Anti-Idiotypic Nanobodies Mimicking an Epitope of the Needle Protein of the Chlamydial Type III Secretion System for Targeted Immune Stimulation.

The development of new approaches and drugs for effective control of the chronic and complicated forms of urogenital chlamydia caused by Chlamydia trachomatis, which is suspected to be one of the main causes of infertility in both women and men, is an urgent task. We used the technology of single-domain antibody (nanobody) generation both for the production of targeting anti-chlamydia molecules and for the subsequent acquisition of anti-idiotypic nanobodies (ai-Nbs) mimicking the structure of a given epitope of the pathogen (the epitope of the Chlamydial Type III Secretion System Needle Protein). In a mouse model, we have shown that the obtained ai-Nbs are able to induce a narrowly specific humoral immune response in the host, leading to the generation of intrinsic anti-Chlamydia antibodies, potentially therapeutic, specifically recognizing a given antigenic epitope of Chlamydia. The immune sera derived from mice immunized with ai-Nbs are able to suppress chlamydial infection in vitro. We hypothesize that the proposed method of the creation and use of ai-Nbs, which mimic and present to the host immune system exactly the desired region of the antigen, create a fundamentally new universal approach to generating molecular structures as a part of specific vaccine for the targeted induction of immune response, especially useful in cases where it is difficult to prepare an antigen preserving the desired epitope in its native conformation.

Chlamydia trachomatis Growth and Cytokine mRNA Response in a Prostate Cancer Cell Line.

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.

Small Molecule Inhibitor of Type Three Secretion System Belonging to a Class 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones Improves Survival and Decreases Bacterial Loads in an Airway Pseudomonas aeruginosa Infection in Mice.

Pseudomonas aeruginosa is a cause of high mortality in burn, immunocompromised, and surgery patients. High incidence of antibiotic resistance in this pathogen makes the existent therapy inefficient. Type three secretion system (T3SS) is a leading virulence system of P. aeruginosa that actively suppresses host resistance and enhances the severity of infection. Innovative therapeutic strategies aiming at inhibition of type three secretion system of P. aeruginosa are highly attractive, as they may reduce the severity of clinical manifestations and improve antibacterial immune responses. They may also represent an attractive therapy for antibiotic-resistant bacteria. Recently our laboratory developed a new small molecule inhibitor belonging to a class 2,4-disubstituted-4H-[1,3, 4]-thiadiazine-5-ones, Fluorothiazinon (FT), that effectively suppressed T3SS in chlamydia and salmonella in vitro and in vivo. In this study, we evaluate the activity of FT towards antibiotic-resistant clinical isolates of P. aeruginosa expressing T3SS effectors ExoU and ExoS in an airway infection model. We found that FT reduced mortality and bacterial loads and decrease lung pathology and systemic inflammation. In addition, we show that FT inhibits the secretion of ExoT and ExoY, reduced bacteria cytotoxicity, and increased bacteria internalization in vitro. Overall, FT shows a strong potential as an antibacterial therapy of antibiotic-resistant P. aeruginosa infection.

Non-Invasive Immunofluorescence Assessment of Lycopene Supplementation Status in Skin Smears.

Circulating lycopene level is negatively associated with the prevalence of cardiovascular disease, cancers (prostate and breast), type 2 diabetes mellitus, and aging. Traditionally, lycopene is measured in biological specimens by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry methods. Moreover, as we recently reported, tissue/cell lycopene depositions can be observed by the immunohistochemistry method with a newly developed monoclonal antibody (mAb) against lycopene. A main objective of this study is to evaluate the performance of a new noninvasive immunofluorescence (IF) lycopene quantification skin test with mAbs against lycopene versus HPLC lycopene assay of serum lycopene in volunteers subjected to lycopene supplementation which represents a novel approach to lycopene measurement methodology. For this purpose, 32 healthy volunteers, 30-40 years old, were supplemented with lycopene (n = 15) or placebo (n = 17) for a period of 4 weeks. It was found that lycopene supplementation leads to a significant increase in serum lycopene concentration after 2 and 4 weeks by 2.6- and 3.4-fold over control, respectively. This was accompanied by a concordant step-wise rise in IF staining of skin corneocytes and sebum, quantifiable by arbitrary IF scores. Placebo supplementation did not affect serum lycopene values or intensity of IF staining of the skin samples. There was 86.6% agreement in paired HPLC/IF variants for the intermediate time point and 80.0% agreement at the end of the study in the lycopene group. Intraclass correlation between paired values in this group was +0.49 for the 2-week time point and +0.63 for the end point. These results indicate that the new antibody-based skin assay can be used for rapid detection of lycopene deficiencies. Moreover, the noninvasive nature of the skin swab test would allow using it to monitor, optimize, and personalize lycopene supplementation protocol of risk groups in the general population.

Association with Monoclonal Antibody Promotes Intracellular Delivery of Lycopene.

Incubation of B10.MLM cells, a cell line of alveolar macrophages, with lycopene, a carotenoid, leads to an increase of lycopene content in their microsomal fraction. That increase was higher and developed faster when the cells were incubated with immune complexes formed by lycopene and mAb 6B9 (L-6B9 mAb), a monoclonal hapten-specific antibody raised against lycopene, as compared with dimethyl sulfoxide (DMSO)-dissolved lycopene (DMSO-L). Moreover, incubation of B10.MLM cells with L-6B9 mAb complexes was accompanied by more efficient accumulation of lipid droplets in the cultured cells and more significant inhibition of mRNA for 3-hydroxy-3-methylglutaryl-coenzyme (HMG-CoA) reductase, a rate-limiting enzyme of cholesterol biosynthesis known to be targeted by lycopene. Additionally, there was a better inhibition of Chlamydia trachomatis infection in B10.MLM cells infected with the pathogen and incubated thereafter with L-6B9 mAb complexes as compared with DMSO-L. Altogether, the results suggest that association with monoclonal antibody promotes intracellular delivery of lycopene in cultured cells possibly through Fc-receptor mediated uptake.

Identification of chlamydial T3SS inhibitors through virtual screening against T3SS ATPase.

Chlamydia trachomatis is a widespread sexually transmitted pathogen that resides within a special vacuole inside host cells. Although acute infection can be treated with antibiotics, chlamydia can enter persistent state, leading to chronic infection that is difficult to cure. Thus, novel anti-chlamydial compounds active against persistent chlamydia are required. Chlamydiae rely upon type III secretion system (T3SS) to inject effector proteins into host cell cytoplasm, and T3SS inhibitors are viewed as promising compounds for treatment of chlamydial infections. C. trachomatis ATPase SctN is an important T3SS component and has not been targeted before. We thus used virtual screening against homology modeled SctN structure to search for SctN inhibitors. Selected compounds were tested for their ability to inhibit chlamydial survival and development within eukaryotic cells, and for the ability to suppress normal T3SS functioning. We identified two compounds that were able to block normal protein translocation through T3SS and inhibit chlamydial survival within eukaryotic cells in 50-100 µm concentrations. These two novel T3SS inhibitors also possessed relatively low toxicity toward eukaryotic cells. A small series of derivatives was further synthesized for the most active of two inhibitors to probe SAR properties.

Lycopene Inhibits Propagation of Chlamydia Infection.

Chlamydiaceae is a family of obligate intracellular pathogenic bacteria with similar developmental cycles and cell biology responsible for a wide range of diseases in different hosts including genital and eye inflammatory diseases, arthritis, and inflammatory diseases of the respiratory and cardiovascular systems. In the present paper, we report that lycopene, one of the main dietary carotenoids, which is present in tomato and some other fruits, has a strong inhibitory effect on C. trachomatis and C. pneumoniae infections in alveolar macrophages. This finding was documented by both immunofluorescence analysis and electron microscopy. It was noted that lycopene treatment inhibited intracellular phase of the chlamydial developmental cycle and resulted in a significant loss of infectious progeny. The antichlamydial effect of lycopene was also confirmed in a clinical setting. There was a significant reduction of IgG antibodies against C. pneumoniae in the serum of volunteers treated for a month with oral ingestion of 7 mg of lycopene. Additional studies are needed to further explore the antichlamydial activity of lycopene and its possible effect on C. pneumoniae in relation to antichlamydial activity of lycopene to mechanisms of atherosclerosis.

Chlamydial Type III Secretion System Needle Protein Induces Protective Immunity against Chlamydia muridarum Intravaginal Infection.

Chlamydia trachomatis imposes serious health problems and causes infertility. Because of asymptomatic onset, it often escapes antibiotic treatment. Therefore, vaccines offer a better option for the prevention of unwanted inflammatory sequelae. The existence of serologically distinct serovars of C. trachomatis suggests that a vaccine will need to provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of structural and effector proteins which is an essential virulence factor. In this study, we expressed the T3SS needle protein of Chlamydia muridarum, TC_0037, an ortholog of C. trachomatis CdsF, in a replication-defective adenoviral vector (AdTC_0037) and evaluated its protective efficacy in an intravaginal Chlamydia muridarum model. For better immune responses, we employed a heterologous prime-boost immunization protocol in which mice were intranasally primed with AdTC_0037 and subcutaneously boosted with recombinant TC_0037 and Toll-like receptor 4 agonist monophosphoryl lipid A mixed in a squalene nanoscale emulsion. We found that immunization with TC_0037 antigen induced specific humoral and T cell responses, decreased Chlamydia loads in the genital tract, and abrogated pathology of upper genital organs. Together, our results suggest that TC_0037, a highly conserved chlamydial T3SS protein, is a good candidate for inclusion in a Chlamydia vaccine.

Generation and Application of Monoclonal Antibody Against Lycopene.

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.

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.

Small molecule inhibitor of type three secretion suppresses acute and chronic Chlamydia trachomatis infection in a novel urogenital Chlamydia model.

Previously, we reported that a compound from a group of thiohydrazides of oxamic acids, CL-55, possessed antichlamydial activity in vitro that was accompanied by a decreased translocation of the type three secretion effector, IncA, into the host cell. In this study, the antichlamydial activity of CL-55 was tested in vivo in DBA/2 mice infected with C. trachomatis serovar D. We found that intravaginal inoculation of DBA/2 mice with the clinically relevant strain, C. trachomatis serovar D, results in a course of infection and pathology similar to that observed in humans. The early stage of infection in this model was characterized by a shedding of Chlamydia in vaginal secretions followed by an ascending infection and inflammation in the upper genital tract. We found that CL-55 possessed antibacterial activity in vivo and was able to control C. trachomatis vaginal shedding, ascending infection, and inflammation in the upper genital organs in DBA/2 mice. Our data provide a proof of concept for the protective effect of the thiadiazinon, CL-55, against chlamydial infection in vivo and support the feasibility of further studies of its potential therapeutic applications.

Roquefort cheese proteins inhibit Chlamydia pneumoniae propagation and LPS-induced leukocyte migration.

Inflammation in atherosclerosis, which could be associated with some subclinical infections such as C. pneumoniae, is one of the key factors responsible for the development of clinical complications of this disease. We report that a proprietary protein extract isolated from Roquefort cheese inhibits the propagation of C. pneumoniae in a human HL cell line in a dose-dependent manner, as revealed by the immunofluorescence analysis. These changes were accompanied by a significant reduction in the infective progeny formation over the protein extract range of 0.12-0.5 µg/mL. Moreover, short term feeding of mice with Roquefort cheese (twice, 10 mg per mouse with an interval of 24 hours) led to the inhibition of the migration of peritoneal leukocytes caused by intraperitoneal injection of E. coli lipopolysaccharide. These changes were complemented by a reduction in neutrophil count and a relative increase in peritoneal macrophages, suggesting that ingestion of Roquefort could promote regenerative processes at the site of inflammation. The ability of this protein to inhibit propagation of Chlamydia infection, as well as the anti-inflammatory and proregenerative effects of Roquefort itself, may contribute to the low prevalence of cardiovascular mortality in France where consumption of fungal fermented cheeses is the highest in the world.

Detection of C. trachomatis in the serum of the patients with urogenital chlamydiosis.

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 × 10(2)-10(3) 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.