Aerosol delivery of star polymer-siRNA nanoparticles as a therapeutic strategy to inhibit lung tumor growth.

Lung cancer is a major contributor to cancer-related death worldwide. siRNA nanomedicines are powerful tools for cancer therapeutics. However, there are challenges to overcome to increase siRNA delivery to solid tumors, including penetration of nanoparticles into a complex microenvironment following systemic delivery while avoiding rapid clearance by the reticuloendothelial system, and limited siRNA release from endosomes once inside the cell. Here we characterized cell uptake, intracellular trafficking, and gene silencing activity of miktoarm star polymer (PDMAEMA-POEGMA) nanoparticles (star nanoparticles) complexed to siRNA in lung cancer cells. We investigated the potential of nebulized star-siRNA nanoparticles to accumulate into orthotopic mouse lung tumors to inhibit expression of two genes [ßIII-tubulin, Polo-Like Kinase 1 (PLK1)] which: 1) are upregulated in lung cancer cells; 2) promote tumor growth; and 3) are difficult to inhibit using chemical drugs. Star-siRNA nanoparticles internalized into lung cancer cells and escaped the endo-lysosomal pathway to inhibit target gene expression in lung cancer cells in vitro. Nebulized star-siRNA nanoparticles accumulated into lungs and silenced the expression of ßIII-tubulin and PLK1 in mouse lung tumors, delaying aggressive tumor growth. These results demonstrate a proof-of-concept for aerosol delivery of star-siRNA nanoparticles as a novel therapeutic strategy to inhibit lung tumor growth.

Alpha-toxin is produced by skin colonizing Staphylococcus aureus and induces a T helper type 1 response in atopic dermatitis.

BACKGROUND: Staphylococcus aureus is a well known trigger factor of atopic dermatitis (AD). Besides the superantigens, further exotoxins are produced by S. aureus and may have an influence on the eczema. OBJECTIVE: To explore the impact of staphylococcal alpha-toxin on human T cells, as those represent the majority of skin infiltrating cells in AD. METHODS: Adult patients with AD were screened for cutaneous colonization with alpha-toxin producing S. aureus. As alpha-toxin may induce necrosis, CD4(+) T cells were incubated with sublytic alpha-toxin concentrations. Proliferation and up-regulation of IFN-gamma on the mRNA and the protein level were assessed. The induction of t-bet translocation in CD4(+) T cells was detected with the Electrophoretic Mobility Shift Assay. RESULTS: Thirty-four percent of the patients were colonized with alpha-toxin producing S. aureus and alpha-toxin was detected in lesional skin of these patients by immunohistochemistry. Sublytic alpha-toxin concentrations induced a marked proliferation of isolated CD4(+) T cells. Microarray analysis indicated that alpha-toxin induced particularly high amounts of IFN-gamma transcripts. Up-regulation of IFN-gamma was confirmed both on the mRNA and the protein level. Stimulation of CD4(+) T cells with alpha-toxin resulted in DNA binding of t-bet, known as a key transcription factor involved into primary T helper type 1 (Th1) commitment. CONCLUSION: alpha-toxin is produced by S. aureus isolated from patients with AD. We show here for the first time that sublytic alpha-toxin concentrations activate T cells in the absence of antigen-presenting cells. Our results indicate that alpha-toxin is relevant for the induction of a Th1 like cytokine response. In AD, this facilitates the development of Th1 cell dominated chronic eczema.

Prospective investigation of autonomic cardiac neuropathy in diabetes mellitus.

Forty-five patients with clinically manifest diabetes mellitus were investigated (25 male, 20 female, 48 +/- 10 yrs, 14 diabetes type 1, 31 type 2). Duration of manifestation was 12.2 +/- 9.7 yrs.Vibration thresholds and thermal thresholds were assessed. Respiratory sinus arrhythmia (RSA) was measured during deep respiration at 6/min. The QTc-interval was assessed according to Bazett's formula. MIBG-SPECT was carried out in all 45 cases. Patients with abnormal MIBI perfusion scintigraphy had previously been excluded from the study. RSA was abnormal in 12/45 patients. The MIBG-SPECT was abnormal in 28/45 cases with dorso-septal lack of activity. No difference was seen between type 1 and 2 diabetics with regard to either vibration and thermal thresholds or RSA and MIBG-SPECT. Abnormal MIBG-SPECT was correlated with vibration threshold and abnormal heart RSA tests but not with abnormality in QTc. The mean QTc-interval was 419 +/- 24 ms (QTc normal in 36, abnormal > or = 440ms in 9). It was longer in female than in male patients. There exists no significant correlation of QTc-interval results with either heart rate variability or MIBG-SPECT. The QTc-interval is not a sensitive parameter of autonomic cardiac denervation.

Pancreatic islets express a Ca2+-independent phospholipase A2 enzyme that contains a repeated structural motif homologous to the integral membrane protein binding domain of ankyrin.

Pancreatic islets express a Ca2+-independent phospholipase A2 (CaI-PLA2) activity that is sensitive to inhibition by a haloenol lactone suicide substrate that also attenuates glucose-induced hydrolysis of arachidonic acid from islet phospholipids and insulin secretion. A cDNA has been cloned from a rat islet cDNA library that encodes a protein with a deduced amino acid sequence of 751 residues that is homologous to a CaI-PLA2 enzyme recently cloned from Chinese hamster ovary cells. Transient transfection of both COS-7 cells and Chinese hamster ovary cells with the cloned islet CaI-PLA2 cDNA resulted in an increase in cellular CaI-PLA2 activity, and this activity was susceptible to inhibition by haloenol lactone suicide substrate. The domain of the islet CaI-PLA2 from amino acid residues 150-414 is composed of eight stretches of a repeating sequence motif of approximately 33-amino acid residues in length that is highly homologous to domains of ankyrin that bind both tubulin and integral membrane proteins, including several proteins that regulate ionic fluxes across membranes. These findings complement previous pharmacologic observations that suggest that CaI-PLA2 may participate in regulating transmembrane ion flux in glucose-stimulated beta-cells.

Characterization of expression of phosphofructokinase isoforms in isolated rat pancreatic islets and purified beta cells and cloning and expression of the rat phosphofructokinase-A isoform.

Phosphofructokinase (PFK) plays a key role in regulating glycolytic flux, and the mammalian enzyme is a tetramer. Three monomeric isoforms are encoded by separate genes, are differentially expressed in specific tissues, and are designated by tissues in which they are most abundant (A, muscle; B, liver; and C, brain). Glucose-induced insulin secretion from pancreatic islets requires glucose transport into islet beta-cells and glycolytic metabolism. Little is known about islet PFK isozymes, but the possibility that PFK-A is expressed in beta-cells is of interest because that isoform is thought to govern glycolytic oscillations and to interact with a metabolically activated beta-cell phospholipase A2 enzyme. Using as probe a PCR product generated from rat islet RNA with primers designed from the human PFK-A sequence, we have cloned a full-length PFK-A cDNA from a rat islet cDNA library. The rat PFK-A deduced amino-acid sequence is 96% identical to that of human PFK-A, and all residues thought to participate in substrate or allosteric effector binding are conserved between the two sequences. The rat PFK-A amino-acid sequence is 69% and 68% identical to those for rat PFK-B and rat PFK-C, respectively, and differences in residues involved in binding of allosteric effectors were observed among the three isoforms. Rat PFK-A expressed as a glutathione-S-transferase fusion protein was recognized by antibodies raised against a peptide in the PFK-A sequence. Expression of PFK isoform mRNA species was examined by RT-PCR in rat islets, in purified populations of beta-cells prepared by fluorescence-activated cell sorting (FACS), and in RIN-m5F insulinoma cells, all of which expressed mRNA species for PFK-A, -B, and -C isoforms. PFK-A mRNA was expressed at much lower levels in an islet alpha-cell-enriched population. Interleukin-1 impairs islet glucose metabolism and insulin secretion and was found to induce a specific decline in islet expression of PFK-A mRNA. These findings establish the sequence of rat PFK-A, demonstrate that it is expressed in FACS-purified islet beta-cells, and suggest that its expression is regulated by a cytokine which influences insulin secretion.

Isotope dilution mass spectrometric measurements indicate that arachidonylethanolamide, the proposed endogenous ligand of the cannabinoid receptor, accumulates in rat brain tissue post mortem but is contained at low levels in or is absent from fresh tissue.

Arachidonylethanolamide (AEA) isolated from porcine brain binds to cannabinoid receptors, mimics cannabinoid pharmacologic effects, and has been proposed as an endogenous cannabinoid receptor ligand. Demonstration of co-distribution of AEA and cannabinoid receptors in various brain regions could provide supportive evidence for this role. We have performed isotope dilution mass spectrometric measurements of AEA and have demonstrated AEA production by rat tissue homogenates in vitro from exogenous arachidonate and ethanolamine. No detectable endogenous AEA (<3.5 pmol/g of tissue) was observed in fresh rat brain, whether or not inhibitors of AEA hydrolysis were present during tissue processing. AEA (>1 nmol/g) was produced during saponification of brain phospholipid extracts. This appears not to reflect hydrolysis of N-arachidonylethanolamine phospholipid precursors of AEA, because Streptomyces chromfucsis phospholipase D, which is active against NAPE, failed to generate AEA from brain phospholipids despite substantial conversion of phospholipids to phosphatidic acid. Such experiments suggested that the abundance of N-arachidonylethanolamine phospholipid in fresh rat brain may be less than 1 in 10(6) phospholipid molecules. AEA generated during saponification of tissue phospholipids appears to arise from base-catalyzed aminolysis of arachidonate-containing glycerolipids, because AEA was produced from synthetic (1-stearoyl, 2-arachidonoyl)-phosphatidylethanolamine under saponification conditions, and the amount produced increased 300-fold when free ethanolamine was included in the hydrolysis solution. Although AEA was not detectable (<0.17 pmol/mg of protein) in fresh rat brain, AEA accumulated post mortem to levels of 126 pmol/mg of brain protein. These findings do not exclude the possibility that AEA is rapidly synthesized and degraded locally in vivo, but they indicate that the AEA content of fresh rat brain and of NAPE precursors from which AEA might be derived are exceedingly low and that AEA can be produced artifactually from biological materials.

Responsiveness of an SV40-immortalized hepatocyte cell line to growth hormone.

The response of an SV40-immortalized hepatocyte cell line (CWSV-1) derived from adult male rat hepatocytes to human growth hormone (hGH) was investigated. CWSV-1 cells, which have been characterized extensively, retain certain differentiated functions of normal liver (Woodworth and Isom, Mol Cell Biol 7: 3740-3748, 1987). This cell line consists of tightly associated polygonal, mononucleated cells that grow as monolayers. These cells showed no significant morphological changes with the addition of hGH. Northern blot analysis showed that continuous treatment of the CWSV-1 cells with hGH induced the expression of insulin-like growth factor I (IGF-I) and 5 alpha-reductase RNAs. In addition, continuous exposure to hGH resulted in the induction of expression of the growth hormone receptor/growth hormone binding protein (GHR/GHBP) genes. This study indicates that the CWSV-1 cells may serve as a valuable in vitro model system for studying the signaling pathway of GH.

Enrichment of selected serum fatty acids after a small oral dosage of (1-13C)- and (8-13C)triolein in human volunteers analysed by gas chromatography/combustion isotope ratio mass spectrometry.

In this study the fate of 100 mg orally administered (1-13C)- and (8-13C)triolein was traced in the serum lipids of four healthy human subjects. After an overnight fast the subjects consumed hourly meals of a liquid formula diet over 12 h. Ninety minutes after the first meal in the first study (1-13C)triolein was given and in the repeat study the same subject received (8-13C)triolein. Triacylglycerol (TG), phospholipid (PL) and cholesterol ester (CE) were isolated from serum sampled prior to and in intervals after isotope administration. Fatty acid composition of serum lipids was measured using gas chromatography/mass spectrometry. 13C enrichment in palmitic, stearic, oleic and linoleic acids of these fractions was determined by gas chromatography/isotope ratio mass spectrometry. With (8-13C)triolein a significantly higher enrichment (peak +17.1 +/- 14.3/1000 delta 13C) was found in the oleic acid of TG fraction than with (1-13C)triolein (peak -7.1 +/- 4.2/1000), which may be due to a faster elimination of (1-13C)oleic acid from serum TG. 13C enrichments in the other fatty acids of the TG fraction as well as of PL and CE fractions were in the range of natural 13C abundance (-25 to -32/1000).

[Follow-up studies of patients with negative TRH test and normal thyroid hormone levels]. / Verlaufsbeobachtungen bei Patienten mit negativem TRH-Test und normalen Schilddrüsenhormonspiegeln.

The aim of the study was to assess the clinical significance of the association of a negative TRH-test with normal thyroid hormone levels. Therefore, we repeated the investigation in 63 patients one year after the first examination. In 6 patients (10%) hyperthyroidism had developed. Euthyroid autonomy was diagnosed in 11 patients (17%). A prolonged normalisation period of the pituitary-thyroid regulation after having discontinued the thyroid hormone drugs was found in 7 patients (11%). In 40% of the patients the TRH-test remained negative and in 22% of the patients the TSH-response became positive without any known reason. The findings show that a negative TRH-test and normal thyroid hormone levels do not necessarily imply subclinical hyperthyroidism.

Dependence of the carbon-isotope contents of breath carbon dioxide, milk, serum and rumen fermentation products on the delta 13C value of food in dairy cows.

Six dairy cows of two breeds were fed during three alternating periods with products from C3- and C4- plants to yield different natural 13C enrichments of the diet (delta 13C range: -28.0 to -13.7%). The resulting changes in the 13C enrichment of breath carbon dioxide, serum and milk of the animals followed the 13C:12C of the food, in agreement with the individual biological half-lives of those products, and established isotope discriminations. Breath CO2 was more enriched in 13C than expected. This could be related to isotope discriminations during rumen fermentation. From these results an isotopic balance model for the breath CO2 could be established.

Ligatin binds phosphohexose residues on acidic hydrolases.

Ligatin, a receptor that recognizes phosphorylated sugars, was isolated from plasma membranes of mouse macrophages, rat ileum, and rat brain. Several acidic hydrolases including N-acetyl beta-D-glucosaminidase (beta-NAG) were solubilized with this receptor. The solubilized beta-NAG bound to ligatin in vitro as demonstrated by affinity chromatography using the immobilized receptor. beta-N-Acetyl D-glucosaminidase-ligatin complexes were dissociated by low concentrations of mannose 6-phosphate (Man6P) and/or glucose 1-phosphate (Glc 1P). The effectiveness of these two phosphomonosaccharides varied depending on the source of the enzyme: ileal beta-NAG-ligatin complexes showed a four-fold preferential dissociation with Man6P; macrophage complexes showed a 160-fold preferential dissociation with Glc 1P. Brain complexes dissociated with nearly equal preference for Man6P and Glc 1P. Heterologous complexes displayed the specificity characteristic of the source of the enzyme regardless of the source of the ligatin. Treatment of the solubilized hydrolases with endoglucosaminidase H released phosphorous-32 label from these enzymes and prevented binding of beta-NAG to ligatin. However, treatment of the solubilized hydrolases with alkaline phosphatase reduced the binding of beta-NAG to ligatin by no more than 30%. This apparent resistance of beta-NAG to dephosphorylation was consistent with the chromatographic behavior of QAE of 3H-labeled acidic oligosaccharides isolated from the solubilized hydrolases. The oligosaccharides that contain phosphorylated hexose were less acidic than phosphomonoesters and were insensitive to alkaline phosphatase until subjected to acid hydrolysis. These results suggested the presence of a phosphodiester on beta-NAG analogous to the NAC glucosamine 1 P6 mannose present on beta-glucuronidase isolated from mouse lymphoma cells (Tabas I, Kornfield, S: J Biol Chem 255: 6633, 1980).