Bryostatin-1 Attenuates Ischemia-Elicited Neutrophil Transmigration and Ameliorates Graft Injury after Kidney Transplantation.

Ischemia reperfusion injury (IRI) is a form of sterile inflammation whose severity determines short- and long-term graft fates in kidney transplantation. Neutrophils are now recognized as a key cell type mediating early graft injury, which activates further innate immune responses and intensifies acquired immunity and alloimmunity. Since the macrolide Bryostatin-1 has been shown to block neutrophil transmigration, we aimed to determine whether these findings could be translated to the field of kidney transplantation. To study the effects of Bryostatin-1 on ischemia-elicited neutrophil transmigration, an in vitro model of hypoxia and normoxia was equipped with human endothelial cells and neutrophils. To translate these findings, a porcine renal autotransplantation model with eight hours of reperfusion was used to study neutrophil infiltration in vivo. Graft-specific treatment using Bryostatin-1 (100 nM) was applied during static cold storage. Bryostatin-1 dose-dependently blocked neutrophil activation and transmigration over ischemically challenged endothelial cell monolayers. When applied to porcine renal autografts, Bryostatin-1 reduced neutrophil graft infiltration, attenuated histological and ultrastructural damage, and improved renal function. Our novel findings demonstrate that Bryostatin-1 is a promising pharmacological candidate for graft-specific treatment in kidney transplantation, as it provides protection by blocking neutrophil infiltration and attenuating functional graft injury.

Nanoparticle-Encapsulated Bryostatin-1 Activates α-Secretase and PKC Isoforms In vitro and Facilitates Acquisition and Retention of Spatial Learning in an Alzheimer's Disease Mouse Model.

BACKGROUND: Alzheimer's disease (AD) animal models have revealed neuroprotective actions of Bryostatin-1 mediated by activation of novel PKC isoforms, suppression of beta-amyloid and downregulation of inflammatory and angiogenic events, making Bryostatin-1 an attractive candidate for attenuating AD-associated neural, vascular, and cognitive disturbances. OBJECTIVE: To further enhance Bryostatin-1 efficacy, nanoparticle-encapsulated Bryostatin-1 formulations were prepared. METHODS: We compared nano-encapsulated and unmodified Bryostatin-1 in in vitro models of neuronal PKC-d, PKC-e isoforms, α-secretase and studied nano-encapsulated Bryostatin-1 in an AD mouse model of spatial memory (BC3-Tg (APPswe, PSEN1 dE9) 85Dbo/J mice). RESULTS: We found that nanoencapsulated Bryostatin-1 formulations displayed activity greater or equal to that of unmodified Bryostatin-1 in PKC-δ and -ε and α-secretase activation assays. We next evaluated how treatment with a nanoencapsulated Bryostatin-1 formulation facilitated spatial learning in the Morris water maze. AD transgenic mice (6.5 to 8 months of age) were treated with nanoparticle encapsulated Bryostatin-1 formulation (1, 2.5, or 5 µg/mouse) three times the week before testing and then daily for each of the 5 days of testing. Across the acquisition phase, mice treated with nanoencapsulated Bryostatin-1 had shorter latencies, increased % time in the target zone and decreased % time in the opposite quadrant. The mice were given retention testing after a 2-week period without drug treatment. Mice treated with nanoencapsulated Bryostatin-1 had shorter latencies to find the escape platform, indicating retention of spatial memory. CONCLUSION: These data suggest that cognitive deficits associated with AD could be treated using highly potent nanoparticle-encapsulated formulations of Bryostatin-1.

Interferon-gamma depresses human intestinal smooth muscle cell contractility: Relevance to inflammatory gut motility disturbances.

AIM/BACKGROUND: In addition to absorptive disturbances, inflammatory bowel diseases (IBD) perturb normal contractility of intestinal smooth muscle. Such motility disturbances may reflect both nervous alterations and increased abundance of cytokines (e.g. IL-1ß, TNF-α, and IFN-γ), which impair normal intestinal smooth muscle structure and function. In a previous study, we reported that IL-1ß decreased mesenteric muscular contractility, consistent with cytokine-mediated changes in contraction present in IBD. Here, we considered the impact of pro-inflammatory cytokines on human intestinal smooth muscle cell (HISMC) contractility in vitro, which might provide a method for evaluating treatments for IBD. MATERIALS AND METHODS: We used an in vitro tonic contraction assay to study how HISMC contractility was affected by cell density, serum, and cytokines (IL-1ß, TNF-α, and IFN-γ). MTT (3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide) and wound healing analyses were also used to measure cell proliferation and migration in HISMC in response to IFN-γ. KEY FINDINGS: We found that IFN-γ (but not IL-1ß or TNF-α) significantly depressed HISMC tonic contractility over six days. IFN-γ also decreased HISMC proliferation, migration, and smooth muscle F-actin expression in a dose-dependent manner (studied at 4 days). SIGNIFICANCE: Our studies indicate that IFN-γ dose and time-dependently reduces normal HISMC contractility, motility and proliferation which may contribute to dysmotility observed in GI inflammatory disorders and that IFN-γ therapeutics might restore normal HISMC contractility impaired in IBD.

Bryostatin-1 for latent virus reactivation in HIV-infected patients on antiretroviral therapy.

OBJECTIVE: The protein kinase C (PKC) agonist bryostatin-1 has shown significant ex-vivo potency to revert HIV-1 latency, compared with other latency reversing agents (LRA). The safety of this candidate LRA remains to be proven in treated HIV-1-infected patients. METHODS: In this pilot, double-blind phase I clinical-trial (NCT 02269605), we included aviraemic HIV-1-infected patients on triple antiretroviral therapy to evaluate the effects of two different single doses of bryostatin-1 (10 or 20 µg/m) compared with placebo. RESULTS: Twelve patients were included, four in each arm. Bryostatin-1 was well tolerated in all participants. Two patients in the 20 µg/m arm developed grade 1 headache and myalgia. No detectable increases of cell-associated unspliced (CA-US) HIV-1-RNA were observed in any study arm, nor differences in HIV-1 mRNA dynamics between arms (P = 0.44). The frequency of samples with low-level viraemia did not differ between arms and low-level viraemia did not correlate with CA-US HIV-1-RNA levels (P = 0.676). No changes were detected on protein kinase C (PKC) activity and in biomarkers of inflammation (sCD14 and interleukin-6) in any study arm. After the single dose of bryostatin-1, plasma concentrations were under detection limits in all the patients in the 10 µg/m arm, and below 50 pg/ml (0.05 nmol/l) in those in the 20 µg/m arm. CONCLUSION: Bryostatin-1 was safe at the single doses administered. However, the drug did not show any effect on PKC activity or on the transcription of latent HIV, probably due to low plasma concentrations. This study will inform next trials aimed at assessing higher doses, multiple dosing schedules or combination studies with synergistic drugs.

Bryostatin-1 synergizes with histone deacetylase inhibitors to reactivate HIV-1 from latency.

The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication on patients treated with HAART. It has been suggested that successful depletion of such latent reservoirs will require a combination of therapeutic agents that can specifically and efficiently act on cells harboring latent HIV-1 provirus. Using Jurkat-LAT-GFP cells, a tractable model of HIV-1 latency, we have found that bryostatin -1 reactivates HIV-1 through a classical PKC-dependent pathway. Bryostatin-1 also activates MAPKs and NF-κB pathways and synergizes with HDAC inhibitors to reactivate HIV-1 from latency. Bryostatin-1 downregulates the expression of the HIV-1 co-receptors CD4 and CXCR4 and prevented de novo HIV-1 infection in susceptible cells. We applied proteomic methods to investigate major changes in protein expression in Jurkat-LAT-GFP under latency and reactivation conditions. We identified up-regulation of proteins that may be involved in the innate anti-HIV-1 response (NKEF-A and MHD2) and in different cell functions (i.e. cofilin-1 and transgelin-2) of the host cells. PKC agonists may represent a valuable pharmacological approach to purge latent HIV from cellular reservoirs and at the moment, the only clinically available PKC agonist is bryostatin-1. This drug has been tested in numerous clinical trials and its pharmacokinetics and toxicity in humans is well known. Moreover, bryostatin-1 potently synergizes with other HDAC inhibitors commonly used in the medical practice such as valproic acid. Therefore, bryostatin-1, alone or in combination with HDAC inhibitors, could be used in HAART treated patients to validate the hypothesis that reactivating HIV-1 from latency could purge HIV-1 reservoirs.

Phospholipid nanosomes.

Phospholipid nanosomes are small, uniform liposomes manufactured utilizing supercritical fluid technologies. Supercritical fluids are first used to solvate phospholipid raw materials, and then decompressed to form phospholipid nanosomes that can encapsulate hydrophilic molecules such as proteins and nucleic acids. Hydrophobic therapeutics are co-solvated with phospholipid raw materials in supercritical fluids that, when decompressed, form phospholipid nanosomes encapsulating these drugs in their lipid bilayers. Mathematical modeling and semi-empirical experiments indicate that the size and character of phospholipid nanosomes depend on the several process parameters and material properties including the size and design of decompression nozzle, bubble size, pressure and the rate of decompression, interfacial forces, charge distribution and the nature of compound being encapsulated. Examples are presented for the encapsulation of a protein and hydrophobic drugs. In vitro and in vivo data on breast cancer cells and xenografts in nude mice indicate that paclitaxel nanosomes are less toxic and much more effective than paclitaxel in Cremophor EL (Taxol). Camptothecin nanosomes demonstrate that the normally very water-insoluble camptothecin can be formulated in a biocompatible aqueous medium while retaining in vivo efficacy against lymphoma xenografts in nude mice. In vitro data for betulinic acid nanosomes demonstrate enhanced efficacy against HIV-1 (EC50 of 1.01 microg/ml versus 6.72 microg/ml for neat betulinic acid). Phospholipid nanosomes may find utility in the enhanced delivery of hydrophilic drugs such as recombinant proteins and nucleic acid as well as hydrophobic anticancer and anti-HIV drugs.