Mohamed L. Salem

EpCAM Is Overexpressed in Breast Cancer and Is a Potential Target for Breast Cancer Gene Therapy

EpCAM (epithelial cell adhesion molecule) is a cell surface molecule that is known to be highly expressed in colon and other epithelial carcinomas. EpCAM is involved in cell-to-cell adhesion and has been the target of antibody therapy in several clinical trials. To assess the value of EpCAM as a novel target for breast cancer gene therapy, we performed real-time reverse transcription-PCR to quantify the level of EpCAM mRNA expression in normal breast tissue and primary and metastatic breast cancers. We found that EpCAM is overexpressed 100- to 1000-fold in primary and metastatic breast cancer. Silencing EpCAM gene expression with EpCAM short interfering RNA (siRNA) resulted in a 35–80% decrease in the rate of cell proliferation in four different breast cancer cell lines. EpCAM siRNA treatment decreased cell migration by 91.8% and cell invasion by 96.4% in the breast cancer cell line MDA-MB-231 in vitro. EpCAM siRNA treatment was also associated with an increase in the detergent-insoluble protein fraction of E-cadherin, α-catenin, and β-catenin, consistent with the known biology of EpCAM as a regulator of cell adhesion. Our hypothesis is that modulation of EpCAM expression can affect cell migration, invasion, and proliferation by enhancing E-cadherin-mediated cell-to-cell adhesion. These data provide compelling evidence that EpCAM is a potential novel target for breast cancer gene therapy and offer insights into the mechanisms associated with EpCAM gene silencing.

Estrogen, a double-edged sword: modulation of TH1- and TH2-mediated inflammations by differential regulation of TH1/TH2 cytokine production.

Estrogen appears to play a central role in the immune response and immune-mediated diseases. Estrogen receptors are expressed in a variety of immunocompetent cells, including CD4(+) and CD8(+) T cells and macrophages. Clinical observations indicate that some autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis, frequently remit during pregnancy but exacerbate, or have their onset during the postpartum period. Pharmacological levels of estrogen also appear to ameliorate certain autoimmune diseases. In addition, estrogen is known to suppress certain infectious diseases, as well as T cell-mediated responses toward oxazolone, keyhol lympet hemocyanin, Listeria soluble protein and purified protein derivatives. The immune basis for these phenomena is poorly understood. Based on a distinctive profile of cytokine production, data accumulated thus far have revealed modulatory effects for estrogen on the TH1-type and TH2-type cells, which represent two polarized forms of the effector specific immune response. Recent evidence indicates that estrogens inhibit the production of TH1 proinflammatory cytokines, such as IL-12, TNF-alpha and IFN-gamma, whereas they stimulate the production of TH2 anti-inflammatory cytokines, such as IL-10, IL-4, and TGF-beta. This can explain why estrogen suppresses and potentiates TH1- and TH2-mediated diseases, respectively. We hypothesize that exacerbation or suppression of inflammatory diseases by estrogen is mediated by skewing TH1-type to TH2-type response. This view represents a novel mechanism for the modulatory effect of estrogen on certain inflammatory diseases that can lead to beneficial or detrimental impacts depending on the type of immune involved. Such a concept is valuable when considering the application of combination therapies that include estrogen.

Defining the antigen-specific T-cell response to vaccination and poly(I:C)/TLR3 signaling: evidence of enhanced primary and memory CD8 T-cell responses and antitumor immunity.

Poly(I:C), a synthetic double-stranded RNA polymer and a TLR3 agonist, can be used as a vaccine adjuvant to enhance adaptive immunity. However, the antigen-specific CD8 T-cell response to peptide vaccination and poly(I:C) has not been clearly defined. Here, the authors characterized the antigen-specific CD8 T-cell response to peptide vaccination and poly(I:C) and specifically addressed the hypothesis that poly(I:C) can enhance antitumor immunity. To define the antigen-specific T-cell response, the authors established a model based on the adoptive transfer of T cells from the OT-1 T-cell receptor transgenic mouse. In this model, vaccination with peptide alone resulted in a limited, transient expansion of antigen-specific CD8 T cells. In contrast, peptide vaccination with concomitant administration of poly(I:C) resulted in a dramatic sustained increase in the number of antigen-specific CD8 T cells. This increase in cell number was associated with an increase in CD8 T-cell function, as defined by specific IFN-γ and TNF-α production, and protection from tumor challenge. The adjuvant effects of poly(I:C) appear to be at least partially dependent on an increase in the transcription of the anti-apoptotic molecules Bcl-3 and Bcl-xL and a concomitant decrease in apoptosis during the contraction phase of the primary T-cell response. In addition, administration of poly(I:C) enhanced the response to a nonimmunogenic self-antigen in a dendritic cell vaccine-based vaccine strategy. Collectively, these results confirm the potential of poly(I:C) as a vaccine adjuvant and suggest that targeting of TLR3 is likely to be a valuable addition to peptide-based vaccination strategies.

Defining the ability of cyclophosphamide preconditioning to enhance the antigen-specific CD8+ T-cell response to peptide vaccination: creation of a beneficial host microenvironment involving type I IFNs and myeloid cells.

Although cyclophosphamide (CTX) has been clearly shown to enhance active specific and adoptive immunotherapies, the mechanism(s) underlying these beneficial effects have not been clearly defined. To define the impact of CTX preconditioning on the antigen-specific CD8 T-cell response to peptide vaccination, we used an adoptive transfer model based on the OT-1 T-cell receptor transgenic mouse. CTX preconditioning dramatically enhanced the antigen-specific CD8 T-cell response to peptide vaccination. Specifically, CTX significantly enhanced the expansion and function of responding CD8 T cells as demonstrated by flow cytometry and cytokine production. In parallel experiments, we attempted to define the mechanism(s) underlying these beneficial effects of CTX therapy. CTX therapy increased the relative number and activation status of myeloid dendritic cells, and was associated with the induction of significant levels of the inflammatory cytokines interferon-α, monocyte chemoattractant protein-1, and IL-6. Adoptive transfer experiments into type I IFNR−/− and CR3−/− mice confirmed that the beneficial effects of CTX were at least partially dependent on type I interferons and myeloid cells. Adoptive transfer of up to 150×106 naive spleen cells at the time of antigen-specific CD8 T-cell transfer did not abrogate the effects of CTX therapy, suggesting that the creation of a niche in the immune system may not be required. CTX decreased the absolute, but not relative number of CD4+CD25+ Treg cells, consistent with the possibility that regulatory T cells may be targeted by CTX therapy. Of note, combination therapy with CTX and a synthetic TLR3 agonist further enhanced the antigen-specific CD8+ T-cell response. Taken together, our data suggest that CTX modulates specific components of the innate immune system resulting in a beneficial host microenvironment. Specific targeting of these components may enhance the effectiveness of CTX preconditioning for adoptive immunotherapy.

Systemic Administration of IL-15 Augments the Antigen-Specific Primary CD8+ T Cell Response Following Vaccination with Peptide-Pulsed Dendritic Cells

The systemic administration of IL-2 can act as a potent adjuvant for T cell-directed vaccine strategies. However, not only is the administration of IL-2 potentially toxic, but recent evidence suggests that it may also paradoxically limit the duration and magnitude of the cytotoxic T cell response. A recently identified cytokine, IL-15, shares many properties with IL-2 and may provide a preferential means of augmenting T cell-directed vaccine responses. Although well characterized in vitro, there are few data on the ability of IL-15 to augment T cell-mediated responses in vivo. We therefore evaluated the ability of systemic IL-15 to function as a T cell adjuvant in a murine vaccine model. To establish a population of easily identifiable Ag-responsive T cells, naive CD8+ (OT-1) T cells were first adoptively transferred into mice. Vaccination with peptide-pulsed dendritic cells induced a modest expansion of OT-1 T cells. The addition of systemic IL-15 for 7 days following vaccination resulted in a significant increase in the expansion of responding T cells in the PBL, spleen, and lymph nodes. Importantly, the responding T cells were cytotoxic and maintained a Tc1-biased phenotype. We did not observe either enhanced resistance to activation-induced cell death or preferential generation of memory T cells as a result of treatment with IL-15 compared with IL-2. These studies show for the first time that IL-15 is capable of augmenting the primary CD8+ T cell response to vaccination and contribute to the basis for future experiments exploring the clinical role of IL-15.

Recovery from Cyclophosphamide-Induced Lymphopenia Results in Expansion of Immature Dendritic Cells Which Can Mediate Enhanced Prime-Boost Vaccination Antitumor Responses In Vivo When Stimulated with the TLR3 Agonist Poly(I:C)

Recent preclinical studies suggest that vaccination following adoptive transfer of CD8+ T cells into a lymphopenic host can augment the therapeutic antitumor responses of the transferred cells. However, the mechanism by which the lymphopenic microenvironment benefits Ag-specific CD8+ T cell responses remains elusive. We show herein that induction of lymphodepletion by a single 4 mg cyclophosphamide (CTX) treatment induces a marked expansion of immature dendritic cells (DCs) in the peripheral blood on days 8–16 post-CTX (termed restoration phase). In vitro, these DCs were functional, because they showed normal phagocytosis and effective Ag presentation capability upon activation. In vivo, administration of the TLR3 agonist poly(I:C) at the peak of DC expansion (day 12 postlymphopenia) induced inflammatory cytokine production and increases in the number of activated DCs in lymph nodes. Importantly, boosting with gp10025–33 melanoma peptide combined with poly(I:C) 12 days after an initial priming with the same regimen significantly increased the expansion and the antitumor efficacy of adoptively transferred pmel-1 CD8+ T cells. These responses were abrogated after depletion of activated DCs during Ag boosting. In conclusion, our data show that CTX treatment induces, during the restoration phase, expansion of immature DCs, which are functional and can be exploited in vivo to foster more effective antitumor adoptive immunotherapy strategies.

Protective effects of Nigella sativa oil and thymoquinone against toxicity-induced by the anticancer drug cyclophosphamide.

Abstract Constituents of the Nigella sativa seed are reported to possess potent antioxidant effects. Treatment with anticancer drugs such as cyclophosphamide (CTX) is associated with significant toxicity due to over-production of reactive oxygen species, resulting in increased levels of oxidative stress. The aim of this study is to test whether or not N. sativa L oil (NSO) or its active ingredient, thymoquinone (TQ), can reduce CTX-induced toxicity. Male albino rats were treated with intraperitoneal administration of phosphate buffered saline (PBS) or 200 mg/Kg CTX followed by intragastric administration of NSO or TQ on alternate days for 12 days. Administration of NSO and TQ was initiated 6 h before or after CTX injection. Twenty-four hours after the last NSO and TQ treatment, blood and liver were harvested to analyse toxicity-related parameters. Treatment with CTX induced significant toxicity as shown by decrease in haemoglobin concentration and increases in blood sugar levels, activities of liver enzymes, bilirubin, urea, creatinine, lipids (triglyceride, cholesterol and low-density lipoprotein (LDL)-cholesterol) and lipid peroxidation in the liver. Treatment with NSO or TQ induced significant reduction in overall toxicity. The antitoxic effects of NSO and TQ were associated with induction of antioxidant mechanisms. These results suggest that administration of NSO or TQ can lower CTX-induced toxicity as shown by an up-regulation of antioxidant mechanisms, indicating a potential clinical application for these agents to minimise the toxic effects of treatment with anticancer drugs.

Transfer of TCR Genes into Mature T Cells Is Accompanied by the Maintenance of Parental T Cell Avidity

The adoptive transfer of tumor-specific T cells expanded in vitro can be of significant therapeutic value in select cancer patients. This strategy is limited though, as it is often difficult, if not impossible, to obtain T cells of clinical value. The transfer of TCR genes to mature T cells to generate tumor-reactive T cells provides a potential mechanism to overcome these limitations. To evaluate the feasibility of such an approach and the quality of the resulting T cells, we generated replication-deficient retroviral vectors using the well-characterized OT-1 TCR genes. After transducing murine T cells, we were able to expand large numbers of Ag-specific T cells that were functionally active against tumor cells expressing the relevant Ag. Furthermore, we found that T cells expressing retrovirally encoded TCR had avidity that was similar to that of the parental clone. This maintenance of avidity was despite variable expression of the retrovirally encoded TCR and the presence of potentially competing endogenous TCRs. These results suggest that the inherent qualities of the TCR, as dictated by the coding sequence, are the most critical parameters in the generation of high-avidity T cells.

Cyclophosphamide Induces Dynamic Alterations in the Host Microenvironments Resulting in a Flt3 Ligand-Dependent Expansion of Dendritic Cells

Preconditioning a recipient host with lymphodepletion can markedly augment adoptive T cell therapy. However, the precise mechanisms involved are poorly understood. In a recent study, we observed a significant increase in the circulating levels of dendritic cells (DCs; CD11c+CD11b+) during the recovery from cyclophosphamide (CTX)-induced lymphodepletion. Herein, we demonstrate that the CTX-induced DC expansion was not altered by adjuvant chemotherapy or tumor burden but was augmented by coadministration of granulocyte-colony stimulating factor. Although the increase in the number of DCs was preceded by a systemic expansion of a population expressing the phenotype of myeloid-derived suppressor cells (Gr-1+CD11b+), depletion of these Gr-1+ cells had no effect on the noted expansion. Moreover, when Gr-1highCD11bhigh cells were sorted from CTX-treated mice and adoptively transferred into control or CTX-treated recipients, they did not differentiate into DCs. Post-CTX expansion of DCs was associated with proliferation of DCs in bone marrow (BM) during the lymphopenic phase and in the blood and spleen during the recovery phase. Furthermore, adoptive transfer of BM cells from CTX-treated mice produced equal numbers of DCs in the blood of either CTX-treated or untreated recipients. CTX induced a dynamic surge in the expression of growth factors and chemokines in BM, where CCR2 and Flt3 signaling pathways were critical for DC expansion. In sum, our data suggest that CTX induces proliferation of DCs in BM prior to their expansion in the periphery. Targeting DCs at these phases would significantly improve their contribution to the clinical application of lymphodepletion to adoptive immunotherapy.

Beta-estradiol-induced decrease in IL-12 and TNF-α expression suppresses macrophage functions in the course of Listeria monocytogenes infection in mice

Mice treated with a contraceptive dose of beta-estradiol (E2) demonstrated changes in their macrophage (Mphi) number and functions. While E2 increased and decreased the Mphi number in PBMC and PEC respectively, it enhanced the in vitro phagocytosis of FITC-labeled beads by both cells. E2 treatment also enhanced the phagocytic function of Mphi as assessed by the in vivo carbon clearance assay. In contrast, the in vitro intracellular killing function of adherent cells in peritoneal exudate cells (PEC) against Listeria monocytogenes decreased after E2 treatment. In line with the decrease in the intracellular killing function, the E2-treated mice showed an impaired protection against L. monocytogenes infection. To clarify the mechanism of the E2-mediated suppression of the protective response against L. monocytogenes infection, we next analyzed the cytokine expression by PEC in E2-treated L. monocytogenes-infected mice. On day 5 of the infection, the expression of IL-12, TNF-alpha and IL-10 by adherent PEC from the E2-treated mice was lower than that from the control-infected mice. The decrease in the cytokine expression by adherent PEC of E2-treated mice coincided with the decrease of IFN-gamma expression, and the increase in the IL-4, IL-10 and TGF-beta expressions by non-adherent PEC. These results revealed two aspects of the effects of E2 on Mphi. Even though E2 was found to enhance Mphi phagocytosis, the anti-bacterial function was suppressed. This suppression may be mediated by the inhibition of both IL-12 and TNF-alpha which play important roles in the protective response against intracellular bacteria.