Extracellular vesicles released by myeloid-derived suppressor cells from pregnant women modulate adaptive immune responses.

Immunological pregnancy complications are a main challenge in reproductive medicine. Mechanisms regulating the adaptation of the maternal immune system to pregnancy are incompletely understood and therapeutic options limited. Myeloid derived suppressor cells (MDSC) are immune-modulatory cells expanding during healthy pregnancy and seem to play a crucial role for maternal-fetal tolerance. Recent studies showed that exosomes produced by MDSC have immune-modulatory effects corresponding to their parental cells under different pathological conditions. Here, we investigated immunological effects of exosomes of GR-MDSC during pregnancy. Isolated GR-MDSC exosomes from peripheral blood of pregnant women were tested for functionality in different in vitro assays. We show that GR-MDSC exosomes exhibited profound immune-modulatory effects such as suppression of T-cell proliferation, T helper 2 (Th2)-cell polarization, induction of regulatory T-cells and inhibition of lymphocyte cytotoxicity. Our results confirm that MDSC-derived exosomes functionally correspond to their parental cells and identify them as an interesting therapeutic target for immunological pregnancy complications.

Human Leucocyte Antigen G and Murine Qa-2 Are Critical for Myeloid Derived Suppressor Cell Expansion and Activation and for Successful Pregnancy Outcome.

During pregnancy, maternal immune system has to balance tightly between protection against pathogens and tolerance towards a semi-allogeneic organism. Dysfunction of this immune adaptation can lead to severe complications such as pregnancy loss, preeclampsia or fetal growth restriction. In the present study we analyzed the impact of the murine MHC class Ib molecule Qa-2 on pregnancy outcome in vivo. We demonstrate that lack of Qa-2 led to intrauterine growth restriction and increased abortion rates especially in late pregnancy accompanied by a disturbed trophoblast invasion and altered spiral artery remodeling as well as protein aggregation in trophoblast cells indicating a preeclampsia-like phenotype. Furthermore, lack of Qa-2 caused imbalanced immunological adaptation to pregnancy with altered immune cell and especially T-cell homeostasis, reduced Treg numbers and decreased accumulation and functional activation of myeloid-derived suppressor cells. Lastly, we show that application of sHLA-G reduced abortion rates in Qa-2 deficient mice by inducing MDSC. Our results highlight the importance of an interaction between HLA-G and MDSC for pregnancy success and the therapeutic potential of HLA-G for treatment of immunological pregnancy complications.

Granulocytic Myeloid-Derived Suppressor Cells (GR-MDSC) in Breast Milk (BM); GR-MDSC Accumulate in Human BM and Modulate T-Cell and Monocyte Function.

Nosocomial bacterial infections (NBI) and necrotizing enterocolitis (NEC) are among the main reasons for death in preterm infants. Both are often caused by bacteria coming from the infected infant's gut and feeding with breast milk (BM) seems beneficial in their pathogenesis. However, mechanisms causing the protective effect of BM are only incompletely understood. Myeloid-derived suppressor cells (MDSC) are myeloid cells with suppressive activity on other immune cells, recently described to play a role in mediating maternal-fetal tolerance during pregnancy and immune adaptation in newborns. Until now, nothing is known about occurrence and function of MDSC in BM. We analyzed MDSC in BM and peripheral blood of breastfeeding mothers and found that granulocytic MDSC, but not monocytic MDSC, accumulate in BM, exhibit an activated phenotype and increased suppressive activity and modulate TLR-expression on monocytes. Furthermore, we found that the lactotrophic hormones prolactin and oxytocin do not induce MDSC from peripheral blood. This is the first study to describe MDSC with immune-modulatory properties in human BM. Our results point toward a role for MDSC in local immune modulation in the gut possibly protecting infants from NBI and NEC.

HLA-G promotes myeloid-derived suppressor cell accumulation and suppressive activity during human pregnancy through engagement of the receptor ILT4.

Establishing and maintaining maternal-fetal tolerance is essential for a successful pregnancy; failure of immunological adaptation to pregnancy leads to severe complications such as abortion or preterm delivery. Myeloid-derived suppressor cells (MDSCs) are innate immune cells that suppress T-cell responses, expand during pregnancy and thus may play a role in tolerance induction. Human leucocyte antigen G (HLA-G) is a major histocompatibility complex (MHC) I molecule with immune-modulatory properties, which is expressed during pregnancy. Here, we investigated the impact of HLA-G on MDSCs accumulation and activation in pregnant women. We demonstrate that granulocytic MDSCs (GR-MDSCs) express receptors for HLA-G, namely immunoglobulin-like transcript (ILT) 2 and 4, and that ILT4-expression by GR-MDSCs is regulated during pregnancy. Stimulation with soluble HLA-G (sHLA-G) increased suppressive activity of GR-MDSCs, induced MDSCs from peripheral blood mononuclear cells (PBMCs) and led to phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and induction of indoleamine-2,3-dioxygenase (IDO) in myeloid cells. Effects of sHLA-G on MDSC accumulation were mediated through ILT4. These results suggest an interaction between MDSCs and HLA-G in humans as a potential mechanism for maintaining maternal-fetal tolerance. Modulating MDSC function during pregnancy via HLA-G might provide new opportunities for a therapeutic manipulation of immunological pregnancy complications.

Granulocytic myeloid derived suppressor cells expand in human pregnancy and modulate T-cell responses.

Immune tolerance toward the semiallogeneic fetus plays a crucial role in the maintenance of pregnancy. Myeloid-derived suppressor cells (MDSCs) are innate immune cells characterized by their ability to modulate T-cell responses. Recently, we showed that MDSCs accumulate in cord blood of healthy newborns, yet their role in materno-fetal tolerance remained elusive. In the present study, we demonstrate that MDSCs with a granulocytic phenotype (GR-MDSCs) are highly increased in the peripheral blood of healthy pregnant women during all stages of pregnancy compared with nonpregnant controls, whereas numbers of monocytic MDSCs were unchanged. GR-MDSCs expressed the effector enzymes arginase-I and iNOS, produced high amounts of ROS and efficiently suppressed T-cell proliferation. After parturition, GR-MDSCs decreased within a few days. In combination, our results show that GR-MDSCs expand in normal human pregnancy and may indicate a role for MDSCs in materno-fetal tolerance.

[Screening for mental stress and the wish for psychological support in patients with breast cancer]. / SCreening psychischer Belastungen und Erhebung des Wunsches nach psychosozialen Unterstützungsangeboten bei Patientinnen mit Mammakarzinom.

OBJECTIVES: The study explores whether breast-cancer patients who request psychosocial support have higher psychological strain compared to those who do not. We also explored whether scores on HADS-D and PHQ-D identify the same patients as being stressed as well as the relationship between patient's wish for psychological support and psychological strain. METHODS: 115 inpatients with breast cancer took part in the screening and were asked if they wanted psychosocial support. RESULTS: Above-normal scores in anxiety and depression are reported by 25-29%, with 41% indicating a wish for psychosocial support. These patients showed significantly higher scores on the questionnaires. Their request for psychosocial support shows a similar sensitivity (.733) and specificity (.662) as the "Distress-Thermometer" or "PO-Bado". DISCUSSION: The desire for psychosocial support appears to be a valuable piece of additional information for indicating psychosocial support. However, HADS-D and PHQ-D agree only in 77% of the individual patients.

Loss of Drop1 expression already at early tumor stages in a wide range of human carcinomas.

In a study on gene deregulation in ovarian carcinoma we found a mRNA coding for a 350 kDa protein, Drop1, to be downregulated 20- to 180-fold in the majority of ovarian and mammary carcinomas. The mRNA is encoded by a set of exons in the 5' region of the SYNE1 gene. Immunohistochemical staining for Drop1 protein by a specific monoclonal antibody corresponds to the pattern seen for the mRNA. cDNA arrays of matched pairs of tumor and normal tissue and in situ hybridizations confirmed the drastic loss of Drop1 mRNA as a common feature in uterus, cervix, kidney, lung, thyroid and pancreas carcinomas, already at early tumor stages and in all metastases. Two-hybrid studies suggest a role of this deficiency in the malignant progression of epithelial tumors.

A stable niche supports long-term maintenance of human epidermal stem cells in organotypic cultures.

Stem cells in human interfollicular epidermis are still difficult to identify, mainly because of a lack of definitive markers and the inability to label human beings for label-retaining cells (LRCs). Here, we report that LRCs could be identified and localized in organotypic cultures (OTCs) made with human cells. Labeling cultures for 2 weeks with iododeoxyuridine (IdU) and then chasing for 6-10 weeks left <1% of basal cells retaining IdU label. Whole mounts demonstrated that LRCs were individually dispersed in the epidermal basal layer. Some LRCs, but not all, colocalized with cells expressing melanoma chondroitin sulfate proteoglycan, a putative stem cell marker. Although we found LRCs in both collagen- and scaffold-based OTCs, only the scaffold-OTCs supported long-term survival and regeneration. LRCs ' short survival in collagen-OTCs was not due to loss of appropriate growth factors from fibroblasts. Instead, it was due to expression of metalloproteinases, especially matrix metalloproteinase (MMP)-2 and MMP-14, which caused collagen fragmentation, matrix degradation, and dislocation of specific basement membrane components bound to epidermal integrins. Blocking MMP activation not only abrogated MMP-dependent matrix degradation but also increased longevity of the epidermis and the LRCs in these cultures. Such findings indicate that the stem cell niche, the microenvironment surrounding and influencing the stem cell, is essential for stem cell survival and function, including long-term tissue regeneration. Disclosure of potential conflicts of interest is found at the end of this article.

Clinical and mechanistic aspects of glucocorticoid-induced chemotherapy resistance in the majority of solid tumors.

BACKGROUND: Glucocorticoids have been used widely in conjunction with cancer therapy due to their ability to induce apoptosis in hematological cells and to prevent nausea and emesis. However, recent data including ours, suggest induction of therapy-resistance by glucocorticoids in solid tumors, although it is unclear whether this happens only in few carcinomas or is a more common cell type specific phenomenon. MATERIAL AND METHODS: We performed an overall statistical analysis of our new and recent data with 157 tumor probes evaluated in vitro, ex vivo and in vivo. The effect of glucocorticoids on apoptosis, viability and cell cycle progression under diverse clinically important questions was examined. RESULTS: New in vivo results demonstrate glucocorticoid-induced chemotherapy resistance in xenografted prostate cancer. In an overall statistical analysis we found glucocorticoid-induced resistance in 89% of 157 analysed tumor samples. Resistance is common for several cytotoxic treatments and for several glucocorticoid-derivatives and due to an inhibition of apoptosis, promotion of viability and cell cycle progression. Resistance occurred at clinically achievable peak plasma levels of patients under anti-emetic glucocorticoid therapy and below, lasted for a long time, after one single dose, but was reversible upon removal of glucocorticoids. Two nonsteroidal alternative anti-emetic agents did not counteract anticancer treatment and may be sufficient to replace glucocorticoids in cotreatment of carcinoma patients. CONCLUSION: These data demonstrate the need for prospective clinical studies as well as for detailed mechanistic studies of GC-induced cell-type specific pro- and anti-apoptotic signalling.

Phase I trial of the trifunctional anti-HER2 x anti-CD3 antibody ertumaxomab in metastatic breast cancer.

PURPOSE: Ertumaxomab is an intact bispecific antibody targeting HER2/neu and CD3 with selective binding to activatory Fcgamma type I/III receptors, resulting in the formation of a tri-cell complex between tumor cells, T cells, and accessory cells. Patients with metastatic breast cancer were enrolled into a multicenter phase I dose-escalating trial. EXPERIMENTAL DESIGN: Three ascending doses of ertumaxomab (10-200 microg) were administered i.v. on day 1, 7 +/- 1, and 13 +/- 1. Safety and tolerability were the primary objectives. Secondary objectives were antitumor activity and different immunologic variables. RESULTS: Fifteen out of 17 enrolled patients completed the study. One hundred micrograms was identified as the maximal tolerable single dose. Most drug-related adverse events were mild and transient including fever (94%), rigors (47%), headache (35%), nausea (29%), vomiting (29%). Grades 3 and 4 (Common Toxicity Criteria) were lymphocytopenia (76%) and elevation of liver enzymes (47%). One patient (200 mug dose) developed severe hypotension and respiratory distress syndrome, another patient (150 mug dose) developed a systemic inflammatory response syndrome and acute renal failure. Aggravation of congestive heart failure was seen in one patient with preexisting ventricular dysfunction after administration of the third dose (200 microg). All adverse events were fully reversible. Antitumor response was seen in 5 out of 15 evaluable patients (one with a complete response, two with partial responses, two with stable disease) at dose levels of > or = 100 microg. Measurements of cytokines (interleukin-6, interleukin-2, tumor necrosis factor-alpha, and IFN-gamma) suggest a strong T helper cell type 1-associated immune response. The induction of human anti-mouse/anti-rat antibodies was detected in 5 out of 16 (31%) patients. DISCUSSION: Treatment with triple infusions of ertumaxomab yields a strong immunologic response. Doses up to 100 microg can be safely infused with close monitoring of patients. The observed clinical responses are encouraging and indicate antitumor efficacy.

Glucocorticoid-mediated inhibition of chemotherapy in ovarian carcinomas.

The glucocorticoid dexamethasone is frequently used as a co-treatment in cytotoxic cancer therapy, e.g. to prevent nausea, to protect normal tissue or for other reasons. While the potent pro-apoptotic properties and supportive effects of glucocorticoids to tumour therapy in lymphoid cells are well studied, the impact on the cytotoxic treatment of ovarian carcinoma is unknown. We tested apoptosis-induction, viability, tumour growth and protein expression using established cell lines, primary cell lines freshly isolated from patient material and a xenograft on nude mice. We found a general induction of resistance toward cytotoxic therapy by DEX-co-treatment in most of the examined ovarian cancer cells treated in vitro, ex vivo or in vivo. Resistance occurred independently of cell density and was found at peak plasma levels of dexamethasone and below. Mechanistically, the dexamethasone-induced expression of survival genes may be involved in the resistance. These data show that glucocorticoid-induced resistance is common in ovarian carcinomas implicating that the use of glucocorticoids may be harmful for cancer patients.

Pre-existing T-cell immunity against mucin-1 in breast cancer patients and healthy volunteers.

PURPOSE: There is evidence that some tumor patients are able to generate tumor-associated antigen (TAA)-specific T-cell immunity spontaneously. However, little is understood about the existence and nature of self-reactive T-cells that recognize TAA in healthy donors (HD). METHODS: Human mucin (MUC-1), a highly glycosylated transmembrane protein, is a well characterized TAA expressed by epithelial tumors. We compared endogenous MUC-1-specific T-cell immunity of breast cancer patients (BCP) and healthy volunteers using two MUC-1-derived HLA-A*0201-restricted peptides (MUC-1(950-958), MUC-1(12-20)). Antigen-dependent interferon (IFN)-gamma and Granzyme B expression of T-cells were analysed by a reverse transcription-polymerase chain reaction (qRT-PCR)-based assay. RESULTS: A 32% of BCP and 43% of healthy volunteers revealed pre-existent CD8+ T-cells specific for MUC-1(950-958) but not for MUC-1(12-20). In patients, MUC-1-specific T-cells have been detected mainly in early stage disease prior adjuvant therapy. Those T-cells showed MUC-1-dependent IFN-gamma production after short-term stimulation but no clear Granzyme B expression. However, after repetitive in vitro stimulations using peptide-pulsed CD40-stimulated B-cell lines as autologous antigen presenting cells (APC) T-cell lines exhibited lytic capacity against HLA-A*0201+/MUC-1+ tumor cells. CONCLUSION: MUC-1(950-958) is a dominant tumor antigen against which CD8+ T-cells were found frequently in BCP as well as in HD. Until now, this was only known for MelanA/MART-1. In contrast to previous reports, MUC-1-specific immunity was not linked to gender or number of pregnancies in women. Whether MUC-1(950-958)-related immunity highlights a yet unknown cross-reactivity in HD remains unclear. The presence of MUC-1-specific T-cells in some BCP may reflect a balance between immune tolerance and immune defence during aetiopathology.

L1 on ovarian carcinoma cells is a binding partner for Neuropilin-1 on mesothelial cells.

The progression of ovarian cancer is driven by a variety of cellular factors that are incompletely understood. Binding of tumor cells to normal cells and to soluble factors influence tumor growth, angiogenesis and the stimulation of vascular permeability leading to ascites production. L1 adhesion molecule is overexpressed in ovarian carcinoma and is associated with bad prognosis. One receptor for L1 is Neuropilin-1 (NRP-1) that is also known as a receptor for VEGF(165). In the nervous system a complex of NRP-1 and L1 transmits signals by the neurorepellant Sem3A that is critical for the control of neurite outgrowth. NRP-1 has also been detected in human carcinomas but its function remains unknown. Here, we have examined NRP-1 expression in ovarian carcinoma cell lines and tissue. We report that little NRP-1 protein was detected in primary ovarian carcinoma tissues or established cell lines although mRNA for soluble and transmembrane NRP-1 were detected by RT-PCR. Instead, we observed strong expression of NRP-1 in mesothelial cells, which form the lining of the peritoneum. NRP-1 could serve as an isolation marker for primary mesothelial cells present in ascites fluid. We demonstrate that ovarian cancer cells expressing L1 can bind to NRP-1 overexpressing cells and mesothelial cells. Likewise, soluble L1 isolated from ascites of patients or produced as a fusion protein could bind to NRP-1 overexpressing cells and a direct interaction was demonstrated at the protein level. These findings suggest that L1 can support the binding of ovarian carcinoma cells to mesothelial cells via NRP-1. The L1-NRP-1 binding pathway could contribute to the growth of ovarian carcinomas and to reciprocal signalling between mesothelial cells and tumors.

Preeclampsia: increased expression of soluble ADAM 12.

Preeclampsia is a multisystemic pregnancy-associated disease affecting about 3-7% of pregnancies worldwide and is still a principal cause of fetal and maternal morbidity and mortality. To identify potential markers, we have compared gene expression profiles from control and preeclamptic placental tissues taken at various age-matched gestational stages using complementary DNA microarray analysis. Besides previously identified preeclampsia-associated genes, novel differentially expressed transcripts were found. The soluble form of the disintegrin metalloprotease ADAM 12 (a disintegrin and metalloproteinase 12; meltrin-alpha) represented the most upregulated transcript. This was confirmed by in situ hybridization of sections of preeclamptic placentas and by serum protein analysis of preeclamptic pregnant women. Thus, ADAM 12 could serve as an early biomarker for preeclampsia that may be of predictive and/or functional significance.

Cleavage of L1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells.

PURPOSE: The L1 adhesion molecule (CD171) is overexpressed in human ovarian and endometrial carcinomas and is associated with bad prognosis. Although expressed as a transmembrane molecule, L1 is released from carcinoma cells in a soluble form. Soluble L1 is present in serum and ascites of ovarian carcinoma patients. We investigated the mode of L1 cleavage and the function of soluble L1. EXPERIMENTAL DESIGN: We used ovarian carcinoma cell lines and ascites from ovarian carcinoma patients to analyze soluble L1 and L1 cleavage by Western blot analysis and ELISA. RESULTS: We find that in ovarian carcinoma cells the constitutive cleavage of L1 proceeds in secretory vesicles. We show that apoptotic stimuli like C2-ceramide, staurosporine, UV irradiation, and hypoxic conditions enhance L1-vesicle release resulting in elevated levels of soluble L1. Constitutive cleavage of L1 is mediated by a disintegrin and metalloproteinase 10, but under apoptotic conditions multiple metalloproteinases are involved. L1 cleavage occurs in two types of vesicles with distinct density features: constitutively released vesicles with similarity to exosomes and apoptotic vesicles. Both types of L1-containing vesicles are present in the ascites fluids of ovarian carcinoma patients. Soluble L1 from ascites is a potent inducer of cell migration and can trigger extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: We suggest that tumor-derived vesicles may be an important source for soluble L1 that could regulate tumor cell function in an autocrine/paracrine fashion.

A CD80-transfected human breast cancer cell variant induces HER-2/neu-specific T cells in HLA-A*02-matched situations in vitro as well as in vivo.

Adjuvant treatment is still only working in a small percentage of breast cancer patients. Therefore, new strategies need to be developed. Immunotherapies are a very promising approach because they could successfully attack tumor cells in the stage of dormancy. To assess the feasibility of using an allogeneic approach for vaccination of breast cancer patients, we selected a CD80-transfected breast cancer cell line based on its immunogenic properties. Using CD80+ KS breast cancer cells and human leukocyte antigen (HLA)-A*02-matched peripheral blood mononuclear cells (PBMCs) of breast cancer patients in allogeneic mixed lymphocyte-tumor cell cultures (MLTCs), it was possible to isolate HLA-A*02-restricted cytotoxic T cells (CTLs). Furthermore, a genetically modified KS variant expressing influenza A matrix protein serving as a surrogate tumor-associated antigen (TAA) was able to stimulate flu peptide-specific T cells alongside the induction of alloresponses in MLTCs. KS breast cancer cells were demonstrated to express already known TAAs such as CEA, MUC-1, MAGE-1, MAGE-2, and MAGE-3. To further improve antigenicity, HER-2/neu was added to this panel as a marker antigen known to elicit HLA-A*02-restricted CTLs in patients with breast cancer. Thus, the antigen-processing and antigen-presentation capacity of KS cells was further demonstrated by the stimulation of HER-2/neu-specific CD8+ T cells in PBMCs of breast cancer patients in vitro. These results gave a good rationale for a phase I/II trial, where the CD80+ HER-2/neu-overexpressing KS variant is actually used as a cellular vaccine in patients with metastatic breast cancer. As a proof of principle, we present data from two patients where a significant increase of interferon-gamma (IFN-gamma) release was detected when postvaccination PBMCs were stimulated by allogeneic vaccine cells as well as by HLA-A*02-restricted HER-2/neu epitopes. In whole cell vaccine trials, monitoring is particularly challenging because of strong alloresponses and limited knowledge of TAAs. In this study, a panel of HER-2/neu epitopes, together with the quantitative real time (qRT)-PCR method to analyze vaccine-induced cytokines secreted by T cells, proved to be highly sensitive and feasible to perform an "immunological staging" following vaccination.

Efficient carcinoma cell killing by activated polymorphonuclear neutrophils targeted with an Ep-CAMxCD64 (HEA125x197) bispecific antibody.

Bispecific antibodies (bsAb) have attracted much attention over the past several years as a mean to improve immunotherapy of cancer. Due to their dual specificity, bsAb are able to redirect effector cells against tumor targets. In this study, the development and preclinical testing of a new quadroma-derived bsAb, HEA125x197, recognizing the tumor-associated Ep-CAM antigen and the high affinity Fc receptor for IgG, CD64, is reported. Using granulocyte-colony stimulating factor (G-CSF) and interferon-gamma (IFN-gamma)-stimulated polymorphonuclear neutrophils to induce CD64 expression, bsAb HEA125 x 197 elicited strong cytotoxic activity towards allogeneic and autologous ovarian carcinoma cells. The cytolytic efficiency of this antibody was comparable to that of a previously described bsAb, HEA125 x OKT3, targeting preactivated T lymphocytes against Ep-CAM-carrying tumor cells. Based on the pan-carcinoma specificity and the stable expression of Ep-CAM, bsAb HEA125x197 may broaden the spectrum of bispecific reagents for the treatment of epithelial malignancies.

Intraperitoneal bispecific antibody (HEA125xOKT3) therapy inhibits malignant ascites production in advanced ovarian carcinoma.

Bispecific antibody HEA125 x OKT3 was shown to redirect T lymphocytes toward carcinoma cells and to induce tumor cell lysis in vitro. Preclinical studies have demonstrated that tumor-associated lymphocytes (TAL) derived from malignant ascites can be used as effector cells with a high efficacy and without prior stimulation. These data provided the rationale for a clinical trial to investigate whether bsAb HEA125 x OKT3 is also able to induce tumor cell lysis in vivo and can be used for local treatment of malignant ascites arising from ovarian carcinoma. Ten ovarian carcinoma patients presenting with malignant ascites resistant to chemotherapy were enrolled in the study. They received weekly intraperitoneal injections of 1 mg bsAb diluted in 500 ml NaCl solution to allow homogeneous antibody distribution within the peritoneal cavity. All patients responded clinically well to the therapy. Eight patients experienced a complete and 2 patients a partial reduction of ascites production. A decrease or stabilization of tumor marker CA125 was detected in the sera of 8 patients. Only WHO Grade I and II toxicity was observed including mild fever, chills and allergic eczema. Thus, intraperitoneal application of bsAb HEA125 x OKT3 appears to be an easy and cost effective palliative treatment for ovarian carcinoma with recurrent ascites that leads to a substantially increased quality of life for the patients. During therapy TNF-alpha concentrations raised markedly in the ascites fluid whereas VEGF and sFLT-1 ascites levels declined. This indicates that not only T cell-mediated tumor cell lysis but also changes in vascular permeability due to downregulation of VEGF and its receptors might be responsible for the beneficial therapeutic effect.