Effects of iota-carrageenan on ocular Chlamydia trachomatis infection in vitro and in vivo.

Ocular chlamydial infections with the ocular serovars A, B, Ba, and C of Chlamydia trachomatis represent the world's leading cause of infectious blindness. Carrageenans are naturally occurring, sulfated polysaccharides generally considered safe for food and topical applications. Carrageenans can inhibit infection caused by a variety of viruses and bacteria. To investigate whether iota-carrageenan (I-C) isolated from the red alga Chondrus crispus could prevent ocular chlamydial infection, we assessed if targeted treatment of the conjunctival mucosa with I-C affects chlamydial attachment, entry, and replication in the host cell. Immortalized human conjunctival epithelial cells were treated with I-C prior to C. trachomatis infection and analyzed by flow cytometry and immunofluorescence microscopy. In vivo effects were evaluated in an ocular guinea pig inclusion conjunctivitis model. Ocular pathology was graded daily, and chlamydial clearance was investigated. Our study showed that I-C reduces the infectivity of C. trachomatis in vitro. In vivo results showed a slight reduced ocular pathology and significantly less shedding of infectious elementary bodies by infected animals. Our results indicate that I-C could be a promising agent to reduce the transmission of ocular chlamydial infection and opens perspectives to develop prophylactic approaches to block C. trachomatis entry into the host cell.

Detection of Chlamydiaceae and Chlamydia-like organisms on the ocular surface of children and adults from a trachoma-endemic region.

Trachoma, the leading infectious cause of blindness, is caused by Chlamydia trachomatis (Ct), a bacterium of the phylum Chlamydiae. Recent investigations revealed the existence of additional families within the phylum Chlamydiae, also termed Chlamydia-like organisms (CLOs). In this study, the frequency of Ct and CLOs was examined in the eyes of healthy Sudanese (control) participants and those with trachoma (case). We tested 96 children (54 cases and 42 controls) and 93 adults (51 cases and 42 controls) using broad-range Chlamydiae and Ct-specific (omcB) real-time PCR. Samples positive by broad-range Chlamydiae testing were subjected to DNA sequencing. Overall Chlamydiae prevalence was 36%. Sequences corresponded to unclassified and classified Chlamydiae. Ct infection rate was significantly higher in children (31.5%) compared to adults (0%) with trachoma (p < 0.0001). In general, 21.5% of adults and 4.2% of children tested positive for CLOs (p = 0.0003). Our findings are consistent with previous investigations describing the central role of Ct in trachoma among children. This is the first study examining human eyes for the presence of CLOs. We found an age-dependent distribution of CLO DNA in human eyes with significantly higher positivity in adults. Further studies are needed to understand the impact of CLOs in trachoma pathogenicity and/or protection.

The effect of infectious dose on humoral and cellular immune responses in Chlamydophila caviae primary ocular infection.

Following infection, the balance between protective immunity and immunopathology often depends on the initial infectious load. Several studies have investigated the effect of infectious dose; however, the mechanism by which infectious dose affects disease outcomes and the development of a protective immune response is not known. The aim of this study was to investigate how the infectious dose modulates the local and systemic humoral and the cellular immune responses during primary ocular chlamydial infection in the guinea pig animal model. Guinea pigs were infected by ocular instillation of a Chlamydophila caviae-containing eye solution in the conjunctival sac in three different doses: 1×102, 1×104, and 1×106 inclusion forming units (IFUs). Ocular pathology, chlamydial clearance, local and systemic C. caviae-specific humoral and cellular immune responses were assessed. All inocula of C. caviae significantly enhanced the local production of C. caviae-specific IgA in tears, but only guinea pigs infected with the higher doses showed significant changes in C. caviae-specific IgA levels in vaginal washes and serum. On complete resolution of infection, the low dose of C. caviae did not alter the ratio of CD4+ and CD8+ cells within guinea pigs' submandibular lymph node (SMLN) lymphocytes while the higher doses increased the percentages of CD4+ and CD8+ cells within the SMLN lymphocytes. A significant negative correlation between pathology intensity and the percentage of CD4+ and CD8+ cells within SMLN lymphocyte pool at selected time points post-infection was recorded for both 1×104, and 1×106 IFU infected guinea pigs. The relevance of the observed dose-dependent differences on the immune response should be further investigated in repeated ocular chlamydial infections.

Chlamydia trachomatis Infection Is Associated with E-Cadherin Promoter Methylation, Downregulation of E-Cadherin Expression, and Increased Expression of Fibronectin and α-SMA-Implications for Epithelial-Mesenchymal Transition.

Chlamydia trachomatis (Ct) can induce scarring disease of the ocular mucosa, known as trachoma, the most common infectious cause of blindness worldwide. We hypothesized that epithelial-mesenchymal transition (EMT) contributes to the fibrotic process in trachomatous scarring. Infection of human conjunctival epithelial cells (HCjE) with Ct activated signaling pathways involved in EMT induction, which was correlated with decreased expression of E-cadherin, guardian of the epithelial phenotype. In addition, Ct infection was associated with increased expression of two mesenchymal cell markers: fibronectin and α-SMA. The DNA methylation statuses of selected regions of E-cadherin, fibronectin, and α-SMA genes revealed that Ct infection was accompanied with changes in DNA methylation of the E-cadherin promoter, while the expression of the two mesenchymal markers was not related with this epigenetic event. Our data suggest that Ct infection of conjunctival epithelial cells induces EMT-like changes that go along with modification of the methylation profile of the E-cadherin promoter and could, as one of the earliest events, contribute to processes triggering conjunctival scarring.

Implications for Ophthalmic Formulations: Ocular Buffers Show Varied Cytotoxic Impact on Human Corneal-Limbal and Human Conjunctival Epithelial Cells.

PURPOSE: To investigate toxicity associated with buffers commonly used in topical ocular drug formulations using a human corneal-limbal epithelial (HCLE) and a human conjunctival epithelial (HCjE) cell model. METHODS: HCLE and HCjE cells were incubated for 10, 30, or 60 minutes with 4 different buffers based on borate, citrate, phosphate, and Tris-HCl at 10, 50, and 100 mM concentrations. To detect possible delayed effects on cell viability, after 60 minutes of buffer incubation, cells were further incubated for 24 hours with a cell medium. Cell viability was determined using a colorimetric XTT-based assay. The morphology of cells was also investigated. RESULTS: HCjE cells showed more sensitivity to buffer incubation than HCLE cells. The 100 mM phosphate buffer displayed significant delayed effects on cell viability of HCLE 16.8 ± 4.8% and HCjE 39.2 ± 6.1% cells after 60 minutes of exposure (P < 0.05). HCjE cell viability was reduced after 60 minutes incubations with 50 and 100 mM citrate buffer to 42.8 ± 6.5% and 39.3 ± 7.9%, respectively, and even lower percentages at the delayed time point (both P < 0.05). HCLE cell morphology was distinctly altered by 100 mM phosphate and Tris buffers after 30 minutes, whereas HCjE cells already showed marked changes after 10 minutes of exposure to 100 mM citrate and phosphate buffers. CONCLUSIONS: We observed a time-dependent decrease of viability in both HCLE and HCjE cells exposed to higher buffer concentrations. Therefore, we propose further in vivo studies to translate these finding to humans to discern the real effects of the buffer concentration in eye drops on the ocular surface.

A Probiotic Adjuvant Lactobacillus rhamnosus Enhances Specific Immune Responses after Ocular Mucosal Immunization with Chlamydial Polymorphic Membrane Protein C.

Recent advances in the development of chlamydia vaccines, using live-attenuated or ultraviolet light-inactivated chlamydia, are paving the way for new possibilities to oppose the societal challenges posed by chlamydia-related diseases, such as blinding trachoma. An effective subunit vaccine would mitigate the risks associated with the use of a whole-cell vaccine. Our rationale for the design of an efficient subunit vaccine against Chlamydia trachomatis (Ct) is based on the membrane proteins involved in the initial Ct-host cell contact and on the route of immunization that mimics the natural infection process (i.e., via the ocular mucosa). The first aim of our study was to characterize the specific conjunctival and vaginal immune responses following eye drop immunization in BALB/c mice, using the N-terminal portion of the Ct serovar E polymorphic membrane protein C (N-PmpC) as the subunit vaccine antigen. Second, we aimed to examine the adjuvant properties of the probiotic Lactobacillus rhamnosus (LB) when formulated with N-PmpC. N-PmpC applied alone stimulated the production of N-PmpC- and Ct serovar B-specific antibodies in serum, tears and vaginal washes, whereas the combination with LB significantly enhanced these responses. The N-PmpC/LB combination initiated a T cell response characterized by an elevated percentage of CD25+ T cells and CD8+ effector T cells, enhanced CD4+ T-helper 1 skewing, and increased regulatory T cell responses. Together, these results show that eye drop vaccination with combined use of N-PmpC and a live probiotic LB stimulates specific cellular and humoral immune responses, not only locally in the conjunctiva but also in the vaginal mucosa, which could be a promising approach in Ct vaccine development.

Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers.

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world's leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1-893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/c mice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocular mucosa was well tolerated without signs of inflammation. N-PmpC-specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFNγ immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage.

Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface.

To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs) as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN), whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.

Effect of high-dose simvastatin on brain atrophy and disability in secondary progressive multiple sclerosis (MS-STAT): a randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Secondary progressive multiple sclerosis, for which no satisfactory treatment presently exists, accounts for most of the disability in patients with multiple sclerosis. Simvastatin, which is widely used for treatment of vascular disease, with its excellent safety profile, has immunomodulatory and neuroprotective properties that could make it an appealing candidate drug for patients with secondary progressive multiple sclerosis. METHODS: We undertook a double-blind, controlled trial between Jan 28, 2008, and Nov 4, 2011, at three neuroscience centres in the UK. Patients aged 18-65 years with secondary progressive multiple sclerosis were randomly assigned (1:1), by a centralised web-based service with a block size of eight, to receive either 80 mg of simvastatin or placebo. Patients, treating physicians, and outcome assessors were masked to treatment allocation. The primary outcome was the annualised rate of whole-brain atrophy measured from serial volumetric MRI. Analyses were by intention to treat and per protocol. This trial is registered with ClinicalTrials.gov, number NCT00647348. FINDINGS: 140 participants were randomly assigned to receive either simvastatin (n=70) or placebo (n=70). The mean annualised atrophy rate was significantly lower in patients in the simvastatin group (0·288% per year [SD 0·521]) than in those in the placebo group (0·584% per year [0·498]). The adjusted difference in atrophy rate between groups was -0·254% per year (95% CI -0·422 to -0·087; p=0·003); a 43% reduction in annualised rate. Simvastatin was well tolerated, with no differences between the placebo and simvastatin groups in proportions of participants who had serious adverse events (14 [20%] vs nine [13%]). INTERPRETATION: High-dose simvastatin reduced the annualised rate of whole-brain atrophy compared with placebo, and was well tolerated and safe. These results support the advancement of this treatment to phase 3 testing. FUNDING: The Moulton Foundation [charity number 1109891], Berkeley Foundation [268369], the Multiple Sclerosis Trials Collaboration [1113598], the Rosetrees Trust [298582] and a personal contribution from A Pidgley, UK National Institute of Health Research (NIHR) University College London Hospitals/UCL Biomedical Research Centres funding scheme.