Resveratrol (RESV), an antifungal compound from grapes and other plants, has a distinct ability to inhibit the Chlamydia (C.) trachomatis developmental cycle in McCoy cells, a classic cell line used for chlamydial research. Inoculation of C. trachomatis with increasing amounts of RESV (from 12.5 to 100 μM) gave a dose-dependent reduction in the number of infected McCoy cells visualized by using monoclonal antibodies against chlamydial lipopolysaccharide. A similar trend has been observed with immunoassay for major outer membrane protein (MOMP). Furthermore, there was a step-wise reduction in the number of C. trachomatis infective progenies caused by the increasing concentrations of RESV. The ability of RESV to arrest C. trachomatis growth in McCoy cells was confirmed by a nucleic acid amplification protocol which revealed dose-dependent changes in mRNAs for different genes of chlamydial developmental cycle (euo, incA, and omcB). Although the precise nature of the antichlamydial activity of RESV is yet to be determined and evaluated in future studies, the observed effect of RESV on C. trachomatis infection was not related to its potential effect on attachment/entry of the pathogen into eukaryotic cells or RESV toxicity to McCoy cells. Similar inhibitory effect was shown for C. pneumoniae and C. muridarum.
Extragenital chlamydial complications may be associated with systemic spread of infection, but haematogenous route for C. trachomatis dissemination has not been clearly demonstrated. Here we report that serum specimens obtained from patients with chlamydiosis contain elementary bodies of C. trachomatis shown by culture and immunogold electron microscopy. We have found that 31 of the 52 patients had serum precipitates which were infective to McCoy cells. Immunostaining revealed very small inclusions resembling those reported during persistent C. trachomatis infection in vitro. DNA specimens from 49 (out of 52) patients with chlamydiosis gave positive PCR readings. The viability of the pathogen present in the sera was confirmed by chlamydial RNA detection in the cell monolayer inoculated by the serum precipitates. By using DNA isolation protocol from 1 mL of serum and quantitative TaqMan PCR, it was estimated that bacterial load in patients' sera was 2 × 102–103 GE/mL. These findings for the first time demonstrated that C. trachomatis can be disseminated directly by the plasma, independently from blood cell, which may represent a new possible pathway of the chronic infection development. Therefore, new methodological approaches for detection of C. trachomatis in the serum of patients with complicated and chronic chlamydiosis could be important in the diagnosis of the infection regardless of its anatomical localization.
In the present paper, we report that C. trachomatis can be efficiently propagated and affect mRNA expression for two major cytokines, relevant to tumor progression, in CWR-R1 cells, a malignant prostate cell line. CWR-R1 and McCoy cells, a classic cell line for chlamydial research, were grown and infected with C. trachomatis under similar conditions. Cell monolayers were harvested for RNA analysis and immunostaining with major outer membrane protein (MOMP) antibody at 24, 48, and 72 hours of the postinfection (hpi) period. It was shown that the infectious cycle of chlamydial pathogen in CWR-R1 cells resembles the progression of C. trachomatis infection in McCoy cells but with a few important differences. First of all, the initial stage of C. trachomatis propagation in CWR-R1 cells (24 hpi) was characterized by larger inclusion bodies and more intense, specific immunofluorescent staining of infected cells as compared with McCoy cells. Moreover, there was a corresponding increase in infective progeny formation in CWR-R1 cells along with mRNA for EUO, a crucial gene controlling the early phase of the chlamydial development cycle (24 hpi). These changes were more minimal and became statistically insignificant at a later time point in the infectious cycle (48 hpi). Altogether, these data suggest that the early phase of C. trachomatis infection in CWR-R1 cells is accompanied by more efficient propagation of the pathogen as compared with the growth of C. trachomatis in McCoy cells. Furthermore, propagation of C. trachomatis in CWR-R1 cells leads to enhanced transcription of interleukin-6 and fibroblast growth factor-2, genes encoding two important proinflammatory cytokines implicated in the molecular mechanisms of chemoresistance of prostate cancer and its ability to metastasize. The possible roles of reactive oxygen species and impaired mitochondrial oxidation in the prostate cancer cell line are discussed as factors promoting the early stages of C. trachomatis growth in CWR-R1 cells.
A small-molecule compound belonging to a class of 2,4-disubstituted 1,3,4-thiadiazine-5-ones inhibits intracellular growth and persistence of Chlamydia trachomatis Chlamydia trachomatis is one of the most common sexually transmitted pathogens in the world and often causes chronic inflammatory diseases that are insensitive to antibiotics. The type 3 secretion system (T3SS) of pathogenic bacteria is a promising target for therapeutic intervention aimed at bacterial virulence and can be an attractive alternative for the treatment of chronic infections. Recently, we have shown that a small-molecule compound belonging to a class of 2,4-disubstituted 1,3,4-thiadiazine-5-ones produced through the chemical modification of the thiohydrazides of oxamic acids, designated CL-55, inhibited the intracellular growth of C. trachomatis in a T3SS-dependent manner. To assess the feasibility of CL-55 as a therapeutic agent, our aim was to determine which point(s) in the developmental cycle CL-55 affects. We found that CL-55 had no effect on the adhesion of elementary bodies (EBs) to host cells but significantly suppressed EB internalization. We further found that CL-55 inhibited the intracellular division of reticulate bodies (RBs). An ultrastructural analysis revealed loss of contact between the RBs and the inclusion membrane in the presence of CL-55. Finally, we found that our T3SS inhibitor prevented the persistence of Chlamydia in cell culture and its reversion to the infectious state. Our findings indicate that our T3SS inhibitor may be effective in the treatment of both productive and persistent infections.
A monoclonal antibody (Mab) against lycopene was developed from hybridoma clones obtained from BALB/c mice immunized with trans-isomer of lycopene (t-lycopene, t-LC) conjugated with colloidal gold particles. An alternating immunization schedule which included injection of both formulations of immunogen (without and with Freund's adjuvant) was most effective in the elucidation of a measurable immune response to the t-Lycopene conjugate. Selected hybridoma clones were able to produce an Mab positive in competition assay. In particular, preincubation of 6B9 Mabs with t-LC abolished the ability of 6B9 Mabs to bind LC in the competition assay. Mabs produced by other clones (4F10, 4A3, and 3B12) worked similarly. Analysis of antigen specificity showed that 6B9 Mab raised against t-LC did not recognize other carotenoids such as lutein and carotene. Mab 6B9 was shown to recognize lycopene on a glass surface and in the settings of indirect immunofluorescence experiments performed in cultured hepatocytes and alveolar macrophages incubated with and without lycopene, as well as in sebum and corneocyte specimens from the skin of volunteers supplemented with nutraceutical formulation of lycopene. Newly generated Mabs against lycopene may provide a valuable tool for different analytical assays of lycopene content in various biological, agricultural, and food products.
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