Selection of strains for quality assessment of the disk induction method for detection of inducible clindamycin resistance in Staphylococci: a CLSI collaborative study

A nine-laboratory collaborative study was conducted to select positive and negative quality assessment control strains for the detection of inducible clindamycin resistance in staphylococci. Four strains of Staphylococcus aureus were tested as unknowns on 10 different days in each laboratory using the recently recommended CLSI (formerly NCCLS) disk diffusion method and the inoculum purity control method. Strains contained either macrolide-lincosamide-streptogramin B (MLSB) resistance genes encoded by erm(A) or erm(C) or a macrolide resistance efflux pump encoded by msr(A). Based upon the results of this study, strain UT 32 (now designated ATCC strain BAA-977) containing erm(A) is recommended as the positive control organism for inducible clindamycin resistance. Strain UT 25 (now designated ATCC BAA-976), which harbors the efflux pump encoded by msr(A), is recommended as the negative control organism.     

Differential expression of beta 1 integrins in nonneoplastic smooth and striated muscle cells and in tumors derived from these cells.

Integrins are a superfamily of transmembrane alpha beta heterodimers that play an important role in cell-matrix and cell-cell interactions by acting as receptors for extracellular matrix proteins and for cell adhesion molecules. Using monoclonal antibodies against beta 1, alpha 1 to alpha 6, and alpha v subunits, the in situ distribution pattern of beta 1 integrins was examined immunohistochemically in nonneoplastic smooth and striated muscle cells and in their tumors. Nonneoplastic smooth muscle cells were beta 1+, alpha 1+, alpha 3+, alpha v+ and, in diverse localizations, also alpha 5+ or even alpha 6+. The expression of the beta 1 chain was conserved in all leiomyomas and leiomyosarcomas. The distribution pattern of the alpha subunits by contrast underwent several changes during malignant transformation of smooth muscle cells. These alterations consisted in a neoexpression of alpha 2, alpha 4, and alpha 6 as well as in an abnormal abrogation of alpha 1 and alpha 3 in some leiomyosarcomas. Except for the absence of alpha 5 in the majority of epithelioid leiomyosarcomas, expression of the alpha 5 and alpha v subunits was mainly conserved. In addition, tumors with epithelioid differentiation differed from typical cases by the absence of alpha 1 and the simultaneous presence of alpha 4. Adult striated muscle cells were beta 1+ but alpha 1- to alpha 6- and alpha v-, whereas fetal striated muscle cells were not only beta 1+ but also alpha 3+/-, alpha 4+/-, alpha 5+ and alpha 6+. In all rhabdomyosarcomas the expression of beta 1 was retained. Furthermore, the majority of cases showed the expression of one or more alpha subunits most of which, ie, alpha 4, alpha 5, and alpha 6, were also found in fetal striated muscle cells. In conclusion, beta 1 integrins exhibited a differential expression pattern along the two lines of myogenic differentiation. This integrin profile underwent characteristic changes during malignant transformation. Nevertheless, the compiled distribution patterns of the alpha 1, alpha 3, and alpha v subunits allowed in most instances the discrimination between tumors of smooth (alpha 1+/alpha 3+/alpha v+) and striated muscle (alpha 1-/alpha 3-/alpha v-) differentiation.     

Primary gastric apoptosis-rich T-cell lymphoma co-expressing CD4, CD8, and cytotoxic molecules

In contrast to primary gastric lymphomas of B-cell type, little is known about primary gastric T-cell lymphomas. We describe three cases with remarkably similar features: diffuse growth, epitheliotropism, medium too large cell size, high apoptotic rates, and a CD3+, CD4+, CD8+, CD45RO+ immunophenotype. Clonal TCRγ gene rearrangement was shown in two cases. Epstein-Barr virus infection was excluded in two cases. Taking advantage of fresh-frozen material, we analyzed two cases further, revealing CD5–, CD16+, CD56–, CD57–, CD25+, CD30+, CD103 (αEβ7)+, bcl-2 protein+, CD95+, CD95 ligand(L)–. CD95L, however, was detected in histiocytic and fibroblastoid by stander cells. The lymphomas expressed granzyme B, perforin, and the TIA-1 antigen in various combinations. All three cases had a very unfavorable clinical course characterized by local recurrence and/or dissemination to other epithelial sites, leading to death within 6–12 months after the initial diagnosis despite surgery and aggressive antineoplastic treatment. These data suggest a novel variant of peripheral T-cell lymphoma operationally characterized as primary gastric, apoptosis-rich, CD103+, EBV-, T-cell lymphoma co-expressing CD4, CD8, CD16 and cytotoxic molecules. Received: 20 August 1999 / Accepted: 2 November 1999     

Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca²⁺-dependent regulation. Force then depended on the negative logarithm of Ca²⁺ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa₅₀, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca²⁺, but restoration of Ca²⁺ dependence required incubation with both TnI and TnC. Relaxation at low Ca²⁺ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca²⁺ sensitivity, i.e. the pCa₅₀, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca²⁺ than those reconstituted with cTnC and cTnI (difference in pCa₅₀ approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G¹⁰⁴-K-F-K-R-P-P-L-R-R-V-R¹¹⁵-). The four mutants produced by substitution of T for P¹¹⁰, G for P¹¹⁰, G for L¹¹¹, and G for K¹⁰⁵ were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S₁ ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca²⁺ dependence could be restored when ^gly110sTnI, ^thr110sTnI or ^gly111sTnI was used for reconstitution. The mutant ^gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca²⁺ concentrations and it caused an increase in Ca²⁺ sensitivity. Received: 5 October 1995/Received after revision and accepted: 1 December 1995     

Blutdruck,relatives Körpergewicht,Alkoholkonsum und Elektrolytausscheidung in der BRD und der DDR: Die Intersalt-Studie

Summary The relationships between body mass index (BMI) and age, alcohol consumption, 24-hr urinary electrolyte excretion, and BP were studied in 588 subjects from three German centers participating inIntersalt, a highly standardized, previously reported protocol. Men and women aged 20–59 were sampled in Bernried, FRG; Cottbus, GDR; and Heidelberg, FRG. The subjects from the three centers did not differ in BMI, level of education, physical activity, cigarette- or alcohol-consumption patterns, or urinary Cl excretion. Mean Na excretion was 167, 147, and 172 mmol/24 hr in Bernried, Cottbus, and Heidelberg, while mean K excretion was 72, 55, and 73 mmol/24 hr, respectively. The excretion of these electrolytes was significantly lower in Cottbus than in Bernried or Heidelberg. BMI increased progressively in men with age; in women BMI plateaued until the 5th decade, after which it increased to equal that of men. In individual centers, the excretion of electrolytes was correlated with BML Sodium and chloride excretion were highly correlated. The data from each individual center were fitted to a multiple regression model. Age, BMI, sex, and alcohol consumption entered the model.

     

Capsaicin-sensitive non-cholinergic excitatory innervation of the guinea-pig tracheobronchial smooth muscle

Field stimulation of the isolated main bronchi of the guinea-pig results in a rapid contraction followed by a sustained contractile response. Tetrodotoxin abolished these effects. The first phase was strongly inhibited by hyoscine, indicating that it was mediated mainly by excitation of cholinergic nerves. The lasting contraction was abolished by capsaicin tachyphylaxis but it was resistant to the effects of hyoscine, hexamethonium or physostigmine. It is suggested that capsaicin-sensitive non-cholinergic nerves have major excitatory effect on the guinea-pig bronchial smooth muscle and there is also evidence for their influence on the trachea.     

An anti-GD2 single chain Fv selected by phage display and fused to Pseudomonas exotoxin A develops specific cytotoxic activity against neuroblastoma derived cell lines

Since the disialoganglioside GD2 is abundantly present on the surface of neuroblastoma cells, we constructed a new recombinant immunotoxin for possible clinical use in patients with neuroblastoma. A functional 14.18 scFv-phage was obtained by selection of an anti-GD2 hybridoma derived phage antibody mini-library on the neuroblastoma-derived, GD2-expressing cell line IMR5. By insertion into the bacterial expression vector pBM1.1 the selected scFv was fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Periplasmically expressed 14.18(scFv)-ETA' bound to the GD2 expressing cell line IMR5, but not to the GD2 negative Hodgkin-derived cell line L540Cy as documented by ELISA and flow cytometry. The recombinant immunotoxin (rIT) inhibited cell viability of IMR5 cells by 50% at concentrations (IC(50)) of 0.326 microg/ml. This recombinant immunotoxin will be further investigated in vivo for its value as a new immunotherapeutic agent for the treatment of patients with neuroblastoma.     

Neuromagnetic investigation of somatotopy of human hand somatosensory cortex

Summary In order to investigate functional topography of human hand somatosensory cortex we recorded somatosensory evoked fields (SEFs) on MEG during the first 40 ms after stimulation of median nerve, ulnar nerve, and the 5 digits. We applied dipole modeling to determine the three-dimensional cortial representations of different peripheral receptive fields. Median nerve and ulnar nerve SEFs exhibited the previously described N20 and P30 components with a magnetic field pattern emerging from the head superior and re-entering the head inferior for the N20 component; the magnetic field pattern of the P30 component was of reversed orientation. Reversals of field direction were oriented along the anterior-posterior axis. SEFs during digit stimulation showed analogous N22 and P32 components and similar magnetic field patterns. Reversals of field direction showed a shift from lateral inferior to medial superior for thumb to little finger. Dipole modeling yielded good fits at these peak latencies accounting for an average of 83% of the data variance. The cortical digit representations were arranged in an orderly somatotopic way from lateral inferior to medial superior in the sequence thumb, index finger, middle finger, ring finger, and little finger. Median nerve cortical representation was lateral inferior to that of ulnar nerve. Isofield maps and dipole locations for these components are consistent with neuronal activity in the posterior bank of central fissure corresponding to area 3b. We conclude that SEFs recorded on MEG in conjunction with source localization techniques are useful to investigate functional topography of human hand somatosensory cortex non-invasively.