Bibiana V. Iglesias

Computational Identification of a p38SAPK-Regulated Transcription Factor Network Required for Tumor Cell Quiescence

The stress-activated kinase p38 plays key roles in tumor suppression and induction of tumor cell dormancy. However, the mechanisms behind these functions remain poorly understood. Using computational tools, we identified a transcription factor (TF) network regulated by p38alpha/beta and required for human squamous carcinoma cell quiescence in vivo. We found that p38 transcriptionally regulates a core network of 46 genes that includes 16 TFs. Activation of p38 induced the expression of the TFs p53 and BHLHB3, while inhibiting c-Jun and FoxM1 expression. Furthermore, induction of p53 by p38 was dependent on c-Jun down-regulation. Accordingly, RNAi down-regulation of BHLHB3 or p53 interrupted tumor cell quiescence, while down-regulation of c-Jun or FoxM1 or overexpression of BHLHB3 in malignant cells mimicked the onset of quiescence. Our results identify components of the regulatory mechanisms driving p38-induced cancer cell quiescence. These may regulate dormancy of residual disease that usually precedes the onset of metastasis in many cancers.

Inhibition of Proliferation by PERK Regulates Mammary Acinar Morphogenesis and Tumor Formation

Endoplasmic reticulum (ER) stress signaling can be mediated by the ER kinase PERK, which phosphorylates its substrate eIF2α. This in turn, results in translational repression and the activation of downstream programs that can limit cell growth through cell cycle arrest and/or apoptosis. These responses can also be initiated by perturbations in cell adhesion. Thus, we hypothesized that adhesion-dependent regulation of PERK signaling might determine cell fate. We tested this hypothesis in a model of mammary acini development, a morphogenetic process regulated in part by adhesion signaling. Here we report a novel role for PERK in limiting MCF10A mammary epithelial cell proliferation during acinar morphogenesis in 3D Matrigel culture as well as in preventing mammary tumor formation in vivo. We show that loss of adhesion to a suitable substratum induces PERK-dependent phosphorylation of eIF2α and selective upregulation of ATF4 and GADD153. Further, inhibition of endogenous PERK signaling during acinar morphogenesis, using two dominant-negative PERK mutants (PERK-ΔC or PERK-K618A), does not affect apoptosis but results instead in hyper-proliferative and enlarged lumen-filled acini, devoid of proper architecture. This phenotype correlated with an adhesion-dependent increase in translation initiation, Ki67 staining and upregulation of Laminin-5, ErbB1 and ErbB2 expression. More importantly, the MCF10A cells expressing PERKΔC, but not a vector control, were tumorigenic in vivo upon orthotopic implantation in denuded mouse mammary fat pads. Our results reveal that the PERK pathway is responsive to adhesion-regulated signals and that it is essential for proper acinar morphogenesis and in preventing mammary tumor formation. The possibility that deficiencies in PERK signaling could lead to hyperproliferation of the mammary epithelium and increase the likelihood of tumor formation, is of significance to the understanding of breast cancer.

Mucosal Immunization with an Unadjuvanted Vaccine That Targets Streptococcus pneumoniae PspA to Human Fcγ Receptor Type I Protects against Pneumococcal Infection through Complement- and Lactoferrin-Mediated Bactericidal Activity

ABSTRACT Targeting an antigen to Fc receptors (FcR) can enhance the immune response to the antigen in the absence of adjuvant. Furthermore, we recently demonstrated that intranasal immunization with an FcγR-targeted antigen enhances protection against a category A intracellular mucosal pathogen, Francisella tularensis. To determine if a similar strategy could be applied to the important pathogen Streptococcus pneumoniae, we used an improved mucosal FcR-targeting strategy that specifically targets human FcγR type I (hFcγRI). A humanized single-chain antibody component in which the variable domain binds to hFcγRI [anti-hFcγRI (H22)] was linked in a fusion protein with the pneumococcal surface protein A (PspA). PspA is known to elicit protection against pneumococcal sepsis, carriage, and pneumonia in mouse models when administered with adjuvants. Anti-hFcγRI-PspA or recombinant PspA (rPspA) alone was used to intranasally immunize wild-type (WT) and hFcγRI transgenic (Tg) mice in the absence of adjuvant. The hFcγRI Tg mice receiving anti-hFcγRI-PspA exhibited elevated S. pneumoniae-specific IgA, IgG2c, and IgG1 antibodies in serum and bronchoalveolar lavage fluid. Neither immunogen was effective in protecting WT mice in the absence of adjuvant, but when PspA was targeted to hFcγRI as the anti-hFcγRI-PspA fusion, enhanced protection against lethal S. pneumoniae challenge was observed in the hFcγRI Tg mice compared to mice given nontargeted rPspA alone. Immune sera from the anti-hFcγRI-PspA-immunized Tg mice showed enhanced complement C3 deposition on bacterial surfaces, and protection was dependent upon an active complement system. Immune serum also showed an enhanced bactericidal activity directed against S. pneumoniae that appears to be lactoferrin mediated.

Differential Requirements for Protection against Mucosal Challenge with Francisella tularensis in the Presence versus Absence of Cholera Toxin B and Inactivated F. tularensis

Francisella tularensis is a category A biothreat agent for which there is no approved vaccine and the correlates of protection are not well understood. In particular, the relationship between the humoral and cellular immune response to F. tularensis and the relative importance of each in protection is controversial. Yet, understanding this relationship will be crucial to the development of an effective vaccine against this organism. We demonstrate, for the first time, a differential requirement for humoral vs cellular immunity in vaccine-induced protection against F. tularensis infection, and that the requirement for Ab observed in some protection studies, may be overcome through the induction of enhanced cellular immunity. Specifically, following intranasal/mucosal immunization of mice with inactivated F. tularensis organisms plus the cholera toxin B subunit, we observe increased production of IgG2a/2c vs IgG1 Ab, as well as IFN-γ, indicating induction of a Th1 response. In addition, the requirement for F. tularensis-specific IgA Ab production, observed in studies following immunization with inactivated F. tularensis alone, is eliminated. Thus, these data indicate that enhanced Th1 responses can supersede the requirement for anti-F. tularensis-specific IgA. This observation also has important ramifications for vaccine development against this organism.

Fc receptor-targeted mucosal vaccination as a novel strategy for the generation of enhanced immunity against mucosal and non-mucosal pathogens

Numerous studies have demonstrated that targeting immunogens to Fcγ receptors (FcγR) on antigen (Ag)-presenting cells (APC) can enhance humoral and cellular immunity in vitro and in vivo. FcγR are classified based on their molecular weight, IgG-Fc binding affinities, IgG subclass binding specificity, and cellular distribution and they consist of activating and inhibitory receptors. However, despite the potential advantages of targeting Ag to FcR at mucosal sites, very little is known regarding the role of FcR in mucosal immunity or the efficacy of FcR-targeted mucosal vaccines. In addition, recent work has suggested that FcRn is present in the lungs of adult mice and humans and can transport FcRn-targeted Ag to FcγR-bearing APC within mucosal lymphoid tissue. In this review we will discuss the need for new vaccine strategies, the potential for FcR-targeted vaccines to fill this need, the impact of activating versus inhibitory FcγR on FcR-targeted vaccination, the significance of focusing on mucosal immunity, as well as caveats that could impact the use of FcR targeting as a mucosal vaccine strategy.

Multiple mechanisms mediate enhanced immunity generated by mAb-inactivated F. tularensis immunogen.

We have previously demonstrated that immunization with the inactivated Francisella tularensis, a Category A intracellular mucosal pathogen, combined with IgG2a anti‐F. tularensis monoclonal antibody (Ab), enhances protection against subsequent F. tularensis challenge. To understand the mechanism(s) involved, we examined the binding, internalization, presentation, and in vivo trafficking of inactivated F. tularensis in the presence and absence of opsonizing monoclonal Ab. We found that when inactivated F. tularensis is combined with anti‐F. tularensis monoclonal Ab, presentation to F. tularensis‐specific T cells is enhanced. This enhancement is Fc receptor (FcR)‐dependent, and requires a physical linkage between the monoclonal Ab and the inactivated F. tularensis immunogen. This enhanced presentation is due, in part, to enhanced binding and internalization of inactivated F. tularensis by antigen(Ag)‐presenting cells, and involves interactions with multiple FcR types. Furthermore, targeting inactivated F. tularensis to FcRs enhances dendritic cell maturation and extends the time period over which Ag‐presenting cells stimulate T cells. In vivo trafficking studies reveal enhanced transport of inactivated F. tularensis immunogen to the nasal‐associated lymphoid tissue in the presence of monoclonal Ab, which is FcRn‐dependent. In summary, these are the first comprehensive studies using a single‐vaccine protection model/immunogen to establish the array of mechanisms involved in enhanced immunity/protection mediated by an FcR‐targeted mucosal immunogen. These results demonstrate that multiple cellular/immune mechanisms contribute to FcR‐enhanced immunity.

Molecular biologic assessment of cutaneous specimens of ocular rosacea.

Purpose: To characterize the molecular biologic environment of pathological skin in ocular rosacea and to differentiate the levels of inflammatory molecules in ocular rosacea from those of normal skin. Methods: The concentrations of 48 molecules were assayed in cutaneous biopsies taken from patients with ocular rosacea and from normal controls. Results: There were very few molecular differences between the 2 groups, and 43 of the 48 molecules that were measured in this study were not significantly different between the 2 groups. The concentrations of 5 molecules (interleukin-1&bgr;, interleukin-16, stem cell factor, monocyte chemotactic protein-1, and monokine induced by &ggr;-interferon) were significantly enriched in ocular rosacea. Conclusions: Ocular rosacea is a highly ordered molecular process and involves elevations in the concentrations of specific molecules. The particular pattern of enrichment supports the notion that ocular rosacea represents a disorder of innate immunity. Furthermore, these molecules may represent novel therapeutic targets in the future management of this disorder.

Characterization of the molecular biologic milieu of idiopathic orbital inflammation.

Purpose: To identify the cytokines that are expressed in elevated concentrations in idiopathic orbital inflammation (IOI). Design: Experimental study. Participants and Controls: Biopsy specimens from 8 patients with biopsy-proven IOI and negative serologic and radiographic examinations, versus orbital biopsy samples taken from 8 control patients without clinical evidence or subjective complaint of orbital inflammation. Methods: Quantitative cytokine assays were performed to assess the levels of 9 different molecules for IOI and control patients. Statistical analyses were performed to compare the cytokine concentrations between the 2 groups. Main Outcome Measures: Cytokine concentrations. Results: Six cytokines were statistically significantly elevated in IOI (interleukin-2, -8, -10, -12, gamma interferon, and tumor necrosis factor alpha) (p < 0.05). Gamma interferon and interleukin-12 were expressed at concentrations greater than 10 times higher in patients with IOI. Conclusions: IOI involves discrete elevations of specific cytokines, and individual molecules are clearly implicated in the pathogenesis of this disease. Specifically, gamma interferon and interleukin-12 concentrations are markedly elevated in IOI. These cytokines represent novel therapeutic targets in the management of this inflammatory process. Agents that affect these molecules could be used as potential therapies to control this disease in a more effective fashion with fewer side effects.

Analysis of the molecular biologic milieu of the vitreous in proliferative vitreoretinopathy.

Purpose: Previous investigations have explored molecular differences between proliferative vitreoretinopathy and primary retinal detachment. An exploration of a greater number of molecules might provide novel insight into the biology of this disorder and identify potential therapeutic targets. Methods: Vitreous specimens were obtained from patients with epiretinal membranes or macular puckers (n = 15), patients with a primary retinal detachment without proliferative vitreoretinopathy (n = 15), and patients with retinal detachments and proliferative vitreoretinopathy (n = 15). A multiplex assay was performed to calculate the concentrations of 48 different cytokines and chemokines, and statistical analyses were performed to identify differences between the groups. Results: Of the 48 molecules that were studied, we identified 10 that were statistically significantly different in cases of proliferative vitreoretinopathy, including interleukins 4, 5, 6, and 15; granulocyte–macrophage colony-stimulating factors; stem cell factor; stem cell growth factor; macrophage inflammatory protein 1&agr;; and interferon &ggr;–induced protein 10. Conclusion: Proliferative vitreoretinopathy represents a highly ordered molecular process that involves discrete changes in the concentrations of specific cytokines and chemokines. These molecules may represent novel therapeutic targets.

Toll-like receptors in idiopathic orbital inflammation.

Purpose: A prior investigation has demonstrated that innate immune-specific cytokines are enriched in idiopathic orbital inflammation (IOI). To further document the role of innate immunity in IOI, the authors sought to determine whether toll-like receptors (TLRs) are present in biopsy specimens of this disorder. Methods: Immunohistochemical staining for TLR2, TLR3, and TLR4 was performed on biopsy specimens taken from patients with IOI, and the number of TLR-positive cells was counted across five 40× light microscopic fields. These results were compared with an isotype control and with orbital adipose tissue taken from patients without evidence of inflammation. Results: All IOI specimens demonstrated positivity for all 3 TLRs, and sections stained for isotype controls did not demonstrate any positivity. Furthermore, orbital adipose tissue did not demonstrate any significant signal. The mean number of positive cells was 24.4 cells/high power field (hpf; standard deviation = 11.6 cells/hpf), 7.23 cells/hpf (standard deviation = 5.59 cells/hpf), and 11.7 cells/hpf for TLR2, TLR3, and TLR4, respectively. Conclusions: This study provides the first documentation of TLRs in orbital disease. Toll-like receptors are present in IOI, and IOI may represent an aberrant innate immune response. Interference with TLRs may represent an additional potential therapeutic mechanism in the management of IOI.