Proceedings of the Association of American Physicians最新文献

Most persons infected with Helicobacter pylori strains that produce vacuolating cytotoxin and possess cytotoxin-associated gene A (cagA) genotype nonetheless remain asymptomatic, suggesting that additional genes are important in virulence. We hypothesized that adherence to gastric epithelium provides stimuli that induce expression of some virulence genes. Our aims were to identify expression of H. pylori genes induced by adherence and to determine if such genes were correlated with peptic ulceration, mucosal interleukin-8 (IL-8) levels, and gastric inflammation. RNA was isolated from an ulcer-derived strain and a gastritis-derived strain that were exposed or not exposed to gastric epithelial cells. These RNAs were used for random arbitrarily primed reverse transcription polymerase chain reaction to identify newly expressed transcripts unique to the ulcer-derived strain following adherence. Clinical isolates of H. pylori were characterized for presence of the newly identified gene, and mucosal IL-8 and inflammation were examined in gastric biopsies from the source patients. A novel H. pylori gene, iceA (induced by contact with epithelium), was identified. DNA sequences revealed two families, iceA1 and iceA2. iceA1 strains were significantly associated with peptic ulceration and increased mucosal concentrations of IL-8. Both iceA1 and iceA2 were expressed in vivo by respective H. pylori strains in gastric biopsies. Adherence to gastric epithelial cells in vitro stimulates the transcription of iceA1, an H. pylori gene that is highly correlated with pathological outcome.

Human eosinophils activated by calcium ionophore produce granulocyte-macrophage colony-stimulating factor (GM-CSF). In T lymphocytes GM-CSF messenger RNA (mRNA) stability is regulated by 3' untranslated region (UTR) adenosine-uridine-rich elements (AREs). We show endogenous GM-CSF mRNA is rapidly induced in an eosinophil cell-line (AML14.3D10) after activation with ionomycin. To calculate the decay rate of GM-CSF mRNA in activated cells, eosinophils were transfected with wild-type, full-length GM-CSF mRNA or a mutant version lacking the AUUUA motifs. In unstimulated cells, wild-type GM-CSF mRNA decayed with a half-life time (t1/2) of 6+/-2 min while the mutant decayed with a t1/2 of 20+/-4 min, demonstrating the dominant, destabilizing effect of multiple AUUUA motifs. Within 1 hr of activation by ionomycin, the half-life of transfected wild-type mRNA increased by 2.5-fold, which increased up to 4-fold after 2 hr of activation. The half-life of the mutant GM-CSF was unaffected by ionomycin, demonstrating that ionophore-mediated stabilization requires intact AUUUA motifs. Actinomycin D (ActD) stabilized wild-type GM-CSF mRNA as well, causing poly(A) tail elongation and translation inhibition. These data show that in eosinophil-like cell lines, GM-CSF mRNA is exquisitely unstable but can be markedly stabilized by calcium ionophore. Both effects require intact 3' UTR AREs.

Vasopressin (AVP) is released in response to both osmotic and nonosmotic stimuli. Nonosmotic-stimulated AVP release occurs in cardiac failure, cirrhosis, and pregnancy in response to alterations in arterial circulatory integrity. Cardiac failure in rats is associated with increased plasma AVP and hypothalamic AVP mRNA, and in humans, it is associated with cardiac failure. Plasma AVP concentrations are elevated when measured with a sensitive radioimmunoassay. Urinary concentrations of AVP-responsive aquaporin-2 water channels are also elevated in cardiac failure. V2 receptor antagonists correct the impaired solute-free water excretion seen in rats with low-output cardiac failure and reverse the upregulation of renal aquaporin-2 water channels. Orally active non-peptide-selective V2 receptor antagonists administered to patients with congestive cardiac failure decrease urinary concentrations of aquaporin-2, increase solute-free water clearance, and correct the hyponatremia. Cirrhosis of the liver results in splanchnic arterial vasodilation and increased vascular capacity, most likely secondary to increased nitric oxide production. This relative underfilling of the arterial circulation stimulates nonosmotic AVP release with resultant water retention. Aquaporin-2 gene expression is upregulated in the kidneys of rats with cirrhosis of the liver. AVP-2 receptor antagonists administered to animals with cirrhosis reverse the water retention. Human studies using orally active, non-peptide-selective V2 receptor antagonists in patients with cirrhosis are currently underway. Pregnancy is another state of nitric oxide-mediated arterial vasodilation that is associated with plasma AVP concentrations that are relatively high for the degree of hypoosmolality. Upregulation of the water channel aquaporin-2 in the renal papillae of pregnant rats has also been demonstrated, and this effect is reversed by administration of a V2 receptor antagonist.

Cell-surface expression of endothelial P-selectin increases adhesion and migration of leukocytes and thus may participate in the pathogenesis of reperfusion injury and atherosclerosis. Angiotensin II (Ang II) is also thought to be involved in such disease states. Nitric oxide (NO) downregulates P-selectin expression, and bradykinin (BK) is known to stimulate NO release from endothelial cells. The objective of this study was to determine the effects of 10-min stimulation of cultured human umbilical endothelial cells (HUVECs) with Ang II, BK, or both on P-selectin expression. Ang II (10(-9)-10(-5) M) stimulated P-selectin expression in a concentration-dependent manner, exhibiting a significant effect at 10(-7) M and reaching a plateau at 5 x 10(-5) M. Pretreatment of HUVECs with the AT1 antagonist losartan and the AT1/AT2 antagonist saralasin but not the AT2 antagonist PD123319 (all at 10(-5) M) markedly attenuated the effect of 10(-7) M Ang II. The effects of Ang II on P-selectin expression were not affected by the presence of the NO synthase inhibitor nitro-L-arginine (L-NA, 5 x 10(-4) M) but were abolished by pretreatment with superoxide dismutase (SOD). BK (10(-6) M) abolished the effects of 10(-7) M Ang II on P-selectin expression but did not affect P-selectin expression induced by desmopressin (0.01-10 microM). L-NA obliterated the blunting effect of BK on the Ang II-induced P-selectin membrane expression. BK alone slightly stimulated P-selectin expression, but in the presence of L-NA, BK markedly enhanced P-selectin expression. The effects of BK in the presence of NA were not altered by SOD, indicating that at difference with Ang II, it acts by a mechanism other than superoxide generation. Thus, Ang II acting on AT1 receptors stimulates superoxide generation, which, in turn, induces expression of P-selectin on the endothelial cell surface. BK inhibits the effects of Ang II, likely acting via NO. We conclude that the balance between Ang II, BK, and NO can regulate P-selectin expression on the endothelial cell membrane, an important component of the cascade leading to leukocyte adhesion to the vascular endothelium.

The arginine vasopressin (AVP) precursor gene of mammals contains three exons encoding the principal domains of the polyprotein precursor, including vasopressin (exon A), neurophysin (exon B), and glycopeptide (exon C). The AVP precursor (preprohormone) is processed and transported through the endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles, and finally, mature AVP is secreted from the posterior pituitary into the circulation. The exact steps of these processes during AVP translation and posttranslation events are not yet well elucidated. Defects in peptide processing are associated with several genetic disorders, including central diabetes insipidus (CDI). In the Brattleboro rat with CDI, the mRNA and protein of AVP are present in the hypothalamus, but no circulating AVP is detectable, thus suggesting a processing defect, transport defect, or both. The mutated AVP gene precursor of Brattleboro rat has a deletion of a single base, guanine, in the neurophysin coding region that leads to a frameshift resulting in the loss of the normal stop codon. It has been reported that the mutated precursor is trapped in the ER and does not reach the Golgi apparatus. Recent studies examined AVP secretion in cultured COS cells transfected with various constructs from wild-type and mutated Brattleboro AVP gene precursors. The wild-type in vitro studies demonstrated that intact neurophysin, but not the glycoprotein coding region, is necessary for normal AVP processing and secretion. Next, the results demonstrated that the guanine defect in the neurophysin coding region and the prolonged C-terminus accounted for the processing defect in the Brattleboro rat with CDI. These defects no doubt impair the folding and configuration necessary for normal processing of the AVP gene precursor in the ER. In hereditary CDI in humans, the majority of the mutations have also been shown to occur in the neurophysin coding region. However, in contrast to the recessive defect in the Brattleboro rat, in human CDI, neurotoxicity and denigration of the magnocellular neurons have been observed, and dominant inheritance occurs. Moreover, all mutations are missense, nonsense, or deletions in human CDI rather than the shift in reading frame and preserved neurons that is observed with the Brattleboro rat. Thus, the results from studies in the Brattleboro rat may only be partially applicable to hereditary CDI in humans.

Since the discovery of aquaporin water channels, insight into the molecular mechanism by which rapid osmotic water occurs across cell membranes has greatly improved. Aquaporin-2 is the vasopressin-responsive water channel in the collecting duct, and vasopressin control of water permeability in the collecting duct occurs in two ways: a short-term regulation and a long-term adaptation. In congenital nephrogenic diabetes insipidus, the kidney does not respond to vasopressin. Ninety percent of these patients carry a mutation in the gene coding for the vasopressin V2 receptor located on the X chromosome. Autosomal recessive and dominant forms of nephrogenic diabetes insipidus that are caused by mutations in the aquaporin-2 gene have now been described. This review focuses on recent insight in the molecular and cellular defect in autosomal nephrogenic diabetes insipidus.

A series of recent studies have demonstrated that expression of aquaporin-2 (AQP2), the vasopressin-regulated water channel of the kidney collecting duct, is greatly reduced in acquired forms of nephrogenic diabetes insipidus (NDI). In some forms of NDI, there is also impaired delivery of these channels to the apical plasma membrane, where they permit water reabsorption from the urine. The combination of these factors is likely to underlie the urinary concentrating defect that defines these conditions. Direct infusion of vasopressin causes an increase in AQP2 expression, probably via a rise in cytosolic adenosine 3:5-cyclic phosphate, which also acts as the second messenger, triggering the delivery of AQP2 to the plasma membrane. However, it is clear from the studies described that there are also vasopressin-independent pathways that regulate the expression of AQP2, some of which appear to reflect intranephric changes, whereas others involve systemic signals. These studies also show that recovery of AQP2 expression, even after correction of the underlying condition, can be slow, consistent with the clinical observation that recovery of urinary-concentrating ability often takes weeks or months. An understanding of the cellular signals and mechanisms responsible for the decrease in AQP2 expression may make it possible to develop treatments for this common clinical problem.

In congenital nephrogenic diabetes insipidus, the renal collecting ducts are resistant to the antidiuretic action of arginine vasopressin or to its antidiuretic analog 1-deamino[8-D-arginine] vasopressin (dDAVP). This is a rare, but now well described entity secondary to either mutations in the AVPR2 gene that codes for the vasopressin antidiuretic (V2) receptor or to mutations in the AQP2 gene that codes for the vasopressin-dependent water channel. A majority (> 90%) of congenital nephrogenic diabetes insipidus patients have AVPR2 mutations: Of 115 families with congenital nephrogenic diabetes insipidus, 105 families had AVPR2 mutations, and 10 had AQP2 mutations. When studied in vitro, most AVPR2 mutations lead to receptors that are trapped intracellularly and are unable to reach the plasma membrane. A minority of the mutant receptors reach the cell surface but are unable to bind vasopressin or to trigger an intracellular adenosine 3:5-cyclic phosphate signal properly. Most of the reported mutations are secondary to a complete loss of function of the receptor, and only a few mutations have been associated with a mild phenotype. These advances provide diagnostic tools for physicians caring for these patients because, when the disease causing mutation has been identified, carrier and perinatal testing could be done by mutation analysis.