Prognostic value of Ki67 in localized prostate carcinoma: a multi-institutional study of >1000 prostatectomies.

BACKGROUND: Expanding interest in and use of active surveillance for early state prostate cancer (PC) has increased need for prognostic biomarkers. Using a multi-institutional tissue microarray resource including over 1000 radical prostatectomy samples, we sought to correlate Ki67 expression captured by an automated image analysis system with clinicopathological features and validate its utility as a clinical grade test in predicting cancer-specific outcomes. METHODS: After immunostaining, the Ki67 proliferation index (PI) of tumor areas of each core (three cancer cores/case) was analyzed using a nuclear quantification algorithm (Aperio). We assessed whether Ki67 PI was associated with clinicopathological factors and recurrence-free survival (RFS) including biochemical recurrence, metastasis or PC death (7-year median follow-up). RESULTS: In 1004 PCs (∼4000 tissue cores) Ki67 PI showed significantly higher inter-tumor (0.68) than intra-tumor variation (0.39). Ki67 PI was associated with stage (P<0.0001), seminal vesicle invasion (SVI, P=0.02), extracapsular extension (ECE, P<0.0001) and Gleason score (GS, P<0.0001). Ki67 PI as a continuous variable significantly correlated with recurrence-free, overall and disease-specific survival by multivariable Cox proportional hazard model (hazards ratio (HR)=1.04-1.1, P=0.02-0.0008). High Ki67 score (defined as ⩾5%) was significantly associated with worse RFS (HR=1.47, P=0.0007) and worse overall survival (HR=2.03, P=0.03). CONCLUSIONS: In localized PC treated by radical prostatectomy, higher Ki67 PI assessed using a clinical grade automated algorithm is strongly associated with a higher GS, stage, SVI and ECE and greater probability of recurrence.

Hexagonal magnetite nanoprisms: preparation, characterization and cellular uptake.

The capacity of iron oxide nanocrystals to heat tissue when subjected to an alternating magnetic field (AMF hyperthermia) is shape-selective. Although iron oxide nanostructures with numerous shapes have been synthesized to date, hexagonal Fe3O4 prisms of low toxicity remained elusive. Here, we report the use of a dual ligand system permitting feasible reaction conditions to synthesize nearly perfect hexagonal Fe3O4 nanoplatelet structures, with edge length of 45 ± 5 nm and thickness of 5 to 6 nm. Their Specific Absorption Rate (SAR) is >750 W g(Fe)-1. The Fe3O4 hexagons were coated with a dopamine-based ligand to increase dispersibility in aqueous buffers. The Fe3O4 hexagons were only minimally toxic to RAW264.7 cells, which can be utilized in cell-based cancer targeting approaches.

Acid ceramidase induces sphingosine kinase 1/S1P receptor 2-mediated activation of oncogenic Akt signaling.

Acid ceramidase (AC) is overexpressed in most prostate tumors and confers oncogenic phenotypes to prostate cancer cells. AC modulates the cellular balance between ceramide, sphingosine and sphingosine 1-phosphate (S1P). These bioactive sphingolipids have diverse, powerful and often oppositional impacts on cell signaling, including the activation status of the oncogenic kinase Akt. Our studies show that AC expression correlates with phosphorylation of Akt in human prostate tumors, and elevation of phosphorylated Akt in tumor versus patient-matched benign tissue is contingent upon AC elevation. Investigation of the mechanism for AC-induced Akt activation revealed that AC activates Akt through sphingosine kinase 1 (SphK1)-derived generation of S1P. This signaling pathway proceeds through S1P receptor 2 (S1PR2)-dependent stimulation of PI3K. Functionally, AC-overexpressing cells are insensitive to cytotoxic chemotherapy, however, these cells are more susceptible to targeted inhibition of Akt. AC-overexpressing cells proliferate more rapidly than control cells and form more colonies in soft agar; however, these effects are profoundly sensitive to Akt inhibition, demonstrating increased dependence on Akt signaling for the oncogenic phenotypes of AC-overexpressing cells. These observations may have clinical implications for targeted therapy as PI3K and Akt inhibitors emerge from clinical trials.

Confocal imaging of trans-epithelial trafficking by immune and umbilical cord stem cells in the neonatal porcine intestine.

Maternal colostral leucocytes (CL) and peripheral blood mononuclear cells (PBMC) enter the neonatal circulation after ingestion in pigs and cattle. Porcine umbilical cord matrix stem cells (PUCs) are relatively non-immunogenic after initial allogeneic transplantation. Using intestinal explant cultures incubated with labelled cells and confocal microscopy, we demonstrated trans-epithelial trafficking of exogenous CL, PBMC and PUCs below the luminal surface after 72 h of culture. We orally administered PBMC and PUCs to pre-colostral neonatal pigs and tracked their location 8 or 24 h later. Both PBMC and PUCs were found in the intestinal wall of all samples. Exposure to 25% of acellular colostrum had no detected effect on trafficking. Labelled PUCs and PBMC were detected on the surface of the epithelium and in the lamina propria 8 h post-treatment and PBMC were also in the superficial submucosa. At 24 h, PUCs and PBMC were observed on the surface of the epithelium, in the lamina propria, superficial submucosa and deep submucosa. Our findings show the potential of PUCs for allogeneic engraftment in the neonatal intestine and may lead to cell-based delivery of therapeutics.

Molecular pathology of prostate cancer.

This chapter includes discussion of the molecular pathology of tissue, blood, urine, and expressed prostatic secretions. Because we are unable to reliably image the disease in vivo, a 12 core method that oversamples the peripheral zone is widely used. This generates large numbers of cores that need to be carefully processed and sampled. In spite of the large number of tissue cores, the amount of tumor available for study is often quite limited. This is a particular challenge for research, as new biomarker assays will need to preserve tissue architecture intact for histopathology. Methods of processing and reporting pathology are discussed. With the exception of ductal variants, recognized subtypes of prostate cancer are largely confined to research applications, and most prostate cancers are acinar. Biomarker discovery in urine and expressed prostatic secretions would be useful since these are readily obtained and are proximate fluids. The well-known challenges of biomarker discovery in blood and urine are referenced and discussed. Mediators of carcinogenesis can serve as biomarkers as exemplified by mutations in PTEN and TMPRSS2:ERG fusion. The use of proteomics in biomarker discovery with an emphasis on imaging mass spectroscopy of tissues is discussed. Small RNAs are of great interest, however, their usefulness as biomarkers in clinical decision making remains the subject of ongoing research. The chapter concludes with an overview of blood biomarkers such as circulating nucleic acids and tumor cells and bound/free isoforms of prostate specific antigen (PSA).

Characterization of PacMetUT1, a recently isolated human prostate cancer cell line.

BACKGROUND: Existing prostate cancer cell lines have limitations. METHODS: Cells were characterized using Western blotting, immunohistochemistry, invasion into Matrigel, and by studying xenograft tumors. RESULTS: We describe a cell line (PacMetUT1) isolated from a lymph node of a 57-year-old male with prostate cancer. Compared to existing prostate cancer cell lines, the growth rate of PacMetUT1 xenograft tumors is slower with tumors occurring at injection sites and with metastases to lung and liver. Androgen receptor (AR) was detected in vivo by Western blotting and the cells responded to methyltrienolone (R1881). PacMetUT1 cells are more invasive in Matrigel than DU-145, PC-3, and LNCaP cells, and showed greater anchorage-independent growth in soft agar. The cells do not express prostate specific antigen (PSA) in vitro or in xenografts. However, the green fluorescent protein (GFP) gene was introduced and stably expressed in PacMetUT1 cells, allowing tumor imaging in vivo. Xenograft tumors show epithelial features and are positive for keratin, epithelial membrane antigen, EGF receptor, and E cadherin. In contrast, fibroblast markers vimentin, desmin, and Factor VIII, were negative. Karyotyping showed losses of 6p, 7q, 8p, 18q, and 22q, and gains of 8q and 9q; additional genetic material was observed at 2q and 12p. CONCLUSION: The PacMetUT1 cell line allows metastases to be assessed using a single animal model. Because of its slower growth, PacMetUT1 more closely mimics the human disease. Studies of tumor progression or metastasis can be conducted over a longer period of time.

Development of human umbilical cord matrix stem cell-based gene therapy for experimental lung tumors.

Umbilical cord matrix stem (UCMS) cells are unique stem cells derived from Wharton's jelly, which have been shown to express genes characteristic of primitive stem cells. To test the safety of these cells, human UCMS cells were injected both intravenously and subcutaneously in large numbers into severe combined immunodeficiency (SCID) mice and multiple tissues were examined for evidence of tumor formation. UCMS cells did not form gross or histological teratomas up to 50 days posttransplantation. Next, to evaluate whether UCMS cells could selectively engraft in xenotransplanted tumors, MDA 231 cells were intravenously transplanted into SCID mice, followed by intravenous transplantation of UCMS cells 1 and 2 weeks later. UCMS cells were found near or within lung tumors but not in other tissues. Finally, UCMS cells were engineered to express human interferon beta--designated 'UCMS-IFN-beta'. UCMS-IFN-beta cells were intravenously transplanted at multiple intervals into SCID mice bearing MDA 231 tumors and their effect on tumors was examined. UCMS-IFN-beta cells significantly reduced MDA 231 tumor burden in SCID mouse lungs indicated by wet weight. These results clearly indicate safety and usability of UCMS cells in cancer gene therapy. Thus, UCMS cells can potentially be used for targeted delivery of cancer therapeutics.

Differential alterations in 5alpha-reductase type 1 and type 2 levels during development and progression of prostate cancer.

BACKGROUND: In the prostate, conversion of testosterone to dihydrotestosterone (DHT), by the enzymes 5alpha-reductase types 1 and 2 (5alphaR1, 5alphaR2) is required for normal growth and probably also for development of prostate cancer (PCa). Finasteride, a 5alphaR2 inhibitor, was shown to reduce the prevalence of PCa in the Prostate Cancer Prevention Trial. However, inhibition of both 5alphaR isoenzymes causes a greater decrease in serum DHT. The aim of this study was to assess differential expression of these enzymes at various stages of PCa development. METHODS: Immunostaining for 5alphaR1 and 5alphaR2, using specific, well-validated antibodies, was evaluated in 26 benign prostatic hyperplasia (BPH) (16 for 5alphaR2), 53 primary PCa (21 for 5alphaR2), 18 prostatic intraepithelial neoplasia (PIN), 12 primary PCa treated with neoadjuvant androgen ablation, 15 locally recurrent PCa specimens, and 18 PCa metastases. RESULTS: The mean area of moderate plus high intensity staining for 5alphaR1 increased from 4.8 +/- 2.8% of total epithelial area in BPH, to 18.9 +/- 5.7% in PIN, 17.0 +/- 3.2% in primary cancer, 38.0 +/- 7.3% in recurrent cancer, and 55.8 +/- 8.5% in PCa metastases. The mean staining area for 5alphaR2 decreased from 58.8 +/- 7.2% in BPH, to 21.1 +/- 5.5% in PIN and 34.8 +/- 6.7% in primary PCa. Staining for 5alphaR2 was increased in recurrent cancer and PCa metastases compared to primary PCa, at 58.7 +/- 5.2% and 69.2 +/- 8.7%, respectively. CONCLUSIONS: 5alphaR1 immunostaining is increased and 5alphaR2 immunostaining is decreased during development of PCa. In addition, there is increased expression of both 5alphaR isozymes in recurrent and metastatic cancers, suggesting that both isozymes may be important in the development and progression of PCa.

Transplantation of pig stem cells into rat brain: proliferation during the first 8 weeks.

Previous work indicated that pig umbilical cord matrix (pUCM) cells are a type of primitive stem cell and that these cells could be recovered after central or peripheral injection into rats that did not receive immune suppression therapy. To determine the safety and proliferation potential of pUCM cells after brain transplantation, approximately 150 pUCM cells were transplanted into the brains of rats that previously received a striatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA). The pUCM cells were previously engineered to express enhanced green fluorescent protein (eGFP); in this way, the graft cells were identified. The rats did not receive immune suppression therapy. There were no postsurgical complications and the animals thrived following transplantation. At 2, 4, 6, and 8 weeks after transplantation, two rats were sacrificed and the morphology, size and number of graft cells, and the percentage of tyrosine hydroxylase (TH)-positive graft cells were determined. The size distribution of the grafted pUCM cells was unimodal and normal, and the average size increased significantly over the 2- to 8-week survival period. The number of pUCM cells increased from approximately 5400 cells at the 2-week survival period post-transplantation to approximately 20,000 cells at the 8-week survival period. There was an increase in the percentage of TH-positive pUCM cells from approximately 1% at the 2-week survival period to approximately 6% at the 8-week survival period. There was no evidence of a significant host immune response at any time; for example, no accumulation of CD-4, CD-8, CD-11b, CD-161 cells in the transplantation site. These results suggest that pUCM cells engraft and proliferate without requiring immune suppression. These findings also suggest that a subset of pUCM cells can differentiate into TH-positive cells within 8 weeks after transplantation into the 6-OHDA lesioned rat brain.

Promise and challenge: Markers of prostate cancer detection, diagnosis and prognosis.

Approximately 1 man in 6 will be diagnosed with prostate cancer during his life lifetime, and over 200,000 men in the U.S. are diagnosed with prostate cancer annually. Since the widespread adoption of PSA testing, about 60-70% of men at risk in the U.S. have had a blood test for prostate cancer. With this, prostate cancer death rates have decreased, yet only slightly. Thirty thousand men still die each year from this disease. PSA testing fails to identify a small but significant proportion of aggressive cancers, and only about 30% of men with a "positive" PSA have a positive biopsy. Additionally, of men who are treated for prostate cancer, about 25% require additional treatment, presumably due to disease recurrence. Also of concern is the growing evidence that there are some prostate cancers for which treatment may not be necessary. Very long-term studies from the U.S. and Europe, following men with prostate cancer have found that some tumors do not progress over time. In these individuals, prostate cancer treatment is unnecessary and harmful as these men do not benefit from treatment but will be at risk of treatment-related side effects and complications. They suggest a fundamental problem with prostate cancer: it is not possible, at this time, to predict the natural history of the disease. It is for these reasons that the most important challenge in prostate cancer today is the inability to predict the behavior of an individual tumor in an individual patient. Here we review issues related to performance and validation of biomarkers with a focus on "doing no harm", and bearing in mind that it is the ultimate goal of early detection to save lives. Improved diagnostic and prognostic biomarkers are needed for prostate cancer, and the use of these markers should ultimately translate into increased life span and quality of life. The ultimate goal would be to not only have accurate biomarkers suitable for early diagnosis, but also biomarkers that identify men at greatest risk of developing aggressive disease. Technology has been brought to bear on this problem, and the major approaches are genomics, expression analysis, and proteomics. Proteomics and DNA methylation assays may soon be used in sensitive and specific diagnostic testing of serum and tissues for cancer. Expression arrays may be used to establish both a more specific diagnosis and prognosis for a particular tumor. The proteome is only beginning to be understood, and alternative splicing and post-translational modifications of proteins such as glycosylation and phosphorylation are challenging areas of study. Finally, risk assessment and prognosis are being pursued through analysis of genomic polymorphisms (single nucleotide polymorphisms, SNPs). This huge task is only beginning, and requires the combined expertise of molecular epidemiologists, oncologists, surgeons, pathologists, and basic scientists.

5alpha-reductase type 1 immunostaining is enhanced in some prostate cancers compared with benign prostatic hyperplasia epithelium.

PURPOSE: In the prostate testosterone is converted to the more potent androgen dihydrotestosterone by the enzymes 5alpha-reductase (5alphaR) types 1 (5alphaR1) and 2 (5alphaR2). Since 5alphaR2 is the dominant prostatic enzyme, the 5alphaR2 selective inhibitor finasteride has been widely used to treat benign prostatic hyperplasia (BPH). However, inhibition of both 5alphaR enzymes provides a greater decrease in serum dihydrotestosterone. We developed a specific antibody to 5alphaR1 and assessed expression in BPH and prostate cancer (pCa) tissue. The presence of this isoenzyme in localized prostate cancer would provide a rationale for assessing the efficacy of dual inhibition for prostate cancer prevention. MATERIALS AND METHODS: A polyclonal antibody to 5alphaR1 was developed and validated using 5alphaR1 and 5alphaR2 transfected COS-1 cells. A total of 26 BPH and 53 pCa specimens were assessed for 5alphaR1 protein expression using immunocytochemical methods. Also, 29 BPH and 37 pCa specimens were assayed for 5alphaR1 and 5alphaR2 enzyme activity. RESULTS: Specificity of the 5alphaR1 antibody was confirmed using transfected COS-1 cells. Cells transfected with 5alphaR1 showed specific staining in immunocytochemistry experiments and on Western blotting of cell lysates the expected 24 kDa band was observed. High intensity immunoreactivity for 5alphaR1 was observed in the tumor epithelium of 28% of pCa specimens. No high intensity epithelial staining was observed in BPH specimens. In 19% of pCa and 7% of BPH specimens 5alphaR1 enzyme activity was detected. CONCLUSIONS: The presence of increased 5alphaR1 in some prostatic malignancies suggests that it is worthwhile to investigate the use of a dual 5alphaR inhibitor to prevent or treat early stage prostate cancer.

Transplantation of porcine umbilical cord matrix cells into the rat brain.

Immune rejection of transplanted material is a potential complication of organ donation. In response to tissue transplantation, immune rejection has two components: a host defense directed against the grafted tissue and an immune response from the grafted tissue against the host (graft vs host disease). To treat immune rejection, transplant recipients are typically put on immunosuppression therapy. Complications may arise from immune suppression or from secondary effects of immunosuppression drugs. Our preliminary work indicated that stem cells may be xenotransplanted without immunosuppression therapy. Here, we investigated the survival of pig stem cells derived from umbilical cord mucous connective tissue (UCM) after transplantation into rats. Our data demonstrate that UCM cells survive at least 6 weeks without immune suppression of the host animals after transplantation into either the brain or the periphery. In the first experiment, UCM cells were transplanted into the rat brain and recovered in that tissue 2-6 weeks posttransplantation. At 4 weeks posttransplantation, the UCM cells engrafted into the brain along the injection tract. The cells were small and roughly spherical. The transplanted cells were positively immunostained using a pig-specific antibody for neuronal filament 70 (NF70). In contrast, 6 weeks posttransplantation, about 10% of the UCM cells that were recovered had migrated away from the injection site into the region just ventral to the corpus callosum; these cells also stained positively for NF70. In our second experiment, UCM cells that were engineered to constitutively express enhanced green fluorescent protein (eGFP) were transplanted. These cells were recovered 2-4 weeks after brain transplantation. Engrafted cells expressing eGFP and positively staining for NF70 were recovered. This finding indicates a potential for gene therapy. In the third experiment, to determine whether depositing the graft into the brain protected UCM cells from immune detection/clearance, UCM cells were injected into the tail vein and/or the semitendinosis muscle in a group of animals. UCM cells were recovered from the muscle or within the kidney 3 weeks posttransplantation. In control experiments, rat brains were injected with PKH 26-labeled UCM cells that had been lysed by repeated sonic disruption. One and 2 weeks following injection, no PKH 26-labeled neurons or glia were observed. Taken together, these data indicate that UCM cells can survive xenotransplantation and that a subset of the UCM cells respond to local signals to differentiate along a neural lineage.

Incorporation of bovine bone marrow stromal cells into porcine foetal tissues after xenotransplantation.

Bovine bone marrow stromal cells (BMSCs) were injected into the liver of foetal pigs at about 40 days of gestation to test whether these cells could populate developing tissue, and if so, which ones. Approximately 40 days after injection, the foetuses were harvested and tissue sections from many areas of the body were analysed for the presence of bovine cells using two different methods. First, using PCR, bovine repetitive DNA was found to be present in DNA extracted from foetal pig tissues. Secondly, using oligonucleotide primed in situ synthesis (PRINS), the in situ presence of bovine cells was found within porcine tissue sections. PRINS-labelled cells were found within cartilage, perichondrium, connective tissue and smooth muscle. These data suggest that bovine BMSCs integrate throughout the foetal pig.

A novel method for preparing histology slides without a microtome.

A study was undertaken to determine if it might be possible to prepare tissue sections on slides without the use of paraffin embedding, microtome sectioning, or cryostat sectioning which involve equipment and training not always available to scholars or professionals wishing to examine tissue microscopically. After evaluating many different reagents, cutting instruments and solid supports, we developed a method involving application of super glue to a slide, adhering a section of tissue to it, cutting the tissue with a disposable microtome blade, staining the tissue and removing the superglue with a commercially available product. The sections are similar to those sectioned on a microtome, but do not at this time equal their quality. However, histoarchitecture is preserved and individual cell morphology is usually good. We conclude that this is a viable method for preparing histology sections without the use of a microtome or cryostat, something long thought impossible. We have dubbed the method 'RAMP' (Rapid Adhesive-Mediated Procedure).

Diabetic nephropathy with interstitial nephritis presenting with a false-positive anti-GBM antibody.

A 56-year-old male with DM and HTN presented with flank pain and nausea. Review of systems was negative, physical examination was notable for mild hypovolemia and laboratory revealed BUN 51 mg/dl, creatinine (Cr) 5.1 mg/dl (baseline 1.5), Westergren ESR 122 mm/h, fractional excretion of sodium 0.2% and UA positive for blood and protein. Despite volume resuscitation the Cr continued to rise. Urine sediment analysis revealed granular casts, renal tubular epithelial cells and a negative Hansel's stain. Hemodialysis was initiated with Cr 13.7 mg/ dl for dyspnea and dysgeusia. Subsequent laboratory data revealed 2 separate positive anti-GBM antibody titers and prednisone therapy was initiated. Renal biopsy was performed for further diagnostic, therapeutic and prognostic information and demonstrated interstitial nephritis with linear IgG and albumin deposition consistent with diabetic nephropathy. Follow-up antibody titers were negative. prednisone was discontinued and Cr stabilized with conservative therapy. Anti-GBM antibody disease is characterized by circulating IgG antibodies directed against the glomerular basement membrane, specifically the alpha-3 (IV) collagen chain. Anti-GBM nephritis is a rapidly progressive, isolated glomerulonephritis in association with circulating anti-GBM antibodies. A positive immunofluorescence (IF) test is considered diagnostic in the appropriate clinical setting. Therapies include immunosuppressive agents to suppress new antibody production and plasmapheresis to eliminate circulating antibodies. Anti-GBM antibody is not rapidly cleared by steroid therapy and the recovery of renal function is rare if initiation Cr is greater than 7 mg/dl. This case demonstrates that the current ELISA for alpha-3 (IV) collagen is not pathognomonic for anti-GBM nephritis and that renal biopsy with IF for IgG and albumin may be indicated to prevent administration of potentially toxic treatment.

Neutrophil collagenase (MMP-8) is expressed during early development in neural crest cells as well as in adult melanoma cells.

Matrix metalloproteinase-8 (MMP-8) is a neutral metalloproteinase of the fibrillar collagenase family that also includes MMP-1 and MMP-13. In contrast to the other collagenases, MMP-8 has a very limited tissue distribution, thought to be restricted to neutrophils and chondrocytes. In a previous study, we observed MMP-8 expression in human melanoma cells. This observation led us to assess in more detail the expression of MMP-8 in normal and malignant melanocytic cells. We found that MMP-8 was expressed by 11 out of 12 human melanoma cell lines tested and all 10 primary melanomas we examined, but was not expressed by four primary neonatal melanocyte strains. Since melanocytes arise from highly motile neural crest cells, we examined the hypothesis that MMP-8 might be expressed by neural crest cells. RT-PCR analysis of post-implantation mouse embryos indicated the presence of MMP-8 transcripts at E9.5. In situ hybridization and immunohistochemistry of mouse embryos between E9.5-E14.5 demonstrated MMP-8 expression in the surface ectoderm, neural crest cells and chondrocytes. MMP-8 was also detected in neural crest cell migration located in the circumference of the neural tube, branchial arches and the notochord. In addition, MMP-8 expression was observed between the somites, in circumscriptive areas of the developing brain, heart, and eye, and in the interdigital zones of the limbs. In summary, we found MMP-8 to be the first fibrillar collagenase expressed during development. In contrast to its restricted tissue expression post-partum, MMP-8 was present in multiple embryonic tissues, including neural crest cells. The production of MMP-8 by migrating neural crest cells may contribute to their ability to degrade fibrillar collagen matrices while in transit.

Life span is prolonged in food-restricted autoimmune-prone (NZB x NZW)F(1) mice fed a diet enriched with (n-3) fatty acids.

Moderate food and/or energy (calorie) restriction delays age-related immune dysfunction and prolongs life span in multiple animal models. The amount and type of dietary fatty acids can also profoundly affect life span. Marine-derived fish oils contain (n-3) fatty acids, which have potent anti-inflammatory properties. We therefore examined the influence of food restriction (40% overall reduction in intake of all dietary components) combined with substitution of fish oil for corn oil in a factorial design. Autoimmune-prone (NZB x NZW)F(1) (B/W) mice, which develop fatal autoimmune renal disease, were used. The food-restricted/fish oil diet maximally extended median life span to 645 d (vs. 494 d for the food-restricted corn oil diet). Similarly, fish oil prolonged life span in the ad libitum-fed mice to 345 d (vs. 242 for the ad libitum/corn oil diet). Increased life span was partially associated with decreased body weight, blunting renal proinflammatory cytokine (interferon-gamma, interleukins-10 and -12 and tumor necrosis factor-alpha) levels and lower nuclear factor-kappaB (NF-kappaB). Reductions in NF-kappaB were preceded by enhanced superoxide dismutase, catalase and glutathione peroxidase activities. These findings demonstrate the profound additive effects of food restriction and (n-3) fatty acids in prolonging life span in B/W mice. These observations may have additional implications in the management of obesity, diabetes, cancer and/or the aging process.

Demonstration of the extrinsic coagulation pathway in teleostei: identification of zebrafish coagulation factor VII.

It is not known whether the mammalian mechanism of coagulation initiation is conserved in fish. Identification of factor VII is critical in providing evidence for such a mechanism. A cDNA was cloned from a zebrafish (teleost) library that predicted a protein with sequence similarity to human factor VII. Factor VII was shown to be present in zebrafish blood and liver by Western blot analysis and immunohistochemistry. Immunodepletion of factor VII from zebrafish plasma selectively inhibited thromboplastin-triggered thrombin generation. Heterologous expression of zebrafish factor VII demonstrated a secreted protein (50 kDa) that reconstituted thromboplastin-triggered thrombin generation in immunodepleted zebrafish plasma. These results suggest conservation of the extrinsic coagulation pathway between zebrafish and humans and add credence to the zebrafish as a model for mammalian hemostasis. The structure of zebrafish factor VIIa predicted by homology modeling was consistent with the overall three-dimensional structure of human factor VIIa. However, amino acid disparities were found in the epidermal growth factor-2/serine protease regions that are present in the human tissue factor-factor VIIa contact surface, suggesting a structural basis for the species specificity of this interaction. In addition, zebrafish factor VII demonstrates that the Gla-EGF-EGF-SP domain structure, which is common to coagulation factors VII, IX, X, and protein C, was present before the radiation of the teleosts from the tetrapods. Identification of zebrafish factor VII significantly narrows the evolutionary window for development of the vertebrate coagulation cascade and provides insight into the structural basis for species specificity in the tissue factor-factor VIIa interaction.