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91.
Mannering SI Dromey JA Morris JS Thearle DJ Jensen KP Harrison LC 《Journal of immunological methods》2005,298(1-2):83-92
T-cell clones are valuable tools for investigating T-cell specificity in infectious, autoimmune and malignant diseases. T cells specific for clinically-relevant autoantigens are difficult to clone using traditional methods. Here we describe an efficient method for cloning human autoantigen-specific CD4+ T cells pre-labelled with CFSE. Proliferating, antigen-responsive CD4+ cells were identified flow cytometrically by their reduction in CFSE staining and single cells were sorted into separate wells. The conditions (cytokines, mitogens and tissue culture plates) for raising T-cell clones were optimised. Media supplemented with IL-2+IL-4 supported growth of the largest number of antigen-specific clones. Three mitogens, PHA, anti-CD3 and anti-CD3+anti-CD28, each stimulated the growth of similar numbers of antigen-specific clones. Cloning efficiency was similar in flat- and round-bottom plates. Based on these findings, IL-2+IL-4, anti-CD3 and round-bottom plates were used to clone FACS-sorted autoantigen-specific CFSE-labelled CD4+ T cells. Sixty proinsulin- and 47 glutamic acid decarboxylase-specific clones were obtained from six and two donors, respectively. In conclusion, the CFSE-based method is ideal for cloning rare, autoantigen-specific, human CD4+ T cells. 相似文献
92.
93.
Dibasic staining of large epoxy tissue sections and applications to surgical pathology 总被引:3,自引:0,他引:3
A variety of normal and pathologic aldehyde-fixed osmium postfixed human tissues were prepared as large sections embedded in Spurr epoxy. They were stained with a sequential basic fuchsin--methylene blue stain which gives "hematoxylin- and eosin-like" staining and additionally functions as several special stains. This technic also allows for electron microscopy directly on the embedded tissue. The histologic and cytologic preservation and overall staining was superior to tissue embedded in glycol methacrylate. The methods and technics presented in this article have important applications in diagnostic surgical pathology and histology in general. 相似文献
94.
Geographical and temporal conservation of antibody recognition of Plasmodium falciparum variant surface antigens 下载免费PDF全文
Nielsen MA Vestergaard LS Lusingu J Kurtzhals JA Giha HA Grevstad B Goka BQ Lemnge MM Jensen JB Akanmori BD Theander TG Staalsoe T Hviid L 《Infection and immunity》2004,72(6):3531-3535
The slow acquisition of protection against Plasmodium falciparum malaria probably reflects the extensive diversity of important antigens. The variant surface antigens (VSA) that mediate parasite adhesion to a range of host molecules are regarded as important targets of acquired protective immunity, but their diversity makes them questionable vaccine candidates. We determined levels of VSA-specific immunoglobulin G (IgG) in human plasma collected at four geographically distant and epidemiologically distinct localities with specificity for VSA expressed by P. falciparum isolates from three African countries. Plasma levels of VSA-specific IgG recognizing individual parasite isolates depended on the transmission intensity at the site of plasma collection but were largely independent of the geographical origin of the parasites. The total repertoire of immunologically distinct VSA thus appears to be finite and geographically conserved, most likely due to functional constraints. Furthermore, plasma samples frequently had high IgG reactivity to VSA expressed by parasites isolated more than 10 years later, showing that the repertoire is also temporally stable. Parasites from patients with severe malaria expressed VSA (VSASM) that were better recognized by plasma IgG than VSA expressed by other parasites, but importantly, VSASM-type antigens also appeared to show substantial antigenic homogeneity. Our finding that the repertoire of immunologically distinct VSA in general, and in particular that of VSASM, is geographically and temporally conserved raises hopes for the feasibility of developing VSA-based vaccines specifically designed to accelerate naturally acquired immunity, thereby enhancing protection against severe and life-threatening P. falciparum malaria. 相似文献
95.
Multimers of soluble major histocompatibility complex class I and II molecules have proven to be useful reagents in quantifying and following specific T cell populations. This study describes the design, generation, and characterization of a novel, single chain I-A(b) molecule which utilizes a unique linker derived from the murine invariant chain. A fragment of the invariant chain, residues 58-85, binds to a region proximal to the class II peptide binding groove and stabilizes occupancy of the class II invariant chain-associated peptide. We have utilized this fragment, replacing CLIP with the Ealpha peptide sequence, to lock the attached peptide into the class II binding groove. The single chain I-A(b) molecule was recognized by a panel of conformation-sensitive, I-A(b)-specific, monoclonal antibodies. Membrane-bound and soluble forms of the single chain I-A(b) stimulated an antigen-specific T cell hybridoma, and tetramers made from soluble monomers stained these cells. The unique features of this molecule may be useful in the design of recombinant T cell receptor ligands containing peptides with low affinity for MHC. 相似文献
96.
97.
Bacterial persistence and immunity in goats vaccinated with a purE deletion mutant or the parental 16M strain of Brucella melitensis. 下载免费PDF全文
N F Cheville S C Olsen A E Jensen M G Stevens A M Florance H S Houng E S Drazek R L Warren T L Hadfield D L Hoover 《Infection and immunity》1996,64(7):2431-2439
To evaluate host responses, young goats were inoculated subcutaneously with a genetic deletion mutant (deltapurE201) of Brucella melitensis (n = 6), its virulent parental strain 16M (n = 6), or saline (n = 6). No clinical evidence of brucellosis was seen in any goat. Serum antibody titers peaked at postinoculation day (PID) 14. Bacteria in lymph nodes that drained sites of vaccination reached peak numbers of >10(6) CFU/g in both infected groups at PID 7 and progressively declined to PID 84. At necropsy, bacteria were present in mammary lymph nodes or spleen of 33% of goats given virulent 16M but in none of goats given the purE mutant. Lymphadenitis, most severe in goats given 16M, involved depletion of lymphocytes and germinal centers, proliferation of lymphoblasts, and vasculitis. By PID 28, lymph node architecture was restored; there was marked germinal center formation and medullary plasmacytosis. Brucellar antigens, detected with immunoperoxidase techniques, were prominent in capsular granulomas but not in lymph node cortices. Ultrastructurally, bacteria were found in macrophages (>97%) and small lymphocytes (<3%) but not in large lymphocytes. Bacteria were intact in small lymphocytes but in macrophages were in various stages of degradation. The deltapurE phenotype of deltapurE201 was preserved during infection of goat lymph nodes. Unlike Salmonella spp. purE mutants, strain deltapurE201 may be a candidate for efficacy testing; it produced immune responses, was cleared from visceral tissues, and produced less severe pathologic changes than its wild-type parent. 相似文献
98.
K. S. Kristensen S. Norn F. Espersen P. Stahl Skov J. Holst Pedersen J. Sparup N. M. Jensen H. Permin 《Inflammation research》1993,38(3-4):C218-C220
Some cytokines are known to affect IgE-mediated basophil histamine release. The effect of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) on human basophil and masct cell histamine release was studies further. Blood leukocytes with approximately 2% basophils from 9 healthy individuals were incubated with recombinant human GM-CSF (0.3–30 U/ml) in combination with A23187 (10–7–10–6
M) or washedStaphylococcus aureus whole bacteria (0.3–2.5 mg/ml). Histamine release was measured spectrofluorometrically. GM-CSF in itself did not induce histamine release. The addition of GM-CSF to cells stimulated with A 23187 caused a dose-dependent enhancement amounting to mean 70% at 3 U/ml and mean 170% at 30 U/ml (P<0.05). GM-CSF enhanced the bacteria-induced histamine release by 30% at U/ml and by 65% at 3 U/ml (P<0.05). The enhancement did not depend on cell-bound IgE or LPS contamination. In preliminary mast cell experiments with lung tissue we did not find an enancing effect of GM-CSF on IgE-mediated histamine release. 相似文献
99.
Use of 125I-secretin to identify and characterize high-affinity secretin receptors on pancreatic acini 总被引:1,自引:0,他引:1
R T Jensen C G Charlton H Adachi S W Jones T L O'Donohue J D Gardner 《The American journal of physiology》1983,245(2):G186-G195
We prepared 125I-secretin and studied the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini prepared from guinea pig pancreas. Iodinated secretin retained intrinsic biological activity in that it was as effective but 2.5-times less potent than native secretin in its ability to bind to pancreatic acini and to increase cellular cAMP. Scatchard analysis of binding of 125I-secretin indicated that each pancreatic acinar cell has approximately 93,000 binding sites, half of which are occupied by 11 nM iodinated secretin. Binding of 125I-secretin was rapid, reversible, saturable, specific, and temperature dependent. Binding of 125I-secretin was inhibited by secretin, vasoactive intestinal peptide, PHI, and Gila monster venom but not by glucagon, gastric inhibitory polypeptide, cholecystokinin, caerulein, gastrin, bovine pancreatic polypeptide, somatostatin, neurotensin, leucine-enkephalin, methionine-enkephalin, carbachol, bombesin, litorin, eledoisin, physalaemin, or substance P. With agonists (secretin, vasoactive intestinal peptide, PHI, or Gila monster venom), as well as antagonists (C-terminal fragments of secretin), there was a close correlation between their relative potencies for inhibiting binding of 125I-secretin and their relative potencies for increasing cAMP (agonists) or inhibiting the secretin-induced increase in cAMP (antagonists). For a given agonist, however, a 40-fold higher concentration was required for half-maximal inhibition of binding of 125I-secretin than was required to produce a half-maximal increase in cellular cAMP. Thus, maximal stimulation of cellular cAMP occurs when approximately one-third of the secretin receptors are occupied by an agonist. 相似文献
100.
Transient and sustained types of long-term potentiation in the CA1 area of the rat hippocampus 总被引:2,自引:0,他引:2
Synaptic potentiation induced by high frequency stimulation was investigated by recording field excitatory postsynaptic potentials (f-EPSPs) in rat hippocampal slices. Potentiation consisted of a transient period of decaying f-EPSPs (short-term potentiation, STP) that led to a plateau of continuously potentiated f-EPSPs (long-term potentiation, LTP). Here we show that a previously unknown type of transient, use-dependent, long-lasting potentiation (t-LTP) can account for STP. t-LTP could be stored for more than 6 h and its decay was caused by synaptic activation. Both the expression and the decay of t-LTP were input specific. t-LTP was induced differently from conventional LTP in that the amplitude of t-LTP was dependent upon the stimulation frequency, whereas the magnitude of LTP was dependent on the number of stimuli in the induction train. The decay of t-LTP could not be prevented by the blockage of glutamate receptors, but was prevented by the blockage of stimulus-evoked neurotransmitter release, suggesting that t-LTP is expressed presynaptically. Paired-pulse stimulation experiments showed that the decay of t-LTP was mediated by a decrease in the probability of neurotransmitter release. The decline of t-LTP could be prolonged by the activation of NMDA receptors. Hence, both single and paired-pulse stimuli prolonged the decline of the t-LTP. This decline could be prevented by high frequency burst stimulation (200 Hz). We conclude that t-LTP allows dynamic modulation of synaptic transmission by providing not only spatial association but also temporal convergence between synaptic inputs. Therefore, t-LTP might be a substrate for the encoding of synaptic memory. 相似文献