Safety, tolerability and immunogenicity of a novel 24-valent pneumococcal vaccine in toddlers: A phase 1 randomized controlled trial.

BACKGROUND: Pneumococcal conjugate vaccines (PCVs) significantly reduced pneumococcal disease burden. Nevertheless, alternative approaches for controlling more serotypes are needed. Here, the safety, tolerability, and immunogenicity of a 24-valent (1/2/3/4/5/6A/6B/7F/8/9N/9V/10A/11A/12F/14/15B/17F/18C/19A/19F/20B/22F/23F/33F) pneumococcal vaccine based on Multiple Antigen-Presenting System (MAPS) technology (Pn-MAPS24v) was assessed in toddlers. METHODS: In this phase 1, blinded, dose-escalation, active-controlled multicenter study conducted in the United States (September/2020-April/2022), 12-15-month-old toddlers primed with three doses of 13-valent PCV (PCV13) were randomized 3:2 to receive a single dose of one of three Pn-MAPS24v dose levels (1 µg/2 µg/5 µg per polysaccharide) or PCV13 intramuscularly. Reactogenicity (within 7 days), treatment-emergent adverse events (TEAEs, within 180 days), serious/medically attended adverse events (SAEs/MAAEs, within 180 days), and immunogenicity (serotype-specific anti-capsular polysaccharide immunoglobulin G [IgG] and opsonophagocytic activity [OPA] responses at 30 days post-vaccination) were assessed. RESULTS: Of 75 toddlers enrolled, 74 completed the study (Pn-MAPS24v 1 µg/2 µg/5 µg: 15/14/16, PCV13: 29). Frequencies of local (60 %/67 %/31 %) and systemic events (67 %/67 %/75 %) in the Pn-MAPS24v 1 µg/2 µg/5 µg and the PCV13 (55 %, 79 %) groups were in similar ranges. TEAEs were reported by 47 %/40 %/63 % of Pn-MAPS24v 1 µg/2 µg/5 µg recipients and 52 % of PCV13 recipients. No vaccine-related SAE was reported. At 30 days post-vaccination, for each of the 13 common serotypes, ≥93 % of participants in each group had IgG concentrations ≥0.35 µg/mL; >92 % had OPA titers ≥lower limit of quantitation (LLOQ), except for serotype 1 (79 %). For 7/11 unique serotypes (2/8/9N/11A/17F/22F/33F), at all dose levels, ≥78 % of Pn-MAPS24v recipients in each group had IgG concentrations ≥0.35 µg/mL and 80 %-100 % had OPA titers ≥LLOQ. CONCLUSIONS: In 12-15-month-old toddlers, a single dose of Pn-MAPS24v showed an acceptable safety profile, regardless of dose level; AEs were reported at similar frequencies by Pn-MAPS24v and PCV13 recipients. Pn-MAPS24v elicited IgG and OPA responses to all common and most unique serotypes. These results support further clinical evaluation in infants.

Safety and immunopharmacology of ASP0892 in adults or adolescents with peanut allergy: two randomized trials.

BACKGROUND: New treatment options with improved safety and novel mechanisms of actions are needed for patients with peanut allergy. OBJECTIVES: To evaluate the safety, tolerability, and immunogenicity of ASP0892, a peanut DNA vaccine, after intradermal (id) or intramuscular (im) administration in adult or adolescent patients with peanut allergy in two phase 1 studies. METHODS: ASP0892 or placebo was administered every 2 weeks for a total of 4 doses. The doses were 1 mg or 4 mg id or 4 mg im for adults, and 1 mg or 4 mg id for adolescents. Immunologic parameters were assessed longitudinally. RESULTS: Thirty-one adults (mean age 24.3 years, 17 males) received ASP0892 (9, 8, 8 patients for 1 mg id, 4 mg id or 4 mg im, respectively) or placebo (2 patients/group). Twenty adolescents (mean age 14.2 years, 11 males) received ASP0892 (8 patients/group) or placebo (2 patients/group). In both studies, the most common treatment-emergent adverse event (TEAE) was injection site pruritus. No deaths or treatment withdrawal were related to TEAEs. No serious TEAEs related to treatment were observed in adult or adolescent patients. ASP0892 treatment led to modest increases in allergen-specific IgG and/or IgG4 in adults (1 mg id, 4 mg im) and adolescents (1 mg id, 4 mg id). No improvements in clinical outcomes, including double-blind placebo-controlled food challenge, were found after ASP0892 treatment. CONCLUSIONS: In two phase 1 studies, ASP0892 was well tolerated with modest but not clinically relevant changes in immune responses. GOV IDENTIFIERS: NCT02851277, NCT03755713.

Distinct trajectories distinguish antigen-specific T cells in peanut-allergic individuals undergoing oral immunotherapy.

BACKGROUND: Despite similar clinical symptoms, peanut-allergic (PA) individuals may respond quite differently to the same therapeutic interventions. OBJECTIVE: This study aimed to determine whether inherent qualities of cell response at baseline could influence response to peanut oral immunotherapy (PnOIT). METHODS: We first performed ex vivo T-cell profiling on peanut-reactive CD154+CD137+ T (pTeff) cells from 90 challenge-confirmed PA individuals. We developed a gating strategy for unbiased assessment of the phenotypic distribution of rare pTeff cells across different memory CD4+ T-cell subsets to define patient immunotype. In longitudinal samples of 29 PA participants enrolled onto the IMPACT trial of PnOIT, we determined whether patient immunotype at baseline could influence response to PnOIT. RESULTS: Our data emphasize the heterogeneity of pTeff cell responses in PA participants with 2 mutually exclusive phenotypic entities (CCR6-CRTH2+ and CCR6+CRTH2-). Our findings lead us to propose that peanut allergy can be classified broadly into at least 2 discrete subtypes, termed immunotypes, with distinct immunologic and clinical characteristics that are based on the proportion of TH2A pTeff cells. PnOIT induced elimination of TH2A pTeff cells in the context of the IMPACT clinical trial. Only 1 PA patient with a low level of TH2A pTeff cells at baseline experienced long-lasting benefit of remission after PnOIT discontinuation. CONCLUSION: Dividing PA patients according to their individual peanut-specific T-cell profile may facilitate patient stratification in clinical settings by identifying which immunotypes might respond best to different therapies.

Phase 1/2 study of a novel 24-valent pneumococcal vaccine in healthy adults aged 18 to 64 years and in older adults aged 65 to 85 years.

BACKGROUND: Pneumococcal diseases remain prevalent despite available polysaccharide and conjugate vaccines. This phase 1/2 study evaluated safety/tolerability and immunogenicity of a novel 24-valent pneumococcal vaccine (ASP3772) based on high-affinity complexing of proteins and polysaccharides. METHODS: Pneumococcal vaccine-naïve adults aged 18-85 years were randomized to receive either ASP3772 or PCV13 (13-valent conjugate vaccine). Participants received a single intramuscular injection of ASP3772 (1-, 2-, or 5-µg dose per polysaccharide) or PCV13. A separate, nonrandomized group of PCV13-vaccinated participants (65-85 years) received PPSV23 (23-valent polysaccharide vaccine). Assessments were obtained through Day 7 for reactogenicity, through Day 30 for safety and tolerability, and through Month 6 for serious adverse events. Immunogenicity was measured at Day 30 using assays for functional opsonophagocytic activity (OPA) and pneumococcal serotype-specific anticapsular polysaccharide immunoglobulin G for each serotype. RESULTS: In both age cohorts, the most frequently reported local reactions were self-limited tenderness and pain after ASP3772 at all dose levels or after PCV13, occurring within 2-3 days. Fatigue, headache, and myalgia were the most frequently reported systemic reactions following either vaccine. Robust OPA responses for all serotypes were observed across all ASP3772 dose groups in both age cohorts. Older adults (aged 65-85 years) who received ASP3772 had significantly higher immune responses to several PCV13 serotypes and all non-PCV13 serotypes than participants who received PCV13. OPA responses to the ASP3772 5-µg dose were significantly higher for several serotypes in naïve participants than in older adults with prior exposure to PCV13 who were administered PPSV23 in this study. CONCLUSIONS: These results demonstrate that ASP3772 is well tolerated, highly immunogenic, and in adults may offer significantly broader protection than existing pneumococcal vaccines. CLINICALTRIALS: gov: NCT03803202.

Development of MGD007, a gpA33 x CD3-Bispecific DART Protein for T-Cell Immunotherapy of Metastatic Colorectal Cancer.

We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART protein designed to redirect T cells to target gpA33 expressing colon cancer. The gpA33 target was selected on the basis of an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic colorectal cancer specimens, including putative cancer stem cell populations. MGD007 displays the anticipated-bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T cells. Xenograft studies showed tumor growth inhibition at doses as low as 4 µg/kg. Both CD8 and CD4 T cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme and perforin. Notably, suppressive T-cell populations could also be leveraged to mediate lysis of gpA33-expressing tumor cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Following prolonged exposure to MGD007 and gpA33 positive tumor cells, T cells express PD-1 and LAG-3 and acquire a memory phenotype but retain ability to support potent cell killing. In cynomolgus monkeys, 4 weekly doses of 100 µg/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Taken together, MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design and support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Mol Cancer Ther; 17(8); 1761-72. ©2018 AACR.

Tailoring CD19xCD3-DART exposure enhances T-cells to eradication of B-cell neoplasms.

Many patients with B-cell malignancies can be successfully treated, although tumor eradication is rarely achieved. T-cell-directed killing of tumor cells using engineered T-cells or bispecific antibodies is a promising approach for the treatment of hematologic malignancies. We investigated the efficacy of CD19xCD3 DART bispecific antibody in a broad panel of human primary B-cell malignancies. The CD19xCD3 DART identified 2 distinct subsets of patients, in which the neoplastic lymphocytes were eliminated with rapid or slow kinetics. Delayed responses were always overcome by a prolonged or repeated DART exposure. Both CD4 and CD8 effector cytotoxic cells were generated, and DART-mediated killing of CD4+ cells into cytotoxic effectors required the presence of CD8+ cells. Serial exposures to DART led to the exponential expansion of CD4 + and CD8 + cells and to the sequential ablation of neoplastic cells in absence of a PD-L1-mediated exhaustion. Lastly, patient-derived neoplastic B-cells (B-Acute Lymphoblast Leukemia and Diffuse Large B Cell Lymphoma) could be proficiently eradicated in a xenograft mouse model by DART-armed cytokine induced killer (CIK) cells. Collectively, patient tailored DART exposures can result in the effective elimination of CD19 positive leukemia and B-cell lymphoma and the association of bispecific antibodies with unmatched CIK cells represents an effective modality for the treatment of CD19 positive leukemia/lymphoma.

Integrated Pharmacokinetic/Pharmacodynamic Model of a Bispecific CD3xCD123 DART Molecule in Nonhuman Primates: Evaluation of Activity and Impact of Immunogenicity.

Purpose: Flotetuzumab (MGD006 or S80880) is a bispecific molecule that recognizes CD3 and CD123 membrane proteins, redirecting T cells to kill CD123-expressing cells for the treatment of acute myeloid leukemia. In this study, we developed a mathematical model to characterize MGD006 exposure-response relationships and to assess the impact of its immunogenicity in cynomolgus monkeys.Experimental Design: Thirty-two animals received multiple escalating doses (100-300-600-1,000 ng/kg/day) via intravenous infusion continuously 4 days a week. The model reflects sequential binding of MGD006 to CD3 and CD123 receptors. Formation of the MGD006/CD3 complex was connected to total T cells undergoing trafficking, whereas the formation of the trimolecular complex results in T-cell activation and clonal expansion. Activated T cells were used to drive the peripheral depletion of CD123-positive cells. Anti-drug antibody development was linked to MGD006 disposition as an elimination pathway. Model validation was tested by predicting the activity of MGD006 in eight monkeys receiving continuous 7-day infusions.Results: MGD006 disposition and total T-cell and CD123-positive cell profiles were well characterized. Anti-drug antibody development led to the suppression of T-cell trafficking but did not systematically abolish CD123-positive cell depletion. Target cell depletion could persist after drug elimination owing to the self-proliferation of activated T cells generated during the first cycles. The model was externally validated with the 7-day infusion dosing schedule.Conclusions: A translational model was developed for MGD006 that features T-cell activation and expansion as a key driver of pharmacologic activity and provides a mechanistic quantitative platform to inform dosing strategies in ongoing clinical studies. Clin Cancer Res; 24(11); 2631-41. ©2018 AACR.

Targeting CD123 in acute myeloid leukemia using a T-cell-directed dual-affinity retargeting platform.

T-cell-directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123(+) AML.

Development of PF-06671008, a Highly Potent Anti-P-cadherin/Anti-CD3 Bispecific DART Molecule with Extended Half-Life for the Treatment of Cancer.

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART®) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (-4.4 days in human FcRn knock-in mice), high stability (Tm1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.

A CD3xCD123 bispecific DART for redirecting host T cells to myelogenous leukemia: preclinical activity and safety in nonhuman primates.

Current therapies for acute myeloid leukemia (AML) are largely ineffective, and AML patients may benefit from targeted immunotherapy approaches. MGD006 is a bispecific CD3xCD123 dual-affinity re-targeting (DART) molecule that binds T lymphocytes and cells expressing CD123, an antigen up-regulated in several hematological malignancies including AML. MGD006 mediates blast killing in AML samples, together with concomitant activation and expansion of residual T cells. MGD006 is designed to be rapidly cleared, and therefore requires continuous delivery. In a mouse model of continuous administration, MGD006 eliminated engrafted KG-1a cells (an AML-M0 line) in human PBMC (peripheral blood mononuclear cell)-reconstituted NSG/ß2m(-/-) mice at doses as low as 0.5 µg/kg per day for ~7 days. MGD006 binds to human and cynomolgus monkey antigens with similar affinities and redirects T cells from either species to kill CD123-expressing target cells. MGD006 was well tolerated in monkeys continuously infused with 0.1 µg/kg per day escalated weekly to up to 1 µg/kg per day during a 4-week period. Depletion of circulating CD123-positive cells was observed as early as 72 hours after treatment initiation and persisted throughout the infusion period. Cytokine release, observed after the first infusion, was reduced after subsequent administrations, even when the dose was escalated. T cells from animals with prolonged in vivo exposure exhibited unperturbed target cell lysis ex vivo, indicating no exhaustion. A transient decrease in red cell mass was observed, with no neutropenia or thrombocytopenia. These studies support clinical testing of MGD006 in hematological malignancies, including AML.

Cytoskeletal modulation of lipid interactions regulates Lck kinase activity.

The actin cytoskeleton promotes clustering of proteins associated with cholesterol-dependent rafts, but its effect on lipid interactions that form and maintain rafts is not understood. We addressed this question by determining the effect of disrupting the cytoskeleton on co-clustering of dihexadecyl-(C(16))-anchored DiO and DiI, which co-enrich in ordered lipid environments such as rafts. Co-clustering was assayed by fluorescence resonance energy transfer (FRET) in labeled T cells, where rafts function in the phosphoregulation of the Src family kinase Lck. Our results show that probe co-clustering was sensitive to depolymerization of actin filaments with latrunculin B (Lat B), inhibition of myosin II with blebbistatin, and treatment with neomycin to sequester phosphatidylinositol 4,5-bisphosphate. Cytoskeletal effects on lipid interactions were not restricted to order-preferring label because co-clustering of C(16)-anchored DiO with didodecyl (C(12))-anchored DiI, which favors disordered lipids, was also reduced by Lat B and blebbistatin. Furthermore, conditions that disrupted probe co-clustering resulted in activation of Lck. These data show that the cytoskeleton globally modulates lipid interactions in the plasma membrane, and this property maintains rafts that function in Lck regulation.

Cytoplasmic domain of P-selectin glycoprotein ligand-1 facilitates dimerization and export from the endoplasmic reticulum.

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation, reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization, and the cytoplasmic domain propagates signals that activate ß(2) integrins to slow rolling on integrin ligands. Leukocytes from knock-in "ΔCD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly, they have 10-fold less PSGL-1 on their surfaces than WT leukocytes. Using glycosidases, proteases, Western blotting, confocal microscopy, cell-surface cross-linking, FRET, and pulse-chase metabolic labeling, we demonstrate that deleting the cytoplasmic domain impaired dimerization and delayed export of PSGL-1 from the endoplasmic reticulum (ER), markedly increasing a monomeric precursor in the ER and decreasing mature PSGL-1 on the cell surface. A monomeric full-length PSGL-1 made by substituting the transmembrane domain with that of CD43 exited the ER normally, revealing that dimerization was not required for ER export. Thus, the transmembrane and cytoplasmic domains cooperate to promote dimerization of PSGL-1. Furthermore, the cytoplasmic domain provides a key signal to export precursors of PSGL-1 from the ER to the Golgi apparatus en route to the cell surface.

T cell signal regulation by the actin cytoskeleton.

T cells form an immunological synapse (IS) that sustains and regulates signals for cell stimulation. Enriched in the IS is the Src family kinase Lck. Conversely, the membrane phosphatase CD45, which activates Src family kinases, is excluded, and this is necessary to avoid quenching of T cell receptor phosphosignals. Data suggest that this arrangement occurs by an enrichment of cholesterol-dependent rafts in the IS. However, the role of rafts in structuring the IS remains unclear. To address this question, we used fluorescence resonance energy transfer (FRET) to interrogate the nanoscopic structure of the IS. The FRET probes consisted of membrane-anchored fluorescent proteins with distinct affinities for rafts. Both the raft and nonraft probes exhibited clustering in the IS. However, co-clustering of raft donor-acceptor pairs was 10-fold greater than co-clustering of raft-nonraft pairs. We measured the effect of disrupting rafts in the IS on CD45 localization and Lck regulation by treating stimulated T cells with filipin. The filipin specifically disrupted co-clustering of the raft FRET pairs in the IS and allowed targeting of CD45 to the IS and dephosphorylation of the regulatory tyrosine of Lck. Clustering of the raft probes was also sensitive to latrunctulin B, which disrupts actin filaments. Strikingly, enriching the cortical cytoskeleton using jasplakinolide maintained raft probe co-clustering, CD45 exclusion, and Lck regulation in the IS following the addition of filipin. These data show the actin cytoskeleton maintains a membrane raft environment in the IS that promotes Lck regulation by excluding CD45.

Cytoskeleton-membrane interactions in membrane raft structure.

Cell membranes are structurally heterogeneous, composed of discrete domains with unique physical and biological properties. Membrane domains can form through a number of mechanisms involving lipid-lipid and protein-lipid interactions. One type of membrane domain is the cholesterol-dependent membrane raft. How rafts form remains a current topic in membrane biology. We review here evidence of structuring of rafts by the cortical actin cytoskeleton. This includes evidence that the actin cytoskeleton associates with rafts, and that many of the structural and functional properties of rafts require an intact actin cytoskeleton. We discuss the mechanisms of the actin-dependent raft organization, and the properties of the actin cytoskeleton in regulating raft-associated signaling events. We end with a discussion of membrane rafts and the actin cytoskeleton in T cell activation, which function synergistically to initiate the adaptive immune response.

Compartmentalization of phosphatidylinositol 4,5-bisphosphate signaling evidenced using targeted phosphatases.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a prevalent phosphoinositide in cell membranes, with important functions in cell signaling and activation. A large fraction of PIP(2) associates with the detergent-resistant membrane "raft" fraction, but the functional significance of this association remains controversial. To measure the properties of raft and nonraft PIP(2) in cell signaling, we targeted the PIP(2)-specific phosphatase Inp54p to either the raft or nonraft membrane fraction using minimal membrane anchors. Interestingly, we observed that targeting Inp54p to the nonraft fraction resulted in an enrichment of raft-associated PIP(2) and striking changes in cell morphology, including a wortmannin-sensitive increase in cell filopodia and cell spreading. In contrast, raft-targeted Inp54p depleted the raft pool of PIP(2) and produced smooth T cells void of membrane ruffling and filopodia. Furthermore, raft-targeted Inp54p inhibited capping in T cells stimulated by cross-linking the T cell receptor, but without affecting the T cell receptor-dependent Ca(2+) flux. Altogether, these results provide evidence of compartmentalization of PIP(2)-dependent signaling in cell membranes such as predicted by the membrane raft model.

Early and dynamic polarization of T cell membrane rafts and constituents prior to TCR stop signals.

Polarization of membrane rafts and signaling proteins to form an immunological synapse is a hallmark of T cell stimulation. However, the kinetics of raft polarization and associated proteins in relation to the initial contact of the T cell with the APC are poorly defined. We addressed this question by measuring the distribution of membrane-targeted fluorescent protein markers during initial T cell interactions with B cell APCs. Experiments with unpulsed B cells lacking cognate Ag demonstrated an MHC class II-independent capping that was specific to membrane raft markers and required actin rearrangements and signals from Src kinases and PI3K. By live cell imaging experiments, we identified a similar specific polarization of membrane raft markers before TCR-dependent stop signals, and which occurred independently of cognate peptide-MHC class II. T cells conjugated to unpulsed B cells exhibited capping of CD4 and microclusters of the TCR zeta-chain, but only the CD4 enrichment was cholesterol dependent. Furthermore, raft association of CD4 was necessary for its efficient targeting to the Ag-independent caps. Interestingly, anergic Vbeta8(+) T cells isolated from staphylococcal enterotoxin B-injected mice did not exhibit Ag-independent capping of membrane rafts, showing that inhibition of these early, Ag-independent events is a property associated with tolerance. Altogether, these data show that membrane raft capping is one of the earliest events in T cell activation and represents one avenue for promoting and regulating downstream peptide-MHC-dependent signaling within the T cell.

Clustering of membrane raft proteins by the actin cytoskeleton.

Cell membranes are laterally organized into functionally discrete domains that include the cholesterol-dependent membrane "rafts." However, how membrane domains are established and maintained remains unresolved and controversial but often requires the actin cytoskeleton. In this study, we used fluorescence resonance energy transfer to measure the role of the actin cytoskeleton in the co-clustering of membrane raft-associated fluorescent proteins (FPs) and FPs targeted to the nonraft membrane fraction. By fitting the fluorescence resonance energy transfer data to an isothermal binding equation, we observed a specific co-clustering of raft-associated donor and acceptor probes that was sensitive to latrunculin B (Lat B), which disrupts the actin cytoskeleton. Conversely, treating with jasplakinolide to enhance actin polymerization increased co-clustering of the raft-associated FPs over that of the nonraft probes. We also observed by immunoblotting experiments that the actin-dependent co-clustering coincided with regulation of the raft-associated Src family kinase Lck. Specifically, Lat B decreased the phosphorylation of the C-terminal regulatory tyrosine of Lck (Tyr505), and combining the Lat B with filipin further decreased the Tyr505 phosphorylation. Furthermore, the Lat B-dependent changes in Lck regulation required CD45 because no significant changes occurred in treated T cells lacking CD45 expression. These data define a role for the actin cytoskeleton in promoting co-clustering of raft-associated proteins and show that this property is important toward regulating raft-associated signaling proteins such as Lck.

Conversion of beta-carotene to retinal pigment.

Vitamin A and its active metabolite retinoic acid (RA)(1) play a major role in development, differentiation, and support of various tissues and organs of numerous species. To assure the supply of target tissues with vitamin A, long-lasting stores are built in the liver from which retinol can be transported by a specific protein to the peripheral tissues to be metabolized to either RA or reesterified to form intracellular stores. Vitamin A cannot be synthesized de novo by animals and thus has to be taken up from animal food sources or as provitamin A carotenoids, the latter being converted by central cleavage of the molecule to retinal in the intestine. The recent demonstration that the responsible beta-carotene cleaving enzyme beta,beta-carotene 15,15'-monooxygenase (Bcmo1) is also present in other tissues led to numerous investigations on the molecular structure and function of this enzyme in several species, including the fruit fly, chicken, mouse, and also human. Also a second enzyme, beta,beta-carotene-9',10'-monooxygenase (Bcmo2), which cleaves beta-carotene eccentrically to apo-carotenals has been described. Retinal pigment epithelial cells were shown to contain Bcmo1 and to be able to cleave beta-carotene into retinal in vitro, offering a new pathway for vitamin A production in another tissue than the intestine, possibly explaining the more mild vitamin A deficiency symptoms of two human siblings lacking the retinol-binding protein for the transport of hepatic vitamin A to the target tissues. In addition, alternative ways to combat vitamin A deficiency of specific targets by the supplementation with beta-carotene or even molecular therapies seem to be the future.

Protective effects of tomato extract with elevated beta-carotene levels on oxidative stress in ARPE-19 cells.

Epidemiological studies show that dietary products rich in carotenoids delay the progression of age-related macular degeneration. Experimental evidence from cellular studies on the antioxidant actions of carotenoids in the retinal pigment epithelium is still, however, fragmentary. The present study examined the uptake and protective potential of dietary carotenoids from tomato on the human retinal pigment epithelial cell line ARPE-19. ARPE-19 cells were incubated in medium supplemented with tomato extract containing high levels of beta-carotene, lycopene and traces of lutein. The cellular uptake of carotenoids was analysed by reverse-phase HPLC. Oxidative stress was induced by treatment with 1 mm-H2O2. Nitrotyrosine was detected by immunocytochemistry, and oxidised proteins (protein carbonyls) were measured by a quantitative ELISA method. Lipid peroxidation was assessed by quantifying thiobarbituric acid reactive substances. ARPE-19 cells preferentially accumulated lutein and beta-carotene rather than lycopene. Nitrotyrosine formation was considerably reduced in cells incubated with tomato extract compared with controls after H2O2 treatment. Protein carbonyls were reduced by 30 % (P = 0.015), and the formation of thiobarbituric acid-reactive substances was reduced by 140 % (P = 0.003) in cells incubated with tomato extract. The present study provides the experimental evidence for protective effects of dietary tomatoes rich in carotenoids on oxidative stress in the retinal pigment epithelium.