Antibiotic susceptibility testing (agar disk diffusion and agar dilution) of clinical isolates of Corynebacterium jeikeium

Thirty-three clinical isolates of Corynebacterium jeikeium were examined for susceptibility to 27 antimicrobial drugs with the agar dilution test. Sheep-blood-supplemented Mueller-Hinton agar performed better than Wilkins-Chalgren agar. Disk susceptibility (Bauer-Kirby) tests were carried out in parallel with 24 of the chemotherapeutic agents. All isolates were susceptible to teicoplanin and vancomycin. All isolates resisted fosfomycin, mupirocin, and trimethoprim-sulfamethoxazole. The isolates varied in susceptibility to ciprofloxacin, doxycycline, fusidic acid, ofloxacin, and tetracycline; most were susceptible to rifampin. Surprisingly few discrepancies between agar dilution and disk diffusion tests were encountered when utilizing NCCLS interpretive criteria currently valid for enterococcal isolates.     

Aminoglycoside-induced nephrotoxicity

In severe gram-negative infections aminoglycosides generally remain the first-line antibiotic. Their use is limited by the high risk of side effects and, especially, nephrotoxicity. High peak levels are crucial for antibacterial activity, whereas toxic side effects are determined by the more prolonged trough levels. Thus, aminoglycosides should not be given by intramuscular injection because the peak levels achieved are inadequate, whilst long-lasting elevated plasma trough levels result. On administration of a daily single dose intravenously high antibacterial efficacy can be combined with low nephrotoxicity. Besides the dose-dependent bactericidal effect, the post-antibiotic effect of aminoglycosides is of importance. The main site of nephrotoxicity are the proximal tubule epithelial cells. Renal toxicity is usually reversible after discontinuation of drug therapy. Toxic acute renal failure is not uncommon (5-35%) and usually dependent on the underlying disease, preexisting renal function, hydration state, age, cumulative dose, additional medication, previous therapy with aminoglycosides and the choice of the specific aminoglycoside. By implementing a single daily dose regimen in conjunction with adequate hydration, alkalization therapy with bicarbonate, monitoring of plasma trough levels and minimization of the duration of therapy (5 days), development of renal impairment can be prevented in the large majority of patients. Hence, acute renal failure has become an avoidable, and much less frequently observed complication of aminoglycoside therapy due to these measures.     

Pharmacokinetics of recombinant human erythropoietin in rabbits and 3/4 nephrectomized rats

We present a minimal kinetic model for excitatory synaptic transmission to cerebellar Purkinje cells. The main components are a kinetic model for a single glutamate receptor, which is calibrated with the help of patch clamp data, and a mean field approximation for the dynamics of a population of channels, which generate an EPSC. The resulting minimal model of the parallel fiber-Purkinje cell synapse is used to estimate the dynamics of glutamate in the synaptic cleft and to clarify the role of receptor desensitization in synaptic transmission. We also apply the model to different aspects of synaptic modulation, like long-term depression and potentiation by pharmacological application of ampakines. In the framework of the minimal model these effects can be understood as the result of modified receptor kinetics.     

Stable, high-level expression of human serotonin receptors in L929 cells using an inducible expression system

Heterologous expression of cloned receptor subtypes for screening programs has become a real necessity for a modern pharmaceutical company. As the expression levels obtained so far are often low or unstable, we addressed this problem by using an inducible promoter system, i.e. the interferon-inducible mouse Mxl promoter. Using the gene coding for chloramphenicol acetyltransferase (CAT) as a reporter gene, we tested the inducibility of this promoter in the murine cell line L929. We found that background expression was low and that a distinct interferon-induced expression could be obtained. CAT expression reached its maximum at approximately 15 ng CAT/mg protein after induction for 24 hr with 1000 U/ml murine interferon-beta; the induction ratio was 150-fold. Next, L929 cells were transfected with four different human serotonin (5HT) receptor cDNAs (5HT1A, 5HT2A, 5HT1D beta and 5HT1E) under the control of the same Mxl promoter fragment. Also in this case well-regulated serotonin receptor-expressing clones were isolated. Bmax values varied from 3100 fmol/mg protein for the 5HT2A receptor, 3300 fmol/mg protein for the 5HT1D beta receptor, 9800 fmol/mg protein for the 5HT1E receptor, and even up to 10,400 fmol/mg protein for the 5HT1A receptor. Furthermore, the expression levels were shown to remain stable during serial propagation for at least one year, demonstrating the usefulness of this expression system. In fact, the 5HT1D beta receptor-expressing cells were used in the characterization of a new antimigraine agent, viz. alniditan.     

Validation of multi-frequency bioelectrical impedance analysis in detecting changes in fluid balance of geriatric patients

OBJECTIVES: Multi-Frequency Bioelectrical Impedance Analysis (MFBIA) is a quick, simple, and inexpensive method to assess body fluid compartments. This study aimed at determining the validity of MFBIA in detecting clinically relevant changes of fluid balance in geriatric patients. DESIGN: A prospective, observational study. SETTING: The 22-bed Geriatric Department of the University Hospital Nijmegen. PARTICIPANTS: Hospitalized patients were eligible if they did not have a pacemaker, were not suffering from terminal illnesses, and did not have psychogeriatric diseases likely to interfere with capacity to consent or comply. During a 16-months period, 218 patients were admitted, of whom 78 patients were eligible and 53 consented to participate. MEASUREMENTS: Each subject's fluid balance was diagnosed twice a week as dehydrated, overhydrated, or euvolemic, based on standardized physical examination, laboratory tests, and weight evaluation. Changes in fluid balance were quantified by measuring total body water (TBW) and extracellular fluid (ECF) applying deuterium- and bromide-dilution techniques. Impedance at 1, 5, 50, and 100 kHz and body weight were measured daily. Sensitivity and Guyatt's responsiveness indexes of MFBIA in detecting dehydration and overhydration were determined. RESULTS: In total, 1071 MFBIA measurements were performed, during which 14 transitions from dehydration to euvolemia and 13 transitions from overhydration to euvolemia were monitored. Rehydration of dehydrated patients caused an increase in TBW and ECF of 3.4 +/- 1.8 L and 1.9 +/- 1.9 L, respectively, which resulted in significant decreases in impedance of 133 +/- 67 omega at 1 kHz and 93 +/- 61 omega at 100 kHz (P = .001). Treatment of overhydrated patients caused a TBW and ECF loss of 3.8 +/- 4.2 L and 3.1 +/- 3.8 L, respectively, which resulted in significant increases in impedance of 104 +/- 72 omega at 1 kHz and 81 +/- 68 omega at 100 kHz (P < .001). Sensitivity of a single MFBIA in diagnosing dehydration and overhydration was 14% and 17%, respectively. Responsiveness indexes of weighing and MFBIA for dehydration and overhydration were similar at all frequencies and greater than one. CONCLUSION: The sensitivity of a single impedance measurement in detecting dehydration and overhydration was low. However, responsiveness of serial measurements to intra-individual changes in fluid balance was good. Therefore, this noninvasive technique may be used in clinical practice to improve monitoring fluid balance in geriatric patients, especially when daily weighing is difficult.     

Accreditation: who's counting?

Active physician involvement and leadership in their accreditation process can produce a cubic win for patients, payors, and providers. For health care quality to improve and everyone win, physicians need to understand the accountability system, the what and why of data collection, and be involved in short- and long-term performance assessments.     

Lymphocyte-dependent inhibition of growth of virulent Mycobacterium tuberculosis H37Rv within human monocytes: requirement for CD4+ T cells in purified protein derivative-positive, but not in purified protein derivative-negative subjects

Protective human immunity to Mycobacterium tuberculosis (M. tb) has proven difficult to characterize, in part because of technical obstacles to in vitro infection of human cells with virulent M. tb. We established a reproducible method of infecting human monocytes (MN) with the virulent M. tb strain H37Rv that did not reduce MN viability. TNF-alpha had no effect on replication of H37Rv within MN, and IFN-gamma mediated only a 1.9-fold reduction in bacterial growth. In contrast, nonadherent cells (NAC) from purified protein derivative (PPD)-positive and PPD-negative subjects reduced intracellular growth of H37Rv by 6- and 10.6-fold, respectively (p = 0.007 and p = 0.005). CD4+ T cells were essential to growth inhibition mediated by NAC of PPD-positive subjects, whereas containment of M. tb by NAC of PPD-negative subjects did not require CD4+ cells. CD8+ T cells did not contribute to protection mediated by NAC of either group. Supernatants of cocultured H37Rv-infected MN and NAC only partially reduced intracellular growth of M. tb despite containing nanogram concentrations of TNF-alpha and IFN-gamma. Neutralizing antibodies to TNF-alpha, IFN-gamma, and IL-12 failed to affect the NAC-mediated growth limitation. NAC treated with emetine retained approximately 40% of their capacity to contain intracellular H37Rv, however. These studies indicate that protective human recall responses to M. tb are mediated primarily by CD4+ T cells, whereas CD4-CD8- lymphocytes may contribute to innate immunity to M. tb. The ability of NAC to activate M. tb-infected MN is only partly attributable to soluble mediators and may also involve contact-mediated mechanisms.     

CG island methylation changes near the GSTP1 gene in prostatic carcinoma cells detected using the polymerase chain reaction: a new prostate cancer biomarker

Cancer-associated somatic genome alterations offer great promise as cancer biomarkers. Here we describe a new biomarker for human prostate cancer: extensive methylation of deoxycytidine nucleotides distributed throughout a 5' "CG island" region of the pi-class glutathione S-transferase gene (GSTP1). Using the PCR to amplify a GSTP1 promoter sequence fragment containing 12 recognition sites for HpaII and MspI, 52 of 57 (91%) prostatic carcinoma DNA specimens demonstrated extensive somatic increases in deoxycytidine methylation, detected as amplification of target GSTP1 promoter sequences following HpaII digestion, but not following MspI treatment. Using nested primer sets, a sensitive PCR assay for extensive GSTP1 CG island methylation changes was developed that was capable of detecting 200 pg of prostate cancer cell DNA among 1 microgram of normal leukocyte DNA. This GSTP1 CG island DNA methylation assay, which targets a somatic genome change present in most prostate cancer cells but not in normal cells, may serve as a new molecular diagnosis and staging tool to aid in prostate cancer detection and treatment.