Expression of Vascular Notch Ligand Delta-Like 4 and Inflammatory Markers in Breast Cancer

Delta-like ligand 4 (Dll4) is a Notch ligand that is predominantly expressed in the endothelium. Evidence from xenografts suggests that inhibiting Dll4 may overcome resistance to antivascular endothelial growth factor therapy. The aims of this study were to characterize the expression of Dll4 in breast cancer and assess whether it is associated with inflammatory markers and prognosis. We examined 296 breast adenocarcinomas and 38 ductal carcinoma in situ tissues that were represented in tissue microarrays. Additional whole sections representing 10 breast adenocarcinomas, 10 normal breast tissues, and 16 angiosarcomas were included. Immunohistochemistry was then performed by using validated antibodies against Dll4, CD68, CD14, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), CD123, neutrophil elastase, CD31, and carbonic anhydrase 9. Dll4 was selectively expressed by intratumoral endothelial cells in 73% to 100% of breast adenocarcinomas, 18% of in situ ductal carcinomas, and all lactating breast cases, but not normal nonlactating breast. High intensity of endothelial Dll4 expression was a statistically significant adverse prognostic factor in univariate (P = 0.002 and P = 0.01) and multivariate analyses (P = 0.03 and P = 0.04) of overall survival and relapse-free survival, respectively. Among the inflammatory markers, only CD68 and DC-SIGN were significant prognostic factors in univariate (but not multivariate) analyses of overall survival (P = 0.01 and 0.002, respectively). In summary, Dll4 was expressed by endothelium associated with breast cancer cells. In these retrospective subset analyses, endothelial Dll4 expression was a statistically significant multivariate prognostic factor.The growth of tumors requires angiogenesis,¹ which is the consequence of increased expression of proangiogenic factors (for example, vascular endothelial growth factor A [VEGF]^2,3). The expression of VEGF in cancer is controlled by oncogenic signaling,⁴ hypoxia,⁵ and inflammatory cells.⁶ Although there is redundancy among proangiogenic factors in advanced cancer,⁷ many in vivo early stage cancer models show VEGF dependence.^8,9This observation has been exploited clinically, where the addition of an anti-VEGF antibody (bevacizumab) to first line taxane-based chemotherapy in recurrent/metastatic breast cancer was associated with prolongation of progression free survival (from a median of 5.9 to 11.8 months, P < 0.001).¹⁰ Nevertheless, there was no statistically significant overall survival benefit, and all patients in this trial eventually progressed after 4 years.¹⁰ Furthermore, a trial evaluating the addition of bevacizumab to capecitabine in previously treated metastatic/advanced breast cancer demonstrated only a 10.7% improvement in response rate and no survival benefit.¹¹ To date, there are no validated clinical, radiological, or molecular biomarkers that can predict the survival benefit afforded by bevacizumab.^12,13,14,15 Clinical data suggest that antiangiogenic drugs are active in breast cancer,^10,16 and it may be necessary to identify biomarkers that predict their benefit.Additional agents that disrupt functional angiogenesis have been developed to target tumors resistant to anti-VEGF therapy.^17,18 Recent studies have focused on Delta-like ligand 4 (Dll4), a ligand for Notch receptors 1, 3, and 4^17,18,19 that is predominantly expressed by endothelial cells.^17,18,19 Transgenic mice in which Dll4 was replaced by a reporter gene showed that Dll4 expression is restricted to large arteries during development.^20,21 Furthermore, Dll4 heterozygous knockout mice are reported to have defective arterial development²² and venous malformations.²²Experimental systems^17,23,24 have shown that Dll4-Notch inhibition leads to increased sprouting and branching of vessels in association with gradients of VEGF. Conversely, VEGF blockade causes a reduction in Dll4 expression and vessel sprouting.^{17,18,23,24,25,26,27} In addition, endothelial cells transfected with Dll4 down-regulated VEGF receptors KDR and neuropilin1 and showed reduced proliferative and migratory responses to VEGF.²⁸ The implication of this research is that Dll4-Notch signaling regulates endothelial sprouting and branching to form functional vascular beds, under the control of VEGF and by autoregulation of VEGF signaling.²³Disruption of Dll4 signaling by overexpression or inhibition of Dll4 may impair angiogenesis,^17,18 and blockade of Dll4-Notch signaling results in an increased density of nonfunctional vasculature and is associated with a reduction in the growth of human tumor xenografts.^17,18 Indeed, certain xenografts that are resistant to anti-VEGF therapy are reported to be sensitive to anti-Dll4,^17,18,29 and combination treatment with anti-VEGF and anti-Dll4 has additive inhibitory effects on tumor growth.¹⁸ Together these data provide a rationale to target Dll4 in cancer and suggest that Dll4 may have a role in mediating resistance to anti-VEGF therapies.Besides direct vascular effects, Fung et al³⁰ showed that Dll4-Notch signaling in macrophages stimulates a proinflammatory response, which may be proangiogenic.⁶ Moreover, Shojaei et al^31,32 have reported that bevacizumab resistance in certain preclinical in vivo cancer models is causally associated with tumor infiltration by myeloid cells.The characterization of Dll4 protein expression in human cancer is important for the rational design of clinical trials to test the safety and activity of anti-Dll4 therapy. Defining the pattern of Dll4 expression, in association with markers of inflammation, may identify subgroups with distinct clinical behavior and responses to treatment. The aims of this study were to characterize the in situ expression of Dll4 in breast cancer, to assess the association between Dll4 and established markers of inflammation (CD68, CD14, neutrophil elastase, CD123, and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin [DC-SIGN]) and hypoxia (carbonic anhydrase 9 [CA9]), and to determine the prognostic significance of these markers.     

Rapamycin Extends Maximal Lifespan in Cancer-Prone Mice

Aging is associated with obesity and cancer. Calorie restriction both slows down aging and delays cancer. Evidence has emerged that the nutrient-sensing mammalian target of rapamycin (mTOR) pathway is involved in cellular and organismal aging. Here we show that the mTOR inhibitor rapamycin prevents age-related weight gain, decreases rate of aging, increases lifespan, and suppresses carcinogenesis in transgenic HER-2/neu cancer-prone mice. Rapamycin dramatically delayed tumor onset as well as decreased the number of tumors per animal and tumor size. We suggest that, by slowing down organismal aging, rapamycin delays cancer.Astonishing discoveries in model organisms indicate that lifespan is genetically controlled.¹ In particular, the nutrient-sensing target of rapamycin (TOR) pathway is involved in both mammalian cell senescence² and aging in diverse organisms from worms to mammals.^3,4,5,6 In mammals, TOR (mTOR) controls cell growth and metabolism in response to nutrients (eg, amino acids), insulin, and growth factors such as IGF-1.⁷ Calorie restriction (CR) deactivates mTOR in mice.⁸ Not surprisingly, CR extends lifespan in most species including rodents and primates.^9,10 Furthermore, the TOR inhibitor rapamycin decelerates senescence in both yeast¹¹ and mammalian cells.¹² Based on these findings it was suggested that rapamycin, a clinically approved drug, is an antiaging drug.¹³ Recently it has been demonstrated that rapamycin in fact extends lifespan in mice.¹⁴ However, its effect on longevity of cancer-prone mice has not been addressed. There are several lines of evidence that suggest that suppression of organismal aging may delay carcinogenesis. Thus, cancer is an age-related disease, and the incidence of cancer increases with age in both humans and animals.^15,16 Consistently, carcinogenesis is delayed in slowly aging Ames dwarf mice.^17,18 Cancer is often associated with age-related obesity and metabolic syndrome,¹⁹ and calorie restriction affects both the process of aging (by slowing it down) and carcinogenesis (by delaying the tumor onset in normal and cancer-prone mice^20,21). Interestingly, centenarians, people who age slowly, are endowed with a peculiar resistance to cancer.²² Therefore it is reasonable to hypothesize that by slowing down aging rapamycin could delay cancer. Our data demonstrate that rapamycin not only extends lifespan but also significantly delays the onset of spontaneous carcinogenesis in cancer-prone HER-2/neu transgenic mice.     

Adherence Patterns and Adherence-Related DNA Sequences in Escherichia coli Isolates from Children with and without Diarrhea in S?o Paulo City,Brazil

The correlation between various adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from 1- to 4-year-old children with and without diarrhea in São Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent E. coli did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related daaC probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.The first step in the establishment of the diarrheal diseases caused by the various categories of diarrheagenic Escherichia coli is adherence to epithelial cells of the intestinal mucosa. In vitro assays with eukaryotic cell lines (HeLa and HEp-2 cells) have identified three distinct adherence patterns among fecal isolates of E. coli: localized, diffuse, and aggregative (37, 38, 41). Localized adherence (LA) is characterized by formation of bacterial microcolonies on a restricted area(s) of the cell surface, while diffuse adherence (DA) is the scattered attachment of bacteria over the whole surface of the cell (41). The pattern of aggregative adherence (AA) consists of bacterial attachment to the cells and the intervening cell growth surface in a stacked brick-like lattice (37).The LA pattern was first detected in strains classified as enteropathogenic E. coli (EPEC) among serogroups associated with outbreaks of infantile diarrhea (41). Although E. coli strains exhibiting DA (DAEC) have been isolated at similar frequencies from feces of infants and young children with acute diarrhea and nondiarrheic controls in some populations (3, 10, 11, 14, 18), they were significantly associated with diarrhea in other settings (1, 17, 24, 29, 33). E. coli strains showing AA, termed enteroaggregative E. coli (EAEC), have been linked to sporadic persistent diarrhea (3, 4, 7, 10, 13, 26, 27, 44) and to outbreaks of diarrhea in both developing and developed countries (8, 12, 28, 43). However, the role of EAEC in acute diarrhea is still controversial: some studies have shown a correlation (7, 23, 25, 27, 34, 37), but others (1, 3, 6, 10, 11, 13–15, 17, 18, 24, 26, 29, 33, 44) have not.DNA probes derived from adherence-related sequences have been constructed (2, 5, 16, 31, 36) and used in hybridization assays for the detection of the different established and putative categories of diarrheagenic E. coli in many epidemiological studies.We evaluated the relationship between the LA, DA, and AA patterns and hybridization with adherence-related DNA sequences and tested children 1 to 4 years old with and without acute diarrhea for the presence of adherent E. coli strains.     

Dual Roles of CD40 on Microbial Containment and the Development of Immunopathology in Response to Persistent Fungal Infection in the Lung

Persistent pulmonary infection with Cryptococcus neoformans in C57BL/6 mice results in chronic inflammation that is characterized by an injurious Th2 immune response. In this study, we performed a comparative analysis of cryptococcal infection in wild-type versus CD40-deficient mice (in a C57BL/6 genetic background) to define two important roles of CD40 in the modulation of fungal clearance as well as Th2-mediated immunopathology. First, CD40 promoted microanatomic containment of the organism within the lung tissue. This protective effect was associated with: i) a late reduction in fungal burden within the lung; ii) a late accumulation of lung leukocytes, including macrophages, CD4⁺ T cells, and CD8+ T cells; iii) both early and late production of tumor necrosis factor-α and interferon-γ by lung leukocytes; and iv) early IFN-γ production at the site of T cell priming in the regional lymph nodes. In the absence of CD40, systemic cryptococcal dissemination was increased, and mice died of central nervous system infection. Second, CD40 promoted pathological changes in the airways, including intraluminal mucus production and subepithelial collagen deposition, but did not alter eosinophil recruitment or the alternative activation of lung macrophages. Collectively, these results demonstrate that CD40 helps limit progressive cryptococcal growth in the lung and protects against lethal central nervous system dissemination. CD40 also promotes some, but not all, elements of Th2-mediated immunopathology in response to persistent fungal infection in the lung.CD40, a 48-kDa type I transmembrane protein and member of the tumor necrosis factor receptor family, is a well-described costimulatory molecule expressed on B cells, dendritic cells (DC), macrophages, basophils, and platelets as well as nonhematopoietic cells including fibroblasts, epithelial, and endothelial cells. The ligand for CD40, known as CD154 or CD40L, is a type II transmembrane protein member of the tumor necrosis factor (TNF) superfamily expressed primarily by activated T cells, B cells, and platelets.^1,2,3 CD40 can be induced on DC, monocytes, and macrophages under inflammatory conditions.^4,5 Signaling via the CD40/CD40L pathway exerts numerous biological effects including: i) increased cytokine expression (especially TNF-α and Th1 cytokines interleukin (IL)-12 and interferon (IFN)-α) and nitric oxide production; ii) upregulation of additional costimulatory molecules (CD80 and CD86) on antigen-presenting cells (APC); iii) enhanced cell survival (particularly of B and T cells, DC, and endothelial cells); iv) Ig isotype switching; and v) somatic hypermutation of Ig.^1,4,5The CD40/CD40L signaling pathway contributes to adaptive Th1 immune responses required to clear Leishmanisa spp.,^6,7,8 Trypanosoma spp.,^6,7,8,9 Shistosoma mansoini,¹⁰ and the fungi Candida albicans¹¹ and Pneumocystis spp.¹² The enhanced production of IFN-γ, TNF-α, and nitric oxide associated with CD40/CD40L signaling is thought to be responsible for this protective effect. However, other studies have suggest that CD40/CD40L signaling is not required for successful host defense against Listeria monocytogenes,^13,14 Toxoplasma gondi,¹⁵ lymphocytic choriomeningitis virus,^16,17 or the fungus Hisoplasma capsulatum.^18,19 In models of Mycobacterium spp. infection, CD40 appears dispensable for clearance of an i.v. infection,^20,21 but essential for clearing the organism in response to aerosolized infection in the lungs.^22,23 Thus, the role of CD40 in antimicrobial host defense varies and depends not only on the specific pathogen but also on the primary site of infection.Cryptococcus neoformans, an opportunistic fungal pathogen acquired through inhalation, causes significant morbidity and mortality primarily in patients with AIDS, lymphoid or hematological malignancies, or patients receiving immunosuppressive therapy secondary to autoimmune disease or organ transplantation.^24,25 Infection in non-immunocompromised patients has been reported.^26,27,28 Murine models of cryptococcal infection in CBA/J or BALB/c mice demonstrate that development of a Th1 antigen-specific immune response characterized by IFN-γ production and classical activation of macrophages is required to eradicate the organism.^{29,30,31,32,33,34,35,36,37,38,39,40} In contrast, a model of persistent cryptococcal infection has been developed using C57BL/6 mice;^{41,42,43,44,45,46,47} this model reflects many features observed in humans diagnosed with allergic bronchopulmonary mycosis.⁴⁸ Specifically, these mice fail to clear the organism from the lung and develop characteristic Th2-mediated immunopathology including: i) tissue eosinophilia; ii) airway hyperreactivity, mucus production, and fibrosis; and iii) alternative macrophage activation associated with YM1 crystal deposition.The molecular mechanisms responsible for the immunopathologic response associated with persistent cryptococcal infection are not clearly defined. These features are abrogated in the absence of IL-4,⁴⁵ whereas more severe Th2-mediated lung injury occurs in the absence of IFN-γ.^29,41 TNF-α exerts a protective effect by enhancing IFN- γ production and the subsequent classical activation of lung macrophages.^31,35,49,50 Lymphocytes are critical mediators of this Th2 response as the pathological features of chronic cryptococcal infection are substantially diminished in CD4 T cell-depleted mice despite no change in fungal clearance.⁴²Although interactions between CD4 T cells and APC are critical determinants of T cell polarization in response to cryptococcal lung infection,^{49,51,52,53,54,55} the contribution of specific costimulatory molecules including the CD40/CD40L signaling pathway has not been fully elucidated. In vitro studies suggest that activation of the CD40/CD40L pathway in response to Cryptococcus promotes IFN-γ production by T cells and TNF-α, and nitric oxide (NO) production by monocytes.⁵⁶ In the absence of CD40L, primary pulmonary infection with a weakly virulent strain of C. neoformans was associated with impaired fungal clearance; however, measurements of immune function at the site of infection in the lung or evidence of systemic fungal dissemination were not evaluated.⁵⁷ The potential to target CD40 therapeutically is highlighted by studies showing that treatment of mice with disseminated or intracerebral cryptococcal infection with an agonist antibody to CD40 in combination with IL-2 improves survival.^58,59 In this study, we used gene-targeted CD40-deficient mice (on a C57BL/6 genetic background), a clinically relevant model, and assessments of immune function and histopathology in the lung to identify two unique roles for the CD40-signaling pathway in response to persistent cryptococcal lung infection.     

HIF1A Overexpression Is Associated with Poor Prognosis in a Cohort of 731 Colorectal Cancers

Tissue hypoxia commonly occurs in tumors. Hypoxia- inducible factor (HIF)-1 and HIF-2, which are essential mediators of cellular response to hypoxia, regulate gene expression for tumor angiogenesis, glucose metabolism, and resistance to oxidative stress. Their key regulatory subunits, HIF1A (HIF-1α) and endothelial PAS domain protein 1 (EPAS1; HIF-2α), are overexpressed and associated with patient prognosis in a variety of cancers. However, prognostic or molecular features of colon cancer with HIF expression remain uncertain. Among 731 colorectal cancers in two prospective cohort studies, 142 (19%) tumors showed HIF1A overexpression, and 322 (46%) showed EPAS1 overexpression by immunohistochemistry. HIF1A overexpression was significantly associated with higher colorectal cancer-specific mortality in Kaplan-Meier analysis (log-rank test, P < 0.0001), univariate Cox regression (hazard ratio = 1.84; 95% confidence interval, 1.37 to 2.47; P < 0.0001) and multivariate analysis (adjusted hazard ratio = 1.72; 95% confidence interval, 1.26 to 2.36; P = 0.0007) that adjusted for clinical and tumoral features, including microsatellite instability, TP53 (p53), PTGS2 (cyclooxygenase-2), CpG island methylator phenotype, and KRAS, BRAF, PIK3CA, and LINE-1 methylation. In contrast, EPAS1 expression was not significantly associated with patient survival. In addition, HIF1A expression was independently associated with PTGS2 expression (P = 0.0035), CpG island methylator phenotype-high (P = 0.013), and LINE-1 hypomethylation (P = 0.017). EPAS1 expression was inversely associated with high tumor grade (P = 0.0017) and obesity (body mass index ≥ 30 kg/m²) (P = 0.039). In conclusion, HIF1A expression is independently associated with poor prognosis in colorectal cancer, suggesting HIF1A as a biomarker with potentially important therapeutic implications.Tissue hypoxia commonly occurs in tumor, and adaptation to tissue hypoxia appears to be one of important characteristics of malignant cells.^1,2 Hypoxia-inducible factor (HIF)-1 and HIF-2 play a key role in cellular adaptation to hypoxia and regulate the expression of genes responsible for glucose metabolism, angiogenesis, and cell survival.^1,2,3 Thus, HIF and related pathways are potential therapeutic targets.^4,5 Cellular HIF levels are regulated not only by the oxygen-dependent pathway (eg, VHL and prolyl hydroxylase, EGLN) but also by the oxygen-independent pathway (eg, glycogen synthase kinase 3, the phosphatidylinositol 3-kinase pathway, the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway).^6,7 HIF and hypoxia signaling influence a wide variety of pathways including those related to vascular endothelial growth factor (VEGF), cyclins, and MTOR.^1,2 Thus, cellular HIF levels may modify responsiveness to drugs targeting those pathways or hypoxia signaling, and it is of particular interest to examine HIF expression in human cancers. Key regulatory subunits of HIF, HIF1A (the official symbol for HIF-1α), and endothelial PAS domain protein 1 (EPAS1; the official symbol for HIF-2α) are differentially overexpressed^8,9 and have distinct functions in human cancers.^9,10,11 HIF1A expression leads to increased tumor growth and metastasis in some studies,^12,13,14,15 whereas HIF1A inhibits tumor growth by cell cycle arrest or apoptosis induction in other studies.^16,17,18,19 Similar paradoxical effects of EPAS1 have also been reported; EPAS1 appears to promote cancer development and progression in neuroblastoma and renal carcinoma,^20,21,22 whereas it appears to inhibit tumor growth in other cancers including colon cancer.^23,24,25Previous data on HIF1A, EPAS1, and clinical outcome in colorectal cancer have been inconclusive. A study of 90 rectal cancer patients showed poor prognosis associated with HIF1A but not with EPAS1.²⁶ In contrast, another study of 87 colorectal cancer patients reported poor prognosis associated with EPAS1 but not with HIF1A.²⁷ Among studies assessing only HIF1A, some reported its independent prognostic effect^28,29 whereas others did not.^30,31 However, all previous studies^{26,27,28,29,30,31} were limited by small sample sizes (N <136). Considering the increasing importance of the HIF pathway as a potential target for cancer treatment,^1,2,6 the assessment of HIF1A and EPAS1 expression and clinical outcome using a large number of colorectal cancers is needed.We therefore examined prognostic effects of HIF1A and EPAS1 expression among 731 colorectal cancer patients identified in two prospective cohort studies. Moreover, because we concurrently assessed other important molecular events including mutations in KRAS, BRAF, and PIK3CA, LINE-1 hypomethylation, microsatellite instability (MSI), and the CpG island methylator phenotype (CIMP), we could evaluate the effect of HIF1A or EPAS1 expression after controlling for these potential confounders.     

Molecular Characterization of Preneoplastic Lesions Provides Insight on the Development of Renal Tumors

Kidneys are the second most frequent site for chemically induced cancers in rats. However, there is still limited information on direct effects of carcinogens on pathways involved in the development of kidney tumors. Since transformed tumor cells have different characteristics than their cell of origin, it was hypothesized that healthy tissue and progressing stages of preneoplastic lesions are differentially influenced by chemical carcinogens. To elucidate this question, TSC2^−/− Eker rats were gavaged with genotoxic aristolochic acid or nongenotoxic ochratoxin A for 3 and 6 months, respectively. Histopathology and cell proliferation analysis demonstrated a compound- and sex-specific onset of preneoplastic lesions. In contrast, comparable gene expression profiles of laser-microdissected preneoplastic lesions from carcinogen-treated and control rats, including reduced expression of genes involved in carcinogen uptake and metabolism, point to a compound-independent lesion progression. Gene expression profiles and additional immunostaining suggested that clonal expansion of renal lesions appears primarily driven by disturbed mammalian target of rapamycin complex 1 and mammalian target of rapamycin complex 2 pathway regulation. Finally, prolonged carcinogen exposure resulted in only marginal gene expression changes in tubules with normal morphology, indicating that some tubules may have adapted to the treatment. Taken together, these findings indicate that the final outcome of in vivo carcinogenicity studies is primarily determined by time-restricted initial events, while lesion progression may be a compound-independent process, involving deregulated mTOR signaling in the Eker rat model.Renal tumors experimentally induced in rodent bioassays following exposure to chemicals, hormones, viruses, and radiation^1,2,3 are phenotypically comparable with tumor types observed in humans.⁴ Thus, it may be assumed that the deregulation of some cellular pathways, eg, the AKT-tuberous sclerosis complex 2-mammalian target of rapamycin (AKT-TSC2-mTOR) pathway, appears critical for renal tumor development in both humans and rats.^5,6,7 Indeed, inactivating mutations of the TSC2 tumor suppressor gene were shown in both rodent and human renal tumors^8,9 and were accompanied by activation of the raptor containing mTOR complex 1 (TORC1) and its downstream effectors involved in the control of the translational machinery.^5,7 Proteins activated by TORC1 target several processes involved in cancer such as cell growth and proliferation, angiogenesis, and energy metabolism.^10,11 Recent findings suggest that mTOR can also interact with rictor and SIN1 instead of raptor to form a second complex (TORC2).¹² This complex was previously shown to function as an important regulator of the cytoskeleton,^13,14 and to activate the proto-oncogene AKT by phosphorylating AKT at Ser473.¹⁵ However, the role of TSC2 in TORC2-dependent signaling remains elusive.Despite the gain of knowledge on pathways involved in kidney cancer, their distinct participation in the onset and/or progression of tumors is not well understood. Furthermore, the interaction with pathways involved in the development of renal tumors by genotoxic and nongenotoxic carcinogens remains elusive. Novel and sensitive tools like gene expression profiling have been used to study renal carcinogenesis and a number of recent publications have focused on the molecular classification of the different subtypes of kidney cancers in humans.^16,17,18 However, none of these studies have analyzed gene expression profiles of early preneoplastic lesions and of pathways involved in preneoplastic to neoplastic progression. In addition, chronic effects of genotoxic and nongenotoxic carcinogens in morphologically unaffected tissue or tumor tissue have not been distinguished.Using a novel protocol allowing microarray analyses of microdissected renal preneoplastic lesions from carcinogen-treated rats,¹⁹ it was hypothesized that unaffected tissue as well as different stages of preneoplastic lesions can be distinguished by their gene expression profiles, therefore allowing to study pathways involved in the onset and progression of preneoplastic lesions. In addition, it was hypothesized that renal carcinogens, based on their compound class-specific mode of action (genotoxic versus nongenotoxic), differentially affect pathways in preneoplastic lesions (eg, the mTOR pathway).To elucidate the above hypotheses, Eker rats, carrying a heterozygous mutation in the TSC2 tumor suppressor gene and thus predisposed for the early development of renal lesions,^20,21 appeared an ideal model. Their hereditary basophilic tumors are morphologically comparable with chemically induced tumors in other rat strains as well as to human basophilic epithelial adenomas and carcinomas.²² Moreover, Eker rat renal tumors have a hyperactive TORC1 pathway,⁷ similar to the situation assumed to predominate in human renal tumors.^23,24 More importantly, Eker rats are highly susceptible toward genotoxic and nongenotoxic renal carcinogens.^25,26,27 This animal model thus should allow the evaluation of the influence of genotoxic and nongenotoxic carcinogens in the development and progression of preneoplastic and neoplastic renal lesions. Furthermore, the Eker rat model helps to delineate the involvement and importance of the TSC2-mTor pathway in different stages of preneoplastic renal lesions.Accordingly, male and female Eker rats were treated for 3 and 6 months with relatively low yet carcinogenic doses of the genotoxic plant toxin aristolochic acid (AA), and the nongenotoxic mycotoxin, ochratoxin A (OTA). Both compounds are known potent renal carcinogens in rats.^28,29,30 Furthermore, they are assumed to be involved in the etiology of Balkan endemic nephropathy, associated with renal fibrosis and urothelial tumors in humans.^31,32,33Compound-induced nonneoplastic and neoplastic renal pathology, site-specific renal cell proliferation, incidence, phenotype, and progression stage of preneoplastic and neoplastic lesions were determined at the 3- and 6-months time point in both sexes. Finally, microdissected preneoplastic lesions and healthy tissue of AA- and OTA-treated as well as control male Eker rats were analyzed using microarrays. Thus, pathways specific for various progression stages of preneoplastic lesion and gene expression changes specific for AA and OTA exposure could be evaluated. Activation of TORC1 and TORC2 pathways in carcinogen-treated and control rats were visualized via immunohistochemical detection of respective phosphorylated downstream targets.     

A Mouse Model of Lethal Synergism Between Influenza Virus and Haemophilus influenzae

Secondary bacterial infections that follow infection with influenza virus result in considerable morbidity and mortality in young children, the elderly, and immunocompromised individuals and may also significantly increase mortality in normal healthy adults during influenza pandemics. We herein describe a mouse model for investigating the interaction between influenza virus and the bacterium Haemophilus influenzae. Sequential infection with sublethal doses of influenza and H. influenzae resulted in synergy between the two pathogens and caused mortality in immunocompetent adult wild-type mice. Lethality was dependent on the interval between administration of the bacteria and virus, and bacterial growth was prolonged in the lungs of dual-infected mice, although influenza virus titers were unaffected. Dual infection induced severe damage to the airway epithelium and confluent pneumonia, similar to that observed in victims of the 1918 global influenza pandemic. Increased bronchial epithelial cell death was observed as early as 1 day after bacterial inoculation in the dual-infected mice. Studies using knockout mice indicated that lethality occurs via a mechanism that is not dependent on Fas, CCR2, CXCR3, interleukin-6, tumor necrosis factor, or Toll-like receptor-4 and does not require T or B cells. This model suggests that infection with virulent strains of influenza may predispose even immunocompetent individuals to severe illness on secondary infection with H. influenzae by a mechanism that involves innate immunity, but does not require tumor necrosis factor, interleukin-6, or signaling via Toll-like receptor-4.Infections with influenza virus cause mild to severe respiratory illness and may result in death in vulnerable human populations.^1,2,3,4 On average, influenza causes three to five million cases of severe illness per year worldwide and over 200,000 hospitalizations and 36,000 deaths in the United States alone.¹ 5 to 20% of the US population are infected annually. While healthy adults typically experience only acute uncomplicated infection, influenza virus predisposes the lungs to bacterial co-infections,^5,6,7 which cause significant additional morbidity, particularly in young children, elderly and immunocompromised individuals.^8,9,10,11,12 Secondary bacterial infections may also significantly increase mortality in the population as a whole during influenza pandemics.^13,14,15,16 For example, in the 1918 influenza pandemic, which killed approximately 50 million people worldwide, while infection with the virus alone could be lethal, the majority of deaths appeared to result from secondary bacterial pneumonia.^16,17,18,19 The most common bacterial agents mediating such secondary infections in the U.S. are Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae.^20,21,22,23H. influenzae is a small Gram-negative coccobacillus that exists in capsulated or non-capsulated forms. H. influenzae is a common cause of otitis media, acute sinusitis, bronchitis, pneumonia and exacerbations of chronic obstructive pulmonary disease.^{24,25,26,27,28,29} A vaccine against H. influenzae type b (Hib) has greatly reduced the incidence of invasive disease, such as meningitis, caused by this organism in children under 5 years of age.^30,31,32,33 However, Hib invasive disease in children remains a problem in countries where the vaccine is not widely available.^32,34,35 Furthermore, other encapsulated and non-typable (NTHi) forms are increasing in frequency as causes of illness in young children.^28,29,36 During the 1918 influenza pandemic, H. influenzae was often isolated from the autopsied lungs of young adults, a subpopulation who do not usually die from influenza infection.¹⁶Early studies by Shope³⁷ showed that infection of pigs with both influenza virus and H. influenzae suis resulted in severe disease or death, whereas the individual agents induced only mild infection. Similarly, Orticoni et al³⁸ reported that administration of both filtrates of nasal secretions from 1918 influenza patients and H. influenzae caused a lethal disease in guinea pigs, but there was no effect if either agent was administered alone. Influenza also increases the susceptibility of new-born rats to H. influenzae-induced meningitis³⁹ and synergizes with the bacteria in the development of otitis media in the chinchilla.⁴⁰ A single study conducted in 1945 showed that infection with both influenza virus and H. influenzae killed mice at doses that were sublethal when either agent was administered alone.⁴¹ However, this study pre-dated modern immunological techniques, precluding assessment of the underlying mechanism.To investigate the pathobiological mechanisms further, we established a model of influenza and H. influenzae co-infection in mice. Herein, we report that H. influenzae synergizes with influenza virus to cause more severe disease in immunocompetent adult mice, leading to 100% lethality at doses that cause no mortality when the agents are give individually. The mechanism leading to disease exacerbation does not involve T or B cells, and thus appears to be mediated by innate immunity. However, tumor necrosis factor (TNF), interleukin-6 (IL-6), and Toll-like receptor (TLR)-4 are not essential for synergistic lethality in this model.     

Generation of a Novel Transgenic Mouse Model for Bioluminescent Monitoring of Survivin Gene Activity in Vivo at Various Pathophysiological Processes : Survivin Expression Overlaps with Stem Cell Markers

Survival has been implicated to play an important role in various pathophysiological processes. However, because of a lack of appropriate animal models, the role and dynamic expression of survivin during pathophysiology are not well defined. We generated a human survivin gene promoter-driven luciferase transgenic mouse model (SPlucTg) so that dynamic survivin gene activity can be monitored during various pathophysiological conditions using in vivo imaging. Our results show that, consistent with survivin positivity in testis, luciferase activity in normal SPlucTg mice was detected in the testis of male mice. Furthermore, similar to the known requirement of transient expression of survivin for pathophysiological responses, we observed a transient luciferase expression in castrated SPlucTg male mice after supplement of androgen. Significantly, it was reported that survivin expression turns on during mouse liver injury and regeneration; a transient and dose-dependent luciferase expression in the mouse liver was observed after administration of carbon tetrachloride into SPlucTg mice. We further demonstrated that luciferase activity closely correlates with endogenous survivin expression. We also demonstrated that only a subset of cells expresses survivin, and its expression overlaps with the expression of several stem cell markers tested. Thus, we have generated a unique animal model for analysis of diverse pathophysiological processes and possible stem cell distribution/activity in vivo.Evidence indicates that survivin, an inhibitor of apoptosis (IAP) family protein and a promoter of cell cycle and mitosis,^1,2,3 plays an important role in cancer initiation⁴ and angiogenesis,^5,6,7 tumor progression, drug/radiation resistance,⁸ and tumor relapse.^3,9 Survivin may also play a role in other pathophysiological processes.^10,11,12 These include embryonic development, hematopoietic cell survival and proliferation, T-cell development, multiple sclerosis, brain pathology, and pathophysiology of other organs including liver, pancreas, gastrointestine, testes, endometrium, and placenta.¹¹ However, studies indicated that the behavior of survivin expression, subcellular localization, and/or gene regulation appear to be different during normal tissue turnover or repair versus cancer initiation and progression.¹¹ Furthermore, in some cases, normal cell division, survival, and development happen in the absence of survivin¹³ or with differential requirement of survivin.¹⁴ Additionally, because of increased expression of survivin in cancer, the interaction of survivin molecules with other proteins may differ from normal cells and/or may exclusively occur in cancer cells.^15,16 This partially explains the observations indicating that whereas abrogation of survivin expression or functions triggers significant apoptosis of cancer cells, it has minimal effects on normal cells and tissues.^{17,18,19,20,21,22,23,24} Thus, survivin appears to be an ideal cancer preventive and therapeutic target.Targeting survivin for cancer treatment might cause little toxicity to normal tissues. Indeed, many natural chemopreventive and chemotherapeutic dietary components, such as resveratrol, silibinin, and sulindac²⁵ as well as selenium,²⁶ retinoid,^27,28 and vitamin D (eg, calcitriol)²⁹ down-regulate survivin expression in cancer cells. This is consistent with a role for survivin in oncogenesis. However, because of the lack of an appropriate in vivo animal model system, the role for survivin either in cancer-related or in other in vivo pathological processes¹¹ remains to be elucidated.Here, we report the generation of a novel transgenic mouse model that expresses the luciferase reporter gene as the human survivin gene surrogate under the control of a human survivin gene promoter. We demonstrated that this new mouse model possesses an active and highly responsive luciferase reporter system, which reflects the dynamic expression of endogenous survivin during pathology. We further show that survivin expression overlaps with several stem cell markers, which suggests that survivin may be a good stem cell marker as well. Our model has several unique features for in vivo imaging in comparison with the model previously reported.³⁰ In our model, we used the human survivin promoter with sufficient length (4.5 kb), instead of using a mouse survivin proximal promoter (0.8 kb; as a note, the design in Xia et al’s report³⁰ is good for studying the relationship between survivin expression and cell cycle/mitosis). Importantly, using the luciferase reporter as a surrogate to monitor survivin gene activity during various pathophysiological processes will increase sensitivity by several orders of magnitude in comparison with using GFP as a reporter for in vivo imaging, although GFP can provide strong signals. Therefore, with our model, it is now possible to monitor endogenous survivin gene activity through in vivo imaging of its surrogate luciferase activity during various pathophysiological processes. This mouse model has the potential to delineate the dynamic expression and role of survivin in cancer and other pathophysiological processes.     

Prognostic Significance of Gremlin1 (GREM1) Promoter CpG Island Hypermethylation in Clear Cell Renal Cell Carcinoma

Gremlin1 (GREM1), a bone morphogenetic protein antagonist and putative angiogenesis-modulating gene, is silenced by promoter hypermethylation in human malignancies. Here we study GREM1 methylation in clear cell renal cell carcinoma (ccRCC) and its impact on tumor characteristics and clinical outcome. Three GREM1 promoter CpG island regions (i, ii, iii) were analyzed by methylation-specific PCR and/or bisulfite sequencing in ccRCC cell lines and ccRCCs from two independent patient series. Results were correlated with clinicopathological and angiogenic parameters. Bisulfite sequencing of ccRCC cell lines showed GREM1 methylation, associated with absence of GREM1 mRNA. GREM1 methylation prevalence in ccRCCs varied between regions: 55%, 24%, and 20% for regions i, ii, and iii, respectively. GREM1 region iii methylation was associated with increased tumor size (P = 0.02), stage (P = 0.013), grade (P = 0.04), tumor (P = 0.001), and endothelial cell (P = 0.0001) proliferation and decreased mean vessel density (P = 0.001) in a hospital-based ccRCC series (n = 150). In univariate analysis, GREM1 region iii methylated ccRCCs had a significant worse survival when compared with unmethylated ccRCCs (hazard ratio [HR] = 2.35, 95% confidence interval [CI]:1.29 to 4.28), but not in multivariate analysis (HR = 0.88, 95% CI: 0.45 to 1.74). In a population-based validation series (n = 185), GREM1 region iii methylation was associated with increased Fuhrman grade (P = 0.03) and decreased overall survival (P = 0.001) in univariate and multivariate analysis (HR = 2.32, 95% CI: 1.52 to 3.53 and HR = 2.27, 95% CI: 1.44 to 3.59, respectively). The strong correlation between GREM1 region iii promoter methylation and increased malignancy and its correlation with active angiogenesis indicates a role for GREM1 in ccRCC carcinogenesis and tumor angiogenesis.Clear cell renal cell carcinoma (ccRCC) accounts for ≈75% of all cases of renal cell cancer,¹ and is characterized by increased vascularization and an unclear clinical prognosis. Currently, patient performance status, tumor size, nodal and distant metastasis (TNM)–stage, and Fuhrman nuclear grade are the most useful predictors of patient outcome.² However, interest in additional prognostic molecular markers is growing. Inactivation of the von Hippel-Lindau (VHL) gene has been shown to be a common and early event in the carcinogenesis of ccRCC.^3,4,5 Fifty to 70% of ccRCC tumors harbor a VHL mutation,^5,6 and in 5% to 20% of ccRCCs VHL is silenced by promoter CpG island hypermethylation.^7,8 Although defective VHL functioning is a key event in the development in both sporadic and hereditary ccRCCs, alterations in the structure or regulation of the VHL gene do not appear to be directly associated with tumor cell proliferation and patient prognosis,^9,10 suggesting a complex interplay of additional genetic and epigenetic changes that may accumulate during RCC development.One intriguing candidate gene in this development may be the highly conserved Gremlin1 (GREM1), which we have identified to be expressed on 5′aza-2-deoxycytidine (DAC) treatment in four ccRCC cell lines in a epigenome-wide screen. GREM1 is a secreted glycoprotein that binds and antagonizes bone morphogenetic proteins (BMPs) 2, 4, and 7, thereby preventing the ability of these ligands to interact with their receptors resulting in inhibition of downstream transforming growth factor-β signaling.^11,12,13 BMPs, the largest subfamily of the transforming growth factor-β superfamily, are pleiotropic growth factors serving multiple functions in many cell and tissue types including angiogenesis, proliferation, apoptosis, differentiation, chemotaxis, and extracellular matrix production during development as well as in adult life.¹⁴ BMPs and BMP-antagonists such as Gremlin1 have been demonstrated in regulating renal development^15,16,17,18 and in the pathogenesis of nephropathy.^19,20,21 BMP-independent activities of GREM1 in cancer^12,22 and angiogenesis²³ have also been demonstrated. However, the role of GREM1 in renal cancer pathogenesis and the mechanisms by which GREM1 gene expression is regulated remains incompletely understood.GREM1 has been identified as one of the targets of Polycomb Repressive Complex 2 (PCR2) subunit protein SUZ12,²⁴ which marks repressive chromatin during early stages of embryonic stem cell differentiation. Compared with unmarked stem cell genes, Polycomb group targets have up to 12-fold increased susceptibility to develop cancer-specific promoter CpG island hypermethylation,²⁵ suggesting that epigenetic mechanisms may play a key role in regulating GREM1 expression. Further evidence that this mechanism is important in cancer development has recently been provided by the observations that GREM1 promoter CpG island methylation is prevalent in other tumor types such as gastric,²⁶ bladder, and prostate cancer.²⁷The aim of this study was to investigate GREM1 promoter CpG island methylation and its association with clinicopathological and angiogenesis parameters in ccRCC.     

Characterization of the Roles of Hemolysin and Other Toxins in Enteropathy Caused by Alpha-Hemolytic Escherichia coli Linked to Human Diarrhea

Escherichia coli strains producing alpha-hemolysin have been associated with diarrhea in several studies, but it has not been clearly demonstrated that these strains are enteropathogens or that alpha-hemolysin is an enteric virulence factor. Such strains are generally regarded as avirulent commensals. We examined a collection of diarrhea-associated hemolytic E. coli (DHEC) strains for virulence factors. No strain produced classic enterotoxins, but they all produced an alpha-hemolysin that was indistinguishable from that of uropathogenic E. coli strains. DHEC strains also produced other toxins including cytotoxic necrotizing factor 1 (CNF1) and novel toxins, including a cell-detaching cytotoxin and a toxin that causes HeLa cell elongation. DHEC strains were enteropathogenic in the RITARD (reversible intestinal tie adult rabbit diarrhea) model of diarrhea, causing characteristic enteropathies, including inflammation, necrosis, and colonic cell hyperplasia in both small and large intestines. Alpha-hemolysin appeared to be a major virulence factor in this model since it conferred virulence to nonpathogenic E. coli strains. Other virulence factors also appear to be contributing to virulence. These findings support the epidemiologic link to diarrhea and suggest that further research into the role of DHEC and alpha-hemolysin in enteric disease is warranted.Escherichia coli is one of the major causes of human infectious diseases, partly because of the wide variety of virulence mechanisms and pathotypes (15), and new pathotypes continue to be described. A new pathotype was proposed by Gunzburg et al. after examining diarrheal pathogens in a prospective community-based study among Australian Aboriginal children (22). One group of isolates was significantly (P < 0.05) associated with diarrhea, and these isolates were particularly common among children younger than 18 months. The isolates did not produce any recognized enterotoxin or classic enteric virulence factor, although they exhibited diffuse or aggregative adhesion in a modified adhesion assay (15). All isolates were able to detach HEp-2 cell monolayers and were termed “cell-detaching E. coli.” This property was shown to be mediated by alpha-hemolysin, and we demonstrate below that all cell-detaching E. coli strains produce alpha-hemolysin and that some may also produce cytotoxic necrotizing factor 1 (CNF1) and other toxins. However, neither alpha-hemolysin nor CNF1 has been clearly demonstrated to be an enteric virulence factor, and the role of hemolysin in particular is controversial. We will refer to these isolates as diarrhea-associated hemolytic E. coli (DHEC) isolates.Alpha-hemolytic E. coli strains have been associated with human enteric disease, especially among young children (8, 10–12, 20–22), and the related enterohemolysin of E. coli O157 (35) appears to be involved in enteric disease. There has, however, been no large prospective case-controlled epidemiologic study of the association of alpha-hemolysin with human diarrhea. Alpha-hemolytic bacteria are also associated with enteric disease and diarrhea in pigs, cattle, and dogs (9, 13, 33, 36, 44, 45). Porcine diarrheal strains are almost universally hemolytic (23a), and alpha-hemolysin in these isolates enhanced virulence and colonization (37) but was not itself diarrheagenic. More recent studies have found that Hly⁺ CNF1⁺ strains caused fluid accumulation in piglets (33) and that neonatal pigs were susceptible to challenge with Hly⁺ CNF⁺ strains, which caused bloody diarrhea, enterocolitis, and systemic disease (45).In contrast, some earlier studies were unable to demonstrate a role for hemolysin in enteric disease, since neither hemolytic bacteria nor their supernatants caused fluid accumulation in ileal loops (10, 14, 37). Hemolytic strains may be isolated from the feces of asymptomatic people (26), and, among humans, hemolysin is more commonly associated with strains causing extraintestinal infections (5, 26).The genetics and in vitro mechanisms of alpha-hemolysin are well known. The hlyCABD operon encodes the structural 110-kDa hemolysin protein (HlyA) and proteins involved in processing and export (42). Once secreted, hemolytic activity is short-lived, and this has complicated studies of hemolysin toxigenicity (42). Hemolysin does not require a receptor to bind to target cells, inserting instead into the target cell membrane to form a pore that allows the free flow of cations, sugars, and water. This leads to leakage of intracellular contents and affects the cytoskeleton and metabolism (4, 9, 42, 43). In extraintestinal infections, hemolysin has multiple effects and roles, including resistance to host defense, tissue damage, and lethality, either by direct action or by stimulation of inflammatory mediators and signal transduction pathways (7, 9, 16, 42).CNF is a 114-kDa protein with homology to a family of dermonecrotic toxins (18) and is encoded by the monocistronic cnf gene, which lies just downstream of hly. The CNF1 toxin causes HeLa cells to become large and multinucleated as a result of actin disassembly, which results from activation of Rho (10, 19, 31). Similar to alpha-hemolysin, the role of CNF1 in diarrhea remains unclear. CNF1-producing strains have been isolated from diarrheal stools and have been associated with several outbreaks in humans (8, 10) and animals (13, 33, 44). Unfortunately, no large, prospective, case-controlled studies have been performed, and the best evidence for the pathogenicity of CNF1-toxigenic isolates is the marked virulence in piglet challenge experiments (45), outlined above. Purified CNF1 did not show enterotoxic potential in the suckling mouse or induce fluid accumulation in the rabbit ileal loop (10, 14), in contrast to the related CNF2, which is linked to enteric disease in animals (13, 14, 30). Both CNF toxins are extremely lethal, and have a variety of in vivo effects including tissue necrosis and edema (12–14).In this paper, we characterize DHEC isolates that were obtained from a study where alpha-hemolysin was significantly associated with disease (22) and show that they are able to cause disease in rabbits. Using molecular genetics, we attempt to analyze the role of each gene in pathogenesis.     

In Vivo Regulation of Replicative Legionella pneumophila Lung Infection by Endogenous Interleukin-12

The in vivo role of endogenous interleukin 12 (IL-12) in modulating intrapulmonary growth of Legionella pneumophila was assessed by using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of A/J mice with virulent bacteria (10⁶ L. pneumophila cells per mouse) resulted in induction of IL-12, which preceded clearance of the bacteria from the lung. Inhibition of endogenous IL-12 activity, via administration of IL-12 neutralizing antiserum, resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection (compared to untreated L. pneumophila-infected mice). Because IL-12 has previously been shown to modulate the expression of cytokines, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10, which regulate L. pneumophila growth, immunomodulatory effects of endogenous IL-12 on intrapulmonary levels of these cytokines during replicative L. pneumophila lung infection were subsequently assessed. Results of these experiments demonstrated that TNF-α activity was significantly lower, while protein levels of IFN-γ and IL-10 in the lung were similar, in L. pneumophila-infected mice administered IL-12 antiserum, compared to similarly infected untreated mice. Together, these results demonstrate that IL-12 is critical for resolution of replicative L. pneumophila lung infection and suggest that regulation of intrapulmonary growth of L. pneumophila by endogenous IL-12 is mediated, at least in part, by TNF-α.

Legionella pneumophila, the causative agent of Legionnaires’ disease, is an intracellular pathogen of mononuclear phagocytic cells (MPCs) (37, 43, 45). Pulmonary infection usually develops following inhalation of L. pneumophila-contaminated water aerosols or microaspiration of contaminated water sources (9). Following inhalation, the bacteria invade and replicate in host MPCs, primarily in alveolar MPCs (34, 36, 37, 43, 45). Intracellular growth of L. pneumophila results in eventual lysis of infected MPCs, the release of bacterial progeny, and reinfection of additional pulmonary cells (34, 36). Severe lung damage, mediated by tissue-destructive substances likely derived from both damaged host cells and the bacteria, ensues (20, 21).Previous studies have demonstrated that resistance to primary replicative L. pneumophila lung infection is dependent on the induction of cellular immunity and is mediated in part by cytokines including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) (8, 12, 14, 15, 23, 27, 28, 35, 57). Growth of L. pneumophila within permissive MPCs requires iron. IFN-γ limits MPC iron, thereby converting the MPC intracellular environment from one that is permissive to one that is nonpermissive for L. pneumophila replication (14, 15). IFN-γ in combination with other cytokines including TNF-α facilitates elimination of L. pneumophila from infected MPCs, likely through the induction of effector molecules including nitric oxide (12). In contrast, other cytokines including interleukin 10 (IL-10) facilitate growth of L. pneumophila in permissive MPCs, due in part to IL-10-mediated inhibition of TNF-α secretion and IFN-γ-mediated MPC activation (46).IL-12 is a recently described cytokine with pleiotropic effects on T cells and natural killer (NK) cells which include (i) regulation of expression of cytokines including IFN-γ, TNF-α, and IL-10 by T cells and/or NK cells, (ii) induction of T-cell and/or NK cell proliferation and/or differentiation, and (iii) enhancement of NK cell and T-cell cytotoxic activity (4, 5, 19, 32, 33, 39, 44, 47, 48, 50, 56). While systemic administration of exogenous IL-12 has been demonstrated to increase host resistance to several intracellular pathogens, including Leishmania major, Toxoplasma gondii, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium, and Plasmodium chabaudi, in mice (26, 29, 33, 40, 51, 52, 55), the role of endogenous IL-12 in innate immunity to intracellular pathogens including L. pneumophila has not been thoroughly investigated. We have recently developed a model of replicative L. pneumophila lung infection in A/J mice inoculated intratracheally with virulent bacteria and have used this model system to identify immune responses which mediate host resistance to legionellosis (10–12). Using this murine model of Legionnaires’ disease, we assessed the biologic relevance and immunomodulatory role of endogenous IL-12 in innate immunity to replicative L. pneumophila lung infection.     

Inhibition of NF-κB Signaling Reduces Virus Load and Gammaherpesvirus-Induced Pulmonary Fibrosis

IgA nephropathy (IgAN) and Henoch-Schönlein purpura (HSP) are diseases characterized by IgA deposits in the kidney and/or skin. Both may arise after upper respiratory tract infections, but the pathogenic mechanisms governing these diseases remain unclear. Patients with IgAN (n = 16) and HSP (n = 17) were included in this study aimed at examining whether IgA-binding M proteins of group A streptococci could be involved. As M proteins vary in sequence, the study focused on the IgA-binding-region (IgA-BR) of three different M proteins: M4, M22, and M60. Renal tissue from IgAN and HSP patients and skin from HSP patients were examined for deposits of streptococcal IgA-BR by immunohistochemistry and electron microscopy using specific antibodies, and a skin sample from a HSP patient was examined by mass spectrometry. IgA-BR deposits were detected in 10/16 IgAN kidneys and 7/13 HSP kidneys. Electron microscopy demonstrated deposits of IgA-BRs in the mesangial matrix and glomerular basement membrane, which colocalized with IgA. Skin samples exhibited IgA-BR deposits in 4/5 biopsies, a result confirmed by mass spectrometry in one patient. IgA-BR deposits were not detected in normal kidney and skin samples. Taken together, these results demonstrate IgA-BR from streptococcal M proteins in patient tissues. IgA-BR, would on gaining access to the circulation, encounter circulatory IgA and form a complex with IgA-Fc that could deposit in tissues and contribute to the pathogenesis of IgAN and HSP.Tissue deposits containing IgA characterize IgA nephropathy (IgAN) and Henoch-Schönlein purpura (HSP), two conditions affecting kidney function. IgAN is the most common primary glomerulonephritis worldwide. Its predominant clinical feature is episodic macroscopic hematuria usually coinciding with upper respiratory tract infections. Symptoms may, however, vary from microscopic hematuria to a severe nephritic-nephrotic syndrome. End-stage kidney disease occurs in 30% to 40% of patients within 20 years. Histopathologically IgAN is characterized by mesangial cell proliferation and in progressive cases crescent formation as well as glomerular sclerosis, interstitial fibrosis, and tubular atrophy. Ultramorphologic findings show mesangial deposits of immune complexes containing predominantly IgA.^1,2HSP is the most common form of vasculitis in childhood. It may affect many organs, but usually presents as skin lesions, varying from purpura to bullous intradermal bleedings, arthritis, gastrointestinal involvement with pain and/or bleeding. Renal involvement occurs in up to 50% of cases³ and is known as Henoch-Schönlein nephropathy (HSN). HSN may manifest as microscopic or macroscopic hematuria as well as glomerulonephritis or nephrotic syndrome. Approximately 20% of HSN cases will develop renal failure.⁴ The histopathological lesion termed leukocytoclastic vasculitis is characterized by inflammation of small vessels with perivascular polymorphonuclear leukocyte and mononuclear cell infiltrates. Immune deposits in affected organs contain IgA, and renal pathology resembles that seen in IgAN.^1,3The IgA mesangial deposits in kidneys of patients with IgAN and HSP are primarily composed of galactose-deficient IgA1.^5,6,7 The mechanism by which under-glycosylated IgA1 deposits in the mesangium, possibly in complex with IgG,^8,9 has not been determined. Environmental antigens have been proposed to contribute to the disease but have not been consistently associated with mesangial deposits.⁹ Although the etiology of IgAN and HSP is unclear, these diseases are often preceded by infections, primarily of the upper respiratory tract, and an infectious agent has therefore been suspected. There is circumstantial evidence for involvement of group A streptococcus (GAS, Streptococcus pyogenes),^{10,11,12,13,14,15} but infections with other bacteria^16,17 as well as viruses¹⁸ have been implicated as well.In this study we hypothesized that GAS infection could trigger IgAN and/or HSN, because GAS is a very common cause of upper respiratory tract infection, and because many GAS strains bind IgA-Fc.^19,20,21 The ability of a GAS strain to bind human IgA results from the presence of an IgA-binding region (IgA-BR) in the surface-localized M protein.^22,23 The fibrillar M protein, which is a major virulence factor of GAS, varies in sequence between strains²⁴ allowing classification of GAS isolates into more than 120 M serotypes.²⁵ The exact function of the IgA-BR in an M protein is not known, but there is evidence that it contributes to bacterial phagocytosis resistance.²⁶ The IgA-BR of an M protein represents a distinct domain that can be studied in isolated form, as a peptide that binds IgA.^27,28 Such IgA-binding peptides, designated Sap (streptococcal IgA-binding peptide), were used in the experiments described herein.To analyze whether IgA-binding streptococcal M proteins are present in affected tissues of patients with IgAN and/or HSP, and colocalize with IgA, we used antibodies to the IgA-BR of three different M proteins M4, M22, and M60. Of note, M4 and M22 are among the most common serotypes of clinical GAS isolates.²⁹ As the IgA-BRs of different M proteins vary extensively in sequence,^22,23 the use of antibodies to three different serotypes enhanced our chances to detect tissue deposition of an IgA-BR.     

Phenolic Compounds Prevent Alzheimer’s Pathology through Different Effects on the Amyloid-β Aggregation Pathway

Inhibition of amyloid-β (Aβ) aggregation is an attractive therapeutic strategy for Alzheimer’s disease (AD). Certain phenolic compounds have been reported to have anti-Aβ aggregation effects in vitro. This study systematically investigated the effects of phenolic compounds on AD model transgenic mice (Tg2576). Mice were fed five phenolic compounds (curcumin, ferulic acid, myricetin, nordihydroguaiaretic acid (NDGA), and rosmarinic acid (RA)) for 10 months from the age of 5 months. Immunohistochemically, in both the NDGA- and RA-treated groups, Aβ deposition was significantly decreased in the brain (P < 0.05). In the RA-treated group, the level of Tris-buffered saline (TBS)-soluble Aβ monomers was increased (P < 0.01), whereas that of oligomers, as probed with the A11 antibody (A11-positive oligomers), was decreased (P < 0.001). However, in the NDGA-treated group, the abundance of A11-positive oligomers was increased (P < 0.05) without any change in the levels of TBS-soluble or TBS-insoluble Aβ. In the curcumin- and myricetin-treated groups, changes in the Aβ profile were similar to those in the RA-treated group, but Aβ plaque deposition was not significantly decreased. In the ferulic acid-treated group, there was no significant difference in the Aβ profile. These results showed that oral administration of phenolic compounds prevented the development of AD pathology by affecting different Aβ aggregation pathways in vivo. Clinical trials with these compounds are necessary to confirm the anti-AD effects and safety in humans.Alzheimer’s disease (AD) is the most common form of dementia, resulting in deterioration of cognitive function and behavioral changes.¹ One of the pathological hallmarks of AD is extracellular deposits of aggregated amyloid-β protein (Aβ) in the brain parenchyma (senile plaques) and cerebral blood vessels (cerebral amyloid angiopathy (CAA)).¹ Deposition of high levels of fibrillar Aβ in the AD brain is associated with loss of synapses, impairment of neuronal functions, and loss of neurons.^2,3,4,5 Aβ was sequenced from meningeal vessels and senile plaques of AD patients and individuals with Down’s syndrome.^6,7,8 The subsequent cloning of the gene encoding the β-amyloid precursor protein and its localization to chromosome 21,^9,10,11,12 coupled with the earlier recognition that trisomy 21 (Down’s syndrome) invariably leads to the neuropathology of AD,¹³ set the stage for the proposal that Aβ accumulation is the primary event in AD pathogenesis. In addition, certain mutations associated with familial AD have been identified within or near the Aβ region of the coding sequence of gene of the amyloid precursor proteins,^14,15 presenilin-1 and presenilin-2,¹⁶ which alter amyloid precursor protein metabolism through a direct effect on γ-secretase.^17,18 These findings set the stage for the proposal that Aβ aggregation is the primary event in AD pathogenesis and leading to the proposal that anti-Aβ aggregation is a strategy for AD therapy.^19,20 Furthermore, there have been recent reports^{21,22,23,24,25} that Aβ fibrils are not the only toxic form of Aβ for developing AD, and smaller species of aggregated Aβ, Aβ oligomers, may represent the primary toxic species in AD. Therefore, it is necessary to consider the inhibition of Aβ oligomer formation as well as Aβ fibrils for the treatment of AD.²⁶To date, it has been reported that various compounds inhibit the formation and extension of Aβ fibrils, as well as destabilizing Aβ fibrils in vitro.^{19,20,27,28,29,30,31,32,33,34,35,36} Among the reported compounds, several phenolic compounds, such as wine-related polyphenols (myricetin (Myr), morin, and tannic acid, and so on), curcumin (Cur), ferulic acid (FA), nordihydroguaiaretic acid (NDGA), and rosmarinic acid (RA) had especially strong anti-Aβ aggregation effects in vitro. Furthermore, it was shown recently that a commercially available grape seed polyphenolic extract, MegaNatural-Az, inhibited fibril formation, protofibril formation, and oligomerization of Aβ.³⁷ Moreover, MegaNatural-Az also reduced cerebral amyloid deposition as well as attenuating AD-type cognitive deterioration using transgenic mice.³⁸ In addition to these studies by the current authors, several other researchers have reported similar effects of phenolic compounds.^{26,39,40,41,42,43,44} First, Cur decreased cerebral Aβ plaque burden in vivo,^{39,40,41,42,44} and inhibited the formation of Aβ oligomers in vitro.^26,39 Second, epigallocatechin gallate efficiently inhibited fibril and oligomer formation of Aβ.⁴³ However, a very recent in vitro study²⁶ reported that Cur, Myr, and NDGA inhibited the formation of Aβ oligomers, but Cur and NDGA promoted the formation of Aβ fibrils. This indicated that the effects of these phenolic compounds on Aβ aggregation remain controversial. These different results may reflect different experimental conditions in these studies. To resolve this problem, a systematic in vivo study is required; however, few reports on the effects of phenolic compounds on Aβ aggregation in vivo have been published so far, except for reports about Cur.^{39,40,41,42,44}To elucidate the inhibitory effects of phenolic compounds on Aβ aggregation in vivo, several phenolic compounds, including Cur, FA, Myr, NDGA, and RA, were fed to AD model mice, and the cerebral plaque burden and formation of Aβ oligomers were compared systematically.     

CXCR3/Ligands Are Significantly Involved in the Tumorigenesis of Basal Cell Carcinomas

Basal cell carcinoma (BCC) is the most common skin malignancy encountered worldwide. We hypothesized that CXC chemokines, small cytokines involved in inducing directed leukocyte chemotaxis, could play a key role in the modulation of BCC growth. In this study, quantitative RT-PCR revealed that the chemokines CXCL9, 10, 11, and their receptor CXCR3 were significantly upregulated by an average 22.6-fold, 9.2-fold, 26.6-fold, and 4.9-fold, respectively in BCC tissue samples as compared with nonlesional skin epithelium. Immunohistochemistry analysis revealed that CXCR3, CXCL10, and CXCL11, but not CXCL9, colocalized with cytokeratin 17 (K17) in BCC keratinocytes. In addition, CXCR3 and its ligands were expressed in cells of the surrounding BCC stroma. The chemokines and K17 were also expressed in cultured human immortalized HaCaT keratinocytes. Exposure of HaCaT cells or primary BCC-derived cells to CXCL11 peptides in vitro significantly increased cell proliferation. In primary BCC-derived cell cultures, addition of CXCL11 progressively selected for K17⁺/CXCR3⁺ co-expressing cells over time. The expression of CXCR3 and its ligands in human BCC keratinocytes, the enhancement of keratinocyte cell proliferation by CXCL11, and the homogeneity of K17⁺ BCC cells in human BCC-isolated cell population supported by CXCR3/CXCL11 signaling all suggest that CXCR3 and its ligands may be important autocrine and/or paracrine signaling mediators in the tumorigenesis of BCC.Basal cell carcinoma (BCC), a type of nonmelanoma skin cancer, is the most prevalent neoplasm found in the population.^1,2,3 Approximately one-third of the United States population developed nonmelanoma skin cancer from 1994 to 2002, and 83% of these patients presented with BCC.^1,2,3 The BCC frequency in regional populations is increasing at a rate of 2% to 19% per year, with an annual rate increase of 4% in Canada.^4,5,6 There are approximately 800,000 new cases of BCCs diagnosed in the United States and 70,000 in Canada each year.⁷ The age-adjusted incidence per 100,000 individuals is in the region of 100 to 2000 for men and 80 to 1500 for women, depending on the geographical location and specific genetic background of the individuals studied.^8,9,10,11,12 The rising rate of BCC incidence is likely attributable to a combination of improved diagnosis and reporting, increased longevity, increased sun exposure, changes in clothing style, and increased UV radiation intensity due to ozone depletion.^13,14BCC tumors are composed of proliferating keratinocytes that derive from the basal layers of the epidermis and/or hair follicle bulge.^15,16 There are several BCC subtypes, of which the most common one is nodular, followed by superficial, and morpheiform/infiltrating BCCs.^17,18 Although BCCs rarely metastasize (rate of metastasis ranges from 0.003% to 0.55%) and cause death, they can result in significant patient morbidity.^2,17 Because this cancer typically affects sun-exposed skin of the head and neck, cosmetic disfigurement is common. BCCs can behave aggressively with deep invasion, recurrence, and resistance to standard treatment. The presence of BCC is also associated with increased risk of developing other BCCs or skin cancers, such as malignant melanoma and squamous cell carcinoma.^2,17The fundamental initiation of BCC development is typically due to mutation of genes in the sonic hedgehog (Shh) signaling pathway.^19,20,21 Mutation of the Shh pathway associated genes, SHH, PTCH, SMO, GLI1, or GLI2, can promote BCC tumorigenesis.^{21,22,23,24,25,26} Such genomic alteration has been found in >70% of BCC cases studied.^19,20,21 SHH is a morphogene, and it is essential for the development of various organs including the skin and hair follicles.^19,22 Shh plays a key role in regulating stem cell populations,²² and mutations in genes of this signaling pathway can result in inhibition of epithelial cell cycle arrest, neoplasia, and perturbation of other associated cell-cycle events.^22,26,27In addition to genetic defects, depletion of host immune responses and the development of a more permissive tissue environment may act as pivotal forces for BCC progression and transformation.^2,28 Distinct epithelial-stromal-inflammatory patterns have been identified and correlated with specific BCC subtypes and tumor progression.²⁸ However, the mechanisms that underlie these BCC tumorigenesis-enabling events are still not known, and the molecular profile of the immune response during BCC development has not been fully established.The CXC chemokines, CXCL9, 10, and 11 are IFNγ–induced small secretory proteins that are synthesized and released by leukocytes, as well as epithelial, endothelial, and stromal cells.²⁹ They interact with the heptahelical G protein complex receptor CXCR3 and exert signaling effects in a paracrine or autocrine fashion.^30,31 They have been well established as chemoattractive for activated CXCR3⁺ T cells.²⁹ Recently, increased expression of these chemokines, together with their receptor CXCR3, have been found to be associated with advanced-stage tumors, such as malignant melanoma, ovarian carcinoma, and B-cell lymphoma.^32,33,34 In addition, previous literature has demonstrated that IFNγ-induced production of CXCL9, 10, and 11 in human neonatal foreskin keratinocytes is enhanced during inflammatory dermatoses such as psoriasis.^35,36,37 However, little is known about the roles and mechanisms played by these CXCR3 ligands in the immunoregulation of other skin diseases and cancers.Previously, our laboratory conducted a detailed microarray-based analysis of genes in superficial, nodular, and morpheiform BCC tissues to detect specific gene sets with significant differential expression as compared with normal skin.³⁸ With further examination of gene ontology data from this study (Gene Expression Omnibus database series record {"type":"entrez-geo","attrs":{"text":"GSE6520","term_id":"6520"}}GSE6520), we subsequently identified 27 immunoregulatory genes with significant differential expression in BCCs including several CXC chemokines and their receptors (unpublished data). We hypothesized that the upregulation of CXCR3 and its ligands could be a key BCC growth promotion event. In this study, we investigated the potential impact of CXCR3/ ligand signaling on BCC development. Our findings suggest that the chemokines CXCL9, 10, and 11 are potent signaling mediators via CXCR3 for BCC keratinocyte proliferation, and may be significantly involved in the regulation of BCC tumor development.     

Selective Cell Death of Hyperploid Neurons in Alzheimer’s Disease

Aneuploidy, an abnormal number of copies of a genomic region, might be a significant source for neuronal complexity, intercellular diversity, and evolution. Genomic instability associated with aneuploidy, however, can also lead to developmental abnormalities and decreased cellular fitness. Here we show that neurons with a more-than-diploid content of DNA are increased in preclinical stages of Alzheimer’s disease (AD) and are selectively affected by cell death during progression of the disease. Present findings show that neuronal hyperploidy in AD is associated with a decreased viability. Hyperploidy of neurons thus represents a direct molecular signature of cells prone to death in AD and indicates that a failure of neuronal differentiation is a critical pathogenetic event in AD.Understanding the mechanisms underlying generation of neuronal variability and complexity remains a basic challenge to neuroscience. Structural variation in the human genome is likely to be one important mechanism for neuronal diversity and brain disease.¹ The genetic profile of a cell can be permanently altered by chromosomal aneuploidy (i.e., an abnormal number of copies of a genomic region). A combination of multiple different forms of aneuploid cells due to loss or gain of whole chromosomes (mosaic aneuploidy) giving rise to cellular diversity at the genomic level have been described in neurons of the normal and diseased adult human brain.^{2,3,4,5,6,7,8,9,10,11,12}Cells in normal individuals have basically been assumed to contain identical euploid genomes. Still, earlier hypotheses suggested that a number of mammalian somatic tissues are populated by polyploid cells. Adult neurons of mammals were assumed to be postmitotic cells characterized to some extent by a polyploid chromosome complement. Testing this hypothesis in the past through histochemical methods, however, yielded controversial results through technical limitations.^13,14 However, with the recent development of molecular cytogenic techniques, aneuploid cells in the normal developing and mature brain have clearly been identified, indicating that the maintenance of aneuploid neurons in the adult CNS is a widespread, if not universal, property of organization.^{2,3,4,5,6,7,8,9,10,11,12,15}Recent studies of the embryonic brain have shown that approximately one-third of the dividing cells that give rise to the cerebral cortex have genetic variability, manifested as chromosome aneuploidy.^3,7,12 Neurons that constitute the adult brain arise from mitotic neural progenitor cells in the ventricular zone, a proliferating region where aneuploid cells appear to be generated through various chromosome segregation defects initially.^3,10 While a portion of these aneuploid cells apparently die during development,^3,16,17 aneuploid neurons have been identified in the mature brain in all areas assayed^{2,3,4,5,6,7,8,9,10,11,12,15} indicating that aneuploidy does not necessarily impair viability.¹⁸ Aneuploid neurons in the adult have been shown to make distant connections and express markers associated with neural activity, which indicates that these neurons can be integrated into brain circuitry.^4,12Contrary to this physiological consequences of “low-level” aneuploidy potentially contributing to neuronal diversity, aneuploidy above a critical threshold might be detrimental. Genomic instability and imbalances in gene dosage associated with aneuploidy can lead to developmental abnormalities, decreased cellular and organismal fitness, and increased susceptibility to disease.^19,20Aneuploid cells have typically been associated with pathophysiological conditions such as cancer,²¹ and most aneuploid syndromes present brain phenotypes and show a high vulnerability for psychiatric disorders.²² Mental impairment is a characteristic feature of all recognizable autosomal aneuploidy syndromes.Recent studies have shown an increased rate of aneuploid neurons in Alzheimer’s disease (AD), schizophrenia, autism, and ataxia-telangiectasia.^2,5,23,24,25 So far, however, direct evidence for a pathogenetic role for neuronal aneuploidy in these disorders is lacking. In particular, it remains unclear whether neuronal aneuploidy affects cellular viability, thus contributing directly to neurodegeneration and cell death.Here, we analyzed the fate of hyperploid neurons at the conversion from preclinical to mild AD and during further progression to severe stages of the disease. We can show that neurons with a more-than-diploid content of DNA are increased in preclinical stages of AD and are selectively affected by cell death during progression of the disease. Present findings show that neuronal hyperploidy in AD is associated with a decreased viability and directly linked to cell death.     

Chlamydophila pneumoniae Infection Leads to Smooth Muscle Cell Proliferation and Thickening in the Coronary Artery without Contributions from a Host Immune Response

Chlamydophila pneumonia (C. pneumonia) infection has been associated with the progression of atherosclerosis. It remains unclear, however, whether C. pneumoniae in the absence of an immune response can alone initiate atherogenic events within a complex vessel environment. Left anterior descending coronary arteries isolated from porcine hearts were dissected and placed in culture medium for 72 hours before infection with C. pneumoniae. C. pneumoniae replicated within the arterial wall for the duration of the experiment (up to 10 days). A significant increase in chlamydial-HSP60 protein expression from day 2 to 10 post-infection (pi) indicated the presence of metabolically active C. pneumonia within infected vessels. Significant arterial thickening in infected coronary segments was observed by a considerable decrease in the ratio of lumen to total vessel area (48 ± 3% at day 4 pi versus 23 ± 3% at day 10 pi) and a significant increase in the ratio of media to luminal area (113 ± 16% at day 4 pi versus 365 ± 65% at day 10 pi). Structural changes were accompanied by an up-regulation of host HSP60 and proliferating cell nuclear antigen expression levels. Immunohistochemical staining confirmed proliferating cell nuclear antigen expression to be primarily localized within smooth muscle cells of the medial area. These results demonstrate that C. pneumoniae infection can stimulate arterial thickening in a complex vessel environment without the presence of a host immune response and further supports the involvement of HSP60 in this action.Chlamydophila pneumoniae (C. pneumoniae) is an obligate intracellular parasite that causes respiratory illness in humans. It is a widespread pathogen, and up to 50% of adults by the age of 20 may carry serological evidence of exposure.¹ In the cytoplasm of an infected cell, C. pneumoniae multiplies within membrane bound vacuoles termed as the chlamydial inclusions or reticulate body. Active metabolism occurs in the reticulate body which imports ATP, amino acids, nucleoside (RNA) precursors, and other essential molecules from the host cytoplasm.^2,3,4 At 48 to 72 hours post infection (pi), the reticulate bodies differentiate into infectious progeny particles, elementary bodies. Currently, the molecular mechanisms involved at the various stages of the C. pneumoniae life cycle remain unclear.The clinical significance of C. pneumoniae goes beyond respiratory illness. Several in vitro investigations have suggested that C. pneumoniae, after infecting monocytes of the respiratory tract, travels through the circulatory system and disseminates at the endothelial surface of susceptible arterial lesions.^5,6,7 The first association of C. pneumoniae infection with coronary artery disease (CAD) was reported by Saikku et al in 1988.⁸ Since then, numerous studies have confirmed a strong correlation of C. pneumoniae infection with coronary arterial diseases.^{5,6,9,10,11,12} Furthermore, live C. pneumoniae and C. pneumoniae–specific T-cells were detected in coronary and carotid atherosclerotic plaque,^13,14 but not in neighboring healthy tissues.^15,16,17 The use of antibiotics in patients with CAD has been shown to be beneficial.^18,19,20The mechanism whereby C. pneumoniae infection stimulates CAD is unclear. However, C. pneumoniae infection was shown to induce atherogenesis in vivo in an hypercholesterolemic animal model.²¹ C. pneumoniae infection stimulates foam cell formation in atheroma,^22,23,24 and induces proliferation of vascular smooth muscle cells (VSMCs).^25,26,27 Furthermore, C. pneumoniae infection leads to up-regulation of endogenous heat-shock protein 60 (HSP60),²⁷ shown to be a major contributor of an autoimmune response during the development of atherosclerosis.^28,29,30 Overexpression of endogenous HSP60 directly induced VSMC proliferation,²⁷ an event identified as an important component of the atherogenic process.The contribution of the immune response to the proliferative action of the infectious stimulant has been hypothesized to be an important component of the atherogenic response.³¹ Many other infectious agents have also been shown to stimulate atherosclerosis,^32,33 suggesting a common immune/inflammatory response pathway in the atherogenic action. The study of C. pneumoniae-induced atherogenesis on any in vivo model is inevitably complicated by the contributions of host-immune and inflammatory responses (T-cells and cytokines), which are thought to be strong contributors to the lesions. C. pneumoniae infection can augment the secretion of inflammatory markers by endothelial cells.^25,26,27 The up-regulation of HSP60 in human atheroma and the corresponding elevated levels HSP60 antibodies can also lead to autoimmune injury in the arterial environment.^{29,34,35,36,37} However, the question of whether C. pneumoniae infection directly injures coronary arteries and induces thickening without the effects of the immune system remains unanswered. It is difficult to answer this question in an in vivo environment because the immune system is fully activated by the infectious agent. Data from in vitro studies are not ideal for studying C. pneumoniae induced proliferation and thickening because cell lines are naturally proliferative and lack the structural complexity of a vessel. In the current study, we used a novel ex vivo organ culture model that allowed us to study the direct consequences of the C. pneumoniae infection in the complex arterial environment without the confounding contributions of a host immune system. Using this novel approach, we observed C. pneumoniae replication and the spread of the infection into the medial layer of coronary arteries. Furthermore, C. pneumoniae infection induced arterial wall thickening and expression of proliferation and stress markers. This is the first report that shows that C. pneumoniae infection can stimulate an atherogenic response independently of the host immune reactions.