Ali M. Saeed

Infiltration of Proinflammatory M1 Macrophages into the Outer Retina Precedes Damage in a Mouse Model of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of blindness in the developed world. Oxidative stress and inflammation are implicated in AMD, but precise mechanisms remain poorly defined. Carboxyethylpyrrole (CEP) is an AMD-associated lipid peroxidation product. We previously demonstrated that mice immunized with CEP-modified albumin developed AMD-like degenerative changes in the outer retina. Here, we examined the kinetics of lesion development in immunized mice and the presence of macrophages within the interphotoreceptor matrix (IPM), between the retinal pigment epithelium and photoreceptor outer segments. We observed a significant and time-dependent increase in the number of macrophages in immunized mice relative to young age-matched controls prior to overt pathology. These changes were more pronounced in BALB/c mice than in C57BL/6 mice. Importantly, IPM-infiltrating macrophages were polarized toward the M1 phenotype but only in immunized mice. Moreover, when Ccr2-deficient mice were immunized, macrophages were not present in the IPM and no retinal lesions were observed, suggesting a deleterious role for these cells in our model. This work provides mechanistic evidence linking immune responses against oxidative damage with the presence of proinflammatory macrophages at sites of future AMD and experimentally demonstrates that manipulating immunity may be a target for modulating the development of AMD.

Tropomyosin as a regulator of cancer cell transformation.

Tropomyosins (Tms) are among the most studied structural proteins of the actin cytoskeleton that are implicated in neoplastic-specific alterations in actin filament organization. Decreased expression of specific nonmuscle Tm isoforms is commonly associated with the transformed phenotype. These changes in Tm expression appear to contribute to the rearrangement of microfilament bundles and morphological alterations, increased cell motility and oncogenic signaling properties of transformed cells. Below we review aspects of Tm biology as it specifically relates to transformation and cancer including its expression in culture models of transformed cells and human tumors, mechanisms that regulate Tm expression and the role of Tm in oncogenic signaling.

T cells and macrophages responding to oxidative damage cooperate in pathogenesis of a mouse model of age-related macular degeneration

Age-related macular degeneration (AMD) is a major disease affecting central vision, but the pathogenic mechanisms are not fully understood. Using a mouse model, we examined the relationship of two factors implicated in AMD development: oxidative stress and the immune system. Carboxyethylpyrrole (CEP) is a lipid peroxidation product associated with AMD in humans and AMD-like pathology in mice. Previously, we demonstrated that CEP immunization leads to retinal infiltration of pro-inflammatory M1 macrophages before overt retinal degeneration. Here, we provide direct and indirect mechanisms for the effect of CEP on macrophages, and show for the first time that antigen-specific T cells play a leading role in AMD pathogenesis. In vitro, CEP directly induced M1 macrophage polarization and production of M1-related factors by retinal pigment epithelial (RPE) cells. In vivo, CEP eye injections in mice induced acute pro-inflammatory gene expression in the retina and human AMD eyes showed distinctively diffuse CEP immunolabeling within RPE cells. Importantly, interferon-gamma (IFN-γ) and interleukin-17 (IL-17)-producing CEP-specific T cells were identified ex vivo after CEP immunization and promoted M1 polarization in co-culture experiments. Finally, T cell immunosuppressive therapy inhibited CEP-mediated pathology. These data indicate that T cells and M1 macrophages activated by oxidative damage cooperate in AMD pathogenesis.

The influence of Hispanic ethnicity on nonsmall cell lung cancer histology and patient survival: An analysis of the Survival, Epidemiology, and End Results database

Most studies exploring ethnic/racial disparities in nonsmall cell lung cancer (NSCLC) compare black patients with whites. Currently, the effect of Hispanic ethnicity on the overall survival of NSCLC is poorly understood. Therefore, the authors carried out a large‐scale, population‐based analysis using the Surveillance, Epidemiology, and End Results (SEER) data base to determine the impact of Hispanic ethnicity the survival of patients with NSCLC.

Biophysical analysis of binding of WW domains of the YAP2 transcriptional regulator to PPXY motifs within WBP1 and WBP2 adaptors.

The YAP2 transcriptional regulator mediates a plethora of cellular functions, including the newly discovered Hippo tumor suppressor pathway, by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using isothermal titration calorimery and circular dichroism in combination with molecular modeling and molecular dynamics, we provide evidence that the WW1 and WW2 domains of YAP2 recognize various PPXY motifs within WBP1 and WBP2 in a highly promiscuous and subtle manner. Thus, although both WW domains strictly require the integrity of the consensus PPXY sequence, nonconsensus residues within and flanking this motif are not critical for high-affinity binding, implying that they most likely play a role in stabilizing the polyproline type II helical conformation of the PPXY ligands. Of particular interest is the observation that both WW domains bind to a PPXYXG motif with highest affinity, implicating a preference for a nonbulky and flexible glycine one residue to the C-terminal side of the consensus tyrosine. Importantly, a large set of residues within both WW domains and the PPXY motifs appear to undergo rapid fluctuations on a nanosecond time scale, suggesting that WW-ligand interactions are highly dynamic and that such conformational entropy may be an integral part of the reversible and temporal nature of cellular signaling cascades. Collectively, our study sheds light on the molecular determinants of a key WW-ligand interaction pertinent to cellular functions in health and disease.

Putative role of protein kinase C in neurotoxic inflammation mediated by extracellular heat shock protein 70 after ischemia-reperfusion

BackgroundSterile inflammation occurs in the absence of live pathogens and is an unavoidable consequence of ischemia-reperfusion (IR) injury in the central nervous system (CNS). It is known that toll-like receptor 4 (Tlr4) contributes to damage and sterile inflammation in the CNS mediated by IR. However, the mechanism of Tlr4 activation under sterile conditions in ischemic tissue is poorly understood. We performed this study to clarify the mechanism. To this end, we focused on the extracellular heat shock protein 70 (Hsp70), the prototypic Tlr4 ligand.MethodsTlr4-, Myd88- and Trif-knockout animals, as well as C57BL/6 mice, were used for the wild type control. For the in vivo study, we used a mouse model of retinal IR injury. To test the role of protein kinase C (PKC) in IR injury, IR retinas were treated with the PKC inhibitors (polymyxin B and Gö6976) and retinal damage was evaluated by directly counting neurons in the ganglion cell layer of flat-mounted retinas seven days after IR. Primary retinal neurons (retinal ganglion cells) and glial cells were used for in vitro experiments. Quantitative RT-PCR, ELISA and western blot analysis were used to study the production of pro-inflammatory factors in IR retinas and in primary cell cultures.ResultsWe found significant accumulation of extracellular Hsp70 in a model of retinal IR injury. We noted that PKC was involved in Tlr4 signaling, and found that PKC inhibitors promoted neuroprotection by reducing pro-inflammatory activity in ischemic tissue. To put all of the pieces in the signaling cascade together, we performed an in vitro study. We found that PKC was critical to mediate the Hsp70-dependent pro-inflammatory response. At the same time, the contamination of Hsp70 preparations with low-dose endotoxin was not critical to mediate the production of pro-inflammatory factors. We found that extracellular Hsp70 can promote neuronal death at least, by mediating production of cytotoxic levels of tumor necrosis factor alpha, predominantly due to the Tlr4/Myd88 signaling cascade.ConclusionsOur findings suggest that PKC acts as a switch to amplify the pro-inflammatory activity of Hsp70/Tlr4 signaling, which is sufficient to mediate neuronal death.

The oxidative stress product carboxyethylpyrrole potentiates TLR2/TLR1 inflammatory signaling in macrophages.

Oxidative stress is key in the pathogenesis of several diseases including age-related macular degeneration (AMD), atherosclerosis, diabetes, and Alzheimers disease. It has previously been established that a lipid peroxidation product, carboxyethylpyrrole (CEP), accumulates in the retinas of AMD patients. Retinal infiltrating macrophages also accumulate in the retinas of both AMD patients and in a murine model of AMD. We therefore investigated the ability of CEP-adducts to activate innate immune signaling in murine bone-marrow derived macrophages (BMDMs). We found that CEP specifically synergizes with low-dose TLR2-agonists (but not agonists for other TLRs) to induce production of inflammatory cytokines. Moreover, CEP selectively augments TLR2/TLR1-signaling instead of TLR2/TLR6-signaling. These studies uncover a novel synergistic inflammatory relationship between an endogenously produced oxidation molecule and a pathogen-derived product, which may have implications in the AMD disease process and other oxidative stress-driven pathologies.

Molecular mechanism of WW‐domain binding protein‐2 coactivation function in estrogen receptor signaling

The link between breast cancer and estrogen receptor (ER) is well established. The ER is a hormone‐inducible transcription factor that, upon binding to its ligand, regulates the expression of a variety of genes mainly involved in cell proliferation and differentiation. Coactivators are proteins recruited by the hormone‐activated receptor, which allow or enhance the ER transactivation functions by acting as chromatin remodeling enzymes or adaptors between ER and the transcriptional machinery. Our laboratory has previously identified the WW‐domain binding protein‐2 (WBP‐2) as a bona fide coactivator of ER. However, the molecular mechanism underlying WBP‐2 coactivation function was not clear yet. In this study, we explore and identify the mechanism by which WBP‐2 acts as coactivator of ER. Our data show that WBP‐2 is involved in the regulation of ER target genes, and its expression is required for the proper expression of some ER target genes. To clarify the molecular mechanism by which WBP‐2 regulates ER function, we performed chromatin immunoprecipitation assays. We demonstrate here that WBP‐2 binds to the ER target gene promoter pS2 promoter and is required for the binding of the phosphorylated form of RNA polymerase II (associated with active transcription/elongation) to the same promoter. Furthermore, we also show that WBP‐2 is essential for the recruitment of the histone acetyl transferase p300, an important chromatin modifier enzyme and for histone acetylation at the same target region. Collectively, our data indicate that WBP‐2 enhances ER transactivation function at certain genes by facilitating the recruitment and/or the stabilization of a histone modifier enzyme that favors a relaxed chromatin structure, permissive of transcription.

Recruitment of Donor T Cells to the Eyes During Ocular GVHD in Recipients of MHC-Matched Allogeneic Hematopoietic Stem Cell Transplants

PURPOSE The primary objective of the present study was to identify the kinetics and origin of ocular infiltrating T cells in a preclinical model of graft-versus-host disease (GVHD) that induces eye tissue damage. METHODS Graft-versus-host disease was induced using an major histocompatibility complex (MHC)-matched, minor histocompatibility-mismatched hematopoietic stem cell transplant (HSCT) model. This approach, which utilized congenic and EGFP-labeled donor populations, mimics a matched, clinically unrelated donor (MUD) cell transplant. Systemic and ocular GVHD were assessed at varying time points using clinical examination, intravital microscopy, immune phenotype via flow cytometric analyses, and immunohistochemical staining. RESULTS Following transplant, we observed characteristic changes in GVHD-associated immune phenotype as well as clinical signs present in recipients post transplant. Notably, the kinetics of the systemic changes and the ocular damage paralleled what is observed clinically, including damage to the cornea as well as the conjunctiva and lacrimal gland. Importantly, the infiltrate contained predominantly donor CD4 as well as CD8 T cells with an activated phenotype and macrophages together with effector cytokines consistent with the presence of a TH1 alloreactive population. CONCLUSIONS Overall, the findings here unequivocally demonstrated that donor T cells compose part of the corneal and ocular adnexa infiltrate in animals undergoing ocular GVHD. In total, the results describe a novel and promising preclinical model characterized by both systemic and ocular changes as detected in significant numbers of patients undergoing GVHD following allo-HSCT, which can help facilitate dissecting the underlying immune mechanisms leading to damage associated with ocular GVHD.

Locoregional and Overall Recurrence After Neaodjuvant Endocrine Therapy Versus Chemotherapy in Postmenopausal Women With Estrogen Receptor+ Her2− Breast Cancer

Purpose: We report clinical outcomes in patients treated with neoadjuvant endocrine therapy (NET) versus neoadjuvant cytotoxic chemotherapy (NCT) in a cohort of postmenopausal women with ER+, HER2− breast cancer. Materials and Methods: We retrospectively reviewed 140 patients treated between May 1998 and September 2010 and collected patient, disease, and treatment characteristics, response to neoadjuvant therapy, and clinical outcome. Results: The median age was 59.5 years. Stage group: stage I 2.2%, stage II 26.8%, stage III 71%, the median tumor size 6 cm (range, 1.5 to 19 cm). Fifty-seven (40.7%) received NET and 83 (59.3%) NCT. One patient (1.8%) in the NET group and 7 (8.4%) in the NCT group had a pathologic complete response (P=0.142). The median follow-up was 48.1 months. Five-year cumulative incidence of locoregional recurrence (LRR) among the entire cohort was 4.1% (95% confidence interval [CI]: 1.5, 8.9), and any recurrence 25.3% (95% CI: 17.6, 33.6). There was no difference in cumulative incidence of LRR or overall recurrence between NET and NCT. On multivariate analysis adjusting for receipt of chemotherapy, presenting stage, and positive lymph nodes, the use of adjuvant radiation therapy was associated with decreased risk of LRR (hazard ratio [HR]=0.24, P=0.035), and ypN2 status with higher risk of LRR (HR=4.91, P=0.032). When the same multivariate model was fitted for any recurrence outcome, only ypN2 status was a significant predictor of overall recurrence (HR=3.02, P=0.005). Conclusions: We have demonstrated equivalent locoregional and overall outcomes in patients receiving NET versus NCT in a cohort of postmenopausal women with locally advanced ER+HER2−tumors.