Decidual stromal cell response to paracrine signals from the trophoblast: amplification of immune and angiogenic modulators.

During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54,600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor alpha-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.

Stromal PRs mediate induction of 17beta-hydroxysteroid dehydrogenase type 2 expression in human endometrial epithelium: a paracrine mechanism for inactivation of E2.

Progesterone stimulates the expression of 17beta-hydroxysteroid dehydrogenase (HSD) type 2, which catalyzes the conversion of the potent estrogen, E2, to an inactive form, estrone, in epithelial cells of human endometrial tissue. Various effects of progesterone on uterine epithelium have recently been shown to be mediated by stromal PRs in mice. We describe herein a critical paracrine mechanism whereby progesterone induction of 17beta-HSD type 2 enzyme activity, transcript levels, and promoter activity in human endometrial epithelial cells are mediated primarily by PR in endometrial stromal cells. Medium conditioned with progestin-pretreated human endometrial stromal cells robustly increased 17beta-HSD type 2 enzyme activity (2-fold) and mRNA levels (13.2-fold) in Ishikawa malignant endometrial epithelial cells. In contrast, direct progestin treatment of Ishikawa epithelial cells gave rise to much smaller increases in enzyme activity (1.2-fold) and mRNA levels (4-fold). These results suggest that progesterone- dependent paracrine factors arising from stromal cells are primarily responsible for the induction of epithelial 17beta-HSD type 2 expression in the endometrium. We transfected serial deletion mutants of the -1,244 bp 5'-flanking region of the 17beta-HSD type 2 gene into Ishikawa cells. No progesterone response elements could be identified upstream of the 17beta-HSD type 2 promoter. Stromal PR-dependent induction of the 17beta-HSD type 2 promoter was mediated by a critical regulatory region mapped to the -200/-100 bp sequence. Direct treatment of Ishikawa cells with progestin gave rise to a maximal increase in the activity of -200 bp/Luciferase construct only by 1.2-fold, whereas medium conditioned by progestin-pretreated endometrial stromal cells increased promoter activity up to 2.4-fold in a time- and concentration-dependent manner. The stimulatory effect of medium conditioned by progestin-pretreated stromal cells was enhanced strikingly by increasing stromal cell PR levels with the addition of estrogen. This epithelial-stromal interaction was specific for endometrial epithelial cells, since 17beta-HSD type 2 could not be induced in malignant breast epithelial cells by media conditioned with progestin-treated breast or endometrial stromal cells. In conclusion, progesterone regulates the conversion of biologically active E2 to estrone by inducing the 17beta-HSD type 2 enzyme in human endometrial epithelium primarily via PR in stromal cells, which secrete factors that induce transcription mediated primarily by the -200/-100 bp 5'-regulatory region of the 17beta-HSD type 2 promoter.

Baboon mesenchymal stem cells can be genetically modified to secrete human erythropoietin in vivo.

Human mesenchymal stem cells (MSCs) are capable of differentiating into multiple mesenchymal lineages including chondrocytes, osteocytes, adipocytes, and marrow stromal cells. Using a nonhuman primate model, we evaluated nonhuman primate MSCs as targets for gene therapy. Baboon MSCs (bMSCs) cultured from bone marrow aspirates appeared as a homogeneous population of spindle-shaped cells. bMSCs were capable of differentiating into adipocytes and osteocytes in vitro and chondrocytes in vivo. bMSCs were genetically modified with a bicistronic vector encoding the human erythropoietin (hEPO) gene and the green fluorescent protein (GFP) gene. Transduction efficiencies ranged from 72 to 99% after incubation of MSCs with retroviral supernatant. Transduced cells produced from 1.83 x 10(5) to 7.12 x 10(5) mIU of hEPO per 10(6) cells per 24 hr in vitro before implantation. To determine the capacity of bMSCs to express hEPO in vivo, transduced bMSCs were injected intramuscularly in NOD/SCID mice. In a separate experiment, transduced bMSCs were loaded into immunoisolatory devices (IIDs) and surgically implanted into either autologous or allogeneic baboon recipients. Human EPO was detected in the serum of NOD/SCID mice for up to 28 days and in the serum of five baboons for between 9 and 137 days. NOD/SCID mice experienced sharp rises in hematocrit after intramuscular injection of hEPO-transduced bMSCs. The baboon that expressed hEPO for 137 days experienced a statistically significant (p < 0.04) rise in its hematocrit. These data demonstrate that nonhuman primate MSCs can be engineered to deliver a secreted and biologically active gene product. Therefore, human MSCs may be an effective target for future human gene therapy trials.

Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion.

OBJECTIVE: The human bone marrow contains mesenchymal stem cells capable of differentiating along multiple mesenchymal cell lineages. Using a non-human primate model, we sought to determine whether the systemic infusion of baboon-derived mesenchymal stem cells was associated with toxicity and whether these cells were capable of homing to and persisting within the bone marrow. MATERIALS AND METHODS: Five baboons (Papio anubis) were administered lethal irradiation followed by intravenous autologous hematopoietic progenitor cells combined with either autologous (n = 3) or allogeneic (n = 2) mesenchymal stem cells that had been expanded in culture. In four of these baboons, the mesenchymal stem cells were genetically modified with a retroviral vector encoding either the enhanced green fluorescent protein gene (n = 3) or the human placental alkaline phosphatase gene (n = 1) for tracking purposes. A sixth animal received only intravenous gene marked autologous mesenchymal stem cells but no hematopoietic stem cells or conditioning irradiation. RESULTS: Following culture, baboon mesenchymal stem cells appeared morphologically as a homogeneous population of spindle-shaped cells that were identified by the monoclonal antibodies SH-3 and SH-4. These cells did not express the hematopoietic markers CD34 or CD45. Baboon mesenchymal stem cells isolated from primary culture were capable of differentiating along both adipogenic and osteogenic lineages. There was no acute or chronic toxicity associated with the intravenous infusion of mesenchymal stem cells. In all five recipients of gene marked mesenchymal stem cells, transgene was detected in post-transplant bone marrow biopsies. In two animals receiving autologous mesenchymal stem cells, including the one non-conditioned recipient, transgene could be detected over 1 year following infusion. In one recipient of allogeneic gene marked mesenchymal stem cells, transgene was detected in the bone marrow at 76 days following infusion. CONCLUSION: These data demonstrate that baboon mesenchymal stem cells: 1) are not associated with significant toxicity when administered intravenously, 2) are capable of homing to the bone marrow following intravenous infusion, and 3) have the capacity to establish residence within the bone marrow for an extended duration following systemic administration.

Glycodelin-A expression in the uterine cervix.

OBJECTIVE: Local immunosuppressive factors in the uterine cervix infected by human papillomavirus are felt to facilitate the malignant transformation process. Glycodelin-A is an immunosuppressive peptide found in several tissues of müllerian origin, most notably the pregnant and decidualized endometrium. Its expression in the uterine cervix has not been defined but could theoretically contribute to the immunopermissive environment of the cervix. To determine whether glycodelin-A is found in the cervix we examined the squamous and endocervical epithelia from both normal and neoplastic cervical specimens from 14 women. METHODS: Immunohistochemisty identification of glycodelin-A was performed on archival paraffin-embedded sections from 10 hysterectomies and 4 cone biopsies. Sections were evaluated and staining was scored as negative, positive, or strongly positive with a separate score for the squamous and glandular components of the cervix. RESULTS: Eleven of 14 cases, 79%, demonstrated positive staining of the squamous epithelium. Glycodelin-positive cases included hisologically normal (n = 4; 3 strongly positive, 1 positive) as well as dysplastic (n = 5; 1 strongly positive, 2 positive, and 2 negative) and malignant squamous cells (n = 5; 1 strongly positive, 3 positive, and 1 negative). Normal glandular epithelia were negative in all cases but 1, which demonstrated significant squamous and tubal metaplasia of the endocervical glands involved. CONCLUSION: Glycodelin-A is found in the squamous epithelium of both the histologically normal and the neoplastic cervix. Further characterization of these results will focus on the possible immunosuppressive effect glycodelin-A may have in the cervix.

Immunity to murine breast cancer cells modified to express MUC-1, a human breast cancer antigen, in transgenic mice tolerant to human MUC-1.

The high incidence of breast cancer in women and the severity of the disease have stimulated a need for improved and novel forms of therapy. The product of the MUC-1 gene has been identified as a breast cancer-associated antigen in breast cancer patients. The gene has been cloned and sequenced. Transgenic mice were prepared that express human mucin and are naturally tolerant to the molecule, providing a unique opportunity to investigate immunotherapeutic strategies in experimental animals that might eventually be applied to breast cancer patients. A cell line (410.4) derived from a mouse mammary adenocarcinoma that arose in a BALB/c mouse was transduced with a retroviral vector (R1-MUC1-pEMSVscribe) that encoded MUC-1. After confirmation of the expression of human mucin, the cells (E3) were further modified by transduction with retroviral vectors encoding interleukin (IL)-2, IL-4, IL-12, or IFN-gamma to evaluate the effect of cytokine-secretion on the immunogenic properties of the cells in the MUC-1 transgenic mice. The results indicated that modification of the breast cancer cells to secrete IL-12 reduced and at times eliminated the tumorigenic growth properties of the cells. Under similar circumstances, progressively growing tumors formed in MUC-1 transgenic mice that received injections of unmodified E3 cells or with E3 cells modified to secrete IL-2, IL-4, or IFN-gamma. Immunity to breast cancer developed in MUC-1 transgenic mice that had rejected IL-12-secreting E3 cells because the animals were resistant to challenge with (non-cytokine-secreting) E3 cells. In vitro analyses confirmed the presence of T cell-mediated cytotoxicity toward the breast cancer cells in MUC-1 transgenic mice immunized with the IL-12-secreting cells. Our data obtained in a unique animal model system point toward an analogous form of therapy for breast cancer patients.

Evaluation of Langerhans' cells in the cervical epithelium of women with cervical intraepithelial neoplasia.

OBJECTIVE: Cervical infection with human papillomavirus (HPV) results in a more permissive environment for malignant transformation. In squamous epithelia the Langerhans' cell (LC) is responsible for antigen presentation. Studies that use S-100 immunostaining demonstrate low LCs in cervical intraepithelial neoplasia (CIN) while those that use other methods have shown normal numbers of LCs. This observation led us to postulate that a defect in S-100 proteins, not a simple decrease in LC number, may be the cause of immune suppression. To evaluate this we identified LCs in the cervix of women with HPV/CIN in a prospective fashion using two antibodies, S-100 and CD1, each targeting a different element of the LC. METHODS: Paired biopsies of the cervix were taken, one paraffin embedded for S-100 and the other snap frozen for CD1 staining. LCs were counted and expressed as the number of cells per millimeter of epithelium. Analysis of variance was used to assess differences between counts in normal, low-grade, and high-grade lesions. HPV was tested by hybrid capture. RESULTS: S-100 LCs were significantly reduced in dysplasia, LG 8.6 and HG 6.0, compared to normal at 16.7 cells/mm (P = 0.04). S-100 LCs were reduced in HPV-infected cases at 5.9 vs 12.8 cells/mm in HPV negatives (P = 0.02). Acute inflammatory infiltrates were associated with increased S-100 LCs independent of pathology. CD1 LCs were not significantly altered by any parameters tested. CONCLUSIONS: HPV/CIN may exert an immunosuppressive effect by decreasing the S-100 LCs. The association of S-100-positive LCs coupled with cervical inflammatory changes suggests an important function of the S-100 proteins in the development of an anti-HPV response.

Upregulated expression of VLA proteins and CD29 in peripheral blood lymphocytes of chronic alcoholics without ethanol-related diseases.

To analyze adhesion molecule expression on peripheral blood mononuclear cells (PBMCs) and on different lymphocyte subpopulations (CD2+, CD8+, CD19+, and CD56+ subsets) in chronic alcoholism, 30 well-nourished chronic alcoholics without ethanol-related diseases and 30 matched controls were included in the study. Adhesion molecules that mediate adhesion to other cells and to extracellular matrix proteins, and whose cellular expression is modified during lymphocyte activation, were selected for study. A detailed clinical evaluation, laboratory analysis, nutritional assessment, and study of adhesion molecule expression was performed. A significant higher expression of CD29 (beta1-integrin) (p = 0.001), VLA-3 (p = 0.002), VLA-4 (p = 0.03), and VLA-5 (p = 0.001) were observed on PBMCs of chronic alcoholics, compared with control subjects, whereas no changes were observed in CD18 (beta2-integrin) and CD50 (ICAM-3) expression. The upregulation of CD29 and VLA proteins only affected T lymphocytes (CD2+/CD8+/CD4+ cells). These data confirm that T cells of chronic alcoholics are basally activated and that changes in adhesion molecule expression on PBMCs may be responsible of disturbances of adhesion processes in chronic alcoholics without ethanol-related diseases.

Histologic characteristics of laparoscopic argon beam coagulation.

STUDY OBJECTIVES: To describe histologic effects of laparoscopic argon beam coagulation and determine the extent of tissue necrosis at various power settings and exposure times. DESIGN: Prospective experimental analysis (Canadian Task Force classification II-1). SETTING: University animal laboratory. Subjects. Adult female domestic pigs. INTERVENTIONS: Various power settings (40, 60, 80 W) at increasing exposure times (1, 3, 5 sec) were used during laparoscopic application of argon beam coagulation to different tissues (uterine horn, bladder, ureter, kidney, bowel, liver). Animals were sacrificed within 1 hour of coagulation for histologic tissue preparation. MEASUREMENTS AND MAIN RESULTS: Histologic measurements of both depth and lateral extent of electrosurgical tissue effects (mm +/- SD) were ascertained and evaluated statistically by one-way repeated measures analysis of variance. Depth of tissue necrosis was confined to 1 mm or less in uterine horn, bladder, and ureter. Even at highest power settings, bowel had tissue necrosis no greater than 2 mm. Both liver and kidney showed a deeper histologic effect (4-5 mm). The lateral extent of tissue necrosis ranged from 2 mm (ureter) to 15 mm (liver). CONCLUSION: Laparoscopic argon beam coagulation results in tissue effects that are dependent on both low power setting and duration of application, as well as on electrical and physical characteristics of target tissue. Thermal tissue penetration can be expected to be less than 2 mm in bowel, bladder, and ureter, and less than 5 mm in kidney and liver, even at 5 seconds of exposure time and at a power setting as high as 80 W. As with all thermal modalities used for hemostasis and tissue coagulation, laparoscopic argon beam coagulation can be performed safely as long as the potential for inadvertent thermal injury is understood.

Well-differentiated thyroid carcinomas: p53 mutation status and microvessel density.

BACKGROUND: Risk-stratification schemes exist for well-differentiated thyroid carcinoma and include prognostic factors such as age, sex, extent of tumor, size of tumor, and presence of metastasis. Controversy continues, however, over the aggressiveness of initial surgical intervention because of anecdotal experiences of poor clinical outcomes in low-risk patients. Our objective is to determine the prognostic significance of two biologic tumor markers, the p53 gene mutation and CD34 microvessel density (MVD) count, in well-differentiated tumors of thyroid gland. METHODS: We selected 38 patients with well-differentiated thyroid carcinomas from the University of Illinois Tumor Registry. Patients had an average clinical follow-up of 10 years. Paraffin-embedded tumor specimens were available for all patients. Immunohistochemistry was performed to identify mutations of the p53 gene (Ab 1801) and to determine the MVD count (CD34). RESULTS: There were significant increases in MVD counts within thyroid tumor tissue, when compared with surrounding, normal thyroid tissue. There was no significant correlation noted, however, between increased MVD and histology or recurrence rates. There was a trend toward higher MVD counts in tumor specimens of patients initially seen with metastatic lymphadenopathy. The incidence of p53 mutation expression was 28%, and there was no correlation between p53 status and histology, sex, recurrence rate, or survival. CONCLUSIONS: This study supports the concept of tumor neovascularization but fails to correlate MVD with clinical behavior or pathologic features in well-differentiated thyroid carcinoma. Furthermore, we found that the p53 mutation status was not an independent prognosticator of tumor behavior in these lesions.

Human papillomavirus expression and p53 gene mutations in squamous cell carcinoma.

OBJECTIVES: To determine the incidence of human papillomavirus (HPV) infection and p53 gene mutation expression in squamous cell carcinomas (SCCs) of the oral cavity and tonsils, to correlate the presence of HPV and p53 gene mutation with known clinical and pathological features of SCC, and to determine whether infection with HPV or the presence of p53 gene mutations are independent prognosticators of patient survival. DESIGN: To accomplish this goal, 58 patients with SCCs of the oral cavity and 42 patients with SCCs of the tonsils were randomly examined. The cases examined met the criteria of 5-year clinical follow-up, availability of complete staging information and treatment history, and the presence of paraffin-embedded tumor specimens. Immunohistochemical tests were performed to identify the mutant p53 protein. Human papillomavirus identification was accomplished with polymerase chain reaction, with confirmation via restriction fragment length polymorphisms. RESULTS: The incidence of p53 gene mutation expression for this series was 66%. Human papillomavirus infection was found in 11 patients (11%). There was a trend toward increased p53 gene mutation expression with advancing stage of tumor in the oral cavity cancer group, although this was less evident in the tonsil cancer population. The p53 gene mutation status was found not to correlate with the histological grade of the tumor, patient age or sex, recurrence rates, or survival status. Like p53 expression, there were no correlations found between the presence of HPV and age, sex, histological grade, or recurrence rates. However, a correlation did exist between HPV and survival status in the tonsil cancer group, with improved survival noted among patients with tonsil cancers infected with HPV compared with those not infected with HPV. A significant correlation existed with both p53 gene mutation status and HPV status with respect to alcohol and tobacco use. The presence of the p53 gene mutation positively correlated with increased tobacco and alcohol use, whereas infection with HPV predicted a significantly lower rate of alcohol and tobacco consumption. CONCLUSIONS: Human papillomavirus infection is an independent risk factor for the development of oral cavity and tonsil SCCs in those patients with a relatively low alcohol and tobacco use history. Conversely, there is a strong association between heavy alcohol and tobacco use and mutation of the p53 gene. Neither p53 gene mutation nor HPV infection serve as prognosticators of tumor behavior in SCCs of the oral cavity or tonsils, with the exception of improved survival noted among patients with tonsil cancers infected with HPV.

Expression of epidermal growth factor and androgen receptors in ovarian cancer.

Ovarian cancer is the second most common malignancy of the female reproductive tract. Approximately 50% of ovarian cancers have elevated levels of epidermal growth factor receptor (EGFR). This overexpression is correlated with a poor prognosis for patient survival. Ovarian cancers also express a number of sex steroid receptors. The androgen receptor (AR) is the predominant sex steroid receptor and is expressed in over 80% of ovarian cancers. We investigated whether a relationship exists between EGFR and AR in ovarian cancer. Sixty serous cystadenocarcinomas were analyzed for their relative levels of EGFR and AR by Western blot analysis. Data were analyzed by Student's t test and linear regression analysis for statistical significance. More than 98% of the tumors expressed detectable levels of EGFR, while 65% of the tumors expressed detectable levels of AR. The levels of EGFR (mean +/- SEM) were found to be significantly (P < 0.01) higher in AR+ (516 +/- 15) than in AR- (304 +/- 57) tumors. EGFR levels significantly correlated to AR levels (r = 0.49, P < 0.001). These results demonstrate an association between EGFR and AR levels in ovarian cancer. Whether this association represents a causal or a casual relationship remains to be determined.

Physical and sexual abuse and their relation to psychiatric disorder and suicidal behavior among adolescents who are psychiatrically hospitalized.

Adolescents who were psychiatrically hospitalized (N = 105) were classified as sexually abused, physically abused, both sexually and physically abused, or not abused, and studied to determine the prevalence of suicidal behavior and psychiatric disorders. Self-reports of hopelessness, depression, coping, and self-concept were also examined. No difference in suicidal behavior or psychiatric disorder, based on abuse history, was found, with one exception. Adolescents who were sexually abused, particularly those who experienced the most severe sexual abuse, used negative coping strategies more often than those not sexually abused. Findings suggest that symptomatology of adolescents who are psychiatrically hospitalized does not differ markedly based on history of abuse.

Pediatric soft tissue sarcomas of nonmyogenic origin.

Nonrhabdomyosarcoma soft tissue sarcomas (NRSTS) are relatively rare tumors, which nonetheless constitute 50% of the soft tissue sarcomas seen in the pediatric population. The prognosis for these tumors is good, with 92% of patients in our series alive and 61% free of their disease at 5 years follow-up. The most important prognostic factor among our 35 patients was the grade of the tumor. More than 70% of our patients with grade I or II lesions are disease-free at 5 years, compared to only 39% of patients with grade III lesions. The patients with the best outlook are those who can be treated with surgery alone as the definitive care for this disease. Resection remains the primary treatment modality in NRSTS, whereas, unlike the treatment of rhabdomyosarcomas, the value of radiation therapy and chemotherapy in treating NRSTS remains undefined.