Biomedical subjects

D L Kreutzer

Publications and source records attributed to D L Kreutzer.

At least 19 recordsLinked to original sources

PubMed2003

Use of vascular endothelial cell growth factor gene transfer to enhance implantable sensor function in vivo.

In the current study, we developed and validated a simple, rapid and safe in vivo model to test gene transfer and sensor function in vivo. Using the model, we tested the specific hypothesis that in vivo gene transfer of angiogenic factors at sites of biosensor implantation would induce neovascularization surrounding the sensor and thereby enhance biosensor function in vivo. As the in vivo site for testing of our gene transfer cell and biosensor function systems, the developing chorioallantoic membrane (CAM) of the embryo was utilized. Vascular endothelial cell growth factor (VEGF) was used as a prototype for angiogenic factor gene transfer. A helper-independent retroviral vector derived from Rous sarcoma virus (RSV), designated RCAS, was used for gene transfer of the murine VEGF (mVEGF) gene (mVEGF:RCAS) into the DF-1 chicken cell line (designated mVEGF:DF-1). Initially, the ability of VEGF:DF-1 cells to produce VEGF and RCAS viral vectors containing the mVEGF gene (mVEGF:RCAS) was validated in vitro and in vivo, as was the ability of the mVEGF:DF-1 cells to induce neovascularization in the ex ova CAM model. Using the system, we determined the ability of mVEGF:DF-1 cells to enhance acetaminophen sensor function in vivo, by inducing neovascularization at sites of sensor implantation in the ex ova CAM model. For these studies, acetaminophen sensors were placed on 8-day-old ex ova CAMs, followed by addition of media or cells (mVEGF:DF-1 cells or GFP:DF-1 cells) at the sites of biosensor implantation on the CAM. At 4 to 10 days after sensor placement, the biosensor function was determined by measuring sensor response to an intravenous injection of acetaminophen. Sensors implanted on CAMs with buffer or control cells (GFP:DF-1 cells) displayed no induced neovascularization around the sensor and had minimal/baseline sensor responses to intravenous acetaminophen injection (media, 133.33 +/- 27.64 nA; GFP:DF-1, 187.50 +/- 55.43 nA). Alternatively, the sensors implanted with mVEGF:DF-1 cells displayed massive neovascularization and equally massive sensor response to intravenous injection of acetaminophen (VEGF:DF-1, 1387.50 +/- 276.42 nA). These data clearly demonstrate that enhancing vessel density (i.e., neovascularization) around an implanted sensor dramatically enhances sensor function in vivo.

Acetaminophen↗

PubMed2003

Binding and orientation of fibronectin on polystyrene surfaces using immobilized bacterial adhesin-related peptides.

Fibronectin (FN) is known to bind to bacteria via high affinity receptors on bacterial surfaces known as adhesins. The binding of bacteria to FN is thought to have a key role in foreign device associated infections. For example, previous studies have indicated that Staphylococcus aureus adhesins bind to the 29 kDa NH(3) terminus end of FN, and thereby promote bacteria adherence to surfaces. Recently, the peptide sequences within the S. aureus adhesin molecule that are responsible for FN binding have been identified. Based on these observations, we hypothesize that functional FN can be bound and specifically oriented on polystyrene surfaces using bacterial adhesin-related (BRP-A) peptide. We further hypothesize that monoclonal antibodies that react with specific epitopes on the FN can be used to quantify both FN binding and orientation on these surfaces. Based on this hypothesis, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces using BRP-A peptide. To test this hypothesis, the binding and orientation of the FN to immobilized BRP-A was quantified using (125)I-FN, and monoclonal antibodies. (125)I-FN was used to quantitate FN binding to peptide-coated polystyrene surfaces. The orientation of bound FN was demonstrated by the use of monoclonal antibodies, which are reactive with the amine (N) or carboxyl (C) termini of the FN. The results of our studies demonstrated that when the BRP-A peptide was used to bind FN to surfaces that: 1. functional FN was bound to the peptide; 2. anti-C terminus antibodies bound to the peptide FN; and 3. only limited binding of anti-N terminus antibodies to peptide-bound FN occurred. We believe that the data that indicate an enhanced binding of anti-C antibodies reactive to anti-N antibodies are a result of the FN binding in an oriented manner with the N termini of FN bound tightly to the BRP-A on the polystyrene surface.

Adhesins, Bacterial↗

PubMed2003

Binding and orientation of fibronectin to silanated glass surfaces using immobilized bacterial adhesin-related peptides.

Previously, we have demonstrated the suitability of bacterial adhesin-related peptides, directly immobilized on polystyrene surfaces, to bind and orient fibronectin (FN). For these studies a method to bind the large protein FN in a desired orientation on a solid substratum was developed which utilizes a bacterial adhesin-related peptide (designated BRP-A), which is known to bind specifically to the NH3-terminus end of FN. Glass substrata was first coated with an amine-terminated silane, followed by streptavidin (SA), which was used as an intermediate tether to bind the biotinylated bacterial adhesin-related peptide. The BRP-A peptide, used for these studies was synthesized with a terminal biotin to assure irreversible coupling of the BRP-A to the streptavidin. The biotinylated BRP-A was next immobilized on the SA-silanated glass surfaces. 125I-FN was used to quantify the amount of FN binding to the (BRP-A):SA-silanated glass surface. Monoclonal antibodies, which react with specific epitopes at either the NH3- or -COOH-termini of FN, were used to quantify the binding and orientation of FN. The results of these studies indicated: (1) FN bound to the BRP-A:SA-silanated glass surface; and (2) the bound FN was oriented such that NH2-terminal region of FN was bound towards the glass surface and the COOH-terminus was oriented away from the glass surface. These studies demonstrate that small peptides can be used to specifically bind and orient large proteins such as FN on the surfaces.

Adhesins, Bacterial↗

PubMed2001

Binding and orientation of fibronectin on surfaces with collagen-related peptides. Student Research Award in the Masters Science Degree Candidate category, 27th annual meeting of the Society for Biomaterials, St. Paul, MN, April 24-29, 2001.

Although fibronectin (FN) has been used in a variety of in vitro studies to enhance cell and bacteria adhesion, relatively little is known about the molecular interactions of FN with surfaces, particularly the interactions that can control the binding, conformation, and functionality of FN on these surfaces. Even less is known about approaches needed to control binding, orientation, and functionality of FN bound on surfaces. To begin to fill this gap in our knowledge, we hypothesized that functional FN can be bound and specifically oriented on polystyrene surfaces with FN-specific collagen-related peptides (CRPs). We further hypothesized that monoclonal antibodies that react with specific epitopes on FN can be used to quantify both FN binding and orientation on these surfaces. On the basis of these hypotheses, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces with CRPs. To bind FN to surfaces, we used two different CRPs: CRP-I (TLQPVYEYMVGV) and CRP-II (TGLPVGVGYVVTVLT). The binding and orientation of the FN molecule to these immobilized CRPs was quantified with (125)I-FN and monoclonal antibodies. Monoclonal antibodies used for this study were reactive with specific regions of the FN molecule, that is, the amino (N) terminus (anti-N antibodies) and carboxyl (C) terminus (anti-C antibodies). The results of our studies demonstrated that although CRP-I and CRP-II could be bound directly to polystyrene, these directly immobilized CRPs failed to bind (125)I-FN . Thus, to facilitate FN binding to the CRPs, we used bovine serum albumin (BSA) as a spacer to physically elevate the CRPs away from the polystyrene surface. Thus, CRP-I and CRP-II were covalently linked to BSA via the N and C termini of each CRP (CRP-I-BSA and CRP-II-BSA). (125)I-CRP-BSAs were all found to bind to equivalent levels on polystyrene (1.60-2.60 microg/cm2). When CRP-BSAs were immobilized on polystyrene, they all successfully bound (125)I-FN in a range of 34-72 ng/cm2 (mean). Using monoclonal antibodies to FN to characterize the orientation of FN bound to the various CRP-BSAs, we demonstrated that (1) FN consistently bound to either CRP-I-BSA or CRP-II-BSA; (2) bound FN reacted significantly more with anti-C antibodies than with anti-N antibodies; and (3) the increased reactivity of bound FN to anti-C antibodies was consistent, whether FN was bound by CRP-I or CRP-II or the CRPs were bound to BSA by the C or N termini. These data demonstrated an enhanced binding of anti-C antibodies to immobilized CRP-BSA relative to anti-N antibodies. We interpreted the data to be the result of FN binding in an oriented fashion with N termini of FN bound tightly to the BSA-polystyrene surface. In this position, the C termini of FN are exposed and available for binding by the anti-C antibodies. Alternatively, in this orientation the N termini of the FN would not be available to bind the anti-N antibodies, thereby explaining the decreased reactivity of the CRP immobilized FN to the anti-N antibodies. These studies not only demonstrate the utility of peptides in binding and orienting large molecular weight proteins such as FN on surfaces but underscore the need for well-characterized reagents (e.g., monomeric/functional FN and antibodies) to specifically bind, orient, and characterize large molecular weight proteins immobilized on various surfaces.

Animals↗

PubMed2001

Fibrin induces IL-8 expression from human oral squamous cell carcinoma cells.

In recent studies, we have demonstrated that fibrin is present in association with tumor cells in oral squamous cell carcinoma (OSCC) in vivo. We hypothesized that this fibrin can directly induce the expression of known angiogenic factors from oral tumor cells. Since IL-8 is known to be the major inducer of angiogenesis caused by these cells, we examined the ability of fibrin to stimulate IL-8 expression from OSCC cells in vitro. A physiologically relevant concentration of fibrin was found to cause a dose and time-dependent stimulation of IL-8 expression from oral and pharyngeal tumor cells but not from a non-tumorigenic oral cell line. Fibrinogen, thrombin and collagen were all unable to induce significant IL-8 expression, establishing the specificity of fibrin in causing this response. Gel filtration chromatography confirmed the molecular identity of the IL-8 antigen detected in the ELISA system used. These results suggest that fibrin may promote angiogenesis in oral tumors in vivo by directly upregulating the expression of IL-8 from tumor cells.

Analysis of Variance↗

PubMed2001

Actinobacillus actinomycetemcomitans-induced expression of IL-1alpha and IL-1beta in human gingival epithelial cells: role in IL-8 expression.

Gingival epithelial cells (GEC) are the first cells of the host that encounter the periodontal pathogens. and therefore their role in the initiation of the inflammatory response is critical. We aimed to: 1) characterize the expression of interleukin (IL)- Ialpha and IL-Ibeta in human gingiva and cultured GEC: 2) demonstrate the ability of A. actinomycetemcomitans extracts to upregulate IL-1alpha, IL-1beta and IL-8 expression in GEC in vitro: and 3) characterize the role of IL-1alpha and IL-1beta in the induction of IL-8 expression in GEC in vitro. Ten gingival biopsies (5 inflamed and 5 controls) and cultured GEC were examined for IL-1alpha and IL-Ibeta using immunohistochemical techniques. GEC were also challenged with A. actinomycetemcomitans extracts or IL-1alpha, and secretion of IL-1 and IL-8 was determined by ELISA. In vivo, IL-lalpha and IL-1beta were localized in the gingival epithelium and the infiltrating leukocytes. In vitro, A. actinomycetemcomitans extracts induced a time-dependent expression of IL-1alpha, IL-1beta and IL-8 in GEC. IL-1 inhibitors did not affect A. actinomycetemcomitans-induced IL-8. although they inhibited IL-8 induced by IL-1alpha or IL-1beta. In conclusion, GEC are a major source of IL-1alpha and IL-1beta in the periodontium, which in turn induce additional inflammatory mediators such as IL-8. Therefore GEC can be a potential target for therapeutic intervention in the future.

Aggregatibacter actinomycetemcomitans↗

PubMed2000

Despite large-scale T cell activation, only a minor subset of T cells responding in vitro to Actinobacillus actinomycetemcomitans differentiate into effector T cells.

Recent studies in our laboratory have demonstrated that Actinobacillus actinomycetemcomitans has a potent T cell stimulatory effect, activating more than half of all T cells. However, since the fate of these activated T cells was not known, the present study sought to determine whether all of these T cells differentiate into effector cells. To that end, the intracellular expression of T cell cytokines (IL-2, IFN-gamma, IL-4 and IL-10) in response to A. actinomycetemcomitans was determined by flow cytometry. Results demonstrated a time-dependent increase in the expression of the cytokines, most reaching peak levels at 24-48 h. At 48 h, the proportion of T cells expressing each of the cytokines were as follows: IL-2 (1.7%+/-0.3), IFN-gamma (1.8%+/-0.5), IL-4 (1.0%+/-0.2) and IL-10 (1.5%+/-0.5). These data indicated that only 2-5% of all T cells stimulated with A. actinomycetemcomitans expressed any T cell cytokines. The finding of large-scale T cell activation in the absence of cytokine expression suggests that the activation of T cells in response to A. actinomycetemcomitans is incomplete. To investigate this phenomenon, peripheral blood mononuclear cells (PBMC) were cultured with A. actinomycetemcomitans for 24 h followed by sorting of the activated (CD69+) cells by immunomagnetic separation and restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Results demonstrated that nearly 90% of the T cells were unresponsive to further restimulation. A possible explanation for this unresponsiveness is the induction of clonal anergy among the responding T cells. To determine possible preferential effects of the stimulation on specific cytokines, the expression of each cytokine among T cells responding to A. actinomycetemcomitans was compared to the maximum levels achieved by PMA + ionomycin stimulation. Results showed that number of IL-2+ and IFN-gamma+ T cells observed in response to A. actinomycetemcomitans were between 2% and 7% of those seen in response to PMA + ionomycin. Conversely, the proportions of T cells expressing IL-4 or IL-10 were between 35% and 90% of those following stimulation with PMA + ionomycin. Hence, A. actinomycetemcomitans appears to more preferentially induce T cells expressing IL-4 and IL-10. Collectively, these data suggest that the in vitro stimulation of T cells with A. actinomycetemcomitans leads to partial activation, i.e. only a minor subset of T cells responding to A. actinomycetemcomitans differentiate into effector cells, while a significant proportion become unresponsive to restimulation, suggesting clonal anergy.

Aggregatibacter actinomycetemcomitans↗

PubMed2000

Interleukin-1 family expression in human breast cancer: interleukin-1 receptor antagonist.

We hypothesize that interleukin-1 alpha, beta, and receptor antagonist (IL-1 alpha, IL-1 beta, and IL-1 ra, respectively) are present and tumor cell associated in human breast cancer (HBC). We believe the levels of these cytokines in breast tumor homogenates relate to other known prognosticators of patient survival (i.e., estrogen receptor [ER] status). Our results demonstrated that, immunohistochemically, tumor cells express IL-1 alpha, IL-1 beta, and IL-1 ra in most specimens tested. In breast tissue homogenates, IL-1 alpha levels correlated inversely with ER levels (p < 0.06), whereas IL-1 ra levels correlated directly with both ER levels (p < 0.009) and IL-1 beta levels (p < 0.06). When analyzing cytokine levels for the ER (-) versus ER (+) patient groups, we found that in many instances these groups showed a different cytokine profile. These studies suggest that the IL-1 family of cytokines may be important in regulating protumorigenic activities within the HBC tumor microenvironment.

Biomarkers, Tumor↗

PubMed1999

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.

Endothelium, Vascular↗

PubMed1999

Role of angiogenic factors: coexpression of interleukin-8 and vascular endothelial growth factor in patients with head and neck squamous carcinoma.

OBJECTIVE/HYPOTHESIS: Angiogenesis has been used as a prognostic indicator in a variety of cancers and is believed to be controlled by angiogenic factors, including the cytokines interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF). We hypothesized that the in vivo coexpression of both IL-8 and VEGF in head and neck tumors contributes to perpetuating tumor growth and metastasis by enhancing angiogenesis. METHODS: Immunohistochemical analysis for IL-8 and VEGF was performed using specimens from 33 cancer patients and 6 control patients. We quantitatively evaluated levels of IL-8 and VEGF in tumor tissue homogenates from those same patients using enzyme-linked immunosorbent assay and radioimmunoassay. Comprehensive histories of each patient were taken and later analyzed for clinical correlations with IL-8 or VEGF levels. RESULTS: IL-8 and VEGF were found to be colocalized within the head and neck squamous cell carcinoma (HNSCCA) tumor cells. In the head and neck tumor specimens, IL-8 levels ([38,152+/-1.8]x10(5) pg/mg total protein [TP]) were 22-fold greater than controls (1,721+/-2,122 pg/mg TP). The tumor levels of VEGF (1,304+/-6,037 pg/mg TP) were nearly fourfold higher than the controls (317+/-400 pg/mg TP. Interleukin-8 and VEGF levels were found to have a positive correlation (P< or = .0001). Patients exhibiting high levels in picograms per milligram of TP and/or number of moles of IL-8 and VEGF were found to clinically have more aggressive disease manifested by higher TNM stage, more recurrences, and shorter disease-free intervals (P< or =.03) CONCLUSIONS: Marked increase in HNSCCA of IL-8 and VEGF underscores the importance of these angiogenic factors in this disease. Understanding the roles and interplay of angiogenic factors such as IL-8 and VEGF may have value in the treatment of HNSCCA.

Aged↗

PubMed1998

p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF-alpha or FMLP stimulation.

Mitogen-activated protein (MAP) kinase-mediated signal-transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the roles of MAP kinases in neutrophils are not well understood yet. Protein phosphorylation analysis of cellular MAP kinases indicates that exposure of human neutrophils to chemotactic factor FMLP as well as granulocyte-macrophage CSF, PMA, or ionomycin rapidly induced the activation of p38 and p44/42 MAP kinases, but stimulation with inflammatory cytokine TNF-alpha triggered the activation of p38 MAP kinase only. To study the cellular functions of these MAP kinases, the inhibitor SB20358, which specifically inhibited enzymatic activity of cellular p38 MAP kinase, and the inhibitor PD98059, which specifically blocked the induced protein phosphorylation and activation of p44/42 MAP kinase in intact neutrophils, were utilized. Inhibition of the cellular p38 MAP kinase activation almost completely abolished the TNF-alpha-stimulated IL-8 production and superoxide generation of human neutrophils. In addition, the FMLP-induced neutrophil chemotaxis as well as superoxide generation were suppressed markedly by inhibiting the activation of cellular p38 MAP kinase, but not p44/42 MAP kinase. Moreover, RIA indicates that the activation of cellular p38 MAP kinase was required for the neutrophil IL-8 production stimulated by granulocyte-macrophage CSF or LPS as well as TNF-alpha, but not for that induced by PMA or ionomycin. These results demonstrate that the activation of cellular p38 MAP kinase is indispensable for the TNF-alpha- or FMLP-mediated cellular functions in human neutrophils, and suggest that p38 MAP kinase may play a different role in response to distinct stimulation.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1998

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.

Endothelial Growth Factors↗

PubMed1998

Transforming growth factor-beta expression in otitis media with effusion.

OBJECTIVE: To characterize the existence and role of transforming growth factor-beta (TGF-beta) in otitis media with effusion (OME). STUDY DESIGN: Retrospective. METHODS: The levels of two major TGF-beta isoforms, TGF-beta1 and TGF-beta2, in the middle ear effusions (MEEs) of 44 children were evaluated using enzyme-linked immunospecific assays (ELISAs). Forty-eight MEEs were separated into three clinically relevant groups (i.e., serous, mucoid, and purulent), and TGF-beta levels were correlated with clinical parameters of disease for these MEEs. RESULTS: Both TGF-beta1 and TGF-beta2 were present in the samples. Mean levels of TGF-beta1 (920.36 +/- 437.75 pg/mg total protein) were generally 100-fold greater than those of TGF-beta2 (9.65 +/- 11.19 pg/mg total protein). TGF-beta1 levels were elevated in association with a history of previous tympanostomy tube placements (TTPs) (P = .029) and mucoid effusions (P = .042). TGF-beta2 levels were elevated in association with a history of previous TTPs (P = .100) and chronic (i.e., serous or mucoid) effusions (P = .003). CONCLUSIONS: TGF-beta1 is present in the MEEs of children with OME. Furthermore, TGF-beta1 and TGF-beta2 levels were elevated differentially in the presence of chronic disease indicators in OME, suggesting that these isoforms may have differing roles in the inflammatory processes that characterize OME.

Child↗

PubMed1998

Activation of coagulation and angiogenesis in cancer: immunohistochemical localization in situ of clotting proteins and vascular endothelial growth factor in human cancer.

Thrombin-catalyzed, cross-linked fibrin (XLF) formation is a characteristic histopathological finding in many human and experimental tumors and is thought to be of importance in the local host defense response. Although the pathogenesis of tumor-associated fibrin deposition is not entirely clear, several tumor procoagulants have been described as likely primary stimuli for the generation of thrombin (and XLF) in the tumor microenvironment (TME). In a previous study of a variety of human tumors we have shown that tissue factor (TF) is the major procoagulant. However, the relative contribution to fibrin deposition in the TME of tumor cell TF and host cell TF (eg, macrophage-derived) was not established. In addition, recent evidence has implicated TF in the regulation of the synthesis of the pro-angiogenic factor vascular endothelial growth factor (VEGF) by tumor cells. In the current study we used in situ techniques to determine the cellular localization of XLF, TF, VEGF, and an alternative tumor procoagulant, so-called cancer procoagulant (CP), a cysteine protease that activates clotting factor X. In lung cancer we have found XLF localized predominantly to the surface of tumor-associated macrophages, as well as to some endothelial cells and perivascular fibroblasts in the stromal area of the tumors co-distributed with TF at the interface of the tumor and host cells. Cancer pro-coagulant was localized to tumor cells in several cases but not in conjunction with the deposition of XLF. TF and VEGF were co-localized in both lung cancer and breast cancer cells by in situ hybridization and immunohistochemical staining. Furthermore, a strong relationship was found between the synthesis of TF and VEGF levels in human breast cancer cell lines (r2 = 0.84; P < 0.0001). Taken together, these data are consistent with a highly complex interaction between tumor cells, macrophages, and endothelial cells in the TME leading to fibrin formation and tumor angiogenesis.

Blood Coagulation↗

PubMed1997

Fibrin regulation of interleukin-8 gene expression in human vascular endothelial cells.

Recent studies in our laboratory, as well as others, have suggested that fibrin can regulate cell function in vitro and likely control inflammation in vivo by acting as a potent cell activator. This has led us to hypothesize that during tissue and vascular injury, fibrin can enhance leukocyte recruitment by inducing vascular endothelial cell expression of leukocyte chemotactic factors. To begin to test this hypothesis, we developed an in vitro model of in situ fibrin polymerization on human umbilical vein endothelial cell culture (HUVEC) and determined the ability of fibrin to induce HUVEC expression of the potent leukocyte chemotactic factor interleukin-8 (IL-8). Our initial studies showed that fibrin induced IL-8 expression in a time- and dose-dependent fashion. Fibrin-induced IL-8 expression in HUVEC could be seen as early as 2 hours post-fibrin stimulation. Additionally, fibrin concentrations as low as 30 microg/mL stimulated a detectable level of IL-8 antigen expression from HUVEC. We also showed that this fibrin induced IL-8 had the identical molecular weight and similar antigenic identity as recombinant and monocyte derived IL-8. Northern blot analysis showed that the IL-8 antigen increase seen in fibrin treated HUVEC was due to fibrin induced elevation of steady state mRNA expression in HUVEC. These data clearly support our hypothesis that fibrin is a potent vascular endothelial cell (VEC) activator that can directly contribute to leukocyte recruitment and activation by inducing leukocyte chemotactic factor expression from VEC.

Cells, Cultured↗

PubMed1997

Fibrin induction of tissue factor expression in human vascular endothelial cells.

BACKGROUND: For the present study, we hypothesized that fibrin is an inducer of tissue factor (TF) expression in vascular endothelial cells in vitro and in vivo. METHODS AND RESULTS: To test the in vitro aspect of this hypothesis, human umbilical vein endothelial cells (HUVECs) were cocultured with physiologically relevant concentrations of fibrin (0.03 to 1.0 mg fibrin/mL) for various times (0.5 to 24 hours), and TF expression was compared with that in unstimulated HUVECs (media control). Results demonstrated that fibrin induced a time- and dose-dependent increase in TF antigen expression, functional TF procoagulant activity, and TF mRNA in HUVECs. CONCLUSIONS: These studies demonstrate that fibrin can directly regulate TF expression in HUVECs in vitro.

Blotting, Western↗

PubMed1997

Fibrin induction of ICAM-1 expression in human vascular endothelial cells.

In acute and chronic inflammatory processes, fibrin deposition, and leukocyte accumulation are classic histopathologic hallmarks. Previous studies have shown that fibrin and fibrin degradation products can have biologic effects on vascular endothelial cells and can induce the expression of several endothelial cell-derived factors that may be important in regulating inflammation and tissue repair. We now demonstrate that coculture of human vascular endothelial cells (EC) with fibrin results in the up-regulation of intercellular adhesion molecule-1 (ICAM-1), thus providing a first link between fibrin deposition and adhesion molecule expression, which may lead subsequently to leukocyte accumulation and extravasation. Increased ICAM-1 expression was demonstrated by ELISA, flow cytometry, and functional adhesion assays. EC ICAM-1 expression increased in a time and dose response fashion. Cell surface levels of ICAM-1 induced by fibrin were comparable to, or exceeded, levels induced by IL-1beta. ICAM-1 expression increased beginning at 4 h post-fibrin formation with sustained elevated expression at 48 h. Fibrin-stimulated EC also bound increased numbers of polymorphonuclear neutrophils in cellular adhesion assays. This increase in adhesion could be blocked by Ab to ICAM-1. Inhibition of fibrin polymerization also inhibited the up-regulation of ICAM-1. Culture medium from fibrin-stimulated EC contained elevated levels of soluble ICAM-1. These data suggest that fibrin deposition on vascular EC, in addition to other reported effects on EC metabolism, may also lead to leukocyte accumulation and extravasation through the induction of leukocyte adhesion molecules such as ICAM-1.

Cell Adhesion↗

PubMed1997

Cytokine expression in otitis media with effusion. Tumor necrosis factor soluble receptor.

BACKGROUND: A variety of cytokines, such as interleukin 1 and tumor necrosis factor (TNF), appear to play a key role in the initiation and maintenance of the inflammatory response in a variety of diseases, including chronic otitis media with effusion (COME). Recently, cytokine inhibitors have been isolated and are presumed to regulate the proinflammatory effects of these cytokines. This has lead us to hypothesize that the chronic inflammation seen in COME is the result of an imbalance in the ratio of proinflammatory cytokines and inhibitors that favors the proinflammatory cytokines. DESIGN: Middle ear effusions were evaluated for the cytokine TNF-alpha and the inhibitor TNF soluble receptor (TNFsolR) using a combination of enzyme-linked immunosorbent assays and radioactive immunoassays. We determined the presence and quantified the amounts of TNF-alpha and TNFsolR and correlated this result with clinical parameters in patients with COME. SETTING: University hospital. PATIENTS: To test this hypothesis, 35 middle ear effusions were obtained from 29 children aged 2 to 102 months (mean, 39.6 months) undergoing tympanostomy tube insertion for COME. OUTCOME: Children were followed up for at least 12 months postoperatively and the number of subsequent episodes of otitis media correlated with cytokine levels. RESULTS: The TNF-alpha and TNFsolR were present in 83% and 91% of effusions, respectively. The TNF-alpha mean (+/-SEM) cytokine values were 110.6 +/- 32.1 pg/mg of total protein, and 12886 +/- 3108 pg/mg total protein for TNFsolR. Analysis of correlations in a univariate model revealed a statistically significant relationship for TNF-alpha correlated with TNFsolR (R2 = 0.463; P < .001). The TNF-alpha levels were significantly lower for children with multiple tube insertions (P = .02). Higher levels of TNF-alpha were noted in those children who subsequently developed episodes of otitis media after tube placement (P = .02). The ratio of TNF-alpha to TNFsolR, the mean (+/-SEM) inhibitor index was 430 +/- 220 U. CONCLUSIONS: Our data support the hypothesis that cytokines and their inhibitors are present in a large number of middle ear effusions and in part are likely important in the regulation of inflammatory processes in COME.

Analysis of Variance↗