Tumor-targeted gene therapy: strategies for the preparation of ligand-polyethylene glycol-polyethylenimine/DNA complexes.

Surface-shielded DNA delivery systems have been synthesized with virus-like characteristics that target gene expression into distant tumor tissues. Polyethylenimine (PEI)/DNA complexes ('polyplexes') conjugated with the cell-binding ligand transferrin (Tf) or epidermal growth factor (EGF) were used to achieve receptor-mediated endocytosis. The surface charge of the complexes was masked by covalently linking PEI to polyethylene glycol (PEG). Three alternatives for generating these surface-shielded formulations were utilized, attaching ligand and PEG molecules to PEI either before or after DNA complex formation. The stabilized formulations could be ultra-concentrated, stored frozen, and applied systemically after thawing. Intravenous injection of Tf-PEG-coated polyplexes resulted in gene transfer to subcutaneous Neuro2a neuroblastoma tumors of syngeneic A/J mice; EGF-PEG-coated polyplexes were intravenously applied for targeting human hepatocellular carcinoma xenografts in SCID mice. In these models, luciferase marker gene expression levels in tumor tissues were 10- to 100-fold higher than in other organ tissues. Repeated systemic application of Tf-PEG-PEI/DNA complexes encoding tumor necrosis factor alpha (TNF-alpha) into tumor-bearing mice induced tumor necrosis and inhibition of tumor growth in three murine tumor models of different tissue origin (Neuro2a, M-3 or B16 melanoma).     

TWEAK induces liver progenitor cell proliferation

Progenitor ("oval") cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors.     

A molecular basis for nonsecretory myeloma

The biosynthesis of aberrant immunoglobulin polypeptides by monoclonal plasma cells has been implicated in the pathogenesis of nonsecretory myeloma. Our studies of a patient with this disorder indeed have demonstrated the presence of abnormal kappa light chains that resulted from a frameshift mutation in nucleotides encoding the constant region of the molecule. As a consequence of a 2-base deletion in codon 187 and loss of the normal stop codon, this portion of the kappa chain was composed of 128 amino acids (rather than the expected 106), with a completely anomalous sequence after position 187 that included absence of the cysteines required for intrachain and interchain disulfide bonds. The unusual primary structure of this component was confirmed by mass spectrometric and amino acid sequence analyses of cytoplasmic protein extracts. Our studies provide the first evidence that human nonsecretory myeloma may result from an alteration in the light-chain constant region.     

Biosilica formation in diatoms: characterization of native silaffin-2 and its role in silica morphogenesis

The biological formation of inorganic materials with complex form (biominerals) is a widespread phenomenon in nature, yet the molecular mechanisms underlying biomineral morphogenesis are not well understood. Among the most fascinating examples of biomineral structures are the intricately patterned, silicified cell walls of diatoms, which contain tightly associated organic macromolecules. From diatom biosilica a highly polyanionic phosphoprotein, termed native silaffin-2 (natSil-2), was isolated that carries unconventional amino acid modifications. natSil-2 lacked intrinsic silica formation activity but was able to regulate the activities of the previously characterized silica-forming biomolecules natSil-1A and long-chain polyamines. Combining natSil-2 and natSil-1A (or long-chain polyamines) generated an organic matrix that mediated precipitation of porous silica within minutes after the addition of silicic acid. Remarkably, the precipitate displayed pore sizes in the range 100-1000 nm, which is characteristic for diatom biosilica nanopatterns.     

Patients prescribed injectable heroin or methadone -their opinions and experiences of treatment

Aims To describe the opinions and experiences of treatment of a cohort of patients prescribed injectable opiate treatment (IOT). Design, setting, participants Cross‐sectional survey of all patients on injectable diamorphine or methadone at a tertiary referral clinic in the northwest of England in June 2000. Findings A total of 104 subjects were prescribed IOT, mostly male (87.5%) and with a mean age of 36.3 years (SEM 0.66, range 20–53). The majority (75.0%) were prescribed injectable methadone with the remainder (25.0%) on injectable diamorphine. Most subjects (93.3%) used intravenously, many (58.7%) into the femoral vein. Treatment was sought most frequently in order to procure a drug supply of known dose and purity, to improve family relationships and to avoid trouble with the police. Half were satisfied with their treatment but many wanted to change to injectable diamorphine or to increase their doses. Subjects cited many advantages of injectable diamorphine over injectable methadone, although benefits of injectable methadone were acknowledged. Conclusions Subjects articulated a consistent desire for IOT in order to ‘stabilize’ their lives in a number of ways. This sample was recruited from one of the country's largest specialist IOT clinics. The generalizability of this study's findings to all patients in the United Kingdom currently prescribed IOT, however, was not examined. Nevertheless, these findings suggest that clinicians and policy makers should be aware of many heroin users’ perception of IOT as long‐term treatment and their clear preference for injectable diamorphine. Further investigation of differential outcomes between oral and injectable OT and between different injectable opiates is warranted.     

Comorbid alcohol and nicotine dependence: from the biomolecular basis to clinical consequences

This article represents the proceedings of a symposium presented at the 12th Congress of the International Society for Biomedical Research on Alcoholism held in Heidelberg/Mannheim, Germany, on October 1, 2004. The organizers and cochairs were Nassima Ait‐Daoud, MD, and Gerhard A. Wiesbeck, MD. The presentations included the following: (1) The Role of Nicotinic Acetylcholine Receptors in Alcohol‐Seeking Behavior, by Przemyslaw Bienkowski, MD, PhD; (2) Utilization of Linkage Analysis Combined with Microarray Technology to Identify Genes and Mechanisms Underlying Nicotine and Alcohol Use and Abuse in Humans and Rodents, by Ming D. Li, PhD; (3) Smoking and Alcoholic Chronic Pancreatitis: The Underestimated Risk?, by Roland H. Pfützer, MD; (4) Anticraving Medication in Alcohol and Nicotine Dependence, by Otto M. Lesch, MD, PhD; and (5) Pharmacotherapy for Promotion of Abstinence from Nicotine Among Alcohol‐Dependent Individuals, by Bankole A. Johnson, DSc, MD, PhD.     

High circulating ghrelin: a potential cause for hyperphagia and obesity in prader-willi syndrome

Prader-Willi syndrome (PWS) is a genetic disorder occurring in 1 of 10,000-16,000 live births and is characterized by excessive appetite with progressive massive obesity as well as short stature and mental retardation. Most patients have GH deficiency and hypogonadotropic hypogonadism. The causes of the hyperphagia and abnormal GH secretion are unknown. To determine whether ghrelin, a novel GH secretagogue with orexigenic properties, is elevated in PWS, we measured fasting plasma ghrelin concentration; body composition (dual-energy x-ray absorptiometry); and subjective ratings of hunger (visual analog scale) in seven subjects (6 males and 1 female; age, 26 +/- 7 yr; body fat, 39 +/- 11%, mean +/- SD) with PWS (diagnosis confirmed by genetic test) and 30 healthy subjects (reference population, 15 males and 15 females; age, 32 +/- 7 yr; body fat, 36 +/- 11%) fasted overnight. All subjects were weight stable for at least 6 months before admission to the study. The mean plasma ghrelin concentration was higher in PWS than in the reference population (307 +/- 164 vs. 109 +/- 24 fmol/ml; P < 0.001), and this difference remained significant after adjustment for percentage body fat (P < 0.001). Plasma ghrelin was also higher (P = 0.0004) in PWS than in five healthy subjects fasted for 36 h. A positive correlation was found between plasma ghrelin and subjective ratings of hunger (r = 0.71; P = 0.008). Furthermore, in subjects with PWS, the concentration of the hormone was not different before and after ingestion of 2 ml and a satiating amount of the same liquid meal (ghrelin concentrations: 307 +/- 164 vs. 306 +/- 205 vs. 260 +/- 134 fmol/ml, respectively; ANOVA for repeated measures, P = 0.56). This is the first evidence that ghrelin, a novel orexigenic hormone, is elevated in subjects with PWS. Our finding suggests that ghrelin may be responsible, at least in part, for the hyperphagia observed in PWS.     

Effect of fluvastatin slow-release on low density lipoprotein (LDL) subfractions in patients with type 2 diabetes mellitus: baseline LDL profile determines specific mode of action

The objective of this study was to determine the effect of slow-release (XL) fluvastatin on low density lipoprotein (LDL) subfractions in type 2 diabetes. A multicenter, double-blind, randomized, parallel-group comparison of fluvastatin XL 80 mg (n = 42) and placebo (n = 47), each given once-daily for 8 wk, in 89 patients with type 2 diabetes (HbA1c: 7.2 +/- 1.0%, LDL cholesterol (LDL-C): 3.4 +/- 0.7 mmol/liter, high density lipoprotein cholesterol: 1.1 +/- 0.3 mmol/liter, and triglycerides (TG): 2.4 +/- 1.4 mmol/liter). At baseline and on treatment, plasma lipoproteins were isolated and quantified. Eight weeks of fluvastatin treatment decreased total cholesterol (-23.0%, P < 0.001), LDL-C (-29%, P < 0.001) and TG (-18%, P < 0.001), compared with placebo. At baseline, there was a preponderance of dense LDL (dLDL) (apolipoprotein B in LDL-5 plus LDL-6 > 25 mg/dl) in 79% of patients, among whom fluvastatin decreased all LDL subfractions, reductions in dLDL being greatest (-28%, P = 0.001; cholesterol in dLDL -29%). In patients with low baseline dLDL (apolipoprotein B in LDL-5 plus LDL-6 相似文献   

Requirements for simian immunodeficiency virus antigen-specific in vitro proliferation of T cells from infected rhesus macaques and sooty mangabeys

The measurement of cell-mediated immunity against the etiologic agent of human AIDS (HIV) in the non-human primate model of AIDS (simian immunodeficiency virus, SIV) has been difficult. In general, culture of peripheral blood mononuclear cells from HIV-1- and SIV-infected humans and monkeys, respectively, with purified inactivated HIV and SIV virus preparations has given inconsistent or negative proliferative responses. However, we describe herein an assay which consists of coculturing monocytes that have been pulsed with inactivated SIVsmm with nylon-wool-purified autologous T cells, leading to antigen-specific T-cell proliferation. The proliferative response, which predominantly occurs in CD4+ T cells, is major histocompatibility complex (MHC) class II-restricted and requires antigen processing. This assay will greatly facilitate the identification of the immunodominant epitopes recognized by T cells in sooty mangabeys, which are naturally infected but remain clinically asymptomatic, and in rhesus macaques, in which experimental infection leads to clinical symptomatology similar to human AIDS, eventually resulting in death.     

Stage-Dependent Changes in Adrenal Steroids and Catecholamines during Development inXenopus laevis

Changes in adrenal hormones during the complete developmental cycle from egg to juvenile were investigated in the amphibianXenopus laevis.Whole-body concentrations of the adrenal steroids corticosterone (B), and aldosterone (Aldo) were determined by radioimmunoassay and those of the adrenal catecholamines epinephrine (E), norepinephrine (NE), and dopamine (D) were determined by HPLC. In addition, the catecholamine-synthesizing enzymes tyrosine hydroxylase, dopamine β-hydroxylase, and phenylethanolamineN-methyltransferase were immunocytochemically localized for the characterization of chromaffin adrenal cells. B and Aldo were not detectable in the whole body before hatching. B levels rose earlier than Aldo levels from stage 36 onward. B had already peaked at stage 46, whereas the largest amounts of Aldo were found at stage 54. After peaking, both steroids decreased gradually to 2.7 ± 0.62 (B) and 0.4 ± 0.1 (Aldo) ng/g body wt (mean ± SEM,n = 10) in juvenile animals. E, NE, and D were detected just after hatching, when E and D showed an early peak at stage 40. E and NE increased moderately during development and demonstrated a sharp increase at the end of metamorphosis from stages 62 onward to 14.4 ± 1.7 (E) and 34.1 ± 4.67 (NE) ng/g body wt (mean ± SEM,n = 6). Interestingly, D levels had a distinct pattern, because concentrations of D remained lower than those of NE and E over nearly the complete development, but showed a dramatic rise during the latest stages, reaching 707 ± 54 ng/g body wt in juveniles. This dramatic shift in catecholamine levels was confirmed by immunocytochemistry in parallel. A large increase in chromaffin cells labeled with tyrosine hydroxylase immunoreactivity occurred in the latest developmental stages. The catabolic rates for all catecholaminesin vivowere similar, which indicates that the different levels are due to various rates of synthesis. Thus, adrenal corticosteroids as well as catecholamines may have regulatory effects during premetamorphosis and metamorphic climax.