Pharmacokinetics of single-dose daptomycin in patients with suspected or confirmed neurological infections.

There are currently few or no published data on the amount of cerebrospinal fluid (CSF) penetration of daptomycin in patients with suspected or documented neurosurgical infections. We conducted a prospective study, assessing the pharmacokinetics and CSF penetration of a single intravenous daptomycin dose administered at 10 mg/kg, based on total body weight (TBW), in six neurosurgical patients with indwelling external CSF shunts with suspected or documented meningitis or ventriculitis. Each patient had four blood and CSF samples drawn simultaneously at specific times after the end of infusion: 30 min, 6 h, 12 h, and 24 h. Pharmacokinetic parameters of daptomycin in serum were calculated using standard noncompartmental methods, and daptomycin was assayed using high-performance liquid chromatography (for serum) or liquid chromatography with mass spectrometry (for CSF). The mean (± standard deviation [SD]) maximum measured daptomycin concentrations were 93.7 ± 17.3 mg/liter in serum at 0.5 h postinfusion and 0.461 ± 0.51 mg/liter in CSF at 6 h postinfusion. The mean (± SD) daptomycin minimum concentrations were 13.8 ± 4.8 mg/liter in serum at 24 h postinfusion and 0.126 ± 0.12 mg/liter in CSF at 0.5 h postinfusion. The mean daptomycin penetration, determined by the area under the concentration-time curve in CSF (AUC(CSF))/(AUC(serum) ratio), was 0.8%. Corrected for protein binding, the overall CSF penetration was 11.5%. Additional pharmacokinetic studies evaluating multiple and/or higher dosages of daptomycin are necessary in human subjects to better characterize the CSF penetration of daptomycin in neurosurgical patients.

Long-term screening for primary mitochondrial DNA variants associated with Leber hereditary optic neuropathy: incidence, penetrance and clinical features.

Leber hereditary optic neuropathy (LHON) is a neurodegenerative disorder characterised by bilateral, painless, subacute, central vision loss caused by pathogenic sequence variants in mitochondrial DNA (mtDNA). Over the course of 20 years, 734 people were systematically screened by our diagnostic laboratory for suspected LHON or for being at risk of LHON, with 98 found to harbour one of the three primary pathogenic mtDNA variants. Detection incidences were: 0.95% for NC_012920.1(MT-ND1):m.3460G>A; 9.4% for (MT-ND4):m.11778G>A; and 2.9% for (MT-ND6):m.14484T>C. The median age for symptomatic males was 27.3 years and for females 29.5 years, with a male to female ratio of 4.4:1 (62 males; 14 females). Most pathogenic variant carriers were propositi with the other individuals belonging to one of 14 pedigrees with noteworthy intra-family variability of clinical severity of the disease.

Ceragenins: cholic acid-based mimics of antimicrobial peptides.

The prevalence of drug-resistant bacteria drives the quest for new antimicrobials, including those that are not expected to readily engender resistance. One option is to mimic Nature's most ubiquitous means of controlling bacterial growth, antimicrobial peptides, which have evolved over eons. In general, bacteria remain susceptible to these peptides. Human antimicrobial peptides play a central role in innate immunity, and deficiencies in these peptides have been tied to increased rates of infection. However, clinical use of antimicrobial peptides is hampered by issues of cost and stability. The development of nonpeptide mimics of antimicrobial peptides may provide the best of both worlds: a means of using the same mechanism chosen by Nature to control bacterial growth without the problems associated with peptide therapeutics. The ceragenins were developed to mimic the cationic, facially amphiphilic structures of most antimicrobial peptides. These compounds reproduce the required morphology using a bile-acid scaffolding and appended amine groups. The resulting compounds are actively bactericidal against both gram-positive and gram-negative organisms, including drug-resistant bacteria. This antimicrobial activity originates from selective association of the ceragenins with negatively charged bacterial membrane components. Association has been studied with synthetic models of bacterial membrane components, with bacterial lipopolysaccharide, with vesicles derived from bacterial phospholipids, and with whole cells. Comparisons of the antimicrobial activities of ceragenins and representative antimicrobial peptides suggest that these classes of compounds share a mechanism of action. Rapid membrane depolarization is caused by both classes as well as blebbing of bacterial membranes. Bacteria express the same genes in response to both classes of compounds. On the basis of the antibacterial activities of ceragenins and preliminary in vivo studies, we expect these compounds to find use in augmenting or replacing antimicrobial peptides in treating human disease.

Association of the MELAS m.3243A>G mutation with myositis and the superiority of urine over muscle, blood and hair for mutation detection.

A patient with a known family history of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) due to the MT-TL1 m.3243A>G mutation presented with mild myalgia and very minor upper limb proximal muscle weakness. Muscle histology revealed low levels of cytochrome oxidase-negative fibres and non-specific myositis. Using the last "hot cycle" polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), the MELAS MT-TL1 m.3243A>G mutation was only detected in urine, and not in hair, blood or skeletal muscle. This report highlights the need to screen various tissues to achieve an accurate mitochondrial genetic diagnosis and suggests the likelihood of myositis arising secondary to the MELAS MT-TL1 m.3243A>G mutation.

Potential synergy activity of the novel ceragenin, CSA-13, against clinical isolates of Pseudomonas aeruginosa, including multidrug-resistant P. aeruginosa.

OBJECTIVES: Previous data from our research had shown that the novel ceragenin, CSA-13, demonstrated concentration-dependent bactericidal activity against glycopeptide-resistant Staphylococcus aureus. However, it is unknown whether CSA-13 demonstrates a similar property against Pseudomonas aeruginosa. We evaluated CSA-13 antipseudomonal activity compared with cefepime, meropenem, piperacillin/tazobactam, tobramycin and ciprofloxacin by susceptibility testing as well as in combination with cefepime, tobramycin and ciprofloxacin. METHODS: Fifty clinical isolates of P. aeruginosa were analysed by reference broth microdilution methods. Four strains with various susceptibilities were evaluated by time-killing curve (TKC) analysis at 0.5x, 1x, 2x and 4x MIC using an initial inoculum of 10(6) cfu/mL. For synergy testing, TKC analysis of CSA-13 alone and in combination with cefepime, tobramycin and ciprofloxacin at 0.5x MIC was performed. RESULTS: CSA-13 MIC50 and MBC50 were 16 and 16 mg/L, respectively. TKC analysis demonstrated concentration-dependent activity, with CSA-13 at 4x MIC achieving earliest kill at 1 h (99.9%, detection limit). Combination TKC analysis demonstrated synergy or additive effect with cefepime and ciprofloxacin, in some cases achieving early synergy. The addition of tobramycin to CSA-13 resulted in no difference in kill for two strains. CONCLUSIONS: CSA-13 showed concentration-dependent activity against clinical isolates of P. aeruginosa, including multidrug-resistant P. aeruginosa. The addition of cefepime or ciprofloxacin to CSA-13 enhanced bacterial kill, achieving early synergy.

Effective detection of corrected dystrophin loci in mdx mouse myogenic precursors.

Targeted corrective gene conversion (TCGC) holds much promise as a future therapy for many hereditary diseases in humans. Mutation correction frequencies varying between 0.0001% and 40% have been reported using chimeraplasty, oligoplasty, triplex-forming oligonucleotides, and small corrective PCR amplicons (CPA). However, PCR technologies used to detect correction events risk either falsely indicating or greatly exaggerating the presence of corrected loci. This is a problem that is considerably exacerbated by attempted improvement of the TCGC system using high corrective nucleic acid (CNA) to nuclear ratios. Small fragment homologous replacement (SFHR)-mediated correction of the exon 23 dystrophin (DMD) gene mutation in the mdx mouse model of DMD has been used in this study to evaluate the effect of increasing CPA amounts. In these experiments, we detected extremely high levels of apparently corrected loci and determined that at higher CNA to nuclear ratios the extent of locus correction was highly exaggerated by residual CNA species in the nucleic acids extracted from the treated cells. This study describes a generic locus-specific detection protocol designed to eradicate residual CNA species and avoid the artifactual or exaggerated detection of gene correction.

Enzymatic characterization of a recombinant isoform hybrid of glutamic acid decarboxylase (rGAD67/65) expressed in yeast.

BACKGROUND AND AIMS: Glutamic acid decarboxylase (GAD, EC 4.1.1.15) catalyses the conversion of glutamate to gamma-aminobutyric acid (GABA). The 65 kDa isoform, GAD65 is a potent autoantigen in type 1 diabetes, whereas GAD67 is not. A hybrid cDNA was created by fusing a human cDNA for amino acids 1-101 of GAD67 to a human cDNA for amino acids 96-585 of GAD65; the recombinant (r) protein was expressed in yeast and was shown to have equivalent immunoreactivity to mammalian brain GAD with diabetes sera. We here report on enzymatic and molecular properties of rGAD67/65. METHODS: Studies were performed on enzymatic activity of rGAD67/65 by production of 3H-GABA from 3H-glutamate, enzyme kinetics, binding to the enzyme cofactor pyridoxal phosphate (PLP), stability according to differences in pH, temperature and duration of storage, and antigenic reactivity with various GAD-specific antisera. RESULTS: The properties of rGAD67/65 were compared with published data for mammalian brain GAD (brackets). These included a specific enzyme activity of 22.7 (16.7) nKat, optimal pH for enzymatic activity 7.4 (6.8), K(m) of 1.3 (1.3) mM, efficient non-covalent binding to the cofactor PLP, and high autoantigenic potency. The stability of rGAD67/65 was optimal over 3 months at -80 degrees C, or in lyophilized form at -20 degrees C. CONCLUSIONS: Hybrid rGAD67/65 has enzymatic and other properties similar to those of the mixed isoforms of GAD in preparations from mammalian brain as described elsewhere, in addition to its previously described similar immunoreactivity.

Late-adult onset Leigh syndrome.

We report an illustrative case of a 74-year-old man who, in the absence of intercurrent illness, presented with rapid cognitive decline. MRI showed bilateral, symmetrical, high T2-weighted signal in the anterior basal ganglia and medial thalami, extending to the periaqueductal grey matter, basal ganglia and basal frontal lobes. A (18)F-fluorodeoxyglucose-positron emission tomography scan showed widespread reduction of metabolism in the cortex of the frontal, temporal and parietal lobes, posterior cingulate gyrus, precuneus and caudate nuclei, with sparing of the sensorimotor cortex, thalami and lentiform nuclei. A mild vitamin B12 deficiency was found and despite normal thiamine levels, intravenous (IV) thiamine and vitamin B therapy was commenced, with a short course of IV methylprednisolone and tetracycline. Repeat neuropsychological assessment four weeks following treatment revealed increased alertness and interactiveness but significant cognitive decline persisted. Unexpectedly, the patient suffered a transmural anterior myocardial infarction six weeks after presentation and died within 24hours. An a autopsy showed: global reduction in cytochrome oxidase (COX) activity in all skeletal muscles examined; bilateral, symmetrical, hypervascular, focally necrotizing lesions in the substantia nigra, periaqueductal grey matter, superior colliculi, medial thalami anteriorly and posteriorly, as well as in the putamena but the mammillary bodies were not affected. Biochemical analysis of fresh muscle confirmed selective deficiency of complex IV of the oxidative phosphorylation chain. A diagnosis of late-adult onset Leigh syndrome was made. Multiple genetic studies failed to identify the specific underlying mutation. The relevant literature is reviewed.

Novel single base pair COX III subunit deletion of mitochondrial DNA associated with rhabdomyolysis.

A high number of cytochrome c oxidase (COX)-negative muscle fibres (approximately 45%) without ragged red fibres was found in a 27-year-old male patient with a single unprovoked episode of severe rhabdomyolysis. There was no family history of neuromuscular disorder and sequencing revealed a novel COX III single base pair deletion (MT-CO3{NC_012920.1}:m.[9559delC]). The deletion creates a frame shift and downstream termination codon affecting the last 136 amino acids (MT-CO3{YP_003024032.1}:p.[Pro118GlnfsX124]). The heteroplasmic mutation load in muscle was approximately 58% and single COX-negative fibres harboured significantly greater levels of mutant mitochondrial DNA than COX-positive fibres.

Antimicrobial activities of ceragenins against clinical isolates of resistant Staphylococcus aureus.

The rise in the rates of glycopeptide resistance among Staphylococcus aureus isolates is concerning and underscores the need for the development of novel potent compounds. Ceragenins CSA-8 and CSA-13, cationic steroid molecules that mimic endogenous antimicrobial peptides, have previously been demonstrated to possess broad-spectrum activities against multidrug-resistant bacteria. We examined the activities of CSA-8 and CSA-13 against clinical isolates of vancomycin-intermediate S. aureus (VISA), heterogeneous vancomycin-intermediate S. aureus (hVISA), as well as vancomycin-resistant S. aureus (VRSA) and compared them to those of daptomycin, linezolid, and vancomycin by susceptibility testing and killing curve analysis. We also examined CSA-13 for its concentration-dependent activity, inoculum effect, postantibiotic effect (PAE), and synergy in combination with various antimicrobials. Overall, the MICs and minimal bactericidal concentrations of CSA-13 were fourfold lower than those of CSA-8. Time-kill curve analysis of the VRSA, VISA, and hVISA clinical isolates demonstrated concentration-dependent bactericidal killing. An inoculum effect was also observed when a higher starting bacterial density was used, with the time required to achieve 99.9% killing reaching 1 h with a 6-log10-CFU/ml starting inoculum, whereas it was>or=24 h with a 8- to 9-log10-CFU/ml starting inoculum with 10x the MIC (P

Impact of Enterococcus faecalis on the bactericidal activities of arbekacin, daptomycin, linezolid, and tigecycline against methicillin-resistant Staphylococcus aureus in a mixed-pathogen pharmacodynamic model.

We inoculated an in vitro pharmacodynamic model simultaneously with clinical isolates of methicillin-resistant Staphylococcus aureus and an enterocin-producing enterococcus (vancomycin-resistant Enterococcus faecalis, ampicillin susceptible) at 7 log10 CFU/ml to examine enterocin effects and antimicrobial activity on staphylococci. The investigated antimicrobial regimens were 100 mg arbekacin every 12 h (q12h), 6 mg daptomycin per kg of body weight/day, 600 mg linezolid q12h, and 100 mg tigecycline q24h alone and in combination (daptomycin, linezolid, and tigecycline) with arbekacin. Simulations were performed in triplicate; bacterial quantification occurred over 48 h, and development of resistance was evaluated throughout. When we evaluated the impact of antimicrobial activity against S. aureus alone, daptomycin demonstrated bactericidal activity (>or=3 log10 CFU/ml kill), whereas arbekacin, linezolid, and tigecycline displayed bacteriostatic activities (<3 log10 CFU/ml kill). In the mixed-pathogen model, early and distinctive stunting of S. aureus growth was noted (1.5 log CFU/ml difference) in the presence of enterocin-producing E. faecalis compared to growth controls run individually (P=0.02). Most noteworthy was that in the presence of enterocin-producing E. faecalis, bactericidal activity was observed with arbekacin and tigecycline and with the addition of arbekacin to linezolid. Antagonism was noted for the combination of tigecycline and arbekacin against S. aureus in the presence of enterocin-producing E. faecalis. Our research demonstrates that the inhibitory effect of E. faecalis contributed significantly to its overall antimicrobial impact on S. aureus. This contribution was enhanced or improved compared to the activity of each antimicrobial alone. Further research is warranted to determine the impact of polymicrobial infections on antimicrobial activity.

Protection against experimental autoimmune encephalomyelitis generated by a recombinant adenovirus vector expressing the V beta 8.2 TCR is disrupted by coadministration with vectors expressing either IL-4 or -10.

Adenovirus vectors are increasingly being used for genetic vaccination and may prove highly suitable for intervention in different pathological conditions due to their capacity to generate high level, transient gene expression. In this study, we report the use of a recombinant adenovirus vector to induce regulatory responses for the prevention of autoimmune diseases through transient expression of a TCR beta-chain. Immunization of B10.PL mice with a recombinant adenovirus expressing the TCR Vbeta8.2 chain (Ad5E1 mVbeta8.2), resulted in induction of regulatory type 1 CD4 T cells, directed against the framework region 3 determinant within the B5 peptide (aa 76-101) of the Vbeta8.2 chain. This determinant is readily processed and displayed in an I-A(u) context, on ambient APC. Transient genetic delivery of the TCR Vbeta8.2 chain protected mice from Ag-induced experimental autoimmune encephalomyelitis. However, when the Ad5E1 mVbeta8.2 vector was coadministered with either an IL-4- or IL-10-expressing vector, regulation was disrupted and disease was exacerbated. These results highlight the importance of the Th1-like cytokine requirement necessary for the generation and activity of effective regulatory T cells in this model of experimental autoimmune encephalomyelitis.