The Raf/MEK/ERK pathway and the Bcl-2 family proteins are commonly overexpressed in hematologic malignancies, where they promote proliferation and survival of the neoplastic cells. We have previously ...demonstrated that selective MEK inhibitors (MEK-I) exert potent growth-inhibitory effects in preclinical models of both acute myeloid leukemia (AML) and multiple myeloma (MM) (Blood 2006, 108:254). More recently, we have obtained evidence that ABT-737, a Bcl-2/Bcl-xL (BH3 mimetic) inhibitor (kindly provided by Abbott Laboratories), shows potent in vitro growth-inhibitory and pro-apoptotic activity on MM cell lines and primary CD138+ bone marrow cells from MM patients, regardless of the disease status (Libotte F, ASH 2008). Moreover, in AML models, we and others have reported a highly synergistic pro-apoptotic interaction between inhibitors of the Raf/MEK/ERK pathway and of the Bcl-2 family (Blood 2002, Cancer Cell 2006, Ricciardi ASH 2008). Here, we analyzed the impact of the simultaneous inhibition of these two pathways on cell proliferation and apoptosis in MM cell lines. To this purpose, we exposed different MM cell lines to increasing concentrations of MEK-I and ABT-737, alone and in combination. While single compounds dose-dependently inhibited cell growth, as assessed by trypan blue exclusion counting, we observed that their combination synergistically enhanced this effect with combination indexes (CI), as measured by isobologram analysis (Chou–Talalay method), of 0.28 and 0.12 for KMS18 and KMS27 cells, respectively. We next analyzed the effects of combined MEK and Bcl-2/Bcl-xL inhibition on apoptosis induction. Both MEK-I and ABT-737 induced apoptosis in MM cells at high concentrations, as determined by sub-G1 DNA peak and Annexin V staining. When used at concentrations that induced minimal apoptosis as single agents (7.55% and 6.8% net apoptosis induction in KMS18 cells after 72 hours of exposure to MEK-I and ABT-737, respectively), the combination of MEK-I and ABT-737 was able to induce substantial apoptosis (more than 50% net apoptosis induction after 72 hours in both KMS18 and KMS27 cell lines). Such pro-apoptotic interaction was highly synergistic in nature, with CI, as defined using isobologram analysis, of 0.2 and 0.17 for KMS18 and KMS27 cells, respectively. Mitochondrial membrane depolarization was similarly enhanced by the combination of MEK-I and ABT-737. Conversely, in the MEK-I resistant MM cell line ARH-77, ABT-737 was still able to induce apoptosis (up to 40% of the cells) but its effect was not significantly potentiated by MEK inhibition. Preliminary results on primary CD138+ MM cells exposed to both inhibitors confirmed the higher cell growth inhibition induced by combining MEK-I and Bcl-2/Bcl-xL inhibition. In conclusion, we demonstrated a striking synergistic pro-apoptotic activity with combined inhibition of Raf/MEK/ERK and Bcl-2 signaling in MM cell lines. Simultaneous disruption of these two pathways warrants further investigation as novel therapeutic strategy for this disease.
Smoldering multiple myeloma (SMM) is an asymptomatic plasma-cell disorder with a high risk of progression to a symptomatic multiple myeloma (MM). The time and mechanisms underlying the progression ...are still unclear. The Bcl-2 family proteins are frequently aberrantly expressed in MM and support proliferation, survival and drug-resistance. The activity of inhibitors of the anti-apoptotic Bcl-2 family members is currently under investigation in MM, while their therapeutic potential has never been explored in SMM. In this study, we investigated the constitutive protein expression profile of anti-apoptotic Bcl-2 family members and the functional effects of their pharmacological modulation by the Bcl-2/Bcl-xL inhibitor ABT-737 (kindly provided by Abbott Laboratories) on primary cells from SMM and symptomatic MM at different stages of the disease. Mononuclear cells isolated from BM aspirates of MM patients were fractionated into purified CD138+ (>80%) and CD138- populations. Protein extracts were prepared and subjected to Western blot analysis for anti-apoptotic Bcl-2 family members. Within the CD138+ malignant plasma cells, SMM samples were characterized by elevated Mcl-1 levels and barely detectable expression of Bcl-xL, while symptomatic MM samples exhibited higher levels of both Mcl-1 and Bcl-xL. No significant differences in Bcl-2 level expression were instead observed between SMM and symptomatic MM samples. We, then, investigated the functional effects of ABT-737 on MM cell line and on CD138+ malignant plasma cells from SMM and symptomatic MM samples. MM cell lines exposed to increasing concentrations of ABT-737 (10–1000 nM) for up 72 hours showed a dose- and time-dependent cell growth inhibition (IC50 200–400 nM at 72 hours) due to the loss of mitochondrial membrane potential, caspase activation and significant increase in the proportion of apoptotic cells. In addition, cell cycle analysis demonstrated a significant G1-phase depletion in KMS18 cells exposed to 1000 nM ABT-737 for 72 hours (26.9%±1.4 vs 54.6%±4.5, p=0.021). Western Blot analysis demonstrated that ABT-737 dramatically reduces Bcl-2 protein levels, with appearance of cleaved forms, while Mcl-1 was down-regulated only at higher doses. No effects were seen on Bcl-xL expression. The ABT-737 activity was then examined on CD138+ purified primary cells from 9 SMM samples cultured with ABT-737 (10–1000 nM) for 72 hours. A remarkable reduction in cell growth and increase in the percentage of apoptotic cells was observed in response to 100 nM ABT-737. In particular, a statistically significant pro-apoptotic activity of 100 nM ABT-737 was demonstrated by the increment of the subG1 peak after 24 hours (25.0%±9.8 vs 60.1%±17.0, p=0.00011, and vs 78.1%±14.0, p=0.0003, in the presence of 100 and 1000 nM ABT 737, respectively). Similar results were obtained in the CD138+ fraction from symptomatic MM (7 newly diagnosed and in 9 relapsed/refractory) with a significant increment of the subG1 peak after 24 hours of ABT-737 exposure (27.9%±15.5 vs 53.4%±16.8, p<0.0001, and vs 70.0%±15.4, p<0.0001, in the presence of 100 and 1000 nM ABT 737, respectively). Preliminary results indicate that, as in cell lines, Bcl-2 cleavage also occurs in CD138+ primary cells in response to ABT–737. In conclusion, we present the first evidence that Bcl-2/Bcl-xL inhibition by ABT-737 enhances apoptosis in CD138+ primary cells from SMM. We therefore suggest that this strategy may be developed for therapeutic purposes in different disease stages of MM patients.
An integrated and widespread road system, like the one built during the Roman Empire in Italy, plays an important role today in facilitating the construction of new infrastructure. This paper ...investigates the historical path of Roman roads as main determinant of both motorways and railways in the country. The empirical analysis shows how the modern Italian transport infrastructure followed the path traced in ancient times by the Romans in constructing their roads. Being paved and connecting Italy from North to South, consular trajectories lasted in time, representing the starting physical capital for developing the new transport networks.
Background Evidences from mice and human beings indicate that γδ T cells could be relevant in recognition of stress-induced self and/or yet unidentified inhaled foreign antigens. Their specificity ...differs from classic MHC-restricted αβ T cells and involves the immunoglobulin-like structure of the γδ T-cell receptor with the recognition of small organic molecules, alkylamines, and self lipid compounds presented by CD1+dendritic cells. Objective Because CD1 receptors are mainly devoted to lipid antigen presentation, we sought to determine whether exogenous pollen membrane lipids may act as allergens for CD1-restricted γδ T cells. Methods Peripheral blood and nasal mucosa-associated γδ T cells were cloned from normal controls and cypress-sensitive subjects and tested for their antigen specificity and CD1-restriction with phospholipids extracted from tree pollen grains, as well with other natural or synthetic compounds. Phospholipid reactivity of cloned γδ T cells was measured by mean of proliferative response and cytokine release as well as by testing their helper activity on IgE productionin vitroandin vivo. Results Cloned γδ T lymphocytes from subjects with allergy, but not normal controls, were found to recognize pollen-derived phosphatidyl-ethanolamine (PE) in a CD1d-restricted fashion. Only 16:0/18:2 and 18:2/18:2 PE were stimulatory, whereas no response was recorded for disaturated PE, phosphatidylcholine, neutral lipids, or protein extract. Proliferating clones secreted both TH1-type and TH2-type cytokines and drove IgE productionin vitroandin vivo. Conclusion CD1d-restricted γδ T cells specific for phospholipids can represent a key mucosal regulatory subset for the control of early host reactivity against tree pollens. Clinical implications By knowing how lipid allergen constituents interact with mucosal immune system, we can expand our possibilities in diagnostic and therapeutic interventions.
Evidences from mice and human beings indicate that gammadelta T cells could be relevant in recognition of stress-induced self and/or yet unidentified inhaled foreign antigens. Their specificity ...differs from classic MHC-restricted alphabeta T cells and involves the immunoglobulin-like structure of the gammadelta T-cell receptor with the recognition of small organic molecules, alkylamines, and self lipid compounds presented by CD1+ dendritic cells.
Because CD1 receptors are mainly devoted to lipid antigen presentation, we sought to determine whether exogenous pollen membrane lipids may act as allergens for CD1-restricted gammadelta T cells.
Peripheral blood and nasal mucosa-associated gammadelta T cells were cloned from normal controls and cypress-sensitive subjects and tested for their antigen specificity and CD1-restriction with phospholipids extracted from tree pollen grains, as well with other natural or synthetic compounds. Phospholipid reactivity of cloned gammadelta T cells was measured by mean of proliferative response and cytokine release as well as by testing their helper activity on IgE production in vitro and in vivo.
Cloned gammadelta T lymphocytes from subjects with allergy, but not normal controls, were found to recognize pollen-derived phosphatidyl-ethanolamine (PE) in a CD1d-restricted fashion. Only 16:0/18:2 and 18:2/18:2 PE were stimulatory, whereas no response was recorded for disaturated PE, phosphatidylcholine, neutral lipids, or protein extract. Proliferating clones secreted both T(H)1-type and T(H)2-type cytokines and drove IgE production in vitro and in vivo.
CD1d-restricted gammadelta T cells specific for phospholipids can represent a key mucosal regulatory subset for the control of early host reactivity against tree pollens.
By knowing how lipid allergen constituents interact with mucosal immune system, we can expand our possibilities in diagnostic and therapeutic interventions.
Florida Studies Slate, Claudia; Florida College English Association
2006, 2006-06-01
eBook
Florida was the first region of the United States to be discovered, explored, and, after a fashion, settled by Euroamericans. Its population in the early 21st century is approaching 17 million. ...Within years the number of people living in the state will surpass those living in New York, and the Sunshine State will become the most populous area east of the Mississippi. The first book in English about Florida was written by Jean Ribault. A French adventurer, Ribault established a colony of Huguenots near present-day Jacksonville. He was captured by the very able Spanish commander Pedro Menendez, who ordered his French rival and all his minions killed.The state's long and colorful past is matched by its equally long and colorful literary production. Strangely, critical assessment of Florida literature has lagged far behind. With this volume, the Florida College English Association has formally begun an effort to correct this lamentable oversight. Included are papers on every aspect of Florida literature and history by scholars from every part of the state who are employed in every kind of institution of higher learning. Of special interest are the studies of Florida literature in the 19th century and in the late 20th and early 21st centuries, areas that are generally ignored in national journals. The papers on the contributions of African-American literary figures, such as Zora Hurston and James Weldon Johnson, are noteworthy. Of particular interest are the suggestions for teaching Florida studies in the classroom, which can be adapted for high school as well as college students.
Bioactive food-preserving systems are based on the use of a natural antimicrobial agent loaded in a carrier material, which is able to trigger its release once necessary and to control the rate of ...release, thereby exerting either lethal or inhibitory effects against food pathogens or spoilage microorganisms. In this study the Schiff base of chitosan was synthesized by the reaction with cinnamaldehyde at different concentrations. Cinnamaldehyde is an aromatic α,ß-unsaturated aldehyde, and the major component in essential oils from some cinnamon species. It has been shown to exert antimicrobial activity against a wide range of microorganisms including bacteria, yeasts, and mould. The formation of the Schiff base is reversible under suitable conditions, and this might allow the release of the active cinnamaldehyde from chitosan, used as the carrier. The reaction kinetics was investigated by means of rheological analyses and infrared spectroscopy was used to assess the efficacy of the functionalization. Cinnamaldehyde-functionalized chitosan films were then prepared and tested antifungal properties in vitro. Moreover, the effect of the addition of expanded graphite sonicated stacks was evaluated in term of mechanical properties.
