The Effect of Transplacental Administration of Glucagon-Like Peptide-1 on Fetal Lung Development in the Rabbit Model of Congenital Diaphragmatic Hernia.

OBJECTIVE: Glucagon-like peptide-1 (GLP-1) increases surfactant protein expression in type 2 pneumocytes. Herein, we determine if transplacental GLP-1 treatment accelerates lung growth in the fetal rabbit model of congenital diaphragmatic hernia (DH). METHODS: Time-mated does had an induction of DH on day 23 followed by daily GLP-1 or placebo injection until term. At that time, the does were weighed, fetal blood was obtained for GLP-1 assay, and the lungs were dissected. Fetal outcome measures were lung-to-body-weight ratio (LBWR), morphometry, and Ki67 and surfactant protein B (SPB) expression. RESULTS: Maternal weight loss in the GLP-1 group was 7.1%. Fetal survival was lower in GLP-1 fetuses compared to placebo controls (27/85, 32% vs. 35/57, 61%; p < 0.05). Fetal GLP-1 levels were increased 3.6-fold. The LBWR of GLP-1 DH fetuses fell within the range of DH placebo fetuses (1.166 ± 0.207% vs. 1.312 ± 0.418%), being significantly lower than that of placebo-exposed unoperated fetuses (2.280 ± 0.522%; p < 0.001). GLP-1 did not improve airway morphometry. GLP-1 DH lungs had a reduced adventitial and medial thickness within the range of controls, and lesser muscularization of vessels measuring 30-60 µm. There were no differences in Ki67 and SPB expression. CONCLUSION: GLP-1 at this dosage improves peripheric pulmonary vessel morphology in intra-acinar vessels with no effect on airway morphometry but with significant maternal and fetal side effects. Thus, it is an unlikely medical strategy.

In Vitro Assessment of the Expression and T Cell Immunogenicity of the Tumor-Associated Antigens BORIS, MUC1, hTERT, MAGE-A3 and Sp17 in Uterine Cancer.

BACKGROUND: While immunotherapy moved to the forefront of treatment of various cancers, it remains underexplored for uterine cancer. This might be due to the small patient population with advanced endometrial carcinoma and uterine sarcoma. Data about immunotherapeutic targets are scarce in endometrial carcinoma and lacking in uterine sarcoma. METHODS: Expression of five tumor-associated antigens (TAA) (BORIS, MUC1, hTERT, MAGE-A3 and Sp17) was validated in uterine tumor samples by immunohistochemistry (IHC) and/or quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). TAA immunogenicity was analyzed by determining spontaneous T cell responses towards overlapping peptide pools covering the whole TAA in patient blood. RESULTS: At mRNA level, MAGE-A3 and Sp17 were overexpressed in a minority of patients and BORIS was moderately overexpressed (26% in endometrial carcinoma and 62% in uterine sarcoma). hTERT was overexpressed in the vast majority of tumors. On protein level, MUC1 was upregulated in primary, recurrent and metastatic EMCAR and in metastatic US tumors. hTERT protein was highly expressed in both normal and malignant tissue. Spontaneous TAA-specific T cell responses were detected in a minority of patients, except for hTERT to which T cell responses occurred more frequently. CONCLUSIONS: These data point to MUC1 and hTERT as most suitable targets based on expression levels and T cell immunogenicity for use in immunotherapeutic regimens.

Microsatellite instable and microsatellite stable primary endometrial carcinoma cells and their subcutaneous and orthotopic xenografts recapitulate the characteristics of the corresponding primary tumor.

OBJECTIVE: Well-characterized, low-passage, primary cell cultures established directly from patient tumors are an important tool for drug screening because these cultures faithfully recapitulate the genomic features of primary tumors. Here, we aimed to establish these cell cultures from primary endometrial carcinomas (ECs) and to develop subcutaneous and orthotopic xenograft models as a model to validate promising treatment options for EC in the in vivo setting. METHODS: Primary cell cultures of EC tumors were established and validated by analysing histologic and genetic characteristics, telomerase activity, and in vitro and in vivo growth characteristics. Using these primary cell cultures, subcutaneous and orthotopic mouse models were subsequently established. RESULTS: We established and characterized 7 primary EC cell cultures and corresponding xenograft models of different types of endometrioid tumors. Interestingly, we observed that the chance to successfully establish a primary cell culture seems higher for microsatellite instable than microsatellite stable tumors. For the first time, we also established an orthotopic murine model for EC derived from a primary cell culture. In contrast to EC cell lines, grafted tumor cultures preserved the original tumor structure and mimicked all histologic features. They also established abdominal and distant metastases, reflecting the tumorigenic behavior in the clinical setting. Remarkably, the established cell cultures and xenograft tumors also preserved the genetic characteristics of the primary tumor. CONCLUSIONS: The established EC cultures reflect the epithelial genetic characteristics of the primary tumor. Therefore, they provide an appropriate model to investigate EC biology and apply high-throughput drug screening experiments. In addition, the established murine xenograft models, in particular the orthotopic model, will be useful to validate promising therapeutic strategies in vivo, as the grafted tumors closely resemble the primary tumors from which they were derived. Microsatellite instable status seems to determine the success rate of establishing primary cell cultures.

Mapping the immunosuppressive environment in uterine tumors: implications for immunotherapy.

The major hurdle for cancer vaccines to be effective is posed by tumor immune evasion. Several common immune mechanisms and mediators are exploited by tumors to avoid immune destruction. In an attempt to shed more light on the immunosuppressive environment in uterine tumors, we analyzed the presence of PD-L1, PDL2, B7-H4, indoleamine 2,3-dioxygenase (IDO), galectin- 1, galectin-3, arginase-1 activity and myeloid-derived suppressor cell (MDSC) infiltration. IDO, PD-L1, PD-L2 and B7-H4 were analyzed by immunohistochemistry. PDL2 was mostly expressed at low levels in these tumors. We found high IDO expression in 21 % of endometrial carcinoma samples and in 14 % of uterine sarcoma samples. For PD-L1 and B7-H4, we found high expression in 92 and 90 % of endometrial cancers, respectively, and in 100 and 92 % of the sarcomas. Galectin-1 and 3 were analyzed in tissue lysates by ELISA, but we did not find an increase in both molecules in tumor lysates compared with benign tissues. We detected expression of galectin-3 by fibroblasts, immune cells and tumor cells in single-cell tumor suspensions. In addition, we noted a highly significant increase in arginase-1 activity in endometrial carcinomas compared with normal endometria, which was not the case for uterine sarcomas. Finally, we could demonstrate MDSC infiltration in fresh tumor suspensions from uterine tumors. These results indicate that the PD-1/PD-L1 interaction and B7-H4 could be possible targets for immune intervention in uterine cancer patients as well as mediation of MDSC function. These observations are another step toward the implementation of inhibitors of immunosuppression in the treatment of uterine cancer patients.

Increased expression of the neuroregenerative peptide galanin in the major pelvic ganglion following cavernous nerve injury.

INTRODUCTION: Erectile dysfunction (ED) remains a frequent complication of radical prostatectomy due to injury to the cavernous nerves (CNs). A recent microarray showed the neuropeptide galanin to be one of the most strikingly upregulated genes in the rat major pelvic ganglion (MPG) after bilateral CN crush injury (BCNI). AIM: The aim of this study is to evaluate the temporal regulation of galanin in the MPG after BCNI and its relationship to functional nerve regeneration. METHODS: Changes in galanin, galanin receptor (galR), and c-JUN mRNA expression were assessed in Sprague-Dawley rats after sham operation (n = 10) and at 48 hours (n = 10), 7 (n = 10), 14 (n = 5), 21 (n = 5), 30 (n = 5), and 60 (n = 5) days after BCNI using quantitative PCR. Erectile function was assessed by measuring intracavernous pressure (ICP) divided by mean arterial pressure (MAP) during CN electrostimulation. Immunohistochemistry was performed on the MPG in sham-operated animals and 5 days after BCNI. MAIN OUTCOME MEASURES: ICP/MAP upon CN stimulation; galanin, galR1, -2, -3, and c-JUN mRNA expression at various time points after BCNI; and nNOS, galanin, and galR distribution in the MPG of sham-operated rats and after BCNI. RESULTS: After BCNI, ICP/MAP values quickly deteriorate, while after 60 days, spontaneous restoration of erectile responses to CN stimulation is observed, reflecting CN regeneration. Galanin mRNA in the MPG is up to 186-fold upregulated compared with sham-operated rats at 48 hours and 7 days after BCNI and gradually declines with increasing time from injury, whereas galanin receptor expressions decrease and c-JUN gradually increases. Galanin expression shows a strong inverse correlation with erectile responses to CN stimulation with time from injury. Injured MPGs show a colocalization between galanin- and nNOS-positive neuronal cell population in the MPG. CONCLUSIONS: Galanin is upregulated in the MPG in the early phase after CN injury after which it gradually decreases and is present in nNOS-positive neurons of the ganglion. We hypothesize that galanin upregulation is an important factor in the endogenous neuroregenerative response to CN injury.

Kit gene in endometrial carcinoma: an immunohistochemical and mutational analysis.

OBJECTIVE: : Because the outcome of recurrent disease of endometrial carcinoma is cumbersome, the development of target treatment strategies is critical. We evaluated KIT, a receptor tyrosine kinase, to determine a potential role for imatinib mesylate in the treatment of endometrial carcinoma. MATERIALS AND METHODS: : Immunohistochemical analysis for KIT expression was performed on paraffin sections from 45 patients: 30 primary and 15 recurrent tumors. Fifteen primary cases were available for mutation analysis. RESULTS: : Histopathological distribution of paraffin-embedded tissue was as follows: 30 type I and 15 type II endometrial carcinoma. Histopathological distribution of fresh-frozen tissue was as follows: 8 type I and 7 type II. Cases did not show KIT expression or mutations in mutational hotspot exons of KIT gene. CONCLUSIONS: : On the basis of the absence of KIT expression or mutations, endometrial carcinoma is unlikely to respond to imatinib mesylate.

Expression of ERCC1, p53, and class III ß-tubulin do not reveal chemoresistance in endometrial cancer: results from an immunohistochemical study.

BACKGROUND: In non-small cell lung cancer, expression of excision repair cross-complementation group 1 (ERCC1) and p53 correlates with platinum resistance and class III ß-tubulin with resistance to taxanes. The potential to personalize treatment in endometrial cancer remains uninvestigated. METHODS: Patients received platinum-based chemotherapy, with or without paclitaxel. Patients were divided into 2 groups: group A (n = 33) consisted of patients with early-stage endometrial cancer treated with adjuvant chemotherapy. Group B (n = 116) included cases with primary advanced or recurrent disease. Immunohistochemistry was performed to analyze the expression of ERCC1 and p53, for all cases, and class III ß-tubulin for cases treated with paclitaxel. The findings were correlated with response according to Response Criteria in Solid Tumors; recurrence-free, disease-specific survival; and established prognostic markers. RESULTS: The mean age of 149 patients was 64 years (range, 31-84 years). Distribution of histopathologic subtypes was as follows: 44 endometrioid (30%), 92 serous/clear cell (62%), and 13 carcinosarcomas (8%).In group A, 11 (33%) and 19 patients (58%) showed expression for ERCC1 and p53, respectively. Seven (78%) of nine patients receiving paclitaxel were positive for class III ß-tubulin. There was no correlation between expression of ERCC1, p53, or class III ß-tubulin and recurrence or survival. In group B, 25 (22%) and 61 patients (64%) were positive for ERCC1 and p53, respectively. Fifty-two (74%) of seventy patients receiving paclitaxel were positive for class III ß-tubulin. Only p53 expression correlated with survival (P = 0.01). CONCLUSIONS: In contrast to theoretical assumptions, the current study did not reveal evidence that the expression of ERCC1 and class III ß-tubulin predicts response to cytotoxic treatment and patient outcome in endometrial cancer.

Wilms tumor gene 1 (WT1) is a prognostic marker in high-grade uterine sarcoma.

INTRODUCTION: Wilms tumor gene 1 (WT1) contributes to uterine sarcoma tumor biology. In this study, we aimed to clarify the prognostic value of WT1. METHODS: A retrospective clinical and histopathological review of 71 women with high-grade uterine sarcoma (leiomyosarcoma [n = 24], undifferentiated sarcoma [n = 9]), and carcinosarcoma (n = 38) was performed. Patients were followed up for at least 12 months. Wilms tumor gene 1 expression was determined by immunohistochemistry. Data on recurrence (progression-free survival) and overall survival (OS) were available for all patients. Univariate and multivariate analyses of WT1 expression were carried out using Kaplan-Meier curves and Cox regression, respectively. RESULTS: Forty-nine (69%) tumors were WT1 positive. Forty-seven (66%) patients died of the disease, with a median OS time of 22 months. Wilms tumor gene 1 was a predictor of survival in the univariate analysis: the hazard ratio of WT1 positivity was 2.44 (95% confidence interval, 1.34-4.71) for progression-free survival and 2.48 (95% confidence interval, 1.26-4.90) for OS. Multivariate analysis including stage, age, tumor size, and sarcoma subtype identified only stage and WT1 positivity as independent prognostic markers for survival. CONCLUSIONS: The identification of WT1 as a prognostic marker confirms its role in high-grade uterine sarcoma and carcinosarcoma tumor biology.

The use of lymph vessel markers to predict endometrial cancer outcome.

OBJECTIVE: To evaluate lymphangiogenesis and lymph vessel space involvement in different subsets of endometrial cancer using podoplanin, a specific marker for lymphatic endothelium. METHODS: Sixty-two patients undergoing a hysterectomy with lymphadenectomy were included. Distribution of histopathologic subtypes was as follows: 30 endometrioid (48%), 18 serous (29%), 9 clear cell carcinoma (15%), and 5 carcinosarcomas (8%). Distribution of surgical stage according to the International Federation of Gynecology and Obstetrics 2009 criteria was as follows: 33 stage I (53%), 7 stage II (11%), 1 stage IIIA (2%), 15 stage IIIC1 (24%), and 6 stage IIIC2 (10%). Tumor samples were immunostained for podoplanin and the panendothelial marker, CD31. Peritumoral and intratumoral blood vessel density and lymph vessel density were assessed using an image analysis system that calculated mean vessel cross-sectional area (in micrometer squared) and vessel density (per millimeter squared). Presence of blood vessel space involvement and lymph vessel space involvement was screened for. The findings were linked with clinical outcome using Cox regression. RESULTS: Twenty-one patients (34%) experienced recurrence, and 13 (21%) died of disease. Univariate analysis showed that blood vessel space involvement was related to worse overall survival (hazard ratio, 6.59; 95% confidence interval, 1.30-120). Multivariate analyses confirmed the prognostic importance of this variable for overall survival (hazard ratio, 7.52; 95% confidence interval, 1.32-144). CONCLUSION: Blood vessel space involvement is a prognostic marker for worse survival. Although lymph vessels were stained with the most reliable marker, podoplanin, lymph vessel density and lymphovascular space involvement do not seem to be of prognostic importance.

Porous acellular porcine dermal collagen implants to repair fascial defects in a rat model: biomechanical evaluation up to 180 days.

AIM: To investigate the biomechanical properties of porous collagen matrices in a rat abdominal wall defect model. STUDY DESIGN: 112 rats were implanted with non-cross-linked InteXèn LP, cross-linked Pelvicol, and two investigational acellular collagen matrices (ACMs) sterilized either with ethylene oxide (ACM ETO) or gamma-irradiation (ACM GI). After 14, 30, 90 and 180 days, 7 animals per group were sacrificed to document adhesions, herniation, infection, stress resistance and histology. RESULTS: The 2 sterilization methods did not cause measurable differences between ACMs. Pelvicol was more resistant than ACMs but showed degradation at 90 days without loss of strength. InteXèn LP became remodeled as a thin fibrous scar and was more resistant at all time points; however, some animals developed bulging. CONCLUSIONS: Non-cross-linked InteXèn LP became remodeled by 180 days with remarkable stress resistance. Despite cross-linking Pelvicol showed degradation. Comparable but investigational ACM explants were less resistant without morphologic differences to explain this.

Wilms' tumor gene 1 (WT1) in endometrial carcinoma.

OBJECTIVE: Wilms' tumor gene (WT1), located on chromosome 11, encodes a transcription factor that contributes to the carcinogenesis of uterine sarcomas. To expand the knowledge on the biological role of WT1 in other uterine cancers, we focused on its detection in endometrial carcinoma. METHODS: In total, 36 paraffin-embedded tumors were available for WT1 immunohistochemical (IHC) analysis including endometrial endometrioid carcinoma (n=24), serous carcinoma (n=9) and clear cell carcinoma (n=3). Three slides from different sites of the tumor were analysed. Of these tumors, 32 snap frozen tissue samples were available for RT-PCR (endometrioid carcinoma (23), serous carcinoma (7) and clear cell carcinoma (2)). To compare, WT1 expression was also evaluated by IHC in benign endometrium (12) and benign endometrial polyps (5). RESULTS: WT1 positivity was noticed in tumor cells and endothelial cells, lining the intratumoral blood vessels. Overall, 72% (26/36) of tumors stained positive for WT1. RT-PCR results showed WT1 positivity in 75% (24/32) of samples. Comparing the staining patterns of the 3 different bioptic sites, tumor heterogeneity was demonstrated in the majority (72%) of samples. CONCLUSION: Although WT1 is expressed in a majority of endometrial carcinomas, a heterogeneous staining pattern is observed. This information is important for WT1-directed immunotherapy.

Fate of collagen-based implants used in pelvic floor surgery: a 2-year follow-up study in a rabbit model.

OBJECTIVE: The purpose of this study was to compare the long-term host response to 2 different collagen matrices versus macroporous polypropylene mesh. STUDY DESIGN: Four full-thickness abdominal wall defects in 35 rabbits were reconstructed with either polypropylene (Prolene), porcine dermal (Pelvicol), or small intestine submucosal collagen matrix (SIS). Animals were sacrificed on day 30, 60, 90, 180, 365, 540, and 720 days to evaluate morphologic and biomechanical properties of explants. RESULTS: Prolene provoked a fibrotic reaction within 30 days. SIS was entirely replaced by a thin fibrotic layer within 60 days. Pelvicol was encapsulated, remaining structurally unchanged up to 180 days. Thereafter, half underwent degradation by a foreign body reaction. CONCLUSION: Prolene was integrated by an increasingly organised fibrotic scar while SIS was entirely remodelled within 60 days. Pelvicol implants underwent late onset (> or = 180 days) degradation. After 2 years of implantation there were no differences in tensiometric strength between the 3 different materials.

Acute Drug Effects on the Human Placental Tissue: The Development of a Placental Murine Xenograft Model.

OBJECTIVE: A pilot study was conducted to establish a human placental xenograft, which could serve as a model to evaluate the effect of toxic exposures during pregnancy. STUDY DESIGN: The protocol consisted of engraftment of third-trimester human placental tissue in immunocompromised mice, after induction of a pseudo-pregnancy state by ovariectomy and progesterone supplementation. To validate the model, the placental tissue before and after engraftment was examined by immunohistochemistry, fluorescence-activated cell sorting (FACS), single-nucleotide polymorphism (SNP) genotyping, and whole transcriptome sequencing (WTSS). The human chorion gonadotropin (hCG) production in serum and urine was examined by enzyme-linked immunosorbent assay. RESULTS: Microscopic evaluation of the placental tissue before and after engraftment revealed a stable morphology and preserved histological structure of the human tissue. Viable trophoblast was present after engraftment and remained stable over time. Vascularization and hormonal secretion (hCG) were present till 3 weeks after engraftment. Thirty-one SNPs were equally present, and there was a stable expression level for 56 451 genes evaluated by whole transcriptome sequencing. CONCLUSION: Although this human placental xenograft model cannot copy the unique uterine environment in which the placenta develops and interacts between the mother and the fetus, it could be a suitable tool to evaluate the acute impact and adaptive processes of the placental tissue to environmental changes.

Enhancing sealing of fetal membrane defects using tissue engineered native amniotic scaffolds in the rabbit model.

OBJECTIVE: The purpose of this study was to compare the efficacy of native engineered amniotic scaffolds (AS) and polyesterurethane scaffolds (DegraPol) and document wound healing response when sealing iatrogenic fetal membrane defects in the rabbit model. STUDY DESIGN: Native AS were engineered from freshly harvested membranes of 23 days' gestational age (GA; term = 31-2 d). Acellularity of AS was assessed by histology, light and scanning electron microscopy. Fetal membrane defects were created by 14 gauge-needle puncture at GA 23 days and primarily closed with AS (n = 10) or DegraPol (n = 10) or left unclosed (positive controls; n = 10). Sixty-one sacs served as negative controls. At GA 30 days a second look hysterotomy was performed to assess presence of amniotic fluid (AF) and harvest plugging sites for microscopic evaluation. RESULTS: Engineered AS had a cell-free collagenous fiber network. AF was significantly higher only in the DegraPol group (78%; P < .05) compared to the AF in positive controls (17%). Integration of plugs in the fetal membrane defect was better with AS than DegraPol, with higher reepithelialization rates (AS: 52.5% +/- 6.5%; DegraPol: 11.6% +/- 2.6%; P < .001) and proliferation indices (AS: 0.47 +/- 0.03; DegraPol: 0.28 +/- 0.04; P = .001). In both treatment groups, cell proliferation in the myometrium was increased (P < .05). CONCLUSION: Native AS seal iatrogenic fetal membrane defects better than DegraPol. Within a week, there is abundant reepithelilization and minimal local inflammation. This yields the proof of principle that engineered native, amniotic membrane scaffolds enhance fetal membrane wound healing response.

Potential Targets' Analysis Reveals Dual PI3K/mTOR Pathway Inhibition as a Promising Therapeutic Strategy for Uterine Leiomyosarcomas-an ENITEC Group Initiative.

Purpose: Uterine sarcomas are rare and heterogeneous tumors characterized by an aggressive clinical behavior. Their high rates of recurrence and mortality point to the urgent need for novel targeted therapies and alternative treatment strategies. However, no molecular prognostic or predictive biomarkers are available so far to guide choice and modality of treatment.Experimental Design: We investigated the expression of several druggable targets (phospho-S6S240 ribosomal protein, PTEN, PDGFR-α, ERBB2, and EGFR) in a large cohort of human uterine sarcoma samples (288), including leiomyosarcomas, low-grade and high-grade endometrial stromal sarcomas, undifferentiated uterine sarcomas, and adenosarcomas, together with 15 smooth muscle tumors of uncertain malignant potential (STUMP), 52 benign uterine stromal tumors, and 41 normal uterine tissues. The potential therapeutic value of the most promising target, p-S6S240, was tested in patient-derived xenograft (PDX) leiomyosarcoma models.Results: In uterine sarcomas and STUMPs, S6S240 phosphorylation (reflecting mTOR pathway activation) was associated with higher grade (P = 0.001) and recurrence (P = 0.019), as shown by logistic regression. In addition, p-S6S240 correlated with shorter progression-free survival (P = 0.034). Treatment with a dual PI3K/mTOR inhibitor significantly reduced tumor growth in 4 of 5 leiomyosarcoma PDX models (with tumor shrinkage in 2 models). Remarkably, the 4 responding models showed basal p-S6S240 expression, whereas the nonresponding model was scored as negative, suggesting a role for p-S6S240 in response prediction to PI3K/mTOR inhibition.Conclusions: Dual PI3K/mTOR inhibition represents an effective therapeutic strategy in uterine leiomyosarcoma, and p-S6S240 expression is a potential predictive biomarker for response to treatment. Clin Cancer Res; 23(5); 1274-85. ©2017 AACR.

The Use of Toll-like Receptor 4 Agonist to Reshape the Immune Signature in Ovarian Cancer.

BACKGROUND: Dendritic cell (DC) mono-immunotherapy has not been successful so far in ovarian cancer. The addition of a toll-like receptor (TLR) agonist has the potential to boost the innate immune system, in addition to the adoptive immune response initiated by DCs. MATERIALS AND METHODS: ID8-fLuc C57BL/6 mice were injected with DCs loaded with hypericin-based photodynamic therapy-treated tumor lysate. A TLR4 agonist [lipopolysaccharide (LPS)] was administered by different schedules). After two and three DC vaccinations, immune analysis was performed. RESULTS: There was no survival benefit from therapy with TLR4 agonist. Moreover, if LPS administrations started one week after tumor inoculation, the overall survival was even worse than that of untreated controls. Immune analyses revealed an intratumoral increase in natural killer cells and a decrease in regulatory T-cells, but an immunosuppressive signature in the ascites. CONCLUSION: Addition of LPS as an adjuvant to DC immunotherapy of ovarian cancer does not result in survival benefit.

In Vitro Generation of Murine Dendritic Cells for Cancer Immunotherapy: An Optimized Protocol.

BACKGROUND/AIM: Dendritic cell (DC) immunotherapy induces tumor-reactive T-cells. We optimized the maturation of murine DC against ovarian cancer. MATERIALS AND METHODS: Immature DC were generated from bone-marrow progenitor cells and loaded with hypericin-photodynamic-treated (Hyp-PDT) tumor cells (primary maturation). Lipopolysacharide (LPS 1 µg/ml) was used as a secondary maturation stimulus. After 24 h, maturation was assessed using flow cytometry. For in vivo experiments, C57BL/6 mice were vaccinated subcutaneously with matured, loaded mature DC. Immune response was evaluated through immunohistochemistry and cytometric bead array. RESULTS: Based on the existing protocols for DC generation, therapeutic vaccination of tumor-bearing mice with mature ID8-fLuc Hyp-PDT DC induces an immunogenic response, but provides no survival benefit. As loading with Hyp-PDT lysate induces partial primary maturation, we reduced the dose of LPS to 0.125 µg/ml and the duration of maturation to 6 hours, improving viability of mature DC. CONCLUSION: We optimized Hyp-PDT-based ID8-fLuc DC vaccine by reducing the amount of LPS and the duration of maturation.

In Vitro Validation of Survivin as Target Tumor-associated Antigen for Immunotherapy in Uterine Cancer.

Survivin is an antiapoptotic protein, not expressed in terminally differentiated adult tissues, yet overexpressed in several tumors. Therefore, it is an interesting target for immunotherapeutic strategies. In addition to specific overexpression in tumors, tumor survival is mediated by survivin and hence, tumor survival can be tackled by targeting survivin. Survivin expression in uterine cancer was validated by quantitative real-time polymerase chain reaction and immunohistochemistry. In addition, we evaluated survivin immunogenicity by analyzing spontaneous B-cell and T-cell responses in patients. Survivin as a protein was expressed in only a minority of normal tissues, whereas it was being expressed in all of the currently analyzed uterine cancers, both endometrial carcinoma (n = 52) and uterine sarcoma (n = 52). Survivin RNA transcripts were overexpressed in more aggressive tumors and survivin protein was overexpressed in recurrent endometrial tumors compared with primary tumors. Spontaneous T-cell responses were seen in 10/39 endometrial cancer patients and 3/16 uterine sarcoma patients. In normal controls, T-cell responses were found only in 1 donor (n = 21). Although increased antibody titers were found in more aggressive and far-advanced tumors, no differences in B-cell responses were seen. Overall, when compared with normal controls, a B-cell response was only measured in 1/41 uterine sarcoma patients. In conclusion, we currently validated the presence of survivin in uterine cancer. In addition, spontaneous T-cell responses were found in 23.6% of the total patient population. These data indicate that a survivin-specific immune response may be induced spontaneously in patients, further fortifying the eligibility of survivin as an immunotherapeutic target.

Enrichment of collagen plugs with platelets and amniotic fluid cells increases cell proliferation in sealed iatrogenic membrane defects in the foetal rabbit model.

OBJECTIVES: The purpose of this study was to evaluate cell proliferation in platelet-enriched collagen plugs with and without addition of amniotic fluid-derived heterologous foetal cells to seal an iatrogenic membrane defect in the foetal rabbit model. METHODS: Amniotic fluid cells were harvested from three donor does at 23 days of gestation (term = 32 days) and labelled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). In 42 other does, foetal membrane defects were induced by foetoscopic needle puncture at 23 days of gestation, and closed with either a platelet-enriched collagen plug with (n = 44) or without (n = 32) amniotic fluid cells. At 30 days of gestation, the defects were harvested and assessed microscopically. RESULTS: The plugs enriched with heterologous amniotic fluid cells more commonly had proliferating cells in the centre of the plug than those without cell addition. CFDA-SE labelling confirmed the presence of heterologous amniotic fluid cells over the entire membrane plug. Cell typing showed a mixture of fibroblasts and epithelial cells at the wound edges, whereas in the centre, there was an abundance of fibroblasts. CONCLUSION: When sealing iatrogenic membrane defects in the foetal rabbit model, enrichment of collagen plugs with platelets and amniotic fluid-derived heterologous foetal cells increases local cell proliferation.

Tensile strength and host response towards silk and type i polypropylene implants used for augmentation of fascial repair in a rat model.

OBJECTIVE: We compared host response, architectural integration and tensile strength of two different macroporous silk constructs to a polypropylene type I implant in a rat model for augmentation of primary fascial defect repair. MATERIALS AND METHODS: Animals were sacrificed on days 7, 14, 30 and 90 after implantation. The explants were evaluated macroscopically for infections, herniations and adhesions, mechanically for tensile strength, and histopathologically, to evaluate collagen deposition and inflammatory response. RESULTS: The tensile strength of the explants showed a gradual increase for all materials. All implants uniformly shrank around one fifth by 90 days. In the silk implants, the inflammatory reaction showed a remarkable higher number of foreign body giant cells that characteristically spread from the periphery into implants. Collagen deposition was comparable for all the materials. In Silk a higher grade of neovascularisation was observed. CONCLUSION: Silk explants expressed high tensiometric strength, which was associated with a marked fibrotic process. The silk implants induced a strong foreign body reaction accompanied by microscopic signs of architectural degradation at 90 days. Polypropylene explants showed a more moderate foreign body reaction without architectural disturbance.