The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.     

Paracetamol poisoning in the north east of England: presentation, early management and outcome

1. Paracetamol is increasingly involved in self-poisoning in the United Kingdom and remains a common cause of fatal poisoning. 2. To document the epidemiology and early management of paracetamol poisoning data were collected on consecutive patients with suspected paracetamol poisoning presenting to 6 hospitals in the North East of England over 12 weeks in 1994. 3. There were 400 presentations (attendance rate 1.14/10(3) population/yr) involving 343 persons (45% male). Paracetamol concentrations at 4 h correlated weakly with reported paracetamol dose (R = 0.49, P < 0.0001) and were similar comparing those treated and not treated by gastric decontamination. 4. In 38 (9%) cases paracetamol concentrations were above the appropriate nomogram treatment line, including 3% and 20% of patients who reported ingesting less than and more than 12 g respectively. In 21 patients acetylcysteine treatment was deferred until admission to the ward, the mean delay involved was 2.8 h. 5. One patient died, from arrhythmias caused by co-ingested dothiepin. 6. Paracetamol poisoning is common. Most cases do not have potentially toxic plasma paracetamol concentrations, but those who do often present late and antidotal treatment may be delayed inappropriately.     

The course and outcome of viral hepatitis B in drug-addicted patients

In this paper, characterization is given of clinical and biochemical features of VH B course against the background of narcomania. Recordable in such patient populations are high percentage of delta hepatitis (14.2%), unusual severity of the intoxication syndrome, protracted course with exacerbations (12.2%) and recurrences (8%), outcome being a chronic hepatitis (14.2%), slower normalization of biochemical indicators, persistently low ratio of AIAT activity in diluted and whole blood sera. The persistence of viral markers in drug addicts discharged from the hospital (68%) is fraught with danger of spreading viral hepatitis of prophylactic measures are not strictly observed.     

Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL-11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.     

The carboxyl terminus of interferon-gamma contains a functional polybasic nuclear localization sequence

Cytokines such as interferon-gamma (IFN-gamma), which utilize the well studied JAK/STAT pathway for nuclear signal transduction, are themselves translocated to the nucleus. The exact mechanism for the nuclear import of IFN-gamma or the functional role of the nuclear translocation of ligand in signal transduction is unknown. We show in this study that nuclear localization of IFN-gamma is driven by a simple polybasic nuclear localization sequence (NLS) in its COOH terminus, as verified by its ability to specify nuclear import of a heterologous protein allophycocyanin (APC) in standard import assays in digitonin-permeabilized cells. Similar to other nuclear import signals, we show that a peptide representing amino acids 95-132 of IFN-gamma (IFN-gamma(95-132)) containing the polybasic sequence 126RKRKRSR132 was capable of specifying nuclear uptake of the autofluorescent protein, APC, in an energy-dependent fashion that required both ATP and GTP. Nuclear import was abolished when the above polybasic sequence was deleted. Moreover, deletions immediately NH2-terminal of this sequence did not affect the nuclear import. Thus, the sequence 126RKRKRSR132 is necessary and sufficient for nuclear localization. Furthermore, nuclear import was strongly blocked by competition with the cognate peptide IFN-gamma(95-132) but not the peptide IFN-gamma(95-125), which is deleted in the polybasic sequence, further confirming that the NLS properties were contained in this sequence. A peptide containing the prototypical polybasic NLS sequence of the SV40 large T-antigen was also able to inhibit the nuclear import mediated by IFN-gamma(95-132). This observation suggests that the NLS in IFN-gamma may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Finally, we show that intact IFN-gamma, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the peptide IFN-gamma(95-132) and the SV40 T-NLS peptide, suggesting that intact IFN-gamma was also transported into the nucleus through the Ran/importin pathway. Previous studies have suggested a direct intracellular role for IFN-gamma in the induction of its biological activities. Based on our data in this study, we suggest that a key intracellular site of interaction of IFN-gamma is the one with the nuclear transport mechanism that occurs via the NLS in the COOH terminus of IFN-gamma.     

Feasibility of a long-term low-sodium diet in mild hypertension

The present study set out to assess the feasibility of long-term moderate dietary sodium restriction in patients with mild hypertension in general practice. After screening and a run-in phase of 6-8 weeks, a total of 77 previously undiagnosed mildly hypertensive patients were identified. Half of them were randomized to receive a few simple dietary instructions from their general practitioners in order to reduce salt usage; the others were randomized to receive no advice. The patients were followed up for 12 months with quarterly visits. A total of 56 patients (72.7%) completed the study, 26 on a low-sodium diet (LD) and 30 on their usual diet (UD). At each visit in the diet phase, patients provided 24h urine, which was analysed for volume and sodium concentration in order to assess their sodium intake. Blood pressure, heart the rate and body weight were recorded. The mean urinary sodium excretion for all diet phase visits overlapped in the two groups (177.0 +/- 32.9 vs. 169.3 +/- 49.4 mEq/24h respectively in the LD and UD groups). Nevertheless the mean systolic and diastolic blood pressures for all diet phase visits were significantly lower in the LD than in UD group (144.2 +/- 11.1/91.6 +/- 6.4 and 148.0 +/- 13.7/95.6 +/- 4.7 mmHg respectively, P less than 0.01). Our data suggest that it is not feasible at present to reduce sodium intake in mild hypertensives with simple and inexpensive dietary instructions, the only ones suitable for widespread application in general practice.     

Variations of cardiac cycle length in patients with coronary heart disease before and after aortic coronary bypass surgery

It is well known that variations in cardiac cycle length or heart rates may be used for noninvasive evaluation of autonomic cardiovascular control. The investigation uses an original procedure. The data for analysis of cardiac cycle length variations (CCLV) are those obtained regularly at follow-up intervals, random 24-hour Holter 2-lead ECG recordings for 5 sec, and simultaneously calculated continuous sequence of mean heart rate. With the procedure, the correlations of CCLV with the parameters reflecting the early postoperative status of patients with CHD, including acute myocardial infarction, acute heart failure, rhythm and conduction disturbances, as well as age, operative stress, concurrent diseases. The findings are of both diagnostic and predictive value at subsequent stages of CHD treatment.     

Fasting and post-methionine load homocyst(e)ine values are correlated with microalbuminuria and could contribute to worsening vascular damage in non-insulin-dependent diabetes mellitus patients

The study aim was to assess the relationship between homocyst(e)inemia and microalbuminuria in non-insulin-dependent diabetes mellitus (NIDDM) patients. The study was performed on 33 NIDDM patients (16 males and 17 females), and 16 healthy control subjects (seven males and nine females). Plasma fasting and post-methionine load homocyst(e)ine (tHcy), together with other parameters that could modify tHcy levels, were assessed. There were no significant differences between NIDDM patients and controls for fasting tHcy (8.12 +/- 3.17 v 7.19 +/- 2.40 micromol/L) and post-methionine load tHcy (26.51 +/- 11.50 v 25.06 +/- 10.76 micromol/L). Moreover, there was a significant correlation between urinary albumin excretion (UAE) and fasting tHcy (r = .340, P = .05) and post-methionine load tHcy (r = .502, P = .004) in NIDDM patients. Fasting tHcy was correlated both with post-methionine load tHcy (r = .429, P = .01) and with vitamin B12 (r = -.349, P = .04) in NIDDM patients. Microalbuminuric NIDDM patients had higher fasting tHcy (9.05 +/- 3.83 micromol/L) than normoalbuminurics (7.12 +/- 1.95 micromol/L). In addition, NIDDM patients with complications presented higher fasting tHcy values than the group without complications (9.61 +/- 3.34 v 6.53 +/- 2.09 micromol/L, Kolmogorov-Smirnov two-sample test for nonparametric data [KS] = 1.794, P = .003), without any other significant differences in the parameters considered. tHcy could be an important risk factor worsening the prognosis in NIDDM patients, especially microalbuminuric patients. Microalbuminuric NIDDM patients could be particularly prone to hyperhomocyst(e)inemia, probably due to endothelial or renal dysfunction with a reduction in the scavenging of tHcy.     

A T42A Ran mutation: differential interactions with effectors and regulators, and defect in nuclear protein import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran.GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran.GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin beta (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.