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

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.Frontiers in Cellular and Infection Microbiology (2017), 7(JUN): 25

Chlamydia trachomatis infection and development of epithelial mesenchymal transition in conjunctiva : possible epigenetic mechanisms unveiled

Introduction: Trachoma is the most common cause of infectious blindness worldwide, initiated by repeated infection of the conjunctiva with Chlamydia trachomatis (Ct). The resulting chronic inflammation and formation of fibrotic tissue eventually lead to corneal damage. Based on the facts that epithelial to mesenchymal transition (EMT) plays an important role in the development of fibrosis and that EMT is epigenetically regulated process, the aims of this study were to reveal the capacity of Ct to induce EMT in vitro and to unveil potential underlying epigenetic mechanisms. Methods: Human conjunctival epithelial (HCjE) cells were infected with 107 IFU of Ct for 72 h. EMT-inducing signaling pathways, as well as mRNA and protein expression of EMT markers (E-cadherin, fibronectin and α-SMA) were evaluated by RT-qPCR, Immunoblotting and Immunocytochemistry. DNA methylation patterns of selected regions of E-cadherin, fibronectin and α-SMA genes were examined by Methylation-Specific PCR, High Resolution Melting analysis and Bisulfite Sequencing. Results: Infection with Ct was accompanied with the activation of EMT-inducing signaling pathways, downregulation of epithelial marker E-cadherin and upregulation of mesenchymal markers fibronectin and α-SMA. While DNA methylation status of E-cadherin gene promoter correlated with its expression, methylation status of fibronectin and α-SMA genes couldn’t be related to their expression levels. Conclusion: Ct infection of HCjE cells triggers EMT that goes along with changes in the methylation profile of the E-cadherin promoter. Sequence of events described herein could contribute to scarring process in trachoma and open up possibilities for development of new therapeutic strategies in trachoma treatment.Brajušković G, Đorđević A, editors. CoMBoS. 1st Congress of Molecular Biologists of Serbia; 2017 Sep 20-22; Belgrade, Serbia. Belgrade: University of Belgrade, Faculty of Biology; 2017. p. 70

Chlamydia trachomatis infection and development of epithelial mesenchymal transition in conjunctiva : possible epigenetic mechanisms unveiled

Introduction: Trachoma is the most common cause of infectious blindness worldwide, initiated by repeated infection of the conjunctiva with Chlamydia trachomatis (Ct). The resulting chronic inflammation and formation of fibrotic tissue eventually lead to corneal damage. Based on the facts that epithelial to mesenchymal transition (EMT) plays an important role in the development of fibrosis and that EMT is epigenetically regulated process, the aims of this study were to reveal the capacity of Ct to induce EMT in vitro and to unveil potential underlying epigenetic mechanisms. Methods: Human conjunctival epithelial (HCjE) cells were infected with 107 IFU of Ct for 72 h. EMT-inducing signaling pathways, as well as mRNA and protein expression of EMT markers (E-cadherin, fibronectin and α-SMA) were evaluated by RT-qPCR, Immunoblotting and Immunocytochemistry. DNA methylation patterns of selected regions of E-cadherin, fibronectin and α-SMA genes were examined by Methylation-Specific PCR, High Resolution Melting analysis and Bisulfite Sequencing. Results: Infection with Ct was accompanied with the activation of EMT-inducing signaling pathways, downregulation of epithelial marker E-cadherin and upregulation of mesenchymal markers fibronectin and α-SMA. While DNA methylation status of E-cadherin gene promoter correlated with its expression, methylation status of fibronectin and α-SMA genes couldn’t be related to their expression levels. Conclusion: Ct infection of HCjE cells triggers EMT that goes along with changes in the methylation profile of the E-cadherin promoter. Sequence of events described herein could contribute to scarring process in trachoma and open up possibilities for development of new therapeutic strategies in trachoma treatment.Brajušković G, Đorđević A, editors. CoMBoS. 1st Congress of Molecular Biologists of Serbia; 2017 Sep 20-22; Belgrade, Serbia. Belgrade: University of Belgrade, Faculty of Biology; 2017. p. 70

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: 1x10(2), 1x10(4), and 1x10(6) 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 1x10(4), and 1x10(6) 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

When Bacteria and Viruses Collide: A Tale of <i>Chlamydia trachomatis</i> and Sexually Transmitted Viruses

The global incidence of sexually transmitted infections (STIs) remains high, with the World Health Organization (WHO) estimating that over 1 million people acquire STIs daily. STIs can lead to infertility, pregnancy complications, and cancers. Co-infections with multiple pathogens are prevalent among individuals with an STI and can lead to heightened infectivity and more severe clinical manifestations. Chlamydia trachomatis (CT) is the most reported bacterial STI worldwide in both men and women, and several studies have demonstrated co-infection of CT with viral and other bacterial STIs. CT is a gram-negative bacterium with a unique biphasic developmental cycle including infectious extracellular elementary bodies (EBs) and metabolically active intracellular reticulate bodies (RBs). The intracellular form of this organism, RBs, has evolved mechanisms to persist for long periods within host epithelial cells in a viable but non-cultivable state. The co-infections of CT with the most frequently reported sexually transmitted viruses: human immunodeficiency virus (HIV), human papillomavirus (HPV), and herpes simplex virus (HSV) have been investigated through in vitro and in vivo studies. These research studies have made significant strides in unraveling the intricate interactions between CT, these viral STIs, and their eukaryotic host. In this review, we present an overview of the epidemiology of these co-infections, while specifically delineating the underlying mechanisms by which CT influences the transmission and infection dynamics of HIV and HSV. Furthermore, we explore the intricate relationship between CT and HPV infection, with a particular emphasis on the heightened risk of cervical cancer. By consolidating the current body of knowledge, we provide valuable insights into the complex dynamics and implications of co-infection involving CT and sexually transmitted viruses

Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran

  Background: Parasitological methods for the diagnosis of Visceral leishmania-sis (VL) require invasive procedures, so serological and molecular approaches have been developed but are not generally applicable in the field. We evaluated a loop mediated isothermal amplification (LAMP) assay using blood from VL pa-tients and compared it to nested PCR. Methods: Forty-seven subjects with clinical features (fever, hepatosplenomegaly and anemia) were confirmed positive for VL by the direct agglutination test (DAT) at titers >3200. Forty DAT negative individuals from non-endemic areas with no clinical signs or symptoms of VL served as controls. A LAMP assay was performed using a set of six primers targeting Leishmania infantum kinetoplast DNA (kDNA) minicircle gene under isothermal (64 °C) conditions. For nested PCR we used primers targeting the kDNA minicircle gene. Results: The LAMP assay provided a detection limit of 1 parasite in 1 ml of peripheral blood and detected L. infantum DNA in 44 of 47 DAT-confirmed VL cases, with diagnostic sensitivity of 93.6% (95% CI). No L. infantum DNA was amplified in controls, indicating a specificity of 100%. The nested PCR yielded sensitivity of 96% (95% CI) and a specificity of 100% (95% CI). Conclusion: The LAMP assay gave results similar to those of nested PCR but in a shorter time. The LAMP method is simple; requires no sophisticated equip-ment; has a short reaction time; and results, indicated by turbidity of the reaction mixture, are observable with the naked eye