Colocalization pattern of cocaine- and amphetamine-regulated transcript peptide and parvalbumin immunoreactivity in the hippocampus proper of the chinchilla.

This study set out to investigate, for the first time, the distribution and colocalization pattern of cocaine-and amphetamine-regulated transcript (CART) and one of the calcium binding- -proteins: parvalbumin (PV) in the chinchilla's hippocampus proper (HP). HP, consisting of Ammon's horn (CA) and the dentate gyrus (DG), is an important component of the limbic system, involved in learning and memory processes. CA showed a higher immunoreactivity of CART (-IR) compared to DG. CART-IR neurons were mainly observed in the molecular layer of DG and in the pyramidal layer of CA. CART-IR fibers were present in the granular layer; in the hilus numerous mossy fibers were detected, while in the molecular layer CART-IR fibers were not found. In all CA fields (CA1-CA3), CART-IR fibers were only present in the lacuno- sum-molecular layer. Immunofluorescence with double- labeling showed that only CART-IR cells stained positive for PV, whereas in CART-IR fibers there was no PV-positive reaction. Our research supplements missing knowledge about the distribution and colocalization pattern of CART with PV in the chinchilla's hippocampus, and also provides a better understanding of the similarities and differences among individuals of the same species and also with other mammals.

Functional characterisation and cell specificity of BvSUT1, the transporter that loads sucrose into the phloem of sugar beet (Beta vulgaris L.) source leaves.

Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the ß-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks.

Safety, efficacy and repeatability of a novel house dust mite allergen challenge technique in the Fraunhofer allergen challenge chamber.

BACKGROUND: Efficacy testing of immunotherapy in field studies is often hampered by variation of airborne allergens. Standardized allergen exposure in challenge chamber settings might be an alternative. Therefore, we developed a universal technique to create an atmosphere loaded with allergen particles of adjustable size from aqueous solutions of licensed allergen extracts. OBJECTIVE: The aim of this study was to apply this technique and test the safety and efficacy of challenges with house dust mite (HDM) allergen in the Fraunhofer allergen challenge chamber. METHODS: Aerosol particles carrying HDM allergen were produced by spray-drying of an aqueous solution containing HDM allergen and lactose. In a monocenter, placebo-controlled, single-blind, dose-escalation pilot study, 18 subjects with perennial allergic rhinitis and sensitization to HDM were exposed to HDM allergen for 4 h at either 250, 500, 1000 SQE/m3 or lactose alone (0 SQE/m3 ) 7 days apart. The dose of 500 SQE/m³ was repeated to investigate reproducibility. Total nasal symptom score (TNSS) was the primary endpoint. RESULTS: Exposure to HDM increased TNSS (mean ± SD) to 3.4 ± 1.8, 3.3 ± 2.1, and 3.6 ± 2.0 at 250, 500 and 1000 SQE/m3 , respectively, while lactose alone did not change TNSS (0.7 ± 0.6). The results were reproducible at 500 SQE/m3 . Pulmonary function and adverse event frequency did not change with escalation of allergen dose. CONCLUSION: This HDM allergen particle generation is safe, specific and reproducible and can therefore be used for efficacy testing of immunotherapy and for basic clinical research.

Altered serotonin and dopamine transporter availabilities in brain of depressed patients upon treatment with escitalopram: A [123 I]ß-CIT SPECT study.

Altered SERT and DAT availabilities during treatment with escitalopram were investigated with [(123)I]2ß-carbomethoxy-3ß-(4-iodophenyl)tropane (ß-CIT) SPECT in a series of patients fulfilling the criteria for unipolar major depressive disorder (MDD). 27 patients (10m, 42±16y) with diagnosis of MDD were recruited for the study. All patients underwent neuropsychiatric testing for assessment of Hamilton Depression (HAM-D) and Beck Depression Inventory (BDI) scores. At baseline, [(123)I]ß-CIT SPECT recordings were acquired 4h (SERT-weighted) and 20-24h p.i (DAT-weighted). Follow-up scans and neuropsychiatric testing were performed after six weeks of stable escitalopram medication. Voxel-wise parametric maps of specific/ non-specific ratios-1 (~BPND) were calculated. At baseline, DAT-weighted BPND was 5.06±0.81 in striatum and SERT-weighted BPND was 0.94±0.18 in thalamus. There were significant negative correlations with age for DAT in striatum (R=-0.60; p<0.01) and SERT in thalamus (R=-0.45; p<0.05). Under SSRI treatment there was an apparent 42% occupancy of SERT in thalamus (p<0.0001), whereas DAT availability increased significantly by 20% in striatum (p<0.001); higher apparent SERT occupancy in thalamus was associated with lesser DAT increase in striatum (R=-0.62; p<0.005). The low apparent SERT occupancy may be confounded by alterations in SERT expression during treatment. Thus, [(123)I]ß-CIT SPECT revealed age-dependent declines in DAT and SERT availabilities in un-medicated MDD patients, comparable to that seen previously in healthy controls. At follow-up, the SSRI-evoked increase in DAT was less pronounced in the older patients, even though apparent SERT occupancy and clinical improvement were not age-dependent. Present findings may have implications for escitalopram dosage and side effect profile in younger MDD patients.

Human papillomavirus (HPV) status of non-tobacco related squamous cell carcinomas of the lateral tongue.

OBJECTIVES: The human papillomavirus (HPV) is an important cause of some head and neck squamous cell carcinomas (HNSCCs), but its role in cancer of the lateral tongue is debatable. Suspicion of HPV causation is heightened when these lateral tongue carcinomas arise in patients that are young and/or have never smoked. The purpose of this study was to determine the incidence of transcriptionally active high risk HPV in these tumors, with a particular emphasis on non-smoking patients who are often presumed to have HPV-positive tumors. METHODS: We evaluated 78 HNSCCs of the lateral tongue for the presence of HPV using p16 immunohistochemistry and an RNA in situ hybridization assay targeting HPV E6/E7 mRNA. The study population was enriched for patients without traditional risk factors such as smoking and drinking. RESULTS: P16 overexpression was detected in 9 (11.5%) of 78 cases, but HPV E6/E7 mRNA transcripts were detected in only 1 (1.3%) case (positive predictive value of p16 staining for the presence of transcriptionally active HPV=0.12). HPV mRNA transcripts were not detected in any patient under 40 (n=11), or in patients who had never smoked (n=44), had quit smoking (n=15), and/or were only light consumers of alcohol (n=57). CONCLUSIONS: HPV is not detected in the vast majority of lateral tongue carcinomas. In light of the observation that HPV plays little if any role in the development of these cancers, routine HPV testing is unwarranted , even for patients without traditional risk factors. P16 staining is not a reliable marker for the presence of transcriptionally active HPV at this particular anatomic site.

Therapeutic safety of high myocardial expression levels of the molecular inotrope S100A1 in a preclinical heart failure model.

Low levels of the molecular inotrope S100A1 are sufficient to rescue post-ischemic heart failure (HF). As a prerequisite to clinical application and to determine the safety of myocardial S100A1 DNA-based therapy, we investigated the effects of high myocardial S100A1 expression levels on the cardiac contractile function and occurrence of arrhythmia in a preclinical large animal HF model. At 2 weeks after myocardial infarction domestic pigs presented significant left ventricular (LV) contractile dysfunction. Retrograde application of AAV6-S100A1 (1.5 × 10(13) tvp) via the anterior cardiac vein (ACV) resulted in high-level myocardial S100A1 protein peak expression of up to 95-fold above control. At 14 weeks, pigs with high-level myocardial S100A1 protein overexpression did not show abnormalities in the electrocardiogram. Electrophysiological right ventricular stimulation ruled out an increased susceptibility to monomorphic ventricular arrhythmia. High-level S100A1 protein overexpression in the LV myocardium resulted in a significant increase in LV ejection fraction (LVEF), albeit to a lesser extent than previously reported with low S100A1 protein overexpression. Cardiac remodeling was, however, equally reversed. High myocardial S100A1 protein overexpression neither increases the occurrence of cardiac arrhythmia nor causes detrimental effects on myocardial contractile function in vivo. In contrast, this study demonstrates a broad therapeutic range of S100A1 gene therapy in post-ischemic HF using a preclinical large animal model.

A novel continuous powder aerosolizer (CPA) for inhalative administration of highly concentrated recombinant surfactant protein-C (rSP-C) surfactant to preterm neonates.

BACKGROUND: In pulmonary medicine, aerosolization of substances for continuous inhalation is confined to different classes of nebulizers with their inherent limitations. Among the unmet medical needs is the lack of an aerosolized surfactant preparation for inhalation by preterm neonates, to avoid the risks associated with endotracheal intubation and surfactant bolus instillation. In the present report, we describe a high-concentration continuous powder aerosolization system developed for delivery of inhalable surfactant to preterm neonates. METHODS: The developed device uses a technique that allows efficient aerosolization of dry surfactant powder, generating a surfactant aerosol of high concentration. In a subsequent humidification step, the heated aerosol particles are covered with a surface layer of water. The wet surfactant aerosol is then delivered to the patient interface (e.g., nasal prongs) through a tube. RESULTS: The performance characteristics of the system are given as mass concentration, dose rate, and size distribution of the generated aerosol. Continuous aerosol flows of about 0.84 L/min can be generated from dry recombinant surfactant protein-C surfactant, with concentrations of up to 12 g/m(3) and median particle sizes of the humidified particles in the range of 3 to 3.5 µm at the patient interface. The system has been successfully used in preclinical studies. CONCLUSION: The device with its continuous high-concentration delivery is promising for noninvasive delivery of surfactant aerosol to neonates and has the potential for becoming a versatile disperser platform closing the gap between continuously operating nebulizers and discontinuously operating dry powder inhaler devices.

A cluster of transfusion-associated babesiosis in extremely low birthweight premature infants.

Babesiosis is a parasitic infection of the red blood cells most often acquired by a tick bite. As it has also been known to be transmitted vertically and via transfusion, neonates have occasionally been reported with the infection. Here, we report a series of three premature neonates who acquired babesiosis via blood transfusion from a single donor, one of whom had difficulty clearing the infection and required multiple antimicrobials.

Dopaminergic neurotransmission in patients with schizophrenia in relation to positive and negative symptoms.

Schizophrenia is a complex dynamic disorder comprising a wide range of neurobiological alterations including dopaminergic dysfunction. The aim of the study was to investigate dynamic changes of dopaminergic neurotransmission in patients with schizophrenia (n=8, mean age 25.4 ± 5.8 years) in early stages of the disorder, compared to healthy control subjects (n=7, mean age 23.6 ± 2.7 years). A dynamic IBZM SPECT protocol was used to assess the endogenous dopamine release following an amphetamine challenge. Subjects received a bolus activity of 175 MBq followed by a continuous infusion of 45 MBq/h [123I]IBZM. SPECT scans were performed 2 h after bolus injection, and 1 h following the amphetamine challenge (0.3 mg/kg). Striatal IBZM binding to dopamine D2 receptors was assessed with a volume-of-interest (VOI) technique. The change in IBZM binding between pre- and post-challenge scans was used as a measure of endogenous dopamine release triggered by amphetamine. At baseline, patients showed higher mean striatal IBZM binding compared to controls (0.77 ± 0.09 vs. 0.68 ± 0.07, p=0.07). There was a statistically significant difference in IBZM binding between patients, with a predominance of negative vs. positive symptoms (0.84 ± 0.08 vs. 0.71 ± 0.04, p<0.05). Upon amphetamine challenge, mean IBZM binding decreased by about 4.9 ± 7.6% in controls (n=7) compared to a mean of 12.4 ± 5.8% in subjects with schizophrenia (p<0.05). In patients, paranoid symptoms showed a significant negative correlation with IBZM baseline binding, whereas there was a trend towards positive correlation with the decrease of IBZM binding under challenge. Negative symptoms showed positive associations with baseline IBZM binding. The data are in line with previous reports and contribute to the notion of a dynamic instability of the dopaminergic system in patients with schizophrenia, taking into account the psychopathology with respect to positive or negative symptoms.

Detection of Merkel cell virus and correlation with histologic presence of Merkel cell carcinoma in sentinel lymph nodes.

BACKGROUND: Adjuvant treatment can dramatically improve the survival of patients with metastatic Merkel cell carcinoma (MCC), making early, accurate detection of nodal disease critical. The purpose of this study was to correlate Merkel cell virus (MCV) detection with histopathologic disease in sentinel lymph nodes (SLNs) of MCC. METHODS: Merkel cell carcinoma cases with SLN (n=25) were compared with negative controls (n=27). Viral load was obtained by quantitative polymerase chain reaction (PCR) for regions VP1 and LT3 of MCV. Histopathologic disease and viral load were correlated. RESULTS: Merkel cell virus was detected in 16 out of 17 (94%) of primary MCC (mean viral load (MVL)=1.44 copies per genome). Viral load in the negative controls was <0.01 copies per genome. Merkel cell carcinoma was present in 5 out of 25 (20%) SLN by histopathology, and MCV was detected in 11 out of 25 (44%) MCC SLN (MVL=1.68 copies per genome). In all, 15 out of 25 (60%) SLN showed correlation between histologic and MCV results. In all, 2 out of 25 (8%) samples were histopathologically positive and PCR negative. Of note, 8 out of 25 (32%) samples had detectable MCV without microscopic disease. CONCLUSION: Patients with positive SLN for MCV even if negative by histopathology were identified. The application of molecular techniques to detect subhistologic disease in SLN of MCC patients may identify a subset of patients who would benefit from adjuvant nodal treatment.

Myocardial ß(2) -adrenoceptor gene delivery promotes coordinated cardiac adaptive remodelling and angiogenesis in heart failure.

BACKGROUND AND PURPOSE: We investigated whether ß(2) -adrenoceptor overexpression could promote angiogenesis and improve blood perfusion and left ventricular (LV) remodeling of the failing heart. EXPERIMENTAL APPROACH: We explored the angiogenic effects of ß(2) -adrenoceptor overexpression in a rat model of post-myocardial infarction (MI) heart failure (HF). Cardiac adenoviral-mediated ß(2) -adrenoceptor overexpression was obtained via direct intramyocardial injection 4-weeks post-MI. Adenovirus(Ad)-GFP and saline injected rats served as controls. Furthermore, we extended our observation to ß(2) -adrenoceptor -/- mice undergoing MI. KEY RESULTS: Transgenes were robustly expressed in the LV at 2 weeks post-gene therapy, whereas their expression was minimal at 4-weeks post-gene delivery. In HF rats, cardiac ß(2) -adrenoceptor overexpression resulted in enhanced basal and isoprenaline-stimulated cardiac contractility at 2-weeks post-gene delivery. At 4 weeks post-gene transfer, Ad-ß(2) -adrenoceptor HF rats showed improved LV remodeling and cardiac function. Importantly, ß(2) -adrenoceptor overexpression was associated with a markedly increased capillary and arteriolar length density and enhanced in vivo myocardial blood flow and coronary reserve. At the molecular level, cardiac ß(2) -adrenoceptor gene transfer induced the activation of the VEGF/PKB/eNOS pro-angiogenic pathway. In ß(2) -adrenoceptor-/- mice, we found a ~25% reduction in cardiac capillary density compared with ß(2) -adrenoceptor+/+ mice. The lack of ß(2) -adrenoceptors was associated with a higher mortality rate at 30 days and LV dilatation, and a worse global cardiac contractility compared with controls. CONCLUSIONS AND IMPLICATION: ß(2) -Adrenoceptors play an important role in the regulation of the angiogenic response in HF. The activation of VEGF/PKB/eNOS pathway seems to be strongly involved in this mechanism.

Targeting GRK2 by gene therapy for heart failure: benefits above ß-blockade.

Heart failure (HF) is a common pathological end point for several cardiac diseases. Despite reasonable achievements in pharmacological, electrophysiological and surgical treatments, prognosis for chronic HF remains poor. Modern therapies are generally symptom oriented and do not currently address specific intracellular molecular signaling abnormalities. Therefore, new and innovative therapeutic approaches are warranted and, ideally, these could at least complement established therapeutic options if not replace them. Gene therapy has potential to serve in this regard in HF as vectors can be directed toward diseased myocytes and directly target intracellular signaling abnormalities. Within this review, we will dissect the adrenergic system contributing to HF development and progression with special emphasis on G-protein-coupled receptor kinase 2 (GRK2). The levels and activity of GRK2 are increased in HF and we and others have demonstrated that this kinase is a major molecular culprit in HF. We will cover the evidence supporting gene therapy directed against myocardial as well as adrenal GRK2 to improve the function and structure of the failing heart and how these strategies may offer complementary and synergistic effects with the existing HF mainstay therapy of ß-adrenergic receptor antagonism.

[Structural and functional neuronal connectivity in Alzheimer's disease: a combined DTI and fMRI study]. / Strukturelle und funktionelle neuronale Konnektivität bei der Alzheimer-Krankheit : Eine kombinierte DTI- und fMRT-Studie.

BACKGROUND: Cognitive performance depends on intact cortical connectivity. Important for memory processing in the human brain is the connection between posterior cingulate cortex and hippocampus, directly as well as indirectly via the parahippocampal gyrus. These brain areas are involved early in Alzheimer's disease (AD). At the same time, they belong to the default mode network (DMN), a functional network showing high functional connectivity under resting state conditions. In AD, this connectivity in specifically compromised, offering the possibility to investigate the structural basis of functional brain connectivity. METHODS: We studied 18 patients with mild to moderate AD, 16 patients with mild cognitive impairment (MCI) and 20 healthy control subjects using diffusion tensor imaging (DTI) and resting state fMRI at 3.0 Tesla. We determined the effect of structural integrity in the posterior cingulate as assessed by DTI on the functional connectivity between posterior cingulate, hippocampus and parahippocampus during resting state in these three groups. RESULTS: Structural integrity was reduced in posterior cingulate fibre tracts in patients with AD in the left hemisphere; however, this effect was partly accounted for by age differences. All three groups showed high functional connectivity between posterior cingulate cortex and hippocampus, via both the direct and the indirect pathways. Determination of effective connectivity yielded a negative fractional anisotropy (FA)-moderated correlation on the direct pathway in AD and MCI for both hemispheres, and in healthy controls for the right hemisphere. The indirect pathway showed a negative FA-moderated correlation in AD for the right hemisphere and in MCI for both hemispheres. Healthy controls showed a positive correlation on the indirect pathway for the left hemisphere. CONCLUSION: Our data suggest that under healthy conditions, effective connectivity in the DMN between posterior cingulate cortex and hippocampus is mainly maintained by the indirect pathway via the parahippocampal gyrus. Patients with AD and patients with MCI show changes in this connectivity with a partial allocation to the direct pathway, most likely reflecting early parahippocampal lesions. The combination of DTI and fMRI broadens our understanding of human brain connectivity and its pathological changes with AD.

Gene therapy targets in heart failure: the path to translation.

Heart failure (HF) is the common end point of cardiac diseases. Despite the optimization of therapeutic strategies and the consequent overall reduction in HF-related mortality, the key underlying intracellular signal transduction abnormalities have not been addressed directly. In this regard, the gaps in modern HF therapy include derangement of ß-adrenergic receptor (ß-AR) signaling, Ca(2+) disbalances, cardiac myocyte death, diastolic dysfunction, and monogenetic cardiomyopathies. In this review we discuss the potential of gene therapy to fill these gaps and rectify abnormalities in intracellular signaling. We also examine current vector technology and currently available vector-delivery strategies, and we delineate promising gene therapy structures. Finally, we analyze potential limitations related to the transfer of successful preclinical gene therapy approaches to HF treatment in the clinic, as well as impending strategies aimed at overcoming these limitations.

NID2 and HOXA9 promoter hypermethylation as biomarkers for prevention and early detection in oral cavity squamous cell carcinoma tissues and saliva.

Differentially methylated oral squamous cell carcinoma (OSCC) biomarkers, identified in vitro and validated in well-characterized surgical specimens, have shown poor clinical correlation in cohorts with different risk profiles. To overcome this lack of relevance, we used the HumanMethylation27 BeadChip, publicly available methylation and expression array data, and quantitative methylation specific PCR to uncover differential methylation in OSCC clinical samples with heterogeneous risk profiles. A two stage design consisting of discovery and prevalence screens was used to identify differential promoter methylation and deregulated pathways in patients diagnosed with OSCC and head and neck squamous cell carcinoma. Promoter methylation of KIF1A (κ = 0.64), HOXA9 (κ = 0.60), NID2 (κ = 0.60), and EDNRB (κ = 0.60) had a moderate to substantial agreement with clinical diagnosis in the discovery screen. HOXA9 had 68% sensitivity, 100% specificity, and a 0.81 Area Under the Curve (AUC). NID2 had 71% sensitivity, 100% specificity, and a 0.79 AUC. In the prevalence screen, HOXA9 (κ = 0.82) and NID2 (κ = 0.80) had an almost perfect agreement with histologic diagnosis. HOXA9 had 85% sensitivity, 97% specificity, and a 0.95 AUC. NID2 had 87% sensitivity, 95% specificity, and a 0.91 AUC. A HOXA9 and NID2 gene panel had 94% sensitivity, 97% specificity, and a 0.97 AUC. In saliva, from OSCC cases and controls, HOXA9 had 75% sensitivity, 53% specificity, and a 0.75 AUC. NID2 had 87% sensitivity, 21% specificity, and a 0.73 AUC. This phase I Biomarker Development Trial identified a panel of differentially methylated genes in normal and OSCC clinical samples from patients with heterogeneous risk profiles. This panel may be useful for early detection and cancer prevention studies.

Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo.

We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy.

Physicochemical characterisation of different welding aerosols.

Physicochemical properties important in exposure characterisation of four different welding aerosols were investigated. Particle number size distributions were determined by scanning mobility particle sizer (SMPS), mass size distributions by separation and weighing the individual size fractions of an 11-stage cascade impactor. The size distribution of the primary particles of agglomerates, chemical composition and morphology of the particles were examined by TEM. There were significant differences in the particle number size distributions of the different welding aerosols according to the SMPS determinations. The particle mass size distributions determined gravimetrically were, however, not really different. The dominant range with respect to mass was between 0.1 and 1 µm, regardless of the welding technique. Most of the primary particles in all different welding aerosols had diameters between 5 and 40 nm. All types of primary particles had a tendency to form chainlike agglomerates. A clear size dependence of the particle chemical composition was encountered in the case of manual metal arc welding aerosol. Small particles with diameters below 50 nm were mostly metal oxides in contrast to larger particles which also contained more volatile elements (e.g. potassium, fluorine, sodium, sulphur).

Isolated and interactive impact of common CYP2C19 genetic variants on the antiplatelet effect of chronic clopidogrel therapy.

BACKGROUND: With the cytochrome P450 CYP2C19*2 (*2) allelic variant resulting in complete loss of enzyme function and the CYP2C19*17 (*17) variant being linked to increased transcriptional activity with extensive metabolism of CYP2C19 substrates, two common variants of the CYP2C19 gene have been explored recently. Currently, the isolated and interactive impacts of both variants on the antiplatelet effects of chronic clopidogrel therapy are unknown. OBJECTIVES: The aim of this study was to assess the isolated and interactive impacts of *2 and *17 on clopidogrel responsiveness in patients under clopidogrel maintenance treatment. METHODS: Patients (n=986) eligible for this study were under therapy with coronary stent-related chronic treatment with aspirin and clopidogrel. The ADP-induced platelet aggregation was measured on a Multiplate analyzer (in AU*min), and genotypes were determined with a TaqMan assay. RESULTS: Platelet aggregation values were significantly higher in carriers of at least one *2 allele than in homozygous wild-type allele carriers (P<0.001). For *17, platelet aggregation values were significantly lower in carriers of at least one *17 allele than in homozygous wild-type patients (P=0.01). A gene-dose effect was observed for both variants, with a pronounced effect of the mutant allele (*2 or *17) in homozygous patients being seen. For the interactive effect of both variants on platelet aggregation values, a gradual increase in platelet aggregation values was observed from (+)*17/(-)*2 patients, who exhibited the lowest values (median of 207 AU*min) to (-)*17/(-)*2, (+)*17/(+)*2 and (-)*17/(+)*2 patients, who exhibited the highest values (median of 309 AU*min) (P<0.001). CONCLUSIONS: *2 and *17 allele carriage are independent predictors for the antiplatelet effect of chronic clopidogrel therapy.

A toxicological evaluation of inhaled solid lipid nanoparticles used as a potential drug delivery system for the lung.

Inhalation is a non-invasive approach for both local and systemic drug delivery. This study aimed to define the therapeutic window for solid lipid nanoparticles (SLNs) as a drug delivery system by inhalation from a toxicological point of view. To estimate the toxic dose of SLNs in vitro, A549 cells and murine precision-cut lung slices (PCLS) were exposed to increasing concentrations of SLNs. The cytotoxic effect of SLNs on A549 cells was evaluated by MTT and NRU assays. Viability of lung tissue was determined with WST assay and by life/dead staining using calcein AM/EthD-1 for confocal microscopy (CLSM) followed by quantitative analysis with IMARIS. Inflammation was assessed by measuring chemokine KC and TNF-alpha levels. The in vivo effects were determined in a 16-day repeated-dose inhalation toxicity study using female BALB/c mice, which were daily exposed to different concentrations of SLN30 aerosols (1-200 microg deposit dose). Local inflammatory effects in the respiratory tract were evaluated by determination of total protein content, LDH, chemokine KC, IL-6, and differential cell counts, performed on days 4, 8, 12, and 16 in bronchoalveolar lavage fluid. Additionally, a histopathological evaluation of toxicologically relevant organs was accomplished. The in vitro and ex vivo dose finding experiments showed toxic effects beginning at concentrations of about 500 microg/ml. Therefore, we used 1-200 microg deposit doses/animal for the in vivo experiments. Even after 16 days of challenge with a 200-microg deposit dose, SLNs induced no significant signs of inflammation. We observed no consistent increase in LDH release, protein levels, or other signs of inflammation such as chemokine KC, IL-6, or neutrophilia. In contrast, the particle control (carbon black) caused inflammatory and cytotoxic effects at corresponding concentrations. These results confirm that repeated inhalation exposure to SLN30 at concentrations lower than a 200-microg deposit dose is safe in a murine inhalation model.