Role of antibody and complement in opsonization of group B streptococci.
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A requirement for the classic complement pathway in opsonization of group B streptococci was observed by using both a chemiluminescence and a radiolabeled bacterial uptake technique. The classic pathway increased levels of opsonization for types Ia and II stock and wild strains and for some type III wild strains. In contrast, other type III wild strains and the type III stock strain had accelerated kinetics of uptake in the presence of an intact classic pathway, but the level of opsonization was unchanged from that with antibody alone. We could not demonstrate a significant role for the alternative pathway in opsonizing stock or wild strains of group B streptococci. Futhermore, electrophoretic and complement consumption analysis by hemolytic titration failed to reveal alternative pathway activation by the majority of strains of this group. Therapy aimed at supplying opsonins for these organisms will require the presence of type-specific antibody. (+info)
Calcium fluxes in single muscle fibres measured with a glass scintillator probe.
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1. An intracellular glass scintillator (Caldwell & Lea, 1973) has been used to obtain a continuous record of the influx of 45Ca into single muscle fibres of the barnacle, Balanus nubilus. 2. In the presence of intracellular EGTA (final concentration greater than 3 mM/kg), the scintillator detected an initial fast phase to the influx (half-time = 18.3 min, compartment size = 4.1% fibre volume) followed by a slow, linear phase which gave a value for the Ca influx of 1.2 p-mole . cm-2 . sec-1. The efflux of 45Ca was also measured with the scintillator by transferring a 45Ca-loaded fibre into 45Ca-free saline. Two exponential phases of efflux were detected with half-times of 16.2 and 500 min. 3. The characterisitics of the fast phase of the influx and efflux are similar to those of the influx of the impermeant sucrose and inulin, suggesting that the fast phase represents exchange with the extracellular 'cleft space'. This phase was insensitive to external La3+ (2 mM). 4. The slow phase is considered to represent the flux of Ca across the surface membrane. It was inhibited by external La3+ (2 mM) and stimulated by replacing external Na+ with Li+. 5. When EGTA-injected fibres were depolarized using an axial, intracellular electrode the Ca influx, measured from the slow phase, was increased. At higher concentrations of intracellular EGTA (6--22 mM/kg), the extra Ca influx due to a rectangular, depolarizing current pulse was proportional to the number of Ca spikes it produced. A single Ca spike gave an extra Ca influx of 19--48 p-mole . cm-2. External D600 (5 x 10(-4)M) inhibited both Ca spike and the extra Ca influx. 6. At lower intracellular EGTA concentrations (3.6--11 mM/kg), a 50 mV depolarization of 250 msec duration gave a mean extra Ca influx of 80 p-mole . cm-2. The upper value was 145 p-mole . cm-2 and this would increase the total internal Ca by 4.1 micrometer/kg. It is calculated that if all this extra Ca was bound to the myofibrillar sites for tension, it would only produce 6.2% of the force expected for a similar depolarization in a fibre with no intracellular EGTA. (+info)
Optimization of a new scintillation gas detector used to localize electrons emitted by 99mTc.
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We have developed a scintillation gas detector to localize electrons emitted by 99mTc. This type of detector allows direct quantification of images and so provides a clear advantage over autoradiographic film. We have optimized the device to give an image spatial resolution that closely approximates that of typical autoradiographic film. To improve this resolution, it was necessary to select only low-energy electrons (2 and 15 keV) and to devise novel detection and localization techniques for the ionizing particles. METHODS: A parallel-plate proportional avalanche chamber is subject to a uniform electrical field and amplifies the number of released electrons through collisions of ionizing particles in the gas mixture. Light emitted by the gas scintillator during the avalanche process is collected by a highly intensified charge coupled device camera. The centroid of each resulting light distribution is calculated, resulting in a quantitative mapping of the sample's activity. Insertion of the sample within the gas volume improves the efficiency and so provides a method that is both very sensitive and linear. RESULTS: We have shown that in a parallel-plate structure, the application of a high electrical field to the surface of the sample and the selection of appropriate light spots, according to their morphology, can overcome localization errors due to the particles' trajectories. We have obtained a resolution of the order of 30 microm, using electrons from 99mTc. CONCLUSION: This detection technique allows considerable improvement in image resolution. This "electron camera" is a serious rival to existing autoradiographic techniques, because it provides certain other advantages, including direct quantification, linearity, high dynamic range and low noise levels. Thus, new perspectives are made available in quantitative double tracer autoradiography, because electrons can be selected for imaging as a function of their energy. (+info)
Growth and differentiation of cultured fetal hepatocytes isolated various developmental stages.
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We examined the relationship between cell proliferation and differentiation of cultured rat fetal and newborn hepatocytes isolated from various developmental stages. The albumin production rate increased along with cell growth under in vitro culture and became maximal two days after the growth cessation. AFP was secreted by both fetal and newborn hepatocytes with growth ability. Furthermore, the responses to HGF addition in fetal hepatocyte cultures were observed in terms of growth stimulation and down-regulated of the Met receptor. We also studied the changes in RB and liver enriched transcription factors (C/EBPs) for investigating the mechanism underlying proliferation and differentiation of fetal hepatocytes. Western blot analysis of hepatocytes taken from various gestation stages of rat liver showed that the expression of RB and C/EBP beta increased as gestation stage proceeded. When RB antisense S-oligonucleotide was added to the culture medium, proliferation and AFP expression increased, while C/EBP alpha and albumin expressions decreased. These results indicated that the tumor suppressor gene product RB had a profound role not only in cell proliferation but also hepatocyte differentiation. (+info)
Highly Th2-skewed cytokine profile of beta-lactam-specific T cells from nonatopic subjects with adverse drug reactions.
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A positive lymphocyte transformation test to beta-lactams (beta-L) was found in 12 of 29 subjects with adverse drug reaction (ADR) to beta-L, irrespective of either the type of clinical manifestation or the presence of specific serum IgE. Short-term T cell lines specific for penicillin G, amoxicillin, and ampicillin could be generated only from subjects with ADR (eight with positive and one with negative lymphocyte transformation test), while streptokinase and Dermatophagoides pteronyssinus group 1 (Der p 1)-specific T cells were obtained from all these subjects, from 7 atopic Der p-sensitive donors without history of ADR and 17 healthy nonatopic donors. Streptokinase-specific T cells from all subjects showed intracellular expression of IFN-gamma with poor or no IL-4, whereas Der p 1-specific T cells exhibited IFN-gamma but low or no IL-4 expression in nonatopics, and remarkable IL-4 expression in atopic donors. By contrast, all penicillin G-, ampicillin-, and amoxicillin-specific short-term T cell lines showed high intracellular expression of IL-4, IL-5, and IL-13, but poor or no expression of IFN-gamma, thus exhibiting a clear-cut Th2 profile. Accordingly, most penicillin G-specific T cell clones derived from two subjects with ADR released high concentrations of IL-4 alone or IL-4 and IFN-gamma. These data suggest that cytokines produced by Th2 cells play an important role in all beta-L-induced ADR, even when late clinical manifestations occur and an IgE-mediated mechanism is apparently indemonstrable. (+info)
Human immune responses to the highly repetitive Plasmodium falciparum antigen Pf332.
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The B and T cell responses to EB200, a repetitive part of the Plasmodium falciparum antigen Pf332, were examined in malaria-exposed Senegalese adults. Most donors had high levels of antibodies to recombinant EB200 and 17 overlapping peptides spanning EB200. Taking proliferation and/or cytokine (interferon-gamma and interleukin-4) production as a measure of T cell activation, eight of the EB200-derived peptides induced responses in > 40% of the donors tested. There was no general association between the different types of T cell responses measured, emphasizing the importance of including multiple parameters when analyzing T cell responses and suggesting that EB200 induces functionally distinct T cell responses. The most efficient peptide for induction of proliferative responses was one previously shown to induce T cell responses in five different H-2 congenic mouse strains primed with EB200, suggesting that this is a universal T cell epitope. The presence of multiple B and T cell epitopes in EB200, widely recognized by humans, is important since EB200 has been shown to elicit protective antibody responses in monkeys and may be considered for inclusion in malaria subunit vaccines. (+info)
Determination of different amino sugar 2'-epimerase activities by coupling to N-acetylneuraminate synthesis.
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A new procedure for quantitating the amount of N-acetyl-D-mannosamine (ManNAc) or ManNAc-6-phosphate produced by 2'-epimerase activities involved in sialic acid metabolism has been developed. The ManNAc generated by the action of N-acetyl-D-glucosamine (GlcNAc) and UDP-GlcNAc 2'-epimerases is condensed with pyruvate through the action of N-acetylneuraminate lyase and the sialic acid released is measured by the thiobarbituric acid assay. For the analysis of prokaryotic GlcNAc-6-phosphate 2'-epimerase, ManNAc-6-phosphate can also be evaluated by this coupled assay after dephosphorylation of the sugar phosphate. This system provides a sensitive, rapid, reproducible, specific and simple procedure (feasible with commercial reagents) for measuring amino sugar 2'-epimerases from eukaryotic and prokaryotic sources. The technique reported here permitted us to detect UDP-GlcNAc 2'-epimerase and GlcNAc 2'-epimerase in mammalian cell extracts and GlcNAc-6-phosphate 2'-epimerase in bacterial extracts. (+info)
T-cell mediated autoimmunity to the insulinoma-associated protein 2 islet tyrosine phosphatase in type 1 diabetes mellitus.
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The target molecules of the T-cell response in type 1 diabetes, despite their pathogenic importance, remain largely uncharacterized, especially in humans. Interestingly, molecules such as insulin and glutamic acid decarboxylase (GAD) have been shown to be a target not only of autoantibodies, but also of autoreactive T-lymphocytes both in man and in the non-obese diabetic (NOD) mouse. In the present study we aimed to determine the existence of a specific T-cell response towards the insulinoma-associated protein 2 (IA-2) islet tyrosine phosphatase, a recently identified autoantigen which is the target of autoantibodies strongly associated with diabetes development. Human recombinant IA-2 produced in Escherichia coli, was tested for its reactivity with peripheral blood lymphocytes obtained from 16 newly diagnosed type 1 diabetic patients and from 25 normal controls, 15 of whom were HLA-DR-matched. A T-cell proliferation assay was performed in triplicate employing freshly isolated cells in the absence or in the presence of the antigen to be tested (at two different concentrations: 2 microg/ml and 10 microg/ml). A specific T-cell proliferation (defined as a stimulation index (S.I.) >/=3) was observed against IA-2 used at a concentration of 10 microg/ml (but not of 2 microg/ml) in 8/16 diabetic patients, in 1/15 HLA-DR-matched control subjects (P<0.01 by Fisher exact test) and in 0/10 of the remaining normal individuals. A statistically significant difference (P<0.003 by Mann-Whitney U test) was also observed in S.I. values between patients (3.1+/-1.4) and HLA-DR-matched controls (1.7+/-0.54) employing IA-2 at a concentration of 10 microg/ml. However, when IA-2 was used at a concentration of 2 microg/ml, the difference in S. I. between patients (1.65+/-0.8) and controls (1.0+/-0.3) did not reach statistical significance. In conclusion, these data show the presence of a specific, dose-dependent T-lymphocyte response against the IA-2 islet tyrosine phosphatase at the onset of type 1 diabetes. Consequently, this molecule appears to be a target not only at the B-lymphocyte but also at the T-lymphocyte level, reinforcing the potential pathogenic role of this autoantigen in the islet destructive process. (+info)