-
[show abstract]
[hide abstract]
ABSTRACT: A randomized, 2-way crossover study was conducted in healthy Chinese male volunteers to evaluate the bioequivalence of a new generic formulation of entecavir (CAS 142217-69-4) tablets (test) and the available branded formulation (reference) to meet the requirements for marketing the test product in China. Test and reference tablets were administered as a single dose on 2 treatment days separated by a 2-week washout period. Blood samples were collected for a period of 24 h following drug administration. Plasma concentration of entecavir was determined by a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method. Pharmacokinetic parameters were calculated using a noncompartmental model. Bioequivalence was determined by calculating 90% CIs for the ratios of Cmax, AUC0-t and AUC0-∞ values for the test and reference products. Tolerability was assessed by monitoring vital signs, laboratory tests and interviews with the volunteers before administration and every 2 h during the study. The 90% CIs of entecavir for Cmax, AUC0-t and AUC0-∞ were 95.2-106.9%, 98.4-104.6% and 97.3-104.4%, respectively, which fell within the interval of 80-125%. No clinically important adverse effects were reported. These results suggested that the test formulation of entecavir tablets met the regulatory criterion for bioequivalence to the reference formulation.
Arzneimittel-Forschung 01/2012; 62(3):113-6. · 0.72 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Microbial pathogens have been selected for the capacity to evade or manipulate host responses in order to survive after infection. Chlamydia, an obligate intracellular pathogen and the causative agent for many human diseases, can escape T lymphocyte immune recognition by degrading host transcription factors required for major histocompatibility complex (MHC) antigen expression. We have now identified a chlamydial protease- or proteasome-like activity factor (CPAF) that is secreted into the host cell cytosol and that is both necessary and sufficient for the degradation of host transcription factors RFX5 and upstream stimulation factor 1 (USF-1). The CPAF gene is highly conserved among chlamydial strains, but has no significant overall homology with other known genes. Thus, CPAF represents a unique secreted protein produced by an obligate intracellular bacterial pathogen to interfere with effective host adaptive immunity.
Journal of Experimental Medicine 05/2001; 193(8):935-42. · 13.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have previously shown that infection with the C. pneumoniae AR39 strain once monthly for 9 consecutive months significantly exacerbated atherosclerosis in mice with LDL receptor deficiency (LDLR-/-) in the presence of a high cholesterol diet. To further optimize the LDLR-/- mouse model for studying the mechanisms of C. pneumoniae atherogenesis, we have tested a different infection protocol with intranasal inoculation twice monthly for 6 consecutive months in the present study. We found that C. pneumoniae infection for 6 months was sufficient to produce a 130%, significantly greater exacerbation of aortic atherosclerosis in LDLR-/- mice in the presence of a high cholesterol diet. Mice receiving a high cholesterol diet alone displayed a lesion area index of 18.2 +/- 6.1 (S.D.) while mice treated with both the high cholesterol diet and C. pneumoniae infection had a lesion area index of 41.8 +/- 15.2 (S.D.). However, the chlamydial infection did not significantly alter the mouse serum total cholesterol or the LDL levels induced by the high cholesterol diet. This study not only confirms our previous findings that C. pneumoniae infection can exacerbate aortic atherosclerosis lesion in the LDLR-/- mice, but also further optimizes the LDLR-/- mouse model for future mechanism studies.
Molecular and Cellular Biochemistry 01/2001; 215(1-2):123-8. · 2.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have previously shown that the obligate intracellular pathogen chlamydia can suppress interferon (IFN)-gamma-inducible major histocompatibility complex (MHC) class II expression in infected cells by degrading upstream stimulation factor (USF)-1. We now report that chlamydia can also inhibit both constitutive and IFN-gamma-inducible MHC class I expression in the infected cells. The inhibition of MHC class I molecule expression correlates well with degradation of RFX5, an essential downstream transcription factor required for both the constitutive and IFN-gamma-inducible MHC class I expression. We further demonstrate that a lactacystin-sensitive proteasome-like activity identified in chlamydia-infected cell cytosolic fraction can degrade both USF-1 and RFX5. This proteasome-like activity is dependent on chlamydial but not host protein synthesis. Host preexisting proteasomes may not be required for the unique proteasome-like activity. These observations suggest that chlamydia-secreted factors may directly participate in the proteasome-like activity. Efforts to identify the chlamydial factors are underway. These findings provide novel information on the molecular mechanisms of chlamydial evasion of host immune recognition.
Journal of Experimental Medicine 06/2000; 191(9):1525-34. · 13.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We report that chlamydiae, which are obligate intracellular bacterial pathogens, can inhibit interferon (IFN)-gamma-inducible major histocompatibility complex (MHC) class II expression. However, the IFN-gamma-induced IFN regulatory factor-1 (IRF-1) and intercellular adhesion molecule 1 (ICAM-1) expression is not affected, suggesting that chlamydia may selectively target the IFN-gamma signaling pathways required for MHC class II expression. Chlamydial inhibition of MHC class II expression is correlated with degradation of upstream stimulatory factor (USF)-1, a constitutively and ubiquitously expressed transcription factor required for IFN-gamma induction of class II transactivator (CIITA) but not of IRF-1 and ICAM-1. CIITA is an obligate mediator of IFN-gamma-inducible MHC class II expression. Thus, diminished CIITA expression as a result of USF-1 degradation may account for the suppression of the IFN-gamma-inducible MHC class II in chlamydia-infected cells. These results reveal a novel immune evasion strategy used by the intracellular bacterial pathogen chlamydia that improves our understanding of the molecular basis of pathogenesis.
Journal of Experimental Medicine 07/1999; 189(12):1931-8. · 13.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: As is true for other intracellular pathogens, immunization with live Chlamydia trachomatis generally induces stronger protective immunity than does immunization with inactivated organism. To investigate the basis for such a difference, we studied immune responses in BALB/c mice immunized with viable or UV-killed C. trachomatis mouse pneumonitis (MoPn). Strong, acquired resistance to C. trachomatis infection was elicited by immunization with viable but not dead organisms. Immunization with viable organisms induced high levels of antigen-specific delayed-type hypersensitivity (DTH), gamma interferon production, and immunoglobulin A (IgA) responses. Immunization with inactivated MoPn mainly induced interleukin-10 (IL-10) production and IgG1 antibody without IgA or DTH responses. Analysis of local early cytokine and cellular events at days 3, 5, and 7 after peritoneal cavity immunization showed that high levels of granulocyte-macrophage colony-stimulating factor and IL-12 were detected with viable but not inactivated organisms. Furthermore, enrichment of a dendritic cell (DC)-like population was detected in the peritoneal cavity only among mice immunized with viable organisms. The results suggest that early differences in inducing proinflammatory cytokines and activation and differentiation of DCs may be the key mechanism underlying the difference between viable and inactivated organisms in inducing active immunity to C. trachomatis infection.
Infection and Immunity 05/1999; 67(4):1606-13. · 4.16 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Immunization with dendritic cells pulsed ex vivo with antigens has been successfully used to elicit primary antigen-specific immune responses. We report that mouse bone marrow-derived dendritic cells pulsed with inactivated chlamydial organisms induced strong protection against live chlamydial infection in a mouse lung infection model. Either the dendritic cells or chlamydial organisms alone or macrophages similarly pulsed with chlamydial organisms failed to induce any significant protection. These observations suggest that dendritic cells can efficiently process and present chlamydial antigens to naive T cells in vivo. Mice immunized with the chlamydia-pulsed dendritic cells preferentially developed a Th1 cell-dominant response while mice immunized with the other immunogens did not, suggesting a correlation between a Th1 cell-dominant response and protection against chlamydial infection. We further found that dendritic cells produced a large amount of interleukin 12 (IL-12) upon ex vivo pulsing with inactivated chlamydial organisms, which may allow the dendritic cells to direct a Th1 cell-dominant response. Dendritic cells from mice deficient in the IL-12 p40 gene failed to produce IL-12 after a similar ex vivo pulse with chlamydial organisms, and more importantly, immunization with these dendritic cells failed to induce a Th1 cell-dominant response and did not induce strong protection against chlamydial infection. Thus, the ability of dendritic cells to efficiently process and present chlamydial antigens and to produce IL-12 upon chlamydial-organism stimulation are both required for the induction of protection against chlamydial infection. This information may be useful for the further design of effective chlamydial vaccines.
Infection and Immunity 05/1999; 67(4):1763-9. · 4.16 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Epidemiological investigations have linked Chlamydia pneumoniae infection to atherosclerosis. It is not clear, however, whether C. pneumoniae infection plays a causal role in the development of atherosclerosis. Mice with low-density lipoprotein receptor deficiency were induced to develop atherosclerotic lesions in aorta with a cholesterol-enriched diet that increased serum cholesterol by two- to threefold. Using this mouse model, we found that the chlamydial infection alone with either the C. pneumoniae AR39 or the C. trachomatis MoPn strain failed to induce any significant atherosclerotic lesions in aorta over a period of nine months. However, in the presence of a high-cholesterol diet, infection with the C. pneumoniae AR39 strain significantly exacerbated the hypercholesterolemia-induced atherosclerosis, demonstrating that a hypercholesterolemic condition is required for the C. pneumoniae to aggravate the development of atherosclerosis. Although both AR39 and MoPn antigens were detected in aorta of mice infected with the corresponding strains, only mice infected with the C. pneumoniae strain AR39 displayed enhanced atherosclerotic lesions, suggesting that the C. pneumoniae species may possess a unique atherogenic property. This study may provide a model for further understanding the mechanisms of C. pneumoniae atherogenesis and evaluating chlamydial intervention strategies for preventing the advancement of atherosclerotic lesions enhanced by C. pneumoniae infection.
Journal of Clinical Investigation 04/1999; 103(5):747-53. · 15.39 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We report that chlamydiae, which are obligate intracellular bacterial pathogens, possess a novel antiapoptotic mechanism. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by a wide spectrum of proapoptotic stimuli including the kinase inhibitor staurosporine, the DNA-damaging agent etoposide, and several immunological apoptosis-inducing molecules such as tumor necrosis factor-alpha, Fas antibody, and granzyme B/perforin. The antiapoptotic activity was dependent on chlamydial but not host protein synthesis. These observations suggest that chlamydia may encode factors that interrupt many different host cell apoptotic pathways. We found that activation of the downstream caspase 3 and cleavage of poly (ADP-ribose) polymerase were inhibited in chlamydia-infected cells. Mitochondrial cytochrome c release into the cytosol induced by proapoptotic factors was also prevented by chlamydial infection. These observations suggest that chlamydial proteins may interrupt diverse apoptotic pathways by blocking mitochondrial cytochrome c release, a central step proposed to convert the upstream private pathways into an effector apoptotic pathway for amplification of downstream caspases. Thus, we have identified a chlamydial antiapoptosis mechanism(s) that will help define chlamydial pathogenesis and may also provide information about the central mechanisms regulating host cell apoptosis.
Journal of Experimental Medicine 03/1998; 187(4):487-96. · 13.85 Impact Factor
-
G Zhong
Methods in molecular biology (Clifton, N.J.) 01/1998; 87:165-73.
-
[show abstract]
[hide abstract]
ABSTRACT: Identification of protective determinants from microbial proteins is a necessary step in the rational design of subunit vaccines. We have previously used a synthetic peptide scan (Pepscan) assay to map a panel of eight neutralizing monoclonal antibodies (mAb; designated as C1.1 to C1.8) to a common motif sequence from Chlamydia trachomatis. In the present study, five of the eight mAbs were used to screen phage random peptide libraries. mAbs C1.1 and C1.3 selected a motif sequence of G-L-X-N-D from a pIII-based phage random peptide library and a motif sequence of G-X-X-N-D from a pVIII-based random peptide library while mAbs C1.6 to C1.8 failed to select recognizable motifs from either of the phage libraries. However, C1.6 to C1.8 bound to the same motif sequence displayed on phage when the appropriate conformational constraints were imposed onto the motif sequence. Thus the specificity of the mAbs identified on Pepscan assays correlates with the mAbs' dependence on local epitope constraints displayed on the phage surface.
Journal of Industrial Microbiology and Biotechnology 08/1997; 19(1):71-6. · 2.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Conformational constraints were imposed on a peptide epitope from Chlamydia trachomatis to improve its ability to elicit antibodies that cross-react with native antigen. Appropriate constraints were discovered by a strategy that required no prior knowledge of the epitope's native conformation. First, we constructed a library of 3.2 x 10(5) peptides in which the epitope's contact residues were subject to random conformational constraints, each constrained peptide being fused genetically to the surface of a filamentous phage vector. Next, we selected phage displaying the most native-like peptides in the library by affinity purification with antibodies that bind the epitope only in its native conformation. Finally, we immunized mice with the selected phage and titered the resulting antisera against both whole cells and unconstrained peptide. The ratio of anti-cell titer to anti-peptide titer, which reflects the channeling of the antibody response to the native epitope, was up to five times higher for affinity-selected phage than for unselected peptide phage. In this case, therefore, "antigenic fitness," the ability of a peptide to bind antibodies specific for native epitope, correlated with "immunogenic fitness," its ability to elicit antibodies that are effective against the native antigen on an invading pathogen. If the correlation is general, surveying thousands or millions of peptides for antigenic fitness with phage display technology may be a simple but effective pre-screen for immunogenic fitness, which is costly to assess directly.
Journal of Biological Chemistry 10/1994; 269(39):24183-8. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Two BALB/c mice were immunized with serovar C Chlamydia trachomatis elementary bodies, and 63 hybridomas producing monoclonal antibodies to C. trachomatis were recovered. Eight hybridomas which were specific for an identical peptide epitope (AGLQND) in serovar C major outer membrane protein variable domain I were identified. Detailed immunochemical study of the antigen-antibody interaction and genetic characterization of the antibody variable-region gene sequences showed that distinct B-cell clonal lineages were elicited by the epitope sequence. Since each antibody had a distinct pattern of fine specificity for recognition of the epitope and displayed different degrees of cross-reactivity with a related serovar (serovar A), we conclude that B-cell recognition of an immunodominant neutralization epitope can be pleiotropic. Differences in B-cell recognition of a neutralization epitope may delay the emergence by mutation of antigenic-drift variants of the C. trachomatis major outer membrane protein.
Infection and Immunity 06/1994; 62(5):1576-83. · 4.16 Impact Factor
-
BioTechniques 06/1994; 16(5):838-9. · 2.67 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Lipidic amino acid-based synthetic peptides derived from the variable domains (VD) of Chlamydia trachomatis outer membrane protein 1 were evaluated as potential candidate sequences in a vaccine. A peptide sequence designated P2 from the VD IV of serovar B contained a B cell epitope capable of eliciting antibodies binding to serovar B elementary bodies (EB) and a T helper site capable of presentation by multiple H-2 alleles. Polymerization of the P2 into polylysine to form lipid core peptides (LCP) significantly enhanced immunogenicity compared with P2 monomer alone. The LCP system incorporates lipidic amino acids into the polylysine system and enhances lipophobicity and membrane binding effects of the peptide. A second peptide sequence derived from the VD I of serovar C was cosynthesized with P2 into lipidic polylysine LCP and was designated LCP-H1. Antibodies to this construct reacted at high titer with EB of the three major trachoma causing C. trachomatis serovars A, B, and C. LCP-H1 was immunogenic among four of five murine H-2 alleles. Pepscan analysis showed that the fine specificity of antibodies generated to LCP-H1 were directed to the predetermined neutralizing epitope sequences. An in vitro HAK cell neutralization assay showed that LCP-H1 elicited neutralizing antibodies to serovars A, B, and C, but these were of low titer. Because LCP-H1 antibodies bound to the peptide sequence with 10-100-fold higher titer than to EB, the low neutralization titers most likely result from conformational differences between the synthetic peptide and antigenic sites on the native organism. Modification of LCP-H1 to incorporate a predefined conformation may result in improved antigenic properties.
The Journal of Immunology 11/1993; 151(7):3728-36. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: B-cell peptide epitopes in chlamydial heat shock protein 60 (hsp60) were elucidated with antisera from 13 rabbits immunized with Chlamydia trachomatis serovars B, C, and L2 and antisera from eight women with C. trachomatis-associated ectopic pregnancies. Thirteen major epitopes were identified with the human sera, 10 of which were also observed with rabbit antisera. Seven of the 13 epitopes recognized by human antisera exhibited cross-reactive antibody binding to homologous peptide sequences in human hsp60. Self-reactive B-cell immunity to hsp60 may contribute to chlamydial disease pathogenesis.
Infection and Immunity 04/1993; 61(3):1117-20. · 4.16 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effects of both H-2 and non-H-2 genes on antibody responses to two Chlamydia trachomatis heat shock proteins (hsp60 and hsp70) were investigated. These chlamydial proteins are homologs of Escherichia coli GroEL (hsp60) and DnaK (hsp70) and are highly sequence conserved between bacterial and mammalian sources. Antibody responses among 17 different strains of mice immunized with C. trachomatis serovar B and serovar C elementary bodies were evaluated by immunoblot, radioimmunoprecipitation and enzyme-linked immunosorbent assay. Antibody responses to the two proteins displayed host genetic restriction. Of six distinctive H-2 haplotypes, only H-2d generated high antibody responses to hsp70. Five of the six H-2 haplotypes, i.e., H-2a, H-2d, H-2k, H-2q, and H-2s, produced high antibody responses to hsp60. Only the H-2b-bearing strain had low antibody responses to hsp60. By using congenic and H-2 recombinant strains, the genes responsible for regulating antibody responses to hsp70 and hsp60 were mapped to the K-IA region of the H-2 locus. In F1 hybrid crosses between high and low responders, high responses to hsp60 and hsp70 were dominant traits. Other genes outside the H-2 locus also influenced antibody responses to hsp60 and hsp70, since inbred strains of identical H-2 but different background genes displayed variable antibody responses to the proteins. The genetic control of murine immune responses to C. trachomatis hsp60, a putative chlamydial immunopathologic antigen, suggests that a similar genetic mechanism may also exist in humans, and this observation may help to explain the observed variability in the spectrum of chlamydial diseases seen in humans.
Infection and Immunity 09/1992; 60(8):3143-9. · 4.16 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Both B- and T-cell immunogenicity of a chlamydial 75-kDa protein was analyzed by using 131 partially overlapped decapeptide homologs of the 75-kDa protein from Chlamydia trachomatis serovar L2. Six rabbit antiserum specimens raised with serovars B, C, and L2 were used to assay the antibody reactivities of the decapeptides. Seventy-five of the 131 decapeptides were recognized by at least one antiserum specimen, and two peptides were found to be immunodominant and surface accessible on native organisms. The same set of decapeptides were cleaved from the pins and tested for their T-cell-stimulating activity in an in vitro proliferation assay. A single decapeptide was able to stimulate proliferation of chlamydial antigen-primed lymph node T cells from BALB/c mice.
Infection and Immunity 04/1992; 60(3):1221-4. · 4.16 Impact Factor
