Biomedical subjects
Publications and source records attributed to Q Yi.
This paper applied a typical artificial neural network model, that is Back-Propagation Model, to the diagnose and classify research on functional disease of the esophagus, and achieved good results in clinical practice.
We use mass spectrometry in conjunction with hydrogen-deuterium exchange and NMR to characterize the conformational dynamics of the 62-residue IgG binding domain of protein L under conditions in which the native state is marginally stable. Mass spectra of protein L after short incubations in D2O reveal the presence of two distinct populations containing different numbers of protected protons. NMR experiments indicate that protons in the hydrophobic core are protected in one population, whereas all protons are exchanged for deuterons in the other. As the exchange period is increased, molecules are transferred from the former population to the latter. The absence of molecules with a subset of the core protons protected suggests that exchange occurs in part via a highly concerted transition to an excited state in which all protons exchange rapidly with deuterons. A steady increase in the molecular weight of the population with protected protons, and variation in the exchange rates of the individual protected protons indicates the presence of an additional exchange mechanism. A simple model in which exchange results from rapid (> 10(5)/s) local fluctuations around the native state superimposed upon transitions to an unfolded excited state at approximately 0.06/s is supported by qualitative agreement between the observed mass spectra and the mass spectra simulated according to the model using NMR-derived estimates of the proton exchange rates.
In this paper, the results of some recent studies on idiotype-specific T cells in human multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) are discussed. By using different in vitro measurements such as 3H-thymidine incorporation and ELI-SPOT assay, idiotype-specific T cells have been demonstrated in most of MM and MGUS patients. Based on the cytokine-secretion profiles, idiotype-specific T cells were found to comprise both Th1 and Th2 cells. A Th1 type immunity was found preferentially in indolent disease and a Th2-like response predominated in advanced MM, suggesting a specific T-cell regulation of the tumor B-cell clone. The mode of T-cell recognition of id determinants on M-components has been studied. We found that idiotype-specific T cells recognized processed id determinants presented by MHC class II (HLA-DR) molecules on APC. B cells were much more efficient APC than monocytes. With the aim to induce or to amplify an idiotype-specific T-cell response, we have immunized MM patients with the autologous M-component precipitated in aluminum. Three out of the five patients showed an induction of specific cellular and humoral immunity. Nevertheless, the role for such immunity in controlling the tumor clone remains to be established.
Multiple myeloma is characterized by a proliferation of clonal B lymphocytes and plasma cells. The idiotypic structure of clonal immunoglobulin (Ig) expressed on the tumour B-cell surface can be regarded as a tumour-specific antigen and, as such, a potential target for anti-idiotypic T and B-cells in an immune regulation of the tumour-cell clone. Active immunization using the autologous mono-clonal Ig as a 'vaccine' was shown to induce tumour-specific immunity in murine B-cell tumours and in human B-cell lymphoma. With the aim to induce or amplify an anti-idiotypic response in multiple myeloma, five stage I-III patients were repeatedly immunized with the autologous monoclonal IgG. Induction of idiotype-specific cellular immunity was analysed in vitro by an enzyme-linked immunospot assay (interferon-gamma and interleukin-4 secreting cells). B cells secreting anti-idiotypic IgM antibodies were also analysed. An anti-idiotypic T-cell response was amplified 1.9-5-fold in three of the five patients during immunization. The number of B cells secreting anti-idiotypic antibodies also increased in these three patients. In two of the patients induction of idiotype-specific immunity was associated with a gradual decrease of blood CD19+ B cells. The induced T-cell response was eliminated during repeated immunization. Further studies are warranted to optimize the immunization schedule in order to achieve a long-lasting T-cell immunity against idiotypic determinants on the tumour clone. A role for immunity in controlling the tumour clone remains to be established.
Eighteen percent of patients with myasthenia gravis (MG) have serum antibodies against a synthetic peptide corresponding to the second extracellular loop of the human beta 2AR (residues 172-197). In this study we examined T and B cell responses to the peptide, using assays to detect individual cells secreting interferon-gamma (IFN-gamma) and IL-4 or antibodies against the peptide, and by measuring thymidine incorporation in response to the peptide. The peptide from the beta 2AR induced cytokine secretion from blood mononuclear cells in 67% of MG patients, compared with 14-28% of the control groups. Cells secreting antibodies binding to the peptide were present in 54% of MG patients and in 19-28% of controls. The numbers of beta 2AR-reactive cells were higher in MG patients than in controls. Peptide-induced increase in thymidine incorporation in cells was also more frequently demonstrated in patients (26%) compared with controls (about 10%). Activation of cells was dependent on monocytes and on MHC class II DR antigen. Based on the pattern of the cytokine secretion induced, beta 2AR-reactive T cells comprise both T helper type-1 and type-2 subsets. In addition, control peptide-reactive T and B cells were much less frequently demonstrated in the patients, and the number of such cells did not differ between the groups. Our results show that beta 2AR-reactive cells are present in most patients with MG. Such autoreactive antibodies and cells might play a role in the pathogenesis of the disease by influencing the function of skeletal muscle and immune systems.
An important and yet unresolved question concerns the mode of T cell recognition of idiotypic epitopes on immunoglobulin molecules in humans. Results from murine and human studies show that some idiotype-specific T cells recognize conformational epitopes on immunoglobulin, and such T cells are not MHC-restricted. In the present study T cell stimulation induced by idiotypic determinants on the autologous monoclonal IgG (M-components) from patients with monoclonal gammopathies was studied. In parallel, T cell stimulation in response to a conventional antigen, purified protein derivative, was also examined. It is shown that, as with conventional antigen, idiotype-induced T cell stimulation requires the presence of antigen-presenting cells (APC; monocytes and/or B cells), and is MHC class II (DR)-restricted. B cells, but not monocytes, can present idiotypic determinants to T cells at very low antigen concentrations, while monocytes do so only when antigen is present at high concentrations. Antigen processing and presentation is abrogated by treatment of APC with chloroquine. In conclusion, our study demonstrates that human idiotype-specific T cells recognize processed idiotypic determinants presented by MHC class II (HLA-DR) molecules on APC, and that B cells require about 1000-fold less antigen that monocytes.
The majority of patients with myasthenia gravis were shown to have T and B cells specific for a beta-bungarotoxin binding protein, presynaptic membrane receptor (PsmR). Such autoreactive T cells may be subdivided into different subsets according to the pattern of cytokine production. In this study the authors examined the subpopulation of the T cells by analysing their IFN-gamma and/or IL-4 secretion pattern. T cell response to human muscle acetylcholine receptor (AChR) was examined in parallel. PsmR-stimulated IFN-gamma secretion was found in 60%, and IL-4 secretion in 48% of the patients. Cells stimulated to secrete both IFN-gamma and IL-4 or IFN-gamma only were the most common patterns. Treatment of the cells with a mouse anti-human HLA-DR antibody abolished the secretion of cytokines. There was a positive correlation between the numbers of PsmR-reactive and AChR-reactive T cells. In conclusion, the results show that PsmR-stimulated T cells secreted IFN-gamma and/or IL-4. This T cell response is MHC class II restricted. Thus, this study indicates that both Th1/Th2 or Th0 subsets of the T cells are involved in the autoimmune response in the disease.
An idiotypic network involving T and B cells bearing complementary structures has been suggested to be important for the regulation of immune response in healthy and disease situations. A previous study by the authors has demonstrated the presence of a relatively higher concentration of anti-idiotypic antibodies than of idiotypic in early myasthenia gravis (MG), suggesting that the development of an anti-idiotypic immunity is important in early MG. The present study was conducted to examine the cellular components of the idiotypic network in the same situation. T and B cells reactive to acetylcholine receptor (AChR) or to a disease-related idiotype and to an anti-idiotype were analysed in seven patients with early MG at various times after the start of the disease. The results show that a significant increase in the number of idiotype-reactive interferon-gamma-secreting T cells and a shift from AChR-reactive to idiotype- and/or anti-idiotype-reactive T cells in the patients at 6 month follow-up were noted. Such changes seem to correlate to a clinical improvement in the patients. The enhanced anti-idiotypic T-cell response and the clinical improvement in the patients may speak in favour of a role for the anti-idiotypic immunity in controlling the autoimmune response in MG, i.e., down-regulating autoantibody-producing B cells and idiotypic (AChR-specific) T cells. Thus, an immune intervention towards the enhancement of the anti-idiotypic immunity in patients might be a rewarding approach. Further studies with regard to cell interactions and immune regulations in the network are warranted.
Mutation of the p53 gene which is located on chromosome 17p is the single most frequent alteration observed in human cancer. In this study we evaluate malignant melanoma, the most common intraocular neoplasm in adults, for aberrant p53 expression. Twenty enucleation specimens representing one ciliary body and 17 choroidal melanomas and two choroidal nevi were studied by immunohistochemistry utilizing the D07 anti-p53 antibody and the MIB-1 monoclonal antibody. The tumors included two spindle cell and 16 mixed cell (spindle + epithelioid cell) melanomas and two spindle cell nevi. The MIB-1 labelling index ranged from < 1% (two cases), 1-5% (13 cases) and > 5% (five cases). Of the 18 melanomas, 13 cases showed nuclear p53 staining with the p53 index < 1% (two cases), 1-3% (eight cases) and 4-5% (three cases). No p53 staining was observed in two malignant melanomas of the spindle cell type and in two choroidal nevi. In the 13 malignant melanomas of the mixed cell type, there was no correlation between MIB-1 index and p53 immunoreactivity. Immunopositivity was not found in normal choroidal melanocytes. Our study suggests that p53 alterations may be found in uveal melanomas; in our series, p53 positivity was present only in malignant melanomas of the mixed cell type.
OBJECTIVES: To explore whether the inhalation of coal mine dust increases the risk of premature death in miners, a survival analysis was conducted in a cohort of 2738 patients with simple pneumoconiosis in the Huai-Bei coal mine, in China. METHODS: Age specific mortalities were calculated by disease severity in terms of pneumoconiotic category with the life table method. The progressions from simple pneumoconiosis to death or progressive massive fibrosis (PMF) were analysed with the Cox's regression model with time as the dependent variable to identify risk factors. RESULTS: During a follow up period (mean 8 y) 3.2% of patients with simple pneumoconiosis developed PMF. The patients with development of PMF presented higher age specific mortalities than those remaining in a state of simple pneumoconiosis (SMR: 3.42; P < 0.01). After adjustment for tuberculosis and duration of work, the relative risk of premature death due to development of PMF was 2.4. Tuberculosis was found to be a main risk factor which not only facilitated premature death (relative risk (RR): 2.0; P < 0.01), but was also a strong facilitator for development of PMF (RR: 7.0; P < 0.01). Also, a long term of work underground and drilling as a main job were identified as risk factors for development of PMF. CONCLUSION: The results imply that patients with simple pneumoconiosis will have altered survival, and premature death among them is related to an increased risk of the development of PMF and the complication of tuberculosis.
OBJECTIVE: To evaluate the expression of the bcl-2 proto-oncogene in uveal malignant melanomas. CASE MATERIAL: We studied 20 uveal malignant melanomas (19 choroidal and 1 ciliary body) by immunohistochemistry with the bcl-2 oncoprotein monoclonal antibody and the cell proliferation marker, MIB-1. RESULTS: Expression of bcl-2 was found in 100% of cases and was not correlated with the histologic subtype of melanoma or the MIB-1 proliferative index. Normal choroidal melanocytes were negative for bcl-2. CONCLUSION: Our results suggest that altered expression of bcl-2 is common in uveal melanomas and is not related to histologic grade.
The presence of idiotype-reactive T-cell subsets and their relation to the tumor load were analyzed in 9 patients with monoclonal gammopathy of undetermined significance (MGUS), in 12 patients with multiple myeloma (MM) clinical stage I, and in 9 patients with MM stage II/III. An enzyme-linked immunospot assay was used to identify interferon-gamma (IFN-gamma)-, interleukin-2 (IL-2)-, or IL-4-secreting T cells after stimulation by F(ab')2 fragments of monoclonal IgG. The response to autologous IgG was significantly higher than that induced by isotypic monoclonal IgG. Comparable results were obtained in a proliferation assay (3H-thymidine incorporation). A total of 8 of 9 patients with MGUS, 7 of 12 patients with MM stage I, and 3 of 9 with MM stage II/III had T cells secreting IFN-gamma and/or IL-2 (T helper [Th1] type-1 cells), whereas cells secreting both Th1 and Th2 or Th0 types of cytokines were more frequent in patients with MM, particularly in those with MM stage II/III. The number and frequency of Th1-type cells were significantly higher in MGUS patients as compared with those of MM stage II/III. The results indicate that idiotype-reactive T cells of the Th1 and Th2 or Th0 subsets were present in MGs and might provide indirect evidence that idiotype-reactive Th1-type cells may have a regulatory impact on the human tumor B cells.
Computer simulation was applied to Sartwell's model to examine the impact of competing risks of death on the underlying assumptions and the power to reject both uniform and normal incubation period distributions. Exponential and nonparametric survival functions were imposed onto lognormal, uniform, and normal distributions to create random samples reflecting competing risk. These random samples were evaluated with the Shapiro-Wilk's test to determine the proportion for which the lognormal distribution was rejected. The simulations indicated that competing causes of death do not significantly alter the lognormal distribution of incubation periods. In only approximately 5% of the samples drawn from a lognormal distribution was a lognormal hypothesis rejected with a goodness-of-fit test when sample size varied from 20 to 500. There was generally good power (> 80%) to reject a lognormal distribution if the random samples were generated from a uniform distribution of incubation times, but not when they were generated from a normal distribution, particularly with increasing ages at disease onset. Varying the standard deviation did not significantly change the simulation results if the random samples came from a lognormal or uniform distribution. These conclusions were further supported by application of Sartwell's model to published data on the ages of onset for several chronic diseases.
We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.
Idiotype-specific T cells were characterized in patients with multiple myeloma stage I by analysing idiotype-induced DNA synthesis (3H-thymidine incorporation), IL-2 and IFN-gamma production at the single cell level (ELISPOT) (in vitro tests) and delayed type hypersensitivity (DTH) skin reaction (in vivo test). In seven out of eight patients at least one of the four tests was positive. In five patients three or more tests were positive. One patient was negative in all four tests. Six patients had both IL-2 and IFN-gamma-secreting cells and three of them also a DTH response. Furthermore, those three patients with a proliferative response also had IL-2 and IFN-gamma-secreting cells induced by the idiotype. The data indicate that part of the idiotype-specific T cell fraction belongs to the CD4 Th1 cell population. Whether CD8-specific T cells also were present could not be ruled out. The present study provides further support for the existence of idiotype-specific T cells in multiple myeloma. Such cells might be an important target for an immune-mediated therapeutic approach.
This paper is reported the technology of lung targeting gelatin microspheres of streptomycin sulphate (SMS). The microspheres were prepared with natural biodegradable gelatin as the load material and castor oil as the oil phase. The experimental conditions were optimized, the mean volume diameter obtained being 9.7 microns and the mean rate of encapsulation 15.69%. The content, shape and size of the microspheres showed no remarkable change after storage at 37 degrees C RH 75% for 3 months. Activation energy of heat decomposition E = 75.86kJ/mol. In vitro, the SMS release rate was found to accord with Higuchi equation with t1/2 = 8.6h. In vivo (rabbits) the gelatin microspheres were proved to be concentrated in the lung.
The Na-K-Cl cotransporter of avian salt gland is a membrane-bound 170-kDa protein that is phosphorylated in response to cAMP- and Ca(2+)-dependent secretogogues and is homologous to the Na-K-Cl cotransporter in another Cl-secreting epithelia; the shark rectal gland (Torchia, J., Lytle, C., Pon, D. J., Forbush, B., and Sen, A. K. (1992) J. Biol. Chem. 267, 25444-25450). In the present study we assess the role of Ca2+ and protein kinase C (PKC) activation on the phosphorylation of the Na-K-Cl cotransporter. Although the addition of ionomycin alone did not significantly stimulate cotransporter phosphorylation, concurrent addition of ionomycin plus the tumor promoter phorbol 12-myristate 13-acetate (PMA) resulted in a concentration-dependent increase in phosphorylation. Immunoprecipitation experiments, using a monoclonal antibody which specifically recognizes the cotransporter, suggested that the response to CCh or ionomycin plus PMA was quantitatively similar (5-fold) and was localized exclusively on serine residues. In contrast, when 4 alpha-phorbol was added in the presence of ionomycin, no stimulation was observed. To further assess the involvement of PKC on cotransporter phosphorylation the effects of protein kinase inhibitors were tested. Both staurosporine and calphostin C inhibited phosphorylation of the cotransporter at concentrations known to inhibit PKC, whereas the calmodulin antagonist W-7 had no significant effect. The requirement for Ca2+ was tested further by removing Ca2+ from the incubation medium and stimulating with CCh. Under these conditions, the CCh-stimulated phosphorylation was transient and, furthermore, could be completely inhibited by preloading the cells with the Ca2+ chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid) prior to stimulation. The involvement of protein phosphatases on the phosphorylation of the Na-K-Cl cotransporter was also tested. The addition of okadaic acid stimulated phosphorylation by approximately 3-fold. Taken together these results suggest that the phosphorylation state of the cotransporter involves a dynamic interplay between changes in intracellular Ca2+, PKC, and protein phosphatase activities.
Hydrogen-deuterium (H-D) exchange labeling and proton NMR have been applied to study the protein-protein association between cytochrome c peroxidase (CcP) and yeast iso-1 ferricytochrome c. Specifically, the exchange behavior of individual backbone amide protons of yeast iso-1 ferricytochrome c in both CcP-bound (i.e., complexed) and free (i.e., never in the complex) forms has been investigated and used in an attempt to map the binding site of CcP on yeast iso-1 ferricytochrome c when the noncovalent complex was formed in very low salt solution. The exchange rates of certain amino acid amide protons were significantly slowed down, by up to 40-fold, in the complex compared to the free form. The protected regions on iso-1 ferricytochrome c include parts of the 10's helix and the 70's helix surrounding the cytochrome c heme solvent-exposed edge (the so-called "front side" of iso-1 cytochrome c). These regions are very similar to the cytochrome c peroxidase binding interface on iso-1 ferricytochrome c that has been defined by X-ray crystallographic data. This further supports the direct involvement of the front side of iso-1 cytochrome c in binding with cytochrome c peroxidase. The results from our H-D exchange experiments also indicated that the amide proton exchange rates of Trp59, Asp60, and part of the 90's helix, all of which are located on the opposite side (the "back" side) of ferricytochrome c from the heme solvent-exposed edge, are also retarded upon complex formation.