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.     

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.     

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.     

Long-Term Expression of Tissue-Inhibitor of Matrix Metalloproteinase-1 in the Murine Central Nervous System Does Not Alter the Morphological and Behavioral Phenotype but Alleviates the Course of Experimental Allergic Encephalomyelitis

Tissue inhibitors of metalloproteinases (TIMPs) are a family of closely related proteins that inhibit matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMPs 2, 3, and 4 are constitutively expressed at high levels, whereas TIMP1 can be induced by various stimuli. Here, we studied the effects of constitutive expression of TIMP1 in the CNS in transgenic mice. Transgene expression started prenatally and persisted throughout lifetime at high levels. Since MMP activity has been implicated in CNS development, in proper function of the adult CNS, and in inflammatory disorders, we investigated Timp1-induced CNS alterations. Despite sufficient MMP inhibition, high expressor transgenic mice had a normal phenotype. The absence of compensatory up-regulation of MMP genes in the CNS of Timp1 transgenic mice indicates that development, learning, and memory functions do not require the entire MMP arsenal. To elucidate the effects of strong Timp1 expression in CNS inflammation, we induced experimental allergic encephalomyelitis. We observed a Timp1 dose-dependent mitigation of both experimental allergic encephalomyelitis symptoms and histological lesions in the CNS of transgenic mice. All in all, our data demonstrate that (1) long-term CNS expression of TIMP1 with complete suppression of gelatinolytic activity does not interfere with physiological brain function and (2) TIMP1 might constitute a promising candidate for long-term therapeutic treatment of inflammatory CNS diseases such as multiple sclerosis.Tissue-inhibitor of matrix metalloproteinase-1 (TIMP1), a tightly regulated 28.5-kDa glycoprotein,^1,2 belongs to a family of multifunctional secreted proteins (TIMPs 1 to 4) that regulate the proteolytic activity of matrix metalloproteinases (MMPs). Together, MMPs and TIMPs control the pericellular environment, including the turnover of extracellular matrix proteins, bioavailability of growth factors and cytokines, and shedding of membrane receptors.³ In the central nervous system (CNS), expression of MMP and TIMP genes is highly orchestrated during development and normal brain function.^4,5,6 The spatio-temporal expression pattern of TIMP1 indicates a developmental role in the hippocampus and the cerebellum.^4,7,8,9 TIMP1 was also proposed as a candidate plasticity protein in learning and memory.^10,11 Several MMPs on the other hand are expressed in the developing cerebellum,⁴ and in migrating neural precursors,^12,13 and MMP activity was localized to the growth cone of cultured sympathetic neurons.^14,15,16 In the normal adult CNS, however, MMP expression is low.^17,18 Alterations of the MMP/TIMP ratio are tightly associated with pathological processes and clearly influence the course of various diseases.^12,17,18,19 In autoimmune diseases such as multiple sclerosis and the animal model experimental allergic encephalomyelitis (EAE), elevated activity of several MMPs was detected and suspected to influence the outcome of the disease.^{17,20,21,22,23} Accordingly, synthetic MMP inhibitors blocked the blood-brain-barrier disruption and improved the clinical condition of the animals,^23,24,25 and Timp1 deficient mice revealed increased leukocyte infiltration as well as enhanced spinal cord demyelination.²⁶ Due to serious side-effects, however, only few synthetic MMP-inhibitors have been introduced to human therapy.²⁷Since high levels of Timp1 were expressed by astrocytes around the inflammatory lesions in EAE¹⁷ and to further investigate the role of Timp1 in the CNS, we targeted its expression to astrocytes in a transgenic mouse model.     

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.     

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.     

Neuroprotective Effect of Oligodendrocyte Precursor Cell Transplantation in a Long-Term Model of Periventricular Leukomalacia

Perinatal white matter injury, or periventricular leukomalacia (PVL), is the most common cause of brain injury in premature infants and is the leading cause of cerebral palsy. Despite increasing numbers of surviving extreme premature infants and associated long-term neurological morbidity, our understanding and treatment of PVL remains incomplete. Inflammation- or ischemia/hypoxia-based rodent models, although immensely valuable, are largely restricted to reproducing short-term features of up to 3 weeks after injury. Given the long-term sequelae of PVL, there is a need for subchronic models that will enable testing of putative neuroprotective therapies. Here, we report long term characterization of a neonatal inflammation-induced rat model of PVL. We show bilateral ventriculomegaly, inflammation, reactive astrogliosis, injury to pre-oligodendrocytes, and neuronal loss 8 weeks after injury. We demonstrate neuroprotective effects of oligodendrocyte precursor cell transplantation. Our findings present a subchronic model of PVL and highlight the tissue protective effects of oligodendrocyte precursor cell transplants that demonstrate the potential of cell-based therapy for PVL.Premature, low-birth weight infants are commonly diagnosed with perinatal white matter damage, which leads to long-term neurological deficits including cerebral palsy.^1,2,3,4 Congenital encephalomyelitis was described almost 150 years ago, a disease in newborn children characterized by pale softened zones of degeneration within the deep white matter surrounding lateral ventricles and is now often referred to as periventricular leukomalacia (PVL).⁵ The white matter damage that occurs in preterm infants has more recently been shown to be also accompanied by significant cerebral-cortex and deep-gray matter abnormalities leading to neurodegeneration and altered neurobehavioral performance.⁶PVL is characterized by selective oligodendrocyte precursor cell (OPC) loss resulting in delayed or disrupted myelination, white matter atrophy and ventriculomegaly, neuronal loss, and cyst and scar formation. Extreme premature birth (approximately 23 to 32 weeks) accompanied by inflammation or infection corresponds to the period when immature dividing and differentiating oligodendrocytes predominate in the cerebral white matter.^7,8,9,10 PVL has a complex etiology. The two most important determinants are cerebral hypoperfusion and maternal intrauterine infection. Periventricular white matter is susceptible to hypoperfusion due to the comparative immaturity of the periventricular vasculature of the preterm infant.^11,12 Early oligodendrocyte lineage cells are vulnerable to the consequences of hypoperfusion and subsequent microglial and astrocytic activation on account of amplified glutamate receptor-mediated responses and lack of efficient antioxidant protection.^{13,14,15,16,17,18} Maternal infection, recently implicated as a causative factor in the pathogenesis of PVL,^{19,20,21,22,23} is believed to initiate an inflammatory/cytokine cascade that results in the release of early-response pro-inflammatory cytokines such as tumor necrosis factor–α (TNF-α), interleukin−1 β (IL-1β) and interleukin−6 (IL-6), and causes damage to immature oligodendrocytes.²⁴ TNF-α appears to have a particularly important role in PVL pathogenesis with in vitro evidence suggesting direct damage to OPCs,^25,26 while IL-1β and IL-6 modulate injury indirectly.^27,28Although cells of the oligodendrocyte lineage are regarded as the primary target in the pathogenesis of PVL, there is increasing evidence that neonatal white matter damage is accompanied by gray matter abnormalities, including neuronal loss, impaired axonal guidance, and altered synaptogenesis.^6,29 Premature newborns affected by PVL often have smaller cerebral cortex and deep gray matter volumes, reduced cortical neurons, and alterations in the orientation of central white matter fiber tracts.^6,30,31,32 Together, these data suggest that developing neurons, like immature oligodendrocyte lineage cells, are also vulnerable to injury caused by inflammatory response or hypoxia.Aspects of PVL have been modeled by ischemia/hypoxia and inflammation-mediated rodent models. Animal models of hypoxia-ischemia have clearly shown that following brain injury there is reduced myelination, enlarged ventricles, loss of neurons and damage to axons and dendrites and altered neurobehavioral performances.^{33,34,35,36,37,38,39} Experimentally induced inflammation has been used in a number of studies to model PVL pathology and test potential treatments.^23,40,41,42 Administration of the endotoxin lipopolysaccharide (LPS), a potent inducer of innate immune response and inflammation,^43,44 either intracerebrally during early neonatal period, intrauterine or peritoneally to a pregnant mother results in inflammation and hypomyelination.^20,22,23,38 Moreover, in animal models of LPS-induced PVL neuronal loss and a reduction in neurite length in the parietal cortex has been observed.^42,45 In vitro evidence suggests LPS is not directly toxic to OPCs, but causes injury through the activation of Toll-like receptor 4-positive microglia, which is a source of pro-inflammatory cytokines, nitric oxide, and free radicals.^44,45,46 Furthermore, it has been shown that LPS-induced inflammation increases the susceptibility of white matter to injury in response to otherwise “harmless” subthreshold hypoxic-ischemic insult.⁴⁷ Current models of PVL are typically short term, with studies using either hypoxia-ischemia or inflammation rarely extending analysis beyond 14 days. There is therefore a need to evaluate the longer-term, subchronic consequences of LPS injury and specifically address whether hypomyelination and neuronal injury are self-limiting or indeed spontaneously repair.Cellular therapeutic strategies are predicated on cell replacement and/or tissue protection independent of specific cellular differentiation. Progenitor cells including OPCs have previously been used for cellular replacement of damaged or lost cells in a variety of CNS injury models where damage to myelin and neuronal loss occur.^{48,49,50,51,52,53} Furthermore, there is accumulating evidence that implicates progenitor cell mediated neuroprotection through a variety of mechanisms including graft derived neurotrophic support independent of directed differentiation.^54,55 Moreover, there is evidence to suggest that OPCs secrete factors that are capable of supporting neuronal survival,^{56,57,58,59,60,61} thus suggesting they may be a potential therapeutic source for replacing lost or damaged cells and protecting healthy tissue following neonatal brain injury. In this study we have examined the subchronic effects of LPS induced injury and then examined the putative neuroprotective effects of OPC transplantation.     

A Protein Kinase Cδ-Dependent Protein Kinase D Pathway Modulates ERK1/2 and JNK1/2 Phosphorylation and Bim-Associated Apoptosis by Asbestos

Inhalation of asbestos and oxidant-generating pollutants causes injury and compensatory proliferation of lung epithelium, but the signaling mechanisms that lead to these responses are unclear. We hypothesized that a protein kinase (PK)Cδ-dependent PKD pathway was able to regulate downstream mitogen-activated protein kinases, affecting pro- and anti-apoptotic responses to asbestos. Elevated levels of phosphorylated PKD (p-PKD) were observed in distal bronchiolar epithelial cells of mice inhaling asbestos. In contrast, PKCδ−/− mice showed significantly lower levels of p-PKD in lung homogenates and in situ after asbestos inhalation. In a murine lung epithelial cell line, asbestos caused significant increases in the phosphorylation of PKCδ-dependent PKD, ERK1/2, and JNK1/2/c-Jun that occurred with decreases in the BH3-only pro-apoptotic protein, Bim. Silencing of PKCδ, PKD, and use of small molecule inhibitors linked the ERK1/2 pathway to the prevention of Bim-associated apoptosis as well as the JNK1/2/c-Jun pathway to the induction of apoptosis. Our studies are the first to show that asbestos induces PKD phosphorylation in lung epithelial cells both in vivo and in vitro. PKCδ-dependent PKD phosphorylation by asbestos is causally linked to a cellular pathway that involves the phosphorylation of both ERK1/2 and JNK1/2, which play opposing roles in the apoptotic response induced by asbestos.Asbestos is a group of naturally occurring mineral fibers that are linked to the development of lung cancer, mesothelioma, and pleural and pulmonary fibrosis, ie, asbestosis.^1,2 The mechanisms leading to asbestos-related diseases are still unclear, but oxidative stress due to phagocytosis of longer fibers, iron-driven generation of oxidants from fiber surfaces, and depletion of cellular antioxidants are linked to cell injury and inflammation.^3,4,5,6Bronchiolar and alveolar type II epithelial cells, which first encounter asbestos fibers after inhalation, are key cell types in asbestos-associated inflammation and fibroproliferation.² Initial cell reactions to asbestos include epithelial cell injury, ie, apoptosis and necrosis,^5,6 which may lead to compensatory cell proliferation^7,8 and the production of inflammatory and fibrogenic cytokines.^8,9,10 Asbestos-induced signaling mechanisms governing these cell responses appear to involve a broad variety of cascades including the mitogen-activated protein kinases (MAPK),^3,7,11,12 nuclear factor-κB (NF-κB),^9,13,14 and the protein kinase (PK)C^10,12,15,16 and A families.¹⁷A critical signaling protein involved in asbestos signaling is PKCδ, which is known to be activated in bronchiolar and alveolar epithelial cells in vivo and in vitro^10,12,16 via increased formation of diacylglycerol.¹⁸ We have shown that PKCδ governs apoptosis via an oxidant-dependent mitochondrial pathway after exposure of lung epithelial cells to asbestos fibers.¹⁶ Recent studies comparing PKCδ +/+ and PKCδ −/− mice also reveal an important role of PKCδ in metalloproteinase expression as well as cytokine production in vitro and in vivo.^10,15 A variety of other studies also link PKCδ to either pro-apoptotic or anti-apoptotic events depending on the stimulus and cell type.^19,20In this study, we focused on PKD as a potential link between PKCδ, activation of MAPKs and downstream repercussions such as expression of fos/jun proto-oncogenes and apoptosis in asbestos-exposed lung epithelium. PKD is a serine/threonine protein kinase classified as a subfamily of the Ca²⁺/calmodulin-dependent kinase superfamily.²¹ PKD1, which includes mouse PKD and its human homolog PKCμ, is the most extensively studied PKD.²² The other two members of this family include PKD2²³ and PKD3, (originally PKCν).²⁴ Conserved regions of PKDs include a phosphorylation-dependent catalytic domain, a pleckstrin-homology domain that inhibits the catalytic activity, and cysteine-rich motifs that recruit PKD to the plasma membrane. PKCδ is proposed to interact with the pleckstrin-homology domain of PKD, transphosphorylating its activation loop at Ser744 and Ser748, and leading to PKD activation.²⁵ In addition, PKD can be activated through the Src-Abl pathway by tyrosine phosphorylation of Tyr463 (T463) in the pleckstrin-homology domain after oxidative stress,²⁶ as well as by caspase-mediated proteolytic cleavage 27 and by bone morphogenetic protein 2.²⁸ Downstream targets of PKD signaling include several important signaling molecules such as ERK1/2, JNK1/2, and NF-κB,^21,26,29,30 but how these affect functional ramifications of carcinogens, such as asbestos, are unclear.The BH3-only protein, Bim, is a pro-apoptotic member of the Bcl-2 family that links stress-induced signals to the core apoptotic machinery.^31,32 There are three different splice variants of the Bim gene encoding short, long, and extra-long Bim proteins (BimS, BimL, and BimEL).³³ BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptosis proteins,³⁴ whereas BimL is bound to microtubules and is less cytotoxic.³⁵ Disruption of BimL binding to microtubules via JNK-dependent phosphorylation can cause its redistribution to the mitochondria and induction of pro-apoptotic machinery.³⁶ BimEL is post-translationally regulated by ERK1/2, which promotes its phosphorylation and rapid dissociation from Mcl-1 and Bcl-x(L)³⁷ and proteasomal degradation.³⁸We reveal here that PKD is involved in multiple signaling events after asbestos inhalation and in vitro. Specifically, PKD is a downstream effector of PKCδ and modulates phosphorylation of both ERK1/2 and JNK1/2 in lung epithelial cells after asbestos exposure. Our data also suggest that PKD inhibits apoptosis through an ERK1/2-mediated destabilization of the pro-apoptotic BH3-only protein, BimEL. The fact that PKD is an important signaling molecule in MAPK signaling and survival after cell injury by asbestos may have important therapeutic implications in asbestos-related diseases.     

Interleukin-12 Is Required for Control of the Growth of Attenuated Aromatic-Compound-Dependent Salmonellae in BALB/c Mice: Role of Gamma Interferon and Macrophage Activation

The attenuated S. typhimurium SL3261 (aroA) strain causes mild infections in BALB/c mice. We were able to exacerbate the disease by administering anti-interleukin-12 (IL-12) antibodies, resulting in bacterial counts in the spleens and livers of anti-IL-12-treated mice that were 10- to 100-fold higher than the ones normally observed in premortem mice; yet the animals showed only mild signs of illness. Nevertheless, they eventually died of a slow, progressive disease. Mice infected with salmonellae become hypersusceptible to endotoxin. We found that IL-12 neutralization prevented the death of infected mice following subcutaneous injection of lipopolysaccharide. Granulomatous lesions developed in the spleens and livers of control animals, as opposed to a widespread infiltration of mononuclear cells seen in the organs of anti-IL-12-treated mice. In the latter (heavily infected), salmonellae were seen within mononuclear cells, indicating an impairment of the bactericidal or bacteriostatic ability of the phagocytes in the absence of biologically active IL-12. Gamma interferon (IFN-γ) levels were reduced in the sera and tissue homogenates from anti-IL-12-treated mice compared to those in control animals. Furthermore, fluorescence-activated cell sorter analysis on spleen cells showed that IL-12 neutralization impaired the upregulation of I-A^d/I-E^d antigens on macrophages from infected mice. Inducible nitric oxide synthase and IFN-γ mRNA production was down-regulated in anti-IL-12-treated mice, which also showed an increased production of IL-10 mRNA and a decrease in nitric oxide synthase activity in the tissues. Administration of recombinant IFN-γ to anti-IL-12-treated mice was able to restore host resistance, granuloma formation, and expression of major histocompatibility complex class II antigens in F4/80⁺ and CD11b⁺ spleen cells.Salmonella infections still pose a serious health hazard worldwide, affecting both humans and animals. Salmonella typhi, the agent of human typhoid fever, is not pathogenic for common laboratory animals. Therefore, natural resistance and acquired immunity to Salmonella are studied mainly in the mouse model by using host-adapted salmonellae which cause systemic infections believed to mimic the human disease.In mice, early bacterial growth in the reticuloendothelial system (RES) is controlled by the innate resistance Nramp (Ity) gene, which is expressed in macrophages (22). In lethal infections, salmonellae rapidly reach large numbers in the tissues and death occurs presumably by endotoxin poisoning when bacterial counts reach levels of ca. 10⁸ CFU per organ (30). In sublethal infections, survival requires a host response that suppresses the exponential growth of the organisms in the RES towards the end of the first week, resulting in a plateau phase (17, 25). The establishment of the plateau phase does not require functional T cells. In fact, nude (T-cell-deficient) mice and mice depleted of T cells by administration of anti-CD4 and anti-CD8 antibodies can still suppress Salmonella growth in infected tissues (17). A bone marrow-dependent influx of radiation-sensitive cells is required for the plateau phase and for the formation of granulomas rich in mononuclear cells (17, 32). Most of the salmonellae in the spleens and livers of the infected animals are localized within the phagocytes present in the focal lesions (38). Tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and nitric oxide (NO) derivatives appear to be required for the suppression of salmonella growth in the RES (27, 28, 32, 36, 37, 48). TNF-α is needed for the recruitment of mononuclear cells in the tissues and for granuloma formation (32); IFN-γ can activate macrophages to kill salmonellae in vitro (20).The establishment of the plateau phase coincides with the development of hypersusceptibility to the toxic and lethal effects of bacterial lipopolysaccharide (LPS) (29, 33). We have previously shown that mice immunized with a live attenuated aromatic-dependent Salmonella vaccine strain show transient hypersusceptibility to LPS, which can be prevented by treatment with anti-TNF-α antibodies (29). The role of other cytokines in this phenomenon is not known.Interleukin-12 (IL-12) is a 70-kDa heterodimeric cytokine produced by macrophages, B cells, polymorphonuclear leukocytes, and dendritic cells in response to a variety of stimuli including products of bacterial origin (5, 10). IL-12 mediates resistance to intracellular organisms including Listeria, Toxoplasma, Candida, Leishmania, Mycobacterium tuberculosis, and Brucella abortus (8, 13, 18, 23, 39, 46, 50). IL-12 is generally believed to mediate host resistance by inducing IFN-γ production by NK and T cells as well as by contributing to the establishment of protective Th1 antigen-specific responses (5, 6, 9, 10, 12, 13, 24, 34, 39, 43, 47).Evidence for IL-12 induction in salmonellosis has been provided. IL-12 and IL-12-specific mRNA have been detected in vivo and in vitro in response to Salmonella. Elicited peritoneal mouse macrophages stimulated with Salmonella dublin express elevated levels of IL-12 p40-specific mRNA (4, 7). Oral infection with virulent or live attenuated S. dublin induces early (6 and 20 h postinfection) production of IL-12-specific mRNA in Peyer’s patches and mesenteric lymph nodes (3); biologically active IL-12 in lymph node homogenates has been documented 36 h after S. dublin infection (21). We and others previously reported that in vivo IL-12 neutralization reduces the ability of the host to suppress the growth of virulent salmonellae in the tissues and impairs IFN-γ production (21, 31). A recent report indicates that a mutation in the IL-12 receptors renders humans more susceptible to salmonellosis (11). Nevertheless, the mechanisms by which IL-12 mediates host resistance to Salmonella are still unclear.In the present study, we attempted to clarify the mechanisms by which IL-12 contributes to host resistance in mice infected with Salmonella. We investigated the role of IL-12 in survival, granuloma formation, and macrophage activation in mice infected with an attenuated Salmonella strain that normally causes very mild infections in BALB/c mice. We also investigated the involvement of IL-12 in the toxic and lethal effects of high bacterial loads in the tissues as well as in the expression of hypersusceptibility to LPS normally seen in mice infected with salmonellae. We also wished to clarify the involvement of IFN-γ in IL-12-mediated resistance to salmonellosis.     

Novel EphB4 Monoclonal Antibodies Modulate Angiogenesis and Inhibit Tumor Growth

EphB4 receptor tyrosine kinase and its cognate ligand EphrinB2 regulate induction and maturation of newly forming vessels. Inhibition of their interaction arrests angiogenesis, vessel maturation, and pericyte recruitment. In addition, EphB4 is expressed in the vast majority of epithelial cancers and provides a survival advantage to most. Here, we describe two anti-EphB4 monoclonal antibodies that inhibit tumor angiogenesis and tumor growth by two distinct pathways. MAb131 binds to fibronectin-like domain 1 and induces degradation of human EphB4, but not murine EphB4. MAb131 inhibits human endothelial tube formation in vitro and growth of human tumors expressing EphB4 in vivo. In contrast, MAb47 targets fibronectin-like domain 2 of both human and murine EphB4 and does not alter EphB4 receptor levels, but inhibits angiogenesis and growth of both EphB4-positive and EphB4-negative tumors in a mouse s.c. xenograft model. Combination of MAb47 and bevacizumab enhances the antitumor activity and induces tumor regression. Indeed, humanized antibodies hAb47 and hAb131 showed similar affinity for EphB4 and retained efficacy in the inhibition of primary tumor development and experimental metastasis.Angiogenesis is a process of new blood vessel sprouting from pre-existing vessels. This process includes primary capillary sprouting, branching, and remodeling into a mature blood vessel network. In the embryo, angiogenesis is preceded by vasculogenesis and represents one of the earliest processes in organ development.^1,2 In adulthood, angiogenesis is induced at sites of tissue repair, tissue remodeling such as during menstruation, and in diseases constituting enhanced angiogenesis.¹ Angiogenesis is also induced to varying degrees in cancers.¹ Angiogenesis is triggered by protease-mediated degradation of matrix proteins releasing angiogenic factors such as vascular endothelial growth factor (VEGF), epidermal growth factor, and fibroblast growth factor, followed by the migration of endothelial cells to sprout new vessels.^1,3 One critical step in vessel maturation is the interaction between arterial and venous capillaries leading to the fusion and lumen formation across the two cell types. This step is dependent on the interaction between EphB4 receptor tyrosine kinase expressed on venous endothelial cells and the trans-membrane ligand EphrinB2 expressed on arterial endothelial cells.^4,5EphB4 belongs to the Eph family, the largest family of receptor tyrosine kinases. To date, sixteen members of this receptor family have been characterized.^6,7 Eph receptors are divided into two subgroups: EphA members (10 in number) that bind to ligands (EphrinA1-6) that lack trans-membrane domains and localize to the cell membrane via glycosylphosphatidyl inositol linkage. EphB receptors (6 members) bind to ligands (EphrinB1-3) that contain a trans-membrane domain.^6,7 Receptor-ligand interaction leads to dimerization and phosphorylation of both receptor and ligand, resulting in bidirectional signaling. On stimulation with EphrinB2, Eph receptor “forward” signaling is triggered by autophosphorylation of its intracellular tyrosine kinase domain and downstream signaling.^8,9,10 Ephrin-B “reverse” signaling also depends on tyrosine phosphorylation of the Ephrin cytoplasmic region.¹⁰Targeted disruption of the EphB4 gene in mice is embryonically lethal secondary to the failure of blood vessel maturation and capillary arrest.¹¹ EphrinB2 is the only known ligand for EphB4. Targeted disruption of the EphrinB2 gene has a phenotype similar to EphB4 knockout.^5,11 The role of EphB4 in adult vertebrates is not fully understood, in part because of a lack of appropriate conditional knock-out lines. EphB4 and EphrinB2 are induced in newly forming venous and arterial vessels, respectively, and are thus expected to play an important role in adult angiogenesis.^9,12,13 This hypothesis is supported by the observation that the soluble monomeric form of the extracellular domain of EphB4 receptor (sEphB4) that blocks EphrinB2 interaction with its cognate receptors, blocks bidirectional signaling and inhibits angiogenesis at sites of neovascularization in the adult.^14,15,16Human tumor tissue analysis has shown that EphB4 is overexpressed in many tumor types: breast,¹⁷ colon,^18,19,20 bladder,²¹ endometrium,²² head and neck,²³ prostate,^24,25 and ovary.^26,27 EphB4 directly supports tumor cell survival by inhibiting apoptosis in many of these cancers.^{17,18,21,22,23,24,27} Tumor cell–expressed EphB4 also induces angiogenesis by direct interaction with EphrinB2 on tumor vessels.²⁸ In this study, we describe novel EphB4-specific monoclonal antibodies that inhibit formation and maturation of newly forming vessels and inhibit tumor growth in vivo.     

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.     

d-Lactate Production and [14C]Succinic Acid Uptake by Adherent and Nonadherent Escherichia coli

Escherichia coli isolates of different adherence phenotypes produced different amounts of d-lactate. Alterations of culture conditions did not influence the amount of d-lactate produced. The observed pH decreases in tissue culture medium corresponded with increases in d-lactate concentration. Very little [¹⁴C]succinic acid was incorporated into cells during the in vitro incubation of adherent and nonadherent E. coli with HeLa cells, but the amounts of tracer removed from the culture medium by adherent and nonadherent strains differed. The results are further evidence of a difference in the metabolic behavior of adherent and nonadherent E. coli.One of the virulence associated properties of enteropathogenic Escherichia coli (5, 13, 14) is the ability to adhere to small intestinal mucosa (3, 11, 12, 21, 24, 26, 27). Although this adherence is an important event in the induction of diarrhea, the mechanism by which adherent E. coli mediates pathogenicity remains uncertain (1, 2, 7, 18, 26, 27).Several studies have shown that the in vitro adherence of E. coli to HEp-2 or HeLa cells in tissue culture can be used as a marker of enteroadherence (4, 6, 8, 9, 15, 16, 19, 22, 23, 28, 29). We used the HeLa assay (20) to detect this virulence characteristic in E. coli isolates from infants with acute diarrhea and, during the 3-h assay, observed E. coli-induced changes in the pH of the tissue culture medium (17). The pH changes induced by organisms with different adherence phenotypes differed. Since the characteristic end products of E. coli fermentation include lactic acid, succinic acid, and acetic acid, the pH changes could be explained by differences in the production of organic acids. Other plausible explanations are differences in the removal of organic acids from the medium and interactions between bacteria and HeLa cells during adherence.This paper describes two sets of experiments, one based on the production of lactic acid and the other on the removal of succinic acid from the medium. The objectives were to determine (i) whether there is a metabolic difference between localized, diffuse, and nonadherent isolates in the amount of lactate produced or succinate removed from the incubation medium, (ii) whether E. coli changes from aerobic to anaerobic metabolism during incubation periods of up to 5 h under different culture conditions, (iii) whether an increase in lactate production or succinate removal coincides with the drop in pH previously observed, and (iv) whether the pH changes can be attributed to differences in bacterial growth rates between isolates with different in vitro adherence patterns and nonadherent strains.     

Mechanisms of HIV-tat-Induced Phosphorylation of N-Methyl-d-Aspartate Receptor Subunit 2A in Human Primary Neurons : Implications for NeuroAIDS Pathogenesis

HIV infection of the central nervous system results in neurological dysfunction in a large number of individuals. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence of HIV-infected neurons. Neuronal damage and dropout must therefore be due to indirect effects of HIV infection of other central nervous system cells through elaboration of inflammatory factors and neurotoxic viral proteins, including the viral transactivator, tat. We previously demonstrated that HIV-tat-induced apoptosis in human primary neurons is dependent on N-methyl-d-aspartate receptor (NMDAR) activity. NMDAR activity is regulated by various mechanisms including NMDAR phosphorylation, which may lead to neuronal dysfunction and apoptosis in pathological conditions. We now demonstrate that tat treatment of human neurons results in tyrosine (Y) phosphorylation of the NMDAR subunit 2A (NR2A) in a src kinase–dependent manner. In vitro kinase assays and in vivo data indicated that NR2A Y1184, Y1325, and Y1425 are phosphorylated. Tat treatment of neuronal cultures enhanced phosphorylation of NR2A Y1325, indicating that this site is tat sensitive. Human brain tissue sections from HIV-infected individuals with encephalitis showed an increased phosphorylation of NR2A Y1325 in neurons as compared with uninfected and HIV-infected individuals without encephalitis. These findings suggest new avenues of treatment for HIV-associated cognitive impairment.HIV infection causes varying degrees of cognitive impairment in a significant number of individuals.^1,2 Damage to the central nervous system is a multifactorial process including early viral entry, neuroinflammation, and secretion of toxic factors.³ During this process, neuronal damage and apoptosis occur,⁴ but there is little evidence that neurons are infected with HIV.^5,6 Therefore, the neuronal damage and death characteristic of neuroAIDS must be mediated through indirect mechanisms.³ HIV-tat, the transactivator of the virus, is one such toxic factor that causes apoptosis in cultured human neurons.^7,8,9,10,11Binding of tat to the low density lipoprotein receptor-related protein (LRP) on neurons results in the formation of a macromolecular complex at the neuronal cell membrane between LRP and the N-methyl-d-aspartate receptor (NMDAR), mediated by the scaffolding protein PSD-95.⁹ In this complex, the NMDAR plays a critical role in the process of tat induced apoptosis in human primary neurons, as blocking with specific NMDAR inhibitors abrogated cell death completely.^8,9 The specific mechanisms by which tat alters NMDAR activity are unknown.NMDAR phosphorylation results in alterations in NMDAR activity,^12,13,14 protein-protein interactions,¹⁵ and trafficking.^15,16,17 In pathological conditions, overactivation of the NMDAR results in toxicity.¹⁸ Studies in rodent models indicate that postsynaptic density proteins facilitate the interaction of kinases, such as src,¹⁹ fyn,^20,21 and pyk2^22,23,24 with the NMDAR, resulting in phosphorylation of its different subunits. As PSD-95 is recruited to the NMDAR after tat treatment,⁹ we examined tat induced association of these kinases with the NMDAR subunit 2A (NR2A), the main subunit present in the macromolecular complex induced by tat,⁹ and subsequent changes in NR2A phosphorylation.We demonstrate that tat induces tyrosine phosphorylation of the NR2A subunit by a mechanism that involves recruitment of active src to the receptor. We also identify in vitro three tyrosine (Y) residues phosphorylated on human NR2A in a src-dependent manner. One of these, Y1325, showed significantly enhanced phosphorylation in response to tat treatment of human neurons that was dependent on src activity. We also demonstrate in vivo that phosphorylation of Y1325 is significantly increased in neurons present in HIV encephalitic brain tissue sections as compared with those from uninfected and HIV-infected individuals without encephalitis.