A monoclonal antibody directed against a granule membrane glycoprotein (GMP-140/PADGEM, P-selectin, CD62P) inhibits ristocetin-induced platelet aggregation

P-selectin (also called CD62, GMP-140, PADGEM, CD62P) is a recently described member of a family of vascular adhesion receptors expressed by activated platelets and endothelial cells that are involved in leucocyte cell adhesion. The aim of this study was to characterize a new monoclonal antibody (LYP7) directed against activated human blood platelets that inhibits ristocetin-induced platelet aggregation. Immunoadsorbent affinity chromatography and immunoprecipitation studies showed that LYP7 (IgG₁) bound a surface-labelled glycoprotein (GP) which changed its apparent molecular mass (M_r) on reduction from 138 kD (situated below GPIIb) to 148 kD (above GPIIbα). LYP7 and S12, a monoclonal antibody directed against P-selectin immunoprecipitated the same band. Using ELISA assay, purified P-selectin was shown to bind LYP7 and S12 monoclonal antibodies. Binding sites of ¹²⁵I-labelled LYP7, which was greatly increased on thrombin-stimulated (2 U/ml) washed platelets (10825±2886, mean ±SD) (K_d=1.5±0.5 nm ) compared to resting platelets (2801±1278, mean ±SD) (K_d=1.5±0.6 nm ), was found to be normal on thrombin-stimulated platelets taken from a patient with grey platelet syndrome or a patient with Glanzmann thrombasthenia. LYP7 (IgG₁, F(ab′)₂ or Fab fragments) inhibited ristocetin-induced platelet aggregation of platelets in a dose-dependent fashion without affecting the binding of von Willebrand (vWf ) factor. However, agglutination of formaldehyde-fixed platelets induced by ristocetin was not affected by monoclonal antibody LYP7. In addition, the binding of thrombin-activated platelets to neutrophils was inhibited by monoclonal antibody LYP7. These results strongly suggest that P-selectin, by promoting cell–cell contact, may play an active role in platelet–platelet interactions.     

Solubilization of liposomes by weak electrolyte drugs II. Cefotaxime

The solubilization of dimyristoylphosphatidylcholine (DMPC) liposomes by the weak electrolyte drug, cefotaxime (CFX), has been studied as a function of pH, DMPC, temperature, presence of cholesterol (CHOL), and method of liposome preparation. At 7.5mMCFX the lag time for solubilization increased, the rate of solubilization decreased, and the minimum turbidity reached increased as a function of DMPC at pH 1.0 and 40C. Solubilization was most pronounced at pHs below the pKa but inhibited at least one pH unit above the pKa. The critical mole ratio of unionized CFX:DMPC, Rec, for solubilization was estimated to be 0.12. Reducing the temperature slowed the rate of solubilization as did the addition of CHOL. Encapsulation of CFX in liposomes did not significantly reduce CFX degradation, k1=0.048h-1 at 40C and a complex of DMPC and a degradation product of CFX precipitated as rectangular crystals. As a result, an increase in the turbidity of solubilized systems was observed from about 20h to 48h depending on the conditions. Liposomes in the gel state or with at least 20% CHOL did not undergo an apparent reversal of solubilization. It is concluded that the inclusion of weak electrolyte drugs existing predominantly as the unionized species in liquid crystalline state liposomes may undergo a slow solubilization process not necessarily recognized during characterization.     

Preparation and characterization of ofloxacin microspheres for the eradication of bone associated bacterial biofilm

Biodegradable polymers for localized delivery of antibiotics have emerged as an important approach to treating orthopaedic infections. In chronic forms of osteomyelitis which arethought tobeassociated with bacterial biofilm, localized delivery of a suitable antibiotic is desirable. This paper describes the formulation and in vitro evaluation of biodegradable ofloxacin microspheres for the eradication of bone associated bacterial biofilm infections. Ofloxacin microspheres were formulated using poly(glycolic acid-co-dl-lactic acid) (PGLA) by the emulsion solvent evaporation technique. The effects of process parameters such as phase volume, poly(vinyl alcohol) (PVA) concentration, and viscosity grade of the polymer during preparation on encapsulation efficiency (EEF) and in vitro release profiles were investigated. An increase in the phase volume or volume fraction from 21 to 35% at a constant internal phase volume resulted in an increase in EEF from 34 to 74%. Increasing PVA concentration from 0.25 to 2.5% w/v at a constant phase volume or volume fraction did not have an effect on the EEF. Ofloxacin release from the microsopheres was biphasic with an initial burst release followed by a slow release phase. An optimum slowing down of release was observed when the phase volume was 29%. Above and below this phase volume, release of ofloxacin was higher. The higher the viscosity grade of the polymer used for the preparation of microspheres, the higher the PVA concentration needed to prepare microspheres with slower release. The study indicates that various rates of ofloxacin release is possible by varying formulation conditions. This should provide a means for formulating sustained release microspheres of antibiotics for the treatment of biofilm infections associated with the bone.     

Diagnosis of prosthetic heart valve thrombosis. The respective values of transthoracic and transoesophageal Doppler echocardiography

Early diagnosis of acute prosthetic thrombosis remains a challenge,in 20 patients with 23 thrombosed cardiac valves, we evaluatedthe respective value of transthoracic (TTE) and transoesophageal(TEE) Doppler echocardiography. According to the presence orabsence of prosthetic obstruction by continuous-wave Doppler,prostheses were separated into two groups. Group 1 included nine thrombosed prostheses (8 mitral, 1 aortic)with severe obstruction. All patients presented with severesymptoms of heart failure. Transthoracic Doppler echocardiographyallowed immediate diagnosis of prosthetic thrombosis, even incritically ill patients, showing (1) eccentric transprostheticcolour flow jets in all eight mitral prostheses, (2) severeobstruction on Doppler examination (mean gradient = 18 to 36mmHg in eight mitral prostheses, and 69 mmHg in one aortic valve),and (3) direct echocardiographic evidence of thrombosis (i.e.thrombus or abnormal disc or leaflet motion) in four patients.All nine patients were immediately treated by surgery (n=8)or fibrinolysis (n =1) on the basis of TTE results only. TEEallowed better visualization of thrombus and restricted leafletor disc motion, but had little influence on patient management. Group 2 included 14 thrombosed prostheses (10 mitral, 4 aortic)with mild or absent obstruction, in three patients with massivemitral prosthetic thrombosis, an associated minimal thrombosisof a prosthetic aortic valve was found at surgery, but was detectedneither by TTE, nor by TEE. The 11 remaining patients presentedwith isolated partial mitral (n = 10) or aortic (n = 1) thrombosis.Clinical presentation was fever, cerebral embolism, or milddyspnoea, but no heart failure. TTE was normal in all. Continuous-waveDoppler showed normal prosthetic function in five patients andmild obstruction in six. TEE allowed diagnosis of prostheticthrombosis in all, showing an abnormal mobile echo around theprosthesis, despite normal disc or leaflet motion. In conclusion, transthoracic Doppler echocardiography is thediagnostic procedure of choice in patients with severely obstructiveprosthetic thrombosis, while the transoesophageal approach appearspromising in partial thrombosis with mild or absent obstruction.