Supramolecular assemblies of zwitterionic nanoliposome-polynucleotide complexes as gene transfer vectors: Nanolipoplex formulation and in vitro characterisation.

Synthetic gene transfer vectors based on zwitterionic nanoliposome-DNA assemblies (nanolipoplexes), formed by the mediation of magnesium ions, were prepared by a scalable method without employing volatile solvents, high-shear force treatments or extrusion. The zwitterionic nanolipoplexes (NLP) were formulated with PC (phosphatidylcholine) and DPPC (a natural lung surfactant) incorporating different amounts of cholesterol (CHOL). The resulting structures were characterised in terms of their morphology, size and DNA content. In addition, the toxicity and transfection efficiency of the nanolipoplexes were evaluated in cultured Chinese hamster ovary-K1 (CHO-K1) cells. The effects of the multivalent cation Mg(2+) on nanoliposome-DNA transfection potency were evaluated. Formulations containing 10% CHOL showed maximum transfection efficiency and the optimum amount of Mg(2+) ions for transfection with minimum cytotoxicity was ca. 20 mM. The zwitterionic formulations showed significantly less cytotoxicity compared to a commercially available cationic liposome reagent or polyethylenimine (PEI) while they were superior in terms of gene transfer potency. The zwitterionic vectors formulated in this study avoid the use of toxic cationic lipids as well as toxic solvents and may have potential application in gene therapy. The new method will enable scale-up and manufacture of safe and efficacious transfection vehicles required for preclinical and clinical studies. Based on the advantages and superiority of the formulated nanolipoplexes, this method allows for the acceleration of nanolipoplex formulation, enabling the rapid development and evaluation of novel carrier systems for genes and other drugs.

Subchronic oral toxicity study of mixed tocopheryl phosphates in rats.

Rats were fed diets containing 0%, 1%, 3%, or 5% mixed tocopheryl phosphates for 90 days. No abnormal clinical signs related to treatment appeared. Some statistically significant changes in hematology and clinical chemistry parameters appeared, but the majority were not dose dependent, occurred in only one sex or group, and/or remained within the historical control range for this strain of rat. A statistically significant apparent reduction in blood protein was observed in animals treated with the tocopheryl phosphates, but further investigation showed that the test substance interfered with the protein assay. Repeat analysis using a method unaffected by plasma test substance levels showed no difference in plasma proteins among all groups. Gross necropsy revealed no abnormalities; reduced relative heart and epididymal weights were observed, but were not dose dependent and were considered incidental. Histopathological changes occurred only in the mesenteric lymph node and small intestine. Foreign material in a crystal-like form appeared in macrophages in both organs, and increased in a dose-related fashion. In the lymph node, sinus histiocytosis increased with dose, but the severity was similar between the control and low-dose groups. Foreign-body granulomatous inflammation, associated with Maltese cross birefringence of the crystals was seen in the mid- and high-dose animals, but not the low-dose group. Similarly, the small intestine showed increasing amounts of foreign material and inflammation in the mid- and high-dose but not in the 1% diet. The 1% diet (equivalent to 587 and 643 mg mixed tocopheryl phosphates/kg body weight/day for male and female rats, respectively) was considered the no observed adverse effect level.

Evaluation of the safety of mixed tocopheryl phosphates (MTP) -- a formulation of alpha-tocopheryl phosphate plus alpha-di-tocopheryl phosphate.

The safety of a formulation of mixed tocopheryl phosphates, (MTP) was evaluated in a series of toxicological tests in vivo using rats, mice and rabbits and in vitro using bacterial and mammalian cell cultures. The tests conducted included an oral LD(50) study, three 28-day oral repeat-dose studies, two dermal toxicity tests, an ocular irritation test, mutagenic potential tests, and chromosomal aberrations tests. MTP consists of mono alpha-tocopheryl phosphate (TP) and di-tocopheryl phosphate (T(2)P) and is intended for use as a dietary supplement and for dermal applications in humans and animals. The dermal and oral LD(50) values of MTP were determined to be >1130 mg/kg bw (918 mg tocopherol equivalents/kg bw) in rabbits and rats, respectively. MTP was not a dermal or eye irritant in rabbits and showed no allergenic potential in mice. In the mutagenicity and genotoxicity studies, MTP did not increased the number of revertants in Salmonella typhimurium or Escherichia coli and did not induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells. When administered daily for 28 days by gavage at doses up to 955 mg/kg bw/day (780 mg tocopherol equivalents/kg bw/day), MTP produced no consistent, dose-dependent adverse effects in rats.

The effects of human GH and its lipolytic fragment (AOD9604) on lipid metabolism following chronic treatment in obese mice and beta(3)-AR knock-out mice.

Both human GH (hGH) and a lipolytic fragment (AOD9604) synthesized from its C-terminus are capable of inducing weight loss and increasing lipolytic sensitivity following long-term treatment in mice. One mechanism by which this may occur is through an interaction with the beta-adrenergic pathway, particularly with the beta(3)-adrenergic receptors (beta(3)-AR). Here we describe how hGH and AOD9604 can reduce body weight and body fat in obese mice following 14 d of chronic ip administration. These results correlate with increases in the level of expression of beta(3)-AR RNA, the major lipolytic receptor found in fat cells. Importantly, both hGH and AOD9604 are capable of increasing the repressed levels of beta(3)-AR RNA in obese mice to levels comparable with those in lean mice. The importance of beta(3)-AR was verified when long-term treatment with hGH and AOD9604 in beta(3)-AR knock-out mice failed to produce the change in body weight and increase in lipolysis that was observed in wild-type control mice. However, in an acute experiment, AOD9604 was capable of increasing energy expenditure and fat oxidation in the beta(3)-AR knock-out mice. In conclusion, this study demonstrates that the lipolytic actions of both hGH and AOD9604 are not mediated directly through the beta(3)-AR although both compounds increase beta(3)-AR expression, which may subsequently contribute to enhanced lipolytic sensitivity.

Molecular and cellular actions of a structural domain of human growth hormone (AOD9401) on lipid metabolism in Zucker fatty rats.

A lipolytic domain (AOD9401) of human growth hormone (hGH) which resides in the carboxyl terminus of the molecule and contains the amino acid residues 177-191, has been synthesized using solid-phase peptide synthesis techniques. AOD9401 stimulated hormone-sensitive lipase and inhibited acetyl coenzyme A carboxylase (acetyl CoA carboxylase) in isolated rat adipose tissues, in a similar manner to the actions of the intact hGH molecule. The synthetic lipolytic domain mimicked the effect of the intact growth hormone on diacylglycerol release in adipocytes. Chronic treatment of obese Zucker rats with AOD9401 for 20 days reduced the body weight gain of the animals, and the average cell size of the adipocytes of the treated animals decreased from 110 to 80 microm in diameter. Unlike hGH, synthetic AOD9401 did not induce insulin resistance or glucose intolerance in the laboratory animals after chronic treatment. The results suggest that AOD9401 has the potential to be developed into a therapeutic agent for the control of obesity.

Metabolic studies of a synthetic lipolytic domain (AOD9604) of human growth hormone.

A synthetic analogue (AOD9604) of the lipolytic domain of human growth hormone (hGH) has been studied for its metabolic actions in obese Zucker rats. Daily treatment with an oral dose of AOD9604 of 500 microg/kg body weight for 19 days reduced over 50% (15.8 +/- 0.6 vs. 35.6 +/- 0.8 g) body weight gain of the animals in comparison with the control. The adipose tissues of the AOD9604--treated animals were found to have an increase in lipolytic activity. In contrast to chronic treatment with intact hGH, chronic treatment with AOD9604 showed no adverse effect on insulin sensitivity of the animals, as demonstrated with euglycemic clamp techniques. The results in the present study suggest that the analogue of the hGH lipolytic domain may have the potential to be developed into an orally usable and safe therapeutic agent for obesity.

Effects of genetic and diet-induced obesity on lipid metabolism.

C57BL/6J obese (ob/ob) and lean mice fed ad libitum on a normal mouse chow diet (Normal), were compared with lean mice of the same age and strain fed ad libitum on a high-fat diet, consisting of the Normal diet with the addition of beef lard (Lard), from age 3 months for 34 days. The lard-fed mice were seen to have significantly higher (P<0.05) body weight in this 34-day period than that of the other two groups fed on the Normal diet. Epididymal fat depot and adipocyte cell size were significantly larger (P<0.05) in the Lard-fed lean mice and in the obese (ob/ob) mice than were those of the Normal-fed lean mice. Dietary Lard intake did not significantly affect concentrations of plasma triglyceride although those of plasma cholesterol were significantly increased (P<0.05). The development of obesity in these Lard-fed mice appeared to be accelerated and significant.

Maternal serum screening and trisomy 16 confined to the placenta.

Five cases of trisomy 16 confined to the placenta have been detected by invasive procedures (amniocentesis and chorionic villus sampling) after high-risk results for Down syndrome and neural tube defects in a maternal serum screening programme of 6614 consecutive cases. All five pregnancies displayed unusually elevated levels of human chorionic gonadotropin and four out of five also had raised alpha-fetoprotein values. No structural malformation was present but all five pregnancies were complicated by fetal growth retardation, and one by intrauterine death. From our results, we suggest that both amniocentesis and chorionic villus sampling should be considered in the management of cases with high mid-trimester levels of these analytes.

Characterisation of the tumour-associated carbohydrate epitope recognised by monoclonal antibody 4D3.

The tumour-associated epitope recognised by monoclonal antibody (MAb) 4D3 is expressed on a high m.w. mucin glycoprotein preparation known as small intestinal mucin antigen (SIMA). This epitope is detected in tissue from a high proportion of patients with colorectal cancer, and elevated levels occur in serum from a significant number of such patients, highlighting the potential clinical utility of MAb 4D3. In the present study, insight into the composition and structure of the carbohydrate epitope recognised by MAb 4D3 was gained following characterisation of 2 glycopeptides that co-purified with SIMA. Sequence analysis of 1 of these glycopeptides revealed that it was identical to the glycoprotein alpha-1-anti-chymotrypsin. This glycoprotein was subsequently deglycosylated to yield 5 forms corresponding to alpha-1-anti-chymotrypsin substituted with 4, 3, 2, 1 or no branched glycans. MAb 4D3 was reactive with each of the glycosylated forms, including the form carrying only 1 branched glycan, but did not react with fully deglycosylated alpha-1-anti-chymotrypsin. MAb 4D3 also reacted to different extents with ovine, bovine or porcine submaxillary mucins, each of which has a different amount of the O-linked sialylated disaccharide known as sialosyl Tn. Of these mucins, MAb 4D3 was most reactive with ovine submaxillary mucin, in which almost all of the carbohydrate chains are sialosyl Tn. Reactivity of MAb 4D3 towards isolated glycans, sialosyl Tn and related structures led to the conclusion that the preferred MAb 4D3 epitope involves the sialylated N-acetyl galactosamine disaccharide as well as an additional monosaccharide present on a neighbouring carbohydrate chain. Although the preferred epitope recognised by MAb 4D3 involves this sialylated disaccharide, the specificity of MAb 4D3 was different from that of other MAbs with a reported specificity for sialosyl Tn.

Effect of TGF-beta on differentiated organoids of the colon carcinoma cell line LIM 1863.

The LIM 1863 colon carcinoma cell line grows in suspension as morphologically and functionally organized organoids in serum-containing medium. Addition of TGF-beta caused the organoids to adhere and inhibited DNA synthesis. A 20 min incubation with TGF-beta was sufficient to induce adherence and this could be inhibited by cycloheximide. The adhesion and DNA synthesis inhibition were demonstrated to be separate events. We were not able to detect any changes in matrix or cell membrane antigens. Similarly there were no changes in synthesized proteins (by two-dimensional gel electrophoresis), and no upregulation of proteoglycan. When adhered organoids were lysed from the tissue culture plastic surface, untreated organoids adhered to this surface. This 'conditioned' surface was destroyed by trypsin but not collagenase or medium from normal LIM 1863 cultures. However, the adherent phenotype was prevented when organoids were treated with transforming growth factor-beta (TGF-beta) in the presence of medium conditioned by normal LIM 1863 cultures rather than in fresh medium. The adhesion process was inhibited by an antibody (QE2E5) against beta 1 integrin although no quantitative changes in integrins were observed (by immunoprecipitation or RNA analysis). A second anti-beta 1 integrin antibody (61.2C4) inhibited LIM 1863 adhesion to collagen but not TGF-beta induced adhesion, implying that TGF-beta induced a specific conformational change or interaction of a beta 1 integrin. In this morphologically structured system TGF-beta induced a number of subtle effects including formation of new extracellular matrix and conformational change of a beta 1 integrin, rather than the major quantitative changes in cell/matrix molecules reported previously.

Isolation of intestinal cell peroxisomes by rate-dependent banding in a vertical rotor.

The preparation of peroxisome-enriched fractions from mouse intestinal epithelial cells is possible by ultracentrifugation using the vertical rotor under rate-zonal conditions. The run time is short which results in a low total centrifugal effect to which these fragile organelles are subjected. The banding of peroxisomes occurs in the central region of the sucrose gradient whereas the mitochondria band in the lower regions. Good separation from the mitochondria is achieved with contaminants resulting from brush-border fragments and endoplasmic reticulum. Comparisons are made with preparations using a swing-out rotor and a zonal rotor. Electron microscope examination of the preparations confirms the presence of diaminobenzidine-positive particles which resemble the peroxisomes seen in intact cells. In addition, it has been shown that density gradients can be constructed which prevent large particles from impacting and causing wall effects.

Monoclonal antibody 4D3 detects small intestinal mucin antigen (SIMA)--glycoprotein in the serum of patients with colorectal cancer.

We have developed a sensitive ELISA using MAb 4D3 for the detection of a novel epitope on Small Intestinal Mucin Antigen (SIMA) and report here that SIMA is present in the serum of patients with colorectal cancer. SIMA has been shown to occur in tissue from a high proportion of patients with colorectal cancer. SIMA derived from serum was similar to tissue-derived SIMA: both eluted in the void volume of a Superose 6 column indicating a molecular weight above 5,000 kDa and they exhibited similar buoyant densities on CsCl gradients. The ELISA was most reliable after pre-treatment of serum with 0.4 M perchloric acid to remove interfering substances. The upper limit for SIMA in normal serum was set as the mean plus 2 standard deviations determined from a group of 97 healthy control subjects. In a sample of 113 patients with colorectal cancer, SIMA serum levels were elevated in 15% of patients with Dukes' Stage A, 38% with Stage B, 32% with Stage C and 75% with Stage D colorectal cancer. SIMA serum levels were compared with those of the widely used tumor marker, carcinoembryonic antigen (CEA). The SIMA assay detected a significant number of sera that were not detected by the test for CEA. We propose that SIMA will prove to be a valuable serological tumor marker, in combination with CEA and other tumor markers, for the detection of colorectal cancer.