Anti-chlamydial effects of azelastine hydrochloride and the impact of the histamine H1 receptor on chlamydial development.

Introduction. Azelastine hydrochloride, a second-generation histamine H1 receptor (H1R) antagonist, exhibits anti-chlamydial effects against Chlamydia trachomatis (CT) in HeLa cells (genital infection model).Hypothesis/Gap Statement. Non-antibiotic pharmaceutical interactions with CT are an understudied field and the anti-chlamydial effects of azelastine are a potential interaction requiring further elucidation.Aim. To explore the underlying anti-chlamydial mechanisms of azelastine.Methodology. We assessed the specificity of azelastine for the chlamydial species and host cell type, the timing of azelastine application and whether the anti-chlamydial effects could be reproduced with different H1R-modulating compounds.Results. We observed similar anti-chlamydial azelastine effects for Chlamydia muridarum as well as for an ocular CT strain in human conjunctival epithelial cells (ocular infection model). Pre-incubating host cells with azelastine before infection mildly reduced chlamydial inclusion numbers and infectivity. Incubation of cells with azelastine initiated concomitantly with the chlamydial infection, or initiated several hours post-infection, reduced inclusion size, number and infectivity, and altered chlamydial morphology. These effects were strongest when azelastine was added shortly after or with the infection. Azelastine effects were not alleviated by increased concentrations of culture medium nutrients. Additionally, we did not observe anti-chlamydial effects when incubating cultures either with a different H1R antagonist or agonist, indicating that azelastine effects are probably H1R-independent.Conclusion. Accordingly, we conclude that azelastine anti-chlamydial effects are not restricted to a specific chlamydial species, strain or culture model, and are probably not mediated by H1R antagonism. Thus, it appears likely that off-target mechanisms of azelastine may explain our observations.

Refinement of water-filtered infrared A (wIRA) irradiations of in vitro acute and persistent chlamydial infections.

Water-filtered infrared A (wIRA) alone or in combination with visible light (VIS) exerts anti-chlamydial effects in vitro and in vivo in acute infection models. However, it has remained unclear whether reduced irradiation duration and irradiance would still maintain anti-chlamydial efficacy. Furthermore, efficacy of this non-chemical treatment option against persistent (chronic) chlamydial infections has not been investigated to date. To address this knowledge gap, we evaluated 1) irradiation durations of 5, 15 or 30 min in genital and ocular Chlamydia trachomatis acute infection models, 2) irradiances of 100, 150 or 200 mW/cm2 in the acute genital infection model and 3) anti-chlamydial activity of wIRA and VIS against C. trachomatis serovar B and E with amoxicillin (AMX)- or interferon γ (IFN-γ)-induced persistence. Reduction of irradiation duration reduced anti-chlamydial efficacy. Irradiances of 150 to 200 mW/cm2, but not 100 mW/cm2, induced anti-chlamydial effects. For persistent infections, wIRA and VIS irradiation showed robust anti-chlamydial activity independent of the infection status (persistent or recovering), persistence inducer (AMX or IFN-γ) or chlamydial strain (serovar B or E). This study clarifies the requirement of 30 min irradiation duration and 150 mW/cm2 irradiance to induce significant anti-chlamydial effects in vitro, supports the use of irradiation in the wIRA and VIS spectrum as a promising non-chemical treatment for chlamydial infections and provides important information for follow-up in vivo studies. Notably, wIRA and VIS exert anti-chlamydial effects on persistent chlamydiae which are known to be refractory to antibiotic treatment.

Effects of water-filtered infrared A and visible light (wIRA/VIS) radiation on heat- and stress-responsive proteins in the retina and cornea of guinea pigs.

Water-filtered infrared A and visible light (wIRA/VIS), shown to reduce chlamydial infections in vitro and in vivo, might represent an innovative therapeutic approach against trachoma, a neglected tropical disease caused by ocular infection with the bacterium C. trachomatis. In this in vivo study, we assessed the impact of wIRA radiation in combination with VIS (wavelength range 595-1400 nm, intensity 2100 W/m2) on the retina and cornea in a guinea pig animal model of inclusion conjunctivitis. We investigated the effects 19 days after wIRA/VIS irradiation by comparing a single and double wIRA/VIS treatment with a sham control. By immunolabeling and western blot analyses of critical heat- and stress-responsive proteins, we could not detect wIRA/VIS-induced changes in their expression pattern. Also, immunolabeling of specific retinal marker proteins revealed no changes in their expression pattern caused by the treatment. Our preclinical study suggests wIRA/VIS as a promising and safe therapeutic tool to treat ocular chlamydial infections.

Occurrence of Chlamydiaceae and Chlamydia felis pmp9 Typing in Conjunctival and Rectal Samples of Swiss Stray and Pet Cats.

Chlamydia (C.) felis primarily replicates in feline conjunctival epithelial cells and is an important cause of conjunctivitis in cats. Data on C. felis infection rates in stray cats in Switzerland has been missing so far. We performed a qPCR-based Chlamydiaceae-screening on 565 conjunctival and 387 rectal samples from 309 stray and 86 pet cats followed by Chlamydia species identification and C. felis typing using the gene pmp9, which encodes a polymorphic membrane protein. Overall, 19.1% of the stray and 11.6% of the pet cats were Chlamydiaceae-positive with significantly higher rates in cats displaying signs of conjunctivitis (37.1%) compared to healthy animals (6.9%). Rectal shedding of Chlamydiaceae occurred in 25.0% of infected cats and was mostly associated with concurrent ocular positivity (87.5%). In 92.2% of positive conjunctival and rectal samples, the Chlamydia species was identified as C. felis and in 2.6% as C. abortus. The C. felis pmp9 gene was very conserved in the sampled population with only one single-nucleotide polymorphism (SNP) in one conjunctival sample. In conclusion, C. felis strains are circulating in Swiss cats, are associated with conjunctivitis, have a low pmp9 genetic variability, and are rectally shed in about 16% of positive cases.

Maraviroc, celastrol and azelastine alter Chlamydia trachomatis development in HeLa cells.

Introduction . Chlamydia trachomatis (Ct) is an obligate intracellular bacterium, causing a range of diseases in humans. Interactions between chlamydiae and antibiotics have been extensively studied in the past.Hypothesis/Gap statement: Chlamydial interactions with non-antibiotic drugs have received less attention and warrant further investigations. We hypothesized that selected cytokine inhibitors would alter Ct growth characteristics in HeLa cells.Aim. To investigate potential interactions between selected cytokine inhibitors and Ct development in vitro.Methodology. The CCR5 receptor antagonist maraviroc (Mara; clinically used as HIV treatment), the triterpenoid celastrol (Cel; used in traditional Chinese medicine) and the histamine H1 receptor antagonist azelastine (Az; clinically used to treat allergic rhinitis and conjunctivitis) were used in a genital in vitro model of Ct serovar E infecting human adenocarcinoma cells (HeLa).Results. Initial analyses revealed no cytotoxicity of Mara up to 20 µM, Cel up to 1 µM and Az up to 20 µM. Mara exposure (1, 5, 10 and 20 µM) elicited a reduction of chlamydial inclusion numbers, while 10 µM reduced chlamydial infectivity. Cel 1 µM, as well as 10 and 20 µM Az, reduced chlamydial inclusion size, number and infectivity. Morphological immunofluorescence and ultrastructural analysis indicated that exposure to 20 µM Az disrupted chlamydial inclusion structure. Immunofluorescence evaluation of Cel-incubated inclusions showed reduced inclusion sizes whilst Mara incubation had no effect on inclusion morphology. Recovery assays demonstrated incomplete recovery of chlamydial infectivity and formation of structures resembling typical chlamydial inclusions upon Az removal.Conclusion. These observations indicate that distinct mechanisms might be involved in potential interactions of the drugs evaluated herein and highlight the need for continued investigation of the interaction of commonly used drugs with Chlamydia and its host.

Water-filtered Infrared A and visible light (wIRA/VIS) treatment reduces Chlamydia caviae-induced ocular inflammation and infectious load in a Guinea pig model of inclusion conjunctivitis.

Trachoma is a devastating neglected tropical disease caused by Chlamydia trachomatis and the leading global cause of infectious blindness. Although antibiotic treatment against trachoma is efficient (SAFE strategy), additional affordable therapeutic strategies are of high interest. Water-filtered infrared A and visible light (wIRA/VIS) irradiation has proven to reduce chlamydial infectivity in vitro and ex vivo. The aim of this study was to evaluate whether wIRA/VIS can reduce chlamydial infection load and/or ocular pathology in vivo, in a guinea pig model of inclusion conjunctivitis. Guinea pigs were infected with 1 × 106 inclusion-forming units/eye of Chlamydia caviae via the ocular conjunctiva on day 0. In infected animals, wIRA/VIS irradiation (2100 W/m2) was applied on day 2 (single treatment) and on days 2 and 4 (double treatment) post-infection (pi). wIRA/VIS reduced the clinical pathology score on days 7 and 14 pi and the conjunctival chlamydial load on days 2, 4, 7, and 14 pi in comparison with C. caviae-infected, not irradiated, controls. Furthermore, numbers of chlamydial inclusions were decreased in wIRA/VIS treated C. caviae-infected guinea pigs on day 21 pi compared to C. caviae-infected, non-irradiated, controls. Double treatment with wIRA/VIS (days 2 and 4 pi) was more efficient than a single treatment on day 2 pi. wIRA/VIS treatment did neither induce macroscopic nor histologic changes in ocular tissues. Our results indicate that wIRA/VIS shows promising efficacy to reduce chlamydial infectivity in vivo without causing irradiation related pathologies in the follow-up period. wIRA/VIS irradiation is a promising approach to reduce trachoma transmission and pathology of ocular chlamydial infection.

Ultrasonographic documentation of type-3 abomasal ulcer in a cow with left displacement of the abomasum.

BACKGROUND: Ultrasonographic documentation of perforated abomasal ulcer has not been published till now. This report describes the clinical, ultrasonographic and postmortem findings in a Jersey cow with type-3 abomasal ulcer and left displacement of the abomasum (LDA). CASE REPORT: The main clinical findings were abnormal demeanour, rumen atony and tympany, positive foreign body tests, positive auscultation and simultaneous ballottement on the left side, abdominal guarding and loss of negative pressure in the abdominal cavity. The tentative diagnosis was peritonitis and LDA. Abdominal ultrasonography produced images typical of LDA, and in one location between the abdominal wall and abomasum there was a layer of fibrin, a fibrin clot, a break in the abomasal contour, suggestive of a perforated ulcer, and partial obstruction of this gap with fibrin. The diagnosis of perforated abomasal ulcer with subsequent peritonitis was confirmed during postmortem examination. CONCLUSIONS: The examination of this case shows that under certain circumstances, ultrasonographic imaging of a perforated abomasal ulcer in a cow is possible. Antemortem diagnosis of type-3 abomasal ulcer is preferable to relying on exploratory laparotomy and/or post-mortem examination.

wIRA: hyperthermia as a treatment option for intracellular bacteria, with special focus on Chlamydiae and Mycobacteria.

The emergence of antibiotic-resistant bacteria in the last century is alarming and calls for alternative, nonchemical treatment strategies. Thermal medicine uses heat for the treatment of infectious diseases but its use in facultative and obligate intracellular bacteria remains poorly studied. In this review, we summarize previous research on reducing the infectious burden of Mycobacterium ulcerans and Chlamydia trachomatis by using water-filtered infrared A-radiation (wIRA), a special form of heat radiation with high tissue penetration and low thermal load on the skin surface. Mycobacterium ulcerans is a thermosensitive bacterium causing chronic necrotizing skin disease. Therefore, previous data on wIRA-induced improvement of wound healing and reduction of wound infections is summarized first. Then, pathogenesis and treatment of infections with M. ulcerans causing Buruli ulcer and of those with C. trachomatis infecting the ocular conjunctiva and resulting in blinding trachoma are discussed. Both bacteria cause neglected tropical diseases and have similar geographical distributions. Results of previous in vitro and in vivo studies using wIRA on M. ulcerans and C. trachomatis infections are presented. Finally, technical aspects of using wIRA in patients are critically reviewed and open questions driving future research are highlighted. In conclusion, wIRA is a promising tool for reducing infectious burden due to intracellular bacteria such as M. ulcerans and C. trachomatis.

Perspective: Water-Filtered Infrared-A-Radiation (wIRA) - Novel Treatment Options for Chlamydial Infections?

Water-filtered infrared-A-radiation (wIRA) is a promising therapeutic method, which is particularly used as supportive treatment for wound closure, and wound infection treatment and prevention. High penetration properties of the heat field and beneficial effects on wound healing processes predispose wIRA irradiation to be a non-invasive treatment method for bacterial infections in superficial tissues. Since Chlamydia trachomatis still represents the leading cause of infectious blindness in third world countries (WHO http://www.who.int/topics/trachoma/en/) and wIRA displays beneficial effects on chlamydial infections in vitro without inducing cellular damage in ex vivo eye models and also shows beneficial effects on wound healing, this irradiation technique might represent a promising future treatment for trachoma patients. To this end, further studies investigating shorter irradiation times or irradiation of Chlamydia in chronic infections [the chlamydial stress response (Bavoil, 2014)] as well as safety studies in animal models should clearly be performed.

Water Filtered Infrared A and Visible Light (wIRA/VIS) Irradiation Reduces Chlamydia trachomatis Infectivity Independent of Targeted Cytokine Inhibition.

Chlamydia trachomatis is the major cause of infectious blindness and represents the most common bacterial sexually transmitted infection worldwide. Considering the potential side effects of antibiotic therapy and increasing threat of antibiotic resistance, alternative therapeutic strategies are needed. Previous studies showed that water filtered infrared A alone (wIRA) or in combination with visible light (wIRA/VIS) reduced C. trachomatis infectivity. Furthermore, wIRA/VIS irradiation led to secretion of pro-inflammatory cytokines similar to that observed upon C. trachomatis infection. We confirmed the results of previous studies, namely that cytokine secretion (IL-6, IL-8, and RANTES/CCL5) upon wIRA/VIS treatment, and the subsequent reduction of chlamydial infectivity, are independent of the addition of cycloheximide, a host protein synthesis inhibitor. Reproducible cytokine release upon irradiation indicated that cytokines might be involved in the anti-chlamydial mechanism of wIRA/VIS. This hypothesis was tested by inhibiting IL-6, IL-8, and RANTES secretion in C. trachomatis or mock-infected cells by gene silencing or pharmaceutical inhibition. Celastrol, a substance derived from Trypterygium wilfordii, used in traditional Chinese medicine and known for anti-cancer and anti-inflammatory effects, was used for IL-6 and IL-8 inhibition, while Maraviroc, a competitive CCR5 antagonist and anti-HIV drug, served as a RANTES/CCL5 inhibitor. HeLa cell cytotoxicity and impact on chlamydial morphology, size and inclusion number was evaluated upon increasing inhibitor concentration, and concentrations of 0.1 and 1 µM Celastrol and 10 and 20 µM Maraviroc were subsequently selected for irradiation experiments. Celastrol at any concentration reduced chlamydial infectivity, an effect only observed for 20 µM Maraviroc. Triple dose irradiation (24, 36, 40 hpi) significantly reduced chlamydial infectivity regardless of IL-6, IL-8, or RANTES/CCL5 gene silencing, Celastrol or Maraviroc treatment. Neither gene silencing nor pharmaceutical cytokine inhibition provoked the chlamydial stress response. The anti-chlamydial effect of wIRA/VIS is independent of cytokine inhibition under all conditions evaluated. Thus, factors other than host cell cytokines must be involved in the working mechanism of wIRA/VIS. This study gives a first insight into the working mechanism of wIRA/VIS in relation to an integral component of the host immune system and supports the potential of wIRA/VIS as a promising new tool for treatment in trachoma.