Caspofungin treatment in severely ill, immunocompromised patients: a case-documentation study of 118 patients.

BACKGROUND: Caspofungin has shown efficacy in empirical antifungal therapy in neutropenic patients, refractory invasive Aspergillus infections and invasive candidiasis. Here we report the currently largest series of patients treated with caspofungin outside clinical trials. METHODS: Centres in Germany that were known to treat patients with invasive fungal infections were asked to fill out detailed questionnaires for all patients treated with caspofungin. No effort was made to influence the decision to use caspofungin. RESULTS: A total of 118 patients were evaluable (median age 48 years, interquartile range 38-58), out of which 41 (35%) suffered from acute leukaemia, 31 (26%) had allogeneic stem cell transplants, 16 (14%) lymphoma or myeloma, 8 (7%) autologous stem cell transplants and 22 (19%) other causes for immunosuppression. One hundred and six patients were evaluable for efficacy out of which 68 (64%) patients achieved a complete or partial remission. A total of 81 out of 115 (70%) patients were alive 30 days after the end of caspofungin therapy. Response rates were similar in proven (20/32, 63%) and probable (27/46, 59%) infections, in neutropenic patients (41/55, 75%) and in patients who were (44/70, 63%) or were not (24/36, 67%) refractory to antifungal pre-treatment. The response rate in mechanically ventilated patients was 29% (7/24). Caspofungin was well tolerated, even in 14 patients, who were concomitantly treated with ciclosporin A, no drug-related elevations of bilirubin, alanine aminotransferase or creatinine were found. CONCLUSIONS: This open case study of severely ill patients with invasive fungal infections demonstrates both excellent efficacy and very low toxicity of caspofungin.

Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coliby comparing in vivo and in vitro expression.

The atrazine-specific single-chain variable antibody fragments (scFv) K411B was produced by expression in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the pelB leader was N-terminally fused to scFv, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly. Expression of scFv with leader was 2.3 times higher than that of the protein without leader. This was further investigated by generating the respective translation profiles using coupled in vitro transcription/translation assays, the results of which were in agreement. This comparative approach was also applied to functionality: Periplasmic expression and in vitro expression resulted in only 10% correctly folded scFv, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of scFv with enhanced green fluorescent protein (EGFP) fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile as for scFv. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.

The Platelia Aspergillus ELISA in diagnosis of invasive pulmonary aspergilosis (IPA).

The sensitivity of a sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Aspergillus galactomannan was evaluated with 66 serum samples and 113 specimens of the respiratory tract obtained from 52 patients with pulmonary diseases. The patients were divided into five groups: proven invasive pulmonary aspergillosis (IPA) (five patients), probable IPA (seven patients), Aspergillus colonization (eight patients) or unlikely Aspergillus infection (27 patients). Another five patients with doubtful diagnostic test results are discussed in detail. The results of the Platelia Aspergillus ELISA (Sanofi Pasteur, Freiburg, Germany) in testing specimens of the respiratory tract were 90% sensitivity in proven (serum 38%), 60% in probable (serum 37%) and 71% in Aspergillus colonization (serum 0%). Furthermore, 85% of the Aspergillus spp. from positive cultures of specimens of the respiratory tract were also detected in the ELISA. A total of 57% of the culture negative specimens of patients with a least one positive culture or proven aspergillosis in a series of specimens were positive in the ELISA.

Kinetic modeling and simulation of in vitro transcription by phage T7 RNA polymerase.

This study provides a mathematical model of T7 RNA polymerase (T7 RNAP) kinetics under in vitro conditions targeted at application of this model to simulation of dynamic transcription performance. A functional dependence of transcript synthesis rate is derived based on: (a) essential reactant concentrations, including T7 RNAP and its promoter, substrate nucleotides, and the inhibitory byproduct inorganic pyrophosphate; (b) a distinction among vector characteristics such as recognition sequences regulating transcription initiation and termination, respectively; and (c) specific properties of the nucleotide sequence including both transcript length and nucleotide composition. Inactivation kinetics showed a half-life of T7 RNAP activity of 50 min under the conditions applied in vitro using the isolated enzyme. Model parameters and their precision are estimated using dynamic simulation and nonlinear regression analysis. The particular novelty of this model is its capability to incorporate linear genomic sequence information for simulation of nonlinear in vitro transcription kinetics.

Pigeons (Columba livia) learn to link numerosities with symbols.

After responding to each element in varying, successive numerosity displays, pigeons (Columba livia) had to choose, out of an array of symbols, the symbol designated to correspond to the preceding number of elements. After extensive training, 5 pigeons responded with significant accuracy to the numerosities 1 to 4, and 2 pigeons to the numerosities 1 to 5. Several tests showed that feedback tones accompanying element pecks, the familiarity of element configurations, and the shape of the elements were not crucial to this performance. One test, however, indicated that the number of pecks issued to the elements was important for numerosities above 2. An additional test confirmed that the birds chose the symbol that corresponded to a particular numerosity rather than the positions that the symbols had held during training.

High-cell-density fermentation for production of L-N-carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter.

A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escherichia coli was developed using the positively regulated Escherichia coli rhaBAD promoter. The expression system was improved by reducing of the amount of expensive L-rhamnose necessary for induction of the rhamnose promoter and by increasing the vector stability. Consumption of the inducer L-rhamnose was inhibited by inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia coli W3110, responsible for the first irreversible step in rhamnose catabolism. Plasmid instability caused by multimerization of the expression vector in the recombination-proficient W3110 was prevented by insertion of the multimer resolution site cer from the ColE1 plasmid into the vector. Fermentation experiments with the optimized system resulted in the production of 100 g x L(-1) cell dry weight and 3.8 g x L(-1) of recombinant L-N-carbamoylase, an enzyme, which is needed for the production of enantiomeric pure amino acids in a two-step reaction from hydantoins.

In vitro coupled transcription translation: effects of modification in lysate preparation on protein composition and biosynthesis activity.

Cell-free extracts (lysates) from Escherichia coli were used for protein synthesis in vitro. Essential steps of the lysate preparation were modified and analyzed with respect to their impact on in vitro protein synthesis capacity, using the green fluorescent protein (GFP) as a target protein. Variably manufactured lysates of low, medium and higher protein synthesis activity, were examined by high resolution two-dimensional gel electrophoresis to determine whether the modifications result in substantial alterations in protein composition of the final lysate. The total number of proteins calculated from the gel maps did not vary for lysates with different activity and thus cannot serve as an evaluation parameter. Ribosomal proteins RP-S1, RP-L9, and RP-L10 were found in stoichiometric amounts for each of these lysates and in equal concentrations in comparison among the different lysates. Conversely, depending on the activity profiles, up to 7 different isoforms of the elongation factor EF-Ts were detected in the gel maps.

Clostridium difficile-associated diseases. The clinical courses of 18 fatal cases.

OBJECTIVE: Severe cases of Clostridium difficile-associated diseases with sepsis seem to be rare, as are case reports about the pathogen involved and sepsis. Our objective was to investigate the frequency and the clinical courses of severe cases of C. difficile-associated diseases with a fatal outcome in our hospital. SETTING: Teaching hospital of the University of Kiel (650 beds). DESIGN: We reviewed retrospectively all deceased patients' charts who had prior histological or microbiological evidence of C. difficile-associated diarrhea (CDAD) and revised the available histological specimens of the autopsies. PATIENTS: Over a 4-year period (November 1994-October 1998) we diagnosed 304 cases of C. difficile-associated diseases in our hospital. RESULTS: Eighteen of our cases with C. difficile-associated diseases had a fatal outcome. C. difficile was not likely to be the cause of death in two of the cases. Four of the fatal infections were community-acquired and the reason for admission to the hospital. CDAD is most prevalent in elderly patients with multiple or severe underlying diseases and tends to be overlooked. Sepsis was diagnosed in 15 of our 18 patients with C. difficile-associated diseases. CONCLUSION: Our study shows that severe cases of CDAD or cases with C. difficile-associated sepsis are probably not rare. Routine testing of fecal specimens for the presence of C. difficile toxins should be considered not only in nosocomial gastrointestinal infections but also in community-acquired gastrointestinal infections of elderly people.

Microbial hydantoinases--industrial enzymes from the origin of life?

Hydantoinases are valuable enzymes for the production of optically pure D- and L-amino acids. They catalyse the reversible hydrolytic ring cleavage of hydantoin or 5'-monosubstituted hydantoins and are therefore classified in the EC nomenclature as cyclic amidases (EC 3.5.2.). In the EC nomenclature, four different hydantoin-cleaving enzymes are described: dihydropyrimidinase (3.5.2.2), allantoinase (EC 3.5.2.5), carboxymethylhydantoinase (EC 3.5.2.4), and N-methylhydantoinase (EC 3.5.2.14). Beside these, other hydantoinases with known metabolic functions, such as imidase and carboxyethylhydantoinase and enzymes with unknown metabolic function, are described in the literature and have not yet been classified. An important question is whether the distinct hydantoinases, which are frequently classified as L-, D-, and non-selective hydantoinases depending on their substrate specificity and stereoselectivity, are related to each other. In order to investigate the evolutionary relationship, amino acid sequence data can be used for a phylogenetic analysis. Although most of these enzymes only share limited sequence homology (identity < 15%) and therefore are only distantly related, it can be shown (i) that most of them are members of a broad set of amidases with similarities to ureases and build a protein superfamily, whereas ATP-dependent hydantoinases are not related, (ii) that the urease-related amidases have evolved divergently from a common ancestor and (iii) that they share a metal-binding motif consisting of conserved histidine residues. The difference in enantioselectivity used for the classification of hydantoinases on the basis of their biotechnological value does not reflect their evolutionary relationship, which is to a more diverse group of enzymes than was assumed earlier. This protein superfamily probably has its origin in the prebiotic conditions of the primitive earth.

Investigation of translation dynamics under cell-free protein biosynthesis conditions using high-resolution two-dimensional gel electrophoresis.

A cell-free extract from Escherichia coli, generated through a routine procedure according to Chen and Zubay (Methods Enzymol. 1983, 101, 674-690), was used for an in vitro protein synthesis. High-resolution two-dimensional gel electrophoresis (2-DE) was exploited to investigate the protein composition of the cell-extract and its dynamic development during a 24 h-period of cell-free protein synthesis performed in a membrane reactor device. Green fluorescent protein (GFP) was chosen as a target protein to be produced in a cell-free reactor because of its functional activity, which can easily be monitored by measurement of fluorescence, and because of its high sensitivity. GFP synthesis was observed by a standard fluorescence assay and was correlated to a quantitative assessment of the silver-stained GFP spot appearing on 2-DE gel maps. A constant protein synthesis rate was obtained for at least 8 h of process operation. While declining continuously, protein synthesis stopped entirely after 24 h. Both, the total protein content and total number of detectable spots were found to decrease over the reaction time, due to proteolytic digestion and protein precipitation. Certain proteins taking part in the translation process, such as the elongation factors (EF-Tu, EF-Ts) and the ribosomal protein RP-L9, were identified by Edman N-terminal sequencing and have thus been considered for reaction evaluation. The dynamics obtained during the entire process suggest that these translational factors were likewise affected by proteolytic decay.

A case of cryptic miliary tuberculosis mimicking cholecystitis with sepsis.

Miliary tuberculosis is a rare form of tuberculosis in industrialized countries. We report on a 69-year-old woman presenting a sepsis syndrome caused by cryptic miliary tuberculosis clinically mimicking a case of cholecystitis with sepsis. The patient died of a multi-organ failure on day 6 of her hospital stay.

Molecular evolution of hydantoinases.

The complete amino acid sequence of the hydantoinase from Arthrobacter aurescens DSM 3745 has been derived by automated Edman degradation. This is the first ever reported amino acid sequence of a non-ATP-dependent hydantoinase, which hydrolyzes 5'-monosubstituted hydantoin derivatives L-selectively. A homology search performed in protein and nucleic acid databases retrieved only distantly related proteins. All of these are members of the recently described protein superfamily of amidohydrolases related to ureases (Holm and Sander, Proteins 28: 72-82, 1997). Phylogenetic analysis revealed that the novel hydantoinase forms a new branch separate from other hydantoin cleaving enzymes like dihydropyrimidinases (EC 3.5.2.2) and allantoinases (EC 3.5.2.5). Our results suggests that the enzymes of this protein superfamily have evolved from a common ancestor and therefore are the product of divergent evolution. We show further that the enclosed gene families developed very early in evolution, probably prior to the formation of the three domains, Archaea, Eukarya and Bacteria. Hydantoinases related to ATP-dependent N-methylhydantoinases (EC 3.5.2.14) or 5-oxoprolinases (EC 3.5.2.9) do not belong to this superfamily.

Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745: purification and characterization as new member of cyclic amidases.

A hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity with a yield of 77% using a three-step purification procedure. The active enzyme is a tetramer consisting of four identical subunits, each with a molecular mass of 49670 Da as determined by mass spectrometry. The N-terminal amino acid sequence of the enzyme indicates sequence identities to cyclic amidases involved in the nucleotide metabolism as the D-hydantoinase from Agrobacterium radiobacter (53%), the D-selective dihydropyrimidinase from Bacillus stearothermophilus (38%), the allantoinase from Rana catesbeiana (26%), as well as to the catalytic subunit of the urease from Helicobacter pylori (50%). However, all studies based on substrate-dependent growth, induction and catalytic behavior documented the novelty of the bacterial hydantoinase and that its physiological role is not related to any of these enzymes or known metabolic pathways. Its substrate specificity differs from hydantoinases listed in Enzyme Nomenclature and is rather more predominant for the cleavage of aryl- than for alkyl-hydantoin derivatives. It is shown that the stereoselectivity of this enzyme depends on the substrate used for bioconversion: although it is strictly L-selective for the cleavage of D,L-5-indolylmethylhydantoin, it appears to be D-selective for the hydrolysis of D,L-methylthioethylhydantoin. Due to these findings we conclude that this novel bacterial hydantoinase should be classified as a new member of the EC-group 3.5.2 of cyclic amidases.

Visual discrimination in pigeons impaired by glutamatergic blockade of nucleus accumbens.

The nucleus accumbens septi (Acc) is thought to be involved in the control of cognitive processes and to be implicated in the pathophysiology of schizophrenia. Because perceptual-cognitive distortions are a core symptom in schizophrenia, any evidence that the Acc intervenes in a sensory recognition task in an animal species would be of interest. Pigeons were instrumentally trained to discriminate visual shapes. The acute effects of drug microinjections into the Acc on the discrimination of the training shapes, on the correction responding after errors, and on the generalisation to different shapes were examined. The effects of conduction blockade with lidocaine, glutamatergic blockade with 7-aminophosphonoheptanoic acid, and dopaminergic stimulation with apomorphine on behavioural performance were tested. No effects were observed with lidocaine and apomorphine. A significant and reversible performance disruption to near chance levels was obtained after aminophosphonoheptanoic acid injections into the Acc. It appears that a glutamatergic blockade of the Acc interferes with the visual discrimination processes of pigeons.

Detection of fungemia from blood cultures using the BACTEC 9240 instrument.

From January 1995 to November 1996, 14,623 resin-containing blood culture vials were tested with the BACTEC fluorescent series instrument. A total of 1560 microorganisms were recovered. 48 of the microorganisms were fungi. We could demonstrate the ability of the BACTEC 9240 to detect cases of fungemia with Candida species and moulds such as Aspergillus fumigatus (4 cases) and Fusarium solani (1 case).

Transitive responding in animals and humans: Exaptation rather than adaptation?

In order to survive and reproduce, individual animals need to navigate through a multidimensional utility landscape in a near-optimal way. There is little doubt that the behaviourally more advanced species can bring cognitive competencies to bear on this difficult task. Among the cognitive abilities that are helpful in this context is transitive inference. This is typically the competency to derive the conclusion B>D from the premises A>B, B>C, C>D and D>E that imply the series A>B>C>D>E. In transitive inference tests used with humans, the letters stand for verbal items and the inequality symbols stand for a relational expression. To investigate analogous competencies in non-human animals a non-verbal form of the task is used. The premise pairs are converted into a multiple instrumental discrimination task A+B-, B+C-, C+D- and D+E-, where the letters stand for non-verbal stimuli and the plus and minus symbols indicate that choices of the corresponding stimuli either lead to a reward or to a penalty. When these training pairs are adequately discriminated, transitive responding is tested with intermittent presentations of the novel pair B∘D∘, where the circles indicate that responses to the stimuli are not reinforced. Using variants of this basic conditioning task it has been shown that pigeons, rats, squirrel-monkeys, macaques, chimpanzees, young children, older children and adult humans commonly reveal transitive preferences for B over D. Several theories have been proposed to explain this transitive behaviour. The evidence supporting these various models is reviewed. It is shown that the learning of the premises normally brings about a choice and reinforcement biasing and balancing process that can account for transitive responding. It is argued that a very simple algebraic learning model can satisfactorily simulate many of the results obtained in transitivity experiments, including some produced by human subjects who in principle, could have been applying rational logical rules. It is demonstrated that a value transfer mechanism also assumed to explain transitive responding, is in fact, a real phenomenon based on classical conditioning. However, it is argued that it mostly plays a minor role in transitive responding. It is shown that the algebraic learning model can be easily converted into a neural network model exhibiting an equivalent performance. The model can also be modified to cope with the surprising finding that a proportion of human individuals and a few animals subjects learn to discriminate the premise pairs, but nevertheless fail to respond transitively to the conclusion pair. This modification can simulate the results of experiments using non-linear, in particular circular, relational structures. The evolution of transitive responding is considered within the framework of ecosocial demands and neurobiological constraints. It is concluded that, in agreement with a preadaptation (exaptation) evolutionary origin, it seems to involve little beyond the capacity to learn multiple stimulus discriminations.

False-positive results in premature infants with the Platelia Aspergillus sandwich enzyme-linked immunosorbent assay.

The sensitivity of a sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Aspergillus galactomannan was tested using 783 serum samples obtained from 247 patients (1-15 sera per patient) with severe underlying diseases (haematological malignancies or intensive care unit stay). We selected 146 serum samples from 50 patients for retesting. Serum samples were frozen after routine testing at -18 degrees C until retesting. All patients charts were checked for signs of Aspergillus infection, such as pneumonia or sinusitis. Adult patients were divided into four groups: proven (5), probable (6), suspected (8) or unlikely (25) Aspergillus infection. The results of Platelia ELISA were 100% in proven, 33% in probable and 50% in suspected Aspergillus infection. Patients with unlikely infection had no positive results with Platelia ELISA. Group 5 consists of six paediatric patients with prolonged ICU stay and a birth weight of 400-1320 g. In five out of six infants we found positive results with Platelia ELISA. All positive results in this group of patients are considered as false positive (83.3%).

Crystallization and preliminary X-ray analysis of a hydantoinase from Arthrobacter aurescens DSM 3745.

L-Hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity and crystallized from polyethylene glycol solutions in a form suitable for X-ray diffraction analysis. The crystals have been grown by the sitting-drop variant of the vapour-diffusion method. X-ray diffraction studies show that the crystals belong to the monoclinic space group P2(1) with a = 111.2, b = 74.4, c = 146.5 A and beta = 106.7 degrees. Its asymmetric unit contains four monomers related by 222 symmetry. The crystals diffract to at least 2.6 A.

Formation of bacteriophage MS2 infectious units in a cell-free translation system.

We show that a simple cell-free translation system from Escherichia coli, programmed with phage MS2 RNA, is able to infect F+ E. coli cells. The plaques appearing on the E. coli host strain are morphologically indistinguishable from those derived from normal phage MS2 infection. This effect is strictly translation-dependent, since an incomplete translation system or the system inhibited by antibiotics leads to no infection. The cell-free based infection is maximal under conditions favouring the highest synthesis of maturation protein (one of the four phage-encoded proteins). The infection is abolished when RNase A or trypsin treatment is included before addition of cells. Similarly, due to RNA and maturation protein degradation, the continued incubation of the translation mixture under protein synthesis conditions significantly decreases infectivity. These findings suggest the formation of 'minimal infectious units', simple complexes of MS2 RNA and maturation protein. Here we describe the first example of bacteriophage infectious unit formation directly performed in a cell-free translation system. A possible application of this phenomenon might be the construction of newly designed RNA vector delivery systems and, moreover, could be an approach for molecular evolution studies.