The initial vestibulo-ocular reflex and its visual enhancement and cancellation in humans

The gain (ratio of eye velocity to head velocity) of the initial horizontal vestibulo-ocular reflex (VOR) was calculated in 12 normal subjects over 350 ms during impulsive, unpredictable whole body rotation under three conditions: (1) darkness; (2) visual enhancement of the VOR, while the subjects fixated a stationary target; and (3) visual cancellation of the reflex, while subjects fixated a target that rotated with the head. The gain of the initial 80 ms of compensatory eye movement increased significantly during visual fixation in 5 subjects and decreased during attempted VOR cancellation in 3 subjects, when compared with VOR gain in darkness. Compensatory vestibular smooth eye movements were slowed, becoming curved at the onset of VOR cancellation, at mean latencies ranging from 78 to 149 ms in individual subjects (group mean 128 ms). At about 190 ms, quick phases moved the eyes in the same direction as head and target motion. The subsequent vestibular eye movements were about 50% slower than the initial smooth eye movements, indicating more effective cancellation. Visual enhancement of the VOR can occur prior to the onset of pursuit, providing evidence that fixation and smooth pursuit are distinct ocular motor systems. Visual cancellation of the VOR also begins prior to smooth pursuit initiation and becomes more effective after the latency of smooth pursuit.     

Use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms.

Rhinoviruses and enteroviruses are the major members of the picornavirus genus that cause human disease. We compared the polymerase chain reaction and viral culture for the identification of picornaviruses in nasal aspirates from children during episodes of respiratory symptoms and when asymptomatic and from asymptomatic adults. One hundred eight children, aged 9 to 11 years, completed a year-long study. Within 24 to 48 h of a report of respiratory symptoms, a nasal aspirate was taken in the home. Nasal aspirates were also taken from 65 of the children and from 33 normal adults when they had been free of respiratory symptoms for at least 2 weeks. Picornaviruses were isolated by culture for three passages in Ohio HeLa cells in rolling tubes at 33 degrees C and pH 7.0. For the polymerase chain reaction, duplicate 50-microliters samples were amplified with conserved primers from the 5' noncoding region. Picornaviruses generated approximately 380-bp bands in agarose gel electrophoresis; the specificity of these bands was confirmed by filter hybridization with a conserved internal probe. Picornaviruses were isolated by culture in 47 (46 rhinoviruses) of 292 symptomatic episodes (16%), whereas the polymerase chain reaction identified picornavirus genomic material in 146 episodes (50%), including all but one of the culture-positive episodes. As for asymptomatic samples, eight (12%) children and two (4%) adults were positive by the polymerase chain reaction, whereas only one child's specimen was positive by culture. This polymerase chain reaction assay represents a clear advance in the identification of picornavirus infection, with a detection rate threefold greater than the virus culture method.     

Evaluation of an automated immunodiagnostic assay system for direct detection of herpes simplex virus antigen in clinical specimens.

The Vitek ImmunoDiagnostic Assay System (VIDAS) is a 2 1/3-h automated qualitative enzyme-linked fluorescent immunoassay developed for the direct detection of herpes simplex virus (HSV) antigen in clinical specimens. A total of 356 clinical specimens submitted for HSV isolation were prospectively evaluated with the VIDAS, and the results of the technique were compared with those of both HSV isolation in cell culture and Herpchek, a nonautomated enzyme immunoassay. Compared to cell culture, VIDAS had a sensitivity of 91.6% and a specificity of 89.3%, with positive and negative predictive values of 82.6 and 95.0%, respectively. In comparison to Herpchek, VIDAS had a sensitivity of 93.7% and a specificity of 93.0%, with positive and negative predictive values of 89.4 and 95.9%, respectively. The results demonstrated that the VIDAS required minimal manipulation in order to produce results comparable to those of Herpchek and HSV isolation in cell culture.     

Increased aeroallergen-specific interleukin-4-producing T cells in asthmatic adults

BACKGROUND: Asthma, atopy and some forms of respiratory syncytial virus (RSV) disease are thought to be caused by T cells making IL-4 (Th2 cells). However, not all patients with similar patterns of clinical disease have the same underlying pathogenesis and the ability to detect immunopathogenic T cells by examination of the peripheral blood remains in doubt. With the prospect of specific immunotherapy for diseases caused by T cell subsets, it is important to determine whether peripheral blood mononuclear cell (PBMC) reactivity can be used to establish the presence of immunopathogenic responses and therefore to predict therapeutic effects. OBJECTIVE: To detect IL-4 and IFN-gamma production as markers of Th1 and Th2 responses in the peripheral blood of atopic and asthmatic adults. METHODS: PBMC from 22 adult asthmatics (18 of whom were atopic) and 21 non-asthmatic volunteers (ten of whom were atopic) were stimulated with cat, birch and house dust mite allergens, human rhinovirus, RSV and recombinant chimaeric F/G protein from RSV in vitro. ELISPOT assays were used to enumerate cells producing IL-4 and IFN-gamma. RESULTS: Asthmatics had a sixfold increase in frequencies of IL-4-producing cells to cat and birch allergen (median values: 37 vs. 7 per million PBMC, P < 0.01 and 20 vs. 3 per million PBMC, P < 0.04, respectively) compared to non-asthmatics. By contrast, non-asthmatic atopics showed no specific increase in antigen-specific IL-4 responses and there was no evident correlation between skin prick test reactivity and ELISPOT results. Atopics had significantly more IFN-gamma-producing cells specific for FG than nonatopics. while IFN-gamma and IL-4 responses to other antigens were not significantly different. CONCLUSION: Enhanced IL-4 responses to non-viral aeroallergens are seen in adults with asthma, while enhanced IFN-gamma responses to viral antigen FG were see in atopics. In practical terms, ELISPOT assays for specific cytokines may provide a method that could be used to monitor antigen-specific T cell responses in peripheral blood.     

Peritonsillar abscess caused by Nocardia asteroides.

A 22-year-old man with recurrent pharyngitis developed a peritonsillar abscess from which aspirated material yielded a pure culture of Nocardia asteroides. It is likely that the organism was introduced iatrogenically during a prior tonsillar incision. Although unusual, Nocardia species should be considered and microbiological specimens should be handled appropriately in pharyngeal abscesses that respond poorly to conventional therapy.     

Idiotypic relatedness of human monoclonal IgG cryoglobulins.

The serological relatedness of the idiotypic (ID) determinants of one type lambda light chain dimer and fifteen monoclonal IgG cryoglobulins were assayed. Rabbits were immunized with 9/15 IgG cryoglobulins, and twenty-three antisera were obtained and absorbed to render them specific for the ID determinant of the immunizing IgG cryoglobulin. By use of haemagglutination-inhibition, cross-reactivity was detected among five cryoglobulins. This was localized to the Fab region of the IgG, was not related to identity of the variable region subgroups of the heavy and light chains of the cross-reactive cryoglobulins, and was not detected in eighteen non-cryoprecipitable IgG myeloma proteins. The serological relatedness of the ID determinants suggests that a subset of IgG cryoglobulins may possibly have similar variable region structures and/or antigenic specificities.     

A modified Sindbis vector for prolonged gene expression in neurons

Sindbis viruses have been widely used in neurobiology to express a variety of genes in cultured neurons, in cultured slices, and in vivo. They provide fast onset and high levels of expression of foreign genes, but the expression is limited to a short time window due to a shut-off of host protein synthesis. We have used a mutation in an essential gene (nsP2) of the life cycle of Sindbis, which allows the functional analysis of changes in protein expression for >/=6 days after infection. This Sindbis mutant (nsP2) was used to express enhanced green fluorescent protein (EGFP) in hippocampal neurons in culture and in vivo without any sign of toxicity, based on two-photon imaging and electrophysiology. In addition, the EGFP mutant virus can be injected in vivo to visualize spines and other details of neuronal structure. The Sindbis mutant described here provides an improved tool in neurobiology with reduced cytotoxicity and a prolonged time window of expression for novel applications in imaging and behavior. In addition, the use of this vector for the functional expression of mammalian voltage-gated ion channels in organotypic slices is demonstrated.     

Eosinophil granule lysis in vitro induced by soluble antigen antibody complexes

A simple test system is described, for the demonstration of antigen—antibody reactions capable of causing eosinophil granule lysis in vitro. The antigen preparations used were extracts of the nematode Amplicaecum robertsi and body fluid of Ascaris suum. Antisera were obtained from rats infested with Amplicaecum. Eosinophils were obtained from the peritoneal cavity of normal rats. Centrifugation of the cells to form a cell button was an essential step in the procedure. Lysis of eosinophils occurred with antiserum obtained from the animals between the 12th and 32nd days of infestation with Amplicaecum, and was accompanied by vacuole formation in macrophages and mast cell disruption. The reaction was most pronounced during the 3rd week. Serum from adrenalectomized infested animals caused the most marked changes in eosinophils. Serum from cortisonetreated infested animals failed to cause eosinophil changes.

Attempts at purification of the antigen in Ascaris body fluid resulted in two fractions with marked activity in the test system. The same two fractions were found to form precipitin lines on agarose gel diffusion against rat antiserum.

It is postulated that antigen—antibody complexes soluble in low concentration were responsible for the changes observed in the eosinophils, macrophages and mast cells. One or more labile factors in the serum were found to be necessary for eosinophil granule lysis. The evidence, though incomplete, would favour the suggestion that both labile antibody and complement were necessary.

     

Activation of host phospholipases C and D in macrophages after infection with Listeria monocytogenes

Infection of the J774 murine macrophage-derived cell line with Listeria monocytogenes results in several elevations of intracellular calcium during the first 15 min of infection. These appear to result from the actions of secreted bacterial proteins, including phosphatidylinositol-specific phospholipase C (PI-PLC), a broad-range phospholipase C, and listeriolysin O (LLO) (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770-1778, 1999). We have measured hydrolysis of host PI and the activation of host polyphosphoinositide-specific PLC and host phospholipase D (PLD) during infection with wild-type and mutant L. monocytogenes. Elevated hydrolysis of host PI occurred within the first 10 min of infection and was dependent on both bacterial PI-PLC and LLO, both of which were required for the earliest elevations of intracellular calcium in the host cell. A more rapid hydrolysis of host PI was observed at 30 min after infection, at the time when wild-type bacteria have been internalized. Activation of host PLC, also occurred in the first 10 min of infection but was not dependent on the presence of bacterial PI-PLC. Similar observations were made in murine bone marrow-derived macrophages. In J774 cells, activation of host PLD was observed after 20 min of infection and was dependent on bacterial LLO. Mutants in the bacterial phospholipases produced levels of PLD activation similar to those produced by the wild type. Phorbol myristate acetate (PMA) also activated host PLD, while long-term treatment with PMA resulted in loss of the ability of L. monocytogenes to activate host PLD, suggesting an involvement of protein kinase C (PKC) in the activation of PLD. Rottlerin, an inhibitor of PKC delta in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC betaI and betaII, did not. Pretreatment of J774 cells with the PLD inhibitor, 2, 3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.     

Effects of Gabaculine on the uptake,binding and metabolism of GABA

Gabaculine, a conformationally restricted analogue of GABA, is (i) a moderately potent inhibitor (IC₅₀ 69 μM) of the sodium-dependent uptake of GABA in rat brain slices, (ii) ineffective at 100 μM as an inhibitor of the sodium-independent binding of GABA to membranes from rat brain, (iii) a relatively weak inhibitor (IC₅₀ > 1 mM) of glutamate decarboxylase activity in tracts of rat brain, and (iv) a very potent inhibitor (IC₅₀ 3 μM) of the transamination of GABA catalyzed by extracts of rat brain mitochondria. Inhibition of transamination is time-dependent and follows pseudo-first order kinetics, which is consistent with gabaculine acting as a catalytic inhibitor at the active site.