First description of spontaneous fungal peritonitis caused by Fusarium solani in a critically ill patient with liver cirrhosis.

Fusarium spp., common soil moulds, are emerging fungal pathogens in immunocompromised subjects. We report the first case of Fusarium solani peritonitis in a patient with liver cirrhosis. Because of the high morbidity and mortality associated with fusariosis, an aggressive approach to treatment as well as identification of the species and drug susceptibilities is warranted.

A systemic vaccine based on Escherichia coli O157:H7 bacterial ghosts (BGs) reduces the excretion of E. coli O157:H7 in calves.

Cattle are the main reservoir of enterohemorrhagic Escherichia coli O157:H7, a bacterium that, in humans, causes hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease, especially in children and older people. Therefore, the development of vaccines preventing colonization of cattle by E. coli O157:H7 could be a main tool for an HUS control program. In the present study, we evaluated bacterial ghosts (BGs) of E. coli O157:H7 as an experimental vaccine against this pathogen. BGs are empty envelopes of Gram-negative bacteria, which retain the morphological surface make-up of their living counterparts and are produced by controlled expression of the cloned protein E, which causes loss of all the cytoplasm content. In this work, E. coli O157:H7 BGs were used for subcutaneous immunization of calves. The vaccinated animals elicited significant levels of BG-specific IgG but not IgA antibodies in serum. Low levels of IgA and IgG antibodies against BGs were detected in saliva from vaccinated animals. Following oral challenge with E. coli O157:H7, a significant reduction in both the duration and total bacterial shedding was observed in vaccinated calves compared to the nonimmunized group. We demonstrated that systemic vaccination with E. coli O157 BGs provides protection in a bovine experimental model. Further research is needed to reach a higher mucosal immune response leading to an optimal vaccine.

Escherichia coli ghost production by expression of lysis gene E and Staphylococcal nuclease.

The production of bacterial ghosts from Escherichia coli is accomplished by the controlled expression of phage phiX174 lysis gene E and, in contrast to other gram-negative bacterial species, is accompanied by the rare detection of nonlysed, reproductive cells within the ghost preparation. To overcome this problem, the expression of a secondary killing gene was suggested to give rise to the complete genetic inactivation of the bacterial samples. The expression of staphylococcal nuclease A in E. coli resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments shorter than 100 bp. Two expression systems for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. With regard to the absence of reproductive cells from the ghost fractions, the reduction of viability could be determined as being at least 7 to 8 orders of magnitude. The lysis process was characterized by electrophoretic analysis and absolute quantification of the genetic material within the cells and the culture supernatant via real-time PCR. The ongoing degradation of the bacterial nucleic acids resulted in a continuous quantitative clearance of the genetic material associated with the lysing cells until the concentrations fell below the detection limits of either assay. No functional, released genetic units (genes) were detected within the supernatant during the lysis process, including nuclease expression.

Exacerbated vein graft arteriosclerosis in protein kinase Cdelta-null mice.

Smooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCdelta knockout mice and performed vein bypass grafts on these animals. PKCdelta(-/-) mice developed normally and were fertile. Vein segments from PKCdelta(-/-) mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCdelta(+/+) vein grafts. Arteriosclerotic lesions in PKCdelta(-/-) mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCdelta(-/-) mice. SMCs derived from PKCdelta(-/-) aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCdelta(-/-) relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCdelta maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis.

Age differences in the selection of mental sets: the role of inhibition, stimulus ambiguity, and response-set overlap.

Switching between tasks leads to response-time (RT) costs at switch points (local switch costs) and often to RT costs at no-switch transitions that occur in the context of a task-switching block (global set-selection costs). With trial-to-trial cuing of tasks, moderate age effects were obtained for local switch costs, but large age effects were obtained for global selection costs. In Experiment 1, set-specific inhibition was found to be at least as large in old as in young adults, thus ruling out an inhibition deficit as a reason for age differences in global costs. In Experiment 2, large age differences in global costs were limited to conditions of ambiguous stimuli and full response-set overlap. This pattern of results suggests a greater reliance on set-updating processes in old than in young adults. The role of these processes is to ensure unambiguos internal control settings when ambiguity arises from stimuli and response specifications.

Dissociating retention and access in working memory: an age-comparative study of mental arithmetic.

In two experiments, young and older adults solved arithmetic chain tasks with single-digit operands, with or without a concurrent memory load of three or six digits. Variables in the arithmetic tasks had to be replaced by digits from the screen or from the memory set. A task-irrelevant concurrent load impaired neither speed nor accuracy of arithmetic in younger adults. In Experiment 2, this was also true for older adults. A large decrease in arithmetic performance was observed, however, when variables in the arithmetic task had to be substituted by digits from the memory list. Older adults had specific problems with this condition in Experiment 1, where the substitution involved two successive steps, but not in Experiment 2, where the substitution from memory could be done in a single step. The results are difficult to reconcile with models assuming a common resource for storage and processing. Rather, they are compatible with the hypothesis that a concurrent memory load interferes with a processing task only during the points of access to working memory. Further, even though access to working memory was found to be the critical source of concurrent-load interference, it was found to be insensitive to the effects of adult aging.

Emotional experience in everyday life across the adult life span.

Age differences in emotional experience over the adult life span were explored, focusing on the frequency, intensity, complexity, and consistency of emotional experience in everyday life. One hundred eighty-four people, age 18 to 94 years, participated in an experience-sampling procedure in which emotions were recorded across a 1-week period. Age was unrelated to frequency of positive emotional experience. A curvilinear relationship best characterized negative emotional experience. Negative emotions declined in frequency until approximately age 60, at which point the decline ceased. Individual factor analyses computed for each participant revealed that age was associated with more differentiated emotional experience. In addition, periods of highly positive emotional experience were more likely to endure among older people and periods of highly negative emotional experience were less stable. Findings are interpreted within the theoretical framework of socioemotional selectivity theory.

Activation, stimulation and uptake of bacterial ghosts in antigen presenting cells.

Bacterial ghosts have been shown to be an innovative system to prepare vaccines of various bacteria with all features of the intact bacterial cell envelopes, especially all antigenic epitopes, but also to target recombinant proteins inserted in the cell envelopes of the ghost preparations to specific antigen presenting cells. To investigate the activation of the antigen presenting cell by bacterial ghosts in more detail we studied the uptake of bacterial ghosts in dendritic porcine cells and RAW macrophages and the induction of inflammatory mediators or mediators directing the immune response in THP-1 human macrophage cell line. The synthesis of inflammatory macrophage mediators such as TNFalpha in the THP1 cell line was stimulated by a hundred-fold higher dose of ghosts from Vibrio cholerae than the corresponding LPS using ELISA-analysis. These results confirm in vivo experiments indicating no toxic effects of ghosts in rabbits even after intravenous administration in doses stimulating significant humoral responses. We were also able to see a significant activation of IL-12 indicated by the analysis of IL-12(p70) synthesis and IL-12(p40) mRNA accumulation. This interleukine is of special importance in the activation of cellular TH1 immune responses. A rapid uptake of bacterial ghosts in macrophages within 10-30 min could be confirmed by electron microscopy. As antigen presentation is especially effective in porcine dendritic cells (DC) and even a low capacity of antigen uptake is sufficient for an induction of immune responses we investigated uptake and activation of bacterial ghosts by DC. DC are known to be phagocytic in specific immature stages. We found a significant uptake of bacterial ghosts from Actinobacillus pleuropneumoniae (App) and V. cholerae conjugated with FITC (fluorescinisothiocyanate) within 2 h. These data suggest that bacterial ghosts effectively stimulate monocytes and macrophages for the induction of TH1 directed immune responses and dendritic cells treated with bacterial ghosts may serve as a promising vehicle for active immunization and immunotherapy in situ.

Characterization and immunogenicity of Vibrio cholerae ghosts expressing toxin-coregulated pili.

Bacterial ghosts are attractive for use as non-living vaccines and as carriers of heterologous antigens of vaccine relevance. Ghosts were prepared from Vibrio cholerae strains of O1 or O139 serogroup after growth under culture conditions, which favor or repress the production of toxin-coregulated pili (TCP). Immunoblotting confirmed the TCP status of these V. cholerae ghosts (VCG), which retained the cellular morphology and envelope sub-component profile of viable bacteria. Rabbits were immunized with VCGs prepared from O139 bacteria with TCP-positive or TCP-negative phenotypes and the resulting sera assayed for antibodies to lipopolysaccharide (LPS) and to TCP. Regardless of the TCP status of the VCG preparations used for immunization, all animals produced antibodies to LPS as demonstrated in bactericidal assays. These antibodies were probably responsible for the capacity of the antisera to confer passive immunity to challenge with the homologous O139 strain in the infant mouse cholera model (IMCM). Only following immunization with TCP-positive VCG, however, were antibodies to TCP generated, as judged by the potential of antisera to mediate protection against a challenge strain of heterologous serogroup.

Task-set switching and long-term memory retrieval.

The authors tested the hypothesis of a close relationship between the intentional component of task-set switching ("advance reconfiguration;" R. D. Rogers & S. Monsell, 1995) and long-term memory (LTM) retrieval. Consistent with this hypothesis, switch costs are reported to be larger when the switched-to task involves high retrieval demands (i.e., retrieval of episodic information) than when it involves low retrieval demands (i.e., retrieval of semantic information). In contrast, switch costs were not affected by a primary-task difficulty manipulation unrelated to intentional retrieval demands (Experiment 2). Also, the retrieval-demand effect on switch costs was eliminated when time for advanced preparation or task cues explicitly specifying the task rules were provided (Experiment 3). Overall, results were consistent with the hypothesis that the intentional switch-cost component reflects the time demands of retrieving appropriate task rules from LTM.

Sequence learning in Parkinson's disease: a comparison of spatial-attention and number-response sequences.

The serial reaction time (SRT) task has been frequently used to assess procedural learning of sequences. Patients with Parkinson's disease (PD) have been reported to show deficits on this task, but it is as yet unclear whether this impairment reflects a general sequencing deficit or a deficit in the sequencing of motor-output responses. In order to examine this issue, PD patients and controls were administered an SRT task which allowed the simultaneous and independent assessment of the procedural learning of spatial regularities and the learning of motor-response regularities. PD patients were unimpaired at learning a sequence of spatial locations, but showed a deficit at learning a stimulus-to-motor-response sequence. The results suggest that sequencing impairments in PD are not general, but specific to the type of sequential information inherent in a task.

Mental and psychomotoric retardation in two brothers with pure partial trisomy 7q32-q34 due to a maternal insertion (14;7).

We present two brothers with mental retardation, seizures disorder, generalized muscular hypertonia, kyphoscoliosis, minor anomalies and a prominent midface. GTG-banded chromosome analysis showed a derivative chromosome 14 without clues toward the origin of the rearrangement. Microdissection of the derivative chromosome 14 and subsequent reverse painting demonstrated partial trisomy 7q32-q34 as the unbalanced product of a maternal insertion (14;7). Thus, we identified two cases with pure trisomy 7q32-q34 that allowed further delineation of this aneusomy syndrome.

Changing internal constraints on action: the role of backward inhibition.

Flexible control of action requires the ability to disengage from previous goals or task sets. The authors tested the hypothesis that disengagement during intentional shifts between task sets is accompanied by inhibition of the previous task set ("backward inhibition"). As an expression of backward inhibition the authors predicted increased response times when shifting to a task set that had to be abandoned recently and, thus, suffers residual inhibition. The critical backward inhibition effect on the level of abstractly defined perceptual task sets was obtained across 6 different experiments. In addition, it was shown that backward inhibition can be differentiated from negative priming (Experiment 2), that it is tied to top-down sequential control (Experiment 3), that it can account at least partially for "residual shift costs" in set-shifting experiments (Experiment 4), and that it occurs even in the context of preplanned sequences of task sets (Experiment 5).

Local delivery of platelet-derived growth factor receptor-specific tyrphostin inhibits neointimal formation in rats.

Signal transduction through the platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) system is involved in the process of postangioplasty restenosis. Tyrphostins are low molecular weight inhibitors of protein tyrosine kinases. We assessed the antiproliferative effects of PDGFRbeta-specific tyrphostin AG-1295 in vitro and in vivo. AG-1295 significantly inhibited rat smooth muscle cell growth stimulated by PDGF-BB or FCS. This antiproliferative effect was paralleled by reversible reduction of the total phosphotyrosine level and the degree of PDGFRbeta phosphorylation by the drug in vitro. Local sustained delivery of the drug from perivascularly implanted polymeric matrices resulted in focal AG-1295 levels of 711 and 29.1 ng/mg of dry arterial tissue 1 and 14 days after implantation in rats. AG-1295 delivered from polymeric matrices resulted in a 35% reduction of neointimal formation on day 14 after balloon injury in the rat carotid model. Tyrosine phosphorylation of certain transduction proteins in arterial tissue extracts was significantly upregulated by balloon injury on day 3 but was essentially returned to or below basal levels 14 days after injury. Tyrphostin treatment decreased tyrosine phosphorylation at both time points below the basal levels. Moreover, the enhancement of PDGFRbeta expression 3 and 14 days after arterial injury was strongly inhibited by AG-1295 treatment. It can be concluded that AG-1295 reduces neointimal formation by inhibiting PDGFbeta-triggered tyrosine phosphorylation.

The fast and the slow of skilled bimanual rhythm production: parallel versus integrated timing.

Professional pianists performed 2 bimanual rhythms at a wide range of different tempos. The polyrhythmic task required the combination of 2 isochronous sequences (3 against 4) between the hands; in the syncopated rhythm task successive keystrokes formed intervals of identical (isochronous) durations. At slower tempos, pianists relied on integrated timing control merging successive intervals between the hands into a common reference frame. A timer-motor model is proposed based on the concepts of rate fluctuation and the distinction between target specification and timekeeper execution processes as a quantitative account of performance at slow tempos. At rapid rates expert pianists used hand-independent, parallel timing control. In alternative to a model based on a single central clock, findings support a model of flexible control structures with multiple timekeepers that can work in parallel to accommodate specific task constraints.

Complex semantic processing in old age: does it stay or does it go?

Old adults' (N = 24) and young adults' (N = 24) speed of producing exemplars of semantic categories (i.e., semantic fluency) varying in difficulty was assessed both in a standard condition and in a "set-switching" condition where exemplars had to be produced from 2 categories in an alternating manner. "Retrieval-position function" parameters were used to assess speed of semantic access independent of nonsemantic factors. Results suggested age effects in nonsemantic components but not in semantic retrieval per se. Also, age deficits in set switching were relatively subtle. Findings are discussed with relation to issues of domain specificity of age effects as well as of the role of executive control during semantic retrieval and age differences therein.

The coronary endothelium: a target for vascular endothelial growth factor. Human coronary artery endothelial cells express functional receptors for vascular endothelial growth factor in vitro and in vivo.

Vascular endothelial growth factor (VEGF) is an angiogenic peptide that can stimulate endothelial cell proliferation and migration in vitro and collateral development in ischemic organs in vivo. Although postulated, the expression of functional VEGF receptors in the heart has not been demonstrated yet. To prove this hypothesis and to extend the molecular basis of myocardial angiogenesis, we have characterized the expression and function of VEGF receptors in human coronary artery endothelial cells (HCAEC) and in human heart tissue. VEGF strongly induces proliferation and migration of HCAEC. These cells express transcripts of the two VEGF receptors KDR and Flt-1. Their expression levels are higher in HCAEC as compared with human umbilical vein endothelial cells. In HCAEC, VEGF stimulates phosphorylation of KDR in a concentration-dependent manner proving that KDDR is a functional receptor tyrosine kinase. Scatchard analysis demonstrated the presence of the high affinity receptor Flt-1 in HCAEC with a kd of 8 pM. Flt-1 protein could be visualized as a single band corresponding to a size of 210 kd. In addition mature KDR protein could be detected in adult human heart. Taken together, HCAEC and human heart tissue express high levels of functional VEGF receptors. These results broaden the molecular basis for understanding and manipulating VEGF-induced endothelial function and angiogenesis in the coronary circulation.

A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. A novel candidate for prevention of vascular remodeling.

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.

Coincidence of two novel arylsulfatase A alleles and mutation 459+1G>A within a family with metachromatic leukodystrophy: molecular basis of phenotypic heterogeneity.

In a family with three siblings, one developed classical late infantile metachromatic leukodystrophy (MLD), fatal at age 5 years, with deficient arylsulfatase A (ARSA) activity and increased galactosylsulfatide (GS) excretion. The two other siblings, apparently healthy at 12(1/2) and 15 years, respectively, and their father, apparently healthy as well, presented ARSA and GS values within the range of MLD patients. Mutation screening and sequence analysis disclosed the involvement of three different ARSA mutations being the molecular basis of intrafamilial phenotypic heterogeneity. The late infantile patient inherited from his mother the frequent 0-type mutation 459+1G>A, and from his father a novel, single basepair microdeletion of guanine at nucleotide 7 in exon 1 (7delG). The two clinically unaffected siblings carried the maternal mutation 459+1G>A and, on their paternal allele, a novel cytosine to thymidine transition at nucleotide 2435 in exon 8, resulting in substitution of alanine 464 by valine (A464V). The fathers genotype thus was 7delG/A464V. Mutation A464V was not found in 18 unrelated MLD patients and 50 controls. A464V, although clearly modifying ARSA and GS levels, apparently bears little significance for clinical manifestation of MLD, mimicking the frequent ARSA pseudodeficiency allele. Our results demonstrate that in certain genetic conditions MLD-like ARSA and GS values need not be paralleled by clinical disease, a finding with serious diagnostic and prognostic implications. Moreover, further ARSA alleles functionally similar to A464V might exist which, together with 0-type mutations, may cause pathological ARSA and GS levels, but not clinical outbreak of the disease.