and the p145 interleukin-4 receptor protein (IL-4R) on cells in the decidual stroma; there was no expression of CD25, the p55 IL-2R. The IL-4R was also expressed on the basal face of the glandular epithelial cells. Flow cytometric analysis of antibody-labelled decidual cell dispersions confirmed these results. Double antibody labelling demonstrated that p75 was expressed exclusively on the CD56-positive decidual large granular lymphocytes (LGL), whereas the IL4-R was expressed on some decidual LGL, and most decidual macrophages and T cells. In vitro incubation of decidual cells with IL-2 failed to induce expression of p55 or to increase the expression of either p75 or the p145 IL-4R. Purified decidual LGL proliferated in vitro in response to IL-2, and IL-4 inhibited this IL-2-induced proliferation.

Cell Division↗
PubMed1990

Flow cytometric characterisation of cell populations in human pregnancy decidua and isolation of decidual macrophages.

Methods have been developed for isolating human tissue macrophages from first trimester or term pregnancy decidua. After a two stage enzymic digestion, viable cells were separated from cellular debris by velocity sedimentation at unit gravity or by Percoll centrifugation. Cell populations were analysed by flow cytometry after labelling with monoclonal antibodies. In term decidua, 47% of the cells were of bone marrow origin, comprising 18% macrophages, 3% large granular lymphocytes and 8% T cells. The remaining cells, the proportion of which varied between individuals, were CD16-positive granulocytes. Macrophages were isolated flow cytometrically from both first trimester and term decidual cell dispersions after labelling with an antibody to MHC class II. Yields of up to 4 X 10(6) macrophages, greater than 95% pure, were routinely obtained.

Antibodies, Monoclonal↗
PubMed1990

Localization of anti-endometrial antibody binding in women with endometriosis using a double-labelling immunohistochemical method.

Anti-endometrial antibody binding was localized using a double-labelling immunohistochemical method on frozen sections of endometrium taken from a woman without pelvic disease. Serum from 40 women with endometriosis was tested and, as controls, serum samples from 20 adult males and 20 umbilical cords. The method allowed compensation for endogenous immunoglobulins in endometrium and accurate localization of anti-endometrial antibody binding in the cytoplasm of the glandular epithelium. Significantly more women with endometriosis (14/40) were found to have anti-endometrial antibodies than controls (1/40) (P less than 0.001; chi 2). There was no correlation between disease severity and the presence of anti-endometrial antibodies or the intensity of staining.

Adult↗
PubMed1990

Cell populations in the human early pregnancy decidua: natural killer activity and response to interleukin-2 of CD56-positive large granular lymphocytes.

Large granular lymphocytes (LGL) have been shown previously to be the most abundant cell type in the first trimester human decidua. Purified populations of decidual LGL were prepared by flow cytometry of cell dispersions labelled with NKH1 (CD56), an antibody specific for peripheral blood LGL, and the functional properties of CD56-positive cells, CD56-negative and unsorted decidual cells compared. Both CD56-positive cells and unsorted decidual cells have cytotoxic activity against the natural killer (NK) cell target K562 which was weak compared with that of peripheral blood mononuclear cells (PBMC). The CD56-negative cells had no cytotoxic activity against K562. All three decidual cell populations proliferated in response to recombinant human interleukin-2 (rIL-2), but none produced detectable levels of IL-2 in culture. When unsorted decidual cells were cultured for 7 days in rIL-2 the proportion of CD56-positive cells increased and NK activity against K562 was augmented. The NK activity of purified CD56-positive decidual cells was also augmented by culturing in rIL-2. The potential role of decidual LGL in regulating the development of the semi-allogeneic placenta is discussed.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Antiendometrial antibodies in endometriosis measured by an enzyme-linked immunosorbent assay before and after treatment with danazol and nafarelin.

No previous study has investigated prospectively the quantitative effect of treatment on antiendometrial antibody levels in endometriosis. We measured antiendometrial antibody levels by an enzyme-linked immunosorbent assay in 35 women with laparoscopically proved endometriosis who were treated for 6 months with danazol (N = 11) or nafarelin (N = 24) in a randomized double-blind study. Levels before treatment were significantly higher than in a control group (P less than .001). Levels were lowered by treatment, but to a significant extent only in the nafarelin group (P less than .001) (danazol group: P = .091). Our data suggest that in relation to antiendometrial antibodies, nafarelin has a suppressive effect which has previously been ascribed only to danazol.

Adult↗
PubMed1988

Cell populations in human early pregnancy decidua: characterization and isolation of large granular lymphocytes by flow cytometry.

Cell populations of human pregnancy decidua, obtained by enzymic digestion from first trimester samples, were analysed by flow cytometry after labelling with monoclonal antibodies. The majority of these decidual cells (75%) were of bone marrow origin. The most abundant cell type expressed antigens characteristic of large granular lymphocytes (LGL), although macrophages and small numbers of classical T cells were also present. Three subsets of decidual LGL can be defined by single-and double-antibody labelling. Most decidual LGL are positive for NKH1, a marker of peripheral blood LGL, but negative for CD16, the Fc receptor of NK cells, and for the T-cell markers CD3 and CD5. About half the NKH1-positive cells also express CD2, associated with the E-rosette receptor, and are identical to the CD3-negative/CD2-positive cells reported previously in early pregnancy decidua. The NKH1-positive cells apparently correspond to a minor subset of peripheral blood LGL. The remaining decidual LGL are positive for CD16 and negative or only dimly positive for NKH1, and are similar to the major type of peripheral blood LGL. After purification by flow cytometry, the NKH1-positive cells were demonstrated to be of similar size to, but slightly higher granularity than, lymphocytes, whereas the CD16-positive cells were larger and more granular. The possible role of decidual LGL in modulating placental development is discussed.

Antibodies, Monoclonal↗
PubMed1987

Reactivity of human trophoblast with an antibody to the HLA class II antigen, HLA-DP.

Cytotrophoblast cells in term amniochorion and in first trimester chorionic villi were shown by immunohistology of frozen tissue sections to bind B7/21, an antibody specific for the MHC Class II antigen, HLA-DP. This binding was shown to be specific, as adsorption of the B7/21 antibody with a B cell line expressing HLA-DP prevented subsequent binding to trophoblast. When tested with a variety of other antibodies reacting with HLA-DR, HLA-DQ or the common sequences of HLA-DR, -DQ and -DP, trophoblast was negative, thus confirming previous reports. The significance of this unique pattern of reactivity of trophoblast is discussed.

Antibodies, Monoclonal↗
PubMed1977

The degradation of articular collagen by neutrophil proteinases.

The action of the serine proteinase (EC 3.4.21--)of human neutrophil leucocytes, elastase and cathepsin G, on cartilage and tendon was investigated. With cartilage, both enzymes first degraded the proteoglycan, then solubilized collagen by an attack on the terminal peptides, destroying the inter- and intramolecular cross-links. There was little degradation of the helical region of the type II collagen. Elastase also solubilized type I collagen from tendon, though this was less susceptible than cartilage collagen, and attacked the terminal peptides and perhaps the helical region of type I skin collagen in solution. Cathepsin G had little or no effect on type I collagen of skin or tendon. Since massive infiltration of joint tissues by neutrophil leucocytes is a prominent feature of inflammatory joint disease, it may well be that elastase and cathepsin G make a significant contribution to the tissue damage that occurs.

Achilles Tendon↗