Expression of the 60 kDa and 71 kDa heat shock proteins and presence of antibodies against the 71 kDa heat shock protein in pediatric patients with immune thrombocytopenic purpura

BACKGROUND: Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by platelet destruction resulting from autoantibodies against platelet proteins, particularly platelet glycoprotein IIb/IIIa. Heat shock proteins (Hsp) have been shown to be major antigenic determinants in some autoimmune diseases. Antibodies to Hsps have also been reported to be associated with a number of pathological states. METHODS: Using western blot, we measured the levels of the 60 kDa heat shock protein (Hsp60) and of the inducible 71 kDa member of the Hsp70 family (Hsp71) in lymphocytes and the presence of antibodies against these hsps in plasma of 29 pediatric patients with ITP before the treatment and in 6 other patients before and after treatment. RESULTS: Interestingly only one out of 29 patients showed detectable Hsp60 in lymphocytes while this heat shock protein was detected in the 30 control children. Hsp71 levels were slightly lower in lymphocytes of patients with ITP than in controls (1567.8 ± 753.2 via 1763.2 ± 641.8 integrated optical density (IOD) units). There was a small increase of Hsp71 after recovery from ITP. The titers of plasma antibodies against Hsp60 and Hsp71 were also examined. Antibodies against Hsp71 were more common in ITP patients (15/29) than in control children (5/30). The titer of anti-Hsp71 was also higher in children patients with ITP. The prevalence of ITP children with antibodies against Hsp71 (51.7%) was as high as those with antibodies against platelet membrane glycoproteins (58.3%). CONCLUSIONS: In summary, pediatric patients with ITP showed no detectable expression of Hsp60 in lymphocytes and a high prevalence of antibody against Hsp71 in plasma. These changes add to our understanding of the pathogenesis of ITP and may be important for the diagnosis, prognosis and treatment of ITP

A Novel, “Double-Clamp” Binding Mode for Human Heme Oxygenase-1 Inhibition

The development of heme oxygenase (HO) inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308). Using a carbon monoxide (CO) formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC50 = 0.27±0.07 µM) than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC50 = 4.0±1.8 µM). The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This “double-clamp” binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors

Circulating heat shock protein 70 (HSPA1A) in normal and pathological pregnancies

Heat shock proteins (Hsps) are ubiquitous and phylogenetically conserved molecules. They are usually considered to be intracellular proteins with molecular chaperone and cytoprotective functions. However, Hsp70 (HSPA1A) is present in the peripheral circulation of healthy nonpregnant and pregnant individuals. In normal pregnancy, circulating Hsp70 levels are decreased, and show a positive correlation with gestational age and an inverse correlation with maternal age. The capacity of extracellular Hsp70 to elicit innate and adaptive proinflammatory (Th1-type) immune responses might be harmful in pregnancy and may lead to the maternal immune rejection of the fetus. Decreased circulating Hsp70 level, consequently, may promote the maintenance of immunological tolerance to the fetus. Indeed, elevated circulating Hsp70 concentrations are associated with an increased risk of several pregnancy complications. Elevated Hsp70 levels in healthy pregnant women at term might also have an effect on the onset of labor. In preeclampsia, serum Hsp70 levels are increased, and reflect systemic inflammation, oxidative stress and hepatocellular injury. Furthermore, serum Hsp70 levels are significantly higher in patients with the syndrome of hemolysis, elevated liver enzymes, and low platelet count (HELLP syndrome) than in severely preeclamptic patients without HELLP syndrome. In HELLP syndrome, elevated serum Hsp70 level indicates tissue damage (hemolysis and hepatocellular injury) and disease severity. Increased circulating Hsp70 level may not only be a marker of these conditions, but might also play a role in their pathogenesis. Extracellular Hsp70 derived from stressed and damaged, necrotic cells can elicit a proinflammatory (Th1) immune response, which might be involved in the development of the maternal systemic inflammatory response and resultant endothelial damage in preeclampsia and HELLP syndrome. Circulating Hsp70 level is also elevated in preterm delivery high-risk patients, particularly in treatment-resistant cases, and may be a useful marker for evaluating the curative effects of treatment for preterm delivery. In addition, increased circulating Hsp70 levels observed in asthmatic pregnant patients might play a connecting role in the pathomechanism of asthmatic inflammation and obstetrical/perinatal complications. Nevertheless, a prospective study should be undertaken to determine whether elevated serum Hsp70 level precedes the development of any pregnancy complication, and thus can help to predict adverse maternal or perinatal pregnancy outcome. Moreover, the role of circulating Hsp70 in normal and pathological pregnancies is not fully known, and further studies are warranted to address this important issue