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A microfluidic based system was developed for automated online method for the rapid detection and monitoring of drinking water contamination utilising microbial Adrenosine-5'-Triphosphate (ATP) as a bacterial indicator. The system comprises a polymethyl methacrylate based microfluidic cartridge inserted into an enclosure incorporating the functions of fluid storage and delivery, lysis steps and real-time detection. Design, integration and operation of the resulting automated system are reported, including the lysis method, the design of the mixing circuit, the choices of flow rate, temperature and reagent amount. Calibration curves of both total and free ATP were demonstrated to be highly linear over a range from 2.5-5000 pg/mL with the limit of detection being lower than 2.5 pg/mL of total ATP. The system was trialled in a lab study with different types of water, with lysis efficiency being found to be strongly dependent upon water type. Further development is required before online implementation.

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The initial appearance of subacute cutaneous lupus erythematosus (SCLE) skin lesions in conjunction with Ro/SS-A autoantibodies occurring as an adverse reaction to hydrochlorothiazide [i.e. drug-induced SCLE (DI-SCLE)] was first reported in 1985. Over the past decade an increasing number of drugs in different classes has been implicated as triggers for DI-SCLE. The management of DI-SCLE can be especially challenging in patients taking multiple medications capable of triggering DI-SCLE. Our objectives were to review the published English language literature on DI-SCLE and use the resulting summary data pool to address questions surrounding drug-induced SCLE and to develop guidelines that might be of value to clinicians in the diagnosis and management of DI-SCLE. A systematic review of the Medline/PubMed-cited literature on DI-SCLE up to August 2009 was performed. Our data collection and analysis strategies were prospectively designed to answer a series of questions related to the clinical, prognostic and pathogenetic significance of DI-SCLE. One hundred and seventeen cases of DI-SCLE were identified and reviewed. White women made up the large majority of cases, and the mean overall age was 58·0 years. Triggering drugs fell into a number of different classes, highlighted by antihypertensives and antifungals. Time intervals ('incubation period') between drug exposure and appearance of DI-SCLE varied greatly and were drug class dependent. Most cases of DI-SCLE spontaneously resolved within weeks of drug withdrawal. Ro/SS-A autoantibodies were present in 80% of the cases in which such data were reported and most remained positive after resolution of SCLE skin disease activity. No significant differences in the clinical, histopathological or immunopathological features between DI-SCLE and idiopathic SCLE were detected. There is now adequate published experience to suggest that DI-SCLE does not differ clinically, histopathologically or immunologically from idiopathic SCLE. It should be recognized as a distinct clinical constellation differing clinically and immunologically from the classical form of drug-induced systemic lupus erythematosus.

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BACKGROUND: The Utah Psoriasis Initiative (UPI) is an expanding database that is being used to identify and characterize phenotypic variants of psoriasis and explore genotype-phenotype relationships. We recently reported distinct morphological variants of psoriasis that are characterized by thickness of lesions (induration) in the untreated state. OBJECTIVES: To explore the clinical relevance of these morphological variants. METHODS: For these analyses, we used the phenotypic data from 282 additional subjects gathered at enrollment into the UPI and compared their phenotype with that of the original 500 patients reported previously. The analysis was further expanded via a longitudinal follow-up of 286 subjects from the original 500 case cohort. RESULTS: Firstly, the initial findings were confirmed. Expansion of the cohort used for the original observation by about 50% and reanalysis showed that there was no alteration in the proportions of patients expressing thin- and thick-plaque disease phenotypes. Secondly, analysis of the larger cohort showed that this morphological phenotype had clinical relevance: those patients with thin-plaque disease were more likely to report a complete therapeutic response to topical corticosteroids and phototherapy. In contrast, plaque thickness did not appear to be a factor in response to systemic agents. CONCLUSIONS: Using a patient's baseline plaque morphology to choose a primary treatment modality may result in earlier disease improvement and reduce the cost of therapy.

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The ability of a growth factor antagonist, [D-Arg(6),D-Trp(7,9)-N(me)Phe(8)]-substance P(6-11), named antagonist G, to selectively target polyethylene glycol-grafted liposomes (known as sterically stabilized liposomes) to a human classical small cell lung cancer (SCLC) cell line, H69, was examined. Our results showed that radiolabeled antagonist G-targeted sterically stabilized liposomes (SLG) bound to H69 cells with higher avidity than free antagonist G and were internalized (reaching a maximum of 13000 SLG/cell), mainly through a receptor-mediated process, likely involving clathrin-coated pits. This interaction was confirmed by confocal microscopy to be peptide- and cell-specific. Moreover, it was shown that SLG significantly improved the nuclear delivery of encapsulated doxorubicin to the target cells, increasing the cytotoxic activity of the drug over non-targeted liposomes. In mice, [(125)I]tyraminylinulin-containing SLG were long circulating, with a half-life of 13 h. Use of peptides like antagonist G to promote binding and internalization of sterically stabilized liposomes, with their accompanying drug loads, i.e., anticancer drugs, genes or antisense oligonucleotides, into target cells has the potential to improve therapy of SCLC.

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Endocytosis and recycling of coagulation factor VIIa (VIIa) bound to tissue factor (TF) was investigated in baby hamster kidney (BHK) cells stably transfected with TF or TF derivatives. Cell surface expression of TF on BHK cells was required for VIIa internalization and degradation. Approximately 50% of cell surface-bound VIIa was internalized in one hour, and a majority of the internalized VIIa was degraded soon thereafter. Similar rates of VIIa internalization and degradation were obtained with BHK cells transfected with a cytoplasmic domain-deleted TF variant or with a substitution of serine for cysteine at amino acid residue 245 (C245S). Endocytosis of VIIa bound to TF was an active process. Acidification of the cytosol, known to inhibit the internalization via clathrin-coated pits, did not affect the internalization of VIIa. Furthermore, receptor-associated protein, known to block binding of all established ligands to members of the low-density lipoprotein receptor family, was without an effect on the internalization of VIIa. Addition of tissue factor pathway inhibitor/factor Xa complex did not affect the internalization rate significantly. A substantial portion (20% to 25%) of internalized VIIa was recycled back to the cell surface as an intact and functional protein. Although the recycled VIIa constitutes to only approximately 10% of available cell surface TF/VIIa sites, it accounts for 65% of the maximal activation of factor X by the cell surface TF/VIIa. In summary, the present data provide evidence that TF-dependent internalization of VIIa in kidney cells occurs through a clathrin-independent mechanism and does not require the cytoplasmic domain of TF.

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A single amino acid mutation (G156S) in the putative pore-forming region of the G protein-sensitive, inwardly rectifying K(+) channel subunit, GIRK2, renders the conductance constitutively active and nonselective for monovalent cations. The mutant channel subunit (GIRK2wv) causes the pleiotropic weaver disease in mice, which is characterized by the selective vulnerability of cerebellar granule cells and Purkinje cells, as well as dopaminergic neurons in the mesencephalon, to cell death. It has been proposed that divalent cation permeability through constitutively active GIRK2wv channels contributes to a rise in internal calcium in the GIRK2wv-expressing neurons, eventually leading to cell death. We carried out comparative studies of recombinant GIRK2wv channels expressed in Xenopus oocytes and COS-7 cells to determine the magnitude and relative permeability of the GIRK2wv conductance to Ca(2+). Data from these studies demonstrate that the properties of the expressed current differ in the two systems and that when recombinant GIRK2wv is expressed in mammalian cells it is impermeable to Ca(2+).

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Recent studies have shown a discrepancy between the level of tissue factor (TF) expression and the level of TF procoagulant activity on the apical and basolateral surface domains of polarized epithelial cells. The present investigation was performed to elucidate possible reasons for the discordant expression of TF and its activity on the surface of polarized epithelial cells using a human intestinal epithelial cell line, Caco-2 and Madin-Darby canine kidney epithelial cells, type II (MDCK-II). Functional activity of coagulation factor VIIa (VIIa) in complex with TF was 6- to 7-fold higher on the apical than the basolateral surface in polarized Caco-2 cells. In contrast, no significant difference was found in the formation of TF/VIIa complexes between the apical and basolateral surface. Confocal microscopy of Caco-2 cells showed TF expression on both the apical and the basolateral surface domains. Studies with MDCK-II cells showed that the specific functional activity of TF expressed on the apical cell surface was 5-fold higher than on the basolateral surface. To test whether differential expression of TF pathway inhibitor (TFPI) on the apical and basolateral surface could account for differences in TF/VIIa functional activity, we measured cell-surface-bound TFPI activity in Caco-2 cells. Small but similar amounts of TFPI were found on both surfaces. Further, addition of inhibitory anti-TFPI antibodies induced a similar enhancement of TF/VIIa activity on both surface domains. Because the availability of anionic phospholipids on the outer leaflet of the cell membrane could regulate TF/VIIa functional activity, we measured the distribution of anionic phospholipids on the apical and basolateral surface by annexin V binding and thrombin generation. The results showed that the anionic phospholipid content on the basolateral surface, compared with the apical surface, was 3- to 4-fold lower. Mild acid treatment of polarized Caco-2 cells, which markedly increased the anionic phospholipid content on the basolateral surface membrane, increased the TF/VIIa activity on the basolateral surface without affecting the number of TF/VIIa complexes formed on the surface. Overall, our data suggest that an uneven expression of TF/VIIa activity between the apical and basolateral surface of polarized epithelial cells is caused by differences in anionic phospholipid content between the two surface domains and not from a polar distribution of TFPI.

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The effects of endotoxin injury on lung NMR relaxation times (T1, CPMG T2, and Hahn decay constant (Hahn T2)) were studied in excised unperfused rat lungs. Blinded histologic examination showed no clear-cut separation between endotoxin and control lungs. Morphometric lung tissue volume density and gravimetric lung water content did not differ significantly between the two groups. In contrast, the values of the fast, intermediate, and slow T2 components, obtained by multiexponential analysis of the CPMG decay curve, increased markedly after endotoxin administration, with minimal overlap between endotoxin and control values. The response of Hahn T2 was, in general, in the same direction as that of CPMG T2; however, Hahn T2 may be more affected by measurement errors and may be less sensitive to the presence of lung injury. T1 showed minimal changes after injury. The present data suggest that CPMG T2 measurements can consistently detect the presence of lung injury even when conventional histologic, morphometric, and gravimetric studies provide negative or equivocal results, and that the CMPG T2 method is superior, in this respect, to the Hahn decay method. T1 does not appear to be sensitive to lung injury in the absence of significant lung water accumulation.

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Two amphipatic polymers of the poly(2-oxazoline) family, poly(2-methyl-2-oxazoline) (PMOZ) and poly(2-ethyl-2-oxazoline) (PEOZ), were synthesized with the carboxylic group positioned at either the initiation or termination ends of the polymer chains. Distearoylphosphatidylethanolamine was covalently linked to the carboxyl groups of the polymers, resulting in conjugates which incorporate readily into liposomes. Systematic evaluation of plasma clearance kinetics and biodistribution of liposomes containing hydrogenated soy phosphatidylcholine, cholesterol, and 5 mol % the polymer-lipid conjugates in mice revealed the following. Both polymers, PMOZ and PEOZ, exhibited long plasma lifetimes and low hepatosplenic uptake. PMOZ was more effective at decreasing blood clearance rates than PEOZ. The best results, which were quantitatively comparable to the results obtained with the optimized preparations of methoxypolyethylene glycol(PEG)-2000-grafted liposomes, were obtained with formulations containing PMOZ of molecular weight 3260.

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Several coupling methods for binding antibodies (Ab) to liposomes have previously been developed. We were interested in examining if some of these methods would be suitable for attaching Ab to long-circulating formulations of liposomes (SL), sterically stabilized with poly(ethylene glycol) (PEG). We studied three 'classical' coupling methods in which Ab was attached at the bilayer surface of SL, and two new coupling methods in which Ab was attached at the PEG terminus. Parameters examined including binding efficiency, antibody surface density, the ability of the immunoliposomes to remote-load the anticancer drug doxorubicin, and the specific binding of the resulting immunoliposomes to target cells. The non-covalent biotin-avidin coupling method resulted in low Ab densities at the cell surface, as did a coupling in method in which maleimide-derivatized Ab was attached to the liposome surface through a thiolated phospholipid incorporated into the liposomes. The low levels of Ab achieved in these method was likely due to interference by PEG with the access of the Ab to the liposome surface. However, when a maleimide-derivatized Ab was coupled to thiolated PEG, moving the coupling reaction away from the liposome surface, very high coupling efficiencies were achieved, and these immunoliposomes achieved good specific binding to their target cells. Oxidizing the Fc region of the Ab and coupling it to the PEG terminus through a hydrazone bond was a less efficient coupling method, but had the advantage of retaining Ab orientation. Efficient remote-loading of doxorubicin was found for immunoliposomes in which Ab was attached at the PEG terminus.

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The development of long-circulating formulations of liposomes (S-liposomes), sterically stabilized with lipid derivatives of poly(ethylene glycol) (PEG), has increased the likelihood that these liposomes, coupled to targeting ligands such as antibodies, could be used as drug carriers to deliver therapeutic drugs to specific target cell populations in vivo. We have developed a new methodology for attaching monoclonal antibodies to the terminus of PEG on S-liposomes. A new end-group functionalized PEG-lipid derivative pyridylthiopropionoylamino-PEG- distearoylphosphatidylethanolamine (PDP-PEG-DSPE) was synthesized for this purpose. Incorporation of PDP-PEG-DSPE into S-liposomes followed by mild thiolysis of the PDP groups resulted in formation of reactive thiol groups at the periphery of the lipid vesicles. Efficient attachment of maleimide-derivatized antibodies took place under mild conditions even when the content of the functionalized PEG-lipid in S-liposomes was below 1% of total lipid. The resulting S-immunoliposomes showed efficient drug remote loading, slow drug release rates and increased survival times in circulation compared to liposomes lacking PEG. When antibodies recognizing several different tumor-associated antigens were coupled to the PEG terminus of S-liposomes a significant increase in the in vitro binding of liposomes to the target cells was observed. The binding of S-immunoliposomes containing entrapped doxorubicin to their target cell population resulted in increased cytotoxicity compared to liposomes lacking the targeting antibody.

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The development of long-circulating liposomes containing lipid derivatives of poly(ethylene glycol) (PEG), termed Stealth liposomes, has considerably improved the prospects for therapeutic applications of liposomal drug delivery systems. We have examined the pharmacokinetics and biodistribution of long-circulating, as compared to conventional, liposomes after subcutaneous (sc) administration in mice. Results obtained after subcutaneous administration were compared to those obtained after intravenous (iv) and intraperitoneal (ip) administration. Liposomes, following sc administration, appeared intact in the circulation subsequent to moving down the lymph node chains that drain the site of injection. Liposomes containing PEG-distearoylphosphatidylethanolamine (PEG-DSPE) resulted in the highest levels of small (80-90 nm) liposomes in the blood, with up to 30% of vivo label appearing in the blood at 12 to 24 h post-injection. In the absence PEG-DSPE approx. 4-fold lower levels of liposomes were found in the blood. Small size of the liposomes was critical to their ability to move into the circulation, with liposomes above 110-120 nm not appearing in blood to any significant extent. The presence of PEG-DSPE and cholesterol was important for the in vivo stability of the liposome after sc administration. Although liposome levels were significantly higher in the draining lymph nodes after sc administration, levels associated with other tissues were proportionately reduced relative to the iv and ip routes of administration. Liposomes appeared in blood after ip and sc administration with half-lives of approx. 0.6 and 9 h, respectively, and subsequent to appearing in blood had similar biodistribution, pharmacokinetics and half-lives (20.4 h) to liposomes given by the iv route.

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Chronic lung disease in prematurely born infants, defined as the need for increased inspired oxygen at 28 days of age, was thought to be more common in some institutions than in others. To test this hypothesis, we surveyed the experience in the intensive care nurseries at Columbia and Vanderbilt Universities, the Universities of Texas at Dallas, Washington at Seattle, and California at San Francisco, the Brigham and Women's Hospital in Boston, Texas Children's Hospital in Houston, and Mt Sinai Hospital in Toronto. The survey included 1,625 infants with birth weights of 700 to 1,500 g. We confirmed the relationship of risk to low birth weight, white race, and male sex. Significant differences in the incidence of chronic lung disease were found between institutions even when birth weight, race, and sex were taken into consideration through a multivariate logistic regression analysis. Columbia had one of the best outcomes for low birth weight infants and the lowest incidence of chronic lung disease.

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We have previously documented the development and subsequent disappearance of cytolytic activity mediated by lymphocytes from lymph nodes draining Moloney sarcomas destined either to regress or grow progressively. We now report that these tumour-draining lymphnode cells (LNC) that were no longer cytotoxic, spontaneously regenerated peak levels of killing after culture in vitro for 4 days in the absence of exogenous tumour antigen. Cytolytic activity, which was antigenically specific, was mediated by T lymphocytes. Resurgence of cytolytic activity in vitro was accompanied by proliferative changes (DNA synthesis, blast transformation, cell division) which peaked on the 3rd day of culture. Although normal, nonimmune LNC underwent quantitatively similar proliferative changes in culture, the killing that developed was weak and antigenically nonspecific. Transfer of cultured, tumour-draining LNC to immunologically compromised, syngeneic mice conferred complete protection from Moloney sarcoma progression. Adoptive transfer could be delayed for 6 days after tumour induction without loss of protection. These results suggest that there exists in Moloney sarcoma-bearing mice a mechanism that limits the differentiation of pre-killer cells into cytolytically active T lymphocytes, and that such inhibition is eliminated when LNC are explanted into culture.

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Highly purified suspensions of intratumoral T lymphocytes, recovered 11 and 13 days after induction of regressing or progressing Moloney sarcomas, were compared in their ability to lyse specifically the MSC cells used for tumor induction. Cytolytic activity, expressed in terms of lytic units/10(6) T cells, was similar for intratumoral T cell suspensions obtained 11 days after induction of either regressing (3.1 +/- 1.3 LU/10(6) T cells) or progressing (4.3 +/- 1.8) neoplasms. By 13 days post-induction, regressing tumors contained T lymphocytes with an increased cytolytic activity (11.1 +/- 4.5) whereas those from progressing tumors were strikingly less able to kill MSC cells (less than or equal to 0.2). This dramatic loss in cytotoxicity could not be attributed to errors associated with the enzymatic disaggregation method, inhibition by copurified endogenous tumor cells, or immunosuppression induced by viral infection. The changes in functional activity of intratumoral T lymphocytes from the two types of sarcoma appeared to be correlated with the stage of neoplasia. In this model system, cytolytic activity of T lymphocytes increased during spontaneous tumor regression whereas losses in cytotoxicity occurred coincident with the onset of inexorable progression.

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A combination of two cell separation methods was utilized for the isolation of thymus-derived lymphocytes (TL) from enzymatically disaggregated tumors. Passage through Sephadex G-10-glass bead columns to remove adherent cell types followed by exposure to IgG-coated sheep red blood cell monolayers for removal of Fc receptor-bearing inflammatory cells provided functional TL suspensions of high purity with good percentage recovery.

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Regressing and progressing Moloney sarcomas, induced in BALB/c mice by the injection of cultured sarcoma cells (MSC)1, were sampled for histologic analysis and then disaggregated using mixtures of trypsin, collagenase and DNAse or collagenase and DNAse alone. The types of inflammatory cells (IC) found in resultant cell suspensions were determined 6, 11, 14 and 18 days post inoculation. Inflammatory infiltrates were composed almost exclusively of three cell types; neutrophils, T lymphocytes and macrophages. The extent to which each was found in tumors was related to the time post inoculation. Neutrophils were part of an early acute inflammatory response seen in both developing regressing and progressing sarcomas. The onset of regression was associated histologically with the appearance within tumors of a mononuclear inflammatory infiltrate. T lymphocytes and macrophages were the principal constituents. A higher percentage of T lymphocytes was recovered at all sampling times from regressing, compared to progressing, sarcomas. During development of the mononuclear inflammatory infiltrate there were relatively more large T cells in regressing, than in progressing tumors, and the percentage of macrophages was higher. Thereafter, the proportion of macrophages in the recovered cell population was approximately the same for both types of tumor. Such equality was more apparent than real, however, since IC were restricted to the peripheries of progressing sarcomas after the acute inflammatory phase, but continued to be found throughout regressing neoplasms. The effective ratio of macrophages and T lymphocytes to tumor cells therefore was much lower in progressing sarcomas than was suggested by percentage figures. The data presented support the concept that T lymphocytes are instrumental in causing the regression of Moloney sarcomas, possibly through interactions with macrophages.

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