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Defective TCR stimulation in anergized type 2 T helper cells correlates with abrogated p56(lck) and ZAP-70 tyrosine kinase activities.

Development of IgE-mediated allergic conditions is dependent on the secretion of a Th2 cytokine pattern, including IL-4, IL-5, and IL-13. The induction of anergy would be one mechanism to abrogate cytokine secretion by Th2 cells, which may be pivotal to the allergic response. We demonstrate here that incubation of cloned human CD4+ phospholipase A2 (PLA)-specific Th2 cells with antigenic peptide, in the absence of professional APC, results in a state of nonresponsiveness. The anergic T cells failed to proliferate or secrete IL-4 in response to optimal stimulation with PLA and autologous, professional APC. Secretion of IL-5 and IL-13, however, was only partially inhibited. The anergic state of the Th2 cells was not associated with CD3 or CD28 down-regulation. However, anergy did appear to be closely related to alterations in signaling pathways, mediated through the TCR, of the cells. In contrast to untreated Th2 cells, anergized Th2 cells failed to respond to anti-CD3 mAb with either increased tyrosine kinase activity or increased levels of tyrosine phosphorylation of p56(lck) or ZAP70. A strong and sustained intracellular calcium flux, observed in untreated Th2 cells in response to anti-CD3 mAb, was absent in anergic Th2 cells. Furthermore, the induction of anergy seems to represent an active process, associated with increased levels of basal tyrosine kinase activity, cytokine production, and CD25 up-regulation in anergic Th2 cells. Together, our results indicate that anergy in Th2 cells is associated with defective transmembrane signaling through the TCR.

Antigen Presentation↗

pMPY-ZAP: a reusable polymerase chain reaction-directed gene disruption cassette for Saccharomyces cerevisiae.

Gene disruption is an important method for genetic analysis in Saccharomyces cerevisiae. We have designed a polymerase chain reaction-directed gene disruption cassette that allows rapid disruption of genes in S. cerevisiae without previously cloning them. In addition, this cassette allows recycling of URA3, generating gene disruptions without the permanent loss of the ura3 marker. An indefinite number of disruptions can therefore be made in the same strain.

Base Sequence↗

'Zap axotomy': localized fluorescent excitation of single dye-filled neurons induces growth by selective axotomy.

The response of populations of neurons to axotomy has traditionally been studied by crushing or sectioning whole nerve trunks. The present communication describes a technique by which single neurons can be reliably and selectively axotomized in the absence of damage to other axons and non-neuronal cells within the nerve. To obtain selective axotomy, identified neurons of the buccal ganglia of the snail, Helisoma, were first filled with fluorescent dye. Next, the preparation was positioned in a restricted beam of blue light using low light video fluorescence microscopy. Finally, the selected region of axon was briefly exposed to light levels normally employed for fluorescence microscopy. Shortly after irradiation of the identified neuron 5, antidromic action potentials no longer propagated past the region of exposure in the dye-filled cell, whereas adjacent axons were physiologically intact. Several days after exposure, profuse neurite outgrowth was observed from the proximal region of axon of neuron 5, but never in neighboring axons which were not filled with dye at the time of irradiation. When the axons of both neurons 5R and 5L were spot irradiated neurite outgrowth resulted in the formation of a novel electrical connection between these cells. These changes in growth and connectivity which were induced by selective axotomy of single axons were indistinguishable from the changes which are produced by crushing entire nerve trunks.

Animals↗

Zap! Magnetic tricks on conscious and unconscious vision.

Blindsight, the remarkable capability to react to unseen visual stimuli, has thus far only been demonstrated in patients and monkeys with a lesion to primary visual cortex. A recent study by Boyer, Harrison and Ro demonstrates blindsight in normal human observers, using TMS to block visual processing. Combined with other work, this opens new vistas towards understanding mechanisms of conscious and unconscious vision.

Animals↗

Multicentre assessment of linezolid antimicrobial activity and spectrum in Europe: report from the Zyvox antimicrobial potency study (ZAPS-Europe).

OBJECTIVE: To evaluate the in vitro spectrum and activity of linezolid, a recent oxazolidinone, according to well-controlled surveillance data from 42 medical centers in 13 countries throughout Europe. METHODS: Participants tested the susceptibility of 125 clinical strains of enterococcal and staphylococcal species against 13 drugs using reference broth microdilution trays or the standardized disk diffusion method of the National Committee for Clinical Laboratory Standards (NCCLS). Streptococcal species (n = 25 at each center) were tested against six drugs using E test (AB BIODISK, Solna, Sweden). Quality assurance testing was conducted using NCCLS-recommended strains and verification of resistance to linezolid and other selected agents was performed by retesting strains at the regional (Europe) and international (USA) monitor sites. RESULTS: A total of 5598 strains from throughout Europe (91% compliance) were tested. Vancomycin resistance was reported in only 0.6 and 3.0% of Enterococcus faecalis and E. faecium, respectively. Penicillin resistance occurred in 25.1% of Streptococcus pneumoniae; 4.9% at the high-level (> or =2 mg/L). The MIC90 for linezolid was 1 mg/L for streptococci and 2 mg/L for enterococci and staphylococci. Using the US FDA- and EUCAST-recommended susceptible breakpoints for linezolid, there were no confirmed reports of linezolid resistance [minimum inhibitory concentration (MIC), > or =8 mg/L]. The distribution of linezolid MIC values was unimodal and varied between 0.25 and 1 mg/L for streptococci (>90% of isolates), and between 1 and 2 mg/L for staphylococci (>90%) and enterococci (>95%). There were no differences in linezolid susceptibility in the vancomycin-, oxacillin-, or penicillin-resistant subsets of strains when compared to susceptible organism populations. CONCLUSIONS: Compared to the North American component of this study, there was substantially less vancomycin resistance among E. faecium isolates (Europe 3.0% vs. North America 63.4%). While the occurrence of penicillin-resistant S. pneumoniae in Europe and North America was similar (25.1% vs. 29.7%), the recovery of high-level penicillin-resistant strains was nearly three-fold higher in North America (4.9% vs. 13.2%). Only linezolid was universally active against all the tested Gram-positive isolates at </=4 mg/L.

Acetamides↗