The efficacy of HI-6 DMS in a sustained infusion against percutaneous VX poisoning in the guinea-pig.

Post-exposure nerve agent treatment usually includes administration of an oxime, which acts to restore function of the enzyme acetylcholinesterase (AChE). For immediate treatment of military personnel, this is usually administered with an autoinjector device, or devices containing the oxime such as pralidoxime, atropine and diazepam. In addition to the autoinjector, it is likely that personnel exposed to nerve agents, particularly by the percutaneous route, will require further treatment at medical facilities. As such, there is a need to understand the relationship between dose rate, plasma concentration, reactivation of AChE activity and efficacy, to provide supporting evidence for oxime infusions in nerve agent poisoning. Here, it has been demonstrated that intravenous infusion of HI-6, in combination with atropine, is efficacious against a percutaneous VX challenge in the conscious male Dunkin-Hartley guinea-pig. Inclusion of HI-6, in addition to atropine in the treatment, improved survival when compared to atropine alone. Additionally, erythrocyte AChE activity following poisoning was found to be dose dependent, with an increased dose rate of HI-6 (0.48mg/kg/min) resulting in increased AChE activity. As far as we are aware, this is the first study to correlate the pharmacokinetic profile of HI-6 with both its pharmacodynamic action of reactivating nerve agent inhibited AChE and with its efficacy against a persistent nerve agent exposure challenge in the same conscious animal.

Efficacy of the antinicotinic compound MB327 against soman poisoning - Importance of experimental end point.

Medical countermeasures for acute poisoning by organophosphorus nerve agents are generally assessed over 24h following poisoning and a single administration of treatment. At 24h, the antinicotinic bispyridinium compound MB327 (1,10-(propane-1,3-diyl)bis(4-tert-butylpyridinium)) dimethanesulfonate is as effective as the oxime HI-6 against poisoning by soman, when used as part of a treatment containing atropine and avizafone. In this study, we hypothesised that an earlier endpoint, at 6h, would be more appropriate for the pharmacokinetics and mechanism of action of MB327 and would therefore result in improved protection. MB327 diiodide (33.8mg/kg) or the oxime HI-6 DMS (30mg/kg), in combination with atropine and avizafone (each at 3mg/kg) was administered intramuscularly to guinea pigs 1min following subcutaneous soman and the LD50 of the nerve agent was determined at 6h after poisoning for each treatment. The treatment containing HI-6 gave a similar level of protection at 6h as previously determined at 24h (protection ratios 3.9 and 2.9, respectively). In contrast, the protection achieved by treatment containing MB327 showed a striking increase at 6h (protection ratio >15.4) compared to the 24h end point (protection ratio 2.8). The treatment gave full protection for at least 5h against doses of soman up to 525µg/kg; in contrast, mortality began in animals treated with HI-6 after 1h. This study demonstrates the importance of using an appropriate end point and has shown that treatment including MB327 was far superior to oxime-based treatment for poisoning by soman, when assessed over a pharmacologically-relevant duration. The improved outcome was seen following a single dose of treatment: it is possible that additional doses to maintain therapeutic plasma concentrations would further increase survival time. Antinicotinic compounds therefore offer a promising addition to treatment, particularly for rapidly aging or oxime-insensitive nerve agents.

Bioscavenger is effective as a delayed therapeutic intervention following percutaneous VX poisoning in the guinea-pig.

The prolonged systemic exposure that follows skin contamination with low volatility nerve agents, such as VX, requires treatment to be given over a long time due to the relatively short half-lives of the therapeutic compounds used. Bioscavengers, such as butyrylcholinesterase (BChE), have been shown to provide effective post-exposure protection against percutaneous nerve agent when given immediately on signs of poisoning and to reduce reliance on additional treatments. In order to assess the benefits of administration of bioscavenger at later times, its effectiveness was assessed when administration was delayed for 2h after the appearance of signs of poisoning in guinea-pigs challenged with VX (4×LD50). VX-challenged animals received atropine, HI-6 and avizafone on signs of poisoning and 2h later the same combination with or without bioscavenger. Five out of 6 animals which received BChE 2h after the appearance of signs of poisoning survived to the end of the study at 48h, compared with 6 out of 6 which received BChE immediately on signs. All the animals (n=6+6) that received only MedCM, without the addition of BChE, died within 10h of poisoning. The toxicokinetics of a sub-lethal challenge of percutaneous VX were determined in untreated animals. Blood VX concentration peaked at approximately 4h after percutaneous dosing with 0.4×LD50; VX was still detectable at 36h and had declined to levels below the lower limit of quantification (10pg/mL) by 48h in 7 of 8 animals, with the remaining animal having a concentration of 12pg/mL. These studies confirm the persistent systemic exposure to nerve agent following percutaneous poisoning and demonstrate that bioscavenger can be an effective component of treatment even if its administration is delayed.

Potential use of the alkaline comet assay as a predictor of bladder tumour response to radiation.

Bladder tumours show a variable response to radiotherapy with only about 50% showing good local control; currently there is no test to predict outcome prior to treatment. We have used five bladder tumour cell lines (T24, UM-UC-3, TCC-SUP, RT112, HT1376) to investigate the potential of the alkaline comet assay (ACA) to predict radiosensitivity. Radiation-induced DNA damage and repair were compared to clonogenic survival. When the five cell lines were irradiated and initial DNA damage was plotted against cell survival, at all doses (0-6 Gy), a significant correlation was found (r2=0.9514). Following 4 Gy X-irradiation, all cell lines, except T24, showed a correlation between SF2 vs half-time for repair and SF2 vs residual damage at 5, 10, 20 and 30 min. The T24 cell line showed radioresistance at low doses (0-2 Gy) and radiosensitivity at higher doses (4-6 Gy) using both cell survival and ACA end points, explaining the lack of correlation observed for this cell line. These data indicate that initial DNA damage and residual damage can be used to predict for radiosensitivity. Our data suggest that predictive tests of radiosensitivity, appropriate to the clinical situation, may require the use of test doses in the clinical range.

Relationship between clonogenic radiosensitivity, radiation-induced apoptosis and DNA damage/repair in human colon cancer cells.

The intrinsic radiation sensitivity of normal and tumour tissue is a major determinant of the outcome of radiotherapy. There is currently no established test that can be used routinely to measure the radiosensitivity of the cells in an individual patient's cancer in a manner that can inform treatment planning. The purpose of this study was to evaluate, in four human colorectal adenocarcinoma cell lines, two possible end points as surrogate markers of radiation response--apoptosis and induction of DNA single-strand breaks--and to compare the results with those of a conventional clonogenic assay. Cell lines (SW707 SW480, SW48 and HT29) known to differ in radiosensitivity were exposed to single doses of X-rays ranging from 0.5 to 5 Gy and cell survival was measured using the clonogenic assay. Apoptosis was determined on the basis of morphology under fluorescent microscopy and DNA damage/repair was measured, as tail moment, using an adaptation of the alkaline comet assay. The relationship between surviving fraction at 2 Gy (SF2) and the percentage of apoptotic cells 24 h after the same dose was complex, but apoptosis accurately predicted the order of radiosensitivities as measured by SF2. Initial damage measured after 2 Gy using the alkaline comet assay gave a close correlation with SF2 (r2=0.95), whereas there was no correlation between initial DNA damage repair rate and SF2.

Parental investment and child health in a Yanomamö village suffering short-term food stress.

The 1998 El Niño significantly reduced garden productivity in the Upper Orinoco region in Venezuela. Consequently, parents were forced to allocate food carefully to their children. Nutrition data collected from village children combined with genealogical data allowed the determination of which children suffered most, and whether the patterns of food distribution accorded with predictions from parental investment theory. For boys, three social variables accounted for over 70% of the variance in subcutaneous fat after controlling for age: number of siblings, age of the mother's youngest child, and whether the mother was the senior or junior co-wife, or was married monogamously. These results accord well with parental investment theory. Parents experiencing food stress faced a trade-off between quantity and quality, and between investing in younger versus older offspring. In addition, boys with access to more paternal investment (i.e. no stepmother) were better nourished. These variables did not account for any of the variance in female nutrition. Girls' nutrition was associated with the size of their patrilineage and the number of non-relatives in the village, suggesting that lineage politics may have played a role. An apparent lack of relationship between orphan status and nutrition is also interesting, given that orphans suffered high rates of skin flea infections. The large number of orphans being cared for by only two grandparents suggests that grooming time may have been the resource in short supply.

Description and use of the neuroscience nursing self-efficacy scale.

An instrument for assessing nurses' perceived self-efficacy in implementing a variety of neuroscience nursing tasks was developed. Self-efficacy theory served as the guiding framework. From 1988 to 1998, the instrument was used to assess changes in the perceived self-efficacy of 54 nurses who attended a neuroscience nurse-internship program. Self-efficacy was assessed during clinical orientation, prior to the beginning of the program, and at the end of the program. The results showed that the nurses' confidence in performing a variety of neuroscience nursing skills increased during the 6- to 10-month program. The instrument was also useful in helping program directors identify areas in which nurses could benefit most from the program and refine the program to meet the educational needs of the nurses.

Protein adsorption to polyethylene glycol modified liposomes from fibrinogen solution and from plasma.

Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated.

Evaluation of the Neuroscience Nurse Internship Program: the first decade.

This article describes the evaluation of the Neuroscience Nurse Internship Program (NNIP). The NNIP was initiated in 1988 by the National Institutes of Health Clinical Center to meet the demand for highly skilled nurses to care for persons with nervous system disorders. To determine whether the program was meeting its goals, an evaluation component was incorporated into the program. The evaluation process was based on the RSA Model of Continuing Education for nursing. The RSA model consists of four basic components, three of which were included in the assessment of the NNIP--process, content, and outcome. The evaluation revealed that the nurse-interns were satisfied with the lectures and clinical content of the program. Moreover, the nurse-interns improved their knowledge of neuroscience nursing and their confidence in performing neuroscience nursing skills. Information from the evaluation was used to modify the structure of the program and to refine lecture content and clinical requirements.

Increased repair and cell survival in cells treated with DIR1 antisense oligonucleotides: implications for induced radioresistance.

PURPOSE: To determine whether repression of a recently isolated, X-ray-responsive gene, DIR1, using antisense oligonucleotides could affect clonogenic cell survival and repair of DNA strand breaks and have a possible role in the mechanism underlying the phenomenon of 'induced radioresistance' (IRR). MATERIALS AND METHODS: Three cell lines, V79, RT112 and UM-UC-3, which are known to exhibit low-dose hypersensitivity (HRS) and induced radioresistance (IRR), and the radiosensitive cell line ATBIVA, were transfected with antisense oligonucleotides directed towards the DIR1 gene. Scrambled oligonucleotides were used as controls. DNA single-strand break (ssb) repair, using the alkaline comet assay, and cell survival using a standard clonogenic assay was measured after exposure to X-rays. RESULTS: Following treatment with 4Gy X-rays, the V79, RT112 and UM-UC-3 cell lines all exhibited significantly increased rates of ssb repair after transfection with DIR1 antisense oligonucleotides compared with cells transfected with scrambled oligonucleotides. They also demonstrated significantly enhanced survival after exposure to 2 Gy X-rays; the radiosensitive ATBIVA cells did not show these effects. CONCLUSIONS: Repression of the DIR1 gene product leads to an increase in the rate of repair and cell survival in three radioresistant cells lines but not in the radiosensitive ATBIVA cell line. Because DIR1 is repressed by X-rays in the dose range where IRR is observed, it may represent a candidate gene involved in the IRR phenomenon.

Induction and rejoining of DNA double-strand breaks in bladder tumor cells.

The induction and rejoining of radiation-induced double-strand breaks (DSBs) in cells of six bladder tumor cell lines (T24, UM-UC-3, TCC-SUP, RT112, J82, HT1376) were measured using the neutral comet assay. Radiation dose-response curves (0-60 Gy) showed damage (measured as mean tail moment) for five of the cell lines in the same rank order as cell survival (measured over 0-10 Gy), with the least damage in the most radioresistant cell line. Damage induction correlated well with clonogenic survival at high doses (SF10) for all six cell lines. At the clinically relevant dose of 2 Gy, correlation was good for four cell lines but poor for two (TCC-SUP and T24). The rejoining process had a fast and slow component for all cell lines. The rate of these two components of DNA repair did not correlate with cell survival. However, the time taken to reduce the amount of DNA damage to preirradiated control levels correlated positively with cell survival at 10 Gy but not 2 Gy; radioresistant cells rejoined the induced DSBs to preirradiation control levels more quickly than the radiosensitive cells. Although the results show good correlation between SF10 and DSBs for all six cell lines, the lack of correlation with SF2 for TCC-SUP and T24 cells would suggest that a predictive test should be carried out at the clinically relevant dose. At present the neutral comet assay cannot achieve this.

The Neuroscience Nurse Internship Program: the description.

This article describes the Neuroscience Nurse Internship Program (NNIP) and its implementation at the National Institutes of Health (NIH) Clinical Center in Bethesda, MD. The program, developed in 1988, is designed to prepare nurses to deliver care to persons with nervous system disorders. The program comprises 6 months of lecture and clinical instruction in the country's most well-known biomedical research center. In order to complete the program, nurse-interns must receive a passing grade of 90% or higher on all written tests and achieve satisfactory completion of all clinical assignments. NNIP is cosponsored by the NIH Clinical Center Nursing Department and the National Institute of Neurological Disorders and Stroke (NINDS). Nurses interested in NNIP may apply by submitting an application and a letter of interest to the Clinical Center Human Resources Department. Evaluation of the program is conducted on a regular basis and consists of both process and outcome assessments.

Discovery of RWJ-54428 (MC-02,479), a new cephalosporin active against resistant gram-positive bacteria.

The discovery of RWJ-54428 (MC-02,479), a new cephalosporin displaying promising activity against sensitive and resistant Gram-positive bacteria, is described. Progressive structural modification from the previously reported 3-phenylthiocephem MC-02,331 afforded an overall increase in potency against MRSA while retaining other key properties such as acceptable solubility and serum binding. Evaluation of the in vitro potency and in vivo efficacy of a series of closely related compounds resulted in selection of RWJ-54428 (MC-02,479) for further studies.

A novel human stress response-related gene with a potential role in induced radioresistance.

We have isolated a novel gene, DIR1, from L132 cells that is transiently repressed after exposure to low radiation doses and has a potential role in induced radioresistance. Molecular and cellular characterization of this gene reveals that it is unique but has similarities to a family of heat-shock-related proteins known as immunophilins. These have been implicated in various cellular functions including general stress responses and control of the cell cycle. Antisense strategies have demonstrated that the DIR1 gene also appears to have some involvement in the control of the cell cycle. Furthermore, there appears be a potential role for this gene product in the phenomenon of induced radioresistance through a mechanism that increases the rate of DNA repair in cells exposed to X rays and subsequently increases the cells' resistance to radiation. This is the first description of an immunophilin-like gene that has a possible role in adaptive/inducible responses to X rays in mammalian cells.

Discovery of MC-02,331, a new cephalosporin exhibiting potent activity against methicillin-resistant Staphylococcus aureus.

A systematic approach toward building activity against methicillin-resistant staphylococci into the cephalosporin class of beta-lactam antibiotics is described. Initial work focused on finding the optimal linkage between the cephem nucleus and a biphenyl pharmacophore, which established that a thio linkage afforded potent activity in vitro. Efforts to optimize this activity by altering substitution on the pharmacophore afforded iodophenylthio analog MC-02,002, which although highly potent against MRSA, was also highly bound to serum proteins. Further work to decrease serum protein binding showed that replacement of the iodo substituent by the positively-charged isothiouronium group afforded potent activity and reduced serum binding, but insufficient aqueous solubility. Solubility was enhanced by incorporation of a second positively-charged group into the 7-acyl substituent. Such derivatives (MC-02,171 and MC-02,306) lacked sufficient stability to staphylococcal beta-lactamase enzymes. The second positive charge was incorporated into the cephem 3-substituent in order to utilize the beta-lactamase-stable aminothiazolyl(oximino)acetyl class of 7-substituents. These efforts culminated with the discovery of bis(isothiouroniummethyl)phenylthio analog MC-02,331, whose profile is acceptable with respect to potency against MRSA, serum binding, aqueous solubility, and beta-lactamase stability.

Effects of sildenafil on the relaxation of human corpus cavernosum tissue in vitro and on the activities of cyclic nucleotide phosphodiesterase isozymes.

PURPOSE: Sildenafil, an inhibitor of cGMP-specific phosphodiesterase 5 (PDE5), is currently undergoing evaluation as an oral therapy for penile erectile dysfunction. The aims of this study were to investigate the mechanism of action of sildenafil on the neurogenic relaxation of human corpus cavernosum (HCC) in vitro and to determine the activity of sildenafil against a full range of PDE isozymes. MATERIALS AND METHODS: Strips of HCC tissue were precontracted with phenylephrine. Relaxation responses resulting from electrical field stimulation (EFS) were then determined in the presence and absence of sildenafil. The effects of sildenafil on PDE1 to 5 prepared from human tissues and PDE6 from bovine retina were determined by measuring the conversion of [3H]-cGMP or [3H]-cAMP to their respective [3H]-5'-mononucleotides. RESULTS: Sildenafil (0.001 to 1 microM) enhanced the EFS-induced, nitric oxide (NO) dependent, relaxation of HCC in a concentration-dependent manner to a maximum of 3 times the pretreatment level at 1 microM sildenafil. Compared with zaprinast, an early PDE5 inhibitor, sildenafil was approximately 240-fold more potent, inhibiting PDE5 from HCC with a geometric mean IC50 of 3.5 nM. For sildenafil, IC50 values for inhibition of PDE1 to 4 were 80 to more than 8500 times greater than that for PDE5 and the IC50 for PDE6 (33 nM) was approximately 9-fold greater. CONCLUSIONS: The data support the proposal that enhancement of penile erection by sildenafil in patients with erectile dysfunction involves potentiation of the NO-stimulated cGMP signal mediating relaxation of cavernosal smooth muscle during sexual stimulation. Sildenafil is a potent inhibitor of PDE5 from HCC, with high selectivity for PDE5 relative to other PDE isozymes.

Second 46,XX male with MLS syndrome.

We report on a second 46,XX male with microphthalmia with linear skin defects (MLS) syndrome. In addition to microphthalmia and linear skin streaks, he had a secundum ASD, hypospadias with chordee, anal fistula, and agenesis of corpus callosum with colpocephaly. Biopsy of a linear streak showed smooth muscle hamartomata rather than the presumed dermal aplasia. Detailed ophthalmologic examination did not show retinal lacunae typical of Aicardi syndrome. DNA studies with distal Xp specific probes indicated a deletion in one X chromosome and fluorescence in situ hybridization (FISH) studies with X- and Y-specific probes demonstrated the presence of a derivative X chromosome from an X;Y translocation.

Evaluation of glucose monitoring devices in the hyperbaric chamber.

Diabetic patients may experience fluctuations in whole blood glucose (WBG) levels while receiving hyperbaric oxygen therapy (HBO) resulting in seizure-like activity. Therefore, hyperbaric medical attendants must accurately monitor the WBG levels of these patients during HBO. In addressing this concern, this study evaluated the accuracy and reliability of commercially available glucometers (Glucometer M+, Companion 2, HemoCue, One Touch II, and ExacTech Pen) in the hyperbaric environment. WBG samples were prepared, ranging from 25 to 250 mg/dl, for testing glucometers at ground level and at 2.36 atmospheres absolute (ATA). It was noted that at 2.36 ATA, glucose values increased using the Glucometer M+, but decreased with the Companion 2 and HemoCue. The One Touch II values decreased in the hyperglycemic ranges (> 150 mg/dl), whereas the ExacTech Pen monitor readings increased in the hypoglycemic ranges (< 100 mg/dl). The accuracy of WBG monitors is significantly affected by changes in atmospheric pressure.