Efficacy and safety of antithrombin supplementation in neonates and infants on a continuous heparin infusion.

Background: Antithrombin (AT) is a natural anticoagulant and potent inhibitor of several coagulation proteins, including activated factor X (FXa) and FIIa. The therapeutic activity of heparin depends on the presence of AT. Levels of plasma AT are low in neonates and young infants compared to those in adults. Exogenous AT supplementation is postulated to enhance the activity of heparin and facilitate attainment of therapeutic anticoagulation in infants. Objectives: To describe the efficacy and safety of AT administration in infants on a continuous heparin infusion. Methods: Retrospective cohort study of 50 infants who received AT while on a heparin infusion. The primary efficacy outcome was attainment of therapeutic anticoagulation within 48 hours after AT administration. Secondary outcomes included the percent of partial thromboplastin time (PTT) levels and/or antifactor Xa (anti-FXa) activity within the therapeutic window, attainment of the target AT activity level, the incidence and severity of bleeding, and all-cause in-hospital mortality. A secondary analysis investigated the relationship between simultaneously measured PTT levels and anti-FXa activity used for heparin monitoring. Results: AT supplementation resulted in achievement of at least one therapeutic PTT level or anti-FXa activity in 90% of AT courses, though not sustained. PTT was within the therapeutic window more often than anti-FXa activity. When measured simultaneously, therapeutic anti-FXa levels were associated with supratherapeutic PTT levels. Conclusion: AT supplementation in infants on a continuous heparin infusion may transiently improve the therapeutic effect of heparin, but this is largely dependent on the laboratory parameters used for monitoring.

Prostate cancer as a model system for genetic diversity in tumors.

This chapter will summarize novel understandings of the early molecular events in prostatic carcinogenesis that may underlie both the genetic and clinical heterogeneity. Areas covered include preneoplasia, stem cell concepts, telomere abnormalities, and the nature of tumor-stromal interactions. The oncogenomics of prostate cancer is reviewed with emphasis on androgen signaling, ETS gene family aberrations, and PTEN deletion. The notion that "field cancerization," coupled with genomic instability may explain both the occurrence of multifocal disease, and the recent observations of genetic diversity of ERG alteration in individual tumors are discussed. Collectively, genomic studies are rapidly moving human prostate cancer closer to the promise of personalized medicine, so that specific genetic profiles of individual tumors will determine the best therapeutic approaches.

Nonlinear multicontrast microscopy of hematoxylin-and-eosin-stained histological sections.

Imaging hematoxylin-and-eosin-stained cancerous histological sections with multicontrast nonlinear excitation fluorescence, second- and third-harmonic generation (THG) microscopy reveals cellular structures with extremely high image contrast. Absorption and fluorescence spectroscopy together with second hyperpolarizability measurements of the dyes shows that strong THG appears due to neutral hemalum aggregation and is subsequently enhanced by interaction with eosin. Additionally, fluorescence lifetime imaging microscopy reveals eosin fluorescence quenching by hemalums, showing better suitability of only eosin staining for fluorescence microscopy. Multicontrast nonlinear microscopy has the potential to differentiate between cancerous and healthy tissue at a single cell level.

Predominant expression of CCL2 at the tumor site of prostate cancer patients directs a selective loss of immunological tolerance to CCL2 that could be amplified in a beneficial manner.

We have previously shown that, during inflammatory autoimmune diseases in humans, the immune system develops a neutralizing auto-Ab-based response to a very limited number of inflammatory mediators, and that amplification of each response could be beneficial for the host. Our working hypothesis has been that this selective breakdown of immunological tolerance is due to a predominant expression of an inflammatory mediator at an immune-restricted site undergoing a destructive process. All three conditions also take place in cancer diseases. In this study, we delineate this hypothesis for the first time in a human cancer disease and then explore its clinical implications. We show that in primary tumor sections of prostate cancer subjects, CCL2 is predominantly expressed at the tumor site over other chemokines that have been associated with tumor development, including: CXCL12, CXCL10, CXCL8, CCL3, and CCL5. Subsequently, the immune response selectivity mounts an Ab-based response to CCL2. These Abs are neutralizing Abs. These findings hold diagnostic and therapeutic implications. The current diagnosis of prostate cancer is based on prostate-specific Ag measurements that do not distinguish benign hypertrophy from malignancy. We show in this study that development of anti-CCL2 Abs is selective to the malignant stage. From a clinically oriented perspective, we show, in an experimental model of the disease, that DNA-based amplification of this response suppresses disease, which has implications for a novel way of therapy in humans.

Acceleration of diabetic wound healing by an angiopoietin peptide mimetic.

Angiopathies are one of the leading underlying causes of morbidity in diabetic patients. Poorly managed blood glucose levels contribute to vascular defects that manifest themselves in numerous different clinical conditions, including diabetic retinopathy, nephropathy, peripheral artery disease, and compromised wound healing. The angiopoietin family (Angs 1-4) has been shown to play a critical role in the growth and maintenance of vasculature. Here we evaluate the efficacy of a new Ang-based peptidomimetic compound, Vasculotide, on diabetic-related wound healing. Stimulation of endothelial cells (ECs) with Vasculotide results in activation of the Ang receptor, Tie 2, and its associated signaling pathways. This activation promoted biological responses such as EC survival, migration, and matrix metalloproteinase 2 (MMP2) production. We show that Vasculotide alone and in combination with vascular endothelial growth factor (VEGF) results in the production of well-arborized vessels supported by myogenic cells. Using an excisional skin-wound model produced on the back of diabetic B6.Cg-m(+/+)Lepr(db)/J (db/db) mice, we found that Vasculotide-treated wounds presented with decreased wound closure times (p < 0.05) and dramatic increases in granulation tissue (p < 0.01). Although the potential of this novel proangiogenic compound in treating microvascular dysfunction is not strictly limited to topical administration, we provide mechanistic evidence as a proof of principle in support of its efficacious use in diabetic wound healing.

An eosinophil immune response characterizes the inflammatory skin disease observed in Tie-2 transgenic mice.

Although mouse models of inflammatory skin diseases such as psoriasis and atopic dermatitis fail to completely phenocopy disease in humans, they provide invaluable tools to examine the molecular and cellular mechanisms responsible for the epidermal hyperplasia, inflammation, and excess angiogenesis observed in human disease. We have previously characterized a tyrosine kinase with immunoglobin-like and epidermal growth factor-like domain-2 (Tie-2) transgenic mouse model of an inflammatory skin disease exhibiting these features. More specifically, we demonstrated that the inflammatory component consisted of increased infiltration of CD3-positive T lymphocytes and mast cells in the skin. Here, we further characterize the inflammatory component in the blood and skin of Tie-2 transgenic mice at cellular and molecular levels. We observed increased numbers of CD3-positive T lymphocytes in the blood and increased infiltration of eosinophils in the skin. Furthermore, we characterized cytokine protein and gene expression in the blood and skin, respectively, and observed the deregulated expression of cytokines associated with Th1 and eosinophil immune responses. Interestingly, treatment of Tie-2 transgenic mice with anti-CD4 antibody appeared to resolve aspects of inflammation but did not resolve epidermal hyperplasia, suggesting an important role for eosinophils in mediating the inflammatory skin disease observed in Tie-2 transgenic mice.

Vascular endothelial growth factor-mediated decrease in plasma soluble vascular endothelial growth factor receptor-2 levels as a surrogate biomarker for tumor growth.

Vascular endothelial growth factor (VEGF) is a potent proangiogenic protein that activates VEGF receptor (VEGFR) tyrosine kinases expressed by vascular endothelial cells. We previously showed that one of these receptors, VEGFR-2, has a truncated soluble form (sVEGFR-2) that can be detected in mouse and human plasma. Because activation of VEGFR-2 plays an important role in tumor angiogenesis, clinical interest in monitoring plasma sVEGFR-2 levels in cancer patients has focused on its potential exploitation as a surrogate biomarker for disease progression as well as assessing efficacy/activity of antiangiogenic drugs, particularly those that target VEGF or VEGFR-2. However, no preclinical studies have been done to study sVEGFR-2 during tumor growth or the mechanisms involved in its modulation. Using spontaneously growing tumors and both localized and metastatic human tumor xenografts, we evaluated the relationship between sVEGFR-2 and tumor burden as well as underlying factors governing protein level modulation in vivo. Our results show an inverse relationship between the levels of sVEGFR-2 and tumor size. Furthermore, using various methods of VEGF overexpression in vivo, including cell transfection and adenoviral delivery, we found plasma sVEGFR-2 decreases to be mediated largely by tumor-derived VEGF. Finally, in vitro studies indicate VEGF-mediated sVEGFR-2 modulation is the result of ligand-induced down-regulation of the VEGFR-2 from the cell surface. Taken together, these findings may be pertinent to further clinical exploitation of plasma sVEGFR-2 levels as a surrogate biomarker of VEGF-dependent tumor growth as well as an activity indicator of antiangiogenic drugs that target the VEGFR system.

A gene expression signature for relapse of primary wilms tumors.

Anaplastic histology and metastasis are each associated with higher relapse and mortality rates in Wilms tumor patients. However, not all anaplastic tumors relapse and some nonanaplastic tumors relapse unexpectedly. To identify more accurate early prognostic indicators, we analyzed expression of 4,900 cancer-related genes in 26 primary Wilms tumors. This analysis revealed that expression of a set of four genes predicts future relapse of primary Wilms tumors with high accuracy, independent of anaplasia. Random permutation testing of this prognostic gene expression signature yielded P = 0.003. Real-time reverse transcription-PCR analysis of the four genes in an independent primary tumor set resulted in correct prediction of future relapse with an accuracy of 92%. One of the four genes in the prognostic signature, CCAAT/enhancer binding protein beta (C/EBPB), is expressed at higher levels in both primary relapsing tumors and metastatic tumors than in primary nonrelapsing tumors. Short interfering RNA-mediated down-regulation of C/EBPB expression in WiT49, a cell line derived from a metastatic Wilms tumor, resulted in spontaneous apoptosis. These findings suggest that C/EBPB is a critical survival factor for Wilms tumor cells and that its expression contributes to the prognosis of Wilms tumor patients.

Derivation and characterization of a Wilms' tumour cell line, WiT 49.

Wilms' tumour is a pediatric neoplasm exhibiting histologic features of developing kidney. Although the majority of Wilms' tumour patients are treated effectively, approximately 15% develop metastases and of these, 30% succumb to their disease. The biologic factors governing Wilms' tumour metastasis are largely unknown. Attempts at deriving representative Wilms' tumour cell lines, which could facilitate functional studies, have only been partially successful thus far. We now report on derivation and characterization of a Wilms' tumour cell line, WiT 49, from a first-generation xenograft of a human Wilms' tumour lung metastasis. WiT 49 recapitulates the phenotype of the parent tumours (primary and lung metastasis) and expresses normal WT1, overexpresses IGFII and carries a frequently identified p53 mutation. We recently reported overexpression of hepatocyte growth factor(HGF) and its receptor met in a series of Wilms' tumours with higher levels in homotypic metastatic cases. We therefore examined WiT 49 for expression of HGF/met and for met signaling targets associated with cell adhesion and cytoplasmic mediators of transcription using Western blot, co-immunoprecipitation, immunofluorescence labeling and zymography. Our results show co-expression of HGF and met protein, absence of E-cadherin, high levels of beta-catenin co-immunolocalized to met at the cell membrane and moderate levels of gamma-catenin and ezrin protein expression. After cell fractionation, beta-catenin was detected in the cytoplasm and nuclei of WiT 49 with relatively higher levels in the cytoplasm as compared to nuclei. Examination of MMP expression in WiT 49 showed constitutive activation of MMP 9 and latent MMP 2 supporting possible beta-catenin-mediated transcriptional activation. The WiT 49 cell line responded to recombinant human HGF by an increase in the expression of the met receptor, recruitment of the Gab-1 adapter protein to met and release of bound beta-catenin from met. Our studies therefore establish WiT 49 as a representative Wilms' tumour cell line derived from a lung metastasis that co-expresses HGF/met and shows absence of the cadherin-catenin complex supporting a role for these factors in regulation of the invasive and metastatic phenotype in Wilms' tumour.

The expression of ccn3(nov) gene in musculoskeletal tumors.

The CCN3(NOV) protein belongs to the CCN [cysteine-rich CYR61, connective tissue growth factor (CTGF), nephroblastoma overexpressed gene (Nov)] family of growth regulators, sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Although previous studies have revealed an expression of CCN3 protein in several normal tissues, including kidney, nervous system, lung, muscle, and cartilage, less is known about its expression in tumors. In this study, we analyzed the expression of CCN3 in musculoskeletal tumors, using a panel of human cell lines and tissue samples. An association between CCN3 expression and tumor differentiation was observed in rhabdomyosarcoma and cartilage tumors, whereas, in Ewing's sarcoma, the expression of this protein seemed to be associated with a higher risk to develop metastases. CCN3 expression was found in 15 of 45 Ewing's sarcoma tissue samples. In particular, we did not observe any expression of CCN3 in the 15 primary tumors that did not develop metastases. In contrast, 15 of the 30 primary tumors that developed lung and/or bone metachronous metastases showed a high expression of the protein (P < 0.001, Fisher's test). Our studies indicate that CCN3 is generally expressed in the cells of the musculoskeletal system. This protein may play a role both in normal and pathological conditions. However, the regulation of CCN3 expression varies in the different neoplasms and depends on the type of cells. Thus, as reported for other CCN genes, the biological properties and regulation of expression of CCN3 are dependent on the cellular context and the nature of the cells in which it is produced. Further studies will help to clarify the biological role of this protein in musculoskeletal neoplasms.

Expression and localization of HGF and met in Wilms' tumours.

A number of growth factors and cognate receptors that contribute to normal kidney development have been shown to play roles in the pathogenesis of Wilms' tumours. Expression of both hepatocyte growth factor (HGF) and its tyrosine kinase receptor met has been demonstrated in normal tissues and their neoplastic counterparts, implicating these factors in normal development and tumour progression. HGF and met expression has not been studied in Wilms' tumour. Since HGF and met function in a paracrine fashion by regulating tubulogenesis in normal kidney development, they could be involved in the pathogenesis of Wilms' tumour, in which tubular formation is dysplastic. In the present study, a series of ten homotypic (consisting of blastemal, epithelial, and stromal elements) and ten heterotypic (consisting of triphasic histology and a muscle component) Wilms' tumour cases were examined for expression of HGF and met, using in situ hybridization, immunohistochemistry, and western blot analysis. Relatively high met message and protein expression, compared with normal kidney, were evident in homotypic and heterotypic tumour blastemal, epithelial, and rhabdomyoblastic cells and a 145 kD met polypeptide was found in all tumours, with a few cases also expressing the 170 kD precursor form. No apparent alterations of the met receptor were observed. Similarly, HGF protein was also abundantly expressed in blastemal, epithelial, and rhabdomyoblastic cells of the homotypic and heterotypic Wilms' tumours and a 69 kD HGF polypeptide was demonstrated by western blot analysis. Immunohistochemistry for the Ki-67 proliferation marker indicated that the pattern of Ki-67 expression correlated with the HGF and met pattern of expression in both homotypic and heterotypic tumours. These results reveal, for the first time, significant co-expression of met/HGF in Wilms' tumours, with a correspondingly high proliferative index in the same cell groups.