Differential modulation of cellular phenotype and drug sensitivity by extracellular matrix proteins in primary and metastatic pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is reported to be the third highest cause of cancer-related deaths in the United States. PDAC is known for its high proportion of stroma, which accounts for 90% of the tumor mass. The stroma is made up of extracellular matrix (ECM) and nonmalignant cells such as inflammatory cells, cancer-associated fibroblasts, and lymphatic and blood vessels. Here, we decoupled the effects of the ECM on PDAC cell lines by culturing cells on surfaces coated with different ECM proteins. Our data show that the primary tumor-derived cell lines have different morphology depending on the ECM proteins on which they are cultured, while metastatic lesion-derived PDAC lines' morphology does not change with respect to the different ECM proteins. Similarly, ECM proteins modulate the proliferation rate and the gemcitabine sensitivity of the primary tumor PDAC cell lines, but not the metastatic PDAC lines. Lastly, transcriptomics analysis of the primary tumor PDAC cells cultured on different ECM proteins reveals the regulation of various pathways, such as cell cycle, cell-adhesion molecules, and focal adhesion, including the regulation of several integrin genes that are essential for ECM recognition.

Protein Exaptation by Endogenous Retroviral Elements Shapes Tumor Cell Senescence and Downstream Immune Signaling.

Cancer cell senescence in lung squamous cell carcinoma (LUSC) is associated with a poor response to chemotherapies and immunotherapies due to promotion of an immunosuppressive tumor microenvironment. This environment is shaped by the senescence-associated secretory pathway, which recruits suppressive immune cell populations. In a recent study, Attig and colleagues identified a transcription factor-activated molecular switch that circumvents cellular senescence through increased expression of the calbindin protein. A human endogenous retrovirus (HERV) sequence upstream of the calbindin gene, CALB1, promotes the transcription of an HERVH-CALB1 transcript through a splice event at the third CALB1 exon in a process known as protein exaptation. The KLF5 transcription factor mediates this transcriptional activity by binding at the HERVH sequence, subsequently initiating the chimeric HERVH-CALB1 transcription. This increased expression of calbindin reduces CXCL8 chemokine production and downstream neutrophil recruitment in LUSC tumor cells. CALB1 exaptation by HERVH is one example by which endogenous retroelements (ERE) regulate immunity in human cancers, highlighting the emerging role of EREs in tumor immunity.

Epigenetically programmed resistance to chemo- and immuno-therapies.

Resistance to cancer treatments remains a major barrier in developing cancer cures. While promising combination chemotherapy treatments and novel immunotherapies have improved patient outcomes, resistance to these treatments remains poorly understood. New insights into the dysregulation of the epigenome show how it promotes tumor growth and resistance to therapy. By altering control of gene expression, tumor cells can evade immune cell recognition, ignore apoptotic cues, and reverse DNA damage induced by chemotherapies. In this chapter, we summarize the data on epigenetic remodeling during cancer progression and treatment that enable cancer cell survival and describe how these epigenetic changes are being targeted clinically to overcome resistance.

Inhibiting DNA methylation and RNA editing upregulates immunogenic RNA to transform the tumor microenvironment and prolong survival in ovarian cancer.

BACKGROUND: Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment in OC. Inhibiting DNA methyltransferases activate transcription of double-stranded (ds)RNA, including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger type I interferon (IFN) signaling, recruiting host immune cells to kill the tumor cells. Adenosine deaminase 1 (ADAR1) is induced by IFN signaling and edits mammalian dsRNA with an A-to-I nucleotide change, which is read as an A-to-G change in sequencing data. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 inhibits the type I IFN response in a negative feedback loop. We hypothesized that decreasing ADAR1 editing would enhance the DNMTi-induced immune response. METHODS: Human OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8 Trp53-/- mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8 Trp53-/- cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1, anti-CD8, or anti-NK1.1 antibodies every 3 days. RESULTS: We show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-ß compared with either perturbation alone. Furthermore, DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells. CONCLUSION: In summary, we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus, epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC, a disease that does not respond to current immunotherapies.

Androgen deprivation-induced elevated nuclear SIRT1 promotes prostate tumor cell survival by reactivation of AR signaling.

The NAD+-dependent deacetylase, Sirtuin 1 (SIRT1) is involved in prostate cancer pathogenesis. However, the actual contribution is unclear as some reports propose a protective role while others suggest it is harmful. We provide evidence for a contextual role for SIRT1 in prostate cancer. Our data show that (i) mice orthotopically implanted with SIRT1-silenced LNCaP cells produced smaller tumors; (ii) SIRT1 suppression mimicked AR inhibitory effects in hormone responsive LNCaP cells; and (iii) caused significant reduction in gene signatures associated with E2F and MYC targets in AR-null PC-3 and E2F and mTORC1 signaling in castrate-resistant ARv7 positive 22Rv1 cells. Our findings further show increased nuclear SIRT1 (nSIRT1) protein under androgen-depleted relative to androgen-replete conditions in prostate cancer cell lines. Silencing SIRT1 resulted in decreased recruitment of AR to PSA enhancer selectively under androgen-deprivation conditions. Prostate cancer outcome data show that patients with higher levels of nSIRT1 progress to advanced disease relative to patients with low nSIRT1 levels. Collectively, we demonstrate that lowering SIRT1 levels potentially provides new avenues to effectively prevent prostate cancer recurrence.

Nuclear mechanosensing: mechanism and consequences of a nuclear rupture.

The physical connections between the cytoskeletal system and the nucleus provide a route for the nucleus to sense the mechanical stress both inside and outside of the cell. Failure to withstand such stress leads to nuclear rupture, which is observed in human diseases. In this review, we will go through the recent findings and our current understandings of nuclear rupture. Starting with the triggers of nuclear rupture, including the aberrant nuclear lamina composition and the elevated actomyosin contractility. We will also discuss the role of ESCRT-III in nuclear rupture repair and the biological consequences of nuclear rupture, including the negative impacts on cellular compartmentalization, DNA damage, and cellular differentiation. Recent studies on nuclear rupture provide further insights into the direct mechanistic link between nuclear rupture and several pathological conditions. Such knowledge can guide us in developing potential therapeutic solutions for the patients.

Hematopoietic knockdown of PPARδ reduces atherosclerosis in LDLR-/- mice.

PPARδ (peroxisome proliferator-activated receptor δ) mediates inflammation in response to lipid accumulation. Systemic administration of a PPARδ agonist can ameliorate atherosclerosis. Paradoxically, genetic deletion of PPARδ in hematopoietic cells led to a reduction of atherosclerosis in murine models, suggesting that downregulation of PPARδ expression in these cells may mitigate atherogenesis. To advance this finding forward to potential clinical translation through hematopoietic stem cell transplantation-based gene therapy, we employed a microRNA (miRNA) approach to knock down PPARδ expression in bone marrow cells followed by transplantation of the cells into LDLR-/- mice. We found that knockdown of PPARδ expression in the hematopoietic system caused a dramatic reduction in aortic atherosclerotic lesions. In macrophages, a key component in atherogenesis, knockdown of PPARδ led to decreased expression of multiple pro-inflammatory factors, including monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1ß and IL-6. Expression of CCR2, a receptor for MCP-1, was also decreased. The downregulation of pro-inflammatory factors is consistent with significant reduction of macrophage presence in the lesions, which may also be attributable to elevation of ABCA1 (ATP-binding cassette, subfamily A, member 1) and depression of adipocyte differentiate-related protein. Furthermore, the abundance of both MCP-1 and matrix metalloproteinase-9 proteins was reduced in plaque areas. Our results demonstrate that miRNA-mediated PPARδ knockdown in hematopoietic cells is able to ameliorate atherosclerosis.

Mitochondrial genome instability resulting from SUV3 haploinsufficiency leads to tumorigenesis and shortened lifespan.

Mitochondrial dysfunction has been a hallmark of cancer. However, whether it has a causative role awaits to be elucidated. Here, using an animal model derived from inactivation of SUV3, a mitochondrial helicase, we demonstrated that mSuv3+/- mice harbored increased mitochondrial DNA (mtDNA) mutations and decreased mtDNA copy numbers, leading to tumor development in various sites and shortened lifespan. These phenotypes were transmitted maternally, indicating the etiological role of the mitochondria. Importantly, reduced SUV3 expression was observed in human breast tumor specimens compared with corresponding normal tissues in two independent cohorts. These results demonstrated for the first time that maintaining mtDNA integrity by SUV3 helicase is critical for cancer suppression.

Critical role of chemokine (C-C motif) receptor 2 (CCR2) in the KKAy + Apoe -/- mouse model of the metabolic syndrome.

AIMS/HYPOTHESIS: Chemokines and their receptors such as chemokine (C-C motif) receptor 2 (CCR2) may contribute to the pathogenesis of the metabolic syndrome via their effects on inflammatory monocytes. Increased accumulation of CCR2-driven inflammatory monocytes in epididymal fat pads is thought to favour the development of insulin resistance. Ultimately, the resulting hyperglycaemia and dyslipidaemia contribute to development of the metabolic syndrome complications such as cardiovascular disease and diabetic nephropathy. Our goal was to elucidate the role of CCR2 and inflammatory monocytes in a mouse model that resembles the human metabolic syndrome. METHODS: We generated a model of the metabolic syndrome by backcrossing KKAy ( + ) with Apoe ( -/- ) mice (KKAy ( + ) Apoe ( -/- )) and studied the role of CCR2 in this model system. RESULTS: KKAy ( + ) Apoe ( -/- ) mice were characterised by the presence of obesity, insulin resistance, dyslipidaemia and increased systemic inflammation. This model also manifested two complications of the metabolic syndrome: atherosclerosis and diabetic nephropathy. Inactivation of Ccr2 in KKAy (+) Apoe ( -/- ) mice protected against the metabolic syndrome, as well as atherosclerosis and diabetic nephropathy. This protective phenotype was associated with a reduced number of inflammatory monocytes in the liver and muscle, but not in the epididymal fat pads; circulating levels of adipokines such as leptin, resistin and adiponectin were also not reduced. Interestingly, the proportion of inflammatory monocytes in the liver, pancreas and muscle, but not in the epididymal fat pads, correlated significantly with peripheral glucose levels. CONCLUSIONS/INTERPRETATION: CCR2-driven inflammatory monocyte accumulation in the liver and muscle may be a critical pathogenic factor in the development of the metabolic syndrome.

Macrophage LXRα gene therapy ameliorates atherosclerosis as well as hypertriglyceridemia in LDLR(-/-) mice.

Liver X receptors (LXRs) are implicated in the regulation of cholesterol homeostasis, inflammatory response and atherogenesis. Administration of LXR agonists inhibits the progress of atherosclerosis, and also increases plasma triglyceride levels, representing an obstacle to their use in treating this disease. The objective of this study was to develop an alternative approach that could overcome this obstacle. Eight-week-old low-density lipoprotein receptor-deficient (LDLR(-/-)) mice were transplanted with hematopoietic stem cell (HSC)-enriched bone marrow cells transduced with lentivectors expressing either green fluorescent protein (GFP) (Lenti-SP-GFP, control) or LXRα (Lenti-SP-LXRα) driven by a synthetic macrophage promoter. At 4 weeks post-transplant, the mice were fed with a Western diet for 8 weeks and then killed. Compared with Lenti-SP-GFP mice, the Lenti-SP-LXRα mice had a 30% reduction in atherosclerotic lesions, which was accompanied by increases in levels of macrophage expression of cholesterol efflux genes apolipoprotein E and ATP-binding cassette A1, as well as decreases in plasma inflammatory cytokines interleukin-6 and tumor necrosis factor-α. Intriguingly, a 50% reduction of plasma triglyceride level was also observed. We conclude that HSC-based macrophage LXRα gene therapy ameliorates the development of atherosclerosis along with an unexpected concomitant reduction of plasma triglyceride levels in LDLR(-/-) mice. These findings highlight the potential value of macrophage LXR expression as an avenue for therapeutic intervention against atherosclerosis.

Vaccination with Mage-b DNA induces CD8 T-cell responses at young but not old age in mice with metastatic breast cancer.

BACKGROUND: Elderly individuals react less efficiently to vaccines than do adults, mainly because of T-cell unresponsiveness. In this study, we analysed whether tumour-associated antigen (TAA)-specific CD8 T-cell responses could be induced by vaccination in old mice with metastatic breast cancer. METHODS: The effect of pcDNA-3.1- and Listeria-based vaccines, expressing TAA Mage-b, on Mage-b-specific immune responses was tested in spleens and draining lymph nodes (LNs) of mild (4TO7cg) and aggressive (4T1) syngeneic metastatic mouse breast tumour models at young (3 months) and old (20 months) age. RESULTS: Interferon gamma and interleukin-2 levels increased significantly in draining LNs and spleens of Mage-b-vaccinated mice compared with those in control groups at young but not old age in both mouse tumour models. A significant increase was observed in the number of IFNgamma-producing Mage-b-specific CD8 T cells after Mage-b vaccination in the 4T1 model at young but not old age. This correlated with a reduced protective effect of Mage-b vaccination against metastatic breast cancer at old compared with young age. CONCLUSIONS: The absence of CD8 T-cell responses after Mage-b vaccination and the accompanying reduced protection against metastatic breast cancer in old compared with young mice point towards the need for tailoring cancer vaccination to older age.

Automatic segmentation of thoracic and pelvic CT images for radiotherapy planning using implicit anatomic knowledge and organ-specific segmentation strategies.

Automatic segmentation of anatomical structures in medical images is a valuable tool for efficient computer-aided radiotherapy and surgery planning and an enabling technology for dynamic adaptive radiotherapy. This paper presents the design, algorithms and validation of new software for the automatic segmentation of CT images used for radiotherapy treatment planning. A coarse to fine approach is followed that consists of presegmentation, anatomic orientation and structure segmentation. No user input or a priori information about the image content is required. In presegmentation, the body outline, the bones and lung equivalent tissue are detected. Anatomic orientation recognizes the patient's position, orientation and gender and creates an elastic mapping of the slice positions to a reference scale. Structure segmentation is divided into localization, outlining and refinement, performed by procedures with implicit anatomic knowledge using standard image processing operations. The presented version of algorithms automatically segments the body outline and bones in any gender and patient position, the prostate, bladder and femoral heads for male pelvis in supine position, and the spinal canal, lungs, heart and trachea in supine position. The software was developed and tested on a collection of over 600 clinical radiotherapy planning CT stacks. In a qualitative validation on this test collection, anatomic orientation correctly detected gender, patient position and body region in 98% of the cases, a correct mapping was produced for 89% of thorax and 94% of pelvis cases. The average processing time for the entire segmentation of a CT stack was less than 1 min on a standard personal computer. Two independent retrospective studies were carried out for clinical validation. Study I was performed on 66 cases (30 pelvis, 36 thorax) with dosimetrists, study II on 52 cases (39 pelvis, 13 thorax) with radio-oncologists as experts. The experts rated the automatically produced structures on the scale 1-excellent (no corrections necessary, maximum time saving), 2-good (corrections necessary for up to 1/3 of slices), 3-acceptable (major corrections necessary, but still time saving), 4-not acceptable (manual redrawing more efficient, no time saving). A rating

Spontaneous hepatocellular carcinoma is reduced in transgenic mice overexpressing human O6- methylguanine-DNA methyltransferase.

O(6)-methylguanine (O(6)mG) is a potent mutagenic and procarcinogenic DNA lesion. Organisms have evolved with a DNA repair mechanism that largely ameliorates the deleterious effects of O(6)mG through a direct reversal mechanism by a protein termed O(6)-methylguanine-DNA methyltransferase (MGMT). However, the contribution of O(6)mG to carcinogenesis, in the absence of known exposure to agents that produce it, has not been defined. Nontransgenic C3HeB male mice have a high frequency of spontaneous liver tumors. Transgenic CeHeB/FeJ mice expressing human MGMT (hMGMT) were generated that had elevated hepatic MGMT activity. The spontaneous development of hepatocellular carcinoma was significantly reduced in those mice expressing hMGMT compared with nontransgenic C3HeB/FeJ male mice. No differences were detected in spontaneous mutant frequencies in lacI transgenes in mice carrying hMGMT compared with that without hMGMT but the proportion of GC to AT transition mutations was lower in the transgenic mice carrying hMGMT as well as lacI. Tumors that arose in C3HeB/FeJ transgenic mice were largely deficient in hMGMT protein as determined by immunohistochemistry with a monoclonal antibody directed against hMGMT. Together these data indicate that spontaneous O(6)mG lesions induced hepatocellular carcinogenesis in C3HeB/FeJ male mice. These transgenic mice represent a rare example of reduced spontaneous carcinogenesis.

Estrogen receptor alpha is a major mediator of 17beta-estradiol's atheroprotective effects on lesion size in Apoe-/- mice.

The inhibitory effects of estrogen (17beta-estradiol) on atherosclerosis have been well documented in numerous animal models, and epidemiological evidence supports this protective effect in humans. The detailed mechanisms for this protection are not understood, but most are thought to be mediated through estrogen receptors (ERs), of which two are known (ERalpha and ERbeta). To investigate the role of ERalpha in the atheroprotective effect of 17beta-estradiol (E2), we ovariectomized female mice that lack apoE (AAee) or lack both apoE and ERalpha (alphaalphaee), and treated half of them with E2 for three months. E2 treatment of ovariectomized AAee females dramatically reduced the size of the lesions as well as their histological complexity. Plasma cholesterol was significantly reduced in this group, although the observed extent of protection by E2 was greater than could be explained solely by the change in lipid levels. In contrast, E2 treatment of ovariectomized alphaalphaee females caused minimal reduction in lesion size and no reduction in total plasma cholesterol compared with alphaalphaee mice without E2, demonstrating that ERalpha is a major mediator of the atheroprotective effect of E2. Nevertheless, E2 treatment significantly reduced the complexity of plaques in the alphaalphaee females, although not to the same degree as in AAee females, suggesting the existence of ERalpha-independent atheroprotective effects of E2.

Health span and life span in transgenic mice with modulated DNA repair.

One way to better understand the contribution of DNA repair, DNA damage, and mutagenesis in aging would be to enhance DNA repair activity, lower DNA damage, and lower mutagenesis. Because the repair protein O6-methylguanine-DNA methyltransferase (MGMT) acts alone and stoichiometrically, the human MGMT (hMGMT) cDNA was selected to test the feasibility of enhancing DNA repair activity in transgenic mice. MGMT activity is largely responsible for ameliorating the deleterious effects of O6-methylguanine (O6mG) lesions in DNA in a direct reversal mechanism. A transgene was constructed consisting of a portion of the human transferrin (TF) promoter and hMGMT cDNA such that hMGMT is expressed in transgenic mouse brain and liver. Expression of hMGMT was associated with a significant reduction in the occurrence of an age-related hepatocellular carcinoma in male mice at 15 months of age. Longitudinal and cross-sectional studies were initiated to determine whether the reduced incidence of hepatocellular carcinoma would impact median or maximum life span. The cross-sectional study performed on 15-month-old male animals confirmed the reduced occurrence of spontaneous hepatocellular carcinoma. At 30 months of age, however, the occurrence of hepatocellular carcinoma in at least one transgenic line was similar to that for nontransgenic animals. The longitudinal study is ongoing; however, at present no significant differences in life span have been detected. Tissues expressing the MGMT transgene also displayed greater resistance to alkylation-induced tumor formation. These results suggest that transgenes can be used to direct enhanced DNA repair gene expression and that enhanced expression can protect animals from certain spontaneous and induced tumors.

Preformed membrane-associated stores of interleukin (IL)-12 are a previously unrecognized source of bioactive IL-12 that is mobilized within minutes of contact with an intracellular parasite.

The prevailing paradigm is that production of the interleukin (IL)-12 p70 heterodimer, a critical T helper cell type 1 (Th1)-inducing cytokine, depends on the induced transcription of the p40 subunit. Concordant with this paradigm, we found that dendritic cells (DCs) produced IL-12 p70 only after at least 2-4 h of stimulation with lipopolysaccharide plus interferon gamma. However, using several complementary experimental approaches, including electron and confocal microscopy, we now show that resting murine and human myeloid cells, including macrophages/DCs and DC-rich tissues, contain a novel source of bioactive IL-12 that is preformed and membrane associated. These preformed, membrane-associated IL-12 p70 stores are released within minutes after in vitro or in vivo contact with Leishmania donovani, an intracellular pathogen. Our findings highlight a novel source of bioactive IL-12 that is readily available for the rapid initiation of Th1 host responses to pathogens such as Leishmania species.

CC chemokine receptor (CCR)2 is required for langerhans cell migration and localization of T helper cell type 1 (Th1)-inducing dendritic cells. Absence of CCR2 shifts the Leishmania major-resistant phenotype to a susceptible state dominated by Th2 cytokines, b cell outgrowth, and sustained neutrophilic inflammation.

There is growing evidence that chemokines and their receptors regulate the movement and interaction of antigen-presenting cells such as dendritic cells (DCs) and T cells. We tested the hypothesis that the CC chemokine receptor (CCR)2 and CCR5 and the chemokine macrophage inflammatory protein (MIP)-1alpha, a ligand for CCR5, influence DC migration and localization. We found that deficiency of CCR2 but not CCR5 or MIP-1alpha led to distinct defects in DC biology. Langerhans cell (skin DC) density in CCR2-null mice was normal, and their ability to migrate into the dermis was intact; however, their migration to the draining lymph nodes was markedly impaired. CCR2-null mice had lower numbers of DCs in the spleen, and this was primarily due to a reduction in the CD8alpha(1) T helper cell type 1 (Th1)-inducing subset of DCs. Additionally, there was a block in the Leishmania major infection-induced relocalization of splenic DCs from the marginal zone to the T cell areas. We propose that these DC defects, in conjunction with increased expression of B lymphocyte chemoattractant, a B cell-specific chemokine, may collectively contribute to the striking B cell outgrowth and Th2 cytokine-biased nonhealing phenotype that we observed in CCR2-deficient mice infected with L. major. This disease phenotype in mice with an L. major-resistant genetic background but lacking CCR2 is strikingly reminiscent of that observed typically in mice with an L. major-susceptible genetic background. Thus, CCR2 is an important determinant of not only DC migration and localization but also the development of protective cell-mediated immune responses to L. major.

Enhanced atherosclerosis and kidney dysfunction in eNOS(-/-)Apoe(-/-) mice are ameliorated by enalapril treatment.

Hypertension and atherosclerosis are each important causes of morbidity and mortality in the developed world. We have investigated the interaction between these conditions by breeding mice that are atherosclerotic due to lack of apolipoprotein (apo) E with mice that are hypertensive due to lack of endothelial nitric oxide synthase (eNOS). The doubly deficient mice (nnee) have higher blood pressure (BP) and increased atherosclerotic lesion size but no change in plasma lipoprotein profiles compared with normotensive but atherosclerotic (NNee) mice. The nnee mice also develop kidney damage, evidenced by increased plasma creatinine, decreased kidney weight/body weight ratio, and glomerular lipid deposition and calcification. Enalapril treatment abolishes the deleterious effects of eNOS deficiency on BP, atherosclerosis, and kidney dysfunction in nnee mice. In striking contrast, a genetic lack of inducible NOS, which does not affect BP, has no effect on the development of atherosclerotic lesions in Apoe(-/-) mice. We also observed a positive relationship between BP and size of atherosclerotic lesions These results suggest that the atherogenic effects of eNOS deficiency can be partially explained by an increase in BP and reemphasize the importance of controlling hypertension in preventing atherosclerosis.

Aortic wall damage in mice unable to synthesize ascorbic acid.

By inactivating the gene for L-gulono-gamma-lactone oxidase, a key enzyme in ascorbic acid synthesis, we have generated mice that, like humans, depend on dietary vitamin C. Regular chow, containing about 110 mg/kg of vitamin C, is unable to support the growth of the mutant mice, which require L-ascorbic acid supplemented in their drinking water (330 mg/liter). Upon withdrawal of supplementation, plasma and tissue ascorbic acid levels decreased to 10-15% of normal within 2 weeks, and after 5 weeks the mutants became anemic, began to lose weight, and die. Plasma total antioxidative capacities were approximately 37% normal in homozygotes after feeding the unsupplemented diet for 3-5 weeks. As plasma ascorbic acid decreased, small, but significant, increases in total cholesterol and decreases in high density lipoprotein cholesterol were observed. The most striking effects of the marginal dietary vitamin C were alterations in the wall of aorta, evidenced by the disruption of elastic laminae, smooth muscle cell proliferation, and focal endothelial desquamation of the luminal surface. Thus, marginal vitamin C deficiency affects the vascular integrity of mice unable to synthesize ascorbic acid, with potentially profound effects on the pathogenesis of vascular diseases. Breeding the vitamin C-dependent mice with mice carrying defined genetic mutations will provide numerous opportunities for systematic studies of the role of antioxidants in health and disease.

Defects in the generation of IFN-gamma are overcome to control infection with Leishmania donovani in CC chemokine receptor (CCR) 5-, macrophage inflammatory protein-1 alpha-, or CCR2-deficient mice.

We investigated the immune responses in mice lacking CCR2, CCR5, or macrophage inflammatory protein-1 alpha (MIP-1 alpha), a ligand for CCR5, in two situations: following T cell stimulation or after challenge with Leishmania donovani, an intracellular microbe whose control is dependent on a Th1 immune response. Mice deficient in CCR5, MIP-1 alpha, or CCR2 had reduced IFN-gamma responses following ligation of the TCR. Reduced IFN-gamma responses following PMA and ionomycin were also observed in CD8+ T cells of CCR5-/- and CCR2-/- mice. During the early phases of infection, all three knockout mice had low Ag-specific IFN-gamma responses. However, this reduced IFN-gamma response was overcome during a state of persistent Ag stimulation (chronic infection), and was not associated with an adverse parasitologic outcome in any of the gene-targeted mouse strains. To the contrary, during the late phase of infection, an exaggerated Ag-specific IFN-gamma response was evident in CCR5-/- and MIP-1 alpha-/- mice, and this correlated with an enhanced control of parasite replication. Although granuloma formation was abnormal in each of the knockout mice, there was no correlation between the number or architecture of the granulomas and parasite burden. Collectively, these findings indicate an important role for CCR5, MIP-1 alpha, and CCR2 in granulomatous inflammation, and that CCR5 and MIP-1 alpha, possibly acting through CCR5, might play a deleterious role in the outcome of chronic L. donovani infection. Our data also suggest that there might be cross-talk between TCR and chemokine receptor signaling pathways.