Abstract Background Advanced age and human immunodeficiency virus (HIV) infection are associated with increased pneumococcal disease risk. The impact of these factors on cellular responses to ...vaccination is unknown. Methods HIV-infected (HIV+) individuals 50–65 years old with CD4+ T cells/μl (CD4) >200 on antiretroviral therapy (ART) ≥1 year received either the 13-valent pneumococcal conjugate vaccine followed by the 23-valent pneumococcal polysaccharide vaccine (PCV/PPV) or PPV only. HIV-uninfected (HIV−) controls received PCV/PPV. Phenotype distribution and surface expression of complement receptor CD21 and tumor necrosis factor superfamily receptors (TNFRs) were compared on serotype-specific B cells postvaccination. Results Postvaccination serotype-specific B cell percentages were significantly lower in HIV+ PCV/PPV compared to PPV groups, but similar between HIV+ or HIV− PCV/PPV groups. Transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI)+ serotype-specific B cell percentages were significantly decreased in HIV+ PCV/PPV compared to PPV groups. CD21+ serotype-specific B cells were significantly higher in HIV− compared to HIV+ PCV/PPV groups. Conclusions An initial dose of PCV reduced the frequency, but not phenotype distribution, of serotype-specific B cells and also lowered TACI expression in aging HIV+ subjects postvaccination with PPV. These findings suggest that PCV does not enhance cellular responses to revaccination with PPV.
Clusterin is a secreted glycoprotein with stress-induced expression in various diseased and aged tissues. It shares basic features with small heat shock proteins because it may stabilize proteins in ...a folding-competent state. Besides its presence in all human body fluids, clusterin associates with altered extracellular matrix proteins, such as β-amyloid in Alzheimer senile plaques in the brain. Because dermal connective tissue alterations occur because of aging and UV radiation, we explored the occurrence of clusterin in young, aged, and sun-exposed human skin. Immunohistochemical analysis showed that clusterin is constantly associated with altered elastic fibers in aged human skin. Elastotic material of sun-damaged skin (solar elastosis), in particular, revealed a strong staining for clusterin. Because of the striking co-localization of clusterin with abnormal elastic material, we investigated the interaction of clusterin with elastin in vitro . A chaperone assay was established in which elastin was denatured by UV irradiation in the absence or presence of clusterin. This assay demonstrated that clusterin exerted a chaperone-like activity and effectively inhibited UV-induced aggregation of elastin. The interaction of both proteins was further analyzed by electron microscopy, size exclusion chromatography, and mass spectrometry, in which clusterin was found in a stable complex with elastin after UV exposure.
A synthetic lipid A analog (ONO-4007) exhibits antileishmanial activity by activating Leishmania-infected macrophages in experimental leishmaniasis. In the present in vitro study, ONO-4007 at ...concentrations between 0.01 and 1.00 mg/mL markedly inhibited the proliferation of Leishmania major and L. amazonensis promastigotes. Ultrastructurally, L. major-infected macrophages showed degenerated intracellular amastigotes after exposure to ONO-4007. Leishmania-infected macrophages treated with ONO-4007 showed poorly developed parasitophorous vacuoles. High levels of tumor necrosis factor-alpha were induced by ONO-4007 in Leishmania-infected macrophages. In this in vivo study, L. amazonensis-infected BALB/c mice were treated with a dose of 30 mg/kg of ONO-4007 by perilesional and peritoneal injections. The skin lesion size was assessed before treatment with ONO-4007 and at eight weeks after injection. The lesion size was significantly suppressed in mice perilesionally injected with ONO-4007 (P < 0.01) compared with the controls. The data from our present in vitro and in vivo studies indicate that ONO-4007 has an antileishmanial effect.
Recent clinical trials demonstrated a promising clinical activity of a variety of Histone deacetylase inhibitors (HDACi) in patients with relapsed non-Hodgkin and Hodgkin lymphoma (HL). In this ...study, we examined the in vitro activity of the pan- DAC inhibitor vorinostat and the isotype selective HDAC inhibitor MGCD0103. Using a cell free fluorogenic peptide-based substrate assay, both MGCD0103 and vorinostat were potent inhibitors of class I HDACs, with vorinostat also effectively inhibiting HDAC6. Subsequently, we examined the effect of these two compounds on three HL-derived cell lines (HD-LM2, L-428, and KM-H2). Although these cell lines had a similar expression level of class-I HDAC proteins, as determined by western blot analysis, they were more sensitive to MGCD0103 in short term culture. After 72 hours of incubation, the IC50 of MGCD0103 was consistently <0.5 μM compared with >1.5 μM for vorinostat. This differential antiproliferative effect was also observed at the molecular level. Submicromolar concentrations (as low as 0.1 μM) of MGCD0103 effectively induced the expression of p21 and the acetylation of H3 histones, whereas higher concentration of vorinostat were required to achieve a similar effect. Furthermore, we examined the effect of equimolar concentrations of MGCD0103 and vorinostat on cytokine and chemokine secretion, using a multiplex cytokine array (34 cytokines/chemokines), on a selected panel of gene expression using pathway PCR array, and on selected protein expression using pathway protein array (phospho kinases and apoptosis pathways) and multicolor FACS experiments. While vorinostat and MGCD0103 shared similar effects on a variety of these elements, MGCD0103 was more effective in upregulating p21 expression, activating the intrinsic caspase pathway, and favoring the secretion of Th1-type cytokines. Given the recent reported results from two independent phase-II studies in patients with relapsed HL that demonstrated higher response rate achieved with MGCD0103 compared with vorinostat, our results suggest that cell-based assays may have a better predictor value of the potential clinical activity of HDACi in HL compared with the cell-free assays.
Abstract 1562
Poster Board I-585
SNDX-275 is an oral, class 1 isoform selective HDACi. Phase 1 studies in leukemia demonstrated the agent has a long half-life and that weekly or every other week ...dosing is sufficient for antitumor activity. Based on recent favorable in vitro and in vivo activity of several HDAC inhibitors in HL, we investigated the in vitro activity of SNDX275 in HL-derived cell lines.
For apoptosis and gene expression analysis 05 × 106 cells were incubated with 0.1-2 μM of SNDX-275 for 24-72 hours before they were examined for proliferation and cell death by the MTS assay and the annexin-PI and FACS analysis. For combination studies, cells were incubated with 0.1-2 uM of SNDX-275 and 1-20 nM of either gemcitabine or bortezomib for 48-72 hours. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. SNDX-275 effects on a panel of 30 cytokines and chemokines was assayed on 05 × 106 cells after incubation of 48 hrs using a multiplex assay.
SNDX-275 induced cell death in a dose and time dependent manner with an IC50 of 0.4 μM. At the molecular level, SNDX-275 increased H3 acetylation, up-regulated p21 protein expression, and activated the intrinsic apoptosis pathway by down-regulating the anti-apoptotic X-linked inhibitor or apoptosis (XIAP) protein, which was associated with activation of caspase 9 and 3. Combination studies demonstrated that SNDX-275 had synergistic effects when combined with gemcitabine and bortezomib. To further investigate the potential for SNDX-275 activity in HL we measured the effect of SNDX-275 on pathways that may contribute to an anti-tumor immune response. Dysregulated cytokine/chemokine production has been shown to contribute to HL pathology, including immune tolerance of the cancer cells. SNDX-275 increased IL12 p40-70, IP10, and RANTES, and decreased the level of IL13 and IL4, thus favoring Th1-type cytokines/chemokines. In addition, recent data has demonstrated that a variety of epigenetic-modulating drugs may up-regulate the expression of cancer testis tumor associated antigens, leading to a favorable immune response. None of the lines expressed the CTAs without induction. SNDX275 was able to induce CTA expression of SSX2 in L428 but not HDLM2 whereas MAGE-A was induced in both HL cell lines. NY-ESO expression was not induced.
Our studies demonstrate that SNDS-275 has dual effect on apoptotic and immunomodulatory pathways in HL. Furthermore, this data demonstrates that SNDX-275 may upregulate CTAs suggesting that this treatment may render the tumor more immunogeneic and susceptible to immune mediated killing with tumor-specific cytotoxic T lymphocytes. The selectivity profile of SNDX-275 also suggests that HDAC1 and 2 are the primary targets for HDAC inhibition in these cells. Phase 2 studies with SNDX-275 in HL are ongoing.
Younes:MethylGene: Honoraria, Research Funding.
Background Members of the Tumor Necrosis Factor (TNF)-superfamily have speculated roles in the response against T-independent type II antigens (TI-II) including pneumococcal polysaccharides (PPS). ...Dysregulation in their expression is associated with an enhanced risk for pneumococcal disease in neonates but their expression in other high-risk populations including HIV-positive individuals remains to be elucidated. Objective To investigate signals that contribute towards PPS-response and identify potential anomalies that may account for diminished serological response in HIV-positive individuals post Pneumovax (PPV23) immunization. Methods Markers of inflammation, C-reactive protein (CRP), IL-6, sCD27 and sCD30, were assessed in HIV-positive and -negative individuals as potential predictors of PPV23 response. Serum levels of B cell activating factor (BAFF), transmembrane activator and calcium-modulator and cytophilin ligand interactor (TACI), B cell maturation antigen (BCMA) and B cell expression of BAFF-R, TACI, BCMA, CD40 and CD21 were assessed in total (unselected) and PPS23F (antigen)-specific B cells of PPV23 immunized HIV-positive and -negative individuals. Results CRP, sCD27, sCD30 and BAFF were significantly elevated in the serum of HIV-positive individuals but did not adversely affect PPV23 response. Assessment of PPS-specific B cells revealed enhanced TACI and reduced BAFF-R expression compared to unselected B cells in HIV-positive and -negative individuals. Surface TACI was similar but soluble TACI was significantly lower in HIV-positive compared to HIV-negative individuals. Conclusion Current studies highlight a potential role for TACI in PPV23 response based on its enhanced expression on PPS-specific B cells. Although surface levels of TACI were similar, diminished soluble TACI (sTACI) in HIV-positive compared to HIV-negative individuals could potentially decrease BAFF responsiveness and Ig response. A better understanding of the role of TNF receptors could contribute to the design of improved pneumococcal vaccines. Trial Registration ClinicalTrials.gov NCT02515240
Abstract 3735
Poster Board III-671
Recent clinical trials demonstrated a promising clinical activity of the HDAC inhibitor MGCD0103 in heavily pretreated patients with relapsed HL. However, the ...mechanisms underlying MGCD0103 activity in HL remains unclear. Here we show that MGCD0103 preferentially inhibited HDACs1, 2 with no effect on HDAC6. Using three HL-derived cell lines, the IC50 for MGCD0103 after 72 hour of incubation was in the submicromolar concentrations, and was more effective than vorinostat in inducing cell death. Using gene expression profiling (GEP) studies, pathway PCR array, and western blot analysis, we identified several pathways that are modulated by MGCD0103. MGCD0103 downregulated the X-linked inhibitor of apoptosis (XIAP) protein, activated the intrinsic caspase pathway, and synergized with TRAIL agonistic antibodies. MGCD0103 downregulated CD30 mRNA and surface protein expression in a dose dependent manner, but had no significant effect on B cell-associated proteins, including CD19 and CD20, or on the costimulatory receptors CD40 and CD80. MGCD0103 induced TNF-alfa expression and secretion, which was associated with NF-kB activation, upregulation of the silencer of cytokine signaling (SOCS)-3, downregulated STAT6, and upregulation of STAT1 and 2. Selective inhibition of TNF-alfa expression by short interfering mRNA enhanced MGCD0103-induced cell death. Similarly, inhibition of NF-kB by proteasome inhibitors also potentiated the effect of MGCD0103, thus demonstrating synergy independent of HDAC6 inhibition. These findings demonstrate that MGCD0103 has a potent anti lymphoma activity by modulating the expression of a variety of survival proteins, and provides mechanistic rationale for combining class-I HDAC inhibitors with proteasome inhibitors and TRAIL.
Mamidipudi:Celgene: Employment. Heise:Celgene Corporation: Employment, Equity Ownership. Besterman:Methylgene: Employment. Martell:Methylgene: Employment. MacBeth:Celgene Corporation: Employment, Equity Ownership. Younes:MethylGene: Honoraria, Research Funding.
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