Anti-HLA G Anticorps [G233] (A85532)

BACKGROUND:

Fetal cells are shed from the regressing chorionic villi and it is possible to retrieve extravillous cytotrophoblast cells by transcervical sampling. The abundance of trophoblast cells in transcervical samples suggests that this non-invasive approach could distinguish between normal and abnormal pregnancies, such as an ectopic pregnancy (EP) and blighted ovum (BO). We aim to identify and quantify fetal trophoblast cells in the cervical canal during the first trimester to assess their usefulness to predict an abnormal pregnancy.

METHODS:

Patients, age 18-45, presenting with a normal intrauterine pregnancy (IUP; n = 37), diagnosis of EP (n = 10) or BO (n = 5) were enrolled for collection of transcervical specimens using a cytobrush and fixative rinse. Non-pregnant, nulliparous women (n = 7) were included as negative controls. Cells were cleared of mucus by acidification, prepared on microscope slides and labeled with a monoclonal antibody recognizing the trophoblast marker, human leukocyte antigen (HLA)-G. HLA-G positive and negative cells were counted to calculate the ratio of trophoblast cells to total cervical cells.

RESULTS:

Trophoblast cells were observed in 35/37 normal IUP, 6/10 EP and 4/5 BO specimens. The average frequency of HLA-G positive cells in the normal IUP cervical samples was approximately 1 in 2000, which was 4-fold higher than samples from patients with EP or BO (P < 0.001). Receiver operating characteristic analysis showed that EP and BO pregnancies were distinguishable from normal pregnancies with 93% sensitivity, 95% specificity, 97% positive predictive value and 87% negative predictive value.

CONCLUSIONS:

This pilot study presents evidence that trophoblast cells can be reliably obtained and identified among cervical cells in the first trimester by immunohistochemical staining for HLA-G, and suggests for the first time that abnormal pregnancies may be predictable based on the abundance of trophoblast cells in the cervical canal.

HLA-G is an effective ligand of natural killer (NK) inhibitory receptors, HLA-G transcripts have been detected in several human tumors, and cytokines like gamma interferon (IFN) enable HLA-G molecules to be expressed. These findings are particularly upsetting in case of melanomas: IFN treatment is frequently included in melanoma therapeutic protocols, and downregulation of classical class I molecules occurs in nearly half of these tumors. Therefore, a melanoma cell downregulating classical class I and de novo expressing HLA-G, either constitutively or upon IFN treatment, is probably a stealthy target for the immune system, having inhibited both the cytotoxic T lymphocyte (CTL) and the NK activity. To elucidate this point we have investigated the expression of HLA-G molecules in 45 melanoma cell lines before and after gammaIFN treatment. Analysis was performed by immunofluorescence and flow cytometry, using the anti-HLA-G MoAbs 87G and G233, by Western blot, using the anti-HLA-G MEM/G1 MoAb and PAG1 antiserum, and by RT-PCR analysis. In addition, 8 melanoma tissues from patients free from therapy and 6 nevi were studied by immunohistochemistry using the 87G MoAb. No evidence was gathered of HLA-G expression, neither constitutive nor, in cell lines, after gammaIFN treatment. We therefore conclude that HLA-G expression is an uncommon event in melanomas, and that a therapy including IFNs cannot harm the patient by inducing the de novo expression of HLA-G molecules at least in its G1 isoform.

BACKGROUND:

HLA-G in biological fluids has been proposed to be useful as a tumor marker as both a diagnostic and prognostic factor. Most HLA-G measurement procedures are based on ELISA methods using highly specific antibodies. However, results of published studies are in conflict regarding the clinical utility and even the nature of HLA-G present in circulation.

METHODS:

We collected 118 exudates, 94 from cancer patients and 24 from patients without tumors. We measured HLA-G concentrations by ELISA using MEM-G/9 or G233 as capture antibody. Samples were immunoprecipitated with an anti-HLA-G antibody and analyzed by Western blot using a different anti-HLA-G antibody.

RESULTS:

Discrepancies in HLA-G concentrations in exudates were observed depending on what capture anti-HLA-G antibody was used for ELISA (r = 0.376). These discrepancies were not observed when the ELISAs were performed using culture supernatants from HLA-G1-transfected cells (r = 0.983). Immunoprecipitation and Western blot of cell culture supernatants with 2 different anti-HLA-G antibodies produced the typical band at 39 kDa assigned to HLA-G. When the immunoprecipitation and western blot were performed with exudates, however, there were bands at 53 kDa and 70-76 kDa, higher molecular weights than those usually assigned to HLA-G. These HLA-G-like molecules were associated with β(2)-microglobulin and could also form disulfide bridges with other HLA-G-like molecules.

CONCLUSIONS:

The main HLA-G antigenic molecules in exudates are HLA-G-like complexes, a factor that should be considered when analyzing HLA-G in biological fluids.

OBJECTIVE:

To assess the frequency of cytotrophoblastic cells in endocervical samples collected by lavage at early stages of gestation using a specific anti-HLA-G McAb (G233). From a set of four selected samples, cells identified by immunostaining were collected by laser microdissection and then tested by quantitative fluorescent polymerase chain reaction (QF-PCR) for the presence of paternally derived DNA markers, in order to establish their fetal origin.

METHODS:

Syncytial fragments and cytotrophoblastic cells from 23 transcervical samples were identified by immunostaining with McAb G233 reacting against HLA-G antigen and with antibodies against cytokeratin. Slides from the same samples were also tested by fluorescent in situ hybridization (FISH), while selected samples were analysed by QF-PCR. Slides from four samples retrieved from mothers with male fetuses were immunolabelled and then cytotrophoblastic cells, syncytial fragments and maternal epithelial cells were collected by laser microdissection and tested by QF-PCR.

RESULTS:

All endocervical samples retrieved from mothers with male fetuses were found to contain some cells with chromosome Y-specific signals when tested by FISH. Using McAb anti- HLA-G, cytotrophoblastic cellular elements were detected in about 50% of the samples. From four samples, cellular elements identified by immunostaining as cytotrophoblast or syncytial fragments were collected by laser microdissection and shown to be of fetal origin when tested by QF-PCR for the presence of fetal DNA markers.

CONCLUSIONS:

These results confirm that, during an early phase of gestation, fetal cells are released in the lower uterine cavity and that they can be isolated and analysed for prenatal diagnosis of single gene defects and aneuploidies.

Copyright 2002 John Wiley & Sons, Ltd.