A randomized controlled phase III trial of recombinant human granulocyte colony-stimulating factor (filgrastim) for treatment of severe chronic neutropenia

Patients with idiopathic, cyclic, and congenital neutropenia have recurrent severe bacterial infections. One hundred twenty-three patients with recurrent infections and severe chronic neutropenia (absolute neutrophil count < 0.5 x 10(9)/L) due to these diseases were enrolled in this multicenter phase III trial. They were randomized to either immediately beginning recombinant human granulocyte colony- stimulating factor (filgrastim) (3.45 to 11.50 micrograms/kg/d, subcutaneously) or entering a 4-month observation period followed by filgrastim administration. Blood neutrophil counts, bone marrow (BM) cell histology, and incidence and duration of infection-related events were monitored. Of the 123 patients enrolled, 120 received filgrastim. On therapy, 108 patients had a median absolute neutrophil count of > or = 1.5 x 10(9)/L. Examination of BM aspirates showed increased proportions of maturing neutrophils. Infection-related events were significantly decreased (P < .05) with approximately 50% reduction in the incidence and duration of infection-related events and almost 70% reduction in duration of antibiotic use. Asymptomatic splenic enlargement occurred frequently; adverse events frequently reported were bone pain, headache, and rash, which were generally mild and easily manageable. These data indicate that treatment of patients with severe chronic neutropenia with filgrastim results in a stimulation of BM production and maturation of neutrophils, an increase in circulating neutrophils, and a reduction in infection-related events.     

Abnormal stimulated adherence of neonatal granulocytes: impaired induction of surface Mac-1 by chemotactic factors or secretagogues

To identify possible secretory determinants of impaired hyperadherence and stimulated migration of neonatal granulocytes (NGs), we performed correlative studies of: (a) specific granule content and exocytosis, (b) secretago-gue-mediated upregulation of f-met-leu-phe (fMLP) receptors, (c) the chemotactic induction of the adhesive glycoproteins Mac-1 alpha (complement receptor 3) and beta, and (d) morphometric assessments of specific (peroxidase negative) granule depletion following chemotactic stimulation. Lactoferrin (LF) content of NG suspensions (cord blood or peripheral blood cells) was profoundly diminished (mean +/- SD 51% +/- 18% of normal) as compared with healthy adult granulocytes (AGs). Despite diminished cellular content, LF release by NG suspensions in response to fMLP was comparable to that of AGs. In contrast, LF release by NG suspensions was significantly diminished in response to phorbol myristate acetate (PMA) or calcium ionophor A23187 and/or during stimulated cell spreading, experimental conditions promoting overall greater LF depletion than chemotactic stimuli. In addition, NGs demonstrated an impaired capacity to upregulate fMLP receptors in response to PMA or A23187 when tested under the same experimental conditions. Baseline expression of the adhesive glycoproteins Mac-1 alpha and beta on NG surfaces was normal, but induction or upregulation of these proteins by chemotactic concentrations of fMLP, C5a as well as secretory (high) concentrations of PMA and A23187, was significantly diminished as compared with AGs. In contrast, chemotactic induction of the surface expression of the complement receptor-1 (CR-1) on NGs was normal. An impaired induction of Mac-1 alpha or beta was directly related to an impaired enhancement of adherence of NG in response to fMLP over a chemotactically relevant concentration range (10(-10) to 10(-7) mol/L). Moreover, in blocking- incubation experiments using anti-Mac-1 alpha/beta monoclonal antibodies (MAbs), significantly less inhibition of adherence by these MAbs was evident with fMLP-stimulated NG as compared with AG suspensions. Under selected chemotactic conditions, ultrastructural assessments of NGs demonstrated diminished peroxidase-negative granule loss in association with diminished granule-membrane fusion and the "addition" of plasma membrane. These studies suggest that abnormal expression of multiple surface determinants derived from peroxidase- negative granules or other intracellular pools may contribute to deficient chemotaxis or other inflammatory functions of NGs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Clearance of normal and type 2A von Willebrand factor in the rat

A model for the in vivo clearance of normal and mutant forms of human von Willebrand factor (vWF) has been established using catheterized rats. vWF clearance rates in rat plasma were determined by quantitation of reduced vWF subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and multimeric vWF was analyzed using nondenaturing SDS-agarose gels. Normal vWF derived from human umbilical vein endothelial cells displayed a biphasic pattern of clearance, with half times of 35 minutes (T 1/2 a; SD 15. min.) and 245 minutes (T 1/2 b; SD 76. min.); metabolic clearance rate = 0.65%/minute. High molecular weight multimers of vWF were cleared more rapidly than dimeric vWF. vWF containing the S1613P mutation found in some type 2A von Willebrand disease (vWD) patients was observed to undergo proteolysis in vivo resulting in a reduction of high molecular weight vWF and concomitant appearance of rapidly-migrating satellite species, although the overall clearance rate of vWF antigen was similar to wild type vWF. These results provide direct in vivo evidence that the S1613P mutation causes the characteristic type 2A vWD phenotype. Full-length recombinant vWF produced from transfected Chinese hamster ovary cells was cleared at a similar rate to endothelial cell-derived vWF, and recombinant vWF devoid of O-linked carbohydrates was cleared significantly faster. vWF devoid of sulfate was cleared at a similar rate as wild type vWF, indicating the sulfate moiety of vWF does not regulate in vivo clearance. This animal model should prove useful in subsequent in vivo analysis of additional forms of vWD and in the development of protease inhibitor therapy for 2A vWD.     

Analysis of interphase cells for the Philadelphia translocation using painting probe made by inter-Alu-polymerase chain reaction from a radiation hybrid

Fluorescence in situ hybridization (FISH) probe for the identification of the Philadelphia (Ph) translocation [t(9;22) (q34;q11)] in chronic myelogenous leukemia cells was developed by inter-Alu-polymerase chain reaction of DNA from an interspecific somatic cell hybrid containing approximately 5 Mb of human DNA covering the ABL gene region on human chromosome 9q34. This probe was large enough to be effective in identifying the genomic domains yet small enough to resolve them in more than 90% of bone marrow interphase cells. Combination of the probe with a cosmid contig probe for the BCR region of chromosome 22 in two- color FISH reduced the frequency of false-positive identification of the Ph chromosome to less than 1%. The procedure allows detection of as few as 1% Ph+ cells independent of the cycling status or BCR/ABL expression level of cells, and the quantitation of non-Ph chromosome- containing interphase nuclei in the marrow of patients judged 100% Ph+ by standard cytogenetics.     

Cyclic AMP-mediated chloride secretion is induced by prostaglandin F2α in human isolated colon

Background and purpose:

Prostaglandin F_2α (PGF_2α) is implicated in the pathogenesis of inflammatory bowel disease and colorectal cancer. This study investigates the effects of PGF_2α on electrophysiological parameters in isolated human colonic mucosa.

Experimental approach:

Ion transport was measured as changes in short-circuit current across human colonic epithelia mounted in Ussing chambers. Colonic crypts were isolated by calcium chelation and cyclic adenosine monophosphate (cAMP) was measured by ELISA.

Key Results:

PGF_2α stimulated chloride secretion in a concentration-dependent manner with an EC₅₀ of 130 nM. The PGF_2α induced increase in chloride secretion was inhibited by AL8810 (10 µM), a specific PGF_2α receptor antagonist. In addition, PGF_2α (1 µM) significantly increased levels of cAMP in isolated colonic crypts.

Conclusions and implications:

PGF_2α stimulated chloride secretion in samples of human colon in vitro through a previously unrecognizd cAMP-mediated mechanism. These findings have implications for inflammatory states.     

Interaction between udenafil and tamsulosin in rats: non-competitive inhibition of tamsulosin metabolism by udenafil via hepatic CYP3A1/2

Background and purpose:

Orthostatic hypotension has been observed when PDE 5 (cGMP-specific phosphodiesterase type 5) inhibitors are co-administered with α-adrenoceptor antagonists. Here we assessed the pharmacokinetic and haemodynamic interactions between udenafil and tamsulosin in rats, as both drugs are metabolized via rat hepatic cytochrome P450 3A1/2.

Experimental approach:

Interactions between the two drugs were evaluated in rats after simultaneous 1 or 15 min i.v. infusion or after p.o. administration of udenafil (30 mg·kg⁻¹) and/or tamsulosin (1 mg·kg⁻¹). In vitro metabolism of tamsulosin with udenafil was measured to obtain the inhibition constant (K_i) and [I]/K_i ratio of udenafil.

Key results:

The total area under the plasma concentration–time curve from time zero to time infinity (AUC)s (or AUC_0–4h) of tamsulosin were significantly greater after 15 min of i.v. infusion or after oral administration with udenafil, compared with tamsulosin alone. The hepatic first-pass metabolism of tamsulosin was inhibited by udenafil, and the inhibition in vitro was in a non-competitive mode. The arterial systolic blood pressure was significantly lower at 5, 10 and 60 min after oral co-administration of the drugs.

Conclusions and implications:

The significantly greater AUC of tamsulosin after i.v. and p.o. administration of both drugs may be attributable to non-competitive inhibition of cytochrome P450 3A1/2-mediated hepatic tamsulosin metabolism by udenafil. The inhibition was also observed in human liver S9 fractions, suggesting that a reassessment of the oral dosage of tamsulosin is necessary when udenafil and tamsulosin are co-administered to patients with benign prostatic hyperplasia.     

Estimation of the age at onset in spinocerebellar ataxia type 2 Cuban patients by survival analysis

Almaguer‐Mederosa LE, Falcón NS, Almira YR, Zaldivar YG, Almarales DC, Góngora EM, Herrera MP, Batallán KE, Armiñán RR, Manresa MV, Cruz GS, Laffita‐Mesa J, Cyuz TM, Chang V, Auburger G, Gispert S, Pérez LV. Estimation of the age at onset in spinocerebellar ataxia type 2 Cuban patients by survival analysis. Previous studies have investigated the close association that exists between CAG repeat number and the age at onset in SCA2 = spinocerebellar ataxia type 2. These studies have focused on affected individuals. To further characterize this association and estimate the risk of a carrier developing SCA2 at a particular age as a function of a specific CAG repeat size, we have analyzed a large group of 924 individuals, including 394 presymptomatic and 530 affected individuals with a CAG repeat length of 32–79 units. Using a Kaplan–Meier survival analysis, we obtained cumulative probability curves for disease manifestation at a particular age for each CAG repeat length in the 34–45 range. These curves were significantly different (p < 0.001) and showed small overlap. All these information may be very valuable in predictive‐testing programs, in the planning of studies for the identification of other genetic and environmental factors as modifiers of age at onset, and in the design of clinical trials for people at enlarged risk for SCA2.     

Early massive transfusion in trauma patients: Canadian single-centre retrospective cohort study

Purpose

To determine associations between red blood cell (RBC) transfusion and early and late clinical outcomes in massively transfused adult trauma patients.

Methods

A retrospective cohort study (1992–2001) including 260 patients receiving ≥10 RBC units ≤24 hr after admission to a university-affiliated trauma centre. We extracted demographic and clinical data and used multivariable regression to determine independent effects of RBC transfusion on clinical outcomes.

Results

Patients had a high (mean [standard deviation]) injury severity score (ISS) (42.5 [15.1]), a high admission sequential organ failure assessment (SOFA) score (8.4 [3.8]), and a high hospital mortality (58.5%). They received 38 (25–64) (median [interquartile range]) blood components within 48 hr, including 19 (14–28) RBC units. For 143 patients surviving ≥48 hr, the maximum SOFA score was associated with RBC units transfused before 48 hr (linear regression beta coefficient 0.075, P < 0.0001), lower nadir hemoglobin before 48 hr (0.034, P = 0.03), age (0.032, P = 0.015), and admission SOFA (0.59, P < 0.0001). The RBC units transfused by 48 hr were not associated with either hospital mortality (n = 35) among patients surviving ≥48 hr (independent predictors, age [logistic regression odds ratio (OR) 1.06, 95% confidence interval 1.03–1.10], ISS [OR 1.07, 1.02–1.13], and maximum SOFA score [OR 1.56, 1.27–1.93]) or 48-hr mortality (n = 117) (independent predictors, admission SOFA [1.65, 1.45–1.88] and later year of hospital admission [OR 1.15, 1.02–1.29]).

Conclusions

Hospital mortality is high among massively transfused trauma patients. Among early survivors, 48-hr RBC transfusion volume is associated with increased organ dysfunction, but not hospital mortality. Also, it is not associated with 48-hr mortality. Future research should continue to explore methods to improve hemostasis and minimize the need for RBC transfusion.     

Large conductance calcium-activated potassium channels (BKCa) modulate trigeminovascular nociceptive transmission

Migraine is a common, disabling, neurological problem whose acute management would benefit from the development of purely neurally acting therapies. The trigeminocervical complex is pivotal in nociceptive signaling in migraine, and is an accepted target for putative antimigraine agents. Whole-cell patch-clamp or extracellular recordings were made of trigeminal neurons identified in rat brainstem slices. Bath application of the large conductance calcium-activated potassium (BK_Ca) channel opener NS1619 caused a dramatic decrease of cell firing that could be reversed by the co-application of iberiotoxin. NS1619 hyperpolarized the resting membrane potential and reduced the frequency of spontaneous action potentials in these neurons. These data suggest the presence of BK_Ca channels in the trigeminocervical complex. In vivo in cat l -glutamate-evoked firing was facilitated in nociceptive neurons, also responding to stimulation of the superior sagittal sinus, in the trigeminal nucleus caudalis by the BK_Ca peptide antagonists, iberiotoxin and slotoxin. Of units tested, 70% responded to microiontophoretic application of the blockers, identifying a subpopulation of trigeminal neurons expressing toxin-sensitive BK_Ca channels. NS1619 inhibited 74% of cells tested, and this was reversed by slotoxin, suggesting that the action of NS1619 in these cells was mediated through BK_Ca channels. These data are consistent with the presence of BK_Ca channels in the trigeminal nucleus caudalis that are potential targets for the development of antimigraine treatments, and may also offer insights into receptor mechanisms involved in sensitization and thus allodynia, in migraine.