ABSTRACT
Abstract] Objective To study the roles of IL-12 and IL-23 in the development of protective im-munity and pathological changes during chlamydial urogenital infection.Methods C57BL/6J wild type (wt) mice and mice deficient in IL-12p35 (IL-12p35 KO) or IL-12p40 (IL-12p40 KO)were inoculated in-travaginally with 1×104 IFU of live Chlamydia muridarum ( C.muridarum) organisms.Half mice of each group were reinfected on day 114 after primary infection.Vaginal swabs were taken every 3 or 4 days to mo-nitor live organism shedding.The mice were sacrificed after 114 or 143 days of primary infection and the va-ginal tract and kidney samples were collected for pathological analysis.The numbers of chlamydial inclusion bodies and bacteria in kidney homogenates were titrated after 100 days of primary infection.Results The infection time courses of mice deficient in either IL-12p35 or IL-12p40 were similar after primary infection, but were prolonged as compared with the wild type mice.All mice regardless of genotypes developed severe pathological damages in upper genital tracts with no significant difference among different groups.Almost all IL-12p40 KO mice and some IL-12p35 KO mice showed pathological changes in kidney samples.No obvious abnormality was observed in any of the kidneys from wild type mice.Neither the age-matched IL-12p35 KO nor IL-12p40 KO mice developed any gross pathological changes in kidney in the absence of chlamydial in-fection.C.muridarum inclusions were detected in kidney samples with gross pathological damages from IL-12p35 KO mice and IL-12p40 KO mice.No inclusions were ever detected in kidneys from the wild type mice.The numbers of chlamydial inclusions in the IL-12p40 KO mice were much higher than those of the IL-12p35 KO mice.Live bacteria were detected in mice deficient in either IL-12p35 or IL-12p40, but not in the wild type mice.No significant difference with the number of live bacteria was found between IL-12p35 KO mice and IL-12p40 KO mice.Conclusion IL-12 and IL-23 could inhibit the spread of C.muridarum in-fection from genital tract to kidney.The deficiency of IL-12 or IL-23 might relate to the renal lesions induced by Chlamydia infection.
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Objective To investigate the immune injury in genital tract of BALB/c mice induced by plasmid protein pORF5 of Chlamydia trachomatis and its possible mechanism.Methods GST(glutathione-S-transferases)-pORF5 fusion protein was expressed and digested with PreScission Protease to obtain the target protein without GST tag.After further purification and endotoxin removal,pORF5 protein was injected into the posterior fornix of BALB/c mice on day 1,3 and 6,while the control groups were injected with PBS or GST protein respectively,and then all the mice were sacrificed on day 7 to evaluate genital tract gross pathology and histopathological characterization.The levels of TNF-α,IL-1β and IL-6 in serum,splenocytes culture supernatant and vaginal douche were detected by ELISA.Results Mice in pORF5 group developed different degrees of swelling in isthmic portion and ampulla of uterine tube,connective tissue adhesion and hydrosalpinx in the genital tract tissues,while the PBS group and the GST group did not show any obvious change.The inflammatory score showed that the genital tract pathology in pORF5 group was much more severe than PBS and GST control groups (P＜0.O1).The levels of TNF-α,IL-1β and IL-6 in vaginal douche and splenocytes culture supernatants in pORF5 group were obviously higher than those of PBS and GST groups (P＜0.05).The levels of TNF-α and IL-1β in serum were also higher than those of GST and PBS groups (P＜0.01).Conclusion pORF5 plasmid protein could induce pathological immune response in the genital tract of BALB/c mice,which may be associated with the increase of the production of the inflammatory cytokines TNF-α,IL-1β and IL-6 in BALB/c mice.
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ObjectiveTo construct a mouse model for studying pathophysiology and mechanism of human Chlamydia trachomatis genital infection.MethodsInnate immunity-deficient C3H/HeJ female mice were infected intravaginally with human C.trachomatis serovar D urogenital isolates for screening the highest violent clinical strain.The clinical strain UT0603 as well as standard strain D/UW-3/CX were then used to reinfect na(i)ve mice,the lower genital tract shedding were monitored by swabbing every 3-7 day over the entire infection period by culture.Some mice were sacrificed at early infection stage to detection of in site Chlamydia growth by immunofluorescence assay,then all the mice were sacrificed at later infection stage to evaluate upper genital tract gross pathology and histopathological characterization.ResuIts In the lower genital tract,Chlamydia shedding time course were significantly prolonged in clinical strain infected mice.Chlamydia not only growth in the lower genital tract,the live organism also ascending and growth in the upper genital tissue.The gross appearance under naked eyes and dilation and inflammation scores under microscope all showed that the genital tract pathology from the clinical strain infected mice were much more severe than standard strain infected control mice.Conclusion Together,all these results demonstrated that a mouse model for Chlamydia genital infection was constructed.
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Objective To investigate the specific humoral immune response and cellular immune response induced by DNA vaccine with Neisseria gonorrhoeae porin B (PorB) fused with B subunit of Escherichia coli heat-labile enterotoxin B (LTB) in mice. Methods Target genes of porB, ltB and ltB-porB were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic vector pcDNA3.1(-). The recombinants were identified by PCR, enzyme digestion and DNA sequencing.The vectors were transfected into Hela cells, and expressed proteins were checked by cytoimmunofluorescence. Female BALB/c mice were intranasally immunized with recombination vectors. The humoral immune response and cellular immune response were detected by enzyme linked immunosorbent assay (ELISA) and methyl thiazolyl tetrazolium (MTT) colorimetric assay. The expressions of recombination vectors in intranasal mucosal tissues of the immunized mice were detected by immunohistochemistry. The means between groups were compared by analysis of variance. Results All the three recombinants were expressed in Hela cells and intranasal mucosal tissues. The PorB specific IgG in serum and sIgA in vaginal secretions in DNA vaccine immunized mice were significantly higher than those in controls (P＜0.01 ; P＜0.05). Moreover, the sIgA level in pcDNA3.1 (-)/ltB-porB group was higher than that in peDNA3, 1(-)/porB group (P＝0. 002). The levels of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in the supernatants and stimulation index (SI) of spleen lymphocyte culture in pcDNA3, 1(-)/porB group were (170.04±23.89) pg/mL, (114.68±14.27) pg/mL and 1. 68±0.19, respectively; and those in pcDNA3, 1(-)/ltB-porB group were (161.42±27.50) pg/mL, (124.16±19.04) pg/mL and 1.73±0.28, respectively; which were both higher than those in pcDNA3.1(-)/ phosphate buffered saliae (PBS) group (P＜0. 01; P＜0.05) and pcDNA3.1 (-)/ltB group (all P＜0.05), while there was no significant difference between pcDNA3.1 (-)/ltB-porB group and pcDNA3. 1 (-)/porB group (0. 998, 0. 696, 0. 994; all P＞0.05). Conclusions The constructed DNA vaccines are all successfully expressed in Hela cells and murine intranasal mucosal tissues. The mucosal immunization of the vaccines [pcDNA3. 1 (- )/porB and pcDNA3.1 ( -)/ltBporB] could induce humoral immune response and cellular immune response, especially mucosal immune response. It is confirmed that mucosal adjuvant LTB could promote PorB to induce higher level of mucosal immune response in mice.