Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx.

The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium.

Tumour basement membrane laminin expression predicts outcome following curative resection of pancreatic head cancer.

BACKGROUND: Although widely fragmented BMs have been associated with adverse outcome in several cancer types, comparatively little is known with respect to its effect on the prognosis of pancreatic cancer. The aim of the current study was therefore to determine the prognostic value of tumour basement membrane (BM) continuity in two anatomically closely related, however, prognostically different tumours, pancreatic head- and periampullary cancer. METHODS: Tumour BM continuity was determined by immunohistochemical staining of its two major components, laminin and collagen type IV. Associations were made with recurrence free survival (RFS), cancer-specific survival (CSS), overall survival (OS) and conventional prognostic factors. RESULTS: Fifty-nine and 61% of pancreatic head and periampullary tumours, respectively, showed limited BM laminin expression. Whereas 43% and 41% of pancreatic head and periampullary cancers, respectively, showed limited BM collagen type IV expression. Limited BM laminin was associated with poor outcome following curative resection of pancreatic head cancer (P=0.034, 0.013 and 0.017 for RFS, CSS and OS, respectively). Two and a half times as many patients with ≥ 25% BM laminin were recurrence free and alive 5 years following resection compared with those with limited BM laminin. Although staining patterns of both BM components were weakly correlated with each other, BM collagen type IV expression was not significantly associated with outcome in either tumour type. CONCLUSION: Discontinuous BMs, determined by laminin expression, are associated with poor outcome following curative resection of pancreatic head cancer.

Selective transfer of a lipophilic prodrug of 5-fluorodeoxyuridine from immunoliposomes to colon cancer cells.

A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated derivative of the anticancer drug FUdR as a prodrug in their bilayers. We investigated the in vitro interaction of these liposomes with CC531 target cells and the mechanism by which they deliver the active drug FUdR intracellularly to the cells by monitoring the fate of the liposomal bilayer markers cholesterol-[(14)C]oleate and [(3)H]cholesteryloleylether as well as the (3)H-labeled prodrug and colloidal gold as an encapsulated liposome marker. After binding of the immunoliposomes to the cell surface, only limited amounts were internalized as demonstrated by a low level of hydrolysis of liposomal cholesterol ester and by morphological studies employing colloidal gold-labeled immunoliposomes. By contrast, already within 24 h immunoliposome-incorporated FUdR-dP was hydrolyzed virtually completely to the parent drug FUdR intracellularly. This process was inhibited by a variety of endocytosis inhibitors, indicating that the prodrug enters and is processed by the cells by a mechanism involving an endocytic process, resulting in intracellular FUdR concentrations up to 3000-fold higher than those in the medium. Immunoliposomes containing poly(ethyleneglycol) (PEG) chains on their surface, with the antibody coupled either directly to the bilayer or at the distal end of the PEG chains were able to deliver the prodrug into the tumor cells at the same rate as immunoliposomes without PEG. Based on these observations, we tentatively conclude that during the interaction of the immunoliposomes with the tumor cells the lipophilic prodrug FUdR-dP is selectively transferred to the cell surface and subsequently internalized by constitutive endocytic or pinocytic invaginations of the plasma membrane, thus ultimately delivering the prodrug to a lysosomal compartment where hydrolysis and release of parent drug takes place. This concept allows for an efficient delivery of a liposome-associated drug without the need for the liposome as such to be internalized by the cells.

On the mechanism of hepatic transendothelial passage of large liposomes.

Liposomes of 400 nm in diameter can cross the 100-nm fenestrations in the endothelium of the hepatic sinusoid, provided they contain phosphatidylserine (PS) but not phosphatidylglycerol (PG) [Daemen et al. (1997) Hepatology 26, 416]. We present evidence indicating that (i) the PS effect does not involve a pharmacological action of this lipid on the size of the fenestrations, (ii) fluid-type but not solid-type PS liposomes have access to the hepatocytes and (iii) the lack of uptake of PG liposomes by hepatocytes is not due to a lack of affinity of the hepatocytes for PG surfaces. We conclude that the mechanism responsible for the uptake of large PS-containing liposomes by hepatocytes in vivo involves a mechanical deformation of these liposomes during their passage across the endothelial fenestrations.

Water-soluble biodegradable cationic polyphosphazenes for gene delivery.

Polyphosphazenes bearing cationic moieties were synthesized from poly(dichloro)phosphazene, which in turn was obtained by thermal polymerization of hexachlorocyclotriphosphazene in 1,2,4-trichlorobenzene. Next, either 2-dimethylaminoethanol (DMAE) or 2-dimethylaminoethylamine (DMAEA) side groups were introduced by a substitution reaction. The polymers were purified by dialysis against water and tetrahydrofuran, lyophilized and evaluated as polymeric transfectants. The polyphosphazenes were able to bind plasmid DNA yielding positively charged particles (polyplexes) with a size around 80 nm at a polymer/DNA ratio of 3:1 (w/w). The polyphosphazene-based polyplexes were able to transfect COS-7 cells in vitro with an efficiency comparable to a well-known polymeric transfectant [poly(2-dimethylaminoethyl methacrylate), pDMAEMA]. The toxicity of both polyphosphazenes was lower than pDMAEMA. The transfection efficiency for the poly(DMAE)phosphazene-based polyplexes was about threefold higher in the absence of serum than in the presence of 5.0% fetal bovine serum. This is probably caused by unfavorable interactions of the polyplexes with serum proteins. In contrast, the poly(DMAEA)phosphazene-based polyplexes showed a threefold lower transfection activity in the absence of serum. For this system, serum proteins likely masked the toxicity of the polyplexes, as shown by the XTT cell viability assay and confocal laser scanning microscopy studies. Preliminary degradation studies indicate that the polymers were indeed degradable. The half-life at pH 7.5 and 37 degrees C was around 7 days for poly(DMAE)phosphazenes and 24 days for poly(DMAEA)phosphazenes. This study shows that polyphosphazenes are a suitable and promising new class of biodegradable polymeric carriers for gene delivery.

Uptake of long-circulating immunoliposomes, directed against colon adenocarcinoma cells, by liver metastases of colon cancer.

Radiolabeled ([3H]cholesteryloleyl ether) immunoliposomes directed against rat colon adenocarcinoma CC531 cells were prepared by random coupling of a tumor cell-specific antibody, CC52, via a thio ether bond. In vitro binding experiments demonstrated a saturable and specific interaction of CC52-immunoliposomes, which could be inhibited by free non-coupled CC52 but not by irrelevant antibodies. The in vivo targeting potential of CC52-immunoliposomes, which were pegylated to achieve prolonged circulation times, was tested in an established rat liver CC531 metastasis model. Twenty-four hours after injection of the liposomes, 25% of the CC52-immunoliposomes were still present in the blood, which was comparable with the control liposomes (either with or without antibody). Liposomes were mainly taken up from the blood by the liver and the spleen, although hepatic uptake of the immunoliposomes was higher and splenic uptake was lower as compared to liposomes without antibody. Within the metastatic tumor nodules in the liver, uptake of both the CC52-immunoliposomes and non-specific immunoliposomes was significantly higher than that of control liposomes without antibody. Visualization of fluorescently or gold labeled CC52-immunoliposomes revealed that, although targeting to liver metastases was achieved, the immunoliposomes were mostly not associated with tumor cells but rather localized in tumor associated cells, probably macrophages.

Peptide-targeted PEG-liposomes in anti-angiogenic therapy.

Peptides with the RGD amino acid sequence show affinity for the alpha(v)beta(3) integrin, an integrin which is over-expressed on angiogenic endothelium and involved in cell adhesion. A peptide with the sequence ATWLPPR has been demonstrated to show affinity for the vascular endothelial growth factor (VEGF) receptor, a receptor involved in the proliferation of endothelial cells. By coupling these peptides to liposomes, these liposomes can serve as a site-specific drug delivery system to tumor endothelial cells in order to inhibit angiogenesis. In the present study we demonstrate that the coupling of cyclic RGD-peptides or ATWLPPR-peptides to the surface of PEG-liposomes results in binding of these liposomes to endothelial cells in vitro. Subsequent studies with RGD-peptide targeted liposomes in vivo also demonstrate specific binding to the tumor endothelium.

Uptake and intracellular processing of PEG-liposomes and PEG-immunoliposomes by kupffer cells in vitro 1 *.

Specific targeting of drugs to for instance tumors or sites of inflammation may be achieved by means of immunoliposomes carrying site-specific antibodies on their surface. The presence of these antibodies may adversely affect the circulation kinetics of such liposomes as a result of interactions with cells of the mononuclear phagocyte system (MPS), mainly represented by macrophages in liver and spleen. The additional insertion of poly(ethylene glycol) chains on the surface of the immunoliposomes may, however, attenuate this effect. We investigated the influence of surface-coupled rat or rabbit antibodies and of PEG on the uptake of liposomes by rat Kupffer cells in culture with (3)H-cholesteryloleyl ether as a metabolically stable marker. Additionally, we assessed the effects of surface-bound IgG and PEG on the intracellular processing of the liposomes by the Kupffer cells, based on a double-label assay using the (3)H-cholesteryl ether as an absolute measure for liposome uptake and the hydrolysis of the degradable marker cholesteryl-(14)C-oleate as relative measure of degradation. Attachment of both rat and rabbit antibodies to PEG-free liposomes caused a several-fold increase in apparent size. The uptake by Kupffer cells, however, was 3-4 fold higher for the rat than for the rabbit IgG liposomes. The presence of PEG drastically reduced the difference between these liposome types. Uptake of liposomes without antibodies amounted to only about 10% (non-PEGylated) or less (PEGylated) of that of the immunoliposomes. In contrast to the marked effects of IgG and PEG on Kupffer cell uptake, the rate of intracellular processing of the liposomes remained virtually unaffected by the presence of these substances on the liposomal surface. These observations are discussed with respect to the design of optimally formulated liposomal drug preparations, combining maximal therapeutic efficacy with minimal toxicity.

Expression and prognostic significance of thymidylate synthase (TS) in pancreatic head and periampullary cancer.

BACKGROUND: Pancreatic cancer has a dismal prognosis. Attempts have been made to improve outcome by several 5-FU based adjuvant treatment regimens. However, the results are conflicting. There seems to be a continental divide with respect to the use of 5-FU based chemoradiotherapy (CRT). Furthermore, evidence has been presented showing a different response of pancreatic head and periampullary cancer to 5-FU based CRT. Expression of thymidylate synthase (TS) has been associated with improved outcome following 5-FU based adjuvant treatment in gastrointestinal cancer. This prompted us to determine the differential expression and prognostic value of TS in pancreatic head and periampullary cancer. PATIENTS AND METHODS: TS protein expression was studied by immunohistochemistry on original paraffin embedded tissue from 212 patients following microscopic radical resection (R0) of pancreatic head (n = 98) or periampullary cancer (n = 114). Expression was investigated for associations with recurrence free (RFS), cancer specific (CSS) and overall survival (OS), and conventional prognostic factors. RESULTS: High cytosolic TS expression was present in 26% of pancreatic head tumours and 37% of periampullary tumours (p = .11). Furthermore, TS was an independent factor predicting favourable outcome following curative resection of pancreatic head cancer (p = .003, .001 and .001 for RFS, CSS and OS, respectively). In contrast, in periampullary cancer, TS was not associated with outcome (all p > .10). CONCLUSION: TS, was found to be poorly expressed in both pancreatic head and periampullary cancer and identified as an independent prognostic factor following curative resection of pancreatic head cancer.

Fluorescence in situ hybridization to monitor the intracellular location and accessibility of plasmid DNA delivered by cationic polymer-based gene carriers.

Information about the intracellular trafficking of exogenous DNA delivered by nonviral gene delivery systems is of major importance for optimization of such gene carriers. We used fluorescence in situ hybridization (FISH) as a tool to visualize polyplex-delivered pDNA inside cells. This avoids the need to directly label DNA inside the polyplexes, which may influence their cellular behavior and fate. Using FISH the introduced plasmid DNA could be detected in the cytosol and nucleus of different cell lines. The FISH probe itself did not interact with cells nor different polymers used for condensing the DNA. We further demonstrate differences in accessibility of polyplex-delivered DNA when different polymers were used for DNA complexation. Therefore, FISH is a valuable tool to detect location and accessibility of exogenous plasmid DNA delivered in the cell by cationic polymers.

Cellular uptake of cationic polymer-DNA complexes via caveolae plays a pivotal role in gene transfection in COS-7 cells.

PURPOSE: Knowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake. MATERIALS AND METHODS: The effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy. RESULTS: Both pDMAEMA- and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrin- and caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake. CONCLUSIONS: These results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.

An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems.

BACKGROUND: Transfection with non-viral gene delivery vectors, such as cationic polymers, generally results in low transgene expression in vivo. This is likely due to poor cytoplasmic transport and intra-nuclear DNA delivery. METHODS: In this study two strategies to improve nuclear import were investigated. Linear DNA constructs with or without an NLS peptide were prepared by PCR. Alternatively, linear DNA obtained by enzymatic cleavage followed by capping of both ends with DNA-hairpins was used. An NLS peptide was attached to one of the capped ends of the linear DNA. Both biodegradable (pDMAEAppz) and non-degradable polymers (PEI or pDMAEMA) were used to complex the DNA. Several cell types, dividing and non-dividing, were transfected with the linear DNA constructs containing a SV40-derived NLS peptide. Nuclear import of the DNA constructs was studied using digitonin-permeabilized cells. RESULTS: Linear DNA prepared by PCR proved not useful as it was degraded from the 3'end. Linear DNA capped with hairpins was more successful with regard to stability. However, Cells transfected with linear DNA constructs by electroporation or by using cationic polymers with linear DNA containing a NLS peptide, failed to show significantly higher luciferase expression levels when compared to cells transfected with plasmid DNA or linear DNA without an NLS peptide attached. No nuclear localization was observed in digitonin-permeabilized cells. CONCLUSION: Taken together, these data demonstrate that this nuclear localisation signal when attached to DNA is neither able to improve transfection efficiency of cationic polymers nor the nuclear import of the DNA constructs.

[Phototoxic reactions to 8-methoxypsoralen as occupational dermatosis]. / Phototoxische Reaktion auf 8-Methoxypsoralen als Berufsdermatose.

The case history of a 50-year-old assistant pharmacist, who had been working with 8-methoxypsoralene (8-MOP) without any protection, is discussed. Shortly after this work, he was exposed to sunlight. Within 72 h he developed a typical local reaction with painful erythema and vesiculation on the back of both hands, followed by hyperpigmentation a week later. Light testing on skin dusted with 8-MOP resulted in the same clinical picture. Histopathological examination of a lesion showed the characteristics of a phototoxic dermatitis. Guidelines for the prevention of complications in those who are professionally exposed to 8-MOP are emphasized.

A patient with primary syphilis of the hand.

The case history of a man with primary syphilis of the right hand is described. This type of luetic condition nowadays is rare. The incidence of genital, anorectal, and extragenital primary syphilis in both men and women and in sailors presenting at the Venereological Outpatient Clinic of the Department of Dermatology of the University Hospital Rotterdam-Dijkzigt during a period of seven years was studied. In sailors and women only genital primary lesions were found, whereas in male civilians 6.3% of cases of primary syphilis had anorectal lesions. The primary lesions in this study were different from those reported in earlier literature.

First experiences with single-dose treatment of vaginal trichomoniasis with carnidazole (R 25831).

This paper reports the results of treating 152 women with carnidazole, a new trichomonacidal drug. They were divided into two groups. The first comprised 91 patients who were treated with 2 g, while the second group comprised 61 patients who were treated with 1-5 g carnidazole in a single oral dose. Defaulter rates were 6-6% for the first group and 13-1% for the second. Of the remaining 85 patients in the first group, 76 (89-5%) were negative at the first follow-up one to three weeks after treatment, three (3-5%) were considered to be treatment failures, and six (7-0%) were considered to be reinfected. Of 53 women treated with 1-5 g, 39 (73-6%) were negative at first follow-up, eight (15-1%) were considered to be treatment failures, and six (11-3%) were considered to be reinfected. The difference between the number of patients cured in both groups is statistically significant. Fourteen patients experienced side-effects, but these were of little significance. Carnidazole given in a single oral dose of 2 g in 16 women did not cause consistent changes in any of the haematological and biochemical parameters studied.