Martha N. Brackin

Cytokine Expression by Models of Human Trophoblast as Assessed by a Semiquantitative Reverse Transcription‐Polymerase Chain Reaction Technique

PROBLEM: Cytokines form an important communication network between the mother and fetus. Defining the significance of these factors requires an understanding of their constitutive expression by maternal and fetal tissues. This study examines cytokine expression by human trophoblast.

First‐Trimester Human Chorionic Villi Express Both Immunoregulatory and Inflammatory Cytokines: A Role for Interleukin‐10 in Regulating the Cytokine Network of Pregnancy

PROBLEM: T‐helper 2 (TH2)‐type cytokines [i.e., interleukin (IL)‐6. IL‐10, and IL‐13] and transforming growth factor (TGF)‐β are expressed by the murine decidua and/or placenta and are likely to suppress inflammatory cytokine [i.e., IL‐2, interferon (IFN)‐γ, tumor necrosis factor (TNF)‐α, IL‐1α, and IL‐1β] production at the maternal‐fetal interface. In addition, class I IFNs may protect the fetus from immunologic rejection and viral infections. This study examines the expression of inflammatory/immunoregulatory cytokines and IL‐10 production by first‐trimester chorionic villi.

Cytokine Expression by First‐Trimester Human Chorionic Villi

PROBLEM: Communication at the human maternal‐fetal interface occurs by an intricate cytokine network. This study examines cytokine expression by normal first‐trimester human chorionic villi.

HLA Disease Association and Protection in HIV Infection among African Americans and Caucasians

In a previous investigation, we demonstrated an increased progression of overt AIDS in the African American population compared to the Caucasian population as reflected by the significantly lower absolute number of CD4+ lymphocytes detected in the African American population in an earlier study. The present study elucidates some of the possible genetic factors which may contribute to disease association or protection against HIV infection. The HLA phenotypes expressed as A, B, C, DR and DQw antigens were revealed by the Amos-modified typing procedure. NIH scoring was utilized to designate positive cells taking up trypan blue. A test of proportion equivalent to the chi 2 approximation was used to compare the disease population (n = 62; 38 African Americans, 24 Caucasians) to race-matched normal heterosexual local controls (323 African Americans, 412 Caucasians). Significant p values were corrected for the number of HLA antigens tested. HLA markers associated with possible protection from infection for African Americans were Cw4 and DRw6, whereas Caucasians expressed none. Disease association markers present in the African American population were A31, B35, Cw6, Cw7, DR5, DR6, DRw11, DRw12, DQw6 and DQw7, whereas in the Caucasian population A28, Aw66, Aw48, Bw65, Bw70, Cw7, DRw10, DRw12, DQw6 and DQw7 were demonstrated. The highest phenotypic frequency for a disease association marker in the study was for HLA-DR5 (62.9%) in the HIV-infected African American population without Kaposis sarcoma compared to a frequency of 28.9% for the regional control group (p = 0.0012). We conclude that genetic factors do have a role in HIV infection since only 50-60% of those exposed to the AIDS virus will become infected.

HIV-1 Disease Association with HLA-DQ Antigens in African Americans and Caucasians

Previously, we have shown that CD4 levels in African Americans infected with human immunodeficiency virus-1 (HIV-1) were lower than those in Caucasians. To determine whether or not HLA type is associated with susceptibility to HIV-1 infection, we demonstrated serologically that HLA-DQ6(1) and HLA-DQ7(3) were associated with HIV-1 infection in both African Americans and Caucasians. The present investigation was designed to demonstrate whether or not HLA-DQB1 alleles were associated with HIV-1 infection or protection from infection within these two ethnic groups. Oligonucleotide typing was employed and results were analyzed by chi 2 with Fishers exact test to compare HLA-DQ marker frequencies in the regional control population (98 African Americans, 143 Caucasians) to the disease population (n = 52; 30 African Americans and 22 Caucasians). We found a statistically significant increased risk of HIV infection associated with HLA-DQB1*0605 in African Americans, and with HLA-DQB1*0602 in Caucasians. By contrast, HLA-DQB1*0603 was associated with protection in Caucasians.

Immunosuppression by conditioned media derived from a cloned choriocarcinoma cell line in serum-supplemented and defined media.

PROBLEM: Immunosuppressive factor(s) of trophoblast origin may contribute to the immunological privilege afforded the fetal allograft. Characterization of these immunoregulators in humans has been impeded by a lack of sufficient quantities of early gestational trophoblast for experimentation.

Temporal and cell-specific gene expression by human endometrium after coculture with trophoblast

OBJECTIVE Our purpose was to identify temporal and stage-specific expression of endometrial genes during coculture with trophoblast cells. STUDY DESIGN Endometrial stromal cells were cultured to confluence in the presence of estradiol and progesterone. During these culture conditions the gene expression of 1 tissue specimen that secreted abundant prolactin (415 ng/mL culture medium at 21 days) was compared with a second specimen that did not. These 2 tissues were coincubated with trophoblast tissue in a specialized coculture flask. After 4 and 24 hours of culture messenger ribonucleic acid was extracted and reverse transcribed, and the complementary deoxyribonucleic acid products were amplified by polymerase chain reactions. The reverse transcriptase-polymerase chain reaction products were separated by electrophoresis, and potentially important complementary deoxyribonucleic acid fragments were reamplified, inserted into a plasmid vector, and sequenced after recovery. Sequences were submitted for Basic Local Alignment Search Tool searches of GenBank. RESULTS We observed up-regulation of 6 gene fragments in decidualized endometrium after 4 hours of coculture with choriocarcinoma-derived trophoblast BeWo cells, but only 1 gene fragment was up-regulated after 24 hours of exposure. Conversely, 2 fragments were down-regulated in decidualized stroma that was exposed to BeWo for 4 hours and 2 fragments were underexpressed after the 24-hour exposure. In the parallel experiment stromal cells that failed to secrete prolactin did not elicit the same regulation of expression. The nondecidualized endometrium overexpressed 1 gene fragment after 4 hours of BeWo exposure and overexpressed 4 gene fragments after exposure to BeWo for 24 hours. Underexpression of gene products also occurred with the nondecidualized endometrium, and we observed 2 fragments and 1 fragment to be underexpressed after 4 and 24 hours of BeWo exposure, respectively. To date, 3 of the candidate differential display fragments from these experiments have been cloned and sequenced. An up-regulated fragment (C6225J4EB-1) was 99% identical (167/168 sequences) to a reported nonredundant expressed sequence tags isolated from muscle, brain, ovary, testis, liver, and pregnant uterus tissues. A second up-regulated fragment (C4375J4EB-1) matched 100% identity (117/117) with a reported gene fragment in the expressed sequence tags database of GenBank that was derived from fetal heart and pregnant uterus. Additional characterization of these expressed sequence tags has not been reported. The third up-regulated fragment (C4250J24EB-2) was 100% identical (265/265) to human reduced nicotinamide adenine dinucleotide dehydrogenase III in the nonredundant gene database of GenBank. CONCLUSION This report demonstrates the potential usefulness that endometrial-trophoblast coculture and differential display can offer for the molecular analysis of implantation phenomena. We have recognized both overexpression and underexpression of interesting gene fragments during the early phases of endometrial responses to paracrine regulators derived from BeWo trophoblast cells. These responses appear to be specific to the degree of endometrial transformation (decidualization) before challenge by the trophoblast and to the duration of the BeWo exposure. Sequence data identified 1 gene with an unidentified function, another gene with a known function, and a fragment not previously recognized. We submit that our model of endometrial-trophoblast coculture offers a novel tool to test cellular responses during implantation, and differential display represents a sensitive technique that can identify many of the important elements of genomic signaling during nidation.

Hydatidiform Mole Pregnancy Trophoblast Extracts Differentially Suppress lnterleukin-2-lnduced Proliferation of Human T-Lymphocytes and PHA-Blasts

ABSTRACT: Immunoregulatory factors of trophoblast origin may partially abrogate maternal immune responses to the fetus during pregnancy. We have previously shown that soluble factors extracted from hydatidiform mole trophoblast suppress interleukin‐2 (IL‐2)‐dependent proliferation of a cloned murine cytotoxic T cell line (CTLL‐2). To characterize human T cell responses to this trophoblast extract, we measured the effects of molar tissue extracts (HME) on IL‐2‐stimulated proliferation of human T‐lymphocytes and mitogen (PHA) transformed T‐cell blasts (PHA‐blasts). HME significantly (P<0.05) suppressed T‐lymphocyte proliferation in response to 5 and 10 units/ml of IL‐2 at 500 and 250 μg/ml, while no effect was observed at the 100 μg/ml concentration. Suppression by HME of IL‐2‐stimulated T‐cell proliferation was partially overcome by the addition of excess IL‐2. HME also suppressed (P<0.05) IL‐2‐stimulated proliferation of PHA‐blasts at 500 and 250 μg/well at both 5 and 10 units/ml of IL‐2. As observed with resting T‐cell responses, no suppression of PHA‐blast proliferation was observed using 100 μg/ml of HME. In contrast to the response of the resting T‐cells to excess IL‐2, HME suppression of IL‐2‐stimulated blast proliferation was not affected by increasing the concentration of IL‐2. These results indicate that extracts from hydatidiform mole trophoblast contain immunosuppressive factors that block human T‐cell clonal expansion by inhibiting the utilization and/or production of IL‐2. Furthermore, the effects of HME are not reversed by excess IL‐2 when PHA‐blasts are reacted compared to resting T‐cell responses, which are partially reversed in the presence of excess IL‐2. This suggests that human trophoblast‐derived immunoregulatory factors that are present in hydatidiform mole inhibit human lymphocyte utilization of IL‐2 in a fashion similar to the murine (CTLL) model, and exhibit irreversible effects on lymphocytes containing high affinity IL‐2 receptors (PHA‐blasts) compared to lymphocytes with only low affinity IL‐2 receptors (resting T‐cell). This property of the trophoblast may partially explain how hydatidiform mole, and possibly pregnancy trophoblast in general, escapes lethal immunological challenge by the maternal immune system.

Suppression of lymphocyte proliferation in vitro by macromolecules in the vesicle fluid and tissue extracts of hydatidiform mole

This study examines the effects of vesicle fluid and tissue extracts from hydatidiform mole trophoblast on lymphocyte proliferation in vitro. Samples were obtained by direct aspiration of vesicles (hydatidiform mole vesicle fluid (HMF] or homogenization of molar tissues (hydatidiform mole extract (HME] following therapeutic uterine evacuation of hydatidiform mole. Dialyzed and lyophylized HMF pooled from two patients exhibited a 30% suppression (P less than 0.05) of mitogen-induced lymphocyte proliferation at a concentration of 12.5 micrograms protein/ml. Similarly, lymphocyte transformation was significantly suppressed (P less than 0.05) by HME at concentrations of 500 and 250 micrograms/ml. Molecular weight chromatography of HME resolved 4 protein fractions. Fraction 3 (35--50 kDa) and fraction 4 (less than 35 kDa) significantly suppressed mitogen-induced lymphocyte transformation while fractions 1 and 2 demonstrated no immunosuppression. Heat treatment (56 degrees C, 30 min) abolished the immunosuppressive activity of HME as well as fractions 3 and 4. These results suggest that hydatidiform mole trophoblast contains heat-labile macromolecules which suppress mitogen-mediated lymphocyte transformation. Such trophoblast-derived factors may interfere with maternal rejection of the allograft.

HLA-DQB1 Markers Associated with Human Immunodeficiency Virus Type I Disease Progression

In a previous investigation, we demonstrated that certain human leukocyte antigens (HLA) may be associated with human immunodeficiency virus type I (HIV-1) infection or protection from infection among regional African Americans and Caucasians. We demonstrated that HLA-DQB1*0605 was associated with a possible increased risk of susceptibility to infection in African Americans and that DQB1*0602 was associated with a possible increased risk of infection in Caucasians. The present study was designed to demonstrate possible HLA associations with HIV-1 disease progression and AIDS in regional African American and Caucasian populations. To differentiate rapid from slow progressors, immune parameters of the HIV-1-positive patient population were monitored over a mean follow-up period of 23 +/- 2 months for African Americans (n = 30) and 25 +/- 5 months for Caucasians (n = 22). To determine significance, HLA allele frequencies among rapid progressors were compared to those of slow progressors, separated by race. Results were analyzed by chi 2 analysis, with Fishers exact test where applicable, linear logistic regression and Kaplan-Meier survival analysis. In the HIV-1-positive African American group, a better prognosis was associated with HLA-DQB1*0602. In the HIV-1-positive Caucasian group, HLA-DQB1*0302 was associated with rapid HIV disease progression, but no marker was associated with a more favorable prognosis.