Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by 2 inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.

Exosome-educated macrophages and exosomes differentially improve ligament healing.

Recently, our group used exosomes from mesenchymal stromal/stem cells (MSCs) to simulate an M2 macrophage phenotype, that is, exosome-educated macrophages (EEMs). These EEMs, when delivered in vivo, accelerated healing in a mouse Achilles tendon injury model. For the current study, we first tested the ability of EEMs to reproduce the beneficial healing effects in a different rodent model, that is, a rat medial collateral ligament (MCL) injury model. We hypothesized that treatment with EEMs would reduce inflammation and accelerate ligament healing, similar to our previous tendon results. Second, because of the translational advantages of a cell-free therapy, exosomes alone were also examined to promote MCL healing. We hypothesized that MSC-derived exosomes could also alter ligament healing to reduce scar formation. Similar to our previous Achilles tendon results, EEMs improved mechanical properties in the healing ligament and reduced inflammation, as indicated via a decreased endogenous M1/M2 macrophage ratio. We also showed that exosomes improved ligament remodeling as indicated by changes in collagen production and organization, and reduced scar formation but without improved mechanical behavior in healing tissue. Overall, our findings suggest EEMs and MSC-derived exosomes improve healing but via different mechanisms. EEMs and exosomes each have attractive characteristics as therapeutics. EEMs as a cell therapy are terminally differentiated and will not proliferate or differentiate. Alternatively, exosome therapy can be used as a cell free, shelf-stable therapeutic to deliver biologically active components. Results herein further support using EEMs and/or exosomes to improve ligament healing by modulating inflammation and promoting more advantageous tissue remodeling.

Cultured cardiac fibroblasts and myofibroblasts express Sushi Containing Domain 2 and assemble a unique fibronectin rich matrix.

Cardiac fibroblasts and myofibroblasts assemble and maintain extracellular matrix during normal development and following injury. Culture expansion of these cells yield a bioengineered matrix that could lead to intriguing therapeutic opportunities. For example, we reported that cultured rat cardiac fibroblasts form a matrix that can be used to delivery therapeutic stem cells. Furthermore, we reported that matrix derived from cultured human cardiac fibroblasts/myofibroblasts converted monocytes into macrophages that express interesting anti-inflammatory and pro-angiogenic properties. Expanding these matrix investigations require characterization of the source cells for quality control. In these efforts, we observed and herein report that Sushi Containing Domain 2 (SUSD2) is a novel and consistent marker for cultured human cardiac fibroblast and myofibroblasts.

Extracellular Vesicle-Educated Macrophages Promote Early Achilles Tendon Healing.

Tendon healing follows a complex series of coordinated events, which ultimately produces a mechanically inferior tissue more scar-like than native tendon. More regenerative healing occurs when anti-inflammatory M2 macrophages play a more dominant role. Mesenchymal stromal/stem cells (MSCs) are able to polarize macrophages to an M2 immunophenotype via paracrine mechanisms. We previously reported that coculture of CD14+ macrophages (MQs) with MSCs resulted in a unique M2-like macrophage. More recently, we generated M2-like macrophages using only extracellular vesicles (EVs) isolated from MSCs creating "EV-educated macrophages" (also called exosome-educated macrophages [EEMs]), thereby foregoing direct use of MSCs. For the current study, we hypothesized that cell therapy with EEMs would improve in vivo tendon healing by modulating tissue inflammation and endogenous macrophage immunophenotypes. We evaluated effects of EEMs using a mouse Achilles tendon rupture model and compared results to normal tendon healing (without any biologic intervention), MSCs, MQs, or EVs. We found that exogenous administration of EEMs directly into the wound promoted a healing response that was significantly more functional and more regenerative. Injured tendons treated with exogenous EEMs exhibited (a) improved mechanical properties, (b) reduced inflammation, and (c) earlier angiogenesis. Treatment with MSC-derived EVs alone were less effective functionally but stimulated a biological response as evidenced by an increased number of endothelial cells and decreased M1/M2 ratio. Because of their regenerative and immunomodulatory effects, EEM treament could provide a novel strategy to promote wound healing in this and various other musculoskeletal injuries or pathologies where inflammation and inadequate healing is problematic. Stem Cells 2019;37:652-662.

Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma.

Nuclear factor-κB (NF-κB) is a family of transcription factors that play a key role in cell survival and proliferation in many hematological malignancies, including multiple myeloma (MM). Bortezomib, a proteasome inhibitor used in the management of MM, can inhibit both canonical and noncanonical activation of NF-κB in MM cells. However, we previously reported that a significant fraction of freshly isolated MM cells harbor bortezomib-resistant NF-κB activity. Here, we report that hyaluronan and proteoglycan link protein 1 (HAPLN1) is produced in bone marrow stromal cells from MM patients, is detected in patients' bone marrow plasma, and can activate an atypical bortezomib-resistant NF-κB pathway in MM cells. We found that this pathway involves bortezomib-resistant degradation of the inhibitor of NF-κB (IκBα), despite efficient bortezomib-mediated inhibition of proteasome activity. Moreover, HAPLN1 can also confer bortezomib-resistant survival of MM cells. We propose that HAPLN1 is a novel pathogenic factor in MM that induces an atypical NF-κB activation and thereby promotes bortezomib resistance in MM cells.

Macrophages Educated with Exosomes from Primed Mesenchymal Stem Cells Treat Acute Radiation Syndrome by Promoting Hematopoietic Recovery.

In the setting of radiation-induced trauma, exposure to high levels of radiation can cause an acute radiation syndrome (ARS) causing bone marrow (BM) failure, leading to life-threatening infections, anemia, and thrombocytopenia. We have previously shown that human macrophages educated with human mesenchymal stem cells (MSCs) by coculture can significantly enhance survival of mice exposed to lethal irradiation. In this study, we investigated whether exosomes isolated from MSCs could replace direct coculture with MSCs to generate exosome educated macrophages (EEMs). Functionally unique phenotypes were observed by educating macrophages with exosomes from MSCs (EEMs) primed with bacterial lipopolysaccharide (LPS) at different concentrations (LPS-low EEMs or LPS-high EEMs). LPS-high EEMs were significantly more effective than uneducated macrophages, MSCs, EEMs, or LPS-low EEMs in extending survival after lethal ARS in vivo. Moreover, LPS-high EEMs significantly reduced clinical signs of radiation injury and restored hematopoietic tissue in the BM and spleen as determined by complete blood counts and histology. LPS-high EEMs showed significant increases in gene expression of STAT3, secretion of cytokines like IL-10 and IL-15, and production of growth factors like FLT-3L. LPS-EEMs also showed increased phagocytic activity, which may aid with tissue remodeling. LPS-high EEMs have the potential to be an effective cellular therapy for the management of ARS.

Cornea-Derived Mesenchymal Stromal Cells Therapeutically Modulate Macrophage Immunophenotype and Angiogenic Function.

Macrophages are crucial drivers of inflammatory corneal neovascularization and thus are potential targets for immunomodulatory therapies. We hypothesized that therapeutic use of cornea-derived mesenchymal stromal cells (cMSCs) may alter the function of macrophages. We found that cMSCs can modulate the phenotype and angiogenic function of macrophages. In vitro, cMSCs induce apoptosis of macrophages while preferentially promoting a distinct CD14hi CD16hi CD163hi CD206hi immunophenotype that has significantly reduced angiogenic effects based on in vitro angiogenesis assays. In vivo, application of cMSCs to murine corneas after injury leads to reduced macrophage infiltration and higher expression of CD206 in macrophages. Macrophages cocultured ("educated") by cMSCs express significantly higher levels of anti-angiogenic and anti-inflammatory factors compared with control macrophages. In vivo, injured corneas treated with cMSC-educated macrophages demonstrate significantly less neovascularization compared with corneas treated with control macrophages. Knocking down the expression of pigment epithelial derived factor (PEDF) in cMSCs significantly abrogates its modulating effects on macrophages, as shown by the reduced rate of apoptosis, decreased expression of sFLT-1/PEDF, and increased expression of vascular endothelial growth factor-A in the cocultured macrophages. Similarly, cMSCs isolated from PEDF knockout mice are less effective compared with wild-type cMSCs at inhibiting macrophage infiltration when applied to wild-type corneas after injury. Overall, these results demonstrate that cMSCs therapeutically suppress the angiogenic capacity of macrophages and highlight the role of cMSC secreted PEDF in the modulation of macrophage phenotype and function. Stem Cells 2018;36:775-784.

Human Mesenchymal Stem Cell-Educated Macrophages Are a Distinct High IL-6-Producing Subset that Confer Protection in Graft-versus-Host-Disease and Radiation Injury Models.

Mesenchymal stem cells (MSCs) have immunosuppressive and tissue repair properties, but clinical trials using MSCs to prevent or treat graft-versus-host disease (GVHD) have shown mixed results. Macrophages (MØs) are important regulators of immunity and can promote tissue regeneration and remodeling. We have previously shown that MSCs can educate MØs toward a unique anti-inflammatory immunophenotype (MSC-educated MØs [MEMs]); however, their implications for in vivo models of inflammation have not been studied yet. We now show that in comparison with MØs, MEMs have increased expression of the inhibitory molecules PD-L1, PD-L2, in addition to markers of alternatively activated MØs: CD206 and CD163. RNA-Seq analysis of MEMs, as compared with MØs, show a distinct gene expression profile that positively correlates with multiple pathways important in tissue repair. MEMs also show increased expression of IL-6, transforming growth factor-ß, arginase-1, CD73, and decreased expression of IL-12 and tumor necrosis factor-α. We show that IL-6 secretion is controlled in part by the cyclo-oxygenase-2, arginase, and JAK1/STAT1 pathway. When tested in vivo, we show that human MEMs significantly enhance survival from lethal GVHD and improve survival of mice from radiation injury. We show these effects could be mediated in part through suppression of human T cell proliferation and may have attenuated host tissue injury in part by enhancing murine fibroblast proliferation. MEMs are a unique MØ subset with therapeutic potential for the management of GVHD and/or protection from radiation-induced injury.

Large-scale bioreactor production of extracellular vesicles from mesenchymal stromal cells for treatment of acute radiation syndrome.

BACKGROUND: Hematopoietic acute radiation syndrome (H-ARS) occurring after exposure to ionizing radiation damages bone marrow causing cytopenias, increasing susceptibility to infections and death. We and others have shown that cellular therapies like human mesenchymal stromal cells (MSCs), or monocytes/macrophages educated ex-vivo with extracellular vesicles (EVs) from MSCs were effective in a lethal H-ARS mouse model. However, given the complexity of generating cellular therapies and the potential risks of using allogeneic products, development of an "off-the-shelf" cell-free alternative like EVs may have utility in conditions like H-ARS that require rapid deployment of available therapeutics. The purpose of this study was to determine the feasibility of producing MSC-derived EVs at large scale using a bioreactor and assess critical quality control attributes like identity, sterility, and potency in educating monocytes and promoting survival in a lethal H-ARS mouse model. METHODS: EVs were isolated by ultracentrifugation from unprimed and lipopolysaccharide (LPS)-primed MSCs grown at large scale using a hollow fiber bioreactor and compared to a small scale system using flasks. The physical identity of EVs included a time course assessment of particle diameter, yield, protein content and surface marker profile by flow-cytometry. Comparison of the RNA cargo in EVs was determined by RNA-seq. Capacity of EVs to generate exosome educated monocytes (EEMos) was determined by qPCR and flow cytometry, and potency was assessed in vivo using a lethal ARS model with NSG mice. RESULTS: Physical identity of EVs at both scales were similar but yields by volume were up to 38-fold more using a large-scale bioreactor system. RNA-seq indicated that flask EVs showed upregulated let-7 family and miR-143 micro-RNAs. EEMos educated with LPS-EVs at each scale were similar, showing increased gene expression of IL-6, IDO, FGF-2, IL-7, IL-10, and IL-15 and immunophenotyping consistent with a PD-L1 high, CD16 low, and CD86 low cell surface expression. Treatment with LPS-EVs manufactured at both scales were effective in the ARS model, improving survival and clinical scores through improved hematopoietic recovery. EVs from unprimed MSCs were less effective than LPS-EVs, with flask EVs providing some improved survival while bioreactor EVs provide no survival benefit. CONCLUSIONS: LPS-EVs as an effective treatment for H-ARS can be produced using a scale-up development manufacturing process, representing an attractive off-the-shelf, cell-free therapy.

Exosomes, MDSCs and Tregs: A new frontier for GVHD prevention and treatment.

The development of graft versus host disease (GVHD) represents a long-standing complication of allogeneic hematopoietic cell transplantation (allo-HCT). Different approaches have been used to control the development of GVHD with most relying on variations of chemotherapy drugs to eliminate allo-reactive T cells. While these approaches have proven effective, it is generally accepted that safer, and less toxic GVHD prophylaxis drugs are required to reduce the health burden placed on allo-HCT recipients. In this review, we will summarize the emerging concepts revolving around three biologic-based therapies for GVHD using T regulatory cells (Tregs), myeloid-derived-suppressor-cells (MDSCs) and mesenchymal stromal cell (MSC) exosomes. This review will highlight how each specific modality is unique in its mechanism of action, but also share a common theme in their ability to preferentially activate and expand Treg populations in vivo. As these three GVHD prevention/treatment modalities continue their path toward clinical application, it is imperative the field understand both the biological advantages and disadvantages of each approach.

Exosomes from primed MSCs can educate monocytes as a cellular therapy for hematopoietic acute radiation syndrome.

BACKGROUND: Acute radiation syndrome (ARS) is caused by acute exposure to ionizing radiation that damages multiple organ systems but especially the bone marrow (BM). We have previously shown that human macrophages educated with exosomes from human BM-derived mesenchymal stromal cells (MSCs) primed with lipopolysaccharide (LPS) prolonged survival in a xenogeneic lethal ARS model. The purpose of this study was to determine if exosomes from LPS-primed MSCs could directly educate human monocytes (LPS-EEMos) for the treatment of ARS. METHODS: Human monocytes were educated by exosomes from LPS-primed MSCs and compared to monocytes educated by unprimed MSCs (EEMos) and uneducated monocytes to assess survival and clinical improvement in a xenogeneic mouse model of ARS. Changes in surface molecule expression of exosomes and monocytes after education were determined by flow cytometry, while gene expression was determined by qPCR. Irradiated human CD34+ hematopoietic stem cells (HSCs) were co-cultured with LPS-EEMos, EEMos, or uneducated monocytes to assess effects on HSC survival and proliferation. RESULTS: LPS priming of MSCs led to the production of exosomes with increased expression of CD9, CD29, CD44, CD146, and MCSP. LPS-EEMos showed increases in gene expression of IL-6, IL-10, IL-15, IDO, and FGF-2 as compared to EEMos generated from unprimed MSCs. Generation of LPS-EEMos induced a lower percentage of CD14+ monocyte subsets that were CD16+, CD73+, CD86+, or CD206+ but a higher percentage of PD-L1+ cells. LPS-EEMos infused 4 h after lethal irradiation significantly prolonged survival, reducing clinical scores and weight loss as compared to controls. Complete blood counts from LPS-EEMo-treated mice showed enhanced hematopoietic recovery post-nadir. IL-6 receptor blockade completely abrogated the radioprotective survival benefit of LPS-EEMos in vivo in female NSG mice, but only loss of hematopoietic recovery was noted in male NSG mice. PD-1 blockade had no effect on survival. Furthermore, LPS-EEMos also showed benefits in vivo when administered 24 h, but not 48 h, after lethal irradiation. Co-culture of unprimed EEMos or LPS-EEMos with irradiated human CD34+ HSCs led to increased CD34+ proliferation and survival, suggesting hematopoietic recovery may be seen clinically. CONCLUSION: LPS-EEMos are a potential counter-measure for hematopoietic ARS, with a reduced biomanufacturing time that facilitates hematopoiesis.

Antitumor activity of ribonuclease multimers created by site-specific covalent tethering.

Site-specific cross-linking can generate homogeneous multimeric proteins of defined valency. Pancreatic-type ribonucleases are an especially attractive target, as their natural dimers can enter mammalian cells, evade the cytosolic ribonuclease inhibitor (RI), and exert their toxic ribonucleolytic activity. Here, we report on the use of eight distinct thiol-reactive cross-linking reagents to produce dimeric and trimeric conjugates of four pancreatic-type ribonucleases. Both the site of conjugation and, to a lesser extent, the propinquity of the monomers within the conjugate modulate affinity for RI, and hence cytotoxicity. Still, the cytotoxicity of the multimers is confounded in vitro by their increased hydrodynamic radius, which attenuates cytosolic entry. A monomeric RI-evasive variant of bovine pancreatic ribonuclease (RNase A) inhibits the growth of human prostate and lung tumors in mice. An RI-evasive trimeric conjugate inhibits tumor growth at a lower dose and with less frequent administration than does the monomer. This effect is attributable to an enhanced persistence of the trimers in circulation. On a molecular basis, the trimer is ∼300-fold more efficacious and as well tolerated as erlotinib, which is in clinical use for the treatment of lung cancer. These data encourage the development of mammalian ribonucleases for the treatment of human cancers.

Mesenchymal Stromal Cells and Exosomes: Progress and Challenges.

Due to their robust immunomodulatory capabilities, mesenchymal stem/stromal cells (MSCs) have been used as a cellular therapy for a number of human diseases. Part of the mechanism of action of MSCs is the production of extracellular vesicles (EVs) that contain proteins, nucleic acids, and lipids that transmit signals to recipient cells that change their biologic behavior. This review briefly summarizes the development of MSCs as a treatment for human diseases as well as describes our present understanding of exosomes; how they exert their effects on target cells, and how they are differentiated from other EVs. The current treatment paradigm for acute radiation syndrome (ARS) is discussed, and how MSCs and MSC derived exosomes are emerging as treatment options for treating patients after radiation exposure. Other conditions such as graft-versus-host disease and cardiovascular disease/stroke are discussed as examples to highlight the immunomodulatory and regenerative capacity of MSC-exosomes. Finally, a consideration is given to how these cell-based therapies could possibly be deployed in the event of a catastrophic radiation exposure event.

Use of MSCs and MSC-educated macrophages to mitigate hematopoietic acute radiation syndrome.

PURPOSE OF REVIEW: Innovative and minimally toxic treatment approaches are sorely needed for the prevention and treatment of hematopoietic acute radiation syndrome (H-ARS). Cell therapies have been increasingly studied for their potential use as countermeasures for accidental and intentional ionizing radiation exposures which can lead to fatal ARS. Mesenchymal stem/stromal cells (MSCs) are a cell therapy that have shown promising results in preclinical studies of ARS, and are being developed in clinical trials specifically for H-ARS. MSCs, MSC-educated macrophages (MEMs) and MSC-exosome educated macrophages (EEMs) all have the potential to be used as adoptive cell therapies for H-ARS. Here we review how MSCs have been reported to mitigate inflammation from radiation injury while also stimulating hematopoiesis during ARS. RECENT FINDINGS: We discuss emerging work with immune cell subsets educated by MSCs, including MEMs and EEMs, in promoting hematopoiesis in xenogeneic models of ARS. We also discuss the first placental-derived MSC product to enter phase I trials, PLX-R18, and the challenges faced by bringing MSC and other cell therapies into the clinic for treating ARS. SUMMARY: Although MSCs, MEMs and EEMs are potential cell therapy candidates in promoting hematopoietic HRS, challenges persist in translational clinical development of these products to the clinic. Whether any of these cellular therapies will be sufficient as stand-alone therapies to mitigate H-ARS or if they will be a bridging therapy that insures survival until a curative allogeneic hematopoietic stem cell transplant can be performed are the key questions that will have to be answered.

Cardiac fibroblast derived matrix-educated macrophages express VEGF and IL-6, and recruit mesenchymal stromal cells.

The polarization of monocytes into macrophages that possess anti-inflammatory and pro-angiogenic properties could provide a novel therapeutic strategy for patients who are at a high risk for developing heart failure following myocardial infarction (MI). Here in, we describe a novel method of "educating" monocytes into a distinct population of macrophages that exhibit anti-inflammatory and pro-angiogenic features through a 3-day culture on fibronectin-rich cardiac matrix (CX) manufactured using cultured human cardiac fibroblasts. Our data suggest that CX can educate monocytes into a unique macrophage population termed CX educated macrophages (CXMq) that secrete high levels of VEGF and IL-6. In vitro, CXMq also demonstrate the ability to recruit mesenchymal stromal cells (MSC) with known anti-inflammatory properties. Selective inhibition of fibronectin binding to αVß3 surface integrins on CXMq prevented MSC recruitment. This suggests that insoluble fibronectin within CX is, at least in part, responsible for CXMq conversion.

A novel polyamine analog inhibits growth and induces apoptosis in human breast cancer cells.

Polyamine analogs have demonstrated considerable activity against many important solid tumor models including breast cancer. However, the precise mechanisms of antitumor activities of polyamine analogs are not entirely understood. The cytotoxicity of a newly developed polyamine analog compound, SL11144, against human breast cancer was assessed. Treatment of human breast cancer cell lines in culture with SL11144 decreased cell proliferation and induced programmed cell death in a time- and dose-dependent manner. SL11144 also profoundly inhibited the growth of MDA-MB-231 xenografts in host nude mice without overt toxic effects. Treatment of MDA-MB-435 cells with SL11144 led to the release of cytochrome c from mitochondria into cytosol, activation of caspase-3, and poly(ADP-ribose) polymerase cleavage. SL11144 decreased Bcl-2 and increased Bax protein levels in MDA-MB-231 cells. Furthermore, activator protein 1 transcriptional factor family member c-Jun was up-regulated by SL11144 in MDA-MB-435 and MDA-MB-231 cells, but not in MCF7 cells. In addition, significant inhibition of ornithine decarboxylase activity and a decrease in polyamine pools were demonstrated. These results demonstrate that the novel polyamine analog SL11144 has effective antineoplastic action against human breast cancer cells in vitro and in vivo and that multiple apoptotic mechanisms are associated with its cytotoxic effect in specific human breast cancer cell lines.

Cyclopropane-containing polyamine analogues are efficient growth inhibitors of a human prostate tumor xenograft in nude mice.

Polyamine analogues 7, 10, 18, 27, and 32 containing cyclopropane rings were obtained by chemical synthesis. Their antineoplastic activities were assessed against the cultured human prostate tumor cell lines DU-145, DuPro, and PC-3. Decamines 32 and 27 exhibited variable levels of cytotoxicity against all three cell lines, while 7, 10, and 18 were efficacious against DU-145 and DuPro. Maximum tolerated doses (MTD) for all five compounds in a NCr-nu mouse model were determined at dosing schedules of q1d x 5 (ip) in two cycles with a break of 10 days between cycles. Their antitumor efficacies were then tested against DU-145 tumor xenografts in mice treated with all five agents at their respective MTDs. In addition, the efficacies of 7 and 10 against the same tumor xenograft were assessed at doses below their respective MTDs. In all experiments, administration began two weeks after tumor implantation. All compounds efficiently inhibited tumor growth for up to 50 days postimplantation, with negligible animal body weight loss. Tetramine 10 and hexamine 18 were the most efficient among the five analogues in arresting tumor growth. Tetramine 10 containing two cyclopropane rings had the lowest systemic toxicity as reflected in animal body weight loss. It was further assessed at a weekly administration regimen of (q1w x 4) in two cycles with a four-week break between the cycles. At this dosing schedule, 10 again efficiently arrested tumor growth with negligible effect on animal body weight. Tetramine 10 also arrested the growth of large tumors (ca. 2000 mm(3)) treated 66 days postimplantation. Studies on the metabolism of 10 showed that it accumulates in tumor within 6 h after the end of administration and reached a maximum level 72 h after cessation of dosing. Intracellular concentrations of 10 in liver and kidney were much smaller when compared to those in the tumor when measured 72 h after cessation of dosing. In liver and kidney, the deethyl metabolites of 10 accumulated over a 96 h period after cessation of dosing.

Attenuated strains of Mycobacterium avium subspecies paratuberculosis as vaccine candidates against Johne's disease.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease in ruminants. Johne's disease has a severe economic impact on the dairy industry in the USA and worldwide. In an effort to combat this disease, we screened several transposon mutants that were attenuated in the murine model of paratuberculosis for the potential use as live attenuated vaccines. Using the murine model, two vaccine candidates (pgs1360, pgs3965 with mutations of fabG2_2 and umaA1, respectively) were at or below the limit of detection for tissue colonization suggesting their low level persistence and hence safety. Prior to challenge, both candidates induced a M. paratuberculosis-specific IFN-γ, an indication of eliciting cell-mediated immunity. Following challenge with a virulent strain of M. paratuberculosis, the two vaccine candidates significantly reduced bacterial colonization in organs with reduced histological scores compared to control animals. In addition, one of the vaccine candidates (pgs3965) also induced IL-17a, a cytokine associated with protective immunity in mycobacterial infection. Our analysis suggested that the pgs3965 vaccine candidate is a potential live-attenuated vaccine that could be tested further in ruminant models of paratuberculosis. The analysis also validated our screening strategy to identify effective vaccine candidates against intracellular pathogens.

Human ribonuclease with a pendant poly(ethylene glycol) inhibits tumor growth in mice.

Human pancreatic ribonuclease (RNase 1) is a small secretory protein that catalyzes the cleavage of RNA. This highly cationic enzyme can enter human cells spontaneously but is removed rapidly from circulation by glomerular filtration. Here, this shortcoming is addressed by attaching a poly(ethylene glycol) (PEG) moiety to RNase 1. The pendant has no effect on ribonucleolytic activity but does increase persistence in circulation. The RNase 1-PEG conjugates inhibit the growth of tumors in a xenograft mouse model of human lung cancer. Both retention in circulation and tumor growth inhibition correlate with the size of the pendant PEG. A weekly dose of the 60-kDa conjugate at 1 µmol/kg inhibited nearly all tumor growth without affecting body weight. Its molecular efficacy is ∼5000-fold greater than that of erlotinib, which is a small molecule in clinical use for the treatment of lung cancer. These data demonstrate that the addition of a PEG moiety can enhance the in vivo efficacy of human proteins that act within cells and highlight a simple means of converting an endogenous human enzyme into a cytotoxin with potential clinical utility.

Site-specific PEGylation endows a mammalian ribonuclease with antitumor activity.

Mammalian ribonucleases are emerging as cancer chemotherapeutic agents. Their cationicity engenders cell permeability, and their enzymatic activity destroys the biochemical information encoded by RNA. The pharmacologic potential of ribonucleases is, however, obviated by their high sensitivity to a cytosolic inhibitor protein (RI) and their small size, which limits their residence in serum. We reasoned that site specific conjugation of a poly(ethylene glycol) (PEG) chain could both reduce sensitivity to RI and increase serum half-life. We found that appending a PEG moiety can enable bovine pancreatic ribonuclease (RNase A) to evade RI, depending on the site of conjugation and the length and branching of the chain. Although a pendant PEG moiety decreases antiproliferative activity in vitro, PEGylation discourages renal clearance in vivo and leads to nearly complete tumor growth inhibition in a mouse xenograft model. These data demonstrate that a pendant PEG moiety can be beneficial to the action of proteins that act within the cytosol, and that strategic site-specific PEGylation can endow a mammalian ribonuclease with potent antitumor activity.