Ultraviolet radiation intensity predicts the relative distribution of dermatomyositis and anti-Mi-2 autoantibodies in women.

OBJECTIVE: Because studies suggest that ultraviolet (UV) radiation modulates the myositis phenotype and Mi-2 autoantigen expression, we conducted a retrospective investigation to determine whether UV radiation may influence the relative prevalence of dermatomyositis and anti-Mi-2 autoantibodies in the US. METHODS: We assessed the relationship between surface UV radiation intensity in the state of residence at the time of onset with the relative prevalence of dermatomyositis and myositis autoantibodies in 380 patients with myositis from referral centers in the US. Myositis autoantibodies were detected by validated immunoprecipitation assays. Surface UV radiation intensity was estimated from UV Index data collected by the US National Weather Service. RESULTS: UV radiation intensity was associated with the relative proportion of patients with dermatomyositis (odds ratio [OR] 2.3, 95% confidence interval [95% CI] 0.9-5.8) and with the proportion of patients expressing anti-Mi-2 autoantibodies (OR 6.0, 95% CI 1.1-34.1). Modeling of these data showed that these associations were confined to women (OR 3.8, 95% CI 1.3-11.0 and OR 17.3, 95% CI 1.8-162.4, respectively) and suggests that sex influences the effects of UV radiation on autoimmune disorders. Significant associations were not observed in men, nor were UV radiation levels related to the presence of antisynthetase or anti-signal recognition particle autoantibodies. CONCLUSION: This first study of the distribution of myositis phenotypes and UV radiation exposure in the US showed that UV radiation may modulate the clinical and immunologic expression of autoimmune disease in women. Further investigation of the mechanisms by which these effects are produced may provide insights into pathogenesis and suggest therapeutic or preventative strategies.

The relationship of mutations in alpha- and beta-tubulin to microtubule assembly and anti-mitotic drug resistance

The mechanisms responsible for anti-cancer drug (including Taxol) treatment failure have not been identified. In cell culture model systems, many β-tubulin, but very few α-tubulin, mutations have been associated with resistance to Taxol. To test what, if any, mutations in α-tubulin can cause resistance, we transfected a randomly mutagenized α-tubulin cDNA into Chinese hamster ovary (CHO) cells and isolated drug resistant cell lines. A total of 12 mutations were identified in this way and all of them were confirmed to confer Taxol resistance. Furthermore, all cells expressing mutant α-tubulin had less microtubule polymer. Some cells also had abnormal nuclei and enlarged cell bodies. The data indicate that α-tubulin mutations confer Taxol resistance by disrupting microtubule assembly, a mechanism consistent with a large number of previously described β-tubulin mutations. ^ Because α- and β-tubulin are almost identical in their three dimensional structure, we hypothesized that mutations discovered in one subunit, when introduced into the other, would produce similar effects on microtubule assembly and drug resistance. 9 α- and 2 β-tubulin mutations were tested. The results were complex. Some mutations produced similar changes in microtubule assembly and drug resistance irrespective of the subunit in which they were introduced, but others produced opposite effects. Still one mutation produced resistance when present in one subunit, yet had no effect when present on the other; and one mutation that produced Taxol resistance when present in α-tubulin, resulted in assembly-defective tubulin when it was present in β-tubulin. The results suggest that in most cases, the same amino acid modification in α- and β-tubulin affects the microtubule structure and assembly in a similar way. ^ Finally, we tested whether three β-tubulin mutations found in patient tumors could confer resistance to Taxol by recreating the mutations in a β-tubulin cDNA and transfecting it into CHO cells. We found that all three mutations conferred Taxol resistance, but to different extents. Again, microtubule assembly in the transfectants was disrupted, suggesting that mutations in β-tubulin are a potential problem in cancer therapeutics. ^

Platelets and Anti-Angiogenic Resistance in Ovarian Carcinoma

Background: Resistance to targeted anti-angiogenic therapy is a growing clinical concern given the disappointing clinical impact of anti-angiogenic. Platelets represent a component of the tumor microenvironment that are implicated in metastasis and represent a significant reservoir of angiogenic regulators. Thrombocytosis has been shown to be caused by malignancy and associated with adverse clinical outcomes, however the causal connections between these associations remain to be identified. Materials and Methods: Following IRB approval, patient data were collected on patients from four U.S. centers and platelet levels through and after therapy were considered as indicators of recurrence of disease. In vitro effects of platelets on cancer cell proliferation, apoptosis, and migration were examined. RNA interference was used to query signaling pathways mediating these effects. The necessity of platelet activation for in vitro effect was analyzed. In vivo orthotopic models were used to query the impact of thrombocytosis and thrombocytopenia on the efficacy of cytotoxic chemotherapy, the effect of aspirin on thrombocytosis and cancer, and platelet effect on anti-angiogenic therapy. Results: Platelets were found to increase at the time of diagnosis of ovarian cancer recurrence in a pattern comparable to CA-125. Platelet co-culture increased proliferation, increased migration, and decreased apoptosis in all cell lines tested. RNA interference implicated platelet derived growth factor alpha (PDGFRA) and transforming growth factor beta-receptor 1 (TGFBR1) signaling. Biodistribution studies suggested minimal platelet sequestration of taxanes. Blockade of platelet activation blocked in vitro effects. In vivo, thrombocytosis blocked chemotherapeutic efficacy, thrombocytopenia increased chemotherapeutic efficacy, and aspirin therapy partially blocked the effects of thrombocytosis. In vivo, withdrawal of anti-angiogenic therapy caused loss of therapeutic benefit with evidence of accelerated disease growth. This effect was blocked by use of a small-molecule inhibitor of Focal Adhesion Kinase. Anti-angiogenic therapy was also associated with increased platelet infiltration into tumor that was not seen to the same degree in the control or FAK-inhibitor-treated mice. Conclusions: Platelets are active participants in the growth and metastasis of tumor, both directly and via facilitation of angiogenesis. Blocking platelets, blocking platelet activation, and blocking platelet trafficking into tumor are novel therapeutic avenues supported by this data. Copyright © 2012 Justin Neal Bottsford-Miller, all rights reserved.

Anti-glomerular basement membrane glomerulonephritis: Characterization of an epitope, antibody response, and the potential role of IL-4Ralpha in disease progression

Anti-Glomerular Basement Membrane Glomerulonephritis (anti-GBM GM) is one of the earliest described autoimmune disorders. Patients present with proteinuria, anti-GBM antibodies, and renal failure. Studies have implicated a T Helper 1 (TH1) response in disease induction and a T Helper 2 (TH2) response for disease progression. A 13 amino acid long peptide sequence spanning residues 28 through 40 [pCol(28–40)] of the Collagen IV α3 non-collagen domain (Col IV α3 NCD) is immunogenic and induces anti-GBM GN. In order to fully understand disease initiation, this peptide was further characterized. Peptides were created containing one amino acid substitution for the entire length of pCol(28–40) and induction of anti-GBM GN was monitored. When residues 31, 33, or 34 contained the substitution, anti-GBM GN was unable to be induced. Thus, residues 31, 33, and 34 of pCol(28–40) are required for induction of anti-GBM. Glomerular injury is observed as early as 14 days post anti-GBM GN induction. However, the presence of anti-GBM antibodies is not observed until 20 days post immunization. An enlarged lymph node adjacent to the diseased kidney exhibits B cell activation after renal injury and produces antibodies toward GBM. Thus, anti-GBM antibodies are a consequence of the initial renal injury. Differences between disease susceptible and disease resistant rat strains exist in the expression of IL-4Rα, a major player in the TH2 response. IL-4Rα signaling is regulated by soluble IL-4Rα (sIL-4Rα). Low expression levels of sIL-4Rα result in the stabilization of IL-4 binding, while elevated expression sequesters IL-4. Quantitative PCR experiments noted low siL-4Rα expression levels in disease susceptible rats. Induction of an immune response toward sIL-4Rα in this strain was responsible for delayed disease progression in 15 out of the 17 experimental animals. Antibody transfer and in vivo biological activity experiments confirmed that delayed disease development was due to anti-sIL-4Rα antibodies. Together these experiments indicate that a T-cell epitope is required for activation of a TH1 autoimmune response and anti-GBM antibodies are a consequence of renal injury. More importantly, a role for IL-4Rα signaling is implicated in the progression of anti-GBM GN. ^

The Role of Acidic Fibroblast Growth Factor and Fibroblast Growth Factor Receptor 4 in High Grade Serous Carcinoma

Background: High grade serous carcinoma whether ovarian, tubal or primary peritoneal, continues to be the most lethal gynecologic malignancy in the USA. Although combination chemotherapy and aggressive surgical resection has improved survival in the past decade the majority of patients still succumb to chemo-resistant disease recurrence. It has recently been reported that amplification of 5q31-5q35.3 is associated with poor prognosis in patients with high grade serous ovarian carcinoma. Although the amplicon contains over 50 genes, it is notable for the presence of several members of the fibroblast growth factor signaling axis. In particular acidic fibroblast growth factor (FGF1) has been demonstrated to be one of the driving genes in mediating the observed prognostic effect of the amplicon in ovarian cancer patients. This study seeks to further validate the prognostic value of fibroblast growth receptor 4 (FGFR4), another candidate gene of the FGF/FGFR axis located in the same amplicon. The emphasis will be delineating the role the FGF1/FGFR4 signaling axis plays in high grade serous ovarian carcinoma; and test the feasibility of targeting the FGF1/FGFR4 axis therapeutically. Materials and Methods: Spearman and Pearson correlation studies on data generated from array CGH and transcriptome profiling analyses on 51 microdissected tumor samples were used to identify genes located on chromosome 5q31-35.3 that showed significant correlation between DNA and mRNA copy numbers. Significant correlation between FGF1 and FGFR4 DNA copy numbers was further validated by qPCR analysis on DNA isolated from 51 microdissected tumor samples. Immunolocalization and quantification of FGFR4 expression were performed on paraffin embedded tissue samples from 183 cases of high-grade serous ovarian carcinoma. The expression was then correlated with clinical data to assess impact on survival. The expression of FGF1 and FGFR4 in vitro was quantified by real-time PCR and western blotting in six high-grade serous ovarian carcinoma cell lines and compared to those in human ovarian surface epithelial cells to identify overexpression. The effect of FGF1 on these cell lines after serum starvation was quantified for in vitro cellular proliferation, migration/invasion, chemoresistance and survival utilizing a combination of commercially available colorimetric, fluorometric and electrical impedance assays. FGFR4 expression was then transiently silenced via siRNA transfection and the effects on response to FGF1, cellular proliferation, and migration were quantified. To identify relevant cellular pathways involved, responsive cell lines were transduced with different transcription response elements using the Cignal-Lenti reporter system and treated with FGF1 with and without transient FGFR4 knock down. This was followed by western blot confirmation for the relevant phosphoproteins. Anti-FGF1 antibodies and FGFR trap proteins were used to attempt inhibition of FGF mediated phenotypic changes and relevant signaling in vitro. Orthotopic intraperitoneal tumors were established in nude mice using serous cell lines that have been previously transfected with luciferase expressing constructs. The mice were then treated with FGFR trap protein. Tumor progression was then followed via bioluminescent imaging. The FGFR4 gene from 52 clinical samples was sequenced to screen for mutations. Results: FGFR4 DNA and mRNA copy numbers were significantly correlated and FGFR4 DNA copy number was significantly correlated with that of FGF1. Survival of patients with high FGFR4 expressing tumors was significantly shorter that those with low expression(median survival 28 vs 55 month p< 0.001) In a multivariate cox regression model FGFR expression significantly increased risk of death (HR 2.1, p<0.001). FGFR4 expression was significantly higher in all cell lines tested compared to HOSE, OVCA432 cell line in particular had very high expression suggesting amplification. FGF1 was also particularly overexpressed in OVCA432. FGF1 significantly increased cell survival after serum deprivation in all cell lines. Transient knock down of FGFR4 caused significant reduction in cell migration and proliferation in vitro and significantly decreased the proliferative effects of FGF1 in vitro. FGFR1, FGFR4 traps and anti-FGF1 antibodies did not show activity in vitro. OVCA432 transfected with the cignal lenti reporter system revealed significant activation of MAPK, NFkB and WNT pathways, western blotting confirmed the results. Reverse phase protein array (RPPA) analysis also showed activation of MAPK, AKT, WNT pathways and down regulation of E Cadherin. FGFR trap protein significantly reduced tumor growth in vivo in an orthotopic mouse model. Conclusions: Overexpression and amplification of several members of the FGF signaling axis present on the amplicon 5q31-35.3 is a negative prognostic indicator in high grade serous ovarian carcinoma and may drive poor survival associated with that amplicon. Activation of The FGF signaling pathway leads to downstream activation of MAPK, AKT, WNT and NFkB pathways leading to a more aggressive cancer phenotype with increased tumor growth, evasion of apoptosis and increased migration and invasion. Inhibition of FGF pathway in vivo via FGFR trap protein leads to significantly decreased tumor growth in an orthotopic mouse model.

An examination of the characteristics, concentration and distribution of androgen receptor in rat testis during sexual maturation

Previously reported androgen receptor concentrations in rat testis and testicular cell types have varied widely. In the studies reported here a nuclear exchange assay was established in rat testis in which exchange after 86 hours at 4$\sp\circ$C was greater than 85% complete and receptor was stable. Receptor concentration per DNA measured by exchange declined between 15 and 25 days of age in the rat testis, then increased 4-fold during sexual maturation. Proliferation of germ cells which had low receptor concentration appeared to account for the early decline in testicular receptor concentration, whereas increase in receptor number per Sertoli cell between 25 and 35 days of age contributed to the later increase. Increase in Leydig cell number during maturation appeared to account for the remainder of the increase due to the high receptor concentration in these cells. Detailed studies showed that other possible explanations for changes in receptor number (e.g. shifts in receptor concentration between the cytosol and nuclear subcellular compartments or changes in the affinity of the receptor for its ligands) were not likely.^ Androgen receptor dynamics in testicular cells showed rapid, specific uptake of ($\sp3$H) -testosterone that was easily blocked by unlabeled testosterone (RA of 7 nM in both cell types), and medroxyprogesterone acetate (RA of 28 and 16 nM in Sertoli and peritubular cells, respectively), but not as well by the anti-androgens cyproterone acetate (RA of 116 and 68 nM) and hydroxyflutamide (RA of 300 and 180 nM). The affinity of the receptor for the ligand dimethylnortestosterone was similar in the two cell types (K$\rm\sb{d}$ values of 0.78 and 0.71 nM for Sertoli and peritubular cells) and was virtually identical with the affinity of the whole testis receptor (0.89 nM). Medroxyprogesterone acetate and testosterone significantly increased nuclear androgen receptor concentration relative to untreated controls in Sertoli and peritubular cells, whereas hydroxyflutamide and cyproterone acetate did not. Despite the different embryological origins of peritubular and Sertoli cells, their responses to both androgens and anti-androgens were similar. In addition, these studies suggest that peritubular cells are as likely as Sertoli cells to be primary androgen targets. ^

Structural and functional studies of the human integrin $\alpha 4\beta 1$ (CD49d/CD29)

The integrin receptor $\alpha 4\beta 1$ is a cell surface heterodimer involved in a variety of highly regulated cellular interactions. The purpose of this dissertation was to identify and characterize unique structural and functional properties of the $\alpha 4\beta 1$ molecule that may be important for adhesion regulation and signal transduction. To study these properties and to establish a consensus sequence for the $\alpha 4$ subunit, cDNA encoding $\alpha 4$ was cloned and sequenced. A comparison with previously described human $\alpha 4$ sequences identified several substitutions in the $5\prime$ and $3\prime$ untranslated regions, and a nonsynonymous G to A transition in the coding region, resulting in a glutamine substitution for arginine. Further analysis of this single nucleotide substitution indicated that two variants of the $\alpha 4$ subunit exist, and when compared with three ancestrally-related species, the new form cloned in our laboratory was found to be evolutionarily conserved.^ The expression of $\alpha 4$ cDNA in transfected K562 erythroleukemia cells, and subsequent studies using flow cytofluorometric, immunochemical, and ligand binding/blocking analyses, confirmed $\alpha 4\beta 1$ as a receptor for fibronectin (FN) and vascular cell adhesion molecule-1 (VCAM-1), and provided a practical means of identifying two novel monoclonal antibody (mAb) binding epitopes on the $\alpha 4\beta 1$ complex that may play important roles in the regulation of leukocyte adhesion.^ To investigate the association of $\alpha 4\beta 1$-mediated adhesion with signals involved in the spreading of lymphocytes on FN, a quantitative method of analysis was developed using video microscopy and digital imaging. The results showed that HPB-ALL $(\alpha 4\beta 1\sp{\rm hi},\ \alpha 5\beta 1\sp-)$ cells could adhere and actively spread on human plasma FN, but not on control substrate. Many cell types which express different levels of the $\alpha 4\beta 1$ and $\alpha 5\beta 1$ FN binding integrins were examined for their ability to function in these events. Using anti-$\alpha 4$ and anti-$\alpha 5$ mAbs, it was determined that cell adhesion to FN was influenced by both $\beta 1$ integrins, while cell spreading was found to be dependent on the $\alpha 4\beta 1$ complex. In addition, inhibitors of phospholipase A$\sb2$ (PLA$\sb2$), 5-lipoxygenases, and cyclooxygenases blocked HPB-ALL cell spreading, yet had no effect on cell adhesion to FN, and the impaired spreading induced by the PLA$\sb2$ inhibitor cibacron blue was restored by the addition of exogenous arachidonic acid (AA). These results suggest that the interaction of $\alpha 4\beta 1$ with FN, the activation of PLA$\sb2,$ and the subsequent release of AA, may be involved in lymphocyte spreading. ^

New aspects of estrogen action in the endometrium: Reciprocal interplay with retinoic acid pathway and a novel estrogen-regulated gene

The uterine endometrium is a major target for the estrogen. However, the molecular basis of estrogen action in the endometrium is largely unknown. I have used two approaches to study the effects of estrogen on the endometrium. One approach involved the study of the interaction between estrogen and retinoic acid (RA) pathways in the endometrium. I have demonstrated that estrogen administration to rodents and estrogen replacement therapy (ERT) in postmenopausal women selectively induced the endometrial expression of retinaldehyde dehydrogenase II (RALDH2), a critical enzyme of RA biosynthesis. RALDH2 was expressed exclusively in the stromal cells, especially in the stroma adjacent to the luminal and glandular epithelia. The induction of RALDH2 by estrogen required estrogen receptor and occurred via a direct increase in RALDH2 transcription. Among the three RA receptors, estrogen selectively induced the expression of RARα. In parallel, estrogen also increased the utilization of all-trans retinol (the substrate for RA biosynthesis) and the expression of two RA-regulated marker genes, cellular retinoic acid binding protein II (CRABP2) and tissue transglutaminase (tTG) in the endometrium. Thus estrogen coordinately upregulated both the production and signaling of RA in both the rodent and human endometrium. This coordinate upregulation of RA system appeared to play a role in counterbalancing the stimulatory effects of estrogen on the endometrium, since the depletion of endogenous RA in mice led to an increase in estrogen-stimulated stromal proliferation and endometrial Akt phosphorylation. In addition, I have also used a systematic approach (DNA microarray) to categorize genes and pathways affected by the ERT in the endometrium of postmenopausal women and identified a novel estrogen-regulated gene EIG121. EIG121 was exclusively expressed in the glandular epithelial cells of the endometrium and induced by estrogen in vivo and in cultured cell lines. Compared with the normal endometrium, EIG121 was highly overexpressed in type 1 endometrial cancer, but profoundly suppressed in type 2 endometrial tumors. Taken together, these studies suggested that estrogen regulates the expression of many genes of both the pro-proliferative and anti-proliferative pathways and the abnormality of these pathways may increase the risks for estrogen-dependent endometrial hyperplasia and endometrial cancer. ^

Nitric oxide contributes to LPS/IFN-gamma-induced macrophage apoptosis via JNK and BCL-2 family regulation

Lipopolysaccharide (LPS) and interferon-gamma (IFN) activate macrophages and produce nitric oxide (NO) by initiating the expression of inducible Nitric Oxide Synthase (iNOS). Prolonged LPS/IFN-activation results in the death of macrophage-like RAW 264.7 cells and wild-type murine macrophages. This study was implemented to determine how NO contributes to LPS/IFN-induced macrophage death. The iNOS-specific inhibitor L-NIL protected RAW 264.7 cells from LPS/IFN-activated death, supporting a role for NO in the death of LPS/IFN-activated macrophages. A role for iNOS in cell death was confirmed in iNOS-/- macrophages which were resistant to LPS/IFN-induced death. Cell death was accompanied by nuclear condensation, caspase 3 activation, and PARP cleavage, all of which are hallmarks of apoptosis. The involvement of NO in modulating the stress-activated protein kinase (SAPK)/c-jun N-terminal kinase (JNK) signal transduction pathway was examined as a possible mechanism of LPS/IFN-mediated apoptosis. Western analysis demonstrated that NO modifies the phosphorylation profile of JNK and promotes activation of JNK in the mitochondria in RAW 264.7 cells. Inhibition of JNK with sIRNA significantly reduced cell death in RAW 264.7 cells, indicating the participation of the JNK pathway in LPS/IFN-mediated death. JNK has been demonstrated to induce mitochondrial-mediated apoptosis through modulation of Bcl-2 family members. Therefore, the effect of NO on the balance between pro- and anti-apoptotic Bcl-2 family members was examined. In RAW 264.7 cells, Bim was upregulated and phosphorylated by LPS/IFN independently of NO. However, co-immunoprecipitation studies demonstrated that NO promotes the association of Bax with the BimL splice variant. Examination of Bax phosphorylation by metabolic labeling demonstrated that Bax is basally phosphorylated and becomes dephosphorylated upon LPS/IFN treatment. L-NIL inhibited the dephosphorylation of Bax, indicating that Bax dephosphorylation is NO-dependent. NO also mediated LPS/IFN-induced downregulation of Mcl-1, an anti-apoptotic Bcl-2 family member, as demonstrated by Western blotting for Mcl-1 protein expression. Thus, NO contributes to macrophage apoptosis via a JNK-mediated mechanism involving interaction between Bax and Bim, dephosphorylation of Bax, and downregulation of Mcl-1. ^

Significance of signal transducer and activator of transcription 3 during multistage mouse skin carcinogenesis

Signal transducer and activator of transcription 3 (Stat3) is a signaling molecule that transduces signal from cell surface receptors, itself translocates into the nucleus, binds to consensus promoter sequences and activates gene transcription. Here, we showed that Stat3 is constitutively activated in both premalignant tumors (papillomas) and squamous cell carcinomas of mouse skin that is induced by topical treatment with an initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by a tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Additional data demonstrated that epidermal growth factor signaling contributes to the activation of Stat3 in this model. Using mice where Stat3 function is abrogated in keratinocytes via the Cre-LoxP system (K5Cre.Stat3 flox/flox), we demonstrated that Stat3 is required for de novo carcinogenesis since Stat3 deficiency leads to a complete abrogation of skin tumor development induced by DMBA and TPA. We subsequently showed that Stat3 plays a role in both the initiation and promotion stages of carcinogenesis. During initiation, Stat3 functions as an anti-apoptotic molecule for maintaining the survival of DNA-damaged keratinocyte stem cells. During promotion, Stat3 functions as a critical regulator for G1 to S phase cell cycle progression to confer selective clonal expansion of initiated cells into papillomas. On the other hand, using transgenic mice over-expressing a constitutively dimerized form of Stat3 (Stat3C) in keratinocytes (K5.Stat3C), we revealed a role for Stat3 in tumor progression. After treatment with DMBA and TPA, K5.Stat3C transgenic mice developed skin tumors with a shorter latency when 100% bypassed the premalignant stage and became carcinoma in situ. Histological and immunohistochemical analysis revealed these tumors as highly vascularized and poorly differentiated. More strikingly, these tumors exhibited invasion into surrounding mesenchymal tissue, some of which metastasized into lung. The tumor-mesenchymal front was characterized by partial loss of E-cadherin and elevation of vimentin, markers characterizing epithelial-mesenchymal transition. On the other hand, inhibition of Stat3 via a decoy oligonucleotide led to a significant reduction of tumor size in approximately 50% of all papillomas tested. In conclusion, we demonstrated that Stat3 plays a critical in all three stages (initiation, promotion and progression) of skin carcinogenesis, and it may potentially become a good target for cancer prevention and anti-cancer therapy. ^

Establishment of a complex inflammatory and protumorigenic microenvironment in mouse pancreas through cyclooxygenase-2 overexpression

Chronic inflammation is an established risk factor in the pathogenesis of many cancers. Pancreatic ductal adenocarcinoma, a malignancy with a particularly dismal prognosis, is no exception. Cyclooxygenase-2, a key enzyme induced by tissue injury, has a critical role in the generation of bioactive lipids known as prostaglandins. COX-2 overexpression is a frequent finding in pancreatic cancer, chronic pancreatitis and pancreatic intraepithelial neoplasias. To explore mechanisms through which chronic inflammation establishes and maintains a protumorigenic environment, we designed a mouse model overexpressing COX-2 in pancreatic parenchyma (BK5.COX-2 mice). We discovered that constitutive expression of COX-2 has a number of important sequelae, including upregulation of additional eicosanoid-generating enzymes and proinflammatory cytokines. Many of these molecular alterations precede the onset of significant histopathological changes. Increased levels of prostaglandins E2, D2, and F2α, 5-, 12-, and 15-hydroxyeiosatetraenoic acid (HETEs) were documented in tumors and pancreata of younger transgenic mice. Using a TaqMan™ Mouse Immune Panel, we detected elevated mRNAs for a number of proinflammatory cytokines (e.g., TNFα, IL-1β, IL-6). ^ Histological examination revealed early changes in the pancreas with similarities to human chronic pancreatitis, including loss of acinar cells, appearance of metaplastic ducts, and increased deposition of stroma. As the lesions progress, features typical of dysplastic and neoplastic cells emerged within the metaplastic ductal complexes, including cellular and nuclear atypia, crowding of cells, and loss of normal tissue architecture. The amount of fibroinflammatory stroma increased considerably; numerous small vessels were evident. A number of immunocytes from both the myeloid and lymphoid lineages were identified in transgenic pancreata. Neutrophils were the earliest to infiltrate, followed shortly by macrophages and mast cells. B and T cells generally began to appear by 8–12 weeks, and organized aggregates of lymphoid cells were often found in advanced lesions. ^ We tested the efficacy of several chemopreventive agents in this model, including celecoxib, a COX-2 selective inhibitor, pentoxifylline, a cytokine inhibitor, curcumin, a polyphenol with antioxidant and anti-inflammatory properties, and GW2974, a dual EGFR/ErbB2 inhibitor. Effects on lesion development were modest in the GW2974 and pentoxifylline treated groups, but significant prevention effects were observed with curcumin and celecoxib. ^

In vivo longitudinal studies of spinal cord injury and vascular endothelial growth factor treatment

Approximately 12,000 new cases of spinal cord injury (SCI) are added each year to the estimated 259,000 Americans living with SCI. The majority of these patients return to society, their lives forever changed by permanent loss of sensory and motor function. While there are no FDA approved drugs for the treatment of SCI or a universally accepted standard therapy, the current though controversial treatment includes the delivery of high dosages of the corticosteroid methyliprednisolone sodium succinate, surgical interventions to stabilize the spinal column, and physical rehabilitation. It is therefore critically important to fully understand the pathology of injury and determine novel courses and rationally-based therapies for SCI. ^ Vascular endothelial growth factor (VEGF) is an attractive target for treating central nervous system (CNS) injury and disease because it has been shown to influence angiogenesis and neuroprotection. Preliminary studies have indicated that increased vasculature may be associated with functional recovery; therefore exogenous delivery of a pro-angiogenic growth factor such as VEGF may improve neurobehavioral outcome. In addition, VEGF may provide protection from secondary injury and result in increased survival and axonal sprouting. ^ In these studies, SCI rats received acute intraspinal injections of VEGF, the antibody to VEGF, or vehicle control. The effect of these various agents was investigated using longitudinalmulti-modal magnetic resonance imaging (MRI), neuro- and sensory behavioral assays, and end point immunohistochemistry. We found that rats that received VEGF after SCI had increased tissue sparing and improved white matter integrity at the earlier time points as shown by advanced magnetic resonance imaging (MRI) techniques. However, these favorable effects of VEGF were not maintained, suggesting that additional treatments with VEGF at multiple time points may be more beneficial, Histological examinations revealed that VEGF treatment may result in increased oligodendrogenesis and therefore may eventually lead to remyelination and improved functional outcome. ^ On the neurobehavioral studies, treatments with VEGF and Anti-VEGF did not significantly affect performance on tests of open-field locomotion, grid walk, inclined plane, or rearing. However, VEGF treatment resulted in significantly increased incidence of chronic neuropathic pain. This phenomenon could possibly be attributed to the fact that VEGF treatment may promote axonal sprouting and also results in tissue sparing, thereby providing a substrate for the growth of new axons. New connections made by these sprouting axons may involve components of pathways involved in the transmission of pain and therefore result in increased pain in those animals. ^

Immune response to hepatitis B vaccination in drug using populations: A systematic review and meta-regression analysis

Hepatitis B virus (HBV) is a significant cause of liver diseases and related complications worldwide. Both injecting and non-injecting drug users are at increased risk of contracting HBV infection. Scientific evidence suggests that drug users have subnormal response to HBV vaccination and the seroprotection rates are lower than that in the general population; potentially due to vaccine factors, host factors, or both. The purpose of this systematic review is to examine the rates of seroprotection following HBV vaccination in drug using populations and to conduct a meta-analysis to identify the factors associated with varying seroprotection rates. Seroprotection is defined as developing an anti-HBs antibody level of ≥ 10 mIU/ml after receiving the HBV vaccine. Original research articles were searched using online databases and reference lists of shortlisted articles. HBV vaccine intervention studies reporting seroprotection rates in drug users and published in English language during or after 1989 were eligible. Out of 235 citations reviewed, 11 studies were included in this review. The reported seroprotection rates ranged from 54.5 – 97.1%. Combination vaccine (HAV and HBV) (Risk ratio 12.91, 95% CI 2.98-55.86, p = 0.003), measurement of anti-HBs with microparticle immunoassay (Risk ratio 3.46, 95% CI 1.11-10.81, p = 0.035) and anti-HBs antibody measurement at 2 months after the last HBV vaccine dose (RR 4.11, 95% CI 1.55-10.89, p = 0.009) were significantly associated with higher seroprotection rates. Although statistically nonsignificant, the variables mean age>30 years, higher prevalence of anti-HBc antibody and anti-HIV antibody in the sample population, and current drug use (not in drug rehabilitation treatment) were strongly associated with decreased seroprotection rates. Proportion of injecting drug users, vaccine dose and accelerated vaccine schedule were not predictors of heterogeneity across studies. Studies examined in this review were significantly heterogeneous (Q = 180.850, p = 0.000) and factors identified should be considered when comparing immune response across studies. The combination vaccine showed promising results; however, its effectiveness compared to standard HBV vaccine needs to be examined systematically. Immune response in DUs can possibly be improved by the use of bivalent vaccines, booster doses, and improving vaccine completion rates through integrated public programs and incentives.^

Development of HIF-1α/HIF-1β heterodimerization inhibitors using a novel bioluminescence reporter assay system for in vitro high throughput screening and in vivo imaging

Tumor growth often outpaces its vascularization, leading to development of a hypoxic tumor microenvironment. In response, an intracellular hypoxia survival pathway is initiated by heterodimerization of hypoxia-inducible factor (HIF)-1α and HIF-1β, which subsequently upregulates the expression of several hypoxia-inducible genes, promotes cell survival and stimulates angiogenesis in the oxygen-deprived environment. Hypoxic tumor regions are often associated with resistance to various classes of radio- or chemotherapeutic agents. Therefore, development of HIF-1α/β heterodimerization inhibitors may provide a novel approach to anti-cancer therapy. To this end, a novel approach for imaging HIF-1α/β heterodimerization in vitro and in vivo was developed in this study. Using this screening platform, we identified a promising lead candidate and further chemically derivatized the lead candidate to assess the structure-activity relationship (SAR). The most effective first generation drug inhibitors were selected and their pharmacodynamics and anti-tumor efficacy in vivo were verified by bioluminescence imaging (BLI) of HIF-1α/β heterodimerization in the xenograft tumor model. Furthermore, the first generation drug inhibitors, M-TMCP and D-TMCP, demonstrated efficacy as monotherapies, resulting in tumor growth inhibition via disruption of HIF-1 signaling-mediated tumor stromal neoangiogenesis.

Modulation of cytochrome P450 enzyme activity and PAH -induced skin carcinogenesis by naturally occurring coumarins

The present study was designed to determine the potential anticarcinogenic activity of naturally occurring coumarins and their mechanism of action. The results indicated that several naturally occurring coumarins including bergamottin, coriandrin, imperatorin, isopimpinellin, and ostruthin, to which humans are routinely exposed in the diet, were effective inhibitors and/or inactivators of CYP1A1-mediated ethoxyresorufin-O-dealkylase (EROD) or CYP2B1-mediated pentoxyresorufin-O-dealkylase (PROD) in mouse liver microsomes. In addition, bergamottin and corandrin were also found to be inhibitors of purified human P450 1A1 in vitro. Further studies with coriandrin revealed that this compound was a mechanism-based inactivator of P450 1A1 and covalently bound to the P450 1A1 apoprotein. In cultured mouse keratinocytes, bergamottin and coriandrin effectively inhibited the B(a) P metabolism and significantly decreased covalent binding of B(a) P and DMBA to keratinocyte DNA and anti-diol-epoxide-DNA adducts derived from both B(a) P and DMBA in keratinocytes. The data from in vivo experiments showed that bergamottin and coriandrin were potent inhibitors of covalent binding of B (a) P to epidermal DNA and the formation of (+) anti BPDE-DNA adduct, whereas imperatorin and isopimpinellin were more potent inhibitors of covalent binding of DMBA to epidermal DNA. The ability of coumarins to inhibit covalent binding of B (a) P to DNA in mouse epidermis was positively correlated with their inhibitory effect P450 1A1 in vitro, while the inhibitory effect of coumarins on covalent binding of DMBA to epidermal DNA was positively correlated with their inhibitory effects on P450 2B1 and negatively to their inhibitory activity toward P450 1A1. The data from tumor experiments indicated that bergamottin, ostruthin, and coriandrin inhibited tumor initiation by B (a) P in a two-stage carcinogenesis protocol. Bergamottin was most effective in this regard and produced a dose dependent inhibition of papilloma formation in these experiments. In addition, imperatorin was an effective inhibitor of skin tumorigenesis induced by DMBA in SENCAR mouse skin using both a two-stage and a complete carcinogenesis protocol. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B (a) P. The results to date demonstrate that several naturally occurring coumarins possess the ability to block tumor initiation and tumorigenesis by PAHs such as B (a) P and DMBA through inhibition of the P450s involved in the metabolic activation of these hydrocarbons. A working model for the involvement of specific P450s in the metabolic activation of these two PAHs was proposed. ^