Distinct ERG rearrangement prevalence in prostate cancer: higher frequency in young age and in low PSA prostate cancer.

BACKGROUND: The TMPRSS2-ERG gene fusion resulting in ERG overexpression has been found in around 50% of prostate cancers (PCa) and is a very early event in tumorigenesis. Most studies have reported on selected surgical cohorts with inconsistent results. We hypothesized that ERG gene rearrangements impact tumor development and investigated the frequency of ERG overexpression in the context of clinicopathological tumor characteristics. METHODS: ERG overexpression (ERG+ or ERG-) was determined by immunohistochemistry (IHC) in 1039 radical prostatectomy (RP) tumors and association with PSA, D'Amico risk score, histopathology, biochemical recurrence, body mass index and age of PCa cases was analyzed. RESULTS: ERG+ was associated with younger age at diagnosis (P<0.0001), lower serum PSA (P=0.002) and lower prostate volume (PV) (P=0.001). It was most frequent in the youngest age quartile (≤55 years, 63.9% ERG+) and decreased constantly with increasing age to 40.8% in the oldest age quartile (≥67 years, P<0.0001). In the PSA range <4 ng ml(-1) the frequency of ERG positivity was 60.2% compared with 47.5 and 49.1% in the PSA ranges 4-10 and ≥10 ng ml(-1), respectively. In the first age quartile, ERG+ patients had lower median serum PSA and fPSA% and smaller PV. In the highest age quartile tumor volume (TV) was increased. Similar differences were observed in the low PSA range. Multivariate analysis identified the first age quartile as a predictor for ERG status (odds ratios (OR) 2.05, P=0.007). No association was found with the D'Amico progression risk score and with biochemical tumor recurrence. CONCLUSIONS: ERG+ tumors manifest clinically at lower PSA levels and their prevalence is age dependent. This suggests acceleration of tumor development by ERG overexpression that results in earlier tumor detection in young patients. Long-term results are warranted to determine the impact of ERG overexpression on disease outcome.

Prostate cancer cells (LNCaP) generated after long-term interleukin 6 (IL-6) treatment express IL-6 and acquire an IL-6 partially resistant phenotype.

PURPOSE: The levels of interleukin-6 (IL-6) are frequently elevated in sera from patients with advanced prostate carcinoma. Our main objective was to investigate changes in responsiveness to IL-6 and/or androgen that occur in LNCaP cells after long-term treatment with IL-6. This in vitro model could be of clinical relevance because of its similarity with late-stage prostate carcinoma. EXPERIMENTAL DESIGN: LNCaP human prostate cancer cells were treated with IL-6 at a concentration of 5 ng/ml. After 20 passages, the new subline LNCaP-IL-6+ has been established. Passages 20-40 are referred to as low passages (LP) and passages 41-73 as high passages (HP). LNCaP cells passaged at the same time in the absence of IL-6 were used as controls (LNCaP-IL-6-). Cells were counted after treatment with either IL-6 or the synthetic androgen methyltrienolone (R1881), and cell cycle analysis was performed. Binding of IL-6 or R1881 was assessed by radioligand binding assays. Reporter gene activity was measured by chloramphenicol acetyltransferase assay. Prostate-specific antigen in LNCaP-IL-6+ supernatants was measured by an enzyme immunoassay. Expression of IL-6 mRNA and protein was assessed by reverse transcription-PCR and ELISA, respectively. RESULTS: The basal proliferation rate in HP LNCaP-IL-6+ cells was higher than that in LNCaP-IL-6- cells. IL-6 inhibited proliferation of LNCaP-IL-6- cells but not that of either LP or HP of LNCaP-IL-6+ cells. This inability to elicit a growth-inhibitory response was associated with lack of effect on cell cycle distribution in the LNCaP-IL-6+ subline. In parallel, IL-6 binding decreased gradually during long-term IL-6 treatment and, in HP, reached only 33% of the levels measured in controls. Binding of radiolabeled androgen increased 2-fold in HP LNCaP-IL-6+ cells. Reporter gene assays revealed that R1881, at nanomolar concentrations, was a more potent androgen receptor activator in LNCaP-IL-6+ than in LNCaP-IL-6- cells. However, androgen- and IL-6-induced prostate-specific antigen secretion decreased in long-term IL-6-treated cells. IL-6 cDNA fragments were detected by reverse transcription-PCR in HP LNCaP-IL-6+ cells but not in controls or LP. IL-6 protein was first detected in passage 36 of LNCaP-IL-6+ cells, and it increased in HP. CONCLUSIONS: Long-term treatment of LNCaP human prostate cancer cells with IL-6 leads to abolishment of inhibitory growth response. In contrast to control cells, the LNCaP-IL-6+ subline expresses IL-6 mRNA and protein.

Expression and immunogenicity of oncofetal antigen-immature laminin receptor in human renal cell carcinoma.

PURPOSE: The 32 to 44 kDa. oncofetal antigen-immature laminin receptor (OFA-iLR) is a multifunctional protein expressed by various tumors, including breast, lung, ovary and prostate carcinoma as well as lymphoma. OFA-iLR has been implicated in tumor invasiveness, metastasis and growth. Interferon-gamma producing effector T cells and interleukin (IL)-10 producing suppressor T cells specific for OFA-iLR have been described. MATERIALS AND METHODS: The 43515 IgG2a anti-OFA-iLR monoclonal antibody was used to detect OFA-iLR expression in human renal cell carcinoma tissue by flow cytometry and immunoblotting. Spontaneous or therapy induced immune responses against OFA-iLR were determined in patients with metastatic renal cell carcinoma. Proliferative and cytokine (interferon-gamma and IL-10) responses of peripheral blood mononuclear cells from patients with renal cell carcinoma against recombinant OFA-iLR were assessed. RESULTS: Using flow cytometry OFA-iLR was detected in all 13 tumors tested. Immunoblotting revealed differences in OFA-iLR expression in renal cell carcinoma and normal kidney tissue. OFA-iLR specific proliferative and cytokine responses of mononuclear cells were detected in all 6 patients tested. Importantly evidence was also obtained that treating metastatic renal cell carcinoma with tumor lysate pulsed dendritic cells would enhance OFA-iLR specific immunity. CONCLUSIONS: This study demonstrates that OFA-iLR is an immunogenic tumor associated antigen in human renal cell carcinoma. OFA-iLR specific effector T cells producing interferon-gamma may have a role in the control of tumor growth, whereas suppressor T cells producing IL-10 may promote tumor tolerance and, thus, tumor progression.

The disabled dendritic cell.

Dendritic cells are important antigen-presenting cells of the immune system that induce and modulate immune responses. They interact with T and B lymphocytes as well as with natural killer cells to promote activation and differentiation of these cells. Dendritic cells generated in vitro from monocytes by use of the cytokines GM-CSF and IL-4 are increasingly used clinically to enhance antitumor immunity in cancer patients. However, recent studies revealed that the functional repertoire of monocyte-derived dendritic cells may be incomplete. Important functions of monocyte-derived dendritic cells such as migration or the ability to induce natural killer cell activation or type 2 T helper cell differentiation appear to be impaired. We propose that all these deficiencies relate to a single biochemical deficiency of monocyte-derived dendritic cells. IL-4, which is used to generate monocyte-derived dendritic cells, suppresses phospholipase A2, the enzyme that liberates arachidonic acid from membrane phospholipids and contributes to the synthesis of platelet-activating factor. Monocyte-derived dendritic cells must therefore fail to generate platelet-activating factor as well as arachidonic acid derivatives such as prostaglandins, leukotrienes, and lipoxins, collectively referred to as eicosanoids. Since eicosanoids and platelet-activating factor are known to play an important role in processes such as leukocyte migration, natural killer cell activation, and type 2 T helper cell differentiation, the deficiency in eicosanoid and platelet-activating factor biosynthesis may be responsible for the observed handicaps of monocyte-derived dendritic cells.

A clinically approved oral vaccine against pneumotropic bacteria induces the terminal maturation of CD83+ immunostimulatory dendritic cells.

Dendritic cells (DCs) are important antigen-presenting cells of the immune system that have attracted interest as cellular adjuvants to induce immunity in clinical settings. We have investigated the effects of Broncho-Vaxom, an oral vaccine composed of lysates from eight pneumotropic bacteria, on human monocyte-derived dendritic cells (moDCs). Broncho-Vaxom induced the terminal maturation of CD83+ moDCs. MoDCs stimulated with Broncho-Vaxom displayed a phenotype of activated DCs with high levels of major histocompatibility complex (MHC) molecules and increased levels of adhesion and co-stimulatory molecules. In addition, moDCs activated with Broncho-Vaxom exhibited enhanced T cell-stimulatory capacity in the allogeneic mixed leukocyte reaction. Broncho-Vaxom at 100 microg/ml was as potent as TNF-alpha at 1000 U/ml in activating human moDCs. Neither LPS-like activity nor bacterial DNA was found to be responsible for the maturation-inducing activity of Broncho-Vaxom, suggesting that Broncho-Vaxom contains other bacterial factors that are capable of inducing the terminal maturation of moDCs. In DC-based immunotherapy, Broncho-Vaxom could be used as a stimulus of DC maturation, which meets the standards of good manufacturing practice (GMP). In addition, vaccination with Broncho-Vaxom-loaded moDCs may be an attractive treatment option in preventing recurrent airway infection in predisposed individuals.

Effects of alpha1-adrenoceptor antagonists on cultured prostatic smooth muscle cells.

BACKGROUND: alpha1-adrenoceptor (alpha1-AR) antagonists, used to relieve the lower tract urinary symptoms (LUTS) in benign prostate hyperplasia (BPH) patients, are thought to act in inhibiting the contraction of stromal smooth muscle. An attempt was made using new technology to visualize and quantify the effect of alpha1-AR antagonists in a cell culture model of prostatic smooth muscle cells (SMC). METHODS: Prostatic smooth muscle cells cultured from human prostate tissue were treated with alpha1-AR agonists and antagonists. The effects on cell growth, cell contraction, differentiation status, and apoptosis were determined by means of an MTT cell viability assay, time-lapse video microscopy, RT-PCR analysis, and FACS analysis of annexin V/propidium iodide-stained cells, respectively. RESULTS: Prostatic smooth muscle cells derived from prostate tissue expressed SMC-specific markers. They showed spontaneous contractions, and phenylephrine increased the percentage of contracting cells by 3-fold. alpha1-AR antagonists inhibited spontaneous as well as phenylephrine-induced contractions. Long-term treatment with doxazosin induced differentiation tended towards a contractile phenotype, as indicated by an increase of the ratio of smooth muscle heavy chain myosin subtypes SM2/SM1. There was, however, no effect on cell growth. High concentrations of antagonist (100 microM) induced apoptosis in about 80% of the treated SMC. This effect was not cell-type-specific and was also seen in skin fibroblasts and immortalized prostate epithelial cells. CONCLUSION: In an easy-to-handle cell culture model of prostatic smooth muscle cells, the effects of alpha1-AR antagonists on cell contraction, growth, and differentiation can be investigated. The results indicate that in addition to inhibition of cell contraction, alpha1-AR antagonists have the potential to induce apoptosis.

Inhibition of LncaP prostate cancer cells by means of androgen receptor antisense oligonucleotides.

Currently available methods for treatment of human prostatic carcinoma aim to inactivate the androgen receptor (AR) by androgen deprivation or blockade with anti-androgens. Failure of endocrine therapy and tumor progression is characterized by androgen-independent growth despite high levels of AR expression in metastatic disease. We inhibited AR expression in LNCaP prostate tumor cells by using antisense AR oligodeoxynucleotides (ODNs) and explored whether antisense AR treatment would be conceivable as a therapy for advanced prostate cancer. Among the various AR antisense ODNs tested, a 15-base ODN targeting the CAG repeats encoding the poly-glutamine region of the AR (as750/15) was found to be most effective. Treatment of LNCaP cells with as750/15 reduced AR expression to approximately 2% within 24 hours compared with mock-treated controls. AR down-regulation resulted in significant cell growth inhibition, strongly reduced secretion of the androgen-regulated prostate-specific antigen, reduction of epidermal growth factor receptor expression, and an increase in apoptotic cells. Mis-sense and mismatched control ODNs had no or only slight effects. Antisense inhibition was also very efficient in LNCaP-abl cells, a subline established after long-term androgen ablation of LNCaP cells, resulting in inhibition of AR expression and cell proliferation that was similar to that seen for parental LNCaP cells. This study shows that inhibition of AR expression by antisense AR ODNs may be a promising new approach for treatment of advanced human prostate cancer.

Cellular and humoral immune responses in patients with metastatic renal cell carcinoma after vaccination with antigen pulsed dendritic cells.

PURPOSE: Dendritic cells are the most potent stimulators of immune responses including antitumor responses. We performed a pilot study of cultured antigen loaded dendritic cells in patients with metastatic renal cell carcinoma. MATERIALS AND METHODS: Dendritic cells were obtained by culturing plastic adherent mononuclear cells from peripheral blood for 5 days in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. Day 5 dendritic cells were loaded with cell lysate from cultured autologous tumor cells and with the immunogenic protein keyhole-limpet hemocyanin (KLH) which serves as a helper antigen and as a tracer molecule. During the antigen pulse dendritic cells were activated with a combination of tumor necrosis factor-alpha and prostaglandin E2. Dendritic cells were administered by 3 intravenous infusions at monthly intervals. Cellular and humoral immune responses to KLH and cell lysate were measured in vitro before and after the vaccinations. RESULTS: Preparation of 12 dendritic cell vaccines from patients with advanced renal cell carcinoma was successful. Treatment with fully activated CD83+ dendritic cells was well tolerated with moderate fever as the only side effect. Potent immunological responses to KLH and, most importantly, against cell lysate could be measured in vitro after the vaccinations. CONCLUSIONS: Our data demonstrate that a dendritic cell based vaccine can induce antigen specific immunity in patients with metastatic renal cell carcinoma. Dendritic cell based immunotherapy represents a feasible, well tolerated and promising new approach for the treatment of advanced renal cell carcinoma.

Mature dendritic cells induce T-helper type-1-dominant immune responses in patients with metastatic renal cell carcinoma.

We performed a pilot study on a dendritic cell (DC)-based vaccine in 4 patients with advanced renal cell carcinoma. The vaccine consisted of cultured blood DCs loaded with autologous tumor cell lysate plus keyhole limpet hemocyanin (KLH) and matured with a combination of tumor necrosis factor alpha and prostaglandin E(2). We describe the immune response against KLH induced by DC-based immunization in a patient undergoing an objective partial response and compare it with the responses observed in patients with either stable or progressive disease. The patient with the clinical response developed strong delayed-type hypersensitivity (DTH) against KLH after a single vaccination with antigen-loaded DCs, whereas the other patients failed to develop DTH reactivity even after repeated vaccinations. Antigenic stimulation of mononuclear cells (MNCs) induced proliferation and IFN-gamma but not IL-4 production as well as expression of the chemokine receptor CXCR3 consistent with a T-helper (Th) type-1 bias. Exogenous IL-12 enhanced and exogenous IL-4 diminished IFN-gamma production. In the 2 patients with stable disease two or more vaccinations were required to induce maximal MNC responses. In the patient with progressive disease MNC responses were hardly detectable. Anti-KLH antibodies appeared with different kinetics but could be detected in the serum of all patients. Isotype analysis revealed the presence of IgM, IgG(1), IgG(2) and IgG(3) as well as IgA and complete absence of IgE. The patient with progressive disease also developed IgG(4) antibodies indicative of a deviation towards Th2. Cultured blood DCs can be a potent vaccine for the antigen-specific immunization of patients with advanced kidney cancer. KLH serves as a tracer molecule which allows determination of the magnitude, kinetics and Th bias of the cellular and humoral immune response induced by DC-based immunization. The data also suggest that Th type-1-dominant immune responses involving DTH reaction are required for the induction of tumor regression.

Nordihydroguaiaretic acid blocks secretory and endocytic pathways in human dendritic cells.

Nordihydroguaiaretic acid (NDGA), an antioxidant and inhibitor of the lipoxygenase arm of the arachidonic acid metabolism, was recently demonstrated to inhibit transport of secretory proteins to the Golgi complex. We have investigated the effects of NDGA on the secretory and endocytic activity of cultured human blood dendritic cells (DC). Treatment with NDGA strongly diminished cytokine secretion by DC. Moreover, NDGA reduced in a dose- and time-dependent fashion fluid phase as well as receptor-mediated endocytosis in DC. Zileuton and MK-886, specific inhibitors of 5-lipoxygenase and 5-lipoxygenase-activating protein, respectively, had no effect. Likewise, N-acetyl-L-cysteine, a thiol antioxidant precursor of glutathione, did not affect DC function. Finally, serum remarkably protected the cells from the inhibitory effects of NDGA. Our data demonstrate that NDGA not only disrupts vesicular transport along the secretory route but is also a potent inhibitor of the endocytic pathways in human DC and that NDGA has inhibitory properties different from those described.

Interleukin 1beta mediates the modulatory effects of monocytes on LNCaP human prostate cancer cells.

Proliferative and secretory responses in androgen-sensitive prostate cancer LNCaP cells are regulated by steroid and peptide hormones and by differentiation-promoting substances. In the present study, we evaluated whether peripheral blood monocytes that exhibit anti-tumour activity in haematopoietic and solid tumours influence growth and secretion in the LNCaP cell line. For this purpose, LNCaP cells were incubated with monocyte-conditioned medium (MCM), and proliferation as well as expression of androgen receptor (AR) and secretion of prostate-specific antigen (PSA) were assessed. Conditioned medium from monocytes reduced proliferation in a dose-dependent manner. Incubation with 40% MCM caused a 50% reduction in cell proliferation. AR protein decreased by 70% and PSA levels in supernatants from LNCaP cells were reduced by approximately 80% following treatment with MCM. We focused on the contribution of two major products of activated monocytes, prostaglandin E2 and interleukin 1beta (IL-1beta), to the MCM modulatory action. LNCaP cells treated with prostaglandin E2 showed neither a reduction in proliferation nor a down-regulation of AR and PSA levels. The effects of MCM on cellular proliferation, AR protein and PSA secretion were abolished by pretreatment of MCM with a neutralizing anti-IL-1beta antibody. In addition, recombinant IL-1beta was able to replace MCM for the inhibition of proliferation and down-regulation of AR and PSA proteins. LNCaP cells were shown to express the IL-1beta receptor type 1, which transduces IL-1beta signal. Our findings reveal that monocyte-derived IL-1beta inhibits the proliferation of androgen-responsive prostate tumour cells and reduces AR and PSA levels.

Human monocyte-derived dendritic cells produce macrophage colony-stimulating factor: enhancement of c-fms expression by interleukin-10.

Human monocyte-derived dendritic cells (DC) generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 express c-fms (CD115), the receptor for macrophage-CSF (M-CSF). Expression of c-fms on monocyte-derived DC has been interpreted as the susceptibility of these cells to M-CSF-induced macrophage development. We show here that homogeneous cultures of CD14 DC constitutively produced large amounts of M-CSF. However, presence of M-CSF neither induced macrophage development nor did it prevent terminal maturation into CD83+ DC. M-CSF production by DC was driven by GM-CSF and inhibited by the specific phosphatidylinositol 3-kinase inhibitor wortmannin. M-CSF synthesis was rapidly induced during the first 24 h of DC culture and then declined during the 5-day culture period. Replating of the cells, which was associated by a transient adherence, always induced a strong up-regulation of M-CSF synthesis. Addition of recombinant IL-10 to DC cultures enhanced c-fms expression and induced macrophage development as measured by the strong up-regulation of CD14 expression as well as by enhanced expression of the Fcgamma receptors I, II, and III (CD64, CD32, CD16). Our data demonstrate that immature monocyte-derived DC produce M-CSF which does not induce macrophage development, despite the surface expression of c-fms on DC. IL-10 appears to induce macrophage development by up-regulating c-fms and, thereby, enhancing the sensitivity of the cells to endogenously produced M-CSF.

Differential deactivation of human dendritic cells by endotoxin desensitization: role of tumor necrosis factor-alpha and prostaglandin E2.

The endotoxin (lipopolysaccharide)-induced cytokine response is followed by a state of unresponsiveness to lipopolysaccharide (LPS) referred to as LPS tolerance or endotoxin desensitization. LPS tolerance, which can be experimentally induced in vitro and in vivo, is also known to occur in septic disease. Here, we evaluated whether dendritic cells (DC), the most potent antigen-presenting cells, are also subject to this phenomenon. Single doses of LPS added at the initiation of DC culture inhibited in a dose-dependent fashion the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), and IL-12, but not the production of IL-8, in response to a second LPS challenge in day-5 DC. In addition, the LPS-induced expression of the CD83 maturation antigen was inhibited in these cells. Moreover, the endocytic activity of DC generated in the presence of LPS was dramatically reduced. DC desensitized with LPS were potent stimulators of T-cell proliferation but poor inducers of interferon-gamma (IFN-gamma) production in the allogeneic mixed leukocyte reaction. TNF-alpha and prostaglandin E2, two major products of LPS stimulation, could replace LPS for the induction of tolerance to LPS. Moreover, treatment of desensitized DC with TNF-alpha plus prostaglandin E2 fully restored CD83 expression and partially restored IL-12 production as well as the IFN-gamma-inducing activity of DC in the mixed leukocyte reaction. Our data show that human DC are highly susceptible to the induction of LPS tolerance, which seems to be a state of differential deactivation in which some functions are impaired whereas others are retained. Tolerization at the level of the professional antigen-presenting cell by inflammatory mediators may play an important role in septic disease and in the origin of cancers associated with chronic inflammation.

Activation of human dendritic cells by bacillus Calmette-Guerin.

PURPOSE: Dendritic cells are the most potent antigen presenting cells capable of initiating antitumor immune responses. We previously showed that bacillus Calmette-Guerin (BCG) stimulates cultured human dendritic cells. We extended these studies and tested the ability of cultured human dendritic cells to express interleukin IL-8 in response to BCG. We also investigated the T cell stimulatory potential of BCG treated dendritic cells in mixed leukocyte reactions. MATERIALS AND METHODS: Dendritic cells were obtained by culturing plastic adherent mononuclear cells from peripheral blood for 6 days in the presence of granulocyte-macrophage colony-stimulating factor and IL-4. Spontaneous and BCG stimulated IL-8 protein release into culture supernatants was measured by a quantitative immunoassay. IL-8 gene transcription was assessed by reverse transcription-polymerase chain reaction. Untreated and BCG exposed dendritic cells were compared as stimulators of allogeneic T cell proliferation, measured as [3H]thymidine incorporation. RESULTS: BCG stimulated IL-8 messenger ribonucleic acid expression and IL-8 protein release. IL-8 secretion occurred in a dose and time dependent fashion. BCG induced IL-8 release was further enhanced in the presence of indomethacin. BCG treated dendritic cells were much more potent T cell stimulators than untreated dendritic cells. CONCLUSIONS: These data demonstrate that BCG enhances the production of IL-8, a potent chemokine of T cells and granulocytes, as well as the T cell stimulatory potential of human dendritic cells.

Dendritic cell-based immunotherapy of renal cell carcinoma.

Dendritic cells potently stimulate antigen-specific immune responses and recent data indicate that they are also capable of eliciting antitumor immune responses. We are performing a pilot study which tests the safety and efficacy of antigen-loaded, cultured blood dendritic cells in patients with metastatic renal cell carcinoma. Dendritic cells are simultaneously pulsed with lysate from autologous tumor cells and with the immunogenic protein keyhole limpet hemocyanin. During the pulse, the cells are activated with a combination of tumor necrosis factor-alpha and prostaglandin E2. Patients receive 5-10 X 10(6) dendritic cells per intravenous infusion and up to six infusions at monthly intervals. The first results demonstrate that this treatment modality is very well tolerated and can be associated with strong immunological and clinical responses. The present article discusses the importance of dendritic cell maturation and the role of helper antigens in dendritic cell-based immunotherapy.

Basic fibroblast growth factor levels in cancer cells and in sera of patients suffering from proliferative disorders of the prostate.

BACKGROUND: Both benign and malignant growth of the prostate depend on the induction of a microvasculature. Basic fibroblast growth factor (bFGF), a potent angiogenic factor, is thought to play an important role in this process. METHODS: bFGF expression in prostatic carcinoma was assessed by ELISA, reverse transcription polymerase chain reaction, and immunohistochemistry. RESULTS: DU-145 and PC-3 tumor cells produced bFGF. Almost 80-90% of it was localized in the cytoplasm, and 10-20% was associated with extracellular matrix components. Immunohistochemical analysis of prostatic tissue sections showed that cancer cells stained more intensively as compared to putatively healthy epithelium. In prostate cancer patients, mean bFGF serum levels were significantly elevated when compared to a healthy control group (6.64 pg/ml vs. 1.28 pg/ml). Serum bFGF levels did not correlate with any other clinical marker such as PSA, tumor stage, or grade. Four out of five patients who progressed to a more advanced stage showed an increase in serum bFGF levels. CONCLUSIONS: These results suggest that increased bFGF release may be associated with a more aggressive tumor phenotype.

In vitro generation of CD83+ human blood dendritic cells for active tumor immunotherapy.

There is increasing clinical interest in dendritic cells that are capable of initiating antitumor immune responses. Dendritic cells cultured from human blood mononuclear cells using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) are competent for antigen uptake but express relatively low levels of costimulatory molecules and thus correspond to immature resident tissue dendritic cells. In this study we took advantage of the new dendritic cell-specific marker CD83, which is expressed by mature dendritic cells, to delineate the maturation of cultured human blood dendritic cells. Although dendritic cells cultured with GM-CSF and IL-4 contained transcripts for CD83 as determined by reverse transcription PCR, CD83 protein was barely detectable by flow cytometry, confirming that dendritic cells obtained with this system are immature. However, treatment of dendritic cells with tumor necrosis factor-alpha (TNF-alpha) significantly increased the levels of CD83 transcripts and induced CD83 protein expression in dendritic cells. In contrast to the initiation of dendritic cell culture, which was facilitated by high cell density (5 x 10(6) cells/mL), differentiation into CD83+ dendritic cells required a low cell concentration (0.5 x 10(6) cells/mL). At higher cell density (1 x 10(6) cells/mL), CD83 expression was suppressed and was almost completely prevented at 2 x 10(6) cells/mL. Induction of CD83 expression was accompanied by a strong upregulation of the costimulator B7-2 (CD86) on dendritic cells. While untreated CD83(-) dendritic cells efficiently internalized fluoresceinated Dextran, TNF-alpha treated CD83+ dendritic cells excluded these molecules, confirming that maturation of dendritic cells was associated with the silencing of the antigen-capturing machinery. Morphologically, CD83+ dendritic cells presented with pronounced cytoplasmic projections (veils) characteristic of mature dendritic cells. In summary, we show that cell density critically regulates dendritic cell development. Knowledge of the appropriate conditions for dendritic cell generation and maturation will be important in clinical immunotherapy settings.

Bacillus Calmette-Guérin mycobacteria stimulate human blood dendritic cells.

Bacillus Calmette-Guérin (BCG) mycobacteria have been used as adjuvant in the active immunotherapy of various human cancers. In addition, dendritic cells, which are the most potent antigen-presenting cells, have been shown to be capable of initiating anti-tumor immune responses. Here we investigated the effects of BCG on dendritic cells cultured from human blood. Addition of BCG resulted in rapid homotypic adhesion of dendritic cells. Moreover, BCG concentrations ranging from 10(4) to 10(6) bacteria/ml enhanced expression of the dendritic-cell-maturation antigen CD83 and of the T-cell co-stimulator CD86 (B7-2) in a dose-dependent manner. Concomitant with the increase of CD83 and CD86 expression, the cells lost the ability to capture soluble antigens, as determined by the exclusion of fluoresceinated Dextran molecules. Strikingly, the same dosages of BCG-bacteria stimulated TNF-alpha-gene transcription and TNF-alpha-protein release from dendritic cells in a dose-dependent fashion. BCG infection of dendritic cells in the presence of a neutralizing antibody directed against TNF-alpha inhibited CD83 expression by more than 50% indicating that the BCG-induced maturation of dendritic cells was at least partially mediated by dendritic-cell-derived TNF-alpha. The finding that BCG activates the most potent antigen-presenting cells reveals a plausible immunological mechanism of the occasionally observed anti-tumor activity of BCG.