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Cancer vaccines aim to efficiently prime cytotoxic CD8

Cervical ganglia and nerve root injury with resultant paralysis of the phrenic nerve is proposed as a mechanism of injury in “shaken baby syndrome” or nonaccidental head injury of children. We report autopsy findings of a 22-month-old girl in which cervical ganglia and nerve hemorrhage was present along with the triad of encephalopathy, bilateral retinal hemorrhage, and bilateral subdural, intradural, and subarachnoid hemorrhages. Clear evidence of near immediate respiratory arrest was documented from first responder medical reports, juvenile witness statements, and perpetrator confession. The narrowing of time of injury from the anatomic findings at autopsy allowed investigative efforts to focus on a single individual and cleared several other adults with access to the child. Additional technical notes based on our experience are provided for users of cervical neck dissection. Hemorrhage in the tissues surrounding the vertebral artery raises a potential additional injury site in cases of neck injury.

A CMOS image sensor that can operate in both linear and logarithmic mode is described. Two sets of data are acquired and combined in the readout path to render a high dynamic range image. This is accomplished in real-time without the use of frame memory. A dynamic range in excess of 120 dB was achieved at 26 frames/s (352times288-array). The system addresses the problems of high fixed pattern noise (FPN), slow response time, and low signal-to-noise ratio (SNR) in logarithmic mode. FPN has been effectively reduced by single and two parameter calibration, the latter achieving FPN of 2% per decade. A novel on-chip method of deriving a reference point has been implemented. The system is fabricated in a 0.18-mum 1P4M process and achieves a pixel pitch of 5.6 mum with 7 transistors per pixel

Targeted Delivery of Antisense Oligodeoxynucleotides In Vivo by Means of Coated Cationic LipoplexesEarlier we reported on the massive uptake of liposomes surface-modified with negatively charged aconitylated albumin (Aco-HSA) by liver endothelial cells (EC) in vivo. In the present work we apply this principle for in vivo delivery of antisense oligodeoxynucleotides (ODN) to these cells by means of coated cationic lipoplexes (CCL) (). CCL were prepared by complexing ODN with the cationic lipid DOTAP and subsequent coating of the complex by neutral lipids including a lipid-anchored poly(ethylene glycol). Aco-HSA was covalently coupled.The Aco-HSA-CCLs were 160 nm in size, contained 1.03 ± 0.35 nmol ODN and 54 ± 18 µg Aco-HSA per µ mol total lipid. The Aco-HSA-CCLs were rapidly eliminated from plasma, 60% of the injected dose being recovered in the liver after 30 m. Within the liver, the EC accounted for two thirds of total liver uptake. Non-targeted CCLs were eliminated very slowly: after 30 m >90% of the pa...

=0.90). As the maximum clamp spacing to ensure a particular glue line pressure depends on the distance to the first glue line, a look-up table, based on a minimum glue line pressure of 345 kPa (50 psi), was produced giving the maximum recommended clamp spacings for common clamp face to glue line widths.

Obwohl die tiefe Venenthrombose nicht mehr die schlechte Prognose wie in der Ara vor der Antikoagulation aufweist, bleibt auch heute noch eine Reihe von Patienten mit auf konservativem Weg nicht ausreichend beherrschbarem Ulcus cruris zuruck. Prasentiert wird das Fallbeispiel einer 50jahrigen Patientin mit chronisch venoser Insuffizienz III im Rahmen eines postthrombotischen Syndroms. Darin werden die Fortschritte in der bildgebenden und funktionellen Diagnostik des PTS dargestellt, wodurch eine zuverlassige Differenzierung hinsichtlich Obstruktion bzw. Reflux moglich ist. Die autologe Klappentransplantation wird als zur Behandlung des Reflux geeignetes Therapiekonzept beim Versagen konservativer Masnahmen beschrieben, Mangel und Ergebnisse dieser Methode werden diskutiert.

During the last decades the quality requirements for foodstuffs have become progressively higher. Many research programs have been dedicated to the development of materials with improved packaging and organoleptic performance. In this respect, the shelf life of foodstuffs is a key success factor for the packaging material. In this paper an investigation is presented covering the effects of vitamin E (α-Tocopherol) in extrusion coating LDPE, whereby storage of water in aseptic liquid packaging is simulated. In these experiments LDPE cast films have been aged in water during one year at 40'C. Off taste of the water has been determined by a taste panel, and the film and water samples have been analysed by several analytical techniques. Comparison of the LDPE with and without vitamin E showed a significant improvement of shelf life of the packaged water. The formation of low molecular weight oxidation products of LDPE during processing and during aging was suppressed by the vitamin E, thereby avoiding deterioration of the taste of water.

Nanotechnology-inspired approaches to particle design and formulation, an improved understanding of (patho) physiological processes and biological barriers to drug targeting, as well as the limited input of new chemical entities in the 'pipeline' of pharmaceutical companies, suggest a bright future for targeted nanomedicines as pharmaceuticals. There is an increased consensus to the view that a major limitation hampering the entry of targeted delivery systems into the clinic is that new concepts and innovative research ideas within academia are not being developed and exploited in close collaboration with the pharmaceutical industry. Thus, an integrated 'bench-to-clinic' approach realized within a structural collaboration between industry and academia, will facilitate and promote the progression of targeted nanomedicines towards clinical application. The MediTrans project performed under the EU Framework Program 6, was designed to contribute to this ambition. The objectives of this collaborative initiative were: to apply nanotechnology for development of innovative targeted drug-delivery systems; to optimize targeted nanomedicines by using imaging guidance; to promote structural collaboration between industry and academia; and to forward targeted nanomedicines towards the clinic and the market. In this article, we will briefly address the research content, outcome and impact of the MediTrans project.

The white clot syndrome is a serious ideosyncratic side effect of routine anticoagulation with low dose heparin. Two different courses of the white clot syndrome are illustrated. The different ways of diagnosis and therapy as well as means of prophylaxis are discussed.

This prospective study was designed to establish whether vascular endoscopy would provide more information on the graft lumen than standard completion angiography during infrainguinal bypass surgery. Ninety-nine patients with 102 infragenicular bypass grafts who underwent both angiography and angioscopy intraoperatively were evaluated. In 99 of the 102 patients the indication was critical limb ischaemia. Of the 102 bypass grafts, 81 were autogenous vein. Distally, 24 grafts were anastomosed to the below-knee popliteal segment, 64 extended to the crural and 14 to the pedal arteries. On completion of the distal anastomosis, grafts were first evaluated by angiography and then by angioscopy. The images obtained with the two monitoring modalities were compared by the operating surgeon and re-explorations were performed immediately if necessary. Completion angiography and angioscopy produced images of good quality in 96 and 97 cases, respectively. In 12 cases completion angiography showed abnormalities. Of these, five were located below the distal anastomosis and were not accessible to angioscopic examination. Conduit defects were found in seven instances. In one of them angioscopy showed the angiogram to be false-positive. Of the 90 grafts with normal completion angiograms, seven were found to show significant pathology on angioscopy. Compared to angioscopy, the sensitivity and specificity of angiography to detect abnormalities within the graft was 46% and 98%, respectively. Our results suggest that vascular endoscopy is superior to angiography for disclosing conduit defects, but that it does not provide adequate information about the distal arterial anatomy.

Liposomes are phospholipid nanoparticles that extravasate at sites of increased vascular permeability. They have potential in equine medicine for targeted drug delivery and diagnostic imaging of infectious, inflammatory and neoplastic lesions.This study evaluates the safety and biodistribution of i.v. polyethyleneglycol(PEG) liposomes in normal horses.PEG-liposomes were prepared by the film hydration method and labelled using (99m) Tc-hexamethyl-propylene-amine-oxime. A single dose of 0.24 µmol/kg bwt (99m) Tc-PEG-liposomes and 2.4 µmol/kg bwt unlabelled PEG-liposomes was administered to 10 conscious horses via i.v. infusion at a rate of 6 µmol/min for the first 15 min and 60 µmol/min thereafter. Clinical parameters, haematology, plasma biochemistry and serum complement activity were monitored serially. Scintigraphic imaging was performed at 1, 12 and 21 h post infusion (PI). Six horses were subjected to euthanasia at 24 h PI. The percentage injected dose per kilogram of tissue was calculated for multiple organs. Results were analysed using repeated measures ANOVA.Horses did not demonstrate adverse reactions during or after liposome infusion. There was a significant elevation in heart rate and respiratory rate at 20 and 25 min PI. No significant complement consumption was detected, although a trend for decreased total haemolytic complement values at 20 min PI was present. Scintigraphic studies revealed a prolonged vascular phase that lasted to 21 h PI, with a reproducible pattern of organ distribution. Biodistribution studies revealed the highest concentrations of radiopharmaceutical within the lung, kidney, liver and spleen.Intravenous liposome administration appears to be safe in horses. When administered in combination with PEG-liposomes, (99m) Tc-PEG-liposomes have long circulating characteristics and a reproducible pattern of organ distribution in horses.Radiolabelled liposomes may be useful for detecting infection, inflammation and neoplasia in the horse. Liposomes have significant potential for targeted drug delivery in the horse. This study establishes the scintigraphic findings and tissue distribution of 99mTc-PEG-liposomes after i.v. administration in healthy horses.

Pharmacokinetics of 20 mg isosorbide-5-mononitrate (IS-5-MN) after single and multiple administration of two different tablet formulations were investigated in twelve healthy human subjects using an open, randomized, two-way crossover experimental design. Pentacard 20 mg tablets were compared with Ismo 20 mg tablets. After single-dose administration, both preparations caused a rapid increase in IS-5-MN plasma levels with the peak plasma concentration occurring between 0.5 and 1.5 h. For both formulations, the mean plasma half-life was found to be approximately 5 h after a single dose. In steady state during multiple dosing (t.i.d. at 8 h dosing intervals), a reduced elimination rate was observed. In line with this observation, the area under the plasma concentration-time curve (AUC) for one 8 h dosing interval during multiple dosing was higher than the extrapolated AUC after a single dose. Based on statistical evaluation of the various relevant pharmacokinetic parameters calculated from the plasma concentrations occurring after single and multiple dosing, the tablet formulations are judged to be bioequivalent.

A 352/spl times/288 pixel array achieves >120 dB dynamic range through merging sequential linear and logarithmic images. Calibration is used to match offset and gain. A 7-transistor pixel is built in a 0.18 /spl mu/m 1P4M CMOS process.

Recent studies with PEG liposomes in patients have consistently shown that liposomes can induce side effects (flushing, tightness of the chest). Furthermore, the blood clearance of PEG liposomes was shown to be dose-dependent: at lipid doses lower than 1 micromol/kg, PEG liposomes do not show the long-circulation property but instead are cleared relatively rapidly from the bloodstream. Another remarkable observation was that repeated injections of PEG liposomes led to significant pharmacokinetic changes: the circulatory half-life of a second dose of radiolabeled PEG liposomes dramatically decreased when given from 5 days to up to 4 weeks after a first injection. In these three unexpected phenomena, proteins of the complement system seem to play a key role. Therefore, one has to consider that PEG liposomes are not inert drug-carrying vehicles in vivo. Pharmacological effects can occur, induced solely by using liposomal particles irrespective of the drug content.

To gain insight into the host factors influencing liposome localization at sites of bacterial infection.In a unilateral Klebsiella pneumoniae pneumonia rat model, capillary permeability and number of circulating leukocytes was quantified and related to the degree of liposome target localization.Liposome localization was highest in the hemorrhagic zone of infection, a zone characterized by markedly increased capillary permeability and high bacterial numbers. Both liposome localization and capillary permeability correlated positively with severity of infection. Lung instillation of other inflammatory stimuli, such as lipopolysaccharide or 0.1 M HCl inducing increased capillary permeability, also promoted liposome localization. As liposomal target localization in leukopenic rats was similar to that in immunocompetent rats, contribution of circulating leukocytes seems limited. Intrapulmonary distribution of liposomes shows that leukocytes at the target site are involved in liposome uptake after extravasation.Increased capillary permeability plays a crucial role in liposome localization at the infected site, whereas contribution of leukocytes is limited. These results suggest inflammatory conditions that could benefit from liposomal drug delivery. The involvement of leukocytes in liposome uptake at the target site could be important information in the selection of appropriate drugs.

Antimicrobial agents may interact synergistically. But to ensure synergy in vivo, the drugs should both be present at the site of infection at sufficiently high concentrations for an adequate period of time. Coencapsulation of the drugs in a drug carrier may ensure parallel tissue distributions. Since liposomes localize preferentially at sites of infection, this mode of drug delivery could, in addition, increase drug concentrations at the focus of infection. The therapeutic efficacy of gentamicin and ceftazidime coencapsulated into liposomes was examined by monitoring survival in a rat model of an acute unilateral pneumonia caused by antibiotic-susceptible and antibiotic-resistant Klebsiella pneumoniae strains. It is shown that administration of gentamicin in combination with ceftazidime in the free form either as single dose or as 5-day treatment resulted in an additive effect on rat survival in both models. In contrast, targeted delivery of liposome-coencapsulated gentamicin and ceftazidime resulted in a synergistic interaction of the antibiotics in both models. Consequently, liposome coencapsulation of gentamicin and ceftazidime allowed both a shorter course of treatment at lower cumulative doses compared with administration of the antibiotics in the free form to obtain complete survival of rats. Liposomal coencapsulation of synergistic antibiotics may open new perspectives in the treatment of severe infections.

The integrity of a therapeutic protein has to be safeguarded when formulated in delivery systems such as liposomes. In this study, we investigated the conformational stability of recombinant human interferon gamma (hIFNgamma) on association with and after dissociation from liposomal bilayers using circular dichroism (CD) and steady-state fluorescence spectroscopy as well as time-resolved fluorescence methodology. We used hIFNgamma adsorption to and desorption from empty liposomes as a model for hIFNgamma-containing liposomes prepared via the film hydration method. CD studies indicated that no changes in the secondary and tertiary protein structure occur during and after interaction of hIFNgamma with the liposomes. Steady-state fluorescence emission spectra of untreated and liposome-desorbed hIFNgamma revealed that the environment of the sole Trp residue was not affected by the adsorption/desorption process. The Trp-36 residue remained fully quenchable by acrylamide after desorption of hIFNgamma from the liposomes. Time-resolved fluorescence studies were conducted to probe the local environment and the mobility of Trp-36 before, during, and after interaction of hIFNgamma with the liposomal membrane. Differences in rotational correlation time between free and liposomal hIFNgamma were attributed to immobilization of the protein on adsorption to the liposome bilayer. Disparities were detected between the average lifetimes of liposome-adsorbed hIFNgamma and hIFNgamma-liposomes, indicating that subtle changes in the Trp-36 environment took place during preparation of the liposomes via the film hydration method compared with the adsorption of hIFNgamma to the liposome surface. The results of this study indicate that association of hIFNgamma with negatively charged liposomes results in minimal changes in the secondary and tertiary structure of the protein. We conclude that all techniques used point to a full retention or restoration of the protein conformation after desorption from the liposomes.

To assess whether intraarticular (IA) administration of clodronate liposomes results in local macrophage depletion in patients with rheumatoid arthritis (RA). Primary goals were to address both the immunohistologic and potential toxic effects of this approach. Moreover, the correlation between immunohistologic findings and clinical assessments of disease activity and cartilage damage were assessed.An open study was conducted in consecutive RA patients who were scheduled for knee joint replacement in our department. Synovial biopsy tissue was obtained from the knee joint at 2 weeks before and at the time of surgery. This protocol was controlled for safety and immunohistologic concordance in 6 patients. One week before surgery, 10 patients received a single IA dose of clodronate liposomes. Staining of synovial tissue for cell markers (CD68, CD14, CD3, CD38) and adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1]) was assessed by 2 blinded observers. Local and systemic parameters of disease activity were measured before each intervention. Cartilage damage was scored using standard radiologic techniques at baseline and during surgery.A single IA dose of clodronate liposomes significantly reduced the number of CD68-positive cells (P = 0.005) and the expression of ICAM-1 and VCAM-1 in the synovial lining (P = 0.013 and P = 0.039, respectively). The intervention did not affect fibroblast-like synoviocytes, T cells, or plasma cells. No immunohistologic changes were observed in the control group. The procedure was well tolerated. The levels of ICAM-1 and VCAM-1 in the sublining layers correlated with the extent of macroscopic synovitis (P0.0005 and P0.005, respectively). The expression of ICAM-1 and CD14 in the sublining correlated with the levels of C-reactive protein (P0.0005 and P0.01, respectively). Cartilage destruction was correlated only with the expression of CD68 in the sublining (P = 0.02).A single IA administration of clodronate liposomes leads to macrophage depletion and decreased expression of adhesion molecules in the synovial lining in patients with longstanding RA. The procedure is well tolerated, and its therapeutic potential is currently under investigation. The expression of adhesion molecules in the sublining layers reflects ongoing inflammation.

We are exploring liposomal delivery with the aim to change the pharmacokinetics and biodistribution of SOD to increase its therapeutic activity. From a practical point of view, a convenient route of administration would be the subcutaneous (s.c.) route. Liposomal size has been shown to be the most important factor influencing the rate and extent of drainage of liposomes from the s.c. injection site.To monitor the in vivo fate of the subcutaneous administered SOD-containing liposomes in rats with a chronic arthritis inflammation, the liposomes were labeled by the co-encapsulation of the 111In-DTPA complex in the internal water space.Over the initial 10h-observation period post-injection, the small-sized poly(ethyleneglycol)-liposomes (mean size about 110 nm) left the site of injection to a 2-fold higher extent (45% of the injected dose) as compared to large-sized poly(ethyleneglycol)-liposomes (mean size about 450 nm). Small-sized liposomes gave a 17-fold higher uptake in the inflamed foot than the large-sized liposomes. Comparing the localization in the inflamed foot with the non-inflamed foot, uptake was more than 15-fold higher for the small-sized liposomes as compared to the large-sized liposomes. After s.c. administration, small-sized SOD-liposomes showed substantial higher activity than large-sized SOD-liposomes. S.C. administration of small-sized SOD-liposomes is equally effective as i.v. administration of the same liposomes. I.V. administration of the large-sized SOD-liposomes yielded a significantly higher activity as compared to s.c. administration.These results indicate that small-sized poly(ethyleneglycol)-liposomes can be used for the targeting of SOD to arthritic sites after subcutaneous administration.

Poly(2-(dimethylamino)ethyl methacrylate) (p(DMAEMA)) can be used successfully for in vitro transfection of different cell lines, including the OVCAR-3 human ovarian carcinoma cell line. The aim of this study was to investigate whether it is possible to transfect OVCAR-3 cells in vivo with polyplexes containing p(DMAEMA).In order to understand the generally observed gap between in vitro and in vivo transfection, we gradually went from in vitro to in vivo transfection of OVCAR-3 cells, while keeping the exposure conditions the same, as far as possible. To find the reason for the negligible degree of in vivo transfection, in vitro cultured OVCAR-3 cells were transfected in the presence of peritoneal ascites fluid. Next, the influence of hyaluronic acid, one of the ascites components, on the transfection efficiency was studied.P(DMAEMA)-containing polyplexes can transfect OVCAR-3 cells in vitro with an overall transfection efficiency of 10%. Cells grown in vivo can be transfected ex vivo with p(DMAEMA)/plasmid complexes with an overall transfection efficiency of approximately 1-2%. When transfection complexes are injected i.p. into nude mice bearing OVCAR-3 cells in the peritoneal cavity, the degree of in vivo transfection efficiency achieved is negligible. In vitro cultured OVCAR-3 cells were also transfected with polyplexes in the presence of peritoneal ascites fluid. The results indicate that one or more components of ascites had a negative effect on the transfection efficiency of p(DMAEMA)-containing polyplexes. To elucidate which component(s) of ascites may have interfered, the influence of hyaluronic acid, one of the ascites components, on the transfection efficiency was studied. The outcome suggests that hyaluronic acid may have induced a negative effect on the transfection capability of p(DMAEMA)-containing polyplexes.P(DMAEMA) is an efficient transfectant in vitro and ex vivo. However, transfected cells were not detected in vivo which may be caused by a negative influence of components of the ascites fluid.

Liposomal systems may be useful as a cytokine supplement in tumor cell vaccines by providing a cytokine reservoir at the antigen presentation site. Here, we examined the effect of liposome incorporation of mIFNgamma on its potency as adjuvant in an established tumor cell vaccination protocol in the murine B16 melanoma model. Adjuvanticity of the mIFNgamma-liposomes was compared to that achieved by mIFNgamma-gene transfection of the B16 tumor cells. Furthermore, we studied whether liposomal incorporation of mIFNgamma indeed increases the residence time of the cytokine at the vaccination site.C57B1/6 mice were immunized with i) irradiated IFNgamma-gene transfected B16 melanoma cells or ii) irradiated wild type B16 cells supplemented with (liposomal) mIFNgamma, followed by a challenge with viable B16 cells. The residence time of the (liposomal) cytokine at the subcutaneous (s.c.) vaccination site was monitored using radiolabeled mIFNgamma and liposomes.Immunization with irradiated tumor cells admixed with liposomal mIFNgamma generated comparable protection against B16 challenge as immunization with mIFNgamma-gene modified tumor cells. Irradiated tumor cells admixed with soluble mIFNgamma did not generate any protective responses. Radiolabeling studies indicated that free mIFNgamma rapidly cleared from the s.c. injection site. Association of [125I]-mIFNgamma with liposomes increased the local residence time substantially: liposomal association of mIFNgamma resulted in a prolonged local residence time of the cytokine as reflected by a 4-fold increase of the area under the curve. The amount of released cytokine in the optimal dose range corresponds to the amount released by the gene-transfected cells. Moderate but significant CTL-activity against B16 cells was found for mice immunized with irradiated cells supplemented with mIFNgamma-liposomes compared to untreated control animals.Prolonged presence of mIFNgamma at the site of antigen presentation is crucial for the generation of systemic immune responses in the B16 melanoma model. These studies show that liposomal encapsulation of cytokines is an attractive strategy for paracrine cytokine delivery in tumor vaccine development.

Rheumatoid arthritis (RA) is a prevalent and debilitating autoimmune disease that affects the joints. RA is characterized by an infiltration of the affected joint by blood-derived cells. In response to activation, these cells generate reactive oxygen species, resulting in an oxidative stress situation. One approach to counteract this oxidative stress situation is the use of antioxidants as therapeutic agents. The free radical scavenger enzyme superoxide dismutase (SOD) may be used as a therapeutic agent in rheumatoid arthritis, but its rapid elimination from the circulation is a major limitation. Targeted delivery of SOD may overcome this limitation. In this study, the utility of PEGylated liposomes (PEG-liposomes) for targeting SOD to arthritic sites was explored. The targeting of SOD to arthritic sites following intravenous administration of both PEG-liposomes and positively charged liposomes lacking PEG but containing stearylamine (SA-liposomes) in rats with adjuvant arthritis was studied. At 24 h post injection, the blood levels of long circulating liposomes with a mean size of 0.11 micrometer and 0.20 micrometer were 8- and 3-fold higher, respectively, as compared to the SA-liposomes. The majority of SOD administered in liposomal form remains within the liposomes when they circulate in the bloodstream. The highest target uptake was observed with PEG-liposomes with a mean size of 0.11 micrometer and the lowest uptake with the SA-liposomes. These results demonstrate that SOD can be targeted to inflamed sites most efficiently via small-sized PEG-liposomes. Small-sized PEG-coated liposomes are to be preferred if prolonged circulation and enhanced localization of SOD at arthritic sites are desired.

Vaccination with tumor cells genetically engineered to produce interleukin (IL)-2 is an attractive strategy to enhance antitumor immune responses. The improved antitumor immunity upon vaccination with IL-2 gene-modified tumor cells may be due to the prolonged presence of the cytokine at the vaccination site. Because liposomes have been used for sustained delivery of a variety of agents, we compared the protective effect of vaccines consisting of IL-2 gene-modified B16 melanoma cells to that of vaccines composed of IL-2 liposomes and irradiated melanoma cells. The results indicate that both approaches equally protect against a lethal challenge with B16 melanoma cells. More than 20% of the protected animals developed vitiligo at the vaccination and/or tumor challenge site.

The ability of clodronate-containing liposomes to deplete lymph nodes of macrophages was used as a tool to investigate the fate of liposomes in regional lymph nodes after subcutaneous (s.c.) administration. Reduced lymph node localization of liposomes in macrophage-depleted lymph nodes confirmed that phagocytosis by macrophages plays an important role in lymph node retention of liposomes. Depletion of macrophages had less effect on lymph node localization of small liposomes than on the lymph node localization of large liposomes. Inclusion of distearoylphosphatidylethanolamine (DSPE)-poly(ethyleneglycol) (PEG-PE) into the liposomes, which is known to oppose macrophage uptake, did not affect lymph node localization in macrophage-depleted or control lymph nodes. We conclude that PEG-liposomes retained by lymph nodes are also taken up by lymph node macrophages. Morphological observations visualizing the uptake of PEG-liposomes by lymph node macrophages support this conclusion.

The introduction of long-circulating liposomes sterically stabilized by surface coating with polyethylene glycol has expanded the potential for drug targeting to tumors. In recent clinical studies, evidence of significant antitumor activity has been obtained with the industrially prepared formulation of long-circulating polyethylene glycol-coated liposomes containing doxorubicin, referred to as DOXIL. Previous studies performed in rats showed that doxorubicin-containing liposomes can exert major toxic effects on the liver macrophage population for a considerable period of time; a strong impairment of phagocytic function and even a substantial depletion of the liver macrophage populations were observed. In the present study, the phagocytic function of the mononuclear phagocyte system (MPS) after administration of DOXIL at a clinically relevant dosage schedule was evaluated in rats. Phagocytic function of the MPS was assessed by determining bacterial blood clearance capacity. The observations reported herein show that DOXIL is fairly well tolerated regarding bacterial blood clearance capacity of the MPS when administered in a regimen that resembles the clinical setting closely. This outcome has important implications with regard to the clinical utility of the liposomal drug, especially in the restricted context of immunocompromised cancer patients who easily develop systemic infections and should not be confronted with a therapy-induced reduction of the bacterial blood clearance capacity of the MPS.

Synthetic peptide-based vaccines have been shown to induce potent protective and therapeutic T cell-mediated immunity in preclinical animal models and are now being evaluated in clinical phase I/II studies for their efficacy against tumors or infectious diseases. However, such vaccines might also specifically tolerize T cells causing enhanced tumor outgrowth, as shown by vaccination with two CTL epitopes derived from the adenovirus type 5 early region 1 (Ad5E1) oncogenes. We now report that modification of the Ad5E1 peptide vaccine either through incorporation of the peptides into liposomes or by ligation of the peptides to lipid tails, another vaccine formulation being tested in the clinic, fails to convert immunosuppression into effective antitumor vaccination. Inclusion of a helper T cell epitope into the vaccine likewise induces enhanced tumor outgrowth and thus does not diminish the capacity of the peptides to tolerize Ad5E1-specific CTL. In contrast, the Ad5E1-derived peptides evoke a strong tumor-protective CTL response when presented on dendritic cells (DC), indicating that the in vivo CTL-tolerizing potential of these peptides is converted to specific immunostimulation when presented on DC. These findings have important implications for the development of peptide-based immune intervention strategies and emphasize the superior nature of Ag-pulsed DC over other peptide-based vaccination protocols as well as the crucial importance of the mode of peptide-Ag delivery in setting the balance between T cell stimulation and tolerization.

The aim of the present paper was to assess the effect of inclusion of distearoylphosphatidylethanolamine-poly(ethyleneglycol) (DSPE-PEG) into liposomal bilayers on the lymphatic uptake and lymph node localization of liposomes after subcutaneous administration.[3H]-Cholesteryloleylether labeled liposomes of various composition and sizes were injected s.c. into the dorsal side of the foot of rats. At several time-points after injection, blood levels of liposomes were determined. Lymphatic uptake from the s.c. site of injection and lymph node localization in regional lymph nodes were determined at the end of the 52 h observation period.The results demonstrate that inclusion of DSPE-PEG into several types of liposomes has only a modest effect on lymphatic uptake. Also lymph node localization is only slightly affected by PEG-mediated steric stabilization.Factors other than the presence of a steric barrier are more important in determining lymphatic uptake from the s.c. injection site. The observation that lymph node localization was only slightly affected by PEG-coating strongly suggests that macrophage uptake is not the only important mechanism of lymph node localization of s.c. administered liposomes.

The present paper reports on the results of a systematic study on liposome variables potentially affecting lymphatic disposition and biodistribution of liposomes after sc injection. Liposomal size was found to be the most important factor influencing lymphatic uptake and lymph node localization of sc administered liposomes. Lymphatic uptake from the s.c. injection site of small liposomes (about 0.04 microm) was relatively high (76% of the injected dose (%ID)) as compared to large, non-sized liposomes, which remained almost completely at the site of injection. Small liposomes were less efficiently retained by regional lymph nodes than larger liposomes. Liposomal lipid composition did not influence lymphatic uptake with one exception: Lymphatic uptake was decreased in case of neutral liposomes composed of (DPPC). Lymph node localization was substantially enhanced by inclusion of phosphatidylserine (PS) into the liposomal bilayers. Saturation of lymphatic uptake and lymph node localization did not occur over a large liposomal lipid dose range, illustrating the efficient performance of lymph nodes in capturing s.c. administered particles.

Immuno-enzymosomes are tumor-specific immunoliposomes bearing enzymes on their surface. These enzymes are capable of converting relatively nontoxic prodrugs into active cytostatic agents. The enzyme beta-glucuronidase (GUS)4 was coupled to the external surface of immunoliposomes directed against ovarian carcinoma cells. This study aimed at optimization of the prodrug-activating capacity of these immuno-enzymosomes by increasing the enzyme density on the immunoliposomal surface.To achieve coupling of GUS to the liposomes, introduction of extra thiol groups was required. Two thiolating agents were examined: iminothiolane and SATA.When iminothiolane was used, aggregation of enzymosomes was observed above enzyme densities of 10 micrograms GUS/mumol lipid (TL). An increased electrostatic repulsion of the enzymosomes, created by inclusion of additional negatively charged lipids and by lowering the ionic strength of the external aqueous medium resulted in enzyme densitiesor = 20 micrograms GUS/mumol TL without aggregation. Utilizing SATA,or = 30 micrograms GUS/mumol TL could be coupled without aggregation, even at physiological ionic strength. It was shown that the enzyme density on immuno-enzymosomes, and thus on the tumor cell surface, strongly influences the antitumor effect of the prodrug daunorubicin-glucuronide against in vitro cultured ovarian cancer cells. The antitumor effect of immuno-enzymosomes with enzyme densities of about 20 micrograms GUS/mumol TL was similar to that of the parent drug daunorubicin.SATA-mediated thiolation of GUS-molecules enabled the preparation of immuno-enzymosomes with high enzyme densities while avoiding spontaneous aggregation. In vitro antitumor activity experiments showed that the improved immuno-enzymosome system is able to completely convert the prodrug daunorubicin-glucuronide into its parent compound.

The high incidence of flow reducing graft and inflow/outflow artery lesions remains a major problem in femorodistal reconstructions. To detect these abnormalities which may threaten graft permeability, some authors [6, 15] rely upon ABI measurements alone in certain clinical situations. In contrast, other groups [2, 3, 4, 8, 11] emphasize the importance of duplex scanning and duplex-derived velocity parameters to identify the failing graft.

Ambroxol is known to promote bronchial secretion and is used as an expectorant. Previous studies had suggested that high doses of ambroxol could reduce the plasma uric acid concentration. The present study was undertaken to confirm this finding, to determine its dose-response relationship and to identify the underlying mechanism of action. Using a placebo-controlled, double-blind parallel group design, 48 healthy male volunteers were randomly allocated to receive placebo b.d. and ambroxol 125 mg b.d., 250 mg b.d. or 500 mg b.d. (12 subjects per group). The subjects were hospitalised during a dietary run-in period of 3 days (Days -3 to -1) and a treatment period of 5 days (Days 1 to 5). On Day -1 (baseline) and Days 1 to 5, all urine was collected and blood samples were taken for the analysis of uric acid, creatinine, xanthine and ambroxol. The measurements were repeated four days after treatment had closed. Steady state plasma concentrations of ambroxol (trough levels) were reached after 2 or 3 days and were linearly related to dose. Ambroxol induced a significant, dose-dependent, reduction in plasma uric acid (250 mg b.d. about 20%; and at 500 mg b.d. about 30%). The diurnally fluctuating uric acid clearance was dose dependently increased and there was no notable effect on creatinine clearance. Plasma hypoxanthine levels were not affected by ambroxol. No severe adverse events were reported and no drug induced changes in the clinical laboratory values were observed. It is concluded that ambroxol has an uricosuric action following oral administration of higher doses (250 mg-500 mg b.d.) and it is well tolerated.

The value of novel systemically long-circulating liposomes to prolong the duration of an antidiuretic hormone, arg8-vasopressin (VP), was investigated as a representative of low molecular weight peptides with rapid clearance. Cholesterol content was found to have a controlling effect on VP release in serum. Three types of liposomes were selected for urine production measurements in VP deficient Brattleboro rats. One contained phosphatidylserine (PS), which was rapidly cleared from the circulation. In the other two liposomes, the PS component was replaced by either phosphatidylglycerol or a novel phospholipid derivatized with polyethylene glycol (PEG); both showing prolonged circulation. Free VP (up to 8 micrograms/kg) gave reduced urine production for less than 24 hr. The PG formulation exhibited a dose-dependent prolonged duration of bioactivity of up to 4 days. Substitution of PEG-PE resulted in a 2-day delay followed by a prolonged duration of bioactivity for over 4 days. The duration of the prolonged bioactivity was not dose dependent but the amplitude was. This is attributed to VP release from liposomes which have distributed intact to another compartment without having been taken up by the RES. By balancing liposome circulation time, release rate, and dose, long-circulating liposomes can be applied to prolong the biological activity of a therapeutic peptide.

Specific binding of immunoliposomes to target tumor cells was investigated in a xenograft model (athymic nude mice) of i.p. growing human ovarian carcinoma (OVCAR-3). For the first time, quantitative evidence is presented that attachment of a tumor-specific antibody (OV-TL 3) dramatically enhances the association of liposomes with i.p. growing OVCAR-3 cells. The OV-TL 3-mediated binding of liposomes to the OVCAR-3 cells was rapid; 30 min after i.p. injection approximately 70% of the injected dose of OV-TL 3 immunoliposomes was associated with the OVCAR-3 cells while for unconjugated liposomes a value of only approximately 3% was obtained. At 2 h after injection, a maximal binding level of 84% was achieved in case of the OV-TL 3 immunoliposomes whereas the binding level of unconjugated liposomes was still about 3%. Twenty-four h after injection about 83% of the injected dose OV-TL 3 immunoliposomes still was associated with the OVCAR-3 cells, compared to about 10% of the injected dose of unconjugated liposomes. Accordingly, unconjugated liposomes disappeared from the peritoneal cavity much faster than the OV-TL 3 immunoliposomes. By comparison with immunoliposomes bearing irrelevant antibody, the specificity of the binding of the OV-TL 3 immunoliposomes to the OVCAR-3 cells was demonstrated. In addition, it was observed that the sustained high OV-TL 3 immunoliposome levels found in the peritoneal cavity are the result of both reduced particle clearance from the peritoneal cavity and the tenacious binding of the immunoliposomes to the tumor cells. Finally, data are presented showing that the degree of binding of OV-TL 3 immunoliposomes to OVCAR-3 cells in vitro and in vivo correlates positively with the antibody (Fab') density on the liposomes.

In our institution, 95 infrainguinal arterial reconstructions were prospectively entered into a graft surveillance programme which consisted of a postoperative i.a. DSA and routine assessment of graft flow velocity (GFV) and ankle pressure indices (ABI) during the first postoperative year. An average of 4.1 GFV measurements was obtained during a mean follow-up period of 8.2 months. Abnormal GFV led to arteriography in 29 bypasses identifying--aside from three false positive findings--two graft occlusions and 24 severe (70%) graft stenoses. Of the latter, in only 7 cases a significant decrease in ABI was found. Unheralded graft occlusion occurred in 6 patients. Including the corrections of the above mentioned lesions, secondary patency rates were 97% at 30 days and 89% at one year.

Peripheral outflow resistance (OR) was assessed intraoperatively in 75 infrainguinal bypass procedures to the below knee popliteal artery (20) or a single crural artery (55). OR measurement was done after completion of the distal anastomosis. For that purpose, the graft was perfused with Ringer solution at constant flow rates of 100 and 150 ml/min using a roller pump. During infusion, arterial pressure was recorded. In this way distal (DOR) and total (TOR) outflow resistance in E/S anastomoses was calculated before and after vasodilatation with papaverine. DOR at a flow rate of 100 ml/min was found to be most discriminant as a predictor of early graft thrombosis: 1. In the limbs with a thrombosed graft at 30 days OR was significantly higher than in the limbs with a patent graft (1828 +/- 418 vs. 1472 +/- 221 mPRU; p less than 0.01, Mann-Whitney U-Test). 2. The cut-off value of 1700 mPRU, which was determined by ROC curves, was 100% sensitive and 81% specific in the group of femoro-crural grafts to predict early graft failure. A better discrimination by the application of papaverine could not be achieved. We concluded from our results that OR might be an important factor in early graft thrombosis. But still there is more experience required to select patients for primary amputation or adjunctive procedures (av-fistula, sequential graft) on the basis of the present available data.