Laboratory diagnosis of Clostridium difficile-associated diarrhea and colitis: usefulness of Premier Cytoclone A+B enzyme immunoassay for combined detection of stool toxins and toxigenic C. difficile strains

Detection of Clostridium difficile toxins A and B in stools by Premier Cytoclone A+B enzyme immunoassay (EIA) was compared with detection by stool culture for C. difficile followed by detection of toxigenic isolates using the same EIA. Chart reviews were performed to evaluate the likelihood of C. difficile-associated diarrhea and colitis (CADC) for all patients with at least one positive toxin assay. While the toxins were detected in 58 of 85 consecutive CADC patients by both assays, CADC in 5 patients was detected only by stool toxin assay, and in 22 patients CADC was detected only by toxigenic culture. Our results suggest that for laboratories using a rapid toxin A+B EIA, direct toxin detection in stools should be combined with toxigenic culture in cases in which there is a negative stool toxin assay.     

Pharmacokinetics and pharmacodynamics of L-Dopa after acute and 6-week tolcapone administration in patients with Parkinson's disease

Tolcapone, a central and peripheral catechol O-methyltransferase (COMT) inhibitor, reduces the conversion of L-Dopa into 3-O-methyl-Dopa (3-OMD), thus leading to more stable and sustained L-Dopa plasma levels. This study was designed to evaluate the effects of acute and 6-week tolcapone administration on L-Dopa pharmacokinetics and pharmacodynamics in Parkinson's disease (PD) patients with predictable motor fluctuations. Tapping test, walking time, and tremor, as well as L-Dopa and 3-OMD plasma levels, were assessed before and for 5 hours after the administration of a single L-Dopa dose, alone or in combination with 200 mg tolcapone, in seven patients with PD. This clinical and pharmacokinetic study was repeated after 6 weeks of tolcapone therapy (200 mg three times daily). It was observed that tolcapone, after both acute and chronic administration, prolonged the motor improvement induced by L-Dopa. As a result, at week 6 of tolcapone therapy, the daily hours spent "off" were significantly decreased. Tolcapone significantly increased the area under the curve of L-Dopa plasma levels by slowing down the elimination of L-Dopa from plasma, whereas the maximal concentration of L-Dopa was not modified. 3-OMD levels decreased significantly after acute tolcapone administration, and after 6 weeks of tolcapone therapy, they were approximately one sixth of pre-tolcapone values. The data confirm that tolcapone decreases L-Dopa clearance and prolongs motor response in PD patients with motor fluctuations, and that this effect is maintained after 6 weeks of tolcapone therapy.     

Increased intracellular concentration of an initiator protein markedly reduces the minimal sequence required for initiation of DNA synthesis.

One of the most common sites used for cloning in the filamentous phages f1, fd, and M13 lies within the phage "functional origin," a sequence of 140 nucleotides that is required for phage replication. Even small insertions (four nucleotides) at this location severely reduce origin function. Secondary trans-acting mutations in the phage genome are necessary to restore efficient replication. One of these mutations, present in one of our cloning vectors, R218, has been fully characterized. It consists of a regulatory mutation within gene V that leads to a marked increase in the intracellular level of the phage gene II protein, the "initiator" of viral replication. Increased gene II protein production is sufficient to reduce the minimal sequence required for a functional origin to only 40 nucleotides, while the remaining 100 (containing the cloning site) become entirely dispensable. The general implications of these findings are discussed.     

Differentiation-specific increase in ALA-induced protoporphyrin IX accumulation in primary mouse keratinocytes.

A treatment regimen that takes advantage of the induction of intracellular porphyrins such as protoporphyrin IX (PPIX) by exposure to exogenous 5-amino-laevulinic acid (ALA) followed by localized exposure to visible light represents a promising new approach to photodynamic therapy (PDT). Acting upon the suggestion that the effectiveness of ALA-dependent PDT may depend upon the state of cellular differentiation, we investigated the effect of terminal differentiation upon ALA-induced synthesis of and the subsequent phototoxicity attributable to PPIX in primary mouse keratinocytes. Induction of keratinocyte differentiation augmented intracellular PPIX accumulation in cells treated with ALA. These elevated PPIX levels resulted in an enhanced lethal photodynamic sensitization of differentiated cells. The differentiation-dependent increase in cellular PPIX levels resulted from several factors including: (a) increased ALA uptake, (b) enhanced PPIX production and (c) decreased PPIX export into the culture media. Simultaneously, steady-state levels of coproporphyrinogen oxidase mRNA increased but aminolaevulinic acid dehydratase mRNA levels remained unchanged. From experiments using 12-o-tetradecanoylphorbol-13-acetate, transforming growth factor beta 1 and calcimycin we demonstrated that the increase in PPIX concentration in terminally differentiating keratinocytes is calcium- and differentiation specific. Stimulation of the haem synthetic capacity is seen in primary keratinocytes, but not in PAM 212 cells that fail to undergo differentiation. Interestingly, increased PPIX formation and elevated coproporphyrinogen oxidase mRNA levels are not limited to differentiating keratinocytes; these were also elevated in the C2C12 myoblast and the PC12 adrenal cell lines upon induction of differentiation. Overall, the therapeutic implications of these results are that the effectiveness of ALA-dependent PDT depends on the differentiation status of the cell and that this may enable selective targeting of several tissue types.     

Diagnosis, assessment and management of delusional jealousy in Parkinson’s disease with and without dementia

Patients with Parkinson’s disease (PD) may present delusional jealousy (DJ). In a previous cross-sectional prevalence study we identified 15 cognitively preserved and five demented PD patients with DJ. The current study aimed at evaluating their clinical (motor and non-motor) characteristics and the pharmacological treatments associated with DJ, and its subsequent pharmacological management. Patients were assessed by neurologists and psychiatrists using the Hoehn and Yahr scale, the Unified Parkinson’s Disease Rating Scale, the Brief Psychiatric Rating Scale, the Beck Depression Inventory, the Hamilton Anxiety Scale and the Neuropsychiatric Inventory. Efficacy of DJ management was evaluated in follow-up visits. All patients were in therapy with dopamine agonists. A subgroup of five cognitively preserved patients developed DJ after a short period of treatment of therapy with dopamine agonists, while other patients developed DJ after a longer period of dopaminergic treatment. Psychiatric comorbidities were common in cognitively preserved and in demented patients. The pharmacological management included the interruption of dopamine agonists in two patients and the reduction of dopamine agonist dose plus the use of antipsychotics in other patients. These clinical data suggest that the management of medicated PD patients should include investigation for the presence of DJ and the evaluation of clinical characteristics potentially relevant to the prevention or the early recognition of delusions.     

The role of a mineralocorticoid receptor gene functional polymorphism in the symptom dimensions of persistent ADHD

Attention-deficit/hyperactivity disorder (ADHD) affects approximately 5 % of school-aged children and 2.5 % of adults. Genetic studies in ADHD have pointed to genes in different neurobiological systems, with relatively small individual effects. The mineralocorticoid receptor is the main receptor involved in the initial triggering of stress response. Therefore, its encoding gene (NR3C2) is a candidate for psychiatric disorder studies, including ADHD, and behavioral phenotypes. There is evidence that the Val allele of the MRI180V polymorphism (rs5522) increases the risk of depression, attention and cognitive deficits. We investigated the possible role of the mineralocorticoid receptor gene in the symptom dimensions and susceptibility to persistent ADHD. We compared genotype and allele frequencies in 478 adult patients with ADHD and 597 controls and symptom dimensions in 449 patients and 132 controls. Diagnoses were based on the DSM-IV criteria. ADHD symptom dimensions were investigated with SNAP-IV for ADHD severity and Barkley scales for severity and impairment. Carriers of the Val allele presented higher inattention, hyperactivity/impulsivity and impairment scores, while genotype and allele frequencies did not differ between patients and controls. These results are consistent with a possible link between genetic variations in the HPA axis and inattention and hyperactivity measures.     

Switch in gap junction protein expression is associated with selective changes in junctional permeability during keratinocyte differentiation.

Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels that have different permeability properties. We report here that induction of terminal differentiation in mouse primary keratinocytes by calcium results in a specific switch in gap junction protein expression. Expression of alpha 1 (connexin 43) and beta 2 (connexin 26) gap junction proteins is down-modulated, whereas that of beta 3 (connexin 31) and beta 4 (connexin 31.1) proteins is induced. Although both proliferating and differentiating keratinocytes are electrically coupled, there are significant changes in the permeability properties of the junctions to small molecules. In parallel with the changes in gap junction protein expression during differentiation, the intercellular transfer of the small dyes neurobiotin, carboxyfluorescein, and Lucifer yellow is significantly reduced, whereas that of small metabolites, such as nucleotides and amino acids, proceeds unimpeded. Thus, a switch in gap junction protein expression in differentiating keratinocytes is accompanied by selective changes in junctional permeability that may play an important role in the coordinate control of the differentiation process.     

Cytogenetic analysis oxidative damage in lymphocytes of Parkinson's disease patients

Several lines of evidence support the presence of DNA damage in somatic cells of Parkinson's disease (PD) patients due to the formation of free radical species. In order to detect spontaneous chromosome and primary or oxidative DNA damage, we performed the human lymphocyte micronucleus assay (HLMDA) and comet assay in 19 PD patients and 16 healthy controls. Compared with controls, PD patients showed a significant increase in: (1) spontaneous micronucleus (MN) frequency (p<0.001); (2) single strand break (SSB) levels (p<0.001); and (3) oxidized purine base levels (p<0.05). The chromosome damage and the increased levels of oxidized purine bases observed in our patients support the hypothesis of oxidative stress as a relevant factor in the pathogenesis of PD.