Trophoblastic tumors: ultrastructural comparison of choriocarcinoma and placental-site trophoblastic tumor

Ten trophoblastic tumors, including seven classical choriocarcinomas, two choriocarcinomas with atypical histology, and one placental-site trophoblastic tumor (PSTT), were studied to compare their fine structural features. Ultrastructurally, the classical choriocarcinomas showed well-defined cytotrophoblasts and syncytiotrophoblasts. The cytotrophoblasts were primitive epithelial cells, while the syncytiotrophoblasts were complex cells with multiple nuclei and dense cytoplasm containing dilated endoplasmic reticulum, lysosomes, vesicles, and tonofilaments. The syncytiotrophoblast cell membranes often contained numerous microvilli. In the choriocarcinomas, scattered intermediate trophoblasts showed features transitional between the cytotrophoblasts and the syncytiotrophoblasts, with moderately complex cytoplasm containing some of the organelles found in the syncytiotrophoblasts. Histologically, the atypical choriocarcinomas showed a predominance of mononucleate and binucleate cells and indistinct syncytiotrophoblasts. Ultrastructurally, these atypical tumors were composed largely of intermediate trophoblasts, yet contained scattered syncytiotrophoblasts with microvilli in compressed aggregates. The PSTT was composed primarily of intermediate trophoblasts that contained prominent paranuclear filaments not seen in the intermediate trophoblasts of the choriocarcinomas. Rare cells resembling syncytiotrophoblasts were found in the PSTT, but no cytotrophoblasts were observed. Immunoreactivity for human chorionic gonadotropin and human placental lactogen was found in the intermediate trophoblasts and syncytiotrophoblasts of both the choriocarcinomas and the PSTT, demonstrating functional homology between these tumors despite some ultrastructural differences. These results demonstrate ultrastructural features of trophoblastic cells that correlate with the morphologic diversity seen in these tumors by light microscopy. Furthermore, the comparisons suggest that the PSTT is composed of a distinct form of intermediate trophoblast that appears to reflect its origin from the extravillous trophoblast.     

GLUT1 messenger RNA and protein induction relates to the malignant transformation of cervical cancer

We studied whether induction of glucose transporters (GLUTs) 1 to 4 correlates with human papillomavirus (HPV)-dependent malignant transformation of cervical epithelium. Tissue samples of cervical intraepithelial neoplasia (CIN; grades 1 to 3), invasive carcinomas, and lymph node metastasis were examined. HPV typing was performed. Tissue sections were immunostained with GLUT1 to GLUT4 antibodies. Messenger RNA (mRNA) in situ hybridization confirmed GLUT1 protein expression. Weak expression of GLUT1 was found in nondysplastic HPV-positive and HPV-negative epithelium; significant expression was observed in preneoplastic lesions, correlating with the degree of dysplasia. In CIN 3 high-risk HPV lesions, cervical cancer, and metastasis, GLUT1 was expressed at highest levels with a strong correlation of GLUT1 mRNA and protein expression. Immunostains for GLUT2 to GLUT4 were negative. Cervical tumor cells respond to enhanced glucose utilization by up-regulation of GLUT1. The strong induction of GLUT1 mRNA and protein in HPV-positive CIN 3 lesions suggests GLUT1 overexpression as an early event in cervical neoplasia. GLUT1 is potentially relevant as a diagnostic tool and glucose metabolism as a therapeutic target in cervical cancer.     

Pregnancy-related changes: A retrospective review of 278 cervical smears

Pregnancy-related physiologic changes are well recognized. However, the normal range of changes as reflected in the cervical smear have not been adequately described. Review of 278 abnormal cervical smears from 153 pregnant/preabortal and 125 postpartum/abortal patients revealed the following: 21 high-grade squamous intraepithelial lesion (HGSIL) cases, 46 low-grade squamous intraepithelial lesion (LGSIL) cases, 185 atypical squamous cells of undetermined significance (ASCUS) cases, and 26 atypical glandular cells of undetermined significance (AGUS) cases. Surgical correlation (excluding 18 products of conception and 153 placentas) was available in 98 (35%) of the cases. Dysplasia was confirmed on biopsy of 11 cases cytologically diagnosed as HGSIL (7 CINII/III and 4 CIN I), 19 cases cytologically diagnosed as LGSIL (6 CIN II/III and 13 CIN I), 35 cases of ASCUS (4 CIN II/III and 31 CIN I), and 2 cases of AGUS (1 CIN III and 1 CIN I). Decidualization was present in six cervical and three endometrial biopsies. The remaining 180 cases revealed pregnancy-related changes in most of the atypical groups and a few in the dysplasia groups. With pregnancy, both cervical glands and stroma undergo physiologic changes. These result in squamous metaplasia due to ectropion and cells with hypervacuolated cytoplasm and atypical nuclei reflecting endocervical gland hyperplasia and/or Arias-Stella reaction. The decidual cells are large, with variably staining cytoplasm and a large nucleus. Degenerated decidual or trophoblastic cells can also shed from the endometrium and mimic HGSIL. Despite the caution required in this population, dysplastic changes should not be underestimated. Diagn. Cytopathol. 17:99–107, 1997. © 1997 Wiley-Liss, Inc.     

人母-胎界面滋养细胞来源的CXCL16促进蜕膜γδT细胞产生IL-10及TGF-β

目的:探讨人早孕滋养细胞通过CXCL16/CXCR6途径对蜕膜γδT细胞分泌细胞因子功能的影响。方法:临床收集正常早孕妇女绒毛组织及蜕膜组织各10例,体外分离蜕膜γδT细胞和滋养细胞,原代培养滋养细胞12、24、48、72、96小时,ELISA检测培养上清中CXCL16的浓度;RT-PCR检测蜕膜γδT细胞中CXCR6的表达;建立人早孕蜕膜γδT细胞与滋养细胞的共培养体系,同时加或不加CXCL16的中和性抗体,或者γδT细胞与滋养细胞间接共培养,48小时后流式细胞术检测γδT细胞中IL-10和TGF-β的表达。结果:人早孕滋养细胞能够分泌CXCL16,且其浓度呈现时间累积效应;蜕膜γδT细胞则表达CXCR6。与滋养细胞直接共培养后,γδT细胞表达IL-10和TGF-β显著增高,且明显高于间接共培养组,加入CXCL16的中和性抗体后,IL-10和TGF-β的增加效应被部分抵消。结论:人早孕滋养细胞通过分泌CXCL16促进表达CXCR6的蜕膜γδT细胞产生IL-10和TGF-β,从而可能有利于妊娠期母-胎界面Th2型偏移,进而有利于正常妊娠的维持。     

Chronic radiation effects: A correlative study of smears and biopsies from the cervix and vagina

Cervicovaginal smears and biopsies from patients treated with radiotherapy for cervical carcinoma were examined morphologically and immunochemically to provide information on the tissue derivation of cells characteristic of chronic radiation effect in postirradiation smears. In biopsies, stromal changes, such as fibrosis, vessel changes, and atypical fibroblasts were most common. Ulceration, leucocytic infiltration, multinucleated giant cells, regenerative epithelium, and atypical glandular epithelial cells were also present in some specimens. These changes were reflected in smears collected from the same patients, where multinucleated giant cells, repair cells, and large atypical cells were often present. Correlation of smears and biopsies suggest that repair cells are collected from areas of epithelial regeneration and glandular radiation atypia. Sampling of ulcerative or eroded tissue may produce smears with multinucleated giant cells, atypical stromal cells, endothelial cells, and numerous macrophages. Correct recognition of these cell types and smear patterns may assist in avoiding false positive diagnoses. © 1995 Wiley-Liss, Inc.     

Utility of proliferation-associated marker MIB-1 in evaluating lesions of the uterine cervix.

Various cervical lesions at times may be difficult to distinguish from one another on routine hematoxylin and eosin stains, and immunostaining for the proliferation-associated antigen Ki-67, using monoclonal antibody MIB-1, can aid their distinction. The reduced MIB-1 expression in atrophy and increased MIB-1 expression in dysplasia permits easy distinction between these conditions. Presence of MIB-1 in more than 15% of basal cells and/or in surface half of the epithelium favor a diagnosis of condyloma over squamous metaplasia or inflammatory changes. Normal endocervix shows MIB-1 positivity in less than 10% of the cells, but usually in more than 20% of cells in cervical adenocarcinoma. With increasing grade of dysplasia, the percentage of MIB-1 positive cells is increased, and positive cells are seen in the higher levels of the epithelium. Presence of more than 20% MIB-1 positive cells in Pap smears showing atypical cells of uncertain significance is associated with a diagnosis of dysplasia on subsequent biopsies. Cauterized tissues with dysplasia show MIB-1 expression similar to adjacent noncauterized dysplastic areas. MIB-1 expression is, therefore, useful in evaluating various cervical lesions.     

人早期胎盘绒毛运动神经诱向因子1及其受体的免疫组织化学定位

为了解人胎盘绒毛是不存在运动神经诱向因子及其可能的功能意义，本实验用ＭＮＴＦ１单克隆抗体及抗独特型单克隆抗体在人早期胎盘绒毛石蜡切片上进行免疫组织化学反应，对人早期胎盘绒毛的ＭＮＴＦ１及其受体进行定位。结果显示，胎盘绒毛的细胞滋养层细胞，合体滋养层细胞和基质细胞均呈ＭＮＴＦ１强免疫反尖，ＭＮＴＦ１样免疫反应物质分布在胞质内，胞核内阴性。     

Expression of the protein marker p16INK4a in the cervix uteri cancer

Immunohistochemical study was carried out of 18 cervical carcinomas (13 squamous and 5 adenomatous) and of 3 cases of cervical intraepithelial dysplasia. Formalin-fixed paraffin-embedded tissue samples from biopsies as well as from surgical material were used. Staining was performed with monoclonal antibodies to protein p16INK4a. Cytologic smears of epithelial cervical cells from 7 healthy women were taken as a negative control. The reference group consisted of 5 cancer patients with other tumors (breast cancer, B-cell lymphoma). Overexpression of p16INK4a was registered in cervical cancer.     

Semiautomatic PAPNET analysis of proliferating (MIB-1-positive) cells in cervical cytology and histology

With the event of microwave-antigen retrieval it has become possible to detect proliferating cells (staining positive for MiB-1) in cervical smears containing epithelial fragments and in paraffin sections containing cancerous cervical epithelium. The PAPNET system, using neural network computing, is able to collect from the slides epithelial fragments with positive-staining nuclei. The nuclei in epithelial fragments are semiautomatically quantitated using the PAPNET-digitized images. The parameter PPN (proportion-positive nuclei), in which the nuclear area of the positive nuclei is taken into account, prove to be superior to the proliferation index for distinguishing moderate dysplasia from carcinoma in situ. In repair cells, all four quantitative parameters are close to those of moderate dysplasias, indicating that this method is unfit for distinguishing these two entities. However, we show that MiB-1 staining is valuable for the analysis of “false-negative” and “false-positive” smears, and for quantifying proliferation in sections of carcinoma in situ. © 1995 Wiley-Liss, Inc.     

Apical and basolateral localisation of GLUT2 transporters in human lung epithelial cells

Glucose concentrations of normal human airway surface liquid are ~12.5 times lower than blood glucose concentrations indicating that glucose uptake by epithelial cells may play a role in maintaining lung glucose homeostasis. We have therefore investigated potential glucose uptake mechanisms in non-polarised and polarised H441 human airway epithelial cells and bronchial biopsies. We detected mRNA and protein for glucose transporter type 2 (GLUT2) and glucose transporter type 4 (GLUT4) in non-polarised cells but GLUT4 was not detected in the plasma membrane. In polarised cells, GLUT2 protein was detected in both apical and basolateral membranes. Furthermore, GLUT2 protein was localised to epithelial cells of human bronchial mucosa biopsies. In non-polarised H441 cells, uptake of d-glucose and deoxyglucose was similar. Uptake of both was inhibited by phloretin indicating that glucose uptake was via GLUT-mediated transport. Phloretin-sensitive transport remained the predominant route for glucose uptake across apical and basolateral membranes of polarised cells and was maximal at 5–10 mM glucose. We could not conclusively demonstrate sodium/glucose transporter-mediated transport in non-polarised or polarised cells. Our study provides the first evidence that glucose transport in human airway epithelial cells in vitro and in vivo utilises GLUT2 transporters. We speculate that these transporters could contribute to glucose uptake/homeostasis in the human airway.     

Immunohistochemical characterization of p57(KIP2) expression in early hydatidiform moles

The differentiation of complete mole (CM), an aberrant androgenetic conceptus, from partial mole (PM) and hydropic abortion (HA) in early gestations is very important for patient management. In this study, 10 diploid voluntary artificial abortions (ABs), 20 diploid HAs, 20 triploid PMs, and 44 diploid CMs (including 4 persistent diseases), all of which were in the first trimester, were evaluated by immunohistochemistry of formalin-fixed tissues using a monoclonal antibody against p57(KIP2) protein (p57), a putative paternally imprinted inhibitor gene. DNA ploidy in all cases was analyzed by flow cytometry. In all ABs, nuclear p57 was strongly expressed in cytotrophoblasts, intermediate trophoblasts, villous stromal cells, and decidual stromal cells but was absent in syncytiotrophoblast. In diploid CMs, p57 expression in cytotrophoblasts and villous stromal cells was either absent (37 cases) or very low (7 cases). Villous intermediate trophoblasts stained for p57 in 12 cases of CM. On the other hand, 16 HAs and 19 PMs showed p57 levels comparable to those observed in ABs. Decidual stromal cells provided a reliable internal control in all cases. These findings support the hypothesis that misexpression of p57 is involved in the abnormal development of androgenetic CMs. This immunohistochemical analysis is a useful tool for the differential diagnosis of CMs.     

Expression of the insulin-responsive glucose transporter isoform 4 in blastocysts of C57/BL6 mice

Glucose is the most important energy substrate for mammalian blastocysts. In preimplantation embryos glucose uptake is mainly mediated by facilitative glucose transporter molecules (GLUT). Employing RT-PCR in 3.5-day-old mouse blastocysts of strain C57/BL6 we have investigated the expression of the GLUT isoforms 1–4 and 8. We could not only detect GLUT 1, 3 and 8 but, in contrast to earlier studies, also the insulin-responsive isoform 4. GLUT2 was not expressed. The specificity for GLUT4 amplification was verified by sequence analysis. GLUT4 protein was localized by immunohistochemistry with two GLUT4 antibodies. It was found in ICM and trophoblast cells in the cytoplasmic compartment with a strong perinuclear staining. This is the first report on the expression of the insulin-sensitive GLUT4 isoform in mouse preimplantation embryos.Abbreviations GLUT Glucose transporter - ICM Inner cell mass - TE Trophectoderm     

Hyperosmotic shock induces both activation and translocation of glucose transporters in mammalian cells

The effect of osmotic stress on sugar transport was investigated in Clone 9 epithelial cells, which express the glucose uniporter GLUT1, and in 3T3-L1 adipocytes, which express both GLUT1 and GLUT4. An acute hyperosmotic shock increased the uptake of sugars in both cell types. In Clone 9 cells, this was followed by a regulatory volume increase (RVI) response. Stimulation of transport was rapid and reversible, with half-lives (t 1/2) for stimulation of 2-deoxy-D-glucose uptake of 5.6 +/- 0.9 (n=6) and 22.7 +/- 1.5 (n=4) min for Clone 9 cells and adipocytes respectively. The effect was dose dependent, reaching a maximum at 1.1 osM of 2.9 +/- 0.1-fold (n=3) for Clone 9 cells and 8.2 +/- 0.8-fold (n=3) for adipocytes. In the latter, this stimulation correlated with translocation of the glucose transporter isoform GLUT4 to the cell surface and was not significantly different from that elicited by 160 nM insulin (7.6 +/- 1.2-fold, n=3). The effect of osmotic shock was not, however, influenced by inhibitors of either phosphoinositide 3-kinase (PI 3-kinase) (wortmannin, 250 nM) or of p38 mitogen-activated protein kinase (p38 MAP kinase) (SB203580, 20 microM), which reportedly prevent GLUT4 translocation and/or activation by insulin respectively. These inhibitors also had no effect on the stimulation of transport by osmotic shock in Clone 9 cells. However, in contrast to adipocytes, the effect of osmotic shock on glucose transport in Clone 9 cells reflected primarily a change in the intrinsic activity of cell surface transporters and there was only a minor change in their subcellular distribution as assessed by cell immunostaining or no change as assessed by surface biotinylation. These results indicate that the response of cells to osmotic shock can involve changes both in transporter activity and location. The signal transduction pathways involved include neither PI 3-kinase nor the classical, osmotically-activated component, p38 MAP kinase.     

Significance of MiB-1 staining in smears with atypical glandular cells

MiB-1 immunostaining may facilitate recognition of developing adenocarcinoma in cervical smear screening. In a retrospective analysis of prospectively collected data of 170 patients with atypical endocervical glandular cells and with repeat smears, archival Papanicolaou-stained smears were restained for MiB-1 and classified for the presence of preneoplastic changes of MiB-1 positive epithelial fragments. The results of classification based on MiB-1 positive epithelial fragments corresponded "roughly" with cytomorphological diagnoses. Of the 38 patients in which the primary smear was found to be MiB-1-positive, 12 of these persisted in the repeat smear. Thirty-eight of the 132 repeat smears of patients originally negative for MiB-1 pathology were positive. Of the total 50 MiB-1 positive repeat smears, four showed adenocarcinoma in situ on cytomorphological grounds. MiB-1 staining enhances detection of (pre)neoplastic changes. This approach does not destroy the morphology of the original smear and can be applied to routine material.     

Small G proteins in insulin action: Rab and Rho families at the crossroads of signal transduction and GLUT4 vesicle traffic

Insulin stimulates glucose uptake into muscle and adipose tissues through glucose transporter 4 (GLUT4). GLUT4 cycles between the intracellular compartments and the plasma membrane. GLUT4 traffic-regulating insulin signals are largely within the insulin receptor-insulin receptor substrate-phosphatidylinositol 3-kinase (IR-IRS-PI3K) axis. In muscle cells, insulin signal bifurcates downstream of the PI3K into one arm leading to the activation of the Ser/Thr kinases Akt and atypical protein kinase C, and another leading to the activation of Rho family protein Rac1 leading to actin remodelling. Activated Akt inactivates AS160, a GTPase-activating protein for Rab family small G proteins. Here we review the roles of Rab and Rho proteins, particularly Rab substrates of AS160 and Rac1, in insulin-stimulated GLUT4 traffic. We discuss: (1) how distinct steps in GLUT4 traffic may be regulated by discrete Rab proteins, and (2) the importance of Rac1 activation in insulin-induced actin remodelling in muscle cells, a key element for the net gain in surface GLUT4.     

The Pap test in HIV-positive women]

The aim of this work was to estimate the frequency of abnormal Papanicolau (Pap) smears in a group of HIV-infected women undergoing cervical screening. We re-examined 162 Pap smears from 118 patients infected with HIV. The patients were aged 23-55 years. A total of 108 smears (66.6%) from 80 patients were negative; 14 smears (8.6%) from 14 patients showed inflammation; 3 smears (1.8%) from 3 patients had atypical squamous cells of undetermined significance (ASCUS); 20 smears (13.5%) from 16 patients were abnormal for human papillomavirus (HPV); 13 smears (8.0%) from 9 patients revealed low-grade, squamous intraepithelial lesions; 10 smears (6.2%) from 7 patients were SIL-HG; the diagnosis of carcinoma was made in 3 cases (1.8%) and 2 smears from 2 patients were unsatisfactory. HIV-infected women have an increased rate of abnormal Pap smears for both HPV infections and cervical dysplasia. These results confirm the validity of cervical screening by Pap test.     

From maternal glucose to fetal glycogen: expression of key regulators in the human placenta.

The present study investigated the expression of glycogenin, the protein primer for glycogen synthesis, and the high affinity glucose transporter isoform GLUT3 as a further potential regulator of cellular glycogen metabolism, in first trimester and term human placenta using immunohistochemistry and Western blotting. At term, glycogenin was most abundant in the endothelium of fetal vessels. Trophoblast as well as basal decidual cells were moderately stained. The glycogenin distribution pattern in first trimester placentae resembled that at term, but reactivity was generally less intense. Extravillous trophoblast and villous cytotrophoblast were the major sites of GLUT3 expression. Endothelial cells were also strongly labelled with the GLUT3 antiserum. Western blotting identified both free and glucosylated glycogenin, as well as a 48 kDa band reacting with GLUT3 antiserum in placental villous tissue. Glycogenin immunoreactivity remained unaffected by amylolytic glycogen digestion, although preceding electron microscopical examination demonstrated the presence of glycogen. These data may indicate that placental glycogenin can be recycled from the immature glycogen or that it is located on the surface of the glycogen molecule. In conclusion, the co-expression of glycogenin with GLUT3 might enable glycogen-storing cells to exchange glucose quite effectively according to prevailing metabolic demands of glycogen synthesis or degradation.     

Cellular and subcellular expression of monocarboxylate transporters in the pigment epithelium and retina of the rat

The cellular and subcellular expression of the monocarboxylate transporters MCT1, MCT2 and MCT4 [corresponding to MCT3 of Price N. T. et al. (1998) Biochem. J. 329, 321-328] were investigated in the pigment epithelium and outer retina of rats. Immunofluorescence and postembedding immunogold analyses revealed strong MCT1 labelling in the apical membrane of the pigment epithelial and no detectable signal in the basolateral membrane. In contrast, antibodies to the glucose transporter GLUT1 produced intense labelling in both membranes. Neither MCT1 nor GLUT1 was enriched in intracellular compartments. The monocarboxylate transporter MCT4 was very weakly expressed in the retinal pigment epithelium of adult animals, but occurred at higher concentrations at this site in 14-day-old rats. However, even at the latter stage, the immunolabelling of MCT4 was weak compared to that of MCT1. In the neural retina, the data were consistent with a predominant glial localization of MCT1. Specifically, immunogold particles signalling MCT1 occurred in Müller cell microvilli and in the velate processes between the photoreceptors. No labelling was obtained with antibodies to MCT2. Taken together with previous biochemical analyses, the present findings indicate that MCT1 is involved in the outward transport of lactate through the retinal pigment epithelial cells, and in the transfer of lactate between Müller cells and photoreceptors.     

Elements of diabetic nephropathy in a patient with GLUT 2 deficiency

The Fanconi-Bickel syndrome is caused by homozygosity or compound heterozygosity for mutations of the facilitated glucose transporter 2 gene (GLUT2). Glycogen accumulates in renal tubular cells and they fail to reabsorb multiple filtered solutes because of impairment in GLUT2-mediated efflux of glucose. We describe a 10-year-old male child with GLUT2 deficiency who produced massive amounts of 3-deoxyfructose (3-DF) in the kidneys. Since 3-DF is a detoxification product of a potent glycating agent, 3-deoxyglucosone, a precursor of advanced glycation end-products, this suggests a massive accumulation of glucose within tubular cells probably as a consequence of GLUT2 deficiency. The level of 3-DF in the urine of this atypical patient, who also manifested renal glomerular hyperfiltration, microalbuminuria, and glomerular mesangial expansion, was higher than in any patient examined with diabetes mellitus. Elevated levels of glucose and/or its metabolites in renal tubular cells may be necessary but not sufficient for the development of both the renal tubulopathy and diabetic-like glomerular disease in GLUT2 deficiency.