Effects of the cyclin-dependent kinase inhibitor R-roscovitine on eosinophil survival and clearance.

BACKGROUND: Eosinophils are pro-inflammatory cells implicated in the pathogenesis of asthma and atopy. Apoptosis has been proposed as a potential mechanism underlying the resolution of eosinophilic inflammation and studies have indicated the ability of interventions that induce human eosinophil apoptosis to promote the resolution of eosinophilic inflammation. Recently, the cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to enhance neutrophil apoptosis and promote the resolution of neutrophilic inflammation. OBJECTIVE: The purpose of this study was to examine the expression of CDKs in human blood eosinophils, the effects of R-roscovitine on eosinophil survival in vitro and whether R-roscovitine could influence eosinophilic lung inflammation in vivo. METHODS: Eosinophils were isolated from human peripheral blood and the effects of R-roscovitine on apoptosis, degranulation and phagocytic uptake examined in vitro. The effects of R-roscovitine on eosinophilic lung inflammation in vivo were also assessed using an ovalbumin mouse model. RESULTS: Our data demonstrate that human eosinophils express five known targets for R-roscovitine: CDK1, -2, -5, -7 and -9. R-roscovitine induced eosinophil apoptosis in a time- and concentration-dependent manner but also accelerated transition to secondary necrosis as assessed by microscopy, flow cytometry and caspase activation. In addition, we show that R-roscovitine can override the anti-apoptotic signals of GM-CSF and IL-5. We report that the pro-apoptotic effect of R-roscovitine is associated with suppression of Mcl-1L expression and that this compound enhanced phagocytic clearance of eosinophils by macrophages. Finally, we show that R-roscovitine induces apoptosis in murine peripheral blood and spleen-derived eosinophils; despite this, R-roscovitine did not modulate the tissue and lumen eosinophilia characteristic of the ovalbumin mouse model of airway eosinophilia. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that R-roscovitine is capable of inducing rapid apoptosis and secondary necrosis in eosinophils but does not affect the onset or improve the resolution of eosinophilic airway inflammation in vivo.

Immunohistochemical analysis of nuclear versus cytoplasmic staining of WT1 in malignant mesotheliomas and primary pulmonary adenocarcinomas.

CONTEXT: Previous studies have indicated certain immunohistochemical markers, including WT1, may be helpful in distinguishing adenocarcinomas from mesotheliomas, but to date there are no reliable, widely accepted, commercially available antibodies positive in mesotheliomas and negative in adenocarcinomas. We compared the nuclear and cytoplasmic staining patterns of WT1 in these 2 malignancies using a commercially available antibody and examined the expression of 2 other previously reported positive markers, calretinin and thrombomodulin. METHODS: Sixty-seven mesotheliomas and 51 adenocarcinomas, all paraffin embedded, were retrieved from recent case files. The diagnosis of mesothelioma was based on typical clinical and morphologic features, as well as immunohistochemistry; electron microscopy had been performed on 16 cases. The diagnosis of adenocarcinoma was based on typical light microscopic findings and a positive stain for mucin. Commercially available antibodies to WT1, thrombomodulin, and calretinin were applied. Because of the conflict surrounding calretinin, 2 anticalretinin antibodies (from Chemicon Inc and Zymed Laboratories) were utilized. RESULTS: Fifty of 67 mesotheliomas showed strong nuclear staining with WT1. No adenocarcinomas (0/51) showed nuclear staining. Twenty-three of 67 mesotheliomas were positive for thrombomodulin, and 35 of 67 mesotheliomas were positive for calretinin with the Chemicon antibody. Nine of 15 mesotheliomas were positive for calretinin with the Zymed antibody. CONCLUSIONS: Thrombomodulin and calretinin did not prove useful in discriminating between mesotheliomas and adenocarcinomas. The degree of positivity with calretinin may be dependent on the specific antibody utilized. Nuclear staining for WT1 is highly specific for mesothelioma and, in the appropriate clinical setting, can be a helpful adjunct in the distinction between adenocarcinomas and mesotheliomas.

P-selectin binds to bacterial lipopolysaccharide.

Multiple organ failure associated with disseminated intravascular coagulation is a frequent complication in septic shock patients. Accumulation of platelets and neutrophils in the organs contributes to the manifestation of lipopolysaccharide (LPS)-induced organ failure. Although a direct interaction between LPS and platelets is well documented, the nature of the surface receptor for LPS on platelets is unknown. In this article we show that P-selectin is a receptor for LPS. The binding of LPS to P-selectin is independent of Ca2+, and is blocked by antibodies to P-selectin, lipid A and fucoidan. Platelets pre-treated with thrombin showed fourfold higher binding of fluorescein isothiocyanate (FITC)-conjugated LPS compared to untreated platelets and the binding of FITC-conjugated LPS to platelets was blocked in the presence of anti-P-selectin antibodies. It is likely that the binding of LPS via P-selectin on activated platelets or epithelium could have a significant role in the pathophysiology of organ failure in septic shock.

Inhibition of human platelet aggregation by GR91669, a prototype fibrinogen receptor antagonist.

In order to produce more potent and specific fibrinogen receptor (GpIIb/IIIa) antagonists, the Arg-Gly of a chemical series based upon Arg-Gly-Asp was replaced by alkyl chains of varying lengths. The most potent in this series, GR91669, inhibited aggregation of human gel-filtered platelets (GFP) in vitro induced by ADP or the thromboxane A2 mimetic, U46619, with IC50 values of 200nM and 500nM respectively and was selected for further studies. Its inhibitory effects on GFP were reversed by addition of excess fibrinogen. The compound also inhibited ADP- or U46619-induced platelet aggregation in human whole blood (IC50 values of 700nM in both cases). 125I-Fibrinogen binding to ADP-stimulated platelets was inhibited by GR91669 with an IC50 (65nM) similar to that against platelet aggregation. GR91669 (1mM) did not inhibit U46619-induced platelet shape change or 14C-5HT secretion from platelets stimulated by collagen, U46619 or thrombin. Therefore GR91669 inhibits aggregation but has no significant effect on stimulus-response events, a profile consistent with fibrinogen receptor blockade. In addition, GR91669 (1mM), unlike echistatin or Gly-Arg-Gly-Asp-Ser, did not disrupt vitronectin recptor-dependent attachment of cultured HUVECS in vitro and similarly did not inhibit Mac-1 dependent adhesion of human granulocytes. Thus, of the integrins tested, GR91669 appears to be specific for GpIIb/IIIa. Following intravenous administration to marmosets of 1 or 10 mg/kg GR91669, ADP (10 microM)-induced platelet aggregation ex vivo was abolished for 15 and 60 minutes respectively. Greater than 50% inhibition was maintained for 30 minutes and 2 hours respectively. GR91669, therefore appears to be a potent, specific fibrinogen receptor antagonist in vitro and which is also active in vivo.

Improved potency and specificity of Arg-Gly-Asp (RGD) containing peptides as fibrinogen receptor blocking drugs.

A range of cyclic RGD based peptides have been developed to mimic the conformation of RGD within fibrinogen. These peptides, as well as echistatin (IC50 = 0.05 microM) and GRGDS (IC50 = 25 microM) fully inhibited adenosine diphosphate (ADP) (10 microM)-induced platelet aggregation of human gel-filtered platelets (GFP). RGDF was the most potent linear peptide in inhibiting ADP-induced aggregation (IC50 = 8 microM) but cyclisation, using a 6,5 bicyclic coupling group to produce GR83895, led to an approximately 10-fold increase in potency (IC50 = 0.9 microM). In GFP, ADP-induced 125I-fibrinogen binding was inhibited (> 80%) by echistatin, GRGDS or GR83895 at concentrations (IC50 values 0.05 microM, 25 microM and 1.4 microM respectively) similar to those needed to inhibit aggregation. All three compounds also completely inhibited ADP- and U46619-induced aggregation in both platelet rich plasma (PRP) and whole blood. In contrast to platelet aggregation, U-46619-induced 14C-5HT secretion in PRP was not inhibited by GR83895 or echistatin, indicating that agonist-induced signal transduction is not affected by either agent, a profile consistent with that predicted for a specific fibrinogen receptor blocking drug. To test specificity of action, echistatin, GR83895 and GRGDS were also examined for their ability to detach cultured human umbilical vein endothelial cells attached to plastic through a vitronectin receptor dependent process. GR83895 only caused detachment at concentrations 100-fold greater than those required to inhibit platelet aggregation, in contrast to GRGDS and echistatin which caused cell detachment at concentrations similar to those inhibiting aggregation. In summary, cyclisation of RGD-containing peptides has led to both improved potency and specificity of action. Such specificity of action may prove to be an important consideration for the successful development of a fibrinogen receptor blocking drug as an anti-thrombotic drug.

Effect of GR32191 and other thromboxane receptor blocking drugs on human platelet deposition onto de-endothelialized arteries.

A range of thromboxane A2 receptor blocking (TxRB) drugs, prostacyclin and aspirin have been assessed as inhibitors of human platelet deposition onto rabbit and human de-endothelialized arteries in vitro. Platelet deposition was quantified by measuring the radioactivity associated with de-endothelialized arteries following superfusion with 111indium-labelled human platelets reconstituted in blood. Using rabbit aorta, all of the compounds tested produced a similar maximum inhibition (approximately 70%) of platelet deposition; from scanning EM studies the residual deposition appeared to represent a monolayer of adhered platelets. The potency of the TxRB's for inhibiting deposition was GR32191 greater than or equal to GR36246 greater than SQ29,548 greater than ICI185282 greater than or equal to AH23848 much greater than BM13.177 consistent with their TxRB potency on human platelets. Using human umbilical arteries, the TxRB's achieved a smaller maximum inhibition of deposition (approximately 50%) than did prostacyclin or the fibrinogen receptor blocking peptide Gly-Arg-Gly-Asp-Ser (GRGDS) (60-75%). In addition, using human umbilical arteries, the structurally-related TxRB's GR32191 and GR36246 exhibited a greater than 1000-fold enhancement in potency as inhibitors of platelet deposition over that seen in the rabbit aorta. In preliminary experiments, GR32191 also displayed a similar high potency on human cerebral arteries. In contrast, the structurally unrelated compounds SQ29,548, ICI185282 and BM13.177 exhibited similar potencies on human umbilical arteries to those observed on the rabbit aorta; aspirin and prostacyclin also displayed similar potencies on the two preparations. The enhanced effect of GR32191 and GR36246 on human umbilical arteries therefore appears unrelated to their action as TxRB's on human platelets although the mechanism of this unique action is at present unknown. However, if these drugs exhibited a similar high potency for preventing mural thrombus formation in vivo in man, they may represent a major advance in the treatment of occlusive vascular disease.

A prospective assessment of nutritional status and complications in patients with fractures of the hip.

A group of 40 consecutive patients with hip fractures were studied and confirmed to have a high incidence of protein-calorie malnutrition. The prospective nutritional assessment performed for this study included: serum albumin, serum transferrin, anthropometric measurements, skin testing for delayed hypersensitivity, total lymphocyte count, and a 24-h urine collection for metabolic and nitrogen balance determinations. At 3 months after their hip fracture, 37.5% returned to their premorbid ambulatory status; 42.5% sustained a decrement in their ambulatory status or independence; 12.5% died; 7.5% were lost to follow-up. Of the nutritional parameters studied, albumin was significantly associated with mortality (p = 0.004). Considering those patients with an albumin less than 3.0, a mortality rate of 70% was observed in follow-up (maximum of 11 months), compared with a mortality rate of 18% in patients with an albumin greater than or equal to 3.0. It is concluded that the serum albumin has value as a nutritional index without specialized nutritional parameters, and that a more aggressive approach to nutritional support is needed for the hypoalbuminemic patient with a hip fracture, particularly for those with a serum albumin below 3.0.

The inhibitory effect of GR32191, a thromboxane receptor blocking drug, on human platelet aggregation, adhesion and secretion.

GR32191, a potent selective thromboxane receptor antagonist, has been shown to inhibit completely prostaglandin endoperoxide and thromboxane A2 (TxA2)-induced platelet aggregation, [14C]-serotonin secretion and beta-thromboglobulin secretion. Deposition of human platelets onto damaged rabbit aorta in vitro is reduced in the presence of GR32191 which appears to inhibit aggregation of platelets but not direct adhesion of platelets to subendothelium. The effects of non-prostanoid platelet activating agents whose mode of action requires the biosynthesis of TxA2 are also inhibited by GR32191. Prostanoids which inhibit platelet function, such as prostacyclin or PGD2, retain their inhibitory properties in the presence of GR32191 which does not inhibit phospholipase A2, prostaglandin cyclooxygenase, thromboxane synthase, 12-lipoxygenase or cAMP phosphodiesterase activity. The inhibitory action of GR32191 on platelet aggregation, mural thrombus formation and platelet protein storage granule secretion suggests that it has potential in treating thrombotic disease in man.

Dosimetric study of microwave cataractogenesis.

A closed waveguide method has been used to induce opacities in the rabbit eye. A 30-min exposure to 2.45-GHz radiation such that 8.7 W is incident on the head (5.75 W being absorbed) produces a cataract in half of the exposed eyes of New Zealand white rabbits.