Gelanxinning capsule improves coronary microvascular dysfunction by inhibiting inflammation and restoring endothelial function.

OBJECTIVE: Gelanxinning capsule (GXSC) is a Chinese medicine to cure coronary artery disease (CAD) and a compound of Pueraria lobata, hawthorn extract, and gypenosides. However, whether GXSC could improve coronary microvascular dysfunction (CMD) is unknown. We aimed to demonstrate the therapeutic effect of GXSC on CMD and its underlying mechanisms in CAD patients. PATIENTS AND METHODS: This was a single-center, randomized control trial. A total of 78 patients diagnosed by selective coronary angiography (CAG) participated in this study. Patients' demographics, medical history, medications, and results of laboratory testing were collected. The index of microcirculatory resistance (IMR) and coronary flow reserve (CFR) were obtained by CAG and single-photon emission computed tomography (SPECT) separately. Fasting blood samples were obtained on the morning following the admission day. Concentrations of several molecules of inflammation, endothelial function, and coronary microvascular function were measured by ELISA. Patients were followed-up two months after discharge and fasting blood samples were also acquired. RESULTS: All patients were randomly divided into 2 groups: GXSC, 38 (48.7%), and control, 40 (51.3%). The intergroup comparison revealed no significant differences with respect to all baseline variables. As for inflammation biomarkers, proinflammatory NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and interleukin (IL)-1 were significantly decreased in GXSC compared with the control group (0.71±0.08 vs. 1.04±0.07, p<0.01 and 7.16±0.59 vs. 10.93±1.04, p<0.01). Anti-inflammatory adropin was increased in the GXSC group (7.75±0.59 vs. 5.71±0.68, p=0.03). As for indexes of endothelial function, the concentrations of syndecan (SDC) 1, SDC4 and heparan sulphates (HS) were significantly downregulated in 2 months GXSC treatment (3.31±0.28 vs. 4.85±0.43, p<0.01, 3.79±0.56 vs. 5.69±0.68, p=0.03 and 21.31±2.79 vs. 35.18±4.11 p<0.01). In addition, the level of SIRTUIN 1 (SIRT1), which is a vascular protective protein, was upregulated in GXSC group (5.63±0.30 vs. 4.22±0.37, p<0.01). As for molecules of coronary microvascular function, endocan, soluble urokinase plasminogen activator receptor (suPAR), and growth differentiation factor (GDF)-15 were significantly decreased consistently in GXSC compared with the control group (0.09±0.01 vs. 0.19±0.03, p<0.01, 4.44±0.40 vs. 5.73±0.40, p=0.03 and 2.08±0.17 vs. 2.69±0.18, p=0.02). CONCLUSIONS: In conclusion, GXSC could improve CMD by inhibiting inflammation and restoring endothelial function. GXSC might be an effective drug in CAD patients without obstructive epicardial coronary arteries but suffering from angina.

Competition for BLyS-mediated signaling through Bcmd/BR3 regulates peripheral B lymphocyte numbers.

Striking cell losses occur during late B lymphocyte maturation, reflecting BcR-mediated selection coupled with requisites for viability promoting signals. How selection and survival cues are integrated remains unclear, but a key role for B lymphocyte stimulator (BLyS(TM); trademark of Human Genome Sciences, Inc.) is suggested by its marked effects on B cell numbers and autoantibody formation as well as the B lineage-specific expression of BLyS receptors. Our analyses of the B cell-deficient A/WySnJ mouse have established Bcmd as a gene controlling follicular B cell life span, and recent reports show Bcmd encodes a novel BLyS receptor. Here we show that A/WySnJ B cells are unresponsive to BLyS, affording interrogation of how Bcmd influences B cell homeostasis. Mixed marrow chimeras indicate A/WySnJ peripheral B cells compete poorly for peripheral survival. Moreover, in vivo BrdU labeling shows that (A/WySnJ x BALB/c)F(1) B cells have an intermediate but uniform life span, indicating viability requires continuous signaling via this pathway. Together, these findings establish the BLyS/Bcmd pathway as a dominant mediator of B cell survival, suggesting competition for BLyS/Bcmd signals regulates follicular B cell numbers.

TCR transgenic mice in which usage of transgenic alpha- and beta-chains is highly dependent on the level of selecting ligand.

We have produced a TCR transgenic mouse that uses a TCR derived from a Th1 clone that is specific for residues 64 to 76 of the d allele of murine hemoglobin presented by I-Ek. Examination of these TCR transgenic mice on an H-2(k/k) background that expressed the nonstimulatory s allele of murine hemoglobin revealed that these mice express many endogenous TCR chains from both alpha and beta loci. We found that this transgenic TCR is also very inefficient at mediating beta selection, thereby showing a direct linkage between beta selection and allelic exclusion of TCR beta. We have also examined these mice on MHC backgrounds that have reduced levels of I-Ek and found that positive selection of cells with high levels of the transgenic TCR depends greatly on the ligand density. Decreasing the selecting ligand density is a means of reducing the number of available selecting niches, and the data reveal that the 3.L2 TCR is used sparingly for positive selection under conditions where the number of niches becomes limiting. The results, therefore, show a way that T cells may get to the periphery with two self-restricted TCRs: one that efficiently mediates positive selection, and another that is inefficient at positive selection with the available niches.

TCR recognition of the Hb(64-76)/I-Ek determinant: single conservative amino acid changes in the complementarity-determining region 3 dramatically alter antigen fine specificity.

To identify the structural basis of Ag fine specificity, TCR sequences from a panel of Hb(64-76)/I-Ek-specific T cells were compared and found to be restricted in variable (V) gene usage, predominantly using BV1 or BV15 and AV4 or AV10 genes. TCRA and TCRB junctional sequences were extremely diverse. No conservation of length or position was found, which distinguishes this response from others, but correlates with the range of fine specificities that these T cells display. A remarkable subtlety in the recognition of Hb(64-76) was revealed from the study of the response to the D73 variant of Hb(64-76), which contains a conservative change in an MHC anchor residue not affecting the binding affinity to I-Ek. To one group of T cells this determinant was non-cross-reacting with Hb(64-76), whereas another recognized both Ags. Interesting, they all used a different constellation of TCRBV genes than that found in Hb(64-76) recognition. To limit the variability in the anti-Hb(64-76) TCR repertoire, transgenic mice expressing a fixed TCRB rearrangement from a Hb(64-76)-specific T cell were used. In Hb(64-76)-specific TCR from these mice, the endogenous alpha-chains pairing with the transgenic beta-chain were highly restricted in their AV gene usage. A comparison of two pairs of closely related T cells of these endogenous TCR variants, one differing by a single, conservative substitution in the complementarity-determining region 3 and the other containing a positional switch of two amino acids, revealed dramatically different fine specificities. Overall, these findings highlight the exquisite sensitivity of the TCR- peptide/MHC interaction to subtle alterations in any of the components.

Endogenous altered peptide ligands can affect peripheral T cell responses.

T cells potentially encounter a large number of endogenous self-peptide/MHC ligands in the thymus and the periphery. These endogenous ligands are critical to both positive and negative selection in the thymus; however, their effect on peripheral T cells has not been directly ascertained. Using the murine allelic Hbd (64-76)/I-Ek self-antigen model, we have previously identified altered peptide ligands (APLs) which are able to stimulate some but not all TCR-mediated effector functions. To determine directly the effect of endogenously synthesized APL/MHC complexes on peripheral T cells, we used a TCR transgenic mouse which had reversed our normal antigen system, with Ser69 peptide now being the agonist and Hbd(64-76) being the APL. In this report, we show that the constitutive level of endogenous Hbd(64-76)/I-Ek complexes presented by APCs in vivo is too low to affect the response of Ser69 reactive T cells. However, by increasing the number of Hbd(64-76)/I-Ek complexes expressed by the APCs, TCR antagonism is observed for both primary T cells and T cell hybridomas. In addition, the level of the CD4 coreceptor expressed on T cells and T cell hybridomas. In addition, the level of the CD4 coreceptor expressed on T cells changes the response pattern to endogenously presented Hbd(64-76)/I-Ek ligand. These findings demonstrate that T cells are selected to ignore the constitutive levels of endogenous complexes they encounter in the periphery. T cell responses can be affected by endogenous APLs in the periphery under limited but attainable circumstances which change the efficacy of the TCR/ligand interaction. Thus, endogenous APLs play a role in both the selection of T cells in the thymus and the responses of peripheral T cells.

Modulation of T cell development by an endogenous altered peptide ligand.

T cells potentially encounter numerous endogenous peptides during selection in the thymus and in the periphery. We examined the impact of an endogenous peptide on in vivo T cell development, using a TCR transgenic mouse model based on a hemoglobin-specific T cell clone. In these mice, the transgenic beta chains paired with endogenous alpha chains. This led to a serendipitous primary reactivity to Ser69 peptide, an altered peptide ligand of the Hbd (64-76) epitope of the parent clone. Two Ser69-reactive T cell populations were identified. A smaller population of the Ser69-reactive T cells responded both to Ser69 and Hbd (64-76). A majority reacted only to Ser69, and not to Hbd(64-76); in fact, Hbd(64-76) was a specific TCR antagonist for these Ser69-only-reactive T cells. Thus, in this unique experimental system, Ser69 became an agonist, and Hbd (64-76) was an antagonist. Endogenous presentation of the antagonist ligand in the thymus selectively eliminated the high-avidity cells, while sparing low-avidity cells in the Ser69-reactive T cell repertoire. These results highlight how specificity guides developing T cells through a network of ligands and indicate that the endogenous peptide pool has a profound effect on T cell development and repertoire.

Separation of T helper 1 clone cytolysis from proliferation and lymphokine production using analog peptides.

In this report, we investigate the activation of Th1 clones using altered TCR ligand. By changing the immunogenic peptide, cytolytic function can be separated from proliferative and lymphokine responses. These three responses were examined and dissected in two Th1 clones using analogs of the murine hemoglobin [Hb(64-76)] peptide. This analysis was focused on amino acids in the immunogenic peptide that were possible T cell contact residues. Typically, several amino acids were identified as critical contact residues for a Th1 proliferative response. An examination of lymphokine production (IFN-gamma or IL-3) revealed the same pattern of response to the analog peptides indicating that the proliferative and lymphokine responses were directly related. However, for cytolysis, fewer amino acid residues were identified as critical contact residues for effector function. Thus, some altered peptide ligands allowed the disassociation of the cytolytic function from the proliferative and lymphokine responses in Th1 clones. To extend these findings, the activation of T cell hybridomas created from the Th1 clones were similarly examined using the altered TCR ligands. The lymphokine response (IL-2) of the T cell hybridomas identified the same critical amino acids as did the cytolytic response of the Th1 clones. Thus, analog peptides partially activated the Th1 clones such that cytolysis occurred independent from proliferative and lymphokine responses.

Complete dissection of the Hb(64-76) determinant using T helper 1, T helper 2 clones, and T cell hybridomas.

We have generated cloned Th1 cells, Th2 cells, and T cell hybridomas specific for the single immunogenic peptide from the beta-chain of murine hemoglobin (Hb(64-76)). The availability of these various types of T cells provided us an unique opportunity to examine and dissect the T cell response to an immunogenic peptide. A panel of altered Hb peptides was made by replacing each amino acid in the Hb peptide (positions 64-76) with a conservative amino acid substitution or an alanine. Although none of the eleven T cell clones and hybridomas tested exhibited the same pattern of reactivity to the substituted Hb peptides, some general features were identified for all T cell responses. The primary T cell contact residue of Hb(64-76) was shown to be asparagine 72. For every Hb(64-76) specific T cell, no activation was observed using a peptide containing the conservative substitution of a glutamine for the asparagine at position 72. The flanking glutamic acid at position 73 was also required for a proliferative response for all of the Th1 and Th2 clones. The Th subtypes were not grossly unique in their responses to the substituted Hb peptides, but exhibited minor differences in fine specificity with the Th1 cells identifying more critical amino acids then did the Th2 cells. For the Th1 cells and also the T cell hybridomas, the phenylalanine at position 71 was critical for a T cell response. Analysis of peptide affinity for IEk molecules indicated that position 71 played a role in peptide binding to MHC. Secondary T cell contact residues, which were important for many but not all of the T cells, were identified at positions 69, 70, and 76. Overall T cell responses were minimally affected by changes in the amino acid residues at positions 64-68, 74, and 75. We have also demonstrated that cloned Th1 cells, Th2 cells and T hybridomas can be generated against the same Hb(64-76) determinant.

T-lymphocyte entry into the central nervous system.

The entry of T-lymphocytes into the parenchyma of the central nervous system is a critical early feature in the pathogenesis of many experimental and spontaneously occurring immune-mediated illnesses. The physiological mechanisms controlling this entry have not been elucidated. This study reports that T-cell entry into the rat CNS appears to be primarily dependent upon the activation state of the lymphocytes; T-lymphoblasts enter the CNS (and all other tissues examined) in an apparently random manner while T cells not in blast phase are excluded. Antigen specificity, MHC compatibility, T-cell phenotype, and T-cell receptor gene usage do not appear related to the ability of cells to enter. This study demonstrates that when T-lymphoblasts are introduced into the circulation they rapidly appear in the CNS tissue. Their concentration in the CNS reaches a peak between 9 and 12 hr, and lymphocytes which have entered, exit within 1 to 2 days. Cells capable of reacting with a CNS antigen remain in the tissue or cyclically reenter to initiate inflammation if they are able to recognize their antigen in the correct MHC context. This observation also appears to pertain to the entry of activated T cells into many other tissues, although their concentrations in these non-CNS sites was not quantitated.

Elevation of rat liver mRNA for selenium-dependent glutathione peroxidase by selenium deficiency.

Selenium-dependent glutathione peroxidase (Se-GSH-Px, GSH-H2O2 oxidoreductase EC 1.11.1.9) is the best characterized selenoprotein in higher animals, but the mechanism whereby selenium becomes incorporated into the enzyme protein remains under investigation. To elucidate the mechanism of insertion of selenium into Ge-GSH-Px further, we have systematically analyzed and compared the results of Western blot, in vitro translation immunoprecipitation, and Northern blot experiments conducted with liver proteins and RNAs obtained from rats fed on selenium-deficient and selenium-supplemented diets. The anti-serum employed in this study was raised against an electrophoretically pure Se-GSH-Px preparation obtained from rat livers by a simplified purification procedure involving separation by high performance liquid chromatography on a hydrophobic interaction column. Different forms of Se-GSH-Px, including apo-protein, cross-reacted with this antiserum and Western blot analysis found no Se-GSH-Px protein present in livers from rats fed on selenium-deficient diets. By contrast, a distinct protein band corresponding to purified Se-GSH-Px was detected in livers from selenium-supplemented animals, a result consistent with the finding that the Se-GSH-Px activity was reduced to undetectable levels in livers of selenium-deficient rats. The in vitro translation experiments, however, indicated not only that mRNA for Se-GSH-Px was present during selenium deficiency but also that its translation products contained 2-3-fold as much immunoprecipitable protein as the products of poly(A) RNA from livers of selenium-supplemented rats. This result suggests that the Se-GSH-Px mRNA may be increased in the selenium-deficient state. Elevated levels of Se-GSH-Px mRNA were directly demonstrated in Northern blot experiments employing cDNA clone pGPX1211 as a probe. A similar increase in Se-GSH-Px mRNA was observed in such other tissues as kidney, testis, brain, and lung tissue, in selenium-deficient states. The present data support the co-translational mechanism for the incorporation of selenium into Se-GSH-Px in rat liver.

Expression of glutathione peroxidase I gene in selenium-deficient rats.

We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I. The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E. coli, another selenoenzyme. The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively. The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences. We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected. The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally.

Regulatory elements within the murine leukemia virus enhancer regions mediate glucocorticoid responsiveness.

Enhancer elements within nonleukemogenic (Akv) and T-cell leukemogenic (SL3-3) murine leukemia viruses demonstrate strong cell type preference in transcriptional activity. These transcription elements are additionally regulated by the synthetic glucocorticoid dexamethasone, and this pattern of regulation varies according to cell type. The sequences required for dexamethasone regulation for both Akv and SL3-3 are shown to include a 17-nucleotide consensus sequence previously termed the glucocorticoid response element (GRE). Although the GREs are identical for both viral enhancers, the sequences surrounding these elements differ, as does the spatial arrangement of the GRE sequences with respect to one another. It is proposed that the spatial arrangement of the GREs, as well as their precise sequence context, determines the difference in the response to dexamethasone of the enhancers in different cell types.