Recent advances in understanding Marfan syndrome: should we now treat surgical patients with losartan?

OBJECTIVE: Marfan syndrome is a systemic connective tissue disorder caused by mutations in the fibrillin-1 gene. It was originally believed that Marfan syndrome results exclusively from the production of abnormal fibrillin-1 that leads to structurally weaker connective tissue when incorporated into the extracellular matrix. This effect seemed to explain many of the clinical features of Marfan syndrome, including aortic root dilatation and acute aortic dissection, which represent the main causes of morbidity and mortality in Marfan syndrome. METHODS: Recent molecular studies, most based on genetically defined mouse models of Marfan syndrome, have challenged this paradigm. These studies established the critical contribution of fibrillin-1 haploinsufficiency and dysregulated transforming growth factor-beta signaling to disease progression. RESULTS: It seems that many manifestations of Marfan syndrome are less related to a primary structural deficiency of the tissues than to altered morphogenetic and homeostatic programs that are induced by altered transforming growth factor-beta signaling. Most important, transforming growth factor-beta antagonism, through transforming growth factor-beta neutralizing antibodies or losartan (an angiotensin II type 1 receptor antagonist), has been shown to prevent and possibly reverse aortic root dilatation, mitral valve prolapse, lung disease, and skeletal muscle dysfunction in a mouse model of Marfan syndrome. CONCLUSION: There are indicators that losartan, a drug widely used to treat arterial hypertension in humans, offers the first potential for primary prevention of clinical manifestations in Marfan syndrome.

Tmem27: a cleaved and shed plasma membrane protein that stimulates pancreatic beta cell proliferation

The signals and molecular mechanisms that regulate the replication of terminally differentiated beta cells are unknown. Here, we report the identification and characterization of transmembrane protein 27 (Tmem27, collectrin) in pancreatic beta cells. Expression of Tmem27 is reduced in Tcf1(-/-) mice and is increased in islets of mouse models with hypertrophy of the endocrine pancreas. Tmem27 forms dimers and its extracellular domain is glycosylated, cleaved and shed from the plasma membrane of beta cells. This cleavage process is beta cell specific and does not occur in other cell types. Overexpression of full-length Tmem27, but not the truncated or soluble protein, leads to increased thymidine incorporation, whereas silencing of Tmem27 using RNAi results in a reduction of cell replication. Furthermore, transgenic mice with increased expression of Tmem27 in pancreatic beta cells exhibit increased beta cell mass. Our results identify a pancreatic beta cell transmembrane protein that regulates cell growth of pancreatic islets.

Increased numbers of spontaneous SCLC metastasis in absence of NK cells after subcutaneous inoculation of different SCLC cell lines into pfp/rag2 double knock out mice

Spontaneous metastases in small cell lung cancer (SCLC) occur regularly in patients but seldom if any in conventional xenograft mouse models. To overcome this problem, SCLC cells were grafted subcutaneously onto pore forming protein and recombination activating gene 2 double knock out (pfp/rag2) mice and in severe combined immunodeficient (scid) mice. Primary tumours grew well in both mouse strains, while metastases occurred frequently in the pfp/rag2 mice and infrequently in scid mice. Hence NK cells, which are inactive in pfp/rag2 mice, play an important role in SCLC metastasis formation in xenograft models. This observation is in agreement with clinical studies, where a high NK cell number in the blood is correlated with a better prognosis of the patient.

Human urinary metabolomic profile of PPARalpha induced fatty acid beta-oxidation

Activation of the peroxisome proliferator-activated receptor alpha (PPARalpha) is associated with increased fatty acid catabolism and is commonly targeted for the treatment of hyperlipidemia. To identify latent, endogenous biomarkers of PPARalpha activation and hence increased fatty acid beta-oxidation, healthy human volunteers were given fenofibrate orally for 2 weeks and their urine was profiled by UPLC-QTOFMS. Biomarkers identified by the machine learning algorithm random forests included significant depletion by day 14 of both pantothenic acid (>5-fold) and acetylcarnitine (>20-fold), observations that are consistent with known targets of PPARalpha including pantothenate kinase and genes encoding proteins involved in the transport and synthesis of acylcarnitines. It was also concluded that serum cholesterol (-12.7%), triglycerides (-25.6%), uric acid (-34.7%), together with urinary propylcarnitine (>10-fold), isobutyrylcarnitine (>2.5-fold), (S)-(+)-2-methylbutyrylcarnitine (5-fold), and isovalerylcarnitine (>5-fold) were all reduced by day 14. Specificity of these biomarkers as indicators of PPARalpha activation was demonstrated using the Ppara-null mouse. Urinary pantothenic acid and acylcarnitines may prove useful indicators of PPARalpha-induced fatty acid beta-oxidation in humans. This study illustrates the utility of a pharmacometabolomic approach to understand drug effects on lipid metabolism in both human populations and in inbred mouse models.

Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites

We investigated the protective potential of recombinant his-tagged antigens recNcMIC1, recNcMIC3 and recNcROP2, applied either as single vaccines or as vaccine combinations, in BALB/c mouse models for cerebral and fetal infection. Subsequently, mice were mated and challenged by i.p. inoculation of 2 x 10(6)Neospora caninum tachyzoites at day 7 of pregnancy. The mortality and morbidity of adult mice (non-pregnant and dams) and of the newborn pups was studied for a period of 40 days following birth. Vaccination of non-pregnant mice with recNcROP2 or combinations of recNcROP2 with recNcMIC antigens significantly reduced the numbers of mice suffering from clinical signs, and morbidity was completely prevented with the combination of all three antigens. Of the dams, the groups receiving either recNcROP2 alone or the combination of all three antigens did not exhibit any morbidity, the groups receiving ROP2 mixed with either MIC1 or MIC3 exhibited reduced numbers of deaths, and in the infection control group and the adjuvant group 50% and 43% of mice, respectively, succumbed to disease. For pups, the highest survival rates were noted for the groups receiving recNcROP2 (50%) and recNcROP2/NcMIC1/NcMIC3 (35%), while in the infection- and adjuvant- control groups all pups died, the latest at days 25 and 30, respectively. Quantification of parasite DNA by N. caninum-specific real-time PCR revealed consistently lower parasite burdens in brain tissue of pups from vaccinated groups compared with the controls. However, dense granule antigen 2 (GRA2) real-time reverse transcriptase-PCR on brain tissue of surviving pups (applied here to detect viable parasites) demonstrated that only the pups from the group vaccinated with all three antigens in combination appeared free of viable tachyzoites, while in all other groups viable parasites were still present. Serological analysis of humoral (total IgG, IgG1 and IgG2a) and serum cytokine (IL-4 and IFN-gamma) responses showed that this effect was associated with a Th-2-biased immune response, with a clearly elevated IL-4/IFN-gamma ratio in the mice receiving all three antigens in combination. In conclusion, a mixture of recombinant antigens representing important secretory micronemal and rhoptry proteins leads to a significant protection against vertical transmission of N. caninum in mice.

Loss-of-function mutations in the KCNJ8-encoded Kir6.1 K(ATP) channel and sudden infant death syndrome.

BACKGROUND Approximately 10% of sudden infant death syndrome (SIDS) may stem from cardiac channelopathies. The KCNJ8-encoded Kir6.1 (K(ATP)) channel critically regulates vascular tone and cardiac adaptive response to systemic metabolic stressors, including sepsis. KCNJ8-deficient mice are prone to premature sudden death, particularly with infection. We determined the spectrum, prevalence, and function of KCNJ8 mutations in a large SIDS cohort. METHODS AND RESULTS Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, comprehensive open reading frame/splice-site mutational analysis of KCNJ8 was performed on genomic DNA isolated from necropsy tissue on 292 unrelated SIDS cases (178 males, 204 white; age, 2.9±1.9 months). KCNJ8 mutations were coexpressed heterologously with SUR2A in COS-1 cells and characterized using whole-cell patch-clamp. Two novel KCNJ8 mutations were identified. A 5-month-old white male had an in-frame deletion (E332del) and a 2-month-old black female had a missense mutation (V346I). Both mutations localized to Kir6.1's C-terminus, involved conserved residues and were absent in 400 and 200 ethnic-matched reference alleles respectively. Both cases were negative for mutations in established channelopathic genes. Compared with WT, the pinacidil-activated K(ATP) current was decreased 45% to 68% for Kir6.1-E332del and 40% to 57% for V346I between -20 mV and 40 mV. CONCLUSIONS Molecular and functional evidence implicated loss-of-function KCNJ8 mutations as a novel pathogenic mechanism in SIDS, possibly by predisposition of a maladaptive cardiac response to systemic metabolic stressors akin to the mouse models of KCNJ8 deficiency.

The urea transporter family (SLC14): physiological, pathological and structural aspects

Urea transporters (UTs) belonging to the solute carrier 14 (SLC14) family comprise two genes with a total of eight isoforms in mammals, UT-A1 to -A6 encoded by SLC14A2 and UT-B1 to -B2 encoded by SLC14A1. Recent efforts have been directed toward understanding the molecular and cellular mechanisms involved in the regulation of UTs using transgenic mouse models and heterologous expression systems, leading to important new insights. Urea uptake by UT-A1 and UT-A3 in the kidney inner medullary collecting duct and by UT-B1 in the descending vasa recta for the countercurrent exchange system are chiefly responsible for medullary urea accumulation in the urinary concentration process. Vasopressin, an antidiuretic hormone, regulates UT-A isoforms via the phosphorylation and trafficking of the glycosylated transporters to the plasma membrane that occurs to maintain equilibrium with the exocytosis and ubiquitin-proteasome degradation pathways. UT-B isoforms are also important in several cellular functions, including urea nitrogen salvaging in the colon, nitric oxide pathway modulation in the hippocampus, and the normal cardiac conduction system. In addition, genomic linkage studies have revealed potential additional roles for SLC14A1 and SLC14A2 in hypertension and bladder carcinogenesis. The precise role of UT-A2 and presence of the urea recycling pathway in normal kidney are issues to be further explored. This review provides an update of these advances and their implications for our current understanding of the SLC14 UTs.

Molecular basis of Corynebacterium diphtheriae virulence and infection in the Caenorhabditis elegans model host

Corynebacterium diphtheriae is the causative agent of cutaneous and pharyngeal diphtheria in humans. While lethality is certainly caused by diphtheria toxin, corynebacterial colonization may primarily require proteinaceous fibers called pili, which mediate adherence to specific tissues. The type strain of C. diphtheriae possesses three distinct pilus structures, namely the SpaA, SpaD, and SpaH-type pili, which are encoded by three distinct pilus gene clusters. The pilus is assembled onto the bacterial peptidoglycan by a specific transpeptidase enzyme called sortase. Although the SpaA pili are shown to be specific for pharyngeal cells in vitro, little is known about functions of the three pili in bacterial pathogenesis. This is mainly due to lack of in vivo models of corynebacterial infection. As an alternative to mouse models as mice do not have functional receptors for diphtheria toxin, in this study I use Caenorhabditis elegans as a model host for C. diphtheriae. A simple C. elegans model would be beneficial in determining the specific role of each pilus-type and the literature suggests that C. elegans infection model can be used to study a variety of bacterial species giving insight into bacterial virulence and host-pathogen interactions. My study examines the hypothesis that pili and toxin are major virulent determinants of C. diphtheriae in the C. elegans model host.

THE ROLE OF PLATELETS IN OVARIAN CARCINOMA

Platelets represent one of the largest storage pools of angiogenic and oncogenic growth factors in the human body. The observation that thrombocytosis (platelet count >450,000/uL) occurs in patients with solid malignancies was made over 100 years ago. However, the clinical and biological implications as well as the underlying mechanism of paraneoplastic thrombocytosis associated with ovarian carcinoma remains unknown and were the focus of the current study. Following IRB approval, patient data were collected on 619 patients from 4 U.S. centers and used to test associations between platelet count at initial diagnosis, clinicopathologic factors, and outcome. In vitro effects of plasma-purified platelets on ovarian cancer cell proliferation, docetaxel-induced apoptosis, and migration were evaluated using BrdU-PI flow cytometric and two-chamber chemotaxis assays. In vivo effects of platelet depletion on tumor growth, proliferation, apoptosis, and angiogenesis were examined using an anti-platelet antibody (anti-mouse glycoprotein 1ba, Emfret) to reduce platelets by 50%. Complete blood counts and number of mature megakaryocytes in the spleen and bone marrow were compared between control mice and ovarian cancer-bearing mice. Plasma levels of key megakaryo- and thrombopoietic factors including thrombopoietin (TPO), IL-1a, IL-3, IL-4, IL-6, IL-11, G-CSF, GM-CSF, stem cell factor, and FLT-3 ligand were assayed in a subset of 150 patients at the time of initial diagnosis with advanced stage, high grade epithelial ovarian cancer using immunobead-based cytokine profiling coupled with the Luminex® xMAP platform. Plasma cytokines significantly associated with thrombocytosis in ovarian cancer patients were subsequently evaluated in mouse models of ovarian cancer using ELISA immunoassays. The results of human and mouse plasma cytokine profiling were used to inform subsequent in vivo studies evaluating the effect of siRNA-induced silencing of select megakaryo- and thrombopoietic cytokines on paraneoplastic thrombocytosis. Thirty-one percent of patients had thrombocytosis at initial diagnosis. Compared to patients with normal platelet counts, women with thrombocytosis were significantly more likely to have advanced stage disease (p<0.001) and poor median progression-free (0.94 vs 1.35 years, p<0.001) and overall survival (2.62 vs 4.65 years, p<0.001). On multivariate analysis, thrombocytosis remained an independent predictor of decreased overall survival. Our analysis revealed that thrombocytosis significantly increases the risk of VTE in ovarian cancer patients and that thrombocytosis is an independent predictor of increased mortality in women who do develop a blood clot. Platelets increased ovarian cancer cell proliferation and migration by 4.1- and 2.8-fold (p<0.01), respectively. Platelets reduced docetaxel-induced apoptosis in ovarian cancer cells by 2-fold (p<0.001). In vivo, platelet depletion reduced tumor growth by 50%. Staining of in vivo specimens revealed decreased tumor cell proliferation (p<0.001) and increased tumor and endothelial cell apoptosis (p<0.01). Platelet depletion also significantly decreased microvessel density and pericyte coverage (p<0.001). Platelet counts increase by 31-130% in mice with invasive ovarian cancer compared to controls (p<0.01) and strongly correlate with mean megakaryocyte counts in the spleen and bone marrow (r=0.95, p<0.05). Plasma levels of TPO, IL-6, and G-CSF were significantly increased in ovarian cancer patients with thrombocytosis. Plasma levels of the same cytokines were found to be significantly elevated in orthotopic mouse models of ovarian cancer, which consistently develop paraneoplastic thromocytosis. Silencing TPO, IL-6, and G-CSF significantly abrogated paraneoplastic thrombocytosis in vivo. This study provides new understanding of the clinical and biological significance of paraneoplastic thrombocytosis in ovarian cancer and uncovers key humoral factors driving this process. Blocking the development of paraneoplastic thrombocytosis and interfering with platelet-cancer cell interactions could represent novel therapeutic strategies.

MODELING SPORADIC TUMOR FORMATION DRIVEN BY TELOMERE DYSFUNCTION IN THE GASTROINTESTINAL TRACT

Colorectal cancer is a complex disease that is thought to arise when cells accumulate mutations that allow for uncontrolled growth. There are several recognized mechanisms for generating such mutations in sporadic colon cancer; one of which is chromosomal instability (CIN). One hypothesized driver of CIN in cancer is the improper repair of dysfunctional telomeres. Telomeres comprise the linear ends of chromosomes and play a dual role in cancer. Its length is maintained by the ribonucleoprotein, telomerase, which is not a normally expressed in somatic cells and as cells divide, telomeres continuously shorten. Critically shortened telomeres are considered dysfunctional as they are recognized as sites of DNA damage and cells respond by entering into replicative senescence or apoptosis, a process that is p53-dependent and the mechanism for telomere-induced tumor suppression. Loss of this checkpoint and improper repair of dysfunctional telomeres can initiate a cycle of fusion, bridge and breakage that can lead to chromosomal changes and genomic instability, a process that can lead to transformation of normal cells to cancer cells. Mouse models of telomere dysfunction are currently based on knocking out the telomerase protein or RNA component; however, the naturally long telomeres of mice require multiple generational crosses of telomerase null mice to achieve critically short telomeres. Shelterin is a complex of six core proteins that bind to telomeres specifically. Pot1a is a highly conserved member of this complex that specifically binds to the telomeric single-stranded 3’ G-rich overhang. Previous work in our lab has shown that Pot1a is essential for chromosomal end protection as deletion of Pot1a in murine embryonic fibroblasts (MEFs) leads to open telomere ends that initiate a DNA damage response mediated by ATR, resulting in p53-dependent cellular senescence. Loss of Pot1a in the background of p53 deficiency results in increased aberrant homologous recombination at telomeres and elevated genomic instability, which allows Pot1a-/-, p53-/- MEFs to form tumors when injected into SCID mice. These phenotypes are similar to those seen in cells with critically shortened telomeres. In this work, we created a mouse model of telomere ysfunction in the gastrointestinal tract through the conditional deletion of Pot1a that recapitulates the microscopic features seen in severe telomere attrition. Combined intestinal loss of Pot1a and p53 lead to formation of invasive adenocarcinomas in the small and large intestines. The tumors formed with long latency, low multiplicity and had complex genomes due to chromosomal instability, features similar to those seen in sporadic human colorectal cancers. Taken together, we have developed a novel mouse model of intestinal tumorigenesis based on genomic instability driven by telomere dysfunction.

Role and Regulation of EphA2 in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cancer cause of death in the US. Gemcitabine is the first-line therapy for this disease, but unfortunately it shows only very modest benefit. The focus of the current study was to investigate the role and regulation of EphA2, a receptor tyrosine kinase expressed in PDAC, to further understand this disease and identify new therapeutic targets. The role of EphA2 was determined in PDAC by siRNA mediated silencing. In combination with gemcitabine, silencing of EphA2 caused a dramatic increase in apoptosis even in highly resistant cells in vitro. Furthermore, EphA2 silencing was found to be useful in 2 orthotopic models in vivo: 1) shRNA-pretreated Miapaca-2 cells, and 2) in vivo delivery of siRNA to established MPanc96 tumors. Silencing of EphA2 alone reduced tumor growth in Miapaca-2 cells. In MPanc96, only the combination treatment of gemcitabine plus siEphA2 significantly reduced tumor growth, as well as the number of lung and liver metastases. Taken together, these observations support EphA2 as a target for combination therapies for PDAC. The regulation of EphA2 was further explored with a focus on the role of Ras. K-Ras activating mutations are the most important initiating event in PDAC. We demonstrated that Ras regulates EphA2 expression through activation of MEK2 and phosphorylation of ERK. Downstream of ERK, silencing of the transcription factor AP-1 subunit c-Jun or inhibition of the ERK effector RSK caused a decrease in EphA2 expression, supporting their roles in this process. Further examination of Ras/MEK/ERK pathway modulators revealed that PEA-15, a protein that sequesters ERK to the cytoplasm, inhibited expression of EphA2. A significant inverse correlation between EphA2 and PEA-15 levels was observed in mouse models of PDAC. In cells where an EGFR inhibitor reduced phospho-Erk, expression of EphA2 was also reduced, indicating that changes in EphA2 levels may allow monitoring the effectiveness of anti-Ras/MEK/ERK therapies. In conclusion, EphA2 levels may be a good prognostic factor for anti-EGFR/anti-Ras therapies, and EphA2 itself is a relevant target for the development of new therapies.

Prevalence of Premature Ovarian Failure in Women with Tuberous Sclerosis

Tuberous Sclerosis Complex (TSC) is an autosomal dominant tumor suppressor disorder characterized by hamartomas, or benign growths, in various organ systems. Inactivating mutations in either the TSC1 or the TSC2 gene cause most cases of TSC. Recently, the use of ovarian specific conditional knock-out mouse models has demonstrated a crucial role of the TSC genes in ovarian function. Mice with complete deletion of Tsc1 or Tsc2 showed accelerated ovarian follicle activation and subsequent premature follicular depletion, consistent with the human condition premature ovarian failure (POF). POF is defined in women as the cessation of menses before the age of 40 and elevated levels of follicle stimulating hormone (FSH). The prevalence of POF is estimated to be 1%, affecting a substantial number of women in the general population. Nonetheless, the etiology of most cases of POF remains unknown. Based on the mouse model results, we hypothesized that the human TSC1 and TSC2 genes are likely to be crucial for ovarian development and function. Moreover, since women with TSC already have one inactivated TSC gene, we further hypothesized that they may show a higher prevalence of POF. To test this hypothesis, we surveyed 1000 women with TSC belonging to the Tuberous Sclerosis Alliance, a national support organization. 182 questionnaires were analyzed for information on menstrual and reproductive function, as well as TSC. This self-reported data revealed 8 women (4.4%) with possible POF, as determined by menstrual history report and additional supportive data. This prevalence is much higher than 1% in the general population. Data from all women suggested other reproductive pathology associated with TSC such as a high rate of miscarriage (41.2%) and menstrual irregularity of any kind (31.2%). These results establish a previously unappreciated effect of TSC on women’s reproductive health. Moreover, these data suggest that perturbations in the cellular pathways regulated by the TSC genes may play an important role in reproductive function.

The Role of IL-6 in Adenosine-Mediated Pulmonary Fibrosis

Adenosine is a purinergic signaling molecule that regulates various aspects of inflammation and has been implicated in the pathogenesis of chronic lung diseases. Previous studies have demonstrated that adenosine up-regulates IL-6 production through the engagement of the A2B adenosine receptor in various cell types, including alveolar macrophages. IL-6 is elevated in mouse models and humans with chronic lung disease, suggesting a potential role in disease progression. Furthermore, chronic elevation of adenosine in the lungs of adenosine deaminase deficient (Ada-/-) mice leads to the development of pulmonary inflammation, alveolar destruction, and fibrosis, in conjunction with IL-6 elevation. Thus, it was hypothesized that IL-6 contributes to pulmonary inflammation and fibrosis in this model. To test this hypothesis, Ada/IL-6 double knockout mice (Ada/IL-6-/-) were generated to assess the consequences of genetically removing IL-6 on adenosine-dependent pulmonary injury. Ada/IL-6-/- mice exhibited a significant reduction in inflammation, alveolar destruction, and pulmonary fibrosis. Next, Ada-/- mice were treated systematically with IL-6 neutralizing antibodies to test the efficacy of blocking IL-6 on chronic lung disease. These treatments were associated with decreased pulmonary inflammation, alveolar destruction, and fibrosis. To determine the role of IL-6 in a second model of pulmonary fibrosis, wild type mice and IL-6-/- mice were subjected to intraperitoneal injections of bleomycin twice a week for four weeks. Results demonstrated that IL-6-/- mice developed reduced pulmonary fibrosis. To examine a therapeutic approach in this model, wild type mice exposed to bleomycin were treated with IL-6 neutralizing antibodies. Similar results were observed as with Ada-/- mice, namely diminished pulmonary inflammation and fibrosis. In both models, elevations in IL-6 were associated with increased phosphorylated STAT-3 in the nuclei of numerous cell types in the airways, including type II alveolar epithelial cells (AEC). Genetic removal and neutralization of IL-6 in both models was associated with decreased STAT-3 activation in type II AEC. The mechanism of activation in these cells that lack the membrane bound IL-6Ra suggests IL-6 trans-signaling may play a role in regulating fibrosis. Characterization of this mechanism demonstrated that the soluble IL-6Ra (sIL-6Ra) is upregulated in both models during chronic conditions. In vitro studies in MLE-12 alveolar epithelial cells confirmed that IL-6, in combination with the sIL-6Ra, activates STAT-3 and TWIST in association with enhancement of epithelial-to-mesenchymal transition, which can contribute to fibrosis. Similarly, patients with idiopathic pulmonary fibrosis demonstrated a similar pattern of increased IL-6 expression, STAT-3 activation, and sIL-6Ra increases. These findings demonstrate that adenosine-dependent elevations in IL-6 contribute to the development and progression of pulmonary inflammation and fibrosis. The implications from these studies are that adenosine and/or IL-6 neutralizing agents represent novel therapeutic targets for the treatment of pulmonary disorders where fibrosis is a detrimental component.

Radiolabeled human monoclonal IgM for intracompartmental cancer therapy

Radioimmunotherapy (RIT) with i.v. administered radiolabeled IgG can selectively irradiate tumor cells in vivo. However, it only provides effective therapy for lymphomas. Intracompartmental RIT with radiolabeled human monoclonal IgM may allow curative treatment of solid tumors by increasing tumor deposition of radioactivity, reducing systemic toxicity and allowing repeated administration. This hypothesis was tested in nude mouse models with IgM radiolabeled with indium-111 $\rm(\sp{111}In)$ or yttrium-90 $\rm(\sp{90}Y).$ The use of two radioisotopes, $\rm\sp{111}In$ for imaging and $\rm\sp{90}Y$ for therapy, allow for more quantitative and cautious development of RIT.^ Radiolabled 2B12, an IgM reactive with human ovarian carcinomas was tested by i.v. and intraperitoneal (i.p.) administration in nude mice bearing i.p. nodules of a human ovarian carcinoma cell line (SKOV3 NMP2). Radiolabeled CR4E8, an IgM reactive with human squamous cell carcinomas was tested by i.v. and intralesional (i.l.) administration in nude mice bearing subcutaneous tumors of a human head and neck squamous cell carcinoma cell line (886). These two models were selected to test proof of concept. Radiolabeled irrelevant IgM (CH-1B9), and $\rm\sp{90}Y$-aggregate served as specificity controls. Biodistribution was performed by excising, weighing and then measuring the radioactivity of tumor and normal organs. Therapy was conducted with i.p. $\rm\sp{90}Y$-labeled 2B12 using both single and fractionated administration and with i.l. $\rm\sp{90}Y$-labeled CR4E8 using single administration. Mice were monitored for tumor response, survival and systemic toxicity.^ Intracompartmental administration of radiolabeled IgM produced immediate high and prolonged tumor deposition of radioactivity with low normal tissue uptake. In contrast, i.v. administration resulted in low tumor, but high liver and spleen uptake. Similar biodistributions were demonstrated for $\rm\sp{111}In$- and $\rm\sp{90}Y$-labeled IgM. Intraperitoneal therapy with $\rm\sp{90}Y$-labeled 2B12 increased survival by approximately 12 days for every 100 $\rm\mu Ci$ of activity without significant toxicity for single (0-300 $\rm\mu Ci)$ and fractionated (150-510 $\rm\mu Ci)$ administration. Intralesional therapy with $\rm\sp{90}Y$-labeled CR4E8 (150-400 $\rm\mu Ci)$ induced prolonged complete regressions. Significant local or systemic toxicity was not observed.^ Intracompartmental RIT with radiolabeled tumor-reactive human monoclonal IgM can selectively irradiate tumor cells. Intracompartmental radiolabled IgM can significantly extend the survival of treated mice with minimal toxicity. It deserves further development as a new cancer therapy. ^

Cytochrome P450 Regulation by -Tocopherol in Pxr-Null and PXR-Humanized Mice

The pregnane X receptor (PXR) has been postulated to play a role in the metabolism of α-tocopherol owing to the up-regulation of hepatic cytochrome P450 (P450) 3A in human cell lines and murine models after α-tocopherol treatment. However, in vivo studies confirming the role of PXR in α-tocopherol metabolism in humans presents significant difficulties and has not been performed. PXR-humanized (hPXR), wild-type, and Pxr-null mouse models were used to determine whether α-tocopherol metabolism is influenced by species-specific differences in PXR function in vivo. No significant difference in the concentration of the major α-tocopherol metabolites was observed among the hPXR, wild-type, and Pxr-null mice through mass spectrometry-based metabolomics. Gene expression analysis revealed significantly increased expression of Cyp3a11 as well as several other P450s only in wild-type mice, suggesting species-specificity for α-tocopherol activation of PXR. Luciferase reporter assay confirmed activation of mouse PXR by α-tocopherol. Analysis of the Cyp2c family of genes revealed increased expression of Cyp2c29, Cyp2c37, and Cyp2c55 in wild-type, hPXR, and Pxr-null mice, which suggests PXR-independent induction of Cyp2c gene expression. This study revealed that α-tocopherol is a partial agonist of PXR and that PXR is necessary for Cyp3a induction by α-tocopherol. The implications of a novel role for α-tocopherol in Cyp2c gene regulation are also discussed.