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31.
Dendritic cells pulsed with a recombinant chlamydial major outer membrane protein antigen elicit a CD4(+) type 2 rather than type 1 immune response that is not protective 总被引:3,自引:0,他引:3
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Chlamydia trachomatis is an obligate intracellular bacterium that infects the oculogenital mucosae. C. trachomatis infection of the eye causes trachoma, the leading cause of preventable blindness. Infections of the genital mucosae are a leading cause of sexually transmitted diseases. A vaccine to prevent chlamydial infection is needed but has proven difficult to produce by using conventional vaccination approaches. Potent immunity to vaginal rechallenge in a murine model of chlamydial genital infection has been achieved only by infection or by immunization with dendritic cells (DC) pulsed ex vivo with whole inactivated organisms. Immunity generated by infection or ex vivo antigen-pulsed DC correlates with a chlamydia-specific interleukin 12 (IL-12)-dependent CD4(+) Th1 immune response. Because of the potent antichlamydial immunizing properties of DC, we hypothesized that DC could be a powerful vehicle for the delivery of individual chlamydial antigens that are thought to be targets for more conventional vaccine approaches. Here, we investigated the recombinant chlamydial major outer membrane protein (rMOMP) as a target antigen. The results demonstrate that DC pulsed with rMOMP secrete IL-12 and stimulate infection-sensitized CD4(+) T cells to proliferate and secrete gamma interferon. These immunological properties implied that rMOMP-pulsed DC would be potent inducers of MOMP-specific CD4(+) Th1 immunity in vivo; however, we observed the opposite result. DC pulsed ex vivo with rMOMP and adoptively transferred to naive mice generated a Th2 rather than a Th1 anti-MOMP immune response, and immunized mice were not protected following infectious challenge. We conclude from these studies that the immunological properties of ex vivo pulsed DC are not necessarily predictive of the immune response generated in vivo following adoptive transfer. These findings suggest that the nature of the antigen used to pulse DC ex vivo influences the Th1-Th2 balance of the immune response in vivo. 相似文献
32.
The speed with which insulin alters hepatocyte ultrastructure and glycogen levels in insulin-deficient rats has been studied. Insulin deficiency was induced with alloxan, followed by insulin treatment with regular and NPH insulin. Rats were killed at various times after the insulin injection, blood samples were obtained, plasma glucose levels were determined, and liver samples were prepared for electron microscopy and glycogen determinations. Plasma glucose levels in insulin-deficient rats declined to normal values by 4 hours post insulin, returning to insulin-deficient levels by 8 hours post insulin. Hepatic glycogen was considerably reduced in the insulin-deficient rats. By 1 hour post insulin hepatic glycogen increased, reached maximal levels by 8 hours, then declined to insulin-deficient levels by 36 hours. The ultrastructural appearance of both centrilobular and periportal hepatocytes from insulin-deficient rats showed abundant vesicular smooth endoplasmic reticulum (SER), decreased rough endoplasmic reticulum (RER), and enlarged RER intracisternal spaces. One-half hour post insulin, centrilobular hepatocytes were unchanged. In periportal hepatocytes, however, vesicular SER was no longer visible, the RER intracisternal spaces appeared normal, and the amount of RER had increased. By 1 hour post insulin the centrilobular hepatocytes showed similar ultrastructural changes. These changes became more pronounced in the next few hours and remained through 24 hours. By 36 hours both centrilobular and periportal hepatocytes appeared similar to those in the insulin-deficient rat. These results demonstrate the rapid and lobular-specific effects insulin has on the hepatocyte. 相似文献
33.
The 49,000-dalton (49-kDa) small nuclear inclusion (NI) protein of tobacco etch virus (TEV) has two distinct functions associated with it. An N-terminal segment is covalently attached to the genomic length RNA and likely involved in RNA replication, while the C-terminal half is associated with a proteolytic activity critical for genome expression. The junction delineating these two proteins has not been identified. We have analyzed naturally occurring cleavage products of TEV NI proteins and have identified a possible internal cleavage site between Glu and Gly residues at TEV 49-kDa NI protein amino acids 189-190. Similar 49-kDa-derived products are formed in cell-free translation studies in minor amounts upon the addition of excess amounts of NI protein. Cleavage of the 49-kDa (430 amino acids) protein is predicted to result in the formation of two products, 21-kDa (189 amino acids) and 27 kDa (241 amino acids) in size. Complementary DNA encoding the 27-kDa C-terminal portion of the 49-kDa protein gene was cloned into various DNA sequences. This allowed us to express the 27-kDa protein alone or as part of higher molecular weight polyproteins containing flanking TEV or foreign protein sequences. This 27-kDa amino acid sequence had a proteolytic activity similar to the 49-kDa-associated activity. 相似文献
34.
Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury. 总被引:4,自引:1,他引:4
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M. E. Fini W. C. Parks W. B. Rinehart M. T. Girard M. Matsubara J. R. Cook J. A. West-Mays P. M. Sadow R. E. Burgeson J. J. Jeffrey M. B. Raizman R. R. Krueger J. D. Zieske 《The American journal of pathology》1996,149(4):1287-1302
Delayed re-epithelialization of the cornea after injury usually precedes stromal ulceration. Previous findings using a rat thermal injury model suggested that re-epithelialization is impeded by products of resident corneal cells, which destroy adhesive structures at the basement membrane zone. In this study, we provide additional evidence for this concept. Failure to re-epithelialize was found to correlate with an increase in the amounts of gelatinolytic matrix metalloproteinases present in the rat cornea. One of these gelatinases, gelatinase B, is synthesized by the resident corneal cells, and inhibitions of its synthesis correlated with inhibition of basement membrane dissolution. The matrix metalloproteinases collagenase and stromelysin are also synthesized by resident corneal cells in thermally injured corneas of rabbits, but the timing of bulk enzyme synthesis correlated more closely with deposition of repair tissue in the stroma than with failure to re-epithelialize. Nevertheless, in human corneas with repair defects, gelatinase B and collagenase are synthesized by cells in the basal layer of the epithelium directly adjacent to the basement membrane, suggesting that both could participate in dissolution of this structure. Importantly, treatment of thermally injured corneas with a synthetic inhibitor of matrix metalloproteinases significantly improved basement membrane integrity. These data support the concept that over-expression of matrix metalloproteinases by resident corneal cells impedes re-epithelialization after some types of corneal injury. 相似文献
35.
J Schachter W M McCormack R F Smith R M Parks R Bailey A C Ohlin 《Journal of clinical microbiology》1984,19(1):57-59
An enzyme immunoassay (EIA; Gonozyme [Abbott Laboratories]) for gonococcal antigen was assessed for the rapid diagnosis of gonorrhea. Patients attending two sexually transmitted disease clinics were tested by EIA and culture on Thayer-Martin medium. EIA was highly effective in detecting gonococcal infection among symptomatic men, with 70 of 75 (93.3%) culture-positive men having positive tests and no false-positive reactions. The performance of the test was not as good in detecting cervical gonorrhea; the best result obtained was a sensitivity of 87% (33 of 38) for EIA compared with culture. EIA false-positives occurred at a relatively low rate for women, with the test having a specificity of ca. 97%. The test clearly is capable of detecting gonococcal antigen in cervical and urethral specimens, but its role in routine diagnosis is not clear. Its performance seems equal to that of the Gram stain for men, but it seems to be less sensitive than culture for cervical gonorrhea--a drawback in high-risk populations. The low false-positive rate could be an important issue in screening low-prevalence populations. 相似文献
36.
The proteolytic activity associated with the small nuclear inclusion protein (NIa proteinase) of tobacco etch virus (TEV), a potyvirus, catalyzes several cleavages at sites within the polyprotein derived from the TEV RNA genome. The homologous proteinase of tobacco vein mottling virus (TVMV), a closely related potyvirus, cleaves at similar, yet distinct, recognition sites. We examined these proteinases, in a cell-free cleavage system, in an attempt to define the biochemical basis of substrate specificity. Each proteinase was specific for its own cleavage site sequence in cell-free trans processing reactions, and no processing of the heterologous cleavage site was evident. Domains of the proteinase which were important in determining this substrate specificity were identified by generating hybrid proteinase genes containing both TEV and TVMV NIa proteinase coding sequences. Using site-directed mutagenesis and standard recombinant DNA techniques, plasmids were constructed which contained coding sequences for hybrid TEV-TVMV proteinases. These plasmids were expressed and tested in a cell-free environment for their ability to cleave both TEV and TVMV substrates. The data suggest that the carboxy-terminal 150 amino acids of the NIa protein contain the necessary information to specifically recognize a particular cleavage site sequence, and that specificity determinants are contained in at least three interactive subdomains within this region. 相似文献
37.
Site-directed ELISA with synthetic peptides representing the HIV transmembrane glycoprotein 总被引:7,自引:0,他引:7
Francesca Chiodi Agneta von Gegerfeldt Jan Albert Eva Maria Feny Hans Gaines Madeleine von Sydow Gunnel Biberfeld Elliot Parks Erling Norrby 《Journal of medical virology》1987,23(1):1-9
Two partially overlapping 19 and 22 amino acids long peptides representing a highly immunogenic site of the transmembranous glycoprotein (gp41) of human immunodeficiency virus (HIV) were used as antigen in ELISA tests. The results of antibody determination with this assay were compared with those of three or more conventional ELISAs and Western blot (WB) tests and radioimmunoprecipitation assay. Twenty-six sera from patients with AIDS or LAS and from asymptomatic carriers of HIV infection all showed a pronounced reaction in the peptide ELISA as well as positive results with other tests. In contrast, 27 sera from laboratory workers and blood donors were negative by all tests. A group of 39 blood donor sera, which had shown false positive or ambiguous results in the ELISAs and sometimes in WB tests employed for confirmation, also were negative in all cases with the peptide ELISA. Consecutive samples collected from individuals with primary HIV infection were also analyzed. In 6 out of 9 cases, the peptide ELISA revealed an antibody response within one month after onset of clinical symptoms and sensitivity for antibody detection equaled that of other ELISA tests. Eight sera from five West African persons infected with HIV-related viruses did not react in the peptide ELISA, reflecting differences in properties of the envelope components. The peptide ELISA used in this study appears to represent a simple technique employing chemically synthesized antigen for accurate and sensitive estimation of antibodies to the HIV group of nontransforming human retroviruses. 相似文献
38.
39.
Infection of human cells with the paramyxovirus simian virus 5 (SV5) results in minimal cytopathic effect, and host interferon (IFN) and apoptotic pathways are not activated. We have previously shown that an rSV5 containing six naturally occurring P/V gene substitutions (rSV5-P/V-CPI-) displays premature and elevated expression of viral RNA and protein. In addition, cells infected with rSV5-P/V-CPI- show induction of the IFN-beta promoter as well as activation of IFN signaling and apoptotic pathways. In this article, we have tested the hypothesis that rSV5-WT can supply trans-acting factors that prevent host cell antiviral responses induced by rSV5-P/V-CPI-. During coinfection of human A549 cells, rSV5-WT blocked cell rounding, loss of cell volume, and DNA fragmentation induced by rSV5-P/V-CPI-, three later events in the apoptotic pathway, but was not able to block the loss of mitochondrial membrane potential (DeltaPsi(m)), an early event in the cell death process. As expected, IFN signaling was blocked during coinfections, and this was attributed to the loss of STAT1 induced by the rSV5-WT V protein. Surprisingly, simultaneous infection with rSV5-WT could not suppress the activation of the IFN-beta promoter by rSV5-P/V-CPI- infection. However, the IFN-beta promoter was not activated in cells that were first preinfected for 1 h with rSV5-WT and then subsequently infected with rSV5-P/V-CPI-. A model is proposed for activation of host responses to infection with the rSV5-P/V-CPI- mutant and the steps that are blocked by rSV5-WT. 相似文献
40.
A paramyxovirus SV5 mutant (rSV5-P/V-CPI-) that encodes 6 naturally-occurring P/V gene substitutions is a potent inducer of type I interferon (IFN) and is restricted for low moi growth, two phenotypes not seen with WT SV5. In this study, we have compared the IFN sensitivity of WT SV5 and the rSV5-P/V-CPI- mutant in tumor cell lines and in cultures of normal primary cells. We have tested the hypothesis that differences in IFN induction elicited by WT rSV5 and rSV5-P/V-CPI- are responsible for differences in low moi growth and spread. In contrast to WT SV5, low moi infection of A549 lung carcinoma cells with rSV5-P/V-CPI- resulted in a plateau of virus production by 24-48 h pi when secreted IFN levels were between approximately 100 and 1000 U/ml. Gene microarray and RT-PCR analyses identified IFN genes and IFN-stimulated genes whose expression were increased by infection of A549 cells with WT and P/V mutant viruses. Restricted low moi growth and spread of rSV5-P/V-CPI- in A549 cells was relieved in the presence of neutralizing antibodies to IFN-beta but not TNF-alpha. When A549 or MDA-MB-435 breast tumor cells were pretreated with IFN, both WT and P/V mutant viruses showed delayed spread and approximately 10-fold reduction in virus yield, but infections were not eliminated. Using normal primary human epithelial cells that have undergone limited passage in culture, WT rSV5 and rSV5-P/V-CPI- displayed high moi growth properties that were similar to that seen in A549 cells. However, IFN pretreatment of these primary cells as well as normal human lung cells eliminated low moi spread of both mutant and WT rSV5 infections. Together, these data demonstrate that SV5 growth in normal primary human cells is highly sensitive to IFN compared to growth in some tumor cell lines, regardless of whether the P/V gene is WT or mutant. These results suggest a model in which spread of WT SV5 in normal human cells is dependent on the ability of the virus to prevent IFN synthesis. The implications of these results for the use of recombinant paramyxoviruses as vectors are discussed. 相似文献