Measurement of lumefantrine and its metabolite in plasma by high performance liquid chromatography with ultraviolet detection

Artemether-lumefantrine (ARM-LUM) has in recent years become the first-line treatment for uncomplicated malaria in many Sub-Saharan African countries. Vigorous monitoring of the therapeutic efficacy of this treatment is needed. This requires high-quality studies following standard protocols; ideally, such studies should incorporate measurement of drug levels in the study patients to exclude the possibility that insufficient drug levels explain an observed treatment failure. Several methods for measuring lumefantrine (LUM) in plasma by HPLC are available; however, several of these methods have some limitations in terms of high costs and limited feasibility arising from large required sample volumes and demanding sample preparation. Therefore, we set out to develop a simpler reversed phase high performance liquid chromatography (RP-HPLC) method based on UV detection for simultaneous measurement of LUM and its major metabolite the desbutyl LUM (DL) in plasma. Halofantrine was used as an internal standard. Liquid–liquid extraction of samples was carried out using hexane–ethyl acetate (70:30, v/v). Chromatographic separation was carried out on a Synergi Polar-RP column (250 mm × 300 mm, particle size 4 μm). The mobile phase consisted of acetonitrile–0.1 M ammonium acetate buffer adjusted to pH 4.9 (85:15%, v/v). Absorbance of the compounds was monitored at 335 nm using a reference wavelength of 360 nm. Absolute extraction recovery for LUM and DL were 88% and 90%, respectively. Inter- and intraday coefficients of variation for LUM and DL were ≤10%. The lower limits of quantification for LUM and DL were 12.5 and 6.5 ng/ml, respectively. After validation, the methodology was transferred to a local laboratory in Tanga Tanzania and samples from a small subset of malaria patients were analysed for LUM. The method appears to be applicable in settings with limited facilities.     

TCR down-regulation boosts T-cell-mediated cytotoxicity and protection against poxvirus infections

Cytotoxic T (Tc) cells play a key role in the defense against virus infections. Tc cells recognize infected cells via the T-cell receptor (TCR) and subsequently kill the target cells by one or more cytotoxic mechanisms. Induction of the cytotoxic mechanisms is finely tuned by the activation signals from the TCR. To determine whether TCR down-regulation affects the cytotoxicity of Tc cells, we studied TCR down-regulation-deficient CD3γLLAA mice. We found that Tc cells from CD3γLLAA mice have reduced cytotoxicity due to a specific deficiency in exocytosis of lytic granules. To determine whether this defect was reflected in an increased susceptibility to virus infections, we studied the course of ectromelia virus (ECTV) infection. We found that the susceptibility to ECTV infection was significantly increased in CD3γLLAA mice with a mortality rate almost as high as in granzyme B knock-out mice. Finally, we found that TCR signaling in CD3γLLAA Tc cells caused highly increased tyrosine phosphorylation and activation of the c-Cbl ubiquitin ligase, and that the impaired exocytosis of lytic granules could be rescued by the knockdown of c-Cbl. Thus, our work demonstrates that TCR down-regulation critically increases Tc cell cytotoxicity and protection against poxvirus infection.     

Protodynamic therapy for bladder cancer: in vitro results of a novel treatment concept

OBJECTIVE

To present a novel treatment approach for urinary bladder cancer, protodynamic therapy, which comprises inhibition of cancer cell proliferation by intracellular acidification; cis‐urocanic acid (cis‐UCA) was investigated as a protodynamic drug in bladder cancer cell cultures and compared with conventional chemotherapeutic agents.

MATERIALS AND METHODS

The moderately differentiated cell line 5637 and the poorly differentiated T24 cell line were exposed to cis‐UCA for 0.25–2 h, and to epirubicin, doxorubicin, cisplatin and paclitaxel for 2 h, to simulate drug exposure on intravesical instillation. The combination of cis‐UCA and chemotherapeutic agents was also studied. Cell viability was measured with a colorimetric assay.

RESULTS

cis‐UCA inhibited proliferation and suppressed the survival of cells at an extracellular pH ≤ pK_a2 of 6.65 but to a lesser degree at pH > pK_a2, as suggested by the protodynamic theory. cis‐UCA caused dose‐dependent, irreversible termination of cell proliferation. The number of viable surviving BC cells decreased by >85% with 2%cis‐UCA (P < 0.001). Viable cells disappeared completely with 4% and 6%cis‐UCA after a 2‐h treatment, and by 90% with 6%cis‐UCA within a 15‐min exposure. These effects were associated with distinct morphological changes. The other drugs tested had a clearly lower effect on cell survival. Interestingly, when combined, cis‐UCA markedly enhanced the cytotoxic effect of epirubicin.

CONCLUSION

cis‐UCA is a potent antiproliferative agent in bladder cancer cell cultures. As our previous non‐clinical studies showed that cis‐UCA is locally and systemically well tolerated, protodynamic therapy with cis‐UCA is a promising intravesical treatment for bladder cancer.     

Among the branched-chain amino acids, only valine metabolism is up-regulated in astrocytes during glutamate exposure

Glutamate homeostasis during glutamatergic neurotransmission is predominantly maintained via functioning of the glutamate-glutamine cycle. However, the glutamate-glutamine cycle explains only the fate of the carbon atoms but not that of the accompanying transfer of nitrogen from neurons to astrocytes. In this respect, a putative branched-chain amino acid (BCAA) shuttle has been suggested for transfer of amino nitrogen. Metabolism of BCAAs was investigated in cultured cerebellar astrocytes in a superfusion paradigm employing (15)N-labeled leucine, isoleucine, or valine. Some cultures were exposed to pulses of glutamate (50 microM; 10 sec every 2 min; 75 min in total) to mimic conditions during glutamatergic synaptic activity. (15)N labeling of glutamate, aspartate, glutamine, alanine, and the three BCAAs was determined by using mass spectrometry. Incorporation of (15)N into intracellular glutamate from [(15)N]leucine, [(15)N]isoleucine, or [(15)N]valine amounted to about 40-50% and differed only slightly among the individual BCAAs. Interestingly, label (%) in glutamate from [(15)N]valine was not decreased upon exposure to exogenous glutamate, which was in contrast to a marked decrease in labeling (%) from [(15)N]leucine or [(15)N]isoleucine. This suggests an up-regulation of transamination involving only valine during repetitive exposure to glutamate. It is suggested that valine in particular might have an important function as an amino acid translocated between neuronal and astrocytic compartments, a function that might be up-regulated during synaptic activity.     

Development of atopic dermatitis-like skin disease from the chronic loss of epidermal caspase-8

Atopic dermatitis is an inflammatory skin disease that affects approximately 20% of children worldwide. Left untreated, the barrier function of the skin is compromised, increasing susceptibility to dehydration and infection. Despite its prevalence, its multifactorial nature has complicated the unraveling of its etiology. We found that chronic loss of epidermal caspase-8 recapitulates many aspects of atopic dermatitis, including a spongiotic phenotype whereby intercellular adhesion between epidermal keratinocytes is disrupted, adversely affecting tissue architecture and function. Although spongiosis is generally thought to be secondary to edema, we found that suppression of matrix metalloproteinase-2 activity is sufficient to abrogate this defect. p38 MAPK induces matrix metalloproteinase-2 expression to cleave E-cadherin, which mediates keratinocyte cohesion in the epidermis. Thus, the conditional loss of caspase-8, which we previously found to mimic a wound response, can be used to gain insights into how these same wound-healing processes are commandeered in inflammatory skin diseases.     

An update on the pathophysiology and genetics of restless legs syndrome

This update on restless legs syndrome (RLS) focuses on its pathophysiology, genetics, health impact, and treatment. Although symptoms are exquisitely responsive to dopaminergics, clear delineation of dopaminergic pathology has not emerged. Rather, heuristic models of alterations in spinal sensorimotor circuits and central nervous system iron deficiency are gaining more attention. Genome-wide association studies have recently identified polymorphisms in three genes with no obvious relationship to dopamine that account for 70% of the population risk for RLS. A single variant in the BTBD9 gene on chromosome 6 contributes to 50% of the population risk. Although the functions of BTBD9 remain uncertain, its biological plausibility is evidenced by its dose-dependent relationship to periodic limb movements of sleep, decrements in iron stores, and ethnic differences in RLS prevalence. RLS is also implicated in cardiovascular morbidity. Despite these advances in our knowledge, there remain unanswered questions about the pathophysiology of RLS as well as its genetics, health-related significance, and treatment.     

Changing capacity of electrocardiographic ventricular repolarization in post-myocardial infarction patients with and without nonfatal cardiac arrest

Prolonged and labile ventricular repolarization and decreased heart rate variability may be associated with susceptibility to ventricular fibrillation (VF) after myocardial infarction (MI). The response of ventricular repolarization related to abrupt heart rate changes may also be associated with arrhythmia vulnerability. We investigated whether diurnal maximal values or changing capacities of QT and T-wave peak to T-wave end (TPE) intervals are different in patients after MI with and without a history of VF. With an automated computerized program, Holter recordings from 29 patients after MI resuscitated from VF not associated with new MI (VF group) and 27 patients after MI without clinical ventricular arrhythmias (control group) were analyzed. Maximal QT and maximal TPE intervals were shorter in the VF group than in the control group. Patients with VF exhibited smaller capacity to change QT and TPE intervals, with differences between study groups being greatest at heart rates from 60 to 75 beats/min (p = 0.002 and 0.01, respectively). Capacity to change QT and TPE intervals correlated with vagally mediated measurements of heart rate variability (r from 0.35 to 0.46, p from 0.01 to <0.001, respectively). In conclusion, long maximal QT interval may not be the key factor exposing patients after MI to VF. Impaired capacity to change QT and TPE intervals seems to be associated with risk of VF after MI.     

Signal-averaged P wave duration and the long-term risk of permanent atrial fibrillation

OBJECTIVE: To assess the long-term risk of developing permanent AF in relation to the signal-averaged P wave duration (SAPWD) and clinical and echocardiographic characteristics. DESIGN: In an observational study design we studied 131 patients with earlier ECG-documented AF and successfully restored sinus rhythm attending a long-term, follow-up visit at hospital or at home. Established permanent AF was examined in relation to primary clinical, echocardiographic, and electrophysiological parameters. RESULTS: Only prolonged SAPWD (p=0.006) was associated with an increased risk of development of permanent AF. The risk of permanent AF after 3 years follow-up was 0.72 with an SAPWD equal to 180 ms versus 0.39 with a normal SAPWD (130 ms). We found no prognostic effect of age, gender, dilated left atrium, long duration of AF history, or long duration of the most recent episode of AF. Co-existing hypertension reduced the risk of permanent AF; this could be explained by concomitant treatment with angiotensin-converting-enzyme-inhibitors. CONCLUSION: Prolonged SAPWD (a marker of atrial remodelling) appears to be a risk factor for long-term development of permanent AF.     

Parkinson's disease and RLS: the dopaminergic bridge

Dopamine is a neurotransmitter that modulates diverse waking behaviors including movement, motivation, cognition, reward, and feeding. Interest in dopamine's additional contributions to normal and pathologic sleep-wake states has experienced a recent rebirth originating from two clinical disorders: Parkinson's disease and Restless Legs Syndrome. The former, the prototypical disorder of brain dopamine cell loss, is accompanied by marked sleep disruption and impairments in daytime alertness. The latter is exquisitively responsive to pharmacologic agents that act upon dopamine receptors. The potential neurobiological substrates underlying these observations are reviewed here. Converging lines of evidence suggest that mesocorticolimbic dopamine circuits are involved in promoting wakefulness, while a less studied diencephalospinal dopamine system might underly the sensorimotor dysfunction of Restless Legs Syndrome.     

Long-lasting allergic patch test reaction caused by gold

Allergic contact dermatitis caused by gold is rare, and only isolated cases have been reported. Patch Jesting with gold may cause a long-lasing reaction. The purpose of this study is to describe a well-studied case of gold allergy caused by denial gold crowns. A gold-sensitized patient and a non-sensitized control subject were examined using patch tests, immunohistochemistry. electron microscopy and blast transformation reactions. Sodium thiosulfate, auranofin and sodium thiomalate gave positive patch test reactions. Immunohistochemistry and electron microscopy were performed from biopsies taken from allergic patch lest reactions caused by gold sodium thiosulfate 1 day and 17 days after applying the patches, from normal skin and from a 17-day-old allergic patch test reaction caused by ammonium persulfate. Down-regulation had taken place by 17 days in the allergic ammonium persulfate reaction, but not in the 17-day allergic gold lest reaction. The patient reacted to all but one of the gold-induced blast transformation tests, sodium chloroaurate being non-inductive. The non-sensitized control subject did not exhibit any reactions. In conclusion, gold sodium thiosulfate, gold sodium thiomalate and auranofin can be used as patch test substances for gold allergy, though long-lasting allergic patch test reactions may develop. In vitro gold salt induced blast transformation is an alternative test for gold allergy. The slow down-regulation of the allergic patch test reactions needs to be studied further.