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Amplification of the MYCN oncogene in neuroblastomas is generally associated with a more aggressive clinical course. Recently, 1 of the minichromosome maintenance proteins, MCM7, was found to be a direct target of the MYCN transcription factor in neuroblastoma. To confirm this correlation, chromogenic in situ hybridization (CISH) to detect MYCN amplification and immunohistochemical staining for MCM7 protein expression were performed on paraffin tissue sections of 26 neuroblastomas cases and of 4 recurrences of these tumors. Seven of the primary tumors showed MYCN amplification, and all were stage 3 or 4 tumors. Only 4 of these showed MCM7 overexpression. However, 11 primary tumors overexpressed MCM7. The 4 patients with MCM7 expression associated with MYCN amplification all died from the tumor. In contrast, the 7 patients with MCM7 overexpression but no MYCN amplification were all younger than 1 year of age and have shown good survival. This suggests that MCM7 overexpression by itself is not related to a poorer prognosis as is MYCN amplification. In addition, the 4 pairs of primary and recurrent tumors all showed changes in MCM7 expression from negative to positive, whereas none of them had MYCN amplification. This study showed that MCM7 overexpression is not necessarily correlated with MYCN amplification or an aggressive clinical course. Interpretation of the results of CISH was quite easy and straightforward because the preparations were viewed with an ordinary light microscope with good preservation of the tissue morphology.

Gains in the long arm of chromosome 8 (8q) are believed to be associated with poor outcome and the development of hormone-refractory prostate cancer. Based on a meta-analysis of gene expression microarray data from multiple prostate cancer studies (D. R. Rhodes et al., Cancer Res 2002;62:4427–33), a candidate oncogene, Tumor Protein D52 (TPD52), was identified in the 8q21 amplicon. TPD52 is a coiled-coil motif-bearing protein, potentially involved in vesicle trafficking. Both mRNA and protein levels of TPD52 were highly elevated in prostate cancer tissues. Array comparative genomic hybridization and amplification analysis using single nucleotide polymorphism arrays demonstrated increased DNA copy number in the region encompassing TPD52. Fluorescence in situ hybridization on tissue microarrays confirmed TPD52 amplification in prostate cancer epithelia. Furthermore, our studies suggest that TPD52 protein levels may be regulated by androgens, consistent with the presence of androgen response elements in the upstream promoter of TPD52. In summary, these findings suggest that dysregulation of TPD52 by genomic amplification and androgen induction may play a role in prostate cancer progression.

Aneurysmal bone cyst (ABC) is a locally aggressive osseous lesion that typically occurs during the first two decades of life. ABC was regarded historically as a nonneoplastic process, but recent cytogenetic data have shown clonal rearrangements of chromosomal bands 16q22 and 17p13, indicating a neoplastic basis in at least some ABCs. Herein we show that a recurring ABC chromosomal translocation t(16;17)(q22;p13) creates a fusion gene in which the osteoblast cadherin 11 gene (CDH11) promoter region on 16q22 is juxtaposed to the entire ubiquitin-specific protease USP6 (Tre2) coding sequence on 17p13. CDH11-USP6 fusion transcripts were demonstrated only in ABC with t(16;17) but other ABCs had CDH11 or USP6 rearrangements resulting from alternate cytogenetic mechanisms. CDH11 is expressed strongly in bone, and our findings implicate a novel oncogenic mechanism in which deregulated USP6 transcription results from juxtaposition to the highly active CDH11 promoter.

OBJECTIVE AND IMPORTANCE Invasion of trigeminal and facial perineural spaces is a recognized complication of cutaneous malignancies. Centripetal spread along the trigeminal nerve axis and into the cavernous sinus and the gasserian ganglion is rare. Metastasis to the leptomeninges and cauda equina has not been reported. We report a unique case of perineural spread and central dissemination from an epithelial squamous cell carcinoma (SCC) associated with a tumor biomarker. CLINICAL PRESENTATION After excision of multiple cutaneous SCCs and basal cell carcinomas of the head and neck, a 70-year-old male patient developed successive, right-side, V1 and V2 trigeminal neuropathies and complete right cavernous sinus syndrome during a 5-year period. Concurrently, the right face became paralyzed. Left facial paresis developed during the latter half of this period. Two months before admission, subacute left lower-extremity radicular weakness resulted in falls. Serial magnetic resonance imaging scans obtained in the previous 4 years were unrevealing. At the time of admission, enhancing masses were found in the 1) right cavernous sinus and dura, foramina ovale and rotundum, and Meckel's cave, 2) right subtemporal region and orbital rectus muscles, and 3) cauda equina. Cerebrospinal fluid analysis demonstrated mild pleocytosis and rare carcinoma cells. INTERVENTION Biopsy of the right cavernous sinus mass confirmed moderately differentiated, metastatic SCC. Immunohistochemical staining and fluorescence in situ hybridization revealed epidermal growth factor receptor overexpression and genomic amplification. CONCLUSION The indolent progression of cranial nerve palsy among patients with resected cutaneous SCCs of the head and neck must raise clinical suspicion of perineural spread, even in the absence of radiological changes. Biomarkers predicting aggressive SCC behavior, illustrated here by epidermal growth factor receptor amplification and central invasion, have the potential to guide early therapy.

Lipoblastomas are rare soft tissue tumors that occur primarily in young children. They typically contain variably differentiated adipocytes, primitive mesenchymal cells, myxoid matrix, and fibrous trabeculae. Abnormalities in chromosome 8, leading to rearrangements of the PLAG1 gene, were demonstrated recently in four lipoblastomas. In the present report, we determine the frequency of PLAG1 alterations in 16 lipoblastomas from children aged 13 years or younger, and we also evaluate the stages of lipoblastoma differentiation at which PLAG1 genomic alterations are found. Eleven lipoblastomas (69%), including those with either classic or lipoma-like histology, had rearrangements of the 8q12 PLAG1 region. Another three lipoblastomas had polysomy for chromosome 8 in the absence of PLAG1 rearrangement. Only two cases (13%) lacked a chromosome 8 abnormality. Notably, the lipoblastomas with chromosome 8 polysomy had up to five copies of chromosome 8 as an isolated cytogenetic finding in an otherwise diploid cell. We also demonstrate that PLAG1 alterations are found in a spectrum of mesenchymal cell types in lipoblastomas, including lipoblasts, mature adipocytes, primitive mesenchymal cells, and fibroblast-like cells. This finding is consistent with neoplastic origin in a primitive mesenchymal precursor and with variable differentiation to a mature adipocyte end-point. Hence, our studies provide biological validation for the clinical observation that lipoblastomas can evolve into mature, lipoma-like, lesions. They also suggest that PLAG1 dosage alterations caused by polysomy 8 might represent an alternative oncogenic mechanism in lipoblastoma.

Nonrandom chromosomal aberrations, particularly in cancer, identify pathogenic biological pathways and, in some cases, have clinical relevance as diagnostic or prognostic markers. Fluorescence and colorimetric in situ hybridization methods facilitate identification of numerical and structural chromosome abnormalities. We report the development of robust, unique-sequence in situ hybridization probes that have several novel features: 1) they are constructed from multimegabase contigs of yeast artificial chromosome (YAC) clones; 2) they are in the form of adapter-ligated, short-fragment, DNA libraries that may be amplified by polymerase chain reaction; and 3) they have had repetitive sequences (eg, Alu and LINE elements) quantitatively removed by subtractive hybridization. These subtracted probes are labeled conveniently, and the fluorescence or colorimetric detection signals are extremely bright. Moreover, they constitute a stable resource that may be amplified through at least four rounds of polymerase chain reaction without diminishing signal intensity. We demonstrate applications of subtracted probes for the MYC and EWS oncogene regions, including 1) characterization of a novel EWS -region translocation in Ewing's sarcoma, 2) identification of chromosomal translocations in paraffin sections, and 3) identification of chromosomal translocations by conventional bright-field microscopy.

A two-phase study was undertaken to examine the efficiency of using transcervical cells (TCCs) collccted by uterine lavage and fluorescence in situ hybridization (FISH) for early prenatal diagnosis of fetal chromosome aneuploidy. Uterine lavage was performed in 50 women scheduled for elective termination of pregnancy (TOP, n=35) or chorionic villus sampling (CVS, n=15) between 6 and 11 weeks of gestation. TCCS were dissociated by trypsin and collagenase, and interphase FISH was carried out for chromosomes X. Y, 13/21, and 18. The phase I study comprised 36 women. The FISH results were compared with the cytogenetic analysis from long-term culture of villus samples collected at TOP or CVS. Among the 36 samples, 15 had a normal male karyotype and 21 had a normal female karyotype. FISH on TCCs correctly identified 13 out of the 15 pregnancies with a male fetus. In phase II, uterine lavage was performed on 14 women. The samples were first tested for the presence of trophoblasts with an anti-trophoblast antibody, GB25, by immunohistochemical staining. Among 12 GB25-positi ;e samples, the FISH results corresponded to the fetal karyotype. One of the GB25-positive samples had five signals for the chromosome 13/21 probe. The cytogenetic analysis confirmed that the fetus had a karyotypee of 47.XX, + 21. In the GB25-negative samples, FISH failed to identify one male pregnancy. Follow-up was carried out on 13 ongoing pregnancies and no maternal or fetal complications were discovered. This study demonstrates that fetal chromosome numeration can be carried out using FISH on uterine lavage samples in early pregnancy. However, a specific fctal cell marker, such as specific anti-trophoblast antibody, is necessary to avoid a false-negative result.

The expression patterns of a variety of cytoskeletal antigens were studied in normal human tissues (placenta, umbilical cord, myometrium, colon, mammary gland, testis, skeletal muscle, myocardium) as well as in abnormal human tissues (palmar fibromatosis, fibrocystic disease of the mammary gland, mammary carcinoma). The immunohistochemical binding patterns of the monoclonal antibody GB 42 were compared to those of commercial antibodies directed against vimentin, desmin, smooth muscle myosin, pan actin, alpha-smooth muscle actin and gamma-smooth muscle actin. Methods applied comprised immunohistochemistry on cryostat sections and paraffin sections. Immunogold immunocytochemistry was performed on Lowicryl sections. The patterns of GB 42-binding were confirmed biochemically by SDS-PAGE and Western-blotting, and quantitative amino acid analysis. Our data suggest that the monoclonal antibody GB 42 recognizes an actin isoform which is identical to, or closely related to, gamma-smooth muscle actin. Unlike the commercially available antibody against gamma-smooth muscle actin, GB 42 does not cross-react with alpha-skeletal or alpha-cardiac actins. The GB 42-antigen is expressed in smooth muscle cells, myoepithelial cells and in later stages of differentiation of myofibroblasts, in all the tissues investigated. Throughout the development of smooth muscle cells and myofibroblasts, the appearance of the GB 42-antigen occurs after the expression of vimentin, desmin and alpha-smooth muscle actin, but prior to the expression of smooth muscle myosin. GB 42 is a reliable marker for higher stages of differentiation of smooth muscle cells and myofibroblasts.

GB24, a mouse monoclonal antibody, recognizes a trophoblast-leukocyte cross-reactive antigen (TLX), which is likely identical to the membrane cofactor protein (MCP), a complement regulatory protein. GB24 reacts also with a human acrosomal sperm antigen (Fenichel et al.: J Reprod Fertil 87:699-706, 1989). By immunofluorescence or immunoperoxidase, testicular, epididymal, and ejaculated spermatozoa were found to be positive after fixation by acetone. Motile, suspended spermatozoa became positive only through conditions known to induce acrosome reaction (A23187, follicular fluid, contact with oocytes). Ultrastructural studies with immunogold staining localized this protein on the inner acrosome membrane and in the acrosomal content. By SDS-polyacrylamide gel electrophoresis, GB24 immunoprecipitated a unique protein of 48 kDa from capacitated and A23187-induced spermatozoa under reducing conditions. No cross-reactivity was found with mouse, boar, or ram spermatozoa. Localization of this human sperm antigen recognized by GB24 and its similarity with the TLX-MCP family antigens would suggest a possible role of this molecule during fertilization in sperm-egg binding or immune protection.

To evaluate the association of epidermal growth factor receptor (EGFR) gene copy number with EGFR and k-ras mutation status and tyrosine kinase inhibitor (TKI) sensitivity in non-small cell lung cancer (NSCLC), EGFR gene copy number of 182 NSCLC tumor specimens were analyzed by chromogenic in situ hybridization (CISH). EGFR and k-ras mutation analyses were also performed for, respectively, 176 and 157 of the 182 patients. Additionally, 36 patients in this study had received TKI monotherapy. The tumor was considered to be CISH positive if the gene copy number wasor=5 signals per nucleus inor=40% of tumor cells. CISH-positive tumors were strongly associated with adenocarcinoma (56.8%) compared with squamous cell carcinoma (15.9%) (p0.0001). The CISH-positive tumors were also strongly associated with EGFR mutations (78%) compared with wild type (20.2%) (p0.0001). Only six tumors had k-ras mutations. None had EGFR mutation and only one was CISH positive. In the patients treated with TKI, EGFR mutation was strongly associated with TKI responsiveness (22/25 responders) (p0.0001), but the CISH-positive tumors were only marginally significant (18/25 responders) (p=0.0665). Patients with EGFR mutations or CISH-positive tumors were all associated with longer median survival, although not statistically significant. Our results suggest Increased EGFR copy number was highly correlated with EGFR mutation in adenocarcinoma. Although it is less correlated with TKI responsiveness when compared with EGFR mutations, it still could be a good alternative molecular predictive marker for TKI responsiveness, since CISH can be done on paraffin section and is much quicker than DNA sequencing.

Aneurysmal bone cyst (ABC) is a locally recurrent bone lesion that has been regarded as a reactive process. Recently, a neoplastic basis in primary ABC was evidenced by demonstration of clonal chromosome band 17p13 translocations that place the USP6 (TRE2 or TRE17) oncogene under the regulatory influence of the highly active CDH11 promoter. Herein, we report CDH11 and/or USP6 rearrangements in 36 of 52 primary ABCs (69%), of which 10 had CDH11-USP6 fusion, 23 had variant USP6 rearrangements without CDH11 rearrangement, and three had variant CDH11 rearrangements without USP6 rearrangement. USP6 and CDH11 rearrangements were restricted to spindle cells in the ABC and were not found in multinucleated giant cells, inflammatory cells, endothelial cells, or osteoblasts. CDH11 and USP6 rearrangements did not correlate with recurrence-free survival, or with other clinicopathological features. CDH11 and USP6 rearrangements were not found in any of 17 secondary ABC associated with giant cell tumor, chondroblastoma, osteoblastoma, and fibrous dysplasia. These findings demonstrate that primary ABC are mesenchymal neoplasms exhibiting USP6 and/or CDH11 oncogenic rearrangements. By contrast, secondary ABC lack CDH11 and USP6 rearrangements, and although morphological mimics of primary ABC, appear to represent a non-specific morphological pattern of a diverse group of non-ABC neoplasms.

MITF, TFE3, TFEB, and TFEC comprise a transcription factor family (MiT) that regulates key developmental pathways in several cell lineages. Like MYC, MiT members are basic helix-loop-helix-leucine zipper transcription factors. MiT members share virtually perfect homology in their DNA binding domains and bind a common DNA motif. Translocations of TFE3 occur in specific subsets of human renal cell carcinomas and in alveolar soft part sarcomas. Although multiple translocation partners are fused to TFE3 , each translocation product retains TFE3's basic helix–loop–helix leucine zipper. We have identified the genes fused by the chromosomal translocation t(6;11)(p21.1;q13), characteristic of another subset of renal neoplasms. In two primary tumors we found that Alpha , an intronless gene, rearranges with the first intron of TFEB , just upstream of TFEB's initiation ATG, preserving the entire TFEB coding sequence. Fluorescence in situ hybridization confirmed the involvement of both TFEB and Alpha in this translocation. Although the Alpha promoter drives expression of this fusion gene, the Alpha gene does not contribute to the ORF. Whereas TFE3 is typically fused to partner proteins in subsets of renal tumors, we found that wild-type, unfused TFE3 stimulates clonogenic growth in a cell-based assay, suggesting that dysregulated expression, rather than altered function of TFEB or TFE3 fusions, may confer neoplastic properties, a mechanism reminiscent of MYC activation by promoter substitution in Burkitt's lymphoma. Alpha-TFEB is thus identified as a fusion gene in a subset of pediatric renal neoplasms.

Wilms' tumors affecting adults are rare and are thought to have a worse prognosis than similar stage tumors in the pediatric population. To understand these tumors better, the authors reviewed their multi-institutional experience in a series of nine lesions diagnosed as Wilms' tumors in adults. In addition to histologic and immunohistochemical examination, they performed cytogenetic analysis and fluorescence in situ hybridization. On review, four cases were reclassified: two "blastema only" as Ewing's sarcoma/primitive neuroectodermal tumor and the other two as clear cell sarcoma of soft parts and sarcoma not otherwise specified (NOS). Of the remaining five cases, three exhibited biphasic histology and two were triphasic. In this group, there were three women and two men, and patient age ranged from 17 to 37 years (median age, 26 years). Tumor size was large and ranged from 10 to 31 cm (median tumor size, 12.5 cm). Histologically, the tumors showed the typical features of Wilms' tumors with varying amounts of blastema (n = 5), epithelium (n = 5), and stroma (n = 2). No tumors contained anaplasia, and persistent renal blastema was not identified in the non-neoplastic kidney in any specimen. All tumors were positive for cytokeratins (CK7, n = 3; pankeratin, n = 5), and one tumor was weakly positive for CD99 (0-13). Molecular analysis including dual color fluorescence in situ hybridization (all tumors), and cytogenetic analysis (n = 2) disclosed the presence of isochromosome 7q in three of five tumors whereas all tumors were diploid with respect to chromosome 12. Follow-up data ranged from 6 to 133 months (median follow-up, 82 months) with progression in only one patient who had stage IV disease with lymph node and lung metastases at presentation. The authors conclude that adult Wilms' tumor has been overdiagnosed. Most "blastema-only" tumors in adults are not Wilms' tumors, and in an adult, biphasic morphology should be the minimum criteria for their diagnosis. Using strict diagnostic criteria, adult Wilms' tumors have a relatively favorable prognosis. The characteristic findings of isochromosome 7q, lack of trisomy or tetrasomy for chromosome 12, and absence of persistent renal blastema suggest that the pathogenesis of Wilms' tumors in adults may be different than in the pediatric population. These genetic features may be helpful in distinguishing adult Wilms' tumors from other primary renal tumors.

PROBLEM: To investigate the relation between the complement system and reproduction, expression of complement regulatory proteins (C3b receptors and inhibitor of the membrane attack complex) were screened on unfixed human eggs and preimplantation embryos. METHODS: Unfixed unfertilized oocytes and preimplantation embryos obtained from an in vitro fertilization program were stained by indirect immunofluorescence using monoclonal antibodies raised against membrane cofactor protein, (MCP or CD46), decay accelerating factor (DAF or CD55), protectin (CD59), human C3b/C4b receptor (CR1 or CD35), and major histocompatibility complex class I antigen (MHC class I). RESULTS: CD55 and CD59 were both expressed by the plasma membrane of unfertilized oocytes and pre-implantation embryos. CD46 was not expressed by unfertilized oocytes but appeared at the 6-to-8 cell stage embryo when human gene expression first occurs. CD35 and MHC class I antigens were not expressed at all on oocytes and preimplantation embryos. CONCLUSIONS: Selective expression of complement regulatory proteins (DAF and protectin) associated with the lack of MHC class I antigens may represent an immune protective mechanism by which human oocytes and preimplantation embryos escape complement-mediated damage during their travel through the female genital tract. Furthermore, participation of these complement regulatory proteins including MCP in cell to cell interaction during fertilization and/or implantation cannot be excluded

Protectin (CD59) is a complement regulatory protein which blocks the membrane attack complex during complement activation. CD59 was identifield on the human sperm surface by means of H19, an IgG1 anti-protectin mouse monoclonal antibody. Using Indirect immunofluorescence, flow cytometry and immunoperoxidase, CD59 was found to be present on the whole plasma membrane including the head and tail of fresh ejaculated, capacitated and acrosome-reacted spermatozoa. Immunoperoxidase staining of normal testicular sections indicated that this protein was already present on intraluminal germ cells. Analysis of this sperm protein by gel electrophoresis and immunoblotting revealed that its molecular weight of 20 kDa was comparable to that of CD59 expressed on peripheral blood cells (erythrocytes, lymphocytes) and that it was bound to the membrane through a glycophospholipid tail which could be released after treatment with phosphatidylinositol-specific phospholipase C. Associated to membrane cofactor protein (CD46) and decay accelerating factor (CD55) located in the acrosomal membranes, CD59 may participate to the protection of male gametes against complement-mediated damage as they travel through the female genital tract. Moreover CD59, known as an adhesion molecule involved in lymphocyte rosettes, may also participate in cell to cell adhesion during gametic interaction since H19 inhibited sperm binding and reduced the penetration rate and index during the hamster egg penetration test. © 1994 Wiley-Liss, Inc.

GB24 is a mouse monoclonal antibody raised against a common trophoblast-lymphocyte cross-reactive antigen. GB24 detects the membrane cofactor protein (MCP, CD46), a member of the complement regulatory protein family, which serves as a cofactor for factor 1 mediated cleavage of C3b. This study investigated the reactivity of GB24 on 38 breast carcinomas and 34 normal/benign breast tissues by immunochemistry as well as the reactivity of F2B7-2, an antibody specific to the decay accelerating factor (DAF, CD55) of the complement. GB24 staining was present on both normal tissue and benign lesions, but very strong diffuse reactivity was observed on carcinomas. This reactivity increased with the tumor grade. By contrast, malignant tumor cells lacked DAF expression. F2B7-2 antibody reacted weakly with benign epithelial cells. Results were studied by computer assisted image analysis to accurately define the mean optical densities. The densitometric analysis of MCP positive carcinomas showed a high intensity of the staining. Expression of MCP and DAF on MCF-7 cell lines was analyzed by flow cytometry. MCF-7 cell lines were strongly stained by mAb GB24 only. These data suggest that selectively enhanced expression of the antigen recognized by GB24 is associated with malignant breast disorders. This high expression, which may reflect a protective mechanism by which tumor cells survive complement activation, may prove useful as a marker of malignant transformation.

Membrane cofactor protein (MCP) is a complement regulatory protein that acts as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. We have previously reported the characterization of a functional MCP molecule on the acrosomal membrane. This protein migrated as a single band with a molecular weight of 40,000 Da, which is 10,000–20,000 Da smaller than the known MCP molecules, and is devoid of N-and O-linked sugars. We have proposed that the difference in molecular weight resulted from the lack of sugars. To investigate if this is due to the absence of glycosylation sites, we have characterized a cDNA clone from a human testis cDNA library. This cDNA corresponds to a peculiar MCP form previously described, which is characterized by the presence of the serine/threonine/proline-rich exon C (STPC) and the cytoplasmic tail known as CYT2, and we conclude that the absence of mature oligosaccharide of the sperm MCP cannot be totally attributed to a defect of N- and O-glycosylation sequences but rather reflects an alteration of the mechanisms of glycosylation in spermatozoa. The presence of functional MCP on the acrosomal membrane, as well as the other complement regulatory protein, decay-accelerating factor, strongly suggests that these proteins may act concomitantly to protect the acrosome-reacted spermatozoa from the attack of the complement present in the female genital tract. © 1993 Wiley-Liss, Inc.

This paper describes a new Mab BC-1 which is directed at a cell surface antigen expressed by extravillous cytotrophoblast and not by villous cytotrophoblast, so it is useful for distinguishing between the two populations. The antigen recognised by BC-1 is trypsin-resistant, which allows it to be used for flow cytometric analysis of living trophoblast isolated by enzymic disaggregation. However, BC-1 is not trophoblast-specific but cross-reacts with some other tissues, in particular endothelial cells and peripheral blood monocytes. The nature of this antigen has not been established.

Acrosomal status has been studied on human sperm prepared for in vitro fertilization (IVF) and related to the rate of fertilization.A group of 41 men with normal classical semen parameters, included in the IVF program of University of Nice for feminine tubal obstruction (n = 37) or unexplained infertility (n = 4), were evaluated in a prospective study and compared with a control group of 10 fertile donors.Evaluation of acrosome status (spontaneous and A23187-induced acrosome loss) after 6 hours incubation in Ménézo's B2 medium was made by flow cytometry on suspended cells with a new immunofluorescence test recently reported by the authors based on a monoclonal antibody GB24.Spontaneous acrosome loss remained low even after 6 hours capacitation (mean + 1 SD, 6.5% + 4.9%). Response to A23187 increased with the duration of preincubation with a marked response after 6 hours (29.5% + 8.9%). Low spontaneous acrosome loss (less than mean + 1 SD) and high response to A23187 (greater than mean - 1 SD) were observed in 25 out of 26 cases of group A with a high fertilization rate (greater than 50% fertilized oocytes). A high level of spontaneous acrosome loss and/or a lack of response to A23187 was observed in 2 of 7 cases of group B (fertilization rate less than 50%) and 6 of 8 cases of group C (unexplained unsuccessful fertilization).Impaired acrosomal status can be associated with unexplained unsuccessful fertilization.

Class I MHC mRNA has been identified in both villous and extravillous cytotrophoblast cells in first trimester placentas by in situ hybridization. In this report, we expand those observations to additional morphologically and anatomically distinct subpopulations of trophoblast cells in early placentas using the same experimental approach. In the transition zone of first trimester placental villi, where cytotrophoblast cells are proliferating to form new villi or to migrate into adjacent tissue, both cytotrophoblast cells beneath the uninterrupted syncytium and the proliferating cytotrophoblast cells contained class I mRNA whereas a layer of cytotrophoblast cells proximal to the villus core did not contain class I mRNA. In the placental bed, migrating cytotrophoblast cells contained high levels of class I mRNA as determined by the intensity of staining. In contrast, multinucleated giant trophoblast cells contained little specific message. Alterations in levels of class I mRNA seem therefore to be associated with trophoblast proliferation, migration and differentiation.

Contemporary experimental tools, particularly those of molecular biology, have allowed some insights on the selective display of membrane antigens by trophoblast cells. The unique observations offered by reproduction immunologists may have wider application than expected inasmuch as tumor cells may use some of the same mechanisms to avoid recognition and destruction by host immune cells and antibodies. Thus, further pursuit of the conditions that prevail in pregnancy may lead to a better understanding of the mechanisms used by "foreign" cells to coexist peaceably with host cells, bypassing immune mechanisms designed for their destruction.

We have tested a panel of four monoclonal antibodies to the T cell receptor (TCR) gamma/delta heterodimer (delta TCS-1, TS-8, TCR delta-1, anti- C gamma m 1) in pregnant and non-pregnant uteri. The TCR gamma/delta complex was not detected on stromal lymphocytes, but was localized in the cytoplasm of the endometrial glandular epithelium from most pregnant uteri. These antibodies also reacted with endometrial glandular epithelium in a majority of non-pregnant uteri; the reactivity was more consistent in secretory phase glands than that in the proliferative phase of menstrual cycle. However, three different monoclonal antibodies to CD3 (OKT3, leu 4, UCHT-1) failed to show any reactivity, suggesting that the presence of the TCR gamma/delta complex was not associated with CD3 complex. The TCR gamma/delta-positive glandular epithelial cells did not react with two monoclonal antibodies to the TCR alpha/beta complex, and they were also CD4- and CD8-negative. Together with the loss of the class 1 MHC antigens from these endometrial glandular epithelial cells in early pregnancy, these data suggest that these TCR gamma/delta-bearing endometrial glandular cells may undergo phenotypic alterations under local regulation of gene expression.

GB16 GB18 GB19 and GB22 are mouse monoclonal antibodies produced against full-term human placental microvilli. These antibodies reacted predominantly with the apical surface of the syncytiotrophoblast from first-trimester and full-term placentae, and also reacted with several cell lines derived from non-haematopoietic tissues by immunofluorescence. The radioiodinated BeWo (choriocarcinoma) cell surface proteins were immunoprecipitated with these four antibodies and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results demonstrated that the immunoprecipitates migrated at 18o and go kilodaltons under non-reducing and reducing conditions, respectively. Using enzyme-linked immunosorbent assay, the antigens recognized by GB 16, GB 18, GB 19, and G1322 were able to bind human transferrin. Immunoabsorption studies showed that these four antibodies bound to the same molecule as OKT 9 , an antibody to the transferrin receptor. Moreover, the reactivities of these antibodies with HL-6o (promyelocytic leukaemia) cells diminished following dimethyl sulphoxide-induced differentiation by flow cytometric analysis. These data indicate that these four antibodies recognize the transferrin receptor. By competition assay, GB18 bound to an epitope different from those recognized by GBr6, GB19 and GB22. In addition, GB22 displayed significant growth inhibition against the activated lymphocytes and Daudi cells, but not against HL-6o or Jurkat cells. These data suggest that these four monoclonal anti-transferrin receptor antibodies will provide additional means to investigate the physiological roles played by the transferrin receptor.

Summary This study has shown that fresh amniotic epithelial cells which are negative by indirect immunofluorescence for HLA and Trf receptors become positive for both of these markers following prolonged culture. In addition, the amniotic antigens which are characteristic of fresh amniotic epithelial cells are lost and replaced by trophoblast antigens subsequent to maintenance of the cells in vitro. These observations suggest that, although the expression of HLA and Trf receptors are apparently negative in vivo, such gene products become activated and manifest in plasma membranes following exposure of the cells to culture conditions. In addition, the normal manifestation of the amnion antigens is lost following prolonged periods of culture. These findings indicate that the coding and expression of normally accepted transplantation antigens undergo a significant perturbation in vitro, and that markers of normal extra-embryonic epithelium in vivo are lost after the cells have been cultured in vitro.

The use of a fluorescence method employing propidium iodide to determine nuclear morphology of different cell types by epi-illumination in membrane immunofluorescence is described. Its usefulness is illustrated by differentiating nucleated and anuclear transferrin receptor-bearing cells as well as by simultaneously determining the viability of cell suspension without requiring a change from fluorescence microscopy, this causing no loss of visual accommodation.

GB24 is a mouse monoclonal antibody (IgG1) raised against human term placental microvilli. This antibody displayed pan-trophoblast reactivity pattern. In addition, GB24 recognized normal peripheral leukocytes and transformed cell lines (Daudi, HL-60, Jurkat, AV3, BeWo, HT-29) by using membrane immunofluorescence and flow cytometry. By SDS-polyacrylamide gel electrophoresis, this antibody immunoprecipitated two proteins of 62 kilodaltons (kDa) and 75 kDa from placental microvilli. Isoelectrofocusing analysis revealed that these two bands exhibited different isoelectric points: 4.7 for the 62 kDa protein, 4.6 and 4.4 for the two spots corresponding to the 75 kDa protein. Microvilli from different placentae revealed substantial differences regarding the intensity of the labeling of these two bands, suggesting that the antigens recognized by GB24 are probably allotypic.

The reactivities of Trop 3 and 4 monoclonal antibodies (MAbs) were studied on human term and 7-week extraembryonic membranes, adult tissues, and cell lines. Trop 3 MAb reacted with cells of chorionic villi, decidua, amniotic epithelium, and basal plate trophoblast. Trop 4 MAb reacted only with syncytiotrophoblast. On epithelium, Trop 3 MAb bound to stratified and glandular epithelium, but Trop 4 MAb reactivity was limited to basal keratinocytes. On peripheral blood mononuclear cells, Trop 3 MAb reacted with the majority of cells and Trop 4 MAb with the totality. Most cell lines were positive with both MAbs. However, one chemically induced MHC mutant was negative and another decreased its expression of Trop 3 antigen. Our results suggest Trop 3 MAb might recognize a monomorphic determinant of TLX antigens and Trop 4 is involved in cell proliferation or in cell-to-cell interaction.

Human amniochorion was studied by immunofluorescence with the use of antisera to human trophoblast, amniotic epithelium and transferrin as well as with monoclonal antibodies to beta 2M and HLA. The results showed that cytotrophoblast of amniochorion, like its counterpart in placenta, manifests a trophoblast-specific membrane marker and lacks beta 2M and HLA. This tissue also did not react with antisera to amniotic epithelium, and unlike its placental counterpart no evidence for transferrin receptors could be obtained. Amniotic epithelium was found to lack trophoblast antigens, beta 2M, HLA and transferrin receptors, but contained a plasma membrane marker not found on any other extra-embryonic tissues. These results show that extra-embryonic cells with characteristic membrane markers fail to manifest histocompatibility antigens, almost as though they were mutually exclusive. This is supported by the observation that transformed amnion epithelial cells lose their unique surface antigens, react with monoclonal anti-beta 2M and acquire transferrin receptors, raising the possibility that the histocompatibility neutrality of extra-embryonic cells is maintained by the normal insertion of specific fetal markers into their plasma membranes.

Blood clotting Factor V was studied on fixed and unfixed cryostat sections of term human placentae, basal plates, amniochorions, and first-trimester placentae by using one- and two-colour immunofluorescence. A monoclonal antibody (GB36) which reacts with cytotrophoblast was used to identify cytotrophoblast. Factor V was identified in villous cytotrophoblast of term chorionic villi and very weakly or not at all in first-trimester chorionic villi. Other extra-embryonic tissues were non-reactive with anti-Factor V. These data show that Factor V is a useful marker of term villous cytotrophoblast. Immunopathological consequences of this are considered.

Forty-six human fresh amniochorion membranes obtained at cesarean section were found to contain from 0 to 5% dead cells in the amniotic epithelial layer by direct counting and spectrophotometric analysis of trypan blue extracts. With the use of a double marker system it was discovered that many of the dead cells failed to bind the DNA-chelating fluorochrome propidium iodide but reacted with fluorescein isothiocyanate-labeled antibodies to human plasminogen. In addition, both fresh and cultured human amniotic epithelial cells that had plasma membranes damaged by either cryogenic shock or cytocentrifugation specifically bound plasminogen from serum, plasma, or amniotic fluid to cytoplasmic structures. Binding did not occur in other control proteins, but plasminogen was bound from very dilute solutions, suggesting specific and tight binding inside the cell. We propose that such plasminogen can be activated to plasmin within the cell by either plasminogen activators or lysosomal proteases and that this sets into motion a progression of events that are potentially damaging for the amniochorion, perhaps being of relevance in the pathophysiology of premature rupture of the membranes in human pregnancy.

A monoclonal antibody, GB3, has been raised against human amnion. Not only does GB3 bind to amniotic basement membrane, but it also recognizes an antigenic structure expressed by epidermal as well as by some other human basement membranes. This antigen is synthesized (and excreted) by cultured normal human epidermal keratinocytes. It is expressed to a lesser extent by the A431 epidermoid carcinoma cell line, but is not expressed by the SV40 virus-transformed SVK14 keratinocyte cell line. In ultrastructural studies, this antigen was located in the epidermal basement membrane, both in the lamina densa and in the lamina lucida, associated with hemidesmosomes. It was identified as a protein by in vitro proteolytic cleavage studies. The radio-immunoprecipitates from cultured human keratinocytes, analysed by SDS-PAGE, showed that GB3 recognized five polypeptides of 93.5, 125, 130, 146 and 150 kD under reducing conditions. They were probably linked by disulfide bonds. The tissue distribution of the antigen and the molecular weights (MWs) of its constitutive polypeptides suggest that it is different from other known components of basement membranes. It may provide a biochemical marker for hemidesmosomes. Furthermore, GB3 represents an interesting and original clinical probe, since the antigenic structure recognized by GB3 is lacking in Junctional Epidermolysis Bullosa, a lethal genodermatosis in which a dermo-epidermal splitting occurs at the level of lamina lucida.

A newborn male infant with epidermolysis bullosa letalis died at age 6 weeks of septicemia. He had many congenital ectodermal abnormalities and a rudimentary thymus. The spleen had no germinal centers and the lymph nodes lacked follicles. Cytotrophoblast of the amniochorion was sparse and the amniotic epithelium was distorted. Immunohistologic studies of amniotic epithelium revealed the unprecedented presence of trophoblast antigens. Before birth of the patient, the parents had a 5-year history of infertility; after death of the patient, the mother sustained a ruptured tubal pregnancy that necessitated blood transfusions and surgical removal of the tube. She subsequently became pregnant a third time and was delivered of a normal child. The wife shared four major histocompatibility (HLA) antigens with her husband. The parents' immunogenetic characteristics and the immunopathologic features and extraembryonic membranes of the child are considered in terms of maternal responses to trophoblast antigens.

Monoclonal antibody GB36, which was raised against human term syncytiotrophoblastic microvilli, was found to recognize a novel epithelial antigen. The antibody immunoprecipitated several membrane proteins from BeWo (choriocarcinoma) and HT-29 (colon adenocarcinoma) cells. Under non-reducing conditions, four peptides of 180, 155, 135 and 130 kDa were revealed by SDS-PAGE analysis. Three peptides of slightly higher molecular weights were revealed under reducing conditions; these three peptides had identical pI (6.2) as shown by two-dimensional gel electrophoresis. By immunofluorescence, GB36 reacted with villous cytotrophoblasts and cytotrophoblasts of chorion laeve, as well as with the basal surface of syncytiotrophoblast and amniotic epithelium. Extravillous trophoblasts of cytotrophoblastic shell in the basal plate and the cytotrophoblastic cell island were non-reactive. It is suggested that the antigen of GB36 may play a role in the polarity of epithelial cells and the adhesion of epithelial cells to extracellular matrix.

Biopsy specimens from the beds of leg ulcers of fifteen patients were obtained before and after the application for 5 days of cultured human amnion. After amnion application there was considerable granulation tissue in the ulcer bed and microscopical evidence of thinned connective tissues, vessel development, more compact resolution of vascular basement membranes, and many more factor VIII granules within endothelial cells. These findings suggest the presence of angiogenic factors in human amnion and this could explain the hitherto unexplained success of amniotic membranes in surgical practice.

A monoclonal antibody GB25 which was raised against placental syncytiotrophoblast reacted specifically with villous trophoblasts. It recognized syncytiotrophoblast, villous cytotrophoblasts of the first-trimester placentae, and a majority of the cytotrophoblasts in the chorion laeve, but not the residual cells of cytotrophoblastic shell in the basal plates and the cells in the cytotrophoblastic columns. This antibody will be useful for isolating and differentiating villous from extravillous cytotrophoblasts in the first-trimester placentae.

A monoclonal antibody, GB17, was obtained from a hybridoma produced following murine immunization with isolated human term syncytiotrophoblastic microvilli. Immunohistological studies demonstrated the GB17 antigen to be present on syncytiotrophoblast of human term, first- and second trimester placenta, but not on human extravillous trophoblast or baboon placental trophoblast. Other normal adult human tissues and transformed cell lines tested were non-reactive with this antibody. Radio-iodinated term syncytiotrophoblastic microvillous protein was immunoprecipitated by GB17, and shown by sodium dodecyl sulfate—polyacrylamide gel electrophoresis to migrate as a single protein band at 175 kDa. The restricted tissue specificity suggests that the GB17 antigen could be useful for the development of a contragestational vaccine.

Summary. Amniocnonon ootainea at caesarean section ana vaginal delivery, at 34–36 weeks gestation and at term, have been studied by electron microscopy and immunofluorescence for evidence of amniotic epithelial degenerative changes and the presence of plasminogen. When delivery was by caesarean section between 34–36 weeks, electron microscopy revealed no degenerative changes in four cases, but in two cases they were widespread. All but one membrane obtained at term showed only minimal amniotic epithelial cell degenerative changes, but extensive change was seen when delivery was premature after premature membrane rupture. Plasminogen was seen in amniotic epithelium proportional to the degree of cell degeneration; it was absent from healthy membranes. These findings demonstrate that degenerative changes are extensively present in membranes that rupture prematurely, particularly before the onset of premature labour, and suggest a role for plasminogen in membrane rupture.

The expression of class I major histocompatibility complex (MHC) antigens on different regions of human amnion was studied by the avidin-biotin-complex immunoperoxidase (ABC) technique using monoclonal antibodies. In contrast to previous reports, the use of higher affinity monoclonal antibodies and the sensitive immunoperoxidase method has allowed the identification of class I MHC antigens on the amniotic epithelium. The level of expression is different between cells from various parts of the amnion, with the amniotic epithelium from the edge of the placenta consistently showing the strongest reactivity. Some of the class I MHC antigens expressed by the amnion are similar to those expressed by extravillous trophoblast cells in that both show much weaker or no reactivity with the monoclonal antibody 61D2.

Mouse hybridoma cell lines secreting monoclonal antibodies to human amnion were established. The reactivities of eight of these monoclonal antibodies (GB1, GB3, GB4, GB5, GB6, GB9, GB10 and GB11) on human skin and term extra-embryonic tissues, which included reflected amniochorions, basal plates, placentae, chorionic plates and umbilical cords, are reported. GB1, GB4, GB5, GB6, GB9 and GB11 showed various reactivity patterns on the epithelial cells of amnion, chorion and skin at different stages of differentiation. In addition, GB9 and GB11 showed extracellular reactivities; GB9 detected chorionic villi which were usually surrounded by fibrinoid and GB11 reached with fibrinoid structures in the placentae and chorion laeve. GB10 recognized connective tissues in fetal mesenchyme and adult dermis. This study demonstrates the expression of many shared antigens between tissues derived from the extra-embryonic ectoderm and adult skin. These monoclonal antibodies will provide useful tools for further investigations of epithelial differentiation and transformation.

In the human placenta, the maternal blood flows from the uterine arteries to fill the intervillous space where it is in direct contact with the syncytiotrophoblast of the chorionic villi. On the other hand, the fetal placental vascular structures form a complex network inside the villous stroma. The placental barrier which consists of trophoblast, chorionic stroma, and endothelium of the fetal blood vessels controls the major traffic of the interchange of materials between maternal and fetal blood (Boyd and Hamilton, 1970). The maturation and differentiation of the chorionic villi are related to the development of fetal blood vessels in the stroma (Fox, 1968; Kaufmann et al., 1985; Leiser et al., 1985). Abnormalities of the fetal blood vessels often lead to pathological changes in the chorionic villi (Fox, 1978). Since the placenta contains a very rich source of fetal vascular structures, it is not surprising that several monoclonal antibodies derived from a mouse which was immunized with cell membrane preparations from a full term placenta demonstrated specific reactivities to vascular structures. Although these antibodies are not specific only to placental vascular structures, they offer useful markers for the endothelium and differentiate several different vascular structures in the placenta. The reactivity of four monoclonal antibodies, GB37, GB40, GB41, and GB42 on the term human placenta is reported.

Antigen expression by fetal cells near the mother is of interest because of the possibility that some antigens may stimulate beneficial maternal immune responses or may prevent the development of harmful responses. The recent generation of monoclonal antibodies to amnion and trophoblast antigens together with the finding that class I histocompatibility antigen (HLA) expression by amnion cells can be manipulated provided an opportunity to determine if any relationships exist among expression of HLA, amnion antigens, and trophoblast antigens. The results of the present study demonstrate that: (1) class I HLA and amnion-specific antigens are co-expressed by IFN-γ-modulated amnion cells, so neither prevents expression of the other; (2) EGF enhances expression of two amnion-specific antigens; (3) amnion cells do not express transferrin receptors, placental alkaline phosphatase, or other antigens normally associated with trophoblasts following exposure to IFN-γ and/or EGF. The results demonstrate independent modulation of expression of class I HLA, two amnion-specific antigens, and some trophoblast-associated antigens on amnion epithelial cells.

SUMMARY Sera from five patients with clinically and immunopathologically proven herpes gestationis were studied by complement fixing tmmunofluorescence and complement fixing immunoelectron microscopy using specimens of skin, amniochorion and placenta. The resutts demonstrated that the complement fixation antibody (herpes gestationis factor) could bind to the basement membrane zone of skin, amnion and chorion laeve but not to that of the placental syncytiotrophoblast. These data suggest that the herpes gestationis factor may be induced by the basement membrane zone antigens of extra-villous cytotrophoblasts.

The expression of transferrin receptors, HLA-A,B,C and amnion antigens on breast biopsies from 63 patients were studied by immunofluorescence. The majority of the 36 specimens from normal breast or benign mastopathy did not express transferrin receptors, but were reactive with antibodies to HLA-A,B,C and amnion antigens. Among the specimens from patients with adenocarcinoma, 54.5% of the biopsies expressed transferrin receptors, 78.3% were non-reactive with monoclonal antibodies to common determinant of HLA-A,B,C and 63.6% failed to react with heterologous antiserum to amnion antigens. These data showed that the inverse expression of these antigens between normal and adenocarcinoma may be used as an adjunct for diagnosis of breast adenocarcinoma.

The differentiation processes of the amnion and the chorion take place simultaneously with the development of the human embryo (Boyd and Hamilton, 1970). The amnion has been considered to be derived from the trophoblasts which adjoin to the embryonic ectoderm (Bourne, 1962). At term, the amniotic epithelium of reflected and placental amnion lines the amniotic cavity, wraps around the umbilical cord and continues with the fetal skin. By using polyclonal antibodies to human amnion, three groups of antigens in common with some ectodermal tissues were identified (Hsi et al., 1984a). Recently, eight monoclonal antibodies in human amnion have been reported (Hsi and Yeh, 1986). Among them, GB3 reacted with epithelial basement membranes, but the antigen was not among the known ubiquitous components of basement membranes and GB5 recognized the junctional substances of epithelial cells. These antigens are likely to contribute to the cohesion with the subjacent tissues and to the intercellular binding of the epithelium. We report here the expression of these epithelial antigens by trophoblasts in chorion laeve and basal plate, as well as that of breast tumor cells.