George Aboagye-Mathiesen

Interferon Production by Cultured Human Trophoblasts and Choriocarcinoma Cell Lines Induced by Sendai Virus

Human term-placental trophoblasts in primary culture were studied for an interferon (IFN) response when challenged with Sendai virus and compared to three choriocarcinoma cell lines, placental fibroblasts and placental macrophages. Normal trophoblasts were high producers and released both IFN-alpha and IFN-beta. In contrast, one choriocarcinoma cell line was a low producer and all malignant lines produced only IFN-beta. Circulating monocytes produce IFN-alpha but placental macrophages secreted IFN-beta and some IFN-alpha, suggesting that IFN production may be dependent on the stage of differentiation. A role for trophoblast IFNs in protection of the foetus against virus infections is proposed.

High interferon alpha levels in placenta, maternal, and cord blood suggest a protective effect against intrauterine herpes simplex virus infection

Interferons (IFN) are produced by the placenta during pregnancy, and they can be detected in the maternal and fetal blood. Although the anti‐viral potential of IFNs is well established, it remains unclear whether the IFNs associated with pregnancy can prevent transplacental spread of viral infection. The present study was undertaken in order to determine the possible protective effect of placentally produced IFN‐α on fetal acquisition of herpes simplex virus (HSV). Nine mothers with a known history of genital HSV infection were studied. In five cases IFN‐α was detected in the placenta, maternal, and fetal blood, whereas in three cases IFN‐α could not be detected. In the remaining case, IFN‐α was found only in the maternal blood. As corroborated by the serological evidence of early HSV infection in the cord blood, the single case of vertical HSV transmission was observed in the group of IFN nonproducers. Furthermore, virus transmission did not occur in cases where IFN‐α was present in the placenta and simultaneously in the maternal and fetal circulations. Thus, the present data indicate that high levels of IFN during pregnancy may protect the fetus from acquiring a possibly fatal intrauterine HSV infection. J. Med. Virol. 51:210–213, 1997.

Differential interferon production in human first and third trimester trophoblast cultures stimulated with viruses

Stimulation of human placental first and third trimester trophoblast and syncytiotrophoblast cultures with viruses [Newcastle Disease Virus (NDV) and Sendai virus] led to a high interferon (IFN) production. The magnitude of the production was dependent on the gestational age of the trophoblast, type of inducer and the stage of differentiation of the trophoblast. The data obtained indicated that the first trimester trophoblast cultures produced five to sixfold more IFN than the third trimester trophoblast on per cell basis whereas syncytiotrophoblast at term produced twice as much IFN than the mononuclear term trophoblast when stimulated with the viruses. NDV and Sendai virus produced different levels and composition of IFN-alpha and -beta in both first and third trimester trophoblast and syncytiotrophoblast cultures. Purification of the virus-induced trophoblast interferons (tro-IFNs) by tandem high-performance affinity chromatography resulted in specific activities between 0.7 and 2.7 x 10(8) IU/mg of protein when assayed on human amniotic WISH cells. The tro-IFN-alpha protected both human and bovine MDBK cells from virus infection whereas the tro-IFN-beta protected only the human cell lines tested. The possible roles of the tro-IFNs are discussed in light of the observed differences in trophoblast IFN response.

Functional characteristics of human trophoblast interferons

Human trophoblast populations from first‐ and third‐trimester placentas produce interferons (IFNs) in the presence of growth factors (CSF and PDGF) or when infected with virus. The highly invasive extravillous trophoblast population produced a higher level of IFNs (three‐ to eightfold, P < 0.05) than the noninvasive villous trophoblast population when stimulated with growth factors and/or virus. The level of IFN produced was dependent on the type of trophoblast population, the type of inducer and the stage of differentiation of the trophoblasts. Tandem immunoaffinity chromatography of the virus‐induced trophoblast IFNs resulted in the isolation of trophoblast IFN‐α and ‐β types. The purified trophoblast IFNs have antiviral, antiproliferative and immunoregulatory properties. Furthermore, the trophoblast IFNs inhibited the expression of proto‐oncogenes such as EGF‐R, c‐erbB2 and c‐fms reported to be involved in normal trophoblast growth and differentiation. These data suggest essential roles of interferons in normal human development during pregnancy.

Characterization of Sendai virus-induced human placental trophoblast interferons

Human placental trophoblast cultures produce a mixture of interferons (IFNs) when challenged with Sendai virus. High-performance dye-ligand and immunoaffinity chromatography of a trophoblast IFN (tro-IFN) preparation enabled the isolation of three antigenically distinct IFNs, alpha I, alpha II 1 and beta, with Mrs of 16K, 22K and 24K respectively, by reducing and non-reducing SDS-PAGE. The major IFN, responsible for 75% of the total antiviral activity, was tro-IFN-beta, with the remaining activity being due to tro-IFN-alpha I and tro-IFN-alpha II 1, as determined by an antiviral neutralization test using specific anti-human IFN antibodies. The antiviral activities of the tro-IFNs were stable at pH 2.0 for 24 h and tro-IFN-alpha II 1 and -beta were shown to be glycoproteins. The three tro-IFNs showed different antiviral activities when assayed on human and bovine cell species; tro-IFN-alpha I and alpha II 1 protected both human and bovine (MDBK) cells from virus infection, whereas tro-IFN-beta showed a high degree of species specificity, protecting only the human cell types tested.

Purification and initial characterization of human placental trophoblast interferon induced by polyriboinosinic.polyribocytidylic acid.

Human placental trophoblast interferon (tro-IFN), induced in trophoblast cultures by a superinduction procedure, was purified to a homogeneous product with retention of biological activity. The problems associated with isolation from serum-containing medium were overcome by a combination of Blue Sepharose affinity chromatography and reversed-phase HPLC (RP-HPLC) on Separon SGX C-18. This two-step purification procedure yielded tro-IFN with a specific activity of 3.4 x 10(7) international units/mg of protein. The overall recovery of interferon activity was 66.7%. The purified tro-IFN was shown to be a glycoprotein with an Mr of 24K on native and SDS-PAGE. Its antiviral activity was stable at pH 2.0 at 37 degrees C but was sensitive to heat at 56 degrees C for 1 h and was neutralized by antibodies to human IFN-beta.

Interferon gamma regulates a unique set of proteins in fresh human bladder transitional cell carcinomas

Poly(A) mRNA was isolated from human placental trophoblast cells stimulated with 100 U/mL of interleukin‐2 and 5 μg/mL of phytohemagglutinin and reverse‐transcribed. The cDNA coding for the mature interferon‐gamma (IFN‐γ) protein was amplified using specific primers, cloned into the pGEX‐4T2 vector, and expressed in Escherichia coli. Treatment of four fresh bladder transitional cell carcinoma (TCC) biopsies (TCCs 845‐1, grade II, Ta; TCC 925‐1, grade II, Ta; TCC 919‐1, grade III, T1; TCC 950‐1, grade III, T1) with the purified recombinant trophoblast IFN‐γ (50 U/mL, 20 h), followed by proteome analysis using two‐dimensional gel electrophoresis, revealed several major proteins whose level of expression were affected by this cytokine. Of these, five (tryptophanyl‐tRNA synthetase, the interferon gamma‐inducible protein γ3, mangase superoxide dismutase, and two unknown proteins of apparent molecular masses of 35.8 and 11.2 kDa, respectively) were upregulated in at least 75% of the tumors analyzed while one was downregulated (aldose reductase). Proteins were identified using a combination of techniques that included microsequencing, two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) immunoblotting and comparison with the transitional cell carcinoma isoelectric focusing (IEF) database (http://biobase.dk/cgi‐bin/celis). Proteome profile analysis of primary cultures from a low‐grade lesion (TCC 846‐1, Grade II, Ta) labeled in the presence and absence of IFN‐γ showed that all of the proteins disregulated in vivo were also affected in the cultures. The cultured cells, on the other hand, exhibited additional changes that were not detected in vivo and that may reflect adaptation to the culturing conditions. Taken together, the results provide a first glance at the effect of IFN‐γ on the protein expression profiles of TCCs, and in due course may form the basis for more comprehensive studies aimed at evaluating the usefulness of this cytokine in bladder cancer management.

Human trophoblast interferons

The human placental trophoblasts which constitute the first fetal cells and form the major cell layer of the feto-maternal interface are potent producers of interferons (IFNs). The IFN production is dependent on the gestational age of the trophoblast, type of inducer and the stage of differentiation of the trophoblasts. First trimester trophoblast populations produce higher levels (5-6 times) of IFN than the third trimester trophoblasts when stimulated with viruses. Non-viral inducers, such as poly(rl).poly(rC), induce exclusively IFN-beta whereas viruses such as Sendai and Newcastle Disease Virus (NDV) induce mixtures of IFN-alpha subtypes and IFN-beta. Differentiation of mononuclear cytotrophoblasts into syncytiotrophoblasts in vitro increase the IFN production. High-performance and immunoaffinity chromatography of the virus-induced trophoblast IFN preparations resulted in the isolation of three antigenically distinct IFNs, namely, alpha I, alpha II1 (omega 1), and beta with molecular masses of 16, 22 and 24 kDa, respectively, on SDS-PAGE. The human trophoblast IFNs have physical and antiviral activities characteristic of the Type 1 IFNs. The possible roles of the trophoblast IFNs in human placental and fetal development are also discussed in this review.

Human trophoblast interferons enhance major histocompatibility complex class I antigen expression on human term trophoblast cells in culture

The expression and regulation of major histocompatibility complex class I (MHC class I) antigens by virus-induced human trophoblast interferons (tro-IFNs) were examined in term trophoblast cultures. Flow cytometry studies using fluorescence monoclonal antibodies against MHC class I antigens revealed that isolated cytotrophoblasts can express MHC class I antigens. The expression of these antigens increased with stimulation of trophoblast cultures with tro-IFN-alpha and -beta. One hundred IU tro-IFN-alpha and -beta/ml induced no significant higher levels of MHC class I antigens as compared with the control, whereas 1000 IU tro-IFN-alpha and -beta/ml did. The tro-IFN-enhanced expression of MHC class I antigens may be important as it increases the efficiency of local and viral antigen presentation, cytotoxicity by T cell response and local inflammatory processes, thereby preventing virus spread from mother to fetus.

Enhanced chemiluminescence-based hybridization analysis for PCR-mediated HIV-1 DNA detection offers an alternative to 32P-labelled probes

The efficiency of enhanced chemiluminescence based on a novel generation substrate for alkaline phosphatase, adamantyl-1,2-dioxetane phosphate, was compared with that of 32P-labelled probe for visualization of human immunodeficiency virus type 1 (HTV-1)-specific DNA-DNA hybrids. The probe used for nonisotopic detection was digoxigenin labelled and targeted by anti-digoxigenin antibody Fab-fragments conjugated to alkaline phosphatase. The dot-blot hybridization analysis performed on a dilution series of HIV-1 proviral DNA demonstrated a lower sensitivity limit of 0.5 pg with the nonisotopic method. However, one order of magnitude less DNA could still be detected by a random-primed 32P-labelled probe. The ability of nonradioactive and radioactive probes to detect 590-bp gag gene-specific target sequences generated by the polymerase chain reaction (PCR)-mediated amplification of HIV-1 DNA was also compared. Analysis of 20 samples from individuals at increased risk for HIV infection by using the two assayed systems produced virtually equivalent signal images on corresponding specimens. Furthermore, complete concordance in the performance was found when HIV-1 proviral DNA was investigated by PCR in additional 50 samples of human blood mononuclear cells.