PKA and Apicomplexan Parasite Diseases.

The cAMP-dependent protein kinase PKA is a well-characterized member of the serine-threonine protein AGC kinase family and is the effector kinase of cAMP signaling. As such, PKA is involved in the control of a wide variety of cellular processes including metabolism, cell growth, gene expression and apoptosis. cAMP-dependent PKA signaling pathways play important roles during infection and virulence of various pathogens. Since fluxes in cAMP are involved in multiple intracellular functions, a variety of different pathological infectious processes can be affected by PKA signaling pathways. Here, we highlight some features of cAMP-PKA signaling that are relevant to Plasmodium falciparum-infection of erythrocytes and present an update on AKAP targeting of PKA in PGE2 signaling via EP4 in Theileria annulata-infection of leukocytes and discuss cAMP-PKA signling in Toxoplasma.

Loss of Ikappa B-beta is associated with prolonged NF-kappa B activity in human glial cells.

Nuclear factor-kappaB (NF-kappaB) is an inducible transcription factor central in the regulation of expression of a wide variety of genes and synthesis of several proteins involved in the generation of the immune response and inflammatory processes. In resting cells, NF-kappaB is maintained in an inactive state through cytoplasmic retention by IkappaB inhibitors. Stimulation of cells with a wide variety of inducers results in proteolytic degradation of these IkappaB proteins, leading to activation of NF-kappaB. The present study shows that interleukin-1 (IL-1) causes persistent activation of NF-kappaB in glial cells. Stimulation with IL-1 also causes rapid but transient degradation of IkappaB-alpha and IkappaB-epsilon. However, NF-kappaB remains active even after these IkappaB isoforms have returned to control levels. In contrast, the IkappaB-beta isoform fails to reappear following its initial degradation by IL-1, coincident with sustained activation of NF-kappaB. In addition, in vivo overexpression of the various IkappaB isoforms revealed that IkappaB-beta is the only isoform that has the ability to inhibit IL-1-induced NF-kappaB-driven transcription. The findings also suggest that the inability of IkappaB-alpha and IkappaB-epsilon to modulate NF-kappaB activity is due to their modification in vivo. These findings indicate that IkappaB-beta is the key regulator of the activity of NF-kappaB in human glial cells.

Drug utilization evaluation of nonprescription H2-receptor antagonists and alginate-containing preparations for dyspepsia.

AIMS: To evaluate the use, efficacy and adverse effects of nonprescription H2-receptor antagonists and alginate-containing preparations obtained from community pharmacies. METHODS: Questionnaires were distributed to customers from 39 pharmacies in Scotland and Wales. RESULTS: Of 767 customers recruited, 608 (79.3%) returned an initial questionnaire and 472 (61.5%) customers a second questionnaire. The vast majority of respondents (424, 69.7%) had suffered their symptoms on three or more occasions and 369 (60.7%) had previously tried medicines to relieve their symptoms. Referrals to a doctor were less frequent than recommended in guidelines and few of those who were referred actually saw a doctor. Over a quarter of those returning the second questionnaire claimed to be taking more than one product simultaneously for symptom control. Eight customers who were taking prescribed ulcer-healing drugs obtained H2-receptor antagonists. The majority of respondents (355/472, 75. 2%) obtained some or complete symptom relief using the product obtained and 369/472 (78.2%) were completely satisfied with their product. H2-receptor antagonists were more likely to produce complete relief of symptoms than alginate-containing preparations (P < 0.05). Only 14 respondents (3.0%) reported side-effects from the product used which were mostly gastro-intestinal. CONCLUSIONS: The study demonstrated that drug utilization studies are feasible to carry out in a community pharmacy setting. While the results support published evidence of the efficacy and minimal toxicity of these products, they also highlight the possibility of H2-receptor antagonists being used outwith their licenced indications.

Over-the-counter emergency contraception: a feasible option.

BACKGROUND: The high number of unintended pregnancies and terminations in Britain indicates that women who could use emergency contraception do not. Knowledge of access to sources of emergency contraception is limited. Oral administration of combined oestrogen-progestogen is safe and does not require routine physical administration, and there are proposals to re-regulate this from a prescription-only medicine to a pharmacy medicine, available over the counter in community pharmacies under the supervision of a pharmacist. OBJECTIVES: We aimed to demonstrate that the availability of combined, oral oestrogen-progestogen under the supervision of the community pharmacist would be safe and effective. METHOD: Guidelines were developed by a multidisciplinary group incorporating pharmacists, GPs, a pharmacologist and a consultant in family planning. The guidelines were based on published evidence, where possible. CONCLUSION: Guidelines have been developed to accompany the provision of combined, oral oestrogen-progestogen which demonstrate that over-the-counter availability could be a safe and effective method of reducing the number of unwanted pregnancies in Britain.

Prospective study of bone, indium-111-labeled white blood cell, and gallium-67 scanning for the evaluation of osteomyelitis in the diabetic foot.

Twenty-two adult diabetic patients with clinical suspicion of foot and/or ankle infection were prospectively evaluated using radiography, technetium-99m methylene diphosphonate bone scanning (99mTc), indium-111-labeled leukocyte scanning (111In), and gallium-67 scanning (67Ga) to determine the presence of clinically suspected osteomyelitis. Biopsy for culture and histology was performed in 16 patients. The diagnosis of osteomyelitis was confirmed by biopsy in 12 patients. The remaining 10 patients had no evidence of osteomyelitis with long-term follow-up. 99mTc was shown to be of limited valued when used alone in these patients with peripheral neuropathy. 67Ga, either alone or in combination with 99mTc bone scanning, was of little diagnostic value and gave no additional information that was not available from 111In. The combination of three-phase 99mTc and 111In had the highest diagnostic efficacy (100% sensitivity, 80% specificity, and 91% accuracy), followed closely by 111In alone (100% sensitivity, 70% specificity, and 86% accuracy). We conclude that for adult diabetic patients with clinical suspicion of osteomyelitis but no radiographic findings of that disease, 111In alone is an appropriate nuclear medicine evaluation for ruling out infection if it is negative. However, if an area of 111In white blood cell uptake is present, a "simultaneous" 99mTc is often helpful in providing the anatomic correlation to differentiate osteomyelitis from infection that is limited to soft tissue.

Performance of a rapid, on-site human immunodeficiency virus antibody assay in a public health setting.

Rapid, on-site human immunodeficiency virus (HIV) testing has the potential to improve the delivery of prevention services in publicly funded counseling and testing sites. The Single Use Diagnostic System (SUDS) HIV-1 is the only rapid enzyme immunoassay (EIA) approved for diagnostic use in the United States. To evaluate the feasibility of using SUDS in public clinics and to validate the test's performance in a public health laboratory, we conducted blinded SUDS testing on plasma sent for HIV testing. From 19 March through 30 June 1993, 1,923 consecutive samples from a sexually transmitted diseases clinic and an HIV counseling and testing clinic were tested on site with SUDS. Tests done in the first two weeks with a malfunctioning centrifuge n = 402) and those done when there were excessively high temperatures in the laboratory (n = 53) were analyzed separately. Of 1,466 tests, 39 were positive by both SUDS and EIA (with Western blot [immunoblot] confirmation) and 7 were SUDS positive and EIA negative. Western blotting was used as the "gold standard" to adjudicate these discrepancies. There were no SUDS-negative and EIA-positive tests. Compared with that of EIA (with Western blot confirmation), the sensitivity of SUDS was 100% (95% confidence interval, 88.8 to 100%) and the specificity was 99.5% (95% confidence interval, 98.9 to 99.8%). The positive predictive value of SUDS was 88% in the STD clinic and 81% in the HIV counseling and testing clinic. There was a 7.7-fold increase in false positives, from 0.48 to 3.7%, when there was inadequate centrifugation and when the temperature exceeded the manufacturer's recommendations. Rapid, on-site HIV testing by the SUDS assay is feasible and practical in public health settings. The test can be performed accurately, at reasonable cost, and within the time frame of a typical clinic visit. Caution should be used, however, as two conditions adversely affected the accuracy of this test: inadequate specimen preparation and elevated temperature.

Glutathione, cell proliferation, and 1,3-bis-(2-chloroethyl)-1-nitrosourea in K562 leukemia.

We have pursued our findings of glutathione reductase (GSSG-R) deficiency and disturbed glutathione in cancer patients treated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), by investigating how thiol metabolism, cell proliferation, and the nitrosourea interact in human K562 leukemia. Fasting cells arrested in G greatly increased their reduced glutathione (GSH) in response to growth factors. The rise in thiol began after several hours, peaked before DNA synthesis, and resulted from increased production. BCNU inactivated GSSG-R rapidly, and later retarded, doubled, and greatly prolonged GSH formation before stopping DNA synthesis. Pretreatment unlike post treatment with buthionine-S-R-sulfoximine (BSO) diminished BCNU's ability to block GSSG-R. Enzyme inhibition decreased with falling cellular GSH. In the leukemia system as in vivo, sequential BCNU-induced thiol alterations heralded delayed antiproliferative effects. Drug timing markedly affected both thiol and DNA syntheses. By destroying GSSG-R and delaying the upregulation of thiol synthesis while escalating GSH utilization and requirements, the nitrosourea created a striking and previously unrecognized window of vulnerability for GSH-dependent processes. During this period, altered GSH metabolism could contribute indirectly to BCNU's pleiotropic effects by interfering with DNA alkylation repair, glucose decarboxylation, deoxyribose formation, and possibly by influencing other aspects of proliferation. Acquired GSSG-R deficiency was also an early and sensitive marker for prodrug breakdown and activation.

Distribution of amitriptyline and nortriptyline in blood: role of alpha-1-glycoprotein.

To interpret blood levels of tricyclic antidepressants, we studied the distributions of amitriptyline and nortriptyline in human blood and explored their control by plasma factors. Each compound (300 ng/ml) was added to whole adult blood and to cord blood with decreased alpha-1-glycoprotein (AGP). Drugs (250 ng/ml) were also added to washed erythrocytes (RBCs) resuspended in autologous plasma or saline (hematocrit = 0.4) with or without AGP, albumin, or tris(2-butoxyethyl) phosphate (TBEP), used to displace AGP-bound drugs. Plasma AGP was determined in all adult blood donors (n = 17). With adult blood, plasma amitriptyline was 393 +/- 52 ng/ml, RBC amitriptyline was 184 +/- 33 ng/ml. Plasma and RBC nortriptyline were 199 +/- 28 and 288 +/- 39 ng/ml, respectively. With saline, cellular amitriptyline and nortriptyline were 81 +/- 10 and 88 +/- 6%, respectively. With plasma, cellular amitriptyline and nortriptyline were 25 +/- 8 and 49 +/- 10%, respectively. The corresponding cord blood values were 52 +/- 12 and 62 +/- 6%. Graded increments of AGP in saline reproduced the distribution pattern seen with increasing concentrations of plasma. Albumin did not influence drug distribution. TBEP markedly increased erythrocyte amitriptyline in adult but not in cord blood. Plasma AGP correlated positively (p = 0.031) with the RBC/plasma ratio of amitriptyline. Amitriptyline is predominantly distributed in plasma, nortriptyline in RBCs. This differential distribution is dose dependent and reflects the higher binding of amitriptyline to AGP when compared with nortriptyline. Interpretation of tricyclic antidepressant blood levels is clarified by obtaining assays from RBCs and plasma.

Tricyclic antidepressants in red cells and plasma: correlation with impaired intraventricular conduction in acute overdose.

OBJECTIVE: Tricyclic antidepressant levels in red blood cells and plasma in acute overdose and their association with cardiotoxicity were studied. METHODS: This was a prospective study in 15 patients with acute tricyclic antidepressant overdose. Tricyclic antidepressant parent compounds and metabolites were measured in red blood cells and plasma, and tricyclic antidepressant levels were correlated with ECG indexes of toxicity. RESULTS: Plasma levels of the parent compounds were higher than their red blood cell levels on admission (mean +/- SD, 691 +/- 409 and 337 +/- 220 ng/ml, respectively). Admission metabolite levels were higher in red blood cells than in plasma (264 +/- 180 and 190 +/- 164 ng/ml, respectively). QRS duration and the red blood cell levels of the metabolites were significantly correlated at the time of admission (r = 0.77, p < 0.01), as well as at 6 to 10 hours (r = 0.74, p < 0.01). CONCLUSIONS: In acute overdose, a shift of tricyclic antidepressants from plasma to red blood cells and increased levels of red blood cell metabolites reflect tissue redistribution of the drug. Tricyclic antidepressant red blood cell metabolites are the best markers for impaired intraventricular conduction.

Defenses against oxidation in human erythrocytes: role of glutathione reductase in the activation of glucose decarboxylation by hemolytic drugs.

We have used 1,3-bis(2-chloroethyl)-1-nitrosourea, a selective inhibitor of oxidized glutathione reductase (GSSG-R), to examine the role of this enzyme in regulating the hexose monophosphate shunt (HMS) and to explore how a variety of agents influence glucose decarboxylation in intact human red blood cells (RBCs). Substances tested included primaquine and several other drugs that are specially hemolytic and methemoglobinemic in glucose-6-phosphate dehydrogenase (G6PD) deficiency and related disorders. The results allowed us to distinguish and quantitate contrasting modes of HMS stimulation and to clarify how RBCs respond to different classes of oxidants. Some agents like methylene blue (MB), phenazine methosulfate, and pyrroline carboxylate do not require GSSG-R to increase CO2 production; they activate G6PD and 6-phosphogluconic dehydrogenase by directly oxidizing reduced nicotinamide adenine dinucleotide phosphate (NADPH) to oxidized nicotinamide adenine dinucleotide phosphate (NADP). Other compounds, like ascorbate, nitrofurantoin, and doxorubicin, oxidize GSH primarily; CO2 increases indirectly only when GSSG-R, activated by glutathione disulfide (GSSG), raises the level of NADP. Chemicals like primaquine, daunorubicin, and methylphenylazoformate trigger the HMS by independently oxidizing both NADPH and GSH. Unlike MB, most drugs that are hemolytic in G6PD deficiency activate the HMS in a manner that depends to a variable extent on GSSG-R. This variability may explain hitherto puzzling clinical and pharmacogenetic differences between primaquine and diaminodiphenylsulfone-induced hemolysis.

Fluorescent treponemal antibody absorption double-staining test evaluation.

The fluorescent treponemal antibody absorption (FTA-ABS) double-staining (DS) test has been developed for microscopes equipped with incident illumination, and the procedure offers many advantages over the FTA-ABS test when tests are performed with this equipment. In this study, 346 fresh sera, including 35 from patients with syphilis, were evaluated by the FTA-ABS DS test. Parameters for investigation included two readers, each using a different microscope; a new FTA-ABS DS test reporting system; sera heated at 56 degrees C for 30 min versus unheated sera; and sera retested after at least 2 weeks of freezer storage. Agreement for FTA-ABS DS test readings between the two microscopes was 99%. Between-test agreement for the FTA-ABS test with the conventional reporting system and the FTA-ABS DS test with the new reporting system was 95%. Sensitivity calculations based on reactivity for the 35 syphilis sera were 94% for the FTA-ABS DS test and 91% for the FTA-ABS test. Specificity calculations based on non-reactivity of nonsyphilis sera were 98% for the FTA-ABS DS test and 93% for the FTA-ABS test. Differences in percentages appeared to be related to borderline readings in the FTA-ABS test. For example, if the same reporting system was used for the reference FTA-ABS test, the specificity was 97%. When sera were examined within 48 h, no difference was observed in results obtained with heated and unheated sera. Sera frozen for 2 weeks showed comparable results in the FTA-ABS DS test and the FTA-ABS test. These findings strongly support the recommendation that the FTA-ABS DS test be accepted as a confirmatory test for syphilis. The new reporting system for the FTA-ABS DS test would be advantageous for the reference FTA-ABS procedure.

Fluorescent treponemal antibody-absorption double-staining procedure.

The fluorescent treponemal antibody-absorption double-staining step-by-step procedure and proposed reference reagents for the test are described. The test and reagents were evaluated in two separate laboratories on 265 fresh sera, and test results were compared with the reference fluorescent treponemal antibody-absorption test results performed in a third laboratory. The data indicate that the tests are comparable in the areas where the test is recommended for use. Problems with inadequate light filtration occurred, but these could be resolved. This test is recommended for use with microscopes equipped with incident illumination.