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Biomarkers are emerging as potentially important diagnostic tools for cancer and many other diseases. However, many current detection systems for suffer from insufficient sensitivity. To address this concern, we developed a highly sensitive biosensor, featuring monoclonal antibody-coated polystyrene nanobeads assembled in the trenches of a microchip, for the detection of cancer biomarkers. These biosensors detected nucleosomes and carcinoembryonic antigen in serum at concentrations of 62.5 and 15.6 pg/mL, respectively. Very low detection limits that suggest such devices might be beneficial for the early detection of tumors and for monitoring of patients in remission.

Dendritic cells serve as the main immune cells that trigger the immune response. We developed a simple and cost-effective nanovaccine platform based on the α1',2-mannobiose derivative for dendritic cell targeting. In previous work, we have formulated the α1,2-mannobiose-based nanovaccine platform with plasmid DNA and tested it in cattle against BoHV-1 infection. There, we have shown that the dendritic cell targeting using this nanovaccine platform in vivo can boost the immunogenicity, resulting in a long-lasting immunity. In this work, we aim to characterize the α1',2-mannobiose derivative, which is key in the nanovaccine platform. This DC-targeting strategy takes advantage of the specific receptor known as DC-SIGN and exploits its capacity to bind α1,2-mannobiose that is present at terminal ends of oligosaccharides in certain viruses, bacteria, and other pathogens. The oxidative conjugation of α1',2-mannobiose to NH2-PEG2kDa-DSPE allowed us to preserve the chemical structure of the non-reducing mannose of the disaccharide and the OH groups and the stereochemistry of all carbons of the reducing mannose involved in the binding to DC-SIGN. Here, we show specific targeting to DC-SIGN of decorated micelles incubated with the Raji/DC-SIGN cell line and uptake of targeted liposomes that took place in human, bovine, mouse, and teleost fish DCs in vitro, by flow cytometry. Specific targeting was found in all cultures, demonstrating a species-non-specific avidity for this ligand, which opens up the possibility of using this nanoplatform to develop new vaccines for various species, including humans.

To develop transferrin (Tf)-targeted delivery systems for the pro-apoptotic drug, NCL-240, and to evaluate the efficacy of this delivery system in ovarian cancer NCI/ADR-RES cells, grown in vitro in a 3D spheroid model.Tf-targeted PEG-PE-based micellar and ePC/CHOL-based liposomal delivery systems for NCL-240 were prepared. NCI/ADR-RES cells were used to generate spheroids by a non-adhesive liquid overlay technique. Spheroid growth and development were monitored by size (diameter) analysis and HE staining. The targeted formulations were compared to untargeted ones in terms of their degree of spheroid association and penetration. A cell viability analysis with NCL-240-loaded micelles and liposomes was performed to assess the effectiveness of Tf-targeting.Tf-targeted polymeric micelles and Tf-targeted liposomes loaded with NCL-240 were prepared. NCI/ADR-RES cells generated spheroids that demonstrated the presence of a distinct necrotic core along with proliferating cells in the spheroid periphery, partly mimicking in vivo tumors. The Tf-targeted micelles and liposomes had a deeper spheroid penetration as compared to the untargeted delivery systems. Cell viability studies using the spheroid model demonstrated that Tf-mediated targeting markedly improved the cytotoxicity profile of NCL-240.Transferrin targeting enhanced delivery and effectiveness of micelles and liposomes loaded with NCL-240 against NCI/ADR-RES cancer cells in a 3D spheroid model.

Liposome-based pharmaceuticals used within the cardiovascular system are reviewed in this article. The delivery of diagnostic and therapeutic agents by plain liposomes and liposomes with surface-attached targeting antibodies or polyethylene glycol to prolong their circulation time and accumulation at vascular injuries, ischemic zones or sites of thrombi are also discussed. An overview of the advantages and disadvantages of liposome-mediated in vitro, ex vivo and in vivo targeting is presented, including discussion of the targeting of liposomes to pathological sites on the blood vessel wall and a description of liposomes that can be internalized by endothelial cells. Diagnostic liposomes used to target myocardial infarction and the relative importance of liposome size, targetability of immunoliposomes and prolonged circulation time on the efficiency of sealing hypoxia-induced plasma membrane damage to cardiocytes are discussed as a promising approach for therapy. The progress in the use of targeted liposomal plasmids for the transfection of hypoxic cardiomyocytes and myocardium is presented. Stent-mediated liposomal-based drug delivery is also reviewed briefly.

A major obstacle to drug therapy for treatment of potential myocardial infarction is the limited access to the ischemic myocardium by drugs in an active form. Encouraging results have been reported with liposomes loaded with ATP in a variety of in vitro and in vivo models. We describe methods for optimized encapsulation of ATP in liposomes, enhancement of their effectiveness by increasing circulation time, and targeting of injured myocardial cells with surface attached antimyosin. In isolated ischemic rat hearts, ATP-loaded liposomes and ATP-loaded immunoliposomes effectively protected myocardium from ischemia/reperfusion damage as measured by systolic and diastolic functional improvements. In vivo, in rabbits with induced localized myocardial ischemia, liposomal encapsulation of ATP significantly diminished the proportion of ventricular muscle at risk that was irreversibly damaged during reperfusion. Therefore, ATP encapsulated in liposomes can provide an effective exogenous source for in vivo application which can protect ischemically damaged hearts. Copyright © 2009 John Wiley & Sons, Inc. For further resources related to this article, please visit the WIREs website.

Near-infrared (NIR) whole-body imaging is a powerful noninvasive method for the visualization of complex biological phenomena such as tumor and tumor vasculature and for whole-body studies. In this study we introduced a new NIR contrast agent in poly(ethylene glycol)-phospholipid micelle-encapsulated quantum dots (QD-Ms). QD-Ms maximally accumulated in the tumor area within 1 hour as compared to 4 hours for the commercially available PEGylated quantum dots (QD-PEGs) and allowed for the visualization of both tumor and internal organs. QD-Ms showed a signal-to-noise ratio of 15 that allowed the quantification of the micelles' biodistribution using image analysis. The signal obtained with the QD-Ms was higher than with the commercial formulation at half the QD dose. Overall, the QD-Ms seem to be a powerful and rapidly acting nanosized imaging agent that allows for effective visualization of tumors using NIR imaging.

With few exceptions, where local administration is feasible, progress towards broad clinical application of gene therapies requires the development of effective delivery systems. Here we report a novel non-viral gene delivery vector, ‘micelle-like nanoparticle’ (MNP) suitable for systemic application. MNP were engineered by condensing plasmid DNA with a chemical conjugate of phospholipid with polyethylenimine (PLPEI) and then coating the complexes with an envelope of lipid monolayer additionally containing polyethylene glycol–phosphatidyl ethanolamine (PEG–PE), resulting in spherical ‘hard-core’ nanoparticles loaded with DNA. MNP allowed for complete protection of the loaded DNA from enzymatic degradation, resistance to salt-induced aggregation, and reduced cytotoxicity. MNP also demonstrated prolonged blood circulation and low RES accumulation. Intravenous injection of MNP loaded with plasmid DNA encoding for the Green Fluorescence Protein (GFP) resulted in an effective transfection of a distal tumor. Thus, MNP provide a promising tool for systemic gene therapy.

We assessed whether the infusion of Coenzyme Q10-loaded liposomes (CoQ10-L) in rabbits with an experimental myocardial infarction can result in increased intracellular delivery of CoQ10 and thus limit the fraction of the irreversibly damaged myocardium.CoQ10-L, empty liposomes (EL), or Krebs-Henseleit (KH) buffer were administered by intracoronary infusion, followed by 30 min of occlusion and 3 h of reperfusion. Unisperse Blue dye was used to demarcate the net size of the occlusion-induced ischemic zone ("area at risk") while nitroblue tetrazolium staining was used to detect the final fraction of the irreversibly damaged myocardium within the total area at risk.The total size of the area at risk in all experimental animals was approx. 20% wt. of the left ventricle (LV). The final irreversible damage in CoQ10-L-treated animals was only ca. 30% of the total area at risk as compared with ca. 60% in the group treated with EL (p0.006) and ca. 70% in the KH buffer-treated group (p0.001).CoQ10-L effectively protected the ischemic heart muscle by enhancing the intracellular delivery of CoQ10 in hypoxic cardiocytes in rabbits with an experimental myocardial infarction as evidenced by a significantly decreased fraction of the irreversibly damaged heart within the total area at risk. CoQ10-L may provide an effective exogenous source of the CoQ10 in vivo to protect ischemic cells.

Preservation of cell viability using Cytoskeletal-antigen Specific ImmunoLiposomes (antimyosin-CSIL) has been demonstrated in cell cultures. The current study utilized the same CSIL intervention for myocardial protection in an in vivo rabbit model of acute myocardial infarction. Rabbit hearts with experimental left ventricular myocardial infarction were treated with CSIL; control liposomes, [(CL), IgG-liposomes (IgG-L) or plain liposomes (PL)], or vehicle (placebo). Mean myocardial infarct size in rabbit hearts treated in vivo with CSIL was 5 times smaller than in those treated with non-specific CL or vehicle. Treatment of ischemic adult myocardium with CSIL results in significant preservation of myocardial viability by dramatically decreasing the infarct size relative to CL or placebo treatment. Immunohistochemical myocardial preservation of CSIL-treated hearts was confirmed by the lack of contraction band necrosis using histological H&E stains relative to controls. Electrocardiographic confirmation of reduction in myocardial injury after CSIL therapy relative to controls was also observed. Application of CSIL technology to non-cardiac tissues would confirm a broader applicability of this cell membrane lesion sealing technology.

We assessed whether the infusion of ATP-loaded liposomes (ATP-L) can limit the fraction of the irreversibly damaged myocardium in rabbits with an experimental myocardial infarction. ATP-L, empty liposomes (EL), or Krebs–Henseleit (KH) buffer were administered by intracoronary infusion, followed by 30 min of occlusion and 3 h of reperfusion. Unisperse Blue dye was used to demarcate the net size of the occlusion-induced ischemic zone (area at risk) and nitroblue tetrazolium staining was used to detect the final fraction of the irreversibly damaged myocardium within the total area at risk. The total size of the area at risk in all experimental animals was approx. 20% wt. of the left ventricle. The final irreversible damage in ATP-L-treated animals was only ca. 30% of the total area at risk as compared with ca. 60% in the group treated with EL (p < 0.009) and ca. 70% in the KH buffer-treated group (p < 0.003). ATP-L effectively protected the ischemic heart muscle in rabbits with an experimental myocardial infarction as evidenced by a significantly decreased fraction of the irreversibly damaged heart within the total area at risk. ATP-L may provide an effective exogenous source of the ATP in vivo to protect ischemically damaged cells.

Dextran-polyethylene glycol (PEG) conjugates were synthesized by activating dextran hydroxy groups with carbonyldiimidazole, introducing amino groups by attaching ethylenediamine, and reacting amino groups with a succinimidyl-activated derivative of PEG. Conjugates with an average of 12 and 21 PEG (5 kDa) residues per single dextran (73 kDa) molecule were prepared. These conjugates have circulation half-lives of 5.3 h and 7.0 h, respectively, compared to 4.0 h for non-PEGylated dextran. The modification of dextran with PEG inhibits the uptake of polymer by the major organ of the reticuloendothelial system, the liver. Dextran-PEG conjugates may represent a convenient platform for long-circulating pharmaceutical preparations.

Purpose To develop transferrin (Tf)-targeted delivery systems for the pro-apoptotic drug, NCL-240, and to evaluate the efficacy of this delivery system in ovarian cancer NCI/ADR-RES cells, grown in vitro in a 3D spheroid model.

Dendritic cells (DC) are antigen presenting cells (APC) uniquely capable of priming naïve T cells and cross-presenting antigens and determine the type of immune response elicited against an antigen. TAT peptide (TATp), is an amphipathic, arginine-rich, cationic peptide that promotes penetration and translocation of various molecules and nanoparticles into cells. TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane. Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC. DNA-loaded TATp-L increased the in vitro transfection efficiency in DC cultures as evidenced by an enhanced expression of the enhanced green fluorescent protein (EGFP) and bovine herpes virus type 1 (BoHV-1) glycoprotein D (gD). The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L as indicated by the secretion of interleukin 6 (IL-6). Fil: Pappalardo, Juan Sebastian. Northeastern University. Center for Pharmaceutical Biotechnology and Nanomedicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina Fil: Langellotti, Cecilia Ana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Di Giacomo, Sebastián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina Fil: Olivera, Valeria. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina Fil: Quattrocchi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Zamorano, Patricia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina Fil: Hartner, William C.. Northeastern University. Center for Pharmaceutical Biotechnology and Nanomedicine; Estados Unidos Fil: Levchenko, Tatyana S.. Northeastern University. Center for Pharmaceutical Biotechnology and Nanomedicine; Estados Unidos Fil: Torchilin, Vladimir P.. Northeastern University. Center for Pharmaceutical Biotechnology and Nanomedicine; Estados Unidos

A novel strategy to improve the therapeutic index of chemotherapy has been developed by the integration of nanotechnology with phage technique. The objective of this study was to combine phage display, identifying tumor-targeting ligands, with a liposomal nanocarrier for targeted delivery of doxorubicin. Following the proof of concept in cell-based experiments, this study focused on in vivo assessment of antitumor activity and potential side-effects of phage fusion protein-modified liposomal doxorubicin. MCF-7-targeted phage-Doxil treatments led to greater tumor remission and faster onset of antitumor activity than the treatments with non-targeted formulations. The enhanced anticancer effect induced by the targeted phage-Doxil correlated with an improved tumor accumulation of doxorubicin. Tumor sections consistently revealed enhanced apoptosis, reduced proliferation activity and extensive necrosis. Phage-Doxil-treated mice did not show any sign of hepatotoxicity and maintained overall health. Therefore, MCF-7-targeted phage-Doxil seems to be an active and tolerable chemotherapy for breast cancer treatment. From the Clinical Editor The authors of this study successfully combined phage display with a liposomal nanocarrier for targeted delivery of doxorubicin using MCF-7-targeted phage-Doxil nanocarriers in a rodent model. The method demonstrated improved efficiency and reduced hepatotoxicity, paving the way to future clinical trials addressing breast cancer.

Rapamycin (Rapa) monotherapy can promote renal allograft survival in dogs, but it is very toxic. To attempt to augment the effectiveness of Rapa and reduce its toxicity in a tolerance induction protocol, canine renal allograft recipients were treated briefly with antilymphocyte serum (ALS), donor bone marrow cells (BMC), and a limited course of cyclosporine (CsA). Rapa had little effect when CsA-treated recipients were given ALS on days -5 to -1 and BMC on day +1. When combined with CsA given days +13 to +42, ALS on days -5 to +7, and BMC on day +10, Rapa at 0.3 mg/kg on day +8 plus alternate days +15 to +39 significantly increased overall survival and was compatible with long-term survival after immunosuppression (6 grafts, 1 graft > 212 days, 1 graft > 470 days). Rapa appeared to prevent early rejections that can occur during treatment with these ALS/BMC/CsA protocols. Little toxicity of Rapa was observed with any treatment.

Introduction The common goal in using liposomes for diagnostics and therapy is to deliver a pharmaceutic to the injured area. Liposomes are spherical, self-closed structures formed by one or several concentric lipid bilayers with an aqueous phase inside and between the lipid bilayers. Their ability to entrap different water-soluble compounds within the inner aqueous phase and lipophilic agents between liposomal bilayers has made them useful for delivery of different kinds of drugs and for carrying diagnostic agents in all imaging modalities — gamma-scintigraphy, magnetic resonance imaging (MRI), computed tomography (CT) imaging, and sonography. Liposomal modification with polyethylene glycol (PEG) increases their field of usage by enhancing circulation time and attachment of antibodies or different targeting moieties to their surface to target specific affected areas. Such modified liposomes and immunoliposomes can be considered for intravascular drug delivery, using cells and noncellular components (such as endothelial cells, subendothelial structures, and blood components) as the targeted sites for diagnosing and treating the most important cardiac pathologies, including myocardial infarction, coronary thrombosis, and atherosclerosis (Figure 1).

Background Optical imaging is a promising method for the detection of tumors in animals, with speed and minimal invasiveness. We have previously developed a lipid coated quantum dot system that doubles the fluorescence of PEG-grafted quantum dots at half the dose. Here, we describe a tumor-targeted near infrared imaging agent composed of cancer-specific monoclonal anti-nucleosome antibody 2C5, coupled to quantum dot (QD)-containing polymeric micelles, prepared from a polyethylene glycol/phosphatidylethanolamine (PEG-PE) conjugate. Its production is simple and involves no special equipment. Its imaging potential is great since the fluorescence intensity in the tumor is twofold that of non-targeted QD-loaded PEG-PE micelles at one hour after injection. Methods Para-nitrophenol-containing (5%) PEG-PE quantum dot micelles were produced by the thin layer method. Following hydration, 2C5 antibody was attached to the PEG-PE micelles and the QD-micelles were purified using dialysis. 4T1 breast tumors were inoculated subcutaneously in the flank of the animals. A lung pseudometastatic B16F10 melanoma model was developed using tail vein injection. The contrast agents were injected via the tail vein and mice were depilated, anesthetized and imaged on a Kodak Image Station. Images were taken at one, two, and four hours and analyzed using a methodology that produces normalized signal-to-noise data. This allowed for the comparison between different subjects and time points. For the pseudometastatic model, lungs were removed and imaged ex vivo at one and twenty four hours. Results The contrast agent signal intensity at the tumor was double that of the passively targeted QD-micelles with equally fast and sharply contrasted images. With the side views of the animals only tumor is visible, while in the dorsal view internal organs including liver and kidney are visible. Ex vivo results demonstrated that the agent detects melanoma nodes in a lung pseudometastatic model after a 24 hours wash-out period, while at one hour, only a uniform signal is detected. Conclusions The targeted agent produces ultrabright tumor images and double the fluorescence intensity, as rapidly and at the same low dose as the passively targeted agents. It represents a development that may potentially serve to enhance early detection for metastases.

ATP cannot be effectively delivered to most tissues including the ischemic myocardium without protection from degradation by plasma endonucleotidases. However, it has been established that ATP can be delivered to various tissues by its encapsulation within liposomal preparations. We describe here, the materials needed and methods used to optimize the encapsulation of ATP in liposomes, enhance their effectiveness by increasing their circulation time and target injured myocardial cells with liposomal surface anti-myosin antibody. Additionally, we outline methods for ex vivo studies of these ATP liposomal preparations in an isolated ischemic rat heart model and for in vivo studies of rabbits with an induced myocardial infarction. The expectation is that these methods will provide a basis for continued studies of effective ways to deliver energy substrates to the ischemic myocardium.

A major obstacle to drug therapy for treatment of potential myocardial infarction is the limited access to the ischemic myocardium by drugs in an active form. Encouraging results have been reported with liposomes loaded with ATP in a variety of in vitro and in vivo models. We describe methods for optimized encapsulation of ATP in liposomes, enhancement of their effectiveness by increasing circulation time, and targeting of injured myocardial cells with surface attached antimyosin. In isolated ischemic rat hearts, ATP-loaded liposomes and ATP-loaded immunoliposomes effectively protected myocardium from ischemia/reperfusion damage as measured by systolic and diastolic functional improvements. In vivo, in rabbits with induced localized myocardial ischemia, liposomal encapsulation of ATP significantly diminished the proportion of ventricular muscle at risk that was irreversibly damaged during reperfusion. Therefore, ATP encapsulated in liposomes can provide an effective exogenous source for in vivo application which can protect ischemically damaged hearts.

ATP cannot be effectively delivered to most tissues including the ischemic myocardium without protection from degradation by plasma endonucleotidases. However, it has been established that ATP can be delivered to various tissues by its encapsulation within liposomal preparations. We describe here, the materials needed and methods used to optimize the encapsulation of ATP in liposomes, enhance their effectiveness by increasing their circulation time and target injured myocardial cells with liposomal surface anti-myosin antibody. Additionally, we outline methods for ex vivo studies of these ATP liposomal preparations in an isolated ischemic rat heart model and for in vivo studies of rabbits with an induced myocardial infarction. The expectation is that these methods will provide a basis for continued studies of effective ways to deliver energy substrates to the ischemic myocardium.

A novel highly biocompatible near infrared nanosized contrast agent was developed and used for rapid tumor detection and quantification using planar optical imaging and analysis. With this in mind, the near infrared fluorescent dye Alexa 750 was covalently attached to polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate, and double labeled (with Alexa and rhodamine) PEG-PE micelles were injected into mice and observed using planar optical imaging. Pixel intensity data from the tumor site were normalized versus the autofluorescence of the animal at the same time point and normalized as signal to noise over the scattered light from the various tissues of the mice. The detected signal from the tumor was higher than the background noise allowing for rapid detection of the tumor. The tumor was clearly visible within one hour. Some signal was also detected from the abdomen of the mice. As determined by microscopy analysis, other organs of accumulation were the liver and kidney, which corresponded well to the data from the whole body imaging animal studies.

Aristarchos Papagiannaros, Amit Kale, Tatyana S Levchenko, Dmitry Mongayt, William C Hartner, Vladimir P TorchilinDepartment of Pharmaceutical Sciences and Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA, USAAbstract: A novel highly biocompatible near infrared nanosized contrast agent was developed and used for rapid tumor detection and quantification using planar optical imaging and analysis. With this in mind, the near infrared fluorescent dye Alexa 750 was covalently attached to polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate, and double labeled (with Alexa and rhodamine) PEG-PE micelles were injected into mice and observed using planar optical imaging. Pixel intensity data from the tumor site were normalized versus the autofluorescence of the animal at the same time point and normalized as signal to noise over the scattered light from the various tissues of the mice. The detected signal from the tumor was higher than the background noise allowing for rapid detection of the tumor. The tumor was clearly visible within one hour. Some signal was also detected from the abdomen of the mice. As determined by microscopy analysis, other organs of accumulation were the liver and kidney, which corresponded well to the data from the whole body imaging animal studies.Keywords: nanotechnology, nanomedicine, cancer imaging agents, near infrared imaging, micelles

The effect of combined treatment with antilymphocyte serum, fractionated donor bone marrow, and a limited course of cyclosporine on renal allograft survival in mongrel dogs was examined. Recipients were treated with ALS from day -5 to day +7, relative to transplantation on day 0 with an MLR-mismatched donor. Fractionated donor bone marrow cells (BMFr3) obtained by density gradient separation were infused 3-7 days after ALS treatment. CsA treatments were begun either 3-7 days after ALS plus BMFr3 infusion or 3-7 days after treatments with ALS alone. Extended allograft survival was achieved at all CsA doses in BMFr3-infused, ALS-treated recipients. Allografts were sustained throughout the CsA treatment period without rejection in the majority of recipients (6 of 8) receiving ALS plus BMFr3 and CsA at 20 Mg/kg/day for 60 days. By contrast, few grafts were sustained through 30 days of treatment with CsA at 3.2 (1 of 12) or 10 mg/kg/day (2 of 9) in ALS plus BMFr3-treated recipients. Cyclosporine treatment was ineffective at all doses in augmenting allograft prolongation in ALS-treated recipients that did not receive BMFr3. Nearly all (6 of 7) long-term survivors (greater than 180 days) were BMFr3-treated. Peripheral blood lymphocytes or bone marrow cells of these long-term survivors proliferated normally in response to Con A and PWM, and were MLR responsive to third-party cells but did not have reduced MLR responsiveness to donor alloantigen in all cases. These long-term survivors promptly rejected third-party renal allografts without adverse effects on the original transplant's function. These results show that long-term renal allograft survival with specific unresponsiveness to the donor can result from the combined treatment with ALS plus donor BMFr3 and a limited course of CsA.

The treatment of myocardial ischemia using gene therapy is a rather novel but promising approach. Gene delivery to target cells may be enhanced by using double-targeted delivery systems simultaneously capable of extracellular accumulation and intracellular penetration. With this in mind, we have used low cationic liposomes-plasmid DNA complexes (lipoplexes) modified with cell-penetrating transactivating transcriptional activator (TAT) peptide (TATp) and/or with monoclonal anti-myosin monoclonal antibody 2G4 (mAb 2G4) specific toward cardiac myosin, for targeted gene delivery to ischemic myocardium. In vitro transfection of both normoxic and hypoxic cardiomyocytes was enhanced by the presence of TATp as determined by fluorescence microscopy and ELISA. The in vitro transfection was further enhanced by the additional modification with mAb 2G4 antibody in the case of hypoxic, but not normoxic cardiomyocytes. However, we did not observe a synergism between TATp and mAb 2G4 ligands under our experimental condition. In in vivo experiments, we have clearly demonstrated an increased accumulation of mAb 2G4-modified TATp lipoplexes in the ischemic rat myocardium and significantly enhanced transfection of cardiomyocytes in the ischemic zone. Thus, the genetic transformation of normoxic and hypoxic cardiomyocytes can be enhanced by using lipoplexes modified with TATp and/or mAb 2G4. Such complexes also demonstrate an increased accumulation in the ischemic myocardium and effective transfection of hypoxic cardiomyocytes in vivo.

Micelles prepared from polyethyleneglycol/phosphatidyl-ethanolamine conjugates (PEG-PE) with a size of 7-20 nm and zeta-potential of approximately -18 mV were administered i.v. to rabbits with experimental myocardial infarctions. Micelles demonstrated a prolonged circulation in the blood (half-life of 2 h) and accumulated in the infarction zone with efficiency more than 8-fold higher as compared to a non-damaged part of the heart muscle. Obtained results suggest that the enhanced permeability and retention (EPR) effect is the primary mechanism of accumulation of microparticles in the infarct areas, and that drug carriers such as PEG-PE micelles can be used for the delivery of therapeutic or diagnostic agents to an area of myocardial infarction.

Temporary donor-specific unresponsiveness induced by treatment of skin allografted mice with antilymphocyte serum (ALS) and donor bone marrow cells (BMC) can be converted to long-term graft survival and tolerance by adding adjunctive immunosuppressive agents, including rapamycin (Rapa). Rapa was given in a suboptimal dosing schedule to probe several aspects of its use in the promotion of tolerization. Single doses were given at 2 weeks post-transplantation to recipients prepared with ALS and donor BMC. Graft survival was markedly prolonged in a dose-dependent fashion by day +14, Rapa, doses ranging from 0.75 to 6.0 mg/kg. Indefinite (> 300 day) graft survival was observed in 26% of recipeints given the highest Rapa done. Short-term treatment with cyclosporin A (CsA) or deoxyspergualin (DSG) was ineffective when injected at this time. Rapa augmented the tolerizing effect of grafting with skin from newborn mice but had no significant additive or synergistic effects with the short course of CsA or with DSG given on days +1 to +3, even though the latter prolonged graft survival when added to the ALS/BMC protocol. Single doses of Rapa on day +1 also prolonged graft survival, but without any of the grafts surviving indefinitely. Later dosing on day +28 resulted in > 70% of grafts surviving > 300 days. Challenge grafting of these mice after day +300 resulted in delayed rejection of donor strain, but not third-party skin grafts. Rapa was very effective when given as widely spaced doses on days +14 and +49. Also, grafts showing the earliest signs of rejection could be rescued with a single Rapa dose in recipients treated with ALS and BMC but not ALS alone. Transfer of prolonged graft survival with spleen cells from ALS plus BMC treated recipients was not adversely affected by Rapa given to the suppressor-like spleen cell donors approximately 1 week before cell harvest. We conclude that the use of Rapa as an adjunctive agent in allograft recipients treated with ALS plus donor BMC is very flexible in terms of timing of administration, and that the drug can be effectively given as widely spaced doses or as a rescue agent after ALS/BMC treatment. Additionally, an active immunoregulatory mechanism induced by ALS/BMC treatment appears to be spared by Rapa.

This paper describes a microscale in vivo sensor platform device for the simultaneous detection of multiple biomarkers. We designed the polymer-based biosensors incorporating multiple active isolated areas, as small as 70 μm × 70 μm, for antigen detection. The fabrication approach involved conventional micro- and nano-fabrication processes followed by site-specific electrophoretic directed assembly of antibody-functionalized nanoparticles. To ensure precise and large-scale manufacturing of these biosensors, we developed a semi-automated system for the attachment of the 250-μm biosensor to a 300-μm catheter probe. Our fabrication and post-processing procedures should enable large-scale production of such biosensor devices at lower manufacturing cost. The principle of detection with these biosensors involved a simple fluorescence-based enzyme-linked immunosorbent assay. These biosensors exhibit high selectivity (ability to selectively detect multiple biomarkers of different diseases), specificity (ability to target generic to specific disease biomarkers), rapid antigen uptake, and low detection limits (for carcinoembryonic antigen, 31.25 pg mL(-1); for nucleosomes, 62.5 pg mL(-1)), laying the foundation for potential early detection of various diseases.

1. 1. Ground squirrels were chronically prepared for the determination of hypothalamic stimulus thresholds during hibernation at brain temperatures ( T br ) of 9–15°C by observations of heart rate (HR), neck muscle activity (EMG) and T br changes. 2. 2. Preliminary tests on euthermic squirrels showed that the hypothalamic sites were rewarding since animals self-stimulated the sites at a rate proportional to the stimulus intensity. 3. 3. During bouts of hibernation, the threshold for increased HR varied from 2- to 5-fold but did not vary significantly with T br or the percentage of the hibernation bout elapsed. 4. 4. Stimulation of the hypothalamus resulted in rises of T br during hibernation only if the stimulus produced cardioacceleration.

Significant extended survival of C3H/He skin grafts in antilymphocyte serum (ALS)-treated B6AF1 mice can be brought about by the injection of donor-strain bone marrow on day 6 or 7 after grafting. In the present study, survival of the active graft-prolonging bone marrow cells under several storage conditions was investigated. The bone marrow cells retained their effectiveness if stored at 4 degrees C in 10% fetal calf serum for 18 hr prior to injection, but not if maintained at 37 degrees C under standard lymphocyte culture conditions. Freezing the cells for 10 days in a cryoprotective medium preserved the ability of the cells to prolong graft survival. In fact, freeze-thawed cells were more effective than fresh cells. Extension of the ALS-bone marrow treatment protocol to human transplantation is expected to be facilitated by frozen and short-term refrigerated storage of the donor bone marrow.

Antilymphocyte serum (ALS)-treated recipient mice that are infused with donor bone marrow cells have been shown to enjoy prolonged skin graft survival. In the present experiments, discontinuous density gradient centrifugation in Percoll was used to prepare fractions of these cells. Two of four fractions had good graft-prolonging ability. In experiments in which the density of the gradients was varied, we were able to estimate the density of a major portion of the cells active in graft prolongation as being in a range of 1.061 to 1.066 g/ml, and to produce one fraction with superior graft-prolonging activity. This fraction constituted only 10% of the recovered cells, was enriched for small-to-medium lymphocytes, Ia+, Thy-1+, and IgM+ cells, and contained cells bearing a marker known to be present on active cells, Fc gamma R. Additionally, this fraction contained cells capable of suppressing the mixed lymphocyte reaction, although two denser fractions, of poor-to-good graft-prolonging ability, also shared this ability. The effectiveness of this method in bringing about a high-capacity, rapid isolation of cells that can augment graft survival in immunosuppressed recipients makes it well suited for future clinical application.

Responses of normothermic and hibernating marmots to manipulations of the preoptic-hypothalamic temperature (TPO) were studied. Independent variables included alteration of TPO and, during normothermia, room temperature. Hibernation occurred at an ambient of 6 degrees C. Dependent variables include brain, subdermal, and surface temperatures, heart rate, and behavioral, electromyographic cortical, and hippocampal responses. Although normothermic autumn marmots displayed most of the usual mammalian responses to alterations of the TPO, evidence of effective dermal vasomotion was not obtained. Single episodes of water drinking accompanied prolonged raising of the TPO; sleep was not elicited. During hibernation, effective central thermoregulation was not apparent until 3 or 4 days had elapsed. After this, thermoregulation was readily demonstrable in response to both raising and lowering the TPO. The apparent open-loop gains (OLG) for rise in body temperature after lowering of the TPO showed an exponential increase in value at lower prestimulus body temperatures. It was postulated that this could be explained on the basis of the recruitment of cold-sensitive neurones, which in turn would provide an explanation for the hypothesized "alarm temperature."

Posttransplant injection of fractionated donor bone marrow cells results in the prolonged survival of mouse skin allografts. In this investigation, we extended the study to the use of donor bone marrow fractionated by density-gradient centrifugation in a larger animal model, canine renal transplantation. Renal allografts were placed in outbred, histoincompatible dogs treated daily with rabbit or horse antilymphocyte serum (ALS) from days -6 to +7 relative to kidney transplantation on day 0. Fresh, unfractionated donor bone marrow (BM) or a bone marrow fraction (BM Fr3) produced by centrifugation in a discontinuous Percoll density gradient was intravenously infused into recipients on days +13 or +14. Alternatively, frozen/thawed (F/Th) BM or BM Fr3 was infused after storage at -80 degrees C for two weeks. BM Fr3 and unfractionated BM significantly (P less than 0.005) prolonged for median allograft function time (MFT) beyond the controls treated only with ALS (46 and 35 days vs. 18 days). The longer MFT with BM Fr3 was achieved with 20-40% of the unfractionated BM cell dose. In addition, BM Fr3-treated dogs exhibited a slower rate of loss of kidney function. F/Th BM was as effective as fresh BM in prolonging graft survival. A reduced mixed lymphocyte response to donor and third-party cells and markedly reduced responsiveness to Con A, PHA, and pokeweed mitogens at 30-45 days posttransplant suggested that animals were nonspecifically immunosuppressed during this period. By 60 days posttransplant, in two dogs treated with BM Fr3, the MLR to third-party cells, but not to donor cells, and the responsiveness to mitogens had returned to pre-ALS treatment values. Thus, treatment with ALS combined with fresh or F/Th BM that has been fractionated is an effective method for the prolongation of kidney allografts in dogs. The development of specific, long-term immunosuppression induced by fractionated bone marrow and success with F/Th BM in this canine renal allograft model suggests that application to human transplantation may be effective with fresh or F/Th BM fractions obtained from living-related or cadaveric donors.

Posttransplantation administration of donor-specific bone marrow cells to ALS-treated allograft recipients produces graft survival that is greater than that produced by ALS treatment alone. We have now studied the effect of peripheral blood lymphocytes (PBLs) in a mouse skin allograft model using this protocol (C3H skin grafted to B6AF1 mice, day 0; i.p. ALS on days -1 and +2; i.v. bone marrow days +6 or +7). When PBLs are injected in place of bone marrow cells, graft survival is extended beyond that noted for control mice given ALS only. The timing and specificity of this phenomenon suggest that it resembles the effect produced by posttransplant bone marrow administration and not that associated with pretransplant blood transfusion. The PBLs active in graft prolongation are Thy-1-negative and display a density in Percoll gradients similar to that of the active marrow cells. In contrast, when PBLs are injected in combination with bone marrow cells, the length of graft survival is shortened in comparison with that produced by bone marrow alone. The cells associated with this partial abrogation of the effectiveness of bone marrow appear to be mature T cells; this abrogation cannot be produced by PBLs treated with anti-Thy-1 plus complement or by thymocytes, but it is a property of lymph node cells enriched for T cells by nylon-wool fractionation. This study suggests that clinical application of this posttransplantation induction of specific allograft unresponsiveness can be facilitated by the use of peripheral blood lymphocytes rather than marrow, sparing a living organ donor from having to undergo bone marrow harvest. Additionally, the data indicate that contamination of marrow with blood lymphocytes should be minimized. However, the density gradient fractionation method that we have found to be effective in preparing the graft prolonging bone marrow cells simultaneously removes the PBLs deleterious to graft survival.

La survie d'allogreffes de peau chez des rats traites par serum antilymphocytes est prolongee si les receveurs sont traites par perfusion de cellules de moelle de la souche du donneur. L'efficacite est la meme si le donneur de moelle est jeune ou adulte (6,5 semaines a 34,5 semaines)