CD4+ T cells play an important role in the induction and maintenance of an effective antiviral and antitumor immune response. However, standardized monitoring of antigen-specific CD4+ T cells has not been established at the single-cell level. We now present a sensitive, specific, and simple methodology in which purified memory CD4+ T cells are expanded from PBMC in a single cycle of antigen-driven stimulation and quantitatively assayed by interferon-gamma ELISPOT. Issues of nonspecific background in assays were resolved with the use of innovative target cells, autologous PHA-expanded CD4+ T cells (T-APC). Remarkably, T-APC could not only present peptide epitopes from model antigens NY-ESO-1 and influenza nucleoprotein, but could also process full-length antigen endogenously expressed from recombinant fowlpox vector. This approach makes it possible to monitor CD4+ T cells in large series of patients, regardless of HLA haplotype, against the full peptide repertoire of a given antigen. 相似文献
Monoclonal antibody (mAb) MR6 recognises a 200 kDa glycoprotein, gp200-MR6, which is expressed at high levels on the surface of human thymic cortical epithelium. In order to produce further mAbs against the gp200-MR6 molecule, mice were immunised with purified human gp200-MR6, hybridomas produced and supernatants screened for MR6-like reactivity on human thymic sections. Surprisingly this conventional hybridoma technique failed to produce stable hybridoma cells producing MR6-like antibodies. However, antibodies with specificities other than MR6-like were obtained. Three such antibodies (1B2, 3A3 and 4B3) were analysed further. Expression of 1B2-antigen, 3A3-antigen and 4B3-antigen was analysed on skin, tonsil and thymic sections, on cultured thymic epithelial cells (TEC), thymocytes and peripheral blood mononuclear cells (PBMC), and found to be expressed by both lymphocytes and epithelial cell populations. Furthermore, the antigens were also expressed on mouse thymic epithelial cells. The regulation of expression of these antigens was analysed following mitogen or cytokine stimulation of PBMC and cultured TEC, respectively. Expression on T cells was clearly affected by mitogens that mimic activation through the T cell receptor and expression on cultured TEC was affected by T cell-derived cytokines. Thus, the shared epithelial-lymphocyte molecules identified in this study may play a role in the cross-talk between the developing thymocytes and their epithelial microenvironment. The production of mAbs with specificities other than that of purified gp200-MR6 indicates that a wide range of B cells with specificity for components of the human thymic microenvironment exist in the normal mouse. These may detect epitopes that are shared with common pathogens to which the animals are exposed. Alternatively, they may be autoreactive B cells that are normally silent in the absence of T cell help. This help may be provided by T cells specific for human gp200-MR6, or nonspecifically by polyclonal activation induced by the adjuvant. 相似文献
A thermodynamic study is presented of temperature distributions created by an inductively heated 6-mm-diam Ni sphere imbedded in vivo and in vitro into porcine brain tissue. This study was performed in support of the development of a system that creates localized heat-induced lesions in deep-seated brain tumors. In this system, a magnetic "seed" will be remotely repositioned within the brain by an externally produced magnetic field. Convective effects of a hot moving seed will produce a different thermodynamic situation than that arising from an array of static implants. In this work, a study is presented of part of the expected change, in which a static sphere is heated to high temperature. Measurements were made of the temporal and spatial dependence of the temperature rise in the vicinity of the heated sphere, in vivo in four animals and in one that was euthanized immediately prior to experimentation. These results are used for parameter estimation with a theoretical model based on a point source solution to a form of the thermal diffusion equation, i.e., the "bioheat transfer equation." With this model thermal distributions from a power source of arbitrary geometry can be found using appropriate integration methods, and the method has widespread applicability. Estimates of blood flow rates, tissue thermal conductivity, and seed power absorption were found using the parameter estimation algorithm. The estimated blood perfusion exhibits a step increase following the first heating in multiple heating experiments. Thermal conductivity estimated using data from the nonperfused (in vitro) animal is 0.6 W/m degrees C. Seed power absorption is estimated correspondingly to be 0.9 W, a result confirmed independently with calorimetry. Statistical uncertainty is established for the radial decrease of the tissue temperature rise created by this method. This result allows estimation of a cell death boundary uncertainty of 0.6 mm, caused by fluctuations in power delivered to the seed, uncertainty in the temperature probe placements, and thermal properties such as blood perfusion and tissue thermal conductivity. 相似文献
Solid mesoionic 2‐[2‐(isopropenylcarbonyloxy)ethylthio]‐1‐methyl‐6‐oxo‐3‐phenyl‐5‐propyl‐1,6‐dihydropyrimidin‐3‐ium‐4‐olate was complexed in water using β‐cyclodextrin (β‐CD) and randomly methylated β‐CD, which resulted in polymerizable complexes with 2:1 stoichiometry. The β‐CD complex was characterized using 1H NMR, ROESY NMR and UV spectroscopy. Polymerization of the complex prepared from methylated β‐CD led to a photosensitive polymer, which precipitated during polymerization and was nearly free of CD. Polymerization was carried out with a water‐soluble redox initiator. In addition, a copolymer with methyl methacrylate was prepared from the complexes, which showed a different mass‐dependent distribution in the incorporation in comparison to a copolymer prepared without CD in organic solvents.
The chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells. 相似文献
The ability of antigen-specific T-helper (Th) cells to enhance direct plaque-forming cell responses in spleen cells from Trypanosoma cruzi-infected C57BL/6 mice was investigated at various times during the course of infection from day 7 to day 230. The injection of antigen-specific Th cells in vivo or the addition of antigen-specific Th cells in vitro was effective in enhancing direct plaque-forming cell responses, except at the time of the most intense suppression during the acute phase of infection (i.e., day 28). The ability of antigen-specific Th cells to overcome nonspecific immunosuppression was due not only to the activity of antigen-specific Th cells added to Mishel-Dutton cultures but also to activation of resident T cells. Thus, antigen-specific Th cells and resident T cells act in concert to produce enhanced direct plaque-forming cell responses. The effect of plastic-adherent spleen cells from infected mice on the ability of antigen-specific Th cells to stimulate anti-sheep erythrocyte responses of normal spleen cells was examined because macrophages have been shown to have an immunoregulatory role during the course of experimental American trypanosomiasis. Increasing numbers of macrophages from infected mice caused increased immunosuppression of normal spleen cells that could not be overcome with the addition of primed Th cells. It can be concluded from these data that antigen-specific Th cells can potentiate immune responses in mice infected with T. cruzi but that highly active suppressor macrophages can inhibit the expression of these primed Th cells. 相似文献
Gene therapy is an interesting approach for the correction of defective genes, the treatment of cancer and the introduction of immunomodulatory genes. Various techniques for gene transfer into cells or tissues have been developed within the last decade; these can be divided generally into viral and nonviral gene transfer systems. Nonviral techniques include the liposome- or gene gun-mediated introduction of therapeutic genes; however, the efficiency of gene transfer by these applications is still very low. In contrast, viruses have optimised their strategies for efficient infection of virtually any cell type in a mammalian organism. The genetic modification of genomes from different virus families (Adenoviridae, Retroviridae, Herpesviridae) led to the development of gene therapy vectors with a similar capacity to infect cells or tissues as that of wild type viruses. In contrast to wild type viruses, gene therapy vectors are engineered to transfer therapeutic genes into the target cells or tissues. In addition, they have lost their capacity for replication in target cells, because of the removal of essential genes, which allows replication only in specialised packaging cell lines engineered for the production of recombinant viruses. Despite considerable progress over the past decade in the generation of gene transfer systems with reduced immunogenic properties, the remaining immunogenicity of many gene therapy vectors is still the major hurdle, preventing their frequent application in clinical trials. Recombinant adenoviruses have been shown to be promising vectors for gene therapy, since they are able to transduce both quiescent and proliferating cells very efficiently. However, a major disadvantage of adenoviral vectors lies in the activation of both the innate and adaptive parts of the recipient's immune system when applied in vivo. The inflammatory responses induced by adenovirus particles can be very strong and can be fatal in patients treated with these adenoviral constructs. Therefore, many experiments have been performed in the effort to prevent these inflammatory responses mediated by adenoviral particles. The depletion of cell populations responsible for these inflammatory responses as well as the application of immunosuppressive drugs have been investigated. Moreover, the generation of less immunogenic adenoviral vectors by further genetic modification within the adenoviral genome has led to vectors with reduced immunogenic properties. Both strategies to reduce inflammatory responses against adenoviral particles are discussed in this review. 相似文献
Full-length DNA clones representing the 10 double-stranded RNA segments of US bluetongue virus serotype 10 (BTV-10) have been used in a study to determine the genetic relationships among 20 different BTV serotypes. The study was undertaken using Northern blot hybridization techniques involving 32P-labelled DNA probes and total RNA species extracted from BHK-21 cells infected with 20 different BTV serotypes. The results obtained indicate that all the genes representing the nonstructural proteins of BTV (NS1, NS2 and NS3) as well as most of the inner capsid polypeptides are highly conserved (e.g., VP1, VP3, VP4), while VP6 and VP7, the remaining two inner capsid components, are less conserved. The genes representing the two outer capsid polypeptides, VP2 and VP5, vary significantly. When complete DNA clones of RNA segment 2 (representing the VP2 neutralization gene) of 4 other US serotypes (BTV-2, -11, -13 and -17) and one Australian serotype (BTV-1) were used in similar hybridization studies, the data obtained showed that despite geographical distances, a certain BTV serotype exhibits similarities. Some hybridization signals were detected with several of the inner capsid genes and the corresponding RNA segments of epizootic hemorrhagic disease virus (EHDV), a distantly related orbivirus, although none of the BTV outer capsid genes, nor any of the nonstructural genes hybridized with either EHDV-1 or EHDV-2 RNA species. 相似文献
The activities of aldehyde dehydrogenases using benzaldehydeand propionaldehyde as substrates and NADP and NAD as coenzymeswere determined in normal liver, hepatocyte nodules and hepatocellularcarcinomas from male Wistar rats. Hepatocyte nodules were producedby intermittent exposure of rats to 0.05% 2-acetylaminofluoreneor by initiation with diethylnitrosamine followed by selectionusing 2 weeks of dietary exposure to 0.02% 2-acetylaminofluoreneand partial hepatectomy. The activities of propionaldehyde:NAD and benzaldehyde: NADP aldehyde dehydrogenases were increasedin hepatocyte nodules of all types as well as in most hepatocellularcarcinomas. The most prominent elevation of enzyme activitywas found in the cytosol of persistent hepatocyte nodules (3560times) and some hepatocellular carcinomas (92 times) using benzaldehydeand NADP. The benzaldehyde: NADP aldehyde dehydrogenase activityvaried considerably between different nodules suggesting theexistence of a subpopulation of hepatocyte nodules with veryhigh enzymatic activities. The activity of propionaldehyde:NAD aldehyde dehydrogenase activity as well as of -glutamyltransferasedid not show substantial internodular variations. The activityof benzaldehyde: NADP aldehyde dehydrogenase in individual carcinomasinvestigated in these experiments varied extensively. The datadid not support the idea that all hepatomas had been developedfrom pre-neoplastic nodules with very high activity of thisenzyme. 相似文献