Synthesis and biological evaluation of fucoidan-mimetic glycopolymers through cyanoxyl-mediated free-radical polymerization.

The sulfated marine polysaccharide fucoidan has been reported to have health benefits ranging from antivirus and anticancer properties to modulation of high blood pressure. Hence, they could enhance the biological function of materials for biomedical applications. However, the incorporation of fucoidan into biomaterials has been difficult, possibly due to its complex structure and lack of suitable functional groups for covalent anchoring to biomaterials. We have developed an approach for a rapid synthesis of fucoidan-mimetic glycopolymer chains through cyanoxyl-mediated free-radical polymerization, a method suitable for chain-end functionalizing and subsequent linkage to biomaterials. The resulting sulfated and nonsulfated methacrylamido α-L-fucoside glycopolymers' fucoidan-mimetic properties were studied in HSV-1 infection and platelet activation assays. The sulfated glycopolymer showed similar properties to natural fucoidan in inducing platelet activation and inhibiting HSV-1 binding and entry to cells, thus indicating successful syntheses of fucoidan-mimetic glycopolymers.

Enhanced Collagen-like Protein for Facile Biomaterial Fabrication.

We present a collagen-mimetic protein of bacterial origin based upon a modified subdomain of the collagen-like Sc12 protein from Streptococcus pyogenes, as an alternative collagen-like biomaterial platform that is highly soluble, forms stable, homogeneous, fluid-like solutions at elevated concentrations, and that can be efficiently fabricated into hydrogel materials over a broad range of pH conditions. This extended bacterial collagen-like (eBCL) protein is expressed in a bacterial host and purified as a trimeric assembly exhibiting a triple helical secondary structure in its collagen-like subdomain that is stable near physiological solution conditions (neutral pH and 37 °C), as well as over a broad range of pH conditions. We also show how this sequence can be modified to include biofunctional attributes, in particular, the Arg-Gly-Asp (RGD) sequence to elicit integrin-specific cell binding, without loss of structural function. Furthermore, through the use of EDC-NHS chemistry, we demonstrate that members of this eBCL protein system can be covalently cross-linked to fabricate transparent hydrogels with high protein concentrations (at least to 20% w/w). These hydrogels are shown to possess material properties and resistance to enzymatic degradation that are comparable or superior to a type I collagen control. Moreover, such hydrogels containing the constructs with the RGD integrin-binding sequence are shown to promote the adhesion, spreading, and proliferation of C2C12 and 3T3 cells in vitro. Due to its enhanced solubility, structural stability, fluidity at elevated concentrations, ease of modification, and facility of cross-linking, this eBCL collagen-mimetic system has potential for numerous biomedical material applications, where the ease of processing and fabrication and the facility to tailor the sequence for specific biological functionality are desired.

Cholesterol effectively blocks entry of flavivirus.

Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2) are enveloped flaviviruses that enter cells through receptor-mediated endocytosis and low pH-triggered membrane fusion and then replicate in intracellular membrane structures. Lipid rafts, cholesterol-enriched lipid-ordered membrane domains, are platforms for a variety of cellular functions. In this study, we found that disruption of lipid raft formation by cholesterol depletion with methyl-beta-cyclodextrin or cholesterol chelation with filipin III reduces JEV and DEN-2 infection, mainly at the intracellular replication steps and, to a lesser extent, at viral entry. Using a membrane flotation assay, we found that several flaviviral nonstructural proteins are associated with detergent-resistant membrane structures, indicating that the replication complex of JEV and DEN-2 localizes to the membranes that possess the lipid raft property. Interestingly, we also found that addition of cholesterol readily blocks flaviviral infection, a result that contrasts with previous reports of other viruses, such as Sindbis virus, whose infectivity is enhanced by cholesterol. Cholesterol mainly affected the early step of the flavivirus life cycle, because the presence of cholesterol during viral adsorption greatly blocked JEV and DEN-2 infectivity. Flavirial entry, probably at fusion and RNA uncoating steps, was hindered by cholesterol. Our results thus suggest a stringent requirement for membrane components, especially with respect to the amount of cholesterol, in various steps of the flavivirus life cycle.

Direct Lymph Node Vaccination of Lentivector/Prostate-Specific Antigen is Safe and Generates Tissue-Specific Responses in Rhesus Macaques.

Anti-cancer immunotherapy is emerging from a nadir and demonstrating tangible benefits to patients. A variety of approaches are now employed. We are invoking antigen (Ag)-specific responses through direct injections of recombinant lentivectors (LVs) that encode sequences for tumor-associated antigens into multiple lymph nodes to optimize immune presentation/stimulation. Here we first demonstrate the effectiveness and antigen-specificity of this approach in mice challenged with prostate-specific antigen (PSA)-expressing tumor cells. Next we tested the safety and efficacy of this approach in two cohorts of rhesus macaques as a prelude to a clinical trial application. Our vector encodes the cDNA for rhesus macaque PSA and a rhesus macaque cell surface marker to facilitate vector titering and tracking. We utilized two independent injection schemas demarcated by the timing of LV administration. In both cohorts we observed marked tissue-specific responses as measured by clinical evaluations and magnetic resonance imaging of the prostate gland. Tissue-specific responses were sustained for up to six months-the end-point of the study. Control animals immunized against an irrelevant Ag were unaffected. We did not observe vector spread in test or control animals or perturbations of systemic immune parameters. This approach thus offers an "off-the-shelf" anti-cancer vaccine that could be made at large scale and injected into patients-even on an out-patient basis.

Surface-Engineered Contact Lens as an Advanced Theranostic Platform for Modulation and Detection of Viral Infection.

We have demonstrated an entirely new concept of a wearable theranostic device in the form of a contact lens (theranostic lens) with a dual-functional hybrid surface to modulate and detect a pathogenic attack, using a the corneal HSV serotype-1 (HSV-1) model. The theranostic lenses were constructed using a facile layer-by-layer surface engineering technique, keeping the theranostic lenses with good surface wettability, optically transparency, and nontoxic toward human corneal epithelial cells. The theranostic lenses were used to capture and concentrate inflammatory cytokines such as interleukin-1α (IL-1α), which is upregulated during HSV-1 reactivation, for sensitive, noninvasive diagnostics. The theranostic lens also incorporated an antiviral coating to serve as a first line of defense to protect patients against disease. Our strategy tackles major problems in tear diagnostics that are mainly associated with the sampling of a relatively small volume of fluid and the low concentration of biomarkers. The theranostic lenses show effective anti-HSV-1 activity and good analytical performance for the detection of IL-1α, with a limit of detection of 1.43 pg mL(-1) and a wide linear range covering the clinically relevant region. This work offers a new paradigm for "wearable" noninvasive healthcare devices combining "diagnosis" and "protection" against disease, while supporting patient compliance. We believe that this approach holds immense promise as a next-generation point-of-care and decentralized diagnostic/theranostic platform for a range of biomarkers.

Safety and efficacy of composite collagen-silver nanoparticle hydrogels as tissue engineering scaffolds.

The increasing number of multidrug resistant bacteria has revitalized interest in seeking alternative sources for controlling bacterial infection. Silver nanoparticles (AgNPs), are amongst the most promising candidates due to their wide microbial spectrum of action. In this work, we report on the safety and efficacy of the incorporation of collagen coated AgNPs into collagen hydrogels for tissue engineering. The resulting hybrid materials at [AgNPs] < 0.4 µM retained the mechanical properties and biocompatibility for primary human skin fibroblasts and keratinocytes of collagen hydrogels; they also displayed remarkable anti-infective properties against S. aureus, S. epidermidis, E. coli and P. aeruginosa at considerably lower concentrations than silver nitrate. Further, subcutaneous implants of materials containing 0.2 µM AgNPs in mice showed a reduction in the levels of IL-6 and other inflammation markers (CCL24, sTNFR-2, and TIMP1). Finally, an analysis of silver contents in implanted mice showed that silver accumulation primarily occurred within the tissue surrounding the implant.

Effect of Surgical Technique on Corneal Implant Performance.

PURPOSE: Our aim was to determine the effect of a surgical technique on biomaterial implant performance, specifically graft retention. METHODS: Twelve mini pigs were implanted with cell-free, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) cross-linked recombinant human collagen type III (RHCIII) hydrogels as substitutes for donor corneal allografts using overlying sutures with or without human amniotic membrane (HAM) versus interrupted sutures with HAM. The effects of the retention method were compared as well as the effects of collagen concentration (13.7% to 15% RHCIII). RESULTS: All implanted corneas showed initial haze that cleared with time, resulting in corneas with optical clarity matching those of untreated controls. Biochemical analysis showed that by 12 months post operation, the initial RHCIII implants had been completely remodeled, as type I collagen, was the major collagenous protein detected, whereas no RHCIII could be detected. Histological analysis showed all implanted corneas exhibited regeneration of epithelial and stromal layers as well as nerves, along with touch sensitivity and tear production. Most neovascularization was seen in corneas stabilized by interrupted sutures. CONCLUSIONS: This showed that the surgical technique used does have a significant effect on the overall performance of corneal implants, overlying sutures caused less vascularization than interrupted sutures. TRANSLATIONAL RELEVANCE: Understanding the significance of the suturing technique can aid the selection of the most appropriate procedure when implanting artificial corneal substitutes. The same degree of regeneration, despite a higher collagen content indicates that future material development can progress toward stronger, more resistant implants.

Cathelicidin LL-37 and HSV-1 Corneal Infection: Peptide Versus Gene Therapy.

PURPOSE: To evaluate the potential utility of collagen-based corneal implants with anti-Herpes Simplex Virus (HSV)-1 activity achieved through sustained release of LL-37, from incorporated nanoparticles, as compared with cell-based delivery from model human corneal epithelial cells (HCECs) transfected to produce endogenous LL-37. METHODS: We tested the ability of collagen-phosphorylcholine implants to tolerate the adverse microenvironment of herpetic murine corneas. Then, we investigated the efficacy of LL-37 peptides delivered through nanoparticles incorporated within the corneal implants to block HSV-1 viral activity. In addition, LL-37 complementary DNA (cDNA) was transferred into HCECs to confer viral resistance, and their response to HSV-1 infection was examined. RESULTS: Our implants remained in herpetic murine corneas 7 days longer than allografts. LL-37 released from the implants blocked HSV-1 infection of HCECs by interfering with viral binding. However, in pre-infected HCECs, LL-37 delayed but could not prevent viral spreading nor clear viruses from the infected cells. HCECs transfected with the LL-37 expressed and secreted the peptide. Secreted LL-37 inhibited viral binding in vitro but was insufficient to protect cells completely from HSV-1 infection. Nevertheless, secreted LL-37 reduced both the incidence of plaque formation and plaque size. CONCLUSION: LL-37 released from composite nanoparticle-hydrogel corneal implants and HCEC-produced peptide, both showed anti-HSV-1 activity by blocking binding. However, while both slowed down virus spread, neither was able on its own to completely inhibit the viruses. TRANSLATIONAL RELEVANCE: LL-37 releasing hydrogels may have potential utility as corneal substitutes for grafting in HSV-1 infected corneas, possibly in combination with LL-37 producing therapeutic cells.

Human umbilical cord perivascular cells exhibit enhanced cardiomyocyte reprogramming and cardiac function after experimental acute myocardial infarction.

We were interested in evaluating the ability of the mesenchymal stromal cell (MSC) population, human umbilical cord perivascular cells (HUCPVCs), to undergo cardiomyocyte reprogramming in an established coculture system with rat embryonic cardiomyocytes. Results were compared with human bone marrow-derived (BM) MSCs. The transcription factors GATA4 and Mef 2c were expressed in HUCPVCs but not BM-MSCs at baseline and, at 7 days, increased 7.6- and 3.5-fold, respectively, compared with BM-MSCs. Although cardiac-specific gene expression increased in both cell types in coculture, upregulation was more significant in HUCPVCs, consistent with Mef 2c-GATA4 synergism. Using a lentivector with eGFP transcribed from the α-myosin heavy chain (α-MHC) promoter, we found that cardiac gene expression was greater in HUCPVCs than BM-MSCs after 14 days coculture (52±17% vs. 29±6%, respectively). A higher frequency of HUCPVCs expressed α-MHC protein compared with BM-MSCs (11.6±0.9% vs. 5.3±0.3%); however, both cell types retained MSC-associated determinants. We also assessed the ability of the MSC types to mediate cardiac regeneration in a NOD/SCID γ mouse model of acute myocardial infarction (AMI). Fourteen days after AMI, cardiac function was significantly better in cell-treated mice compared with control animals and HUCPVCs exhibited greater improvement. Although human cells persisted in the infarct area, the frequency of α-MHC expression was low. Our results indicate that HUCPVCs exhibit a greater degree of cardiomyocyte reprogramming but that differentiation for both cell types is partial. We conclude that HUCPVCs may be preferable to BM-MSCs in the cell therapy of AMI.

Neonatal transfer of membrane-bound stem cell factor improves survival and heart function in aged mice after myocardial ischemia.

Stem cell mobilization to injured tissue contributes to neovascularization, resulting in regeneration after myocardial infarction (MI). We previously showed that direct cardiac injection of a recombinant lentivirus (LV) that engineers expression of membrane-bound stem cell factor (mSCF) improves outcomes immediately after MI. In this study, we evaluated the effect of neonatal LV/mSCF transduction on MI outcomes in aged mice. We constructed a recombinant LV harboring an α-myosin heavy chain promoter that drives mSCF expression and injected it into the temporal vein of neonatal mice. One year later, sustained expression of mSCF in the adult mouse hearts was detected by genomic and quantitative RT-PCR and immunohistochemistry. To evaluate the contribution of neonatal LV/mSCF delivery to recovery from MI, we induced an MI in adult LV/mSCF-transduced, LV only-transduced, and nontransduced control mice. Strikingly, LV/mSCF transduction reduced infarct scar size, enhanced angiogenesis, improved ventricular function, and significantly increased survival of the mice. Regional overexpression of CD11b, a marker of monocytes and proangiogenic cells, was observed on monocytes isolated from the infarcted hearts of LV/mSCF-transduced mice. Our data suggest a model of neonatal gene delivery that leads to sustained mSCF expression during adulthood to aid recovery from MI and prevent heart failure.

Promoter-specific lentivectors for long-term, cardiac-directed therapy of Fabry disease.

In Fabry disease a deficiency of α-galactosidase A (α-gal A) activity leads to accumulation of globotriaosylceramide (Gb3) in various tissues including the heart. A specific cardiac variant of Fabry disease has also been described. Previously we have demonstrated the feasibility of gene therapy for Fabry disease. Here, to provide efficient transfer and increased specificity of transgene expression, we synthesized lentiviral vectors (LVs) with myocardial-specific promoters including: α-myosin heavy chain (α-MHC), myosin light chain (MLC2v), and cardiac troponin T (cTnT). Initially, neonatal Balb/c mice were injected with such LV constructs engineering expression of luciferase. One month post-injection, we found specific expression of luciferase in hearts of recipient animals when compared with transgene expression driven by the standard EF1-α promoter. To examine the feasibility of long-term therapy specifically targeting the heart, recombinant LV/α-gal A therapeutic vectors with analogous cardiac promoters were generated and injected into numerous neonatal Fabry mice. No immune response against the corrective α-gal A hydrolase was observed in the treated mice. Serum α-gal A activity of 10-week-old Fabry mice was increased in LV/α-gal A-injected animals compared to controls. In 28-week-old Fabry mice we observed significantly decreased Gb3 accumulation. Neonatal injections with LVs harboring cardiac-specific promoters may thus be an effective long-term treatment strategy for heart manifestations and cardiac variant Fabry disease. These results can be also extended to other progressive pathologies of the heart.

Autologous transplantation of lentivector/acid ceramidase-transduced hematopoietic cells in nonhuman primates.

Farber disease is a rare lysosomal storage disorder (LSD) that manifests due to acid ceramidase (AC) deficiencies and ceramide accumulation. We present a preclinical gene therapy study for Farber disease employing a lentiviral vector (LV-huAC/huCD25) in three enzymatically normal nonhuman primates. Autologous, mobilized peripheral blood (PB) cells were transduced and infused into fully myelo-ablated recipients with tracking for at least 1 year. Outcomes were assessed by measuring the AC specific activity, ceramide levels, vector persistence/integration, and safety parameters. We observed no hematological, biochemical, radiological, or pathological abnormalities. Hematological recovery occurred by approximately 3 weeks. Vector persistence was observed in PB and bone marrow (BM) cells by qualitative and quantitative PCR. We did not observe any clonal proliferation of PB and BM cells. Importantly, AC-specific activity was detected above normal levels in PB and BM cells analyzed post-transplantation and in spleens and livers at the endpoint of the study. Decreases of ceramide in PB cells as well as in spleen and liver tissues were seen. We expect that this study will provide a roadmap for implementation of clinical gene therapy protocols targeting hematopoietic cells for Farber disease and other LSDs.

Comparative impact of trastuzumab and cyclophosphamide on HER-2-positive human breast cancer xenografts.

PURPOSE: Metronomic chemotherapy is a minimally toxic and frequently effective new treatment strategy that is beginning to show promising phase II clinical trial results, particularly for metastatic breast cancer when combined with various molecularly targeted antitumor agents. Here, we assessed a treatment strategy that uses trastuzumab plus daily oral metronomic cyclophosphamide on metastatic Her-2-positive human breast cancer models. EXPERIMENTAL DESIGN: Treatments were initiated on orthotopic transplanted primary tumors as well as established visceral metastatic disease of two independent Her-2-positive breast cancer models, both independently derived from the human MDA-MB-231 breast cancer cell line. Outcome was assessed by noninvasive measurements of tumor cell-secreted human choriogonadotropin in the urine as a surrogate marker of relative tumor burden, or by whole body bioluminescent imaging, in addition to prolongation of survival. RESULTS: Orthotopic primary tumors responded to trastuzumab monotherapy with significant growth delays, whereas minimal antitumor effect was observed when mice with metastatic disease were treated. Nevertheless, trastuzumab showed a benefit in this latter setting when combined with metronomic low-dose cyclophosphamide as assessed by prolongation of survival. This benefit was similar to trastuzumab plus maximum tolerated dose cyclophosphamide, but was associated with lesser toxicity. CONCLUSIONS: Trastuzumab combined with metronomic cyclophosphamide may be an effective long-term maintenance strategy for the treatment of Her-2-positive metastatic breast cancer.

Flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection.

Flaviviruses such as dengue virus (DEN) and Japanese encephalitis virus (JEV) are medically important in humans. The lipid kinase, phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt have been implicated in the regulation of diverse cellular functions such as proliferation, and apoptosis. Since JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection, we evaluated the possible roles of the PI3K/Akt signaling pathway in flavivirus-infected cells. We found that Akt phosphorylation was noticeable in the JEV- and DEN serotype 2 (DEN-2)-infected neuronal N18 cells in an early, transient, PI3K- and lipid raft-dependent manner. Blocking of PI3K activation by its specific inhibitor LY294002 or wortmannin greatly enhanced virus-induced cytopathic effects (CPEs), even at an early stage of infection, but had no effect on virus production. This severe CPE was characterized as apoptotic cell death as evidenced by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Mechanically, the initiator and effector caspases involved are mainly caspase-9 and caspase-6, since only a pan-caspase inhibitor and the inhibitors preferentially target caspase-9 and -6, but not the ones antagonizing caspase-8, -3, or -7 alleviated the levels of PARP cleavage after virus infection and PI3K blockage. Furthermore, Bcl-2 appears to be a crucial mediator downstream of PI3K/Akt signaling, since overexpression of Bcl-2 reduced virus-induced apoptosis even when PI3K activation was repressed. Collectively, our results suggest an anti-apoptotic role for the PI3K/Akt pathway triggered by JEV and DEN-2 to protect infected cells from early apoptotic cell death.

Antiviral effects of an iminosugar derivative on flavivirus infections.

Endoplasmic reticulum (ER) alpha-glucosidase inhibitors, which block the trimming step of N-linked glycosylation, have been shown to eliminate the production of several ER-budding viruses. Here we investigated the effects of one such inhibitor, N-nonyl-deoxynojirimycin (NN-DNJ), a 9-carbon alkyl iminosugar derivative, on infection by Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2). In the presence of NN-DNJ, JEV and DEN-2 infections were suppressed in a dose-dependent manner. This inhibitory effect appeared to influence DEN-2 infection more than JEV infection, since lower concentrations of NN-DNJ substantially blocked DEN-2 replication. Secretion of the flaviviral glycoproteins E and NS1 was greatly reduced, and levels of DEN-2 viral RNA replication measured by fluorogenic reverse transcription-PCR were also decreased, by NN-DNJ. Notably, the viral glycoproteins, prM, E, and NS1 were found to associate transiently with the ER chaperone calnexin, and this interaction was affected by NN-DNJ, suggesting a potential role of calnexin in the folding of flaviviral glycoproteins. Additionally, in a mouse model of lethal challenge by JEV infection, oral delivery of NN-DNJ reduced the mortality rate. These findings show that NN-DNJ has an antiviral effect on flavivirus infection, likely through interference with virus replication at the posttranslational modification level, occurring mainly in the ER.