Donald L. Kreutzer

In situ detection of tissue factor in vascular endothelial cells: correlation with the malignant phenotype of human breast disease.

Expression of tissue factor (TF) in the endothelium has been observed only rarely in human disease and has been thought to be elaborated on the surface of vascular endothelial cells (VECs) in vitro as an artifact of tissue culture. Using monoclonal antibodies and a novel probe for functional TF, we have localized TF to the VECs (and tumor cells) within the tumors of seven patients with invasive breast cancer but not in the VECs (or tumor cells) of benign tumors from ten patients with fibrocystic disease of the breast. The potent procoagulant TF was shown to be a marker of the initiation of angiogenesis in human breast cancer. Further evidence that the TF was the demonstration of a similar distribution of cross–linked fibrin only in the VECs of the malignant tumors. We interpret these data as further support for the concept that tumor cells can activate nearby VECs and regulate blood vessel growth in vivo. Large clinicalpathologic studies will be necessary to determine whether TF is a useful marker for the “switch to the angiogenic phenotype” in human breast disease and/or correlates with the thromboembolic complications of breast cancer.

Vascular Endothelial Growth Factor (VEGF) Expression in Human Prostate Cancer: In Situ and in Vitro Expression of VEGF by Human Prostate Cancer Cells

PURPOSE A growing body of literature supports the role of angiogenesis in the development and spread of a variety of human cancers including prostate cancer (Pca). Angiogenesis is controlled by chemical signals known as angiogenic factors (AF) however, little is known about angiogenesis factors in prostate cancer. We evaluated the in situ and in vitro expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, in archival prostate cancer specimens and prostate cancer cell cultures during unstimulated and cytokine stimulated conditions. METHODS Ex-vivo studies involved immunohistochemical analysis for VEGF expression and distribution in 25 archival specimens including, prostate cancer, benign prostatic hyperplasia (BPH) and normal prostate tissue. In-vitro studies utilized prostate cancer cells (DU-145) grown in culture and stimulated with cytokines thought to induce VEGF (i.e. IL-1 alpha, IL-1 beta, TNF-alpha and TNF-beta). Cell culture supernatants were analyzed by ELISA for VEGF levels. RESULTS Immunohistochemical studies demonstrated that in 20 of 25 specimens prostate cancers cells stained positively for VEGF. BPH and normal prostate cells displayed little staining for VEGF. DU-145 prostate cancer cells produced low levels of VEGF in unstimulated conditions. Induction of DU-145 cells with cytokines resulted in differential stimulation whereby TNF was a potent inducer of VEGF, and IL-1 produced lesser but statistically significant increases in VEGF expression. CONCLUSIONS Our immunohistochemical results indicate that significant levels of VEGF are present in prostate cancer, but not in BPH or normal prostate cells in-vivo. In-vitro studies suggest that differential regulation of angiogenesis factor expression by IL-1 and TNF occurs in prostate cancer. Identifying the angiogenesis factors involved in prostate cancer growth and understanding their regulation will lead to the development of anti-angiogenic strategies useful for diagnostic studies and therapeutic interventions.

Angiogenesis and prostate cancer : In vivo and in vitro expression of angiogenesis factors by prostate cancer cells

OBJECTIVES Recently, it was confirmed that angiogenesis is important in the development and spread of a variety of human cancers, including prostate cancer (PCa). Tumor neovascularization is thought to be controlled by chemical signals, known as angiogenic factors (AF). To date, little is known regarding the existence and role of AF in PCa. We previously reported on vascular endothelial growth factor (VEGF) in PCa. Currently, we compare VEGF expression with that of interleukin-8 (IL-8), another putative regulator of angiogenesis. We evaluated the expression of these two important AF in PCa and explored the role of inflammatory cytokines IL-1 and tumor necrosis factor (TNF) in their regulation. METHODS Ex vivo studies involved previously reported immunohistochemical analysis for VEGF and recent evaluation of IL-8 expression and distribution in archival tissue samples of PCa, benign prostatic hyperplasia (BPH), and normal prostate tissue. In vitro studies used PCa cells (DU-145) grown in culture and stimulated with cytokines thought to induce VEGF and IL-8 (ie, IL-1 alpha, IL-1 beta, TNF-alpha, and TNF-beta). After 24 hours, with or without cytokines, cell culture supernatants were analyzed by enzyme-linked immunosorbent assay or radioimmunoassay for VEGF or IL-8 levels. RESULTS Immunohistochemical studies of prostate tissue showed that PCa cells stained positively for VEGF and IL-8. Benign prostatic hyperplasia and normal prostate cells displayed little staining for either AF. Low levels of VEGF and IL-8 were produced by unstimulated DU-145 cells. Induction of DU-145 cells with cytokines resulted in differential stimulation whereby TNF was the predominant inducer of VEGF, whereas IL-1 was the predominant inducer of IL-8. CONCLUSIONS Our results indicate that significant levels of VEGF and IL-8 are present in PCa, but not BPH or normal prostate cells in vivo. In vitro studies suggest that differential regulation of AF expression occurs in PCa. Because IL-1 and TNF are present in the PCa tumor microenvironment, it is likely that differential regulation of AF also occurs in human PCa and contributes to differential tumor growth and metastasis.

Expression of vascular endothelial growth factor receptors in human prostate cancer

OBJECTIVES We recently reported the expression and cytokine regulation of vascular endothelial growth factor (VEGF) in human prostate cancer (PCa). VEGF exerts its angiogenic and pro-tumorigenic properties by way of two high affinity receptors, fms-like tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1). We hypothesized that these receptors are expressed and control VEGF functions in the PCa microenvironment. Herein, we evaluate the expression of these receptors in ex vivo PCa tissue, benign prostatic hypertrophy (BPH) tissue, and cultured PCa cell lines. METHODS Ex vivo PCa specimens were obtained from patients undergoing radical retropubic prostatectomy. Specimens were selected to contain both PCa and BPH tissue (n = 15). Immunohistochemical analysis using antihuman FLT-1 and FLK-1 was performed and specimens were analyzed to characterize the expression and distribution of both receptors. Immunocytochemical analysis for FLT-1 and FLK-1 was also performed on cultured PCa cell lines (DU-145 and LNCaP). RESULTS PCa cells expressed the VEGF receptor FLT-1 in 100% of specimens evaluated. Expression of FLK-1 was variable and related to tumor grade; high-grade tumors displayed little or no FLK-1 expression. Vascular endothelial cells (VECs) within areas of PCa consistently expressed both FLT-1 and FLK-1 receptors. FLT-1 and FLK-1 were both expressed in BPH tissue. FLT-1 was expressed in the glandular epithelial cells in BPH, but in most cases FLK-1 was localized specifically to the basal cell layer of hypertrophic glands. FLT-1, but not FLK-1, was expressed by the DU-145 and LNCaP cell lines. CONCLUSIONS Although they are differentially expressed, both FLT-1 and FLK-1 are present in PCa and BPH. Expression of receptors on VECs of tumor vessels supports the well-established role of VEGF in paracrine stimulation of VECs in the tumor microenvironment. The expression of FLT-1 and FLK-1 on tumor cells themselves suggests a potential autocrine function for VEGF (such as regulating tumor cell proliferation). These findings imply that a novel dual role may exist for VEGF, such that it is involved in tumor cell activation (autocrine), in addition to paracrine actions whereby it regulates endothelial cell functions and subsequent neovascular development.

Dexamethasone/PLGA microspheres for continuous delivery of an anti-inflammatory drug for implantable medical devices

The purpose of this research was to develop polylactic-co-glycolic acid (PLGA) microspheres for continuous delivery of dexamethasone for over a 1-month period, in an effort to suppress the acute and chronic inflammatory reactions to implants such as biosensors, which interfere with their functionality. The microspheres were prepared using an oil-in-water emulsion technique. The oil phase was composed of 9:1 dichloromethane to methanol with dissolved PLGA and dexamethasone. Some microspheres were predegraded for 1 or 2 weeks. Ten percent of polyethylene glycol was added to the oil phase in alternative formulations to delay drug release. The in vitro release studies were performed in a constant temperature (37 C) warm room, in phosphate-buffered saline at sink conditions. Drug loading and release rates were determined by HPLC-UV analysis. The standard microsphere systems did not provide the desired release profile since, following an initial burst release, a delay of 2 weeks occurred prior to continuous drug release. Predegraded microspheres started to release dexamethasone immediately but the rate of release decreased after only 2 weeks. A mixed standard and predegraded microsphere system was used to avoid this delay and to provide continuous release of dexamethasone for 1 month.

Characterization of cytokines present in middle ear effusions

Retention of inflammatory mediators and cells in the middle ear cleft during chronic otitis media with effusion (COME), results in ongoing inflammation with the potential for pathologic changes and hearing loss. Cytokines are glycoproteins produced by macrophages and other cells. Activities of cytokines include fever production, osteoclast, fibroblast, phagocyte and cytotoxic cell activation, regulation of antibody formation, and inhibition of cartilage, bone and endothelial cell growth. Using enzyme-linked immunospecific assays we measured levels of six cytokines in middle ear effusions (MEE) from children with COME. Significant levels of four cytokines: interleukin-1-beta (greater than 50 pg/ml), interleukin-2 (greater than 300 pg/ml), tumor necrosis factor-alpha (greater than 40 pg/ml), and gamma-interferon (greater than 6.25 pg/ml) were found in 51%, 54%, 63%, and 19% of MEE, respectively. In contrast, levels of a fifth cytokine, granulocyte-macrophage colony-stimulating factor, and a sixth cytokine, interleukin-4, were undetectable. Age was observed to have a significant effect on the levels of specific cytokines. Interleukin-1 (IL-1) correlated inversely (P less than .02) with age such that the younger the child, the higher the level of IL-1 in MEE. Tumor necrosis factor-alpha (TNF) correlated directly (P less than .005) with age such that the older the child, the higher the level of TNF in MEE. Children undergoing tympanostomy on multiple occasions had average MEE TNF levels (234.2 +/- 109.1 pg/mg total protein) that were nearly 14 times higher (P less than .005) than those from children undergoing their first tympanostomy (16.9 +/- 3.0 pg/mg total protein). Thus IL-1 correlated with the early stages of COME, while TNF correlated with persistence of disease. The presence of these cytokines in MEE may be responsible for the mucosal damage, bone erosion, fibrosis, and resulting hearing loss seen in some cases of COME.

C5 chemotactic fragment induces leukocyte production of tissue factor activity: a link between complement and coagulation.

Complement-activated human plasma causes generation of tissue factor in human leukocytes. This phenomenon appears to be related to the fifth component of complement (C5) as demonstrated by the use of C5 deficient-plasma and suppression of activity with antibody to C5. Isolation of the chemotactic factor from activated serum or trypsinization of purified C5 reproduces the phenomenon. These data provide evidence for a direct link between complement products and activation of the coagulation system. Because chemotactic peptides from C5 can be generated by a variety of enzymes, our findings suggest a relationship between complement, coagulation, and inflammation.

Relationship of functional and antigenic interleukin 6 to disease activity in inflammatory bowel disease

BACKGROUND Intestinal and peripheral blood mononuclear cell interleukin 6 (IL-6) production in inflammatory bowel disease might present an increased quantity of IL-6 into the systemic circulation. The aim of the present study was to examine the relationship of circulatory IL-6 to the clinical and laboratory expression of inflammatory bowel disease in children. METHODS Sera were obtained from 26 children with ulcerative colitis, 49 with Crohns disease, and 29 control patients. Serum functional IL-6 was measured by a bioassay and antigenic IL-6 by enzyme linked immunosorbent assay. RESULTS Functional and antigenic serum IL-6 levels were higher in Crohns disease than in ulcerative colitis or controls (P < 0.0001) and higher in ulcerative colitis than controls (P < 0.04). In Crohns disease affecting the colon, functional and antigenic serum IL-6 levels were greater than in disease limited to the small bowel (P < 0.002). Increasing disease severity was reflected by increasing antigenic but not functional IL-6 levels in both Crohns disease (P < 0.001) and ulcerative colitis (P < 0.02). Serum antigenic IL-6 levels were related to acute phase reactants in both diseases (P < 0.001) whereas functional levels were not. CONCLUSIONS Our results underscore the importance of using both functional and antigenic methodologies in examining the relationship of circulating cytokines to the clinical manifestations of inflammatory bowel disease.

Nerve growth factor and chronic prostatitis/chronic pelvic pain syndrome.

OBJECTIVES To investigate whether the pain experienced by patients with chronic prostatitis/chronic pelvic pain syndrome (CPPS) may be related to the expression of nerve growth factor (NGF), induced by inflammation and tissue injury experienced as a result of chronic inflammation. CPPS is a disease of unknown pathogenesis. METHODS We measured the levels of NGF and the pro-inflammatory cytokine interleukin (IL)-6 and compared these with the levels of IL-8, interferon-gamma, IL-2, and IL-10 in the seminal plasma of 31 patients with CPPS and 14 controls using enzyme-linked immunosorbent assay technology. Results were correlated with health-related quality of life as measured by the multidimensional pain inventory, the McGill pain questionnaire, and the International Prostate Symptom Score. RESULTS The cytokines analyzed were detectable in the seminal plasma from the patients with CPPS and controls. NGF correlated directly with pain severity (P <0.01) and IL-10 levels (P <0.04), and IL-6 correlated inversely with pain severity (P <0.03). CONCLUSIONS These results suggest that NGF and cytokines that regulate inflammation (IL-6 and IL-10) may play a role in the pain symptoms experienced by patients with CPPS.

Alterations in bone metabolism in children with inflammatory bowel disease: an in vitro study.

BACKGROUND In patients with inflammatory bowel disease (IBD), accelerated bone loss and osteopenia have been found. Potential etiologies of these bone abnormalities have included malnutrition, poor calcium intake or absorption, and the use of corticosteroids. Recent studies have suggested that circulating pro-inflammatory cytokines, which are produced in inflamed bowel, can have a profound effect on bone metabolism, particularly bone resorption. Our aim was to characterize the effects of serum from subjects with IBD on bone metabolism in an in vitro bone culture system. METHODS Organ cultures of fetal rat parietal bones were treated with sera from 9 subjects with Crohns disease, 7 with ulcerative colitis, and 10 controls with functional bowel disease (age range of all subjects 7-16 years). Patients were also classified by disease activity, serum albumin level, erythrocyte sedimentation rate (ESR), and serum interleukin (IL) 6 levels. The effects of sera on bone formation and resorption were quantified. RESULTS Compared with control serum, serum from patients with Crohns disease significantly decreased bone dry weight (p < 0.01) and calcium content (p < 0.001) during 96 h of culture, while serum from ulcerative colitis patients had no effect. While no difference in collagen synthesis was noted between any of the three experimental groups, noncollagen protein synthesis was lower in the ulcerative colitis group than in the control group or those with Crohns disease (p < 0.05). DNA content was similar in all groups. There was no significant effect of serum from any experimental group on bone resorption. There was no demonstrable relationship between clinical disease activity, ESR, or serum IL-6 levels and measures of bone metabolism. Histologic evaluation of cultured bone showed marked differences between control subjects and Crohns disease patients, with the latter being characterized by disorganization of mineral and osteoid and morphologically abnormal osteoblasts. CONCLUSIONS Serum from children with IBD has a significantly different effect than control serum on an in vitro model of bone metabolism. Our data suggest that circulating factors may affect osteoblasts and bone formation, leading to bone loss. Further work will be required to further characterize the nature of these factors and develop treatment strategies to minimize their effects.