Proceedings of the Association of American Physicians最新文献

Evidence is presented that shows that free fatty acids (FFA) are one important link between obesity, insulin resistance, and type 2 diabetes. Plasma FFA levels are elevated in most obese subjects, and physiological elevations of plasma FFA inhibit insulin-stimulated glucose uptake into muscle. This peripheral insulin resistance is caused by an FFA-induced defect, which develops 3-4 hr after raising plasma FFA, in insulin-stimulated glucose transport or phosphorylation, or both. This resistance is also caused by a second defect, which develops after 4-6 hr, consisting of inhibition of glycogen synthase activity. Whether elevated plasma FFA levels inhibit insulin action on endogenous glucose production (EGP), that is, cause central insulin resistance, is more difficult to demonstrate. On the one hand, FFA increase gluconeogenesis, which enhances EGP; on the other hand, FFA increase insulin secretion, which decreases EGP. Basal plasma FFA support approximately one third of basal insulin secretion in diabetic and nondiabetic subjects and, hence, are responsible for some of the hyperinsulinemia in obese, normoglycemic patients. In addition, elevated plasma FFA levels potentiate glucose-stimulated insulin secretion acutely and during prolonged exposure (48 hr). It is hypothesized that obese subjects who are genetically predisposed to develop type 2 diabetes will become partially "lipid blind," that is, unable to compensate for their FFA-induced insulin resistance with FFA-induced insulin oversecretion. The resulting insulin resistance/secretion deficit will then have to be compensated for with glucose-induced insulin secretion, which, because of their partial "glucose blindness," will result in hyperglycemia and eventually in type 2 diabetes.

The effects of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) on T cell expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), the diphtheria toxin (DT) receptor, were investigated in the Tsup-1 cultured line of human CD4+ 8+ 3low T lymphoblastoma cells. Tsup-1 cells bear endothelial differentiation gene (edg)-2 and -4-encoded G protein-coupled receptors (GPCRs) for LPA and Edg-3 and -5 GPCRs for S1P. Suppression by DT of Tsup-1 cell protein synthesis was enhanced by LPA and S1P, with lipid structural specificity similar to that required for their recognition by Edg receptors. LPA and S1P increased the Tsup-1 cell level of immunoreactive HB-EGF, and neutralizing antibodies to HB-EGF inhibited LPA and S1P enhancement of Tsup-1 cell susceptibility to DT. Stabilized transfection of Tsup-1 cells with a combination of plasmids encoding Edg-2 plus -4 antisense mRNA suppressed the levels of Edg-2 and -4, but not Edg-3 and -5, in Western blots and reduced in parallel the increments in HB-EGF and susceptibility to DT evoked by LPA but not S1P. Similar transfection with Edg-3 plus -5 antisense plasmids suppressed Tsup-1 cell levels of immunoreactive Edg-3 and -5, but not Edg-2 or -4, and concurrently reduced S1P-, but not LPA-, induced Tsup-1 cell increases in both HB-EGF and susceptibility to DT. Edg GPCR-mediated LPA and S1P enhancement of T cell sensitivity to DT, thus, may be attributable to increased expression of the DT receptor HB-EGF.

The observation that transgenes can be stably integrated into the genome of fibroblasts using recombinant retroviruses enhanced interest in using these cells as a vector for gene therapy. This enthusiasm has lessened during the past 8 years, not because skin has lost the features that make it attractive for gene therapy, but rather because stable transgene expression in vivo has not been achieved. All investigators who have used genetically modified fibroblasts to study in vivo aspects of gene therapy have shown a decrease in transgene expression with time. This contrasts with transgene expression in similarly transduced fibroblasts in vitro, where expression is not lost or is lost very slowly. We have initiated an approach to bring further understanding to the biology of transgene expression by fibroblasts carrying stably integrated transgenes in an in vivo setting. Experiments described permit the following conclusions. Expression by and survival of genetically modified fibroblasts a) requires a persistent matrix scaffold in in vivo settings; b) is prolonged if the matrix is allowed to mature in vitro; c) is enhanced if the matrix is partially sequestered behind a coating of normal fibroblasts; and d) can be substantively prolonged in vivo by immortalizing the cells. These observations support the notion that prolonged expression of transgenes by fibroblasts can be achieved in vivo and that gene therapy utilizing fibroblasts and other cells of the skin has clinical utility.

One of the most promising applications of recent advances in gene therapy is the development of immunization strategies based on the delivery of antigen-encoding DNA. DNA-based vaccination, also referred to as genetic vaccination or polynucleotide vaccination, offers considerable promise for improvement over existing immunization strategies, and the skin offers unique potential as a target tissue for genetic vaccines. The expression of genetically introduced antigens in a cutaneous microenvironment rich in both professional antigen-presenting cells and accessory cells, which are capable of producing immunostimulatory cytokines, has the potential to overcome the historical limitations of vaccinology and immunotherapy. Though the precise molecular mechanisms of genetic immunization remain unclear, a general working model of the events through which antigen-encoding plasmids introduced into the skin initiate an immune response can be constructed. The finding that Langerhans cells can be transfected in vivo raises the exciting possibility that these migrating professional antigen-presenting cells can be genetically engineered in vivo. By designing strategies to codeliver genes encoding antigens with genes encoding immunoregulatory molecules to the same antigen-presenting cell, it may be possible to either induce or suppress antigen-specific immune responses in the host. Though many aspects of the biology of cutaneous DNA immunization remain unknown, the skin appears to offer unique potential for the application of advances in gene therapy to vaccination and genetic engineering of the immune response.

In vivo gene therapy is a direct and effective way to express genes in the epidermis. Plasmid DNA that contains the desired gene can be injected intradermally, and it is rapidly absorbed and expressed by the epidermis. Because gene expression following plasmid injection is transient, the two principal therapeutic uses of this approach are genetic immunization and the expression of biological response modifiers to treat skin disease.

Glucocorticoids are currently regarded as the drug of choice in the treatment of inflammatory airway and lung diseases, however, they are not routinely effective in fibrotic phases of inflammation. In the current study, glucocorticoids were investigated for their ability to affect fibroblast mediated contraction of a three dimensional collagen gel, a measure of one aspect of tissue remodeling. Dexamethasone, budesonide, hydrocortisone and fluticasone propionate were all able to significantly augment fibroblast contractility in a concentration dependent manner. Glucocorticoids also had an augmentative effect on collagen gel contraction mediated by fibroblasts from bronchi, skin and bone marrow. The increased contractility was not due to cell proliferation or to collagen degradation, since the glucocorticoids did not alter the amounts of DNA and hydroxyproline in the gels. The concentration of prostaglandin E2 (PGE2) in supernatant media was lower from glucocorticoid-treated gels compared to control gels. Consistent with this, addition of exogenous PGE2 to the culture system restored the contractile properties and indomethacin augmented contraction similar to the glucocorticoids suggesting that inhibition of prostaglandins or related eicosanoids may be the mechanism by which the increased contractility occurs. DBcAMP, forskolin and the long lasting beta2-agonist formoterol were able to reverse the effect of the glucocorticoids on fibroblast mediated collagen gel contraction suggesting that enhancers of cAMP can counteract the effect of glucocorticoids. Thus, we provide evidence that glucocorticoids have the ability to directly augment fibroblast contractility by inhibiting fibroblast endogenous PGE synthesis. The findings could be one possible mechanism to explain the poor therapeutic response to glucocorticoids on the later stages of fibrotic diseases.

For more than two decades, it has been known that activation of the plasma kallikrein/kinin system only occurs when it is exposed to artificial, negatively charged surfaces. The existence of physiological, negatively charged surfaces has, however, never been demonstrated in vivo. In this report, we describe current knowledge about how the proteins of the plasma kallikrein/kinin system interact with and become activated on cell membranes. In this model, activation of the plasma kallikrein/kinin system on endothelial cells is not initiated by factor XII autoactivation, as seen on artificial surfaces. On endothelial cells, plasma prekallikrein is activated by a membrane-associated cysteine protease. This activation is dependent on the presence of high molecular weight kininogen and an optimal zinc (Zn2+) concentration. Although the initiation of activation of plasma prekallikrein is independent of factor XII, kallikrein-mediated factor XIIa generation, in turn, accelerates the activation of the system. Further kallikrein formed on endothelial cell membranes is capable of cleaving its receptor and native substrate, high molecular weight kininogen, liberating bradykinin and terminating activation. In addition, the kallikrein formed on the surface of endothelial cells results in kinetically favorable activation of prourokinase and, subsequently, plasminogen. Activation of the plasma kallikrein/kinin system on endothelial cells proceeds by a physiological mechanism to initiate cellular fibrinolysis independent of plasmin, fibrin, and tissue-type plasminogen activator.

Autoimmune diseases include a wide spectrum of disorders, which have been divided into systemic and organ-specific disorders. Lupus, the prototypic systemic autoimmune disease, is characterized by female predominance, multiorgan pathology, and autoantibodies, primarily directed against nuclear antigens. The disease is heterogeneous, with variable organ involvement, serology, and clinical course. Susceptibility to lupus is inherited as a polygenic trait with added contributions from environmental and stochastic variance. Concerted efforts have recently been made by several laboratories to define the genetic basis of this disease in predisposed mice and humans. The identification of the Fas/FasL defects in lpr and gld lupus mice was the first example of spontaneous mutations of apoptosis-promoting genes being associated with systemic autoimmunity. This research was instrumental in clarifying the roles of these genes in tolerance and immunoregulation, and in extrapolating these results to other autoimmune diseases, as well as cancer and transplantation. To these findings have been added those from transgenic and gene knockout mouse studies that have helped to define the systemic autoimmunity-inducing or -modifying effects of specific genes in normal background and lupus-congenic mice. In addition, the findings from genome-wide searches have begun to identify predisposing loci (and ultimately genes) for the spontaneous lupus-like diseases in various mouse strains and in humans. The emerging picture is that multiple genetic contributions can independently lead to systemic autoimmunity in mice, which reinforces the view that human lupus may be similarly composed of diverse genotypes. This complexity underscores the importance of defining the predisposing alleles and mechanisms of action, an undertaking that is certainly feasible given current technologies and future advances in the definition of mammalian genomes.

Continuously renewing tissues, such as the epidermis, are populated by a hierarchy of dividing transient amplifying cells, which are maintained by stem cells. Transient amplifying cells divide to maintain the tissue, but they are limited to a finite number of cell divisions before they differentiate and are sloughed. Only the stem cells remain for the life of the tissue. Thus, it is critical to target stem cells when designing gene therapy regimes for genetically inherited diseases, such as epidermolysis bullosa simplex (EBS). Unfortunately, isolating pure epithelial stem cells has been problematic. In this study, we used rapid adherence to collagen type IV to successfully enrich for epidermal stem cells from adult human skin. These preselected stem cells were slow to proliferate, but they ultimately formed large colonies. When recombined with the dermal substrate AlloDerm, the stem cells re-formed a stratified squamous epidermis within 1 week after raising the AlloDerm to the air-liquid interface. These organotypic cultures grew continuously and, even after 6 weeks in culture, they maintained a proliferative basal layer. When transduced with a retroviral LacZ vector, preselected stem cells formed beta-galactosidase-positive clones in submerged and organotypic cultures. Transduced cells showed persistent expression through 12 weeks in organotypic culture, demonstrating the feasibility of using preselected stem cells for gene therapy. Currently, we are developing two models of EBS to test a gene therapy approach, which is based on the premise that EBS stem cells with a mutant keratin (K)14 gene corrected to wild type will have a growth advantage over noncorrected EBS stem cells.

The range of harms to health from psychoactive substances is considered, and current data are presented on the health burden of alcohol, tobacco, and illicit drugs. Four major strategies for preventing or reducing harm are discussed: education and persuasion, treatment, insulating use from harm, and regulating the availability and conditions of use. Education and treatment are necessary parts of an overall public health approach, but are not very effective in themselves in reducing rates of harm in the population. Insulating use from harm and regulating availability have often proved effective components of a public health approach.