Monitoring the pharmacokinetics of pyridinium aldoximes in the body.

A huge number of organophosphate poisonings occurring in agriculture, and a constant threat of misapplication of organophosphates as warfare agents require antidotes that efficiently improve the health-condition of intoxicated subjects. Pyridinium aldoximes are medically used to reactivate the cholinesterase enzymes inhibited by organophosphates. This paper outlines pharmacokinetics, metabolic disposition and blood-brain-barrier penetration of pyridinium aldoximes into the human and animal body, and the methods of their pharmacological analysis.

Application of in vivo microdialysis for studying the efficacy of protective preparations against sulfur mustard penetrating the skin.

Subcutaneous microdialysis was employed for monitoring thiodiglycol (2,2'-thiodiethanol, TDG) levels with the aim of characterizing the transdermal penetration of topically applied liquid sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) in rats. TDG levels, evaluated in 20 min dialysates collected over a 6 h sampling period, were plotted against time after pooling. Linear correlation was identified between the SM dose and the mean areas under the 0-60 min or the whole curve (AUC(0-60) and AUC, respectively) as well as mean peak concentrations (C(max)) in the range of 1.0-3.0 microl applied volume (7.2-21.7 nmol).A commercially available barrier cream, a perfluoropolyether oil and a vaseline based ointment containing zinc oxide were subsequently tested as topical protectants. Each preparation was layered on the skin surface prior to the application of 2.0 microl SM. The evaluation of the efficacy of the preparations was based on obtained AUC(0-60), AUC and C(max) values. A statistical comparison of these parameters with those obtained when 2.0 microl SM was applied without pretreatment indicated that the barrier cream and the perfluoropolyether oil significantly (P < 0.01) reduced the amount of penetrating SM within the sampling period. In addition, the perfluoropolyether oil almost completely prevented the penetration of SM for 20 min. Pretreatment with the ointment did not prove to be an effective countermeasure as its administration resulted in no significant reduction in AUC(0-60), AUC and C(max) values.

Developments in the production and quality control of poliovirus vaccines -- historical perspectives.

Using virus grown in monkey kidney cells, Salk and his colleagues developed an inactivated poliovirus vaccine (IPV) in 1952. A large-scale field trial showed the vaccine to be safe and highly immunogenic in children, but soon after the vaccine became generally available in 1955, cases of paralytic disease were reported in recipients. Investigations showed that almost all the cases occurred in children who had received vaccine from one particular manufacturer. Extensive studies attributed the disaster to problems with inactivation. Addition of a Seitz filtration step midway during formalin inactivation and extension of the inactivation period resulted in a safe vaccine. No further paralytic cases were observed following the use of several hundred million doses of this improved vaccine. Thus, IPV was safe and caused a dramatic decline in the incidence of poliomyelitis in countries where it was used. A second generation IPV is produced in fermentors using well-characterized cell strains or continuous cell lines. The major breakthrough in the development of live poliovirus vaccine was the application of tissue culture methods for virus attenuation. By 1959 several candidate live oral poliovirus vaccines (OPV) had been developed. These were clinically tested in millions of individuals and found to be safe and effective. Since the attenuated virus strains developed by Koprowski and Cox were more neurotropic in monkeys than the Sabin strains, only the latter was licensed in the USA in 1961 and endorsed shortly after by the World Health Organization (WHO). The widespread use of Sabin's OPV in many countries hastened the development of International Requirements by WHO for OPV in 1962 to define the criteria that ensured the uniformity of batches produced by different manufacturers. These have been updated continuously in light of new information and quality control procedures. Extensive field trials have shown the risk of OPV associated polio to be less than 0.3 per million doses administered.

Quantitative analysis of the sulfur mustard hydrolysis product thiodiglycol (2,2'-sulfobisethanol) in in vivo microdialysates using gas chromatography coupled with pulsed flame photometric detection.

An analytical method employing gas chromatography is presented for assessing the concentrations of the sulfur mustard hydrolysis product thiodiglycol (TDG) in cutaneous in vivo microdialysates. The use of a pulsed flame photometric detector allows for selective detection of the analyte following solvent exchange and derivatization with heptafluorobutyric anhydride. Quantitative assessment is performed using thiodipropanol (TDP) as a surrogate internal standard. A linear relationship and a very significant correlation (r2 = 0.9982) between the ratio of TDG and TDP concentrations and the ratio of the square root of peak heights is demonstrated. The suitability of the analytical method is verified by the evaluation of blank in vivo microdialysates spiked with known amounts of TDG. The limit of detection in microdialysates is 0.200 nmol/mL (24.4 ng/mL) and the limit of quantitation was 0.364 nmol/mL (44.4 ng/mL). The presented method provides selective, sensitive, rapid, and high-throughput analysis of microdialysates containing TDG, providing an efficient alternative for high-performance liquid chromatography and capillary electrophoresis techniques.

Ischemia reperfusion injury of the skeletal muscle after selective deafferentation.

The present study analyzes the effect of selective deafferentation on the reperfusion injury of the skeletal muscle when nociceptive sensory fibers of the left sciatic nerve are selectively damaged by capsaicin pretreatment in a rat model following tourniquet ischemia (ISC) applied for 30 min, 1 h, and 2 h on the left hind limb. The isometric tetanic contractile force of the extensor digitorum longus (EDL) muscle was measured after 1 h, and 1, 3, or 7 days of reperfusion. Contractile force of the damaged muscle was compared to the intact contralateral muscle. In another group, ISC was used without capsaicin pre-treatment. After 30 min of ISC, there was no difference between deafferented and non-pretreated groups. Following 1 h ISC, with the exception of 1 h reperfusion, the non-pretreated group produced stronger contractions than the deafferented group. After 2 h ISC, the contractile force of the deafferented muscle was significantly stronger compared to the non-deafferented muscle force at all reperfusion times. In conclusions, it was found that the absence of peptidergic sensory fibers after long-lasting (2 h) ischemia is beneficial in reperfusion injury, whereas the absence of vasodilator peptides has unfavorable effects if tissue damage is milder (after 1 h ischemia).

Mass selective detection of amphetamine, methamphetamine, and related compounds in urine.

A method is presented for the routine analysis of amphetamine, methamphetamine, and related compounds in urine with gas chromatography coupled with mass spectrometry operated in the selective ion monitoring mode. The analytes are isolated by liquid-liquid extraction and are derivatized with trifluoroacetic anhydride. 3,4-Methylenedioxy-methamphetamine-D(5) is employed as the internal standard. Standard solutions are prepared using spiked urine samples, which are subjected to all phases of sample preparation. Disposable deactivated glass containers are employed throughout the process.

Time course of leukocyte response and free radical release in an early reperfusion injury of the superior mesenteric artery.

The sequence of changes in circulating immune cells and in free radical production was studied during the small intestine reperfusion. Rat small intestine ischemia/reperfusion was induced by a 45 min superior mesenteric artery occlusion followed by a 4-hour reperfusion. Samples of peripheral blood were collected every 20 min during reperfusion. While the number of polymorphonuclear leukocytes increased significantly both in the sham-operated controls and the experimental group (about 400 per cent at the end of reperfusion), a decrease in lymphocyte counts to 60 per cent was observed in the experimental group only. Although there were no changes in the counts of total T lymphocytes, a significant reduction in B cell counts was observed. Flow-cytometrical measurements showed no changes in the Tc subpopulation, while the Th subpopulation increased in the experimental group only. Free radical generation in blood (luminometric measurements) increased gradually and reached an eight-fold level by the end of reperfusion in both groups. Thus, it has been shown that the increase in free radical production is mainly due to the increased number of polymorphonuclear leukocytes mobilized already at the initial stages of reperfusion. The reduction in B lymphocyte population is probably due to homing mechanisms

Gas chromatography-mass spectrometry-single ion monitoring measurement of 11-nor-delta 9-tetrahydrocannabinol-carboxylic acid in urine.

Gas chromatography combined with mass spectrometry is used to determine the urinary elimination of 11-nor-Delta(9)-tetrahydrocannabinol-carboxylic acid. Single ion monitoring of both 11-nor-Delta(9)-tetrahydrocannabinol-carboxylic acid and the internal standard (11-nor-Delta(9)-tetrahydrocannabinol-carboxylic-D9) offers selective and sensitive measurement. In order to check the method and the results, we employ 11-nor-Delta(9)-tetrahydrocannabinol-carboxylic acid-glucuronide as an external standard. Our study also demonstrates that the wall of the glass liner (glass tube) in the injector retards the active compound, which makes it imperative to replace the glass insert after each run. Otherwise, the value of the drug measured in subsequent runs will decrease because more of this compound will adhere to the glass walls.

Effect of alpha-FMH and DPPE on colony-forming properties of human peripheral progenitor cells.

Endogenous histamine regulates the haematopoiesis. Histidine decarboxylase inhibitor decreases the histamine level, and its intracellular antagonist decreases the histamine effect. The effect of histidine decarboxylase inhibitor (alpha-fluoromethyl histidine) and the intracellular antagonist of histamine [N'N-diethyl-2-4-(phenylmethyl) phenoxyethan-amine-HCl] was investigated on the colony-forming ability of human peripheral progenitor cells. Semi-solid culture medium was used both in the presence and in the absence of 3 U/ml erythropoietin. alpha-Fluoromethyl histidine was used in the range of 50 through 150 micro Mol/ml, the concentration of N'N-diethyl-2-4-(phenylmethyl) phenoxyethanamine-HCl was between 5 and 25 micro Mol/ml. The number of both the erythroide and the granulocyte macrophage colony was significantly decreased in a concentration dependent manner by the presence of both N'N-diethyl-2-4-(phenylmethyl) phenoxyethanamine-HCl (in all concentrations used) and &alpha-fluoromethyl histidine (at higher concentration). The inhibitory effect was decreased by erythropoietin.

[Role of the monocye-lymphocye system and of endogenous mediators in the severity of acute pancreatitis and in development of its septic complications]. / A monocita/limfocita rendszer és az endogén mediátorok szerepe az akut pancreatitis súlyosságának és szeptikus szövódményeinek kialakulásában.

The activity of monocyte/lymphocyte system and the splanchnic circulation was investigated in acute pancreatitis. The splanchnic blood flow was characterised indirectly by gastric intramucosal pH changes, which strongly correlated with APACHE-II score, and predicted the bad prognosis. The high CD14/HLA-DR and CD14/CD16 coexpression, the low TNF-alpha production proved to be unfavourable prognostic factor in the early phase of the disease. The expression of IL-2, IL-10 and moderately of IL-4 was elevated, but the expression of INF gamma did not show significant alteration. The thrombocyte aggregation decreased in the early phase. There are bad prognostic signs if its level remains constantly low and ATP-release increases. The activity and index of phagocytosis were higher in comparison with controls, but these parameters were not increasable with higher cell concentration. The ROI production increased, and the increase in the fMLP and o'zymosan indicated LDCL seems to be unfavourable prognostic sign, which well correlated with the APACHE-II score.

fMLP-induced respiratory burst and the intracellular Ca2+ signal are not interrelated in neutrophils.

Reactive oxygen intermediate (ROI) production and the development of the intracellular (IC) Ca2+ ([Ca2+]i) signal by formyl-Met-Leu-Phe (fMLP) stimuli were investigated in neutrophils. When the concentration was varied between 2.3 nM-2.3 microM, ROI production and the [Ca2+]i signal showed different fMLP concentration dependencies. ROI production increased continuously with increasing fMLP concentrations, while the [Ca2+]i signal responses reached a plateau around 230 nM fMLP. Moreover, when a consecutive, 2.3 microM fMLP stimulus was applied 10 min after the first fMLP stimulus, the intensity of the ROI production and that of the [Ca2+]i signal showed a variable dependence on the fMLP concentration of the first stimulus. An initial fMLP dose of 2.3 nM and 23 nM sensitized the cells regarding their ROI production and [Ca2+]i signals. After a first fMLP stimulus of 230 nM, the second stimulus produced an increased [Ca2+]i signal, while no ROI production could be activated. A strong fMLP stimulus of 2.3 microM desensitized the cells regarding both [Ca2+]i signal and ROI production. However, even in these desensitized cells, a high level of ROI production could be evoked by other stimuli like PMA or opsonized zymosan. The differences observed between the fMLP concentration dependence of ROI production and the [Ca2+]i signal strongly suggest that these two phenomena are not interrelated.

Telomerase activity is not altered by regular strenuous exercise in skeletal muscle or by sarcoma in liver of rats.

Telomerase is a specialized ribonucleoprotein enzyme complex which prevents the loss of the telomere. The activity of telomerase can be up- and down-regulated by various oxidative stresses but the effect of physical exercise is not known, whereas the modifying effect of cancer on telomerase activity is well documented. In the first study, we investigated the effect of mild and strenuous exercise training on telomerase activity, assessed by a PCR ELISA kit. No alteration in telomerase activity was detected. In the second investigation, solid sarcoma cells were transplanted to control, exercise trained or exercise trained and still exercising mice. On the 16th day after the transplantation, the size of tumors in the exercise trained group was 72% and in the exercising group 57% (P < 0.05) of that in the controls. Telomerase activity and 8-hydroxy-2'-deoxyguanosine levels in the liver were not significantly altered by exercise and/or sarcoma. We conclude that mild and strenuous exercise training does not significantly affect the activity of telomerase in the systems studied. Exercise training during sarcoma significantly retards the development of tumors and could possibly serve as a positive adjunct to treatment.

[Effect of pentoxifylline on the healing of experimental anastomosis of the left colon in rats]. / A pentoxifyllinum hatása a kísérletes bal colonfél anasztomózis gyógyulására.

The healing of colonic anastomoses is determined by several factors such as microcirculation, the strength of the inflammatory response, and the time required for regeneration. We investigated the effects of pentoxifylline--a drug which improves microcirculation and modulates leukocyte functions--on the healing of experimental anastomosis on the left colon of rats. As a result of drug treatment (0.25 mg/100 g, i.p.) in Group I anastomosis bursting pressure (ABP) was by 56 +/- 17% higher at day 2 than in controls with no pentoxifylline treatment. On the 5th postoperative day in Group I, ABP reached 80 +/- 8% the value for the intact colon (218 +/- 21 mmHg), whereas respective value in the control (untreated) group was only 47 +/- 7%. In Group II (pentoxifylline: 2 mg/100 g, i.p.) ABP was by 55 +/- 10% and by 73 +/- 8% higher than control values at postoperative days 1 and 2, respectively. At day 2, in Group I colonic blood flow measured at the anastomosis line by 86Rb uptake technique was significantly higher than in the untreated controls (0.18 +/- 0.01 ml/min vs. 0.14 +/- 0.01 ml/min, (p < 0.02). Blood flow measured in colon tissue above and below the anastomosis changed differently. Pentoxifylline treatment also suppressed the peritoneal inflammatory response assessed with peritoneal reaction index (2.0 +/- 0.3 vs. 1.1 +/- 0.2, p < 0.01). The results of the present study show that pentoxifylline treatment shortens the time needed for the healing of colonic anastomosis. These observations suggest that pentoxifylline medication can prevent failure of colonic anastomoses.

Azurophil granules are heterogeneous with respect to mobilization induced by different concentrations of fMLP.

Azurophil granules of neutrophils beyond their already known heterogeneity of beta-glucuronidase and myeloperoxidase enzyme contents are heterogeneous with respect to a spontaneous or low concentration (2.3 or 23 nM) of formyl-Met-Leu-Phe-induced mobilization. This suggests that the heterogeneity of azurophil granules is manifested in their functions too.

Histidine decarboxylase expression in human melanoma.

Histamine has been implicated as one of the mediators involved in regulation of proliferation in both normal and neoplastic tissues. Histidine decarboxylase, the only enzyme that catalyzes the formation of histamine from L-histidine, is an essential regulator of histamine levels. In this study, we investigated the gene and protein expression of histidine decarboxylase in melanoma. Reverse transcriptase polymerase chain reaction and in situ hybridization studies of WM-35, WM-983/B, HT-168, and M1 human melanoma cell lines both resulted in positive signals for histidine decarboxylase messenger RNA. A polyclonal chicken antibody was developed against human histidine decarboxylase and protein expression was confirmed by western blot analysis of the cell lysates, revealing a predominant immunoreactive band at approximately 54 kDa corresponding to monomeric histidine decarboxylase. Protein expression of histidine decarboxylase was also shown by flow cytometric analysis and strong punctate cytoplasmic staining of melanoma cell lines. Moreover, both primary and metastatic human melanoma tissues were brightly stained for histidine decarboxylase. When compared with the very weak or no reactions on cultivated human melanocytes both western blot and immunohistochemical studies showed much stronger histidine decarboxylase expression in melanoma cells. These findings suggest that expression of histidine decarboxylase is elevated in human melanoma.

[Calculated gastric intramucosal pH changes in the early phase of acute pancreatitis]. / Számított intramucosalis pH-változások a gyomorban az akut pancreatitis kezdeti szakaszában.

Based on the literature dysfunction of splanchnic circulation may be assumeol in the development of severe acute pancreatitis. Abnormal gut functions investigated by routinely used clinical examination is not available. Gastric tonometry indirectly gives information about gut function. Authors followed prospectively 12 patients who suffered from acute pancreatitis. Four patients recovered without complications, 4 patients had different complications and 4 patients died. Gastric intramucosal pH (pHi) was measured by TRIP NGS catheter and Tonocap monitor. Measurements were started at the time of hospitalisation and repeated every six hours on the first 3 days. Intramucosal acidosis (pHi < 7.3) could be measured independently from aetiology. Gastric pHi showed strong correlation with Acute Physiology and Chronic Health Evaluation II. (APACHE II) score (p = 0.02). APACHE II scores were significantly higher in-group pHi < 7.2 (13.9 +/- 1.7) compared to group pHi > 7.2 (7.33 +/- 1.06) (p = 0.002). 24-hour changes in APACHE II scores were significantly greater in the cases of pHi < 7.2 (3.3 +/- 1.47) versus pHi > 7.2 (-0.6 +/- 0.97) (p = 0.03). Changes of pHi in the early phase of acute pancreatitis indicate that splanchnic circulation is already involved and the pHi may have a prognostic value.

New assays for the quality control of live oral poliovirus vaccine.

The strains of all three types of poliovirus used in the production of live oral poliomyelitis vaccine have been shown to yield vaccines that are both immunogenic and highly attenuated when administered orally to susceptible children and adults. Experience obtained for close to four decades with the vaccines prepared from these strains indicates that laboratory and animal tests described in the World Health Organization's (WHO) Requirements for Poliomyelitis Vaccines (Oral) do ensure the consistency of virus characteristics during vaccine production. Major advances in our understanding of the molecular basis of attenuation and reversion of polioviruses resulted in the development of a new generation of tests. These include an alternative in vivo neurovirulence test in transgenic mice that express the human poliovirus receptor and a new in vitro assay (mutant analysis by polymerase chain reaction and restriction enzyme cleavage, shortly MAPREC) that assess the consistency of vaccine production at a molecular level. A WHO collaborative study was initiated in 1993 with the objective of assessing transgenic mice as potential models for evaluation of the neurovirulence of type 3 vaccines. The results of the study showed that there is a very good correlation between the TgPVR21 mouse assay and the monkey neurovirulence test. Further studies are in progress to develop a statistical model for making regulatory decisions on accepting or rejecting batches of type 3 vaccine. A WHO collaborative study was initiated in 1991 to evaluate the MAPREC assay for type 3 vaccines. The results of this study showed that the MAPREC test was a sensitive, robust and standardized molecular assay suitable for process development for new manufacturers and for monitoring the consistency of existing vaccine production. Screening single virus harvests with MAPREC before pooling them into monovalent bulk vaccine will also improve the quality of the final product.

The trental treatment prevents the reduction of superoxide dismutase enzyme (SOD) activity in rats after colon surgery.

The authors tested the effects of the pentoxiphyllin (px, Trental) pre-treatment upon the superoxide dismutase activity of the left colonpart anastomosis of rats. It has been found that the SOD activity of the proximal (4.9 U/g), the anastomical area (1.9 U/g) and the distal (3.1 U/g) intestinal segments considerably decreased, compared to the control (9.16 U/g). In the operated and pre-treated animals with Trental, the SOD activity of all the three intestinal segments increased (proximal 15.5-anastomosis 5.7-distal 8.9 U/g) compared to the non treated group.