Immunisation safety: a priority of the World Health Organization's Department of Vaccines and Biologicals.

In 1999, the World Health Organization (WHO) Department of Vaccines and Biologicals launched the Immunisation Safety Priority Project to boost its activities in this area, with the aim of establishing a comprehensive system to ensure the safety of all immunisations given in national immunisation programmes. Countries are the primary focus of this project. The WHO has a role to play not only because of its technical and normative role but also because of its privileged relationship with country authorities and other partners, its global vision and mandate, and because it is perceived as free from conflicts of interest. There are four areas of focus in the project: quality control and assessment tools to ensure vaccine safety from clinical trials up to and including the point of use;research and development of safer and simpler delivery systems; access to safer and more efficient systems for vaccine delivery and sharps waste management; and mechanisms to respond promptly and effectively to vaccine safety concerns. The project emphasises the importance of advocating safety and developing necessary infrastructure and human resource to properly deal with immunisation related safety issues at a national level.

Augmentations of glucose uptake and glucose transporter-1 in macrophages following thermal injury and sepsis in mice.

Glucose is the primary metabolic substrate of macrophages, which are critical components of the host response to injury and infection. We have carried out a series of studies to examine macrophage glucose uptake and the status of glucose transporter 1 (GLUT1) at both the mRNA and protein level. Peritoneal macrophages that were obtained from mice undergoing sham burned (S), 15%TBSA burn (B) +/- Pseudomonas aeruginosa burn infection (B + I) and lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) administration. [3H]deoxyglucose uptake was significantly increased (B, 157 +/- 9%; B + I, 243 +/- 19%; S + LPS, 231 +/- 24%; S + TNF-alpha, 379 +/- 18%; B + LPS, 230 +/- 13%; and B + TNF, 305 +/- 23%, P< 0.01 vs. sham). GLUT1 mRNA and protein levels were increased as well (mRNA: B, 135 +/- 13%; B + I, 250 +/- 33%; S + LPS, 282 +/- 29%; S + TNF-alpha, 193 +/- 19%; B + LPS, 378 +/- 20%; and B + TNF-alpha, 204 +/- 16%; protein: B, 159 +/- 27%; B + I, 181 +/- 17%; S + LPS, 219 +/- 26%; S + TNF-alpha, 343 +/- 51%; B + LPS, 366 +/- 41%; and B + TNF-alpha, 415 +/- 44, P< 0.01 vs. sham). Macrophages co-cultured with LPS or TNF-alpha in vitro demonstrated a similar response pattern. Following burn injury and infection, macrophages augment their cellular glucose uptake, which is facilitated by an increased GLUT1 mRNA and protein levels. TNF-alpha elicited by LPS may mediate this enhanced carbohydrate metabolism at the point of glucose entry into the cell.

Alterations of glucose transporter mRNA and protein levels in brain following thermal injury and sepsis in mice.

Since glucose transport and utilization are profoundly influenced by injury and infection, and the brain is an organ which primarily utilizes glucose as its energy source, we examined the status of the facilitative glucose transporters GLUT1 and GLUT3 in brain following thermal injury and infection. BDF1 mice underwent a 15% total body surface area burn with or without Pseudomonas aeruginosa infection. At 4 and 72 h post injury +/- infection, GLUT1 and GLUT3 mRNA abundance was measured by Northern blotting, and the correlative proteins determined using Western blotting. At 4 h, both brain GLUT1 mRNA and protein abundance were significantly increased in burned (mRNA 150 +/- 12%, protein 122 +/- 6%) and burn/infected (mRNA 165 +/- 11%, protein 119 +/- 5%) animals. At 72 h, GLUT1 mRNA and protein levels were also significantly increased in burn (mRNA: 139 +/- 11%, protein: 120 +/- 7%) and burn/infected (mRNA: 145 +/- 14%, protein: 138 +/- 8%) animals. Our studies suggest that alterations of GLUT1 mRNA and protein abundance were primary responses to the burn injury and were not further altered by burn wound infection.

Treatment of insulin-resistant mice with the oral antidiabetic agent pioglitazone: evaluation of liver GLUT2 and phosphoenolpyruvate carboxykinase expression.

Obese KKAy insulin-resistant mice represent a model for the human syndrome of noninsulin-dependent diabetes mellitus. As such, the animals are hyperglycemic and hyperinsulinenic. Treatment of KKAy mice with pioglitazone, a new antihyperglycemic agent, lowered elevated blood glucose and insulin levels to near normal. Since hepatic glucose overproduction is a key abnormality in noninsulin-dependent diabetes mellitus, the aim of the present study was to define the specific effects of pioglitazone on hepatic glucose metabolism and release. To do so, we evaluated the expression of the major liver glucose transporter, GLUT2, and examined the activity and expression of the major rate-limiting enzyme for gluconeogenesis, phosphoenolpyruvate carboxykinase. Our results showed that GLUT2 mRNA abundance was unchanged in diabetic KKAy mice compared to nondiabetic animals, and that no changes were elicited by pioglitazone treatment. Such unaltered GLUT2 levels were consistent with a role for liver GLUT2 in bidirectional transport of glucose during physiological states of uptake or release. In contrast, phosphoenolpyruvate carboxykinase activity and mRNA abundance were concordantly elevated 2-fold in diabetic animals and were returned to normal levels after treatment with pioglitazone. Given that pioglitazone therapy led to decreased hepatic gluconeogenesis while insulin levels were concomitantly lowered, it appeared that pioglitazone acted to restore sensitivity to insulin's normal inhibitory actions.

High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells.

In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10(6) receptors per cell. The cell line with the highest insulin binding (NIH 3T3 HIR3.5) had 6 X 10(6) receptors with a Kd of 10(-9) M. This level was not dependent on exposure to metals but could be increased further to 2 X 10(7) receptors per cell by addition of sodium butyrate to the culture medium. The alpha and beta subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the alpha and beta subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis.

Insulin-ricin B hybrid molecules: receptor binding and biological activity in a minimal-deviation hepatoma cell line.

Hybrid molecules were produced by covalently coupling the hormone insulin to the binding chain B of the plant toxin ricin. Binding of the insulin-ricin B hybrid to minimal-deviation hepatoma cells occurred primarily through ricin-specified cell-surface carbohydrates (galactose, N-acetylgalactosamine) since 125I-insulin-ricin B binding to cells could be 90% displaced by 50 mM lactose. [14C]Glucose incorporation into glycogen was maximally stimulated approximately 80% by insulin, whereas maximum stimulation by insulin-ricin B hybrid was greater than 100%. Ricin B chain alone was non-stimulating at concentrations tested (10(-9)-10(-7) M). Furthermore, the stimulation of [14C]glycogen labeling mediated by the hybrid was markedly inhibited by 1 mM lactose, while this sugar had no effect on the stimulation mediated by native insulin. Additionally, a preparation of ricin B shown to actively displace up to 80% of the binding of 125I-hybrid to cells also inhibited hybrid-mediated [14C]glycogen production. These results indicate that insulin-ricin B hybrid molecules possess toxin-specified binding abilities while evoking the insulin-associated cellular response of stimulated incorporation of [14C]glucose into glycogen. Such results thus suggest the possibility that alternate cell-surface receptors may play a role in conveying insulin's intracellular metabolic-control signals.

Insulin-ricin B hybrid molecules mediate an insulin-associated effect on cells which do not bind insulin.

A hybrid molecule was constructed by covalently linking, by a disulfide bridge, the hormone insulin to the binding subunit B of the plant toxin ricin (specificity: Gal, GalNAc). Monolayer-cultured MDCK cells, which lack detectable levels of specific plasma membrane 125I-insulin binding but which readily bind 125I-insulin-ricin B, were used in these studies. Binding of insulin-ricin B to these cells could be displaced by lactose and ricin B, but not by insulin. The biological activity of the hybrid, as measured by [14C]glucose incorporation into glycogen, was stimulated in a dose-dependent manner by the hybrid (10(-11)-10(-8) M), whereas glycogen production was not stimulated by insulin alone. The stimulated glycogen labeling in response to the hybrid was also inhibited by lactose and ricin B. When ricin B alone was tested over the same range of concentrations, stimulation of glycogen synthesis was not observed, nor was there any evidence for stimulation when insulin and ricin B were added simultaneously. These data suggested that alternate cell surface receptors (i.e. ricin B receptors) may substitute for specific receptors (i.e. insulin receptors) to convey intracellular metabolic control signals in this cell line.