Audit of warfarin reversal using a new Octaplex reduced dose protocol.

INTRODUCTION: Prothrombin Complex Concentrate (PCC) is increasingly used for the emergency reversal of the effects of Vitamin K antagonists due to the increased use of the latter. There is no consensus on dosage protocols for its use. There is evidence that small fixed doses are effective. We report the result of the use of a simple three dose level protocol i.e. 2000 IU for CNS bleeds, 1500 IU for other bleeds and 1000 IU for non bleeders. METHODS: Data was prospectively collected over a 6 month period on all patients receiving PCC (Octaplex). These included clinical indication, dose given, INR test results, delay in treatment, and patients' demographics. RESULTS: The protocol was followed in only 40%; 24% were given a larger dose and 35% a smaller dose than we recommended. Despite this the INR was corrected (≤1.5) in 56 (83.6%) out of the 67 patients studied. The average delay in getting INR results was 1 hour 14 minutes and delay between releasing the PCC from blood bank to infusion was 3 hours. CONCLUSION: A simple three level low fixed dose protocol is cost effective in reversing the majority of patients' anticoagulation. Delay in initiating treatment for the reversal of VKA and adherence to protocols remained problematic.

Mice mutant for Egfr and Shp2 have defective cardiac semilunar valvulogenesis.

Atrioventricular and semilunar valve abnormalities are common birth defects, but how cardiac valvulogenesis is directed remains largely unknown. During studies of genetic interaction between Egfr, encoding the epidermal growth factor receptor, and Ptpn11, encoding the protein-tyrosine-phosphatase Shp2, we discovered that Egfr is required for semilunar, but not atrioventricular, valve development. Although unnoticed in earlier studies, mice homozygous for the hypomorphic Egfr allele waved-2 (Egfrwa2/wa2) exhibit semilunar valve enlargement resulting from over-abundant mesenchymal cells. Egfr-/- mice (CD1 background) have similar defects. The penetrance and severity of the defects in Egfrwa2/wa2 mice are enhanced by heterozygosity for a targeted mutation of exon 2 of Ptpn11 (ref. 3). Compound (Egfrwa2/wa2:Ptpn11+/-) mutant mice also show premature lethality. Electrocardiography, echocardiography and haemodynamic analyses showed that affected mice develop aortic stenosis and regurgitation. Our results identify the Egfr and Shp2 as components of a growth-factor signalling pathway required specifically for semilunar valvulogenesis, support the hypothesis that Shp2 is required for Egfr signalling in vivo, and provide an animal model for aortic valve disease.

The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system.

The transformation of an unpatterned epithelium into a patterned one is a fundamental issue in morphogenesis. This transformation occurs in a dramatic fashion in the developing eye imaginal disc, the primordium of the Drosophila compound eye. Molecular and developmental analyses reveals that the sine oculis (so) locus encodes a homeodomain-containing protein that is expressed and required in the unpatterned epithelium prior to morphogenesis. In mutants, cells undergo apoptosis. These findings argue that so plays an essential role in controlling the initial events of pattern formation in the eye disc. So is also expressed and required for the development of the rest of the fly visual system, including the optic lobes (i.e., those regions of the brain that process visual information). So is expressed in the optic lobe primordium prior to its invagination from the embryonic ectoderm; in so mutants, the optic lobe primordium fails to invaginate.

CA 19-9 and CA 125 levels in the sera of normal blood donors in relation to smoking history.

CA 19-9 and CA 125 serum levels were evaluated among smoking and nonsmoking healthy blood donors. Smoking did not elevate mean levels of either CA 19-9 or CA 125 in the sera of 496 of these blood donors from Philadelphia, PA. Mean CA 19-9 levels were slightly higher among females than among males. Among smokers there was a trend toward slightly increasing CA 19-9 serum levels with increased age, which was significant among the male donors. Trends toward slightly decreased mean serum levels of CA 125 among smokers were of borderline significance. Serum CA 19-9 and CA 125 levels in none of these donor subpopulations was elevated compared to levels reported by others for gastrointestinal or ovarian carcinoma patients, respectively. Therefore, smoking status should not interfere with use of either the CA 19-9 or CA 125 assays for diagnostic or monitoring applications.

Roles of the 5' leader region of the ompA mRNA.

OmpA protein, a major outer membrane protein of Escherichia coli, is synthesized from a messenger RNA containing a 134-nucleotide 5' leader region. The role of this leader region in efficient ompA expression was investigated using a series of ompA-lacZ fusion plasmids. These plasmids differ in the amount of DNA encoding the ompA leader region which is fused to the lacZ structural gene. The fusion containing all but six nucleotides of the ompA leader produced the highest beta-galactosidase activity, while the fusion containing the shortest leader synthesized only 4% as much beta-galactosidase. Fusions with leaders intermediate in length produced between 6% and 24% of the activity found in the most efficient fusion. Quantitation of lacZ mRNA synthesis by DNA-RNA hybridization revealed differences in lacZ mRNA production reflecting the observed differences in beta-galactosidase activity. The primary effect of the ompA leader in maintaining high levels of mRNA is discussed in terms of the roles of mRNA secondary structure.

Adrenocortical function after chronic inhalation of fluocortinbutyl and beclomethasone dipropionate.

Fluocortinbutyl (FCB) is a C-21 ester, topically active corticosteroid; no adrenal suppression has been noted after large doses. We compared safety and effects on adrenocortical function of orally inhaled FCB (40 mg/day), beclomethasone dipropionate (BDP) (2 mg/day), and placebo administered in four monitored divided doses for 4 wk by three groups of five healthy men. Circadian plasma cortisol concentration and daily urinary free cortisol excretion were determined before and after 3- and 4-wk exposure. Although pretreatment mean area under the curve (micrograms . hr . dl-1) for plasma cortisol did not differ among groups, mean values after weeks 3 and 4 of treatment were lower (p less than 0.05) in the BDP group (95.1 and 83) than in the FCB (155.8 and 153.7) and placebo (141 and 135.8) groups. Mean urinary cortisol excretion after week 4 for the BDP group (29 micrograms) was less (p less than 0.05) than in the FCB (59 micrograms) and the placebo (69 micrograms) groups. Slopes of individual regression lines noting time trends in plasma and urinary cortisol in the BDP group were negative and less (p less than 0.05) than those of the other groups. A cosyntropin test given intravenously after 4 wk of exposure resulted in similar plasma cortisol responses among groups. No serious adverse effects were noted. Thus after long-term high-dose treatment BDP but not FCB suppressed basal adrenocortical function, but neither suppressed the adrenocortical response to cosyntropin.

The cation-independent mannose-6-phosphate receptor binds to soluble GPI-linked proteins via mannose-6-phosphate.

The cation-independent mannose-6-phosphate/insulin-like growth factor II receptor has been observed to bind to soluble forms of glycosyl-phosphatidylinositol-linked molecules, one of mammalian origin (rat Thy-1) and two of protozoan origins. Of the two phosphate groups found on the soluble forms of the protozoan glycosyl-phosphatidylinositol-linked molecules: (i) the internal mannose-6-phosphate diester (which forms a part of the ethanolamine bridge) and (ii) the inositol-1,2 cyclic phosphate group (which arises after cleavage of the membrane associated form with phosphatidylinositol-specific phospholipase C), only the former appears to be recognized by the mannose-6-phosphate/insulin-like growth factor II receptor, as mild acid hydrolysis which destroys the latter has been observed not to affect the receptor binding site.

The cation-independent mannose-6-phosphate receptor binds to soluble GPI-linked proteins via mannose-6-phosphate.

The cation-independent mannose-6-phosphate/insulin-like growth factor II receptor has been observed to bind to soluble forms of glycosyl-phosphatidylinositol-linked molecules, one of mammalian origin (rat Thy-1) and two of protozoan origins. Of the two phosphate groups found on the soluble forms of the protozoan glycosyl-phosphatidylinositol-linked molecules: (i) the internal mannose-6-phosphate diester (which forms a part of the ethanolamine bridge) and (ii) the inositol-1,2 cyclic phosphate group (which arises after cleavage of the membrane associated form with phosphatidylinositol-specific phospholipase C), only the former appears to be recognized by the mannose-6-phosphate/insulin-like growth factor II receptor, as mild acid hydrolysis which destroys the latter has been observed not to affect the receptor binding site.

Interpretation of serum phenytoin concentrations in uremia is assay-dependent.

A 25-year-old man with sickle cell disease and chronic renal insufficiency had tonic-clonic seizures treated with phenytoin. Serum phenytoin concentrations, total and free, measured by two homogeneous enzyme immunoassays (EMIT, CAC) were reported to be within the therapeutic range, yet the patient experienced seizures. Values on discharge exceeded the therapeutic range but were not associated with signs or symptoms of toxicity. Reanalysis of serum samples by a more specific, high performance liquid chromatographic (HPLC) method revealed the previous values were spurious, apparently due to phenytoin metabolite cross-reactivity. Values by fluorescence polarization immunoassay (TDX) correlated well with those by HPLC, as well as with the patient's clinical course.

Effect of mic gene structure on repressor activity in the OmpA system.

It has been previously established that micRNA (mRNA-interfering complementary RNA) complementary to an individual mRNA specifically represses the expression of the target mRNA. We have constructed several plasmids which produce micRNAs which are complementary to different regions of the ompA mRNA. The repressor activity of these different micRNAs has been compared to determine the role of mic gene structure in effective micRNA function. The results indicate that micRNAs complementary to regions of the ompA mRNA likely to encounter ribosomes have the highest repressor activities. A clear effect of mic gene dosage was also observed. This was demonstrated using both identical and different mic(ompA) genes.

Recognition of the major cell surface glycoconjugates of Leishmania parasites by the human serum mannan-binding protein.

Activation of complement on the surface of parasitic protozoa of the genus Leishmania appears to be important for parasite infectivity in the mammalian host, as it allows these parasites to attach to and invade macrophages via their surface complement receptors. Serum mannan-binding protein (MBP) is a known activator of complement. Therefore, in the present study, we have investigated whether serum MBP binds to live Leishmania parasites, and to mannose-containing saccharides derived from the parasite cell surface. We have observed by fluorescence microscopy that biotinylated MBP binds to the surface of L. major and L. mexicana promastigotes. At this developmental stage the parasites are coated by a mannose-containing lipophosphoglycan (LPG). We have observed that radioiodinated MBP binds in a mannose-inhibitable manner to purified LPG which has been immobilized in plastic microwells, as well as to purified mannose-terminating di-, tri- and tetrasaccharide fragments ('cap' structures) which have been released by mild acid hydrolysis from the outer chains of the LPG, converted into neoglycolipids and resolved by thin-layer chromatography. 125I-MBP also binds in the chromatogram-binding assay to the mannose-containing glycoinositol-phospholipids that are expressed in high copy number on both the promastigote and the intracellular amastigote stages of most Leishmania species. These data suggest that MBP has the potential to opsonize the major developmental stages of Leishmania parasites, and provide a possible mechanism for the antibody-independent activation of complement on their surface.

Inhibition of recombinant low-voltage-activated Ca(2+) channels by the neuroprotective agent BW619C89 (Sipatrigine).

T-type Ca(2+) currents were recorded in 2 mM Ca(2+) from HEK 293 cells stably expressing recombinant low-voltage-activated Ca(2+) channel subunits. Current-voltage relationships revealed that these currents were low-voltage activated in nature and could be reversibly antagonised by mibefradil, a known T-type channel blocker. At a test potential of -25 mV alpha(1I)-mediated Ca(2+) currents were rapidly and reversibly inhibited by 1-100 microM BW619C89 (IC(50)=14 microM, Hill coefficient 1.3). In contrast to its actions on N-type Ca(2+) channels, a near IC(50) dose (10 microM) of BW619C89 produced no alterations in either the kinetics or voltage-dependence of T-type currents. In additional single dose experiments, currents mediated by rat alpha(1G), human alpha(1H) or human alpha(1I) channel subunits were also inhibited by BW619C89. Overall our data indicate that T-type Ca(2+) channels are more potently blocked by BW619C89 than either type-II Na(+) channels or N-type Ca(2+) channels. It seems, therefore, that inhibition of low-voltage-activated Ca(2+) channels is likely to contribute to the anticonvulsant and neuroprotective actions of this and related compounds.

Distribution and expression of TREK-1, a two-pore-domain potassium channel, in the adult rat CNS.

TREK-1 is a member of the two-pore-domain potassium channel family which is expressed predominantly in the CNS. Using an anti-peptide polyclonal antiserum, we have determined the distribution of TREK-1 in the brain and spinal cord of adult rats. Specificity of the antiserum was tested using a TREK-1-transfected cell line and confirmed with c-myc-tagged TREK-1. In thin tissue sections, immunoreactivity was widespread throughout the rat brain and spinal cord. TREK-1-like signals were observed in the cerebral cortex, basal ganglia, hippocampus, and various other subcortical nuclei in the hypothalamus, thalamus, mesencephalon and rhombencephalon. TREK-1 labelling appeared to be over the entire cell membrane, including the cell body and processes. Cells that morphologically resembled projection neurones and interneurones but not glial cells were labelled. As interneurones and known GABAergic projection neurones were the predominant population labelled, we investigated the possibility that TREK-1 is expressed in GABA-containing neurones using a specific anti-GABA antiserum. Expression of TREK-1 in GABA-containing neurones was observed in a number of areas, including the isocortex, hippocampus and thalamus. Thus, TREK-1 expression defines a unique and specific subset of interneurones and principal cells. These studies indicate a widespread distribution of TREK-1 potassium channels throughout the rat brain and spinal cord, with expression in a number of areas being demonstrated to be present on GABA-containing neurones.

The random transition model of the cell cycle. A critical review.

The random transition model of the cell cycle has received much attention in recent years in attempts to describe and explain variations in cell cycle times. In this review we suggest statistical procedures for fitting the model to experimental data in place of the invalid techniques currently used. However, we also argue that there have been misconceptions about criteria for quality of fit of the model, and consequent biological interpretations. Other models fit just as well, and the analyses we describe do not provide evidence for any particular biological mechanism.

Peritoneal fluid and serum steroids in infertility patients.

Peritoneal fluid and serum were collected from 78 patients at the time of laparoscopy. Twenty-two were fertile controls (CTL), and 56 were infertility patients, who were subdivided into three main groups: endometriosis (EMS), pelvic adhesions (ADH), and ovarian dysfunction (OvDF). Based on control group data, biochemical criteria indicative of the presence of a stigma, S(+), were established: (1) serum progesterone (P) greater than or equal to 2 ng/ml, (2) peritoneal fluid P greater than or equal to 50 ng/ml, and (3) peritoneal fluid/serum ratio of P greater than or equal to 3. Direct visualization by laparoscopy showed that 21% CTL, 75% EMS, 69% ADH, and 56% OvDF subjects had luteinized unruptured follicle (LUF) syndrome. Biochemical criteria, however, demonstrated only 7% CTL, 37% EMS, 23% ADH, and 56% OvDF subjects had LUF. Peritoneal fluid estradiol (E2) and P concentrations and total content were significantly lower in LUF than in non-LUF patients, whereas serum E2 and P concentrations were not different between the two groups. Values for testosterone and androstenedione in peritoneal fluid and serum were similar between these two groups. Endometrial dating in LUF versus non-LUF patients were also similar. The usual indicators of ovulation, i.e., serum P, endometrial dating, and basal body temperature, failed to identify LUF. The diagnosis of LUF can be best made by P assay of peritoneal fluid and serum.

Bayesian reconstructions from emission tomography data using a modified EM algorithm.

A novel method of reconstruction from single-photon emission computerized tomography data is proposed. This method builds on the expectation-maximization (EM) approach to maximum likelihood reconstruction from emission tomography data, but aims instead at maximum posterior probability estimation, which takes account of prior belief about smoothness in the isotope concentration. A novel modification to the EM algorithm yields a practical method. The method is illustrated by an application to data from brain scans.

Differential roles of neuroticism, extraversion, and event desirability for mood in daily life: an integrative model of top-down and bottom-up influences.

Top-down and bottom-up approaches were combined to assess the relative impact of extraversion, neuroticism, and daily events on daily mood. Ninety-six community-residing men completed diaries for 8 consecutive nights. Extraversion predicted positive mood, whereas neuroticism predicted positive and negative mood. Undesirable events predicted negative mood and, more modestly, positive mood. Desirable events predicted positive mood. Negative dispositional and situational factors play a larger role in daily positive affect than positive factors do in daily negative affect.

Loa loa: the use of a blood cell separator to reduce microfilaraemia before specific chemotherapy.

Three years after returning to the U.K. a 58-year-old British engineer, who had worked in the Nigerian oilfields for 24 years, was found to have Loa loa. His midday microfilarial count ranged between 12 X 10(6) and 16 X 10(6) parasites per litre. Before starting treatment, I litre of leucocyte/platelet-rich plasma was removed by means of a blood cell separator. Six intermittent cycles of blood separation were performed during a single 4-h session around midday in order to coincide with the period of maximum microfilarial presence in the peripheral circulation. This resulted in a 50% reduction of the microfilaraemia. Where facilities for blood cell separation are available, this procedure, together with appropriate chemotherapy, should be particularly beneficial in the management of heavy filarial infections.

Idiopathic chronic disseminated intravascular coagulation.

Disseminated intravascular coagulation, whether acute or chronic is usually associated with an underlying causative condition. In this case chronic disseminated intravascular coagulation persisted in one 79 year old man for 3 years with no detectable underlying cause.

Digoxin-like immunoreactive substance: monoclonal and polyclonal RIA and FPIA compared.

Digoxin-like immunoreactive substance (DLIS) was measured in 34 samples obtained from subjects not receiving digoxin: 10 uremic, 10 third trimester pregnancy, seven cord blood and seven normal. DLIS concentration was measured by four commercial polyclonal radioimmunoassay (RIA) systems: Clinical Assay (CA), Corning Immophase (CI), Diagnostic Products Double Antibody (DP), Kallestad Quantitope (KK), a monoclonal antibody (MA) assay and a Fluorescence Polarization Immunoassay (FPIA). In general, the cord blood samples were richer in DLIS. Digoxin immunoassays, MA and DP showed minimal interference by DLIS in all samples, whereas FPIA and CA exhibited the maximal cross-reactivity with DLIS. In cord blood samples, mean +/- SD DLIS concentration ranged from 0.41 +/- 0.13 (by CA) to 0.034 +/- 0.02 ng ml-1 as measured by MA assay. In uremics, the mean DLIS concentration was below the detection limit of all RIA assays. The FPIA method showed a higher degree of cross-reactivity to DLIS, especially in the cord and pregnancy samples (0.42 +/- 0.13 and 0.4 +/- 0.14 ng ml-1, respectively). DLIS in uremics was below the FPIA detection limit of 0.2 ng ml-1. Overall, the degree of interference by DLIS in decreasing order was FPIA greater than CA greater than CI greater than or equal to KQ greater than DP greater than or equal to MA. The cord blood samples were re-analysed by FPIA (Digoxin-II assay) 4 months later, resulting in 2-4-fold higher DLIS concentrations for these samples. This appears to be due to the substitution of 5-sulphosalicylic acid as a protein precipitating reagent and this effect may have been accentuated by freeze-thaw cycles.