Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo.

Targeted silencing of disease-associated genes by synthetic short interfering RNA (siRNA) holds considerable promise as a novel therapeutic strategy. However, unmodified siRNA can be potent triggers of the innate immune response, particularly when associated with delivery vehicles that facilitate intracellular uptake. This represents a significant barrier to the therapeutic development of siRNA due to toxicity and off-target gene effects associated with this inflammatory response. Here we show that immune stimulation by synthetic siRNA can be completely abrogated by selective incorporation of 2'-O-methyl (2'OMe) uridine or guanosine nucleosides into one strand of the siRNA duplex. These noninflammatory siRNA, containing less than 20% modified nucleotides, can be readily generated without disrupting their gene-silencing activity. We show that, coupled with an effective systemic delivery vehicle, 2'OMe-modified siRNA targeting apolipoprotein B (apoB) can mediate potent silencing of its target mRNA, causing significant decreases in serum apoB and cholesterol. This is achieved at therapeutically viable siRNA doses without cytokine induction, toxicity, or off-target effects associated with the use of unmodified siRNA. This approach to siRNA design and delivery should prove widely applicable and represents an important step in advancing synthetic siRNA into a broad range of therapeutic areas.     

Benign intracranial hypertension and recombinant growth hormone therapy in Australia and New Zealand

Benign intracranial hypertension (BIH) is reported in three children from Australia and one from New Zealand, who were being treated with recombinant human growth hormone (rhGH). Three males and one female, aged between 10.5 and 14.2 y, developed intracranial hypertension within 2 weeks to 3 months of starting treatment. A national database, OZGROW, has been prospectively collecting data on all 3332 children treated with rhGH in Australia and New Zealand from January 1986 to 1996. The incidence of BIH in children treated with growth hormone (GH) is small, 1.2 per 1000 cases overall, but appears to be greater with biochemical GHD (<10IUml ^-1), i.e. 6.5/1000 (3 in 465 cases), relative risk 18.4, 95% confidence interval 1.9-176.1, than in all other children on the database. The incidence in patients with Turner's syndrome was 2.3/1000 (1 in 428 cases). No cases in patients with partial GHD (10–20 IUml ^-1) or chronic renal failure were identified. Possible causative mechanisms are discussed. The authors'practice is now to start GH replacement at less than the usual recommended dose of 14IUm-² week^-1 in those children considered to be at high risk of developing BIH. Ophthalmological evaluation is recommended for children before and during the first few months following commencement of rhGH therapy and is mandatory in the event of peripheral or facial oedema, persistent headaches, vomiting or visual symptoms. The absence of papilledema does not exclude the diagnosis.     

Estimation methods for Maximum Residue Limits for pesticides

Maximum Residue Limits (MRLs) are standards that represent the maximum residue concentration expected to be found if a pesticide is applied according to good agricultural practice (GAP). MRLs are established only where the residues in food resulting from particular use patterns of the pesticide pass the public health risk assessment. Foodstuffs are monitored for MRL compliance and MRL exceedance can have economic and trade consequences. There is a trade-off when deciding on values for MRLs. The aim is to establish MRLs at levels that are high enough to prevent chance exceedance but not so high that misuse will not be detected. Small data sets typically available for estimating MRLs present problems for establishing consistent values. A review of MRL estimation methods is presented together with an assessment of the various methods.     

Synthesis-secretion coupling of insulin: effect of aging

Synthesis-secretion coupling of insulin was measured in four age groups of perfused pancreases taken from Sprague-Dawley rats ranging in age from 2-12 months. The effect of long term (6 h) near-maximal glucose stimulation (300 mg/dl) on both insulin secretion and net insulinogenesis demonstrated an age-related increase in both parameters. Net insulinogenesis as well as total insulin secretion increased linearly as a function of aging. Compared to that in 2-month-old rats, total net insulin synthesis was more than 3-fold greater in 12-month-old rats, slightly less than 3-fold greater in 8-month-old rats, and twice as much in 4-month-old rats. Compared to that in 2-month-old rats, total glucose-stimulated insulin secretion was 3-fold greater in 12-month-old rats, approximately 2.2-fold greater in 8-month-old rats, and about 1.7-fold greater in 4-month-old rats. A shorter term (90 min) glucose stimulation at 150 mg/dl produced an age-related increase in insulin secretion which was relatively comparable to the higher glucose stimulus. Of equal importance is that fact that pancreases from the older rats exhibited the same degree of secretory responsiveness to changing glucose levels as did pancreases from the younger rats. Regardless of age, first phase insulin secretion was approximately twice as much in response to the higher glucose level as to the lower. Similarly, second phase insulin secretion was almost 3 times greater regardless of age. When normalized and reported in terms of insulin content, total insulin secretion was no different as a function of aging during the first 1 h of glucose stimulation (i.e. the first two phases of secretion), but it was significantly elevated in the third secretory phase (2-6 h) by the older rat groups. Total 6-h net insulinogenesis was also greater in the older rat groups. When normalized and reported in terms of total body weight, both insulin synthesis and total insulin secretion became comparable and showed no specific age-related difference. Thus, there is no indication that aging results in an uncoupling of relatively long term (6-h) insulin synthesis-secretion, since both glucose-induced responses parallel one another as a function of aging. Furthermore, reporting insulin secretion and synthesis on the basis of body weight, rather than age, totally normalizes synthesis-secretion coupling of insulin.     

Endothelin receptor expression in human decidua

The endothelins are signalling peptides that act via two receptors, ET(A) and ET(B). In the human endometrium, endothelin receptors have been demonstrated in glands and stroma and have been shown to vary during the course of the menstrual cycle. The present study was undertaken to determine whether or not expression of endothelin receptors changes during pregnancy or after administration of exogenous progestagens. The expression of the receptors was correlated with the appearance of basement membrane components during decidualization of the endometrial stroma. Decidual specimens (n = 15) were obtained during the first trimester of pregnancy and 10 at term. Sixteen pairs of endometrial biopsies were obtained from women with menorrhagia before and after exposure to exogenous progestagens. A total of 15 hysterectomy specimens were used as controls for the expression of stromal basement membrane proteins in the absence of decidualization. Autoradiography was carried out with selective ligands for ET(A) ([125I]-PD 151242) and ET(B) ([125I]-BQ3020). The distribution of ligand binding was then compared with the distribution of laminin alpha2 light chain and collagen IV. ET(A), ET(B), laminin alpha2 light chain, and collagen IV were expressed in stromal decidual cells in the first trimester of pregnancy. ET(B) was also found on endometrial glandular epithelium. Quantitative macro-autoradiography and multiple regression analysis demonstrated a highly significant positive correlation (P < 0.001) between expression of ET(B) and laminin alpha2 light chain. In the third trimester qualitative examination suggested a reduction of ET(A) in the stroma. Progestagen-induced decidua exhibited a similar pattern to that found in first trimester decidua. This study has demonstrated up-regulation of ET(B) during the progesterone- dependent process of decidualization and suggests a paracrine or autocrine role for endothelins in the decidua.      

Semiautomated determination of minimum bactericidal concentrations of antibiotics

Using a newly devised 50-channel photometer which records the opacity of growing bacterial cultures, it was shown that the time taken by cultures diluted 1/1000 in fresh broth to reach 50% of the opacity of a fully grown culture was inversely related to the concentration of organisms in the original culture. This relation was used to determine the numbers of survivors after exposure to benzylpenicillin and gentamicin alone and in combination. The procedure is commended as a labour-saving and potentially rapid method of obtaining comprehensive information on the bactericidal action and interaction of antibiotics.