Detection of drug-dependent, platelet-reactive antibodies by antigen- capture ELISA and flow cytometry

The effectiveness of flow cytometry in the detection of drug-dependent, platelet-reactive antibodies was investigated. In studies of seven sera known to contain quinine- or quinidine-dependent, platelet-reactive antibodies, flow cytometry was 5 to 10 times more sensitive in detecting drug-dependent antibodies (DDAbs) than the 51Cr release assay, antigen-capture enzyme-linked immunosorbent assay (ELISA), and indirect immunofluorescence microscopic assay. With flow cytometry, DDAbs could be detected at drug concentrations as low as 0.1 microM, or less than one-tenth the level required with other methods. Antigen-capture ELISA was not as sensitive as flow cytometry in DDAb detection, but it did allow identification of the DDAbs' target molecules. With this assay, five of the seven DDAbs recognized both the glycoprotein Ib/IX (GPIb/IX) and glycoprotein IIb/IIIa (GPIIb/IIIa) complexes, while the remaining two sera reacted only with GPIb/IX. Of 44 consecutive patients who developed thrombocytopenia while taking quinidine, DDAbs were detected by flow cytometry in 11 (25%), more than twice the number detected by other methods. In one patient who developed thrombocytopenia while taking trimethoprim/sulfamethoxazole, DDAbs could be detected only by flow cytometry. It can be concluded that flow cytometry is highly sensitive in detecting DDAbs and allows their detection at pharmacologic concentrations of the drug. Most quinidine-dependent antibodies recognize at least two different glycoprotein complexes in the platelet membrane.     

Recovery and survival in vivo of platelet concentrates prepared with prostaglandin E1

In follow-up to previous studies showing that stimulators of adenylate cyclase inhibit the activation of platelets in platelet concentrates (PC), the posttransfusion recovery and survival of autologous platelets prepared and stored after the addition of prostaglandin E1 (PGE1) to platelet-rich plasma at a concentration of 1.3 X 10(-8) M were investigated. Six normal subjects were studied on the two occasions, using PC stored with and without PGE1 and radiolabeling with 51Cr. The mean recovery of platelets prepared with PGE1 (35.2 +/- 8.1%) was significantly lower (p less than 0.013) than that of routinely prepared platelet concentrates (46.3 +/- 9.4%). The mean life-spans of platelets prepared with and without PGE1 were 7.1 +/- 0.4 and 6.2 +/- 1.0 days, respectively (p = NS). Despite its ability to inhibit the activation of platelets during concentration and storage, prostaglandin E1 appears to reduce posttransfusion recovery of platelets significantly in this experimental model and cannot be recommended at this time as an adjunct for PC preparation.     

Study on Alzheimer’s disease model

Study on Alzheimer’s disease model     

Spinal glial TLR4-mediated nociception and production of prostaglandin E2 and TNF

Background and purpose:

Toll-like receptor 4 (TLR4) expressed on spinal microglia and astrocytes has been suggested to play an important role in the regulation of pain signalling. The purpose of the present work was to examine the links between TLR4, glial activation and spinal release of prostaglandin E₂ (PGE₂) and tumour necrosis factor (TNF), and the role these factors play in TLR4-induced tactile allodynia.

Experimental approach:

Toll-like receptor 4 was activated by intrathecal (i.t.) injection of lipopolysaccharide (LPS) and KDO₂-Lipid A (KDO₂) to rats. Tactile allodynia was assessed using von Frey filaments and cerebrospinal fluid collected through spinal dialysis and lumbar puncture. PGE₂ and TNF levels were measured by mass spectometry and elisa. Minocycline and pentoxifylline (glia inhibitors), etanercept (TNF-blocker) and ketorolac (COX-inhibitor) were given i.t. prior to injection of the TLR4-agonists, in order to determine if these agents alter TLR4-mediated nociception and the spinal release of PGE₂ and TNF.

Key results:

Spinal administration of LPS and KDO₂ produced a dose-dependent tactile allodynia, which was attenuated by pentoxifylline, minocycline and etanercept but not ketorolac. Both TLR4 agonists induced the spinal release of PGE₂ and TNF. Intrathecal pentoxifylline blunted PGE₂ and TNF release, while i.t. minocycline only prevented the spinal release of TNF. The release of PGE₂ induced by LPS and KDO₂ was attenuated by i.t. administration of ketorolac.

Conclusions and implications:

Activation of TLR4 induces tactile allodynia, which is probably mediated by TNF released by activated spinal glia.     

High prolactin and low dehydroepiandrosterone sulphate serum levels in patients with severe systemic sclerosis

The aim was to determine serum levels of prolactin (PRL) and dehydroepiandrosterone sulphate (DHEAS), and to demonstrate a link between PRL or DHEAS and soluble immune mediators in patients with systemic sclerosis (SSc) with different degrees of disease-induced organ involvement. Thirty-one patients with SSc were studied to evaluate 18 possible disease manifestations. In the serum, PRL, DHEAS and soluble immune mediators were determined by ELISA. Compared to SSc with <9 disease manifestations, patients with > or =9 disease manifestations had higher PRL (P = 0.044), higher soluble interleukin 2 receptor (sIL-2R, P = 0.004) and vascular cell adhesion molecule (sVCAM, P = 0.044), and lower DHEAS (P = 0.029). PRL (R(Rank) = 0.490, P = 0.003) and DHEAS (R(Rank) = -0.399, P = 0.013) were significantly correlated with the number of disease manifestations. The inverse correlation between PRL and DHEAS showed a trend (P = 0.059). PRL correlated with sIL-2R (R(Rank) = 0.553, P = 0.001) and sVCAM (R(Rank) = 0.520, P = 0.002). The number of disease manifestations and sIL-2R correlated significantly (R(Rank) = 0.463, P = 0.006). Psychometric variables to examine the presence of depression were not measured, but from the general aspect, the patients were not suffering from major depression which may have influenced our results. In conclusion, this study demonstrates the close association between DHEAS and, particularly, PRL and SSc severity and T-lymphocyte mechanisms.      

Reference values for pulse oximetry at high altitude

OBJECTIVE: To determine reference values for oxygen saturation (Sao2) in healthy children younger than 5 years living at high altitude. DESIGN: One hundred and sixty eight children were examined for Sao2 at 4018 m during well child visits. Physiological state was also noted during the examination. RESULTS: The mean Sao2 was 87.3% (95% confidence intervals (CI) 86.7%, 87.9%) with a median value of 87.7%. A significant difference was observed in Sao2 between children younger than 1 year compared with older children, although the difference was no longer demonstrable when sleeping children were excluded. CONCLUSIONS: This study has provided a reference range of Sao2 values for healthy children under 5 years old so that pulse oximetry may be used as an adjunct in diagnosing acute respiratory infections. Younger children were also shown to have a lower mean Sao2 than older children living at high altitude, which suggests physiological adaptation to high altitude over time. In addition, sleep had a lowering effect on Sao2, although the clinical importance of this remains undetermined.     

MAG3 renal scintigraphy: Improved ability to make anatomical diagnoses in neonates

Technetium-99m mercaptoacetyltriglycine(MAG3) is the most recently introduced renal radiopharmaceutical in Australia and is established as the agent of choice for use in diuresis renography, particularly in neonates and infants. It provides superior anatomical information compared to previously used agents. Three cases are reported in which MAG3 diuresis renography was performed in neonates, who were found to have hydronephrosis detected antenatally. In two neonates, a previously unrecognized horseshoe kidney was demonstrated and in case 3 there were scan features characteristic of a ureterocele. It is highly unlikely that these abnormalities would have been delineated with ^99mTc dimethyltriamine penta-acetic acid (DTPA) study, as confirmed in case 1, because of the relatively poor uptake of DTPA when compared to MAG3.