Longitudinal study of Chlamydia pecorum in a healthy Swiss cattle population.

Chlamydia pecorum is a globally endemic livestock pathogen but prevalence data from Switzerland has so far been limited. The present longitudinal study aimed to get an insight into the C. pecorum prevalence in Swiss cattle and investigated infection dynamics. The study population consisted of a bovine herd (n = 308) located on a farm in the north-eastern part of Switzerland. The herd comprised dairy cows, beef cattle and calves all sampled up to five times over a one-year period. At each sampling timepoint, rectal and conjunctival swabs were collected resulting in 782 samples per sampled area (total n = 1564). Chlamydiaceae screening was performed initially, followed by C. pecorum-specific real-time qPCR on all samples. For C. pecorum-positive samples, bacterial loads were determined. In this study, C. pecorum was the only chlamydial species found. Animal prevalences were determined to be 5.2-11.4%, 38.1-61.5% and 55-100% in dairy cows, beef cattle and calves, respectively. In all categories, the number of C. pecorum-positive samples was higher in conjunctival (n = 151) compared to rectal samples (n = 65), however, the average rectal load was higher. At a younger age, the chlamydial prevalence and the mean bacterial loads were significantly higher. Of all sampled bovines, only 9.4% (29/308) were high shedders (number of copies per µl >1,000). Calves, which tested positive multiple times, either failed to eliminate the pathogen between sampling timepoints or were reinfected, whereas dairy cows were mostly only positive at one timepoint. In conclusion, C. pecorum was found in healthy Swiss cattle. Our observations suggested that infection takes place at an early age and immunity might develop over time. Although the gastrointestinal tract is supposed to be the main infection site, C. pecorum was not present in rectal samples from dairy cows.

Neisseria gonorrhoeae Coinfection during Chlamydia muridarum Genital Latency Does Not Modulate Murine Vaginal Bacterial Shedding.

Chlamydia trachomatis and Neisseria gonorrhoeae are the most frequently reported agents of bacterial sexually transmitted disease worldwide. Nonetheless, C. trachomatis/N. gonorrhoeae coinfection remains understudied. C. trachomatis/N. gonorrhoeae coinfections are more common than expected by chance, suggesting C. trachomatis/N. gonorrhoeae interaction, and N. gonorrhoeae infection may reactivate genital chlamydial shedding in women with latent (quiescent) chlamydial infection. We hypothesized that N. gonorrhoeae would reactivate latent genital Chlamydia muridarum infection in mice. Two groups of C. muridarum-infected mice were allowed to transition into genital latency. One group was then vaginally inoculated with N. gonorrhoeae; a third group received N. gonorrhoeae alone. C. muridarum and N. gonorrhoeae vaginal shedding was measured over time in the coinfected and singly infected groups. Viable C. muridarum was absent from vaginal swabs but detected in rectal swabs, confirming C. muridarum genital latency and consistent with the intestinal tract as a C. muridarum reservoir. C. muridarum inclusions were observed in large intestinal, but not genital, tissues during latency. Oviduct dilation was associated with C. muridarum infection, as expected. Contradicting our hypothesis, N. gonorrhoeae coinfection did not reactivate latent C. muridarum vaginal shedding. In addition, latent C. muridarum infection did not modulate recovery of vaginal viable N. gonorrhoeae. Evidence for N. gonorrhoeae-dependent increased C. muridarum infectivity has thus not been demonstrated in murine coinfection, and the ability of C. muridarum coinfection to potentiate N. gonorrhoeae infectivity may depend on actively replicating vaginal C. muridarum. The proportion of mice with increased vaginal neutrophils (PMNs) was higher in N. gonorrhoeae-infected than in C. muridarum-infected mice, as expected, while that of C. muridarum/N. gonorrhoeae-coinfected mice was intermediate to the singly infected groups, suggesting latent C. muridarum murine infection may limit PMN response to subsequent N. gonorrhoeae infection. IMPORTANCE Our work builds upon the limited understanding of C. muridarum/N. gonorrhoeae coinfection. Previously, N. gonorrhoeae infection of mice with acute (actively replicating) vaginal C. muridarum infection was shown to increase recovery of viable vaginal N. gonorrhoeae and vaginal PMNs, with no effect on C. muridarum vaginal shedding (R. A. Vonck et al., Infect Immun 79:1566-1577, 2011). It has also been shown that chlamydial infection of human and murine PMNs prevents normal PMN responses, including the response to N. gonorrhoeae (K. Rajeeve et al., Nat Microbiol 3:824-835, 2018). Our findings show no effect of latent genital C. muridarum infection on the recovery of viable N. gonorrhoeae, in contrast to the previously reported effect of acute C. muridarum infection, and suggesting that acute versus latent C. muridarum infection may have distinct effects on PMN function in mice. Together, these studies to date provide evidence that Chlamydia/N. gonorrhoeae synergistic interactions may depend on the presence of replicating Chlamydia in the genital tract, while chlamydial effects on vaginal PMNs may extend beyond acute infection.

Falcons From the United Arab Emirates Infected With Chlamydia psittaci/C abortus Intermediates Specified as Chlamydia buteonis by Polymerase Chain Reaction.

Chlamydiaceae are obligate intracellular bacteria with a broad host range. Several studies have found chlamydial species that are genetically intermediate between Chlamydia psittaci and Chlamydia abortus in various avian species. One of these intermediate Chlamydia species, found in a red-shouldered hawk (Buteo lineatus), was recently classified as a new species Chlamydia buteonis. This newly described Chlamydia species has, so far, only been reported in hawks exhibiting clinical signs of conjunctivitis, dyspnea, and diarrhea. In the present study, fecal samples of 5 gyrfalcons (Falco rusticolus), 3 gyr/peregrine falcon hybrids (Falco rusticolus × Falco peregrinus), and 15 falcons of unknown species presented to falcon clinics on the Arabian Peninsula were shipped to the Vetsuisse Faculty, University of Zurich (Zurich, Switzerland), for examination for the presence of Chlamydiaceae. A step-wise diagnostic approach was performed to identify the chlamydial species involved. Chlamydiaceae were detected in 21/23 falcons by a family-specific real-time quantitative PCR (qPCR). Further identification with a 23S ribosomal RNA-based microarray assay and 16S conventional PCR and sequencing yielded inconclusive results, indicating the presence of an intermediate Chlamydia species. Because none of the falcons tested positive for Chlamydia psittaci by specific qPCR, all 23 samples were subjected to a Chlamydia buteonis-specific qPCR, which was positive in 16/23 samples. Detailed information regarding clinical history was available for 8 falcons admitted to a falcon clinic in Dubai, United Arab Emirates. Six of those birds that were presented to the clinic because of loss of performance and poor general condition, including vomiting and diarrhea, were positive for C buteonis. In 2 birds without clinical disease signs admitted for a routine health examination, 1 was positive for C buteonis, and 1 was negative. It is yet unknown whether Chlamydia buteonis causes disease in birds, but the findings in this study indicate that Chlamydia buteonis may be an infectious pathogen in falcon species.

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.

Generation of Tetracycline and Rifamycin Resistant Chlamydia Suis Recombinants.

The Chlamydiaceae are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture in vitro, allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only Chlamydia (C.) suis naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of Chlamydia to exchange DNA and especially antibiotic resistance, C. suis is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination in vitro. With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant C. suis, which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient C. suis field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced in vitro. Here, we describe the protocol with which RifR C. suis strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in rpoB, a gene encoding for the ß-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed C. suis genome plasticity by revealing variable, apparently non-specific areas of recombination.

Chlamydia suis is associated with intestinal NF-κB activation in experimentally infected gnotobiotic piglets.

Chlamydia suis intestinal infection of single-animal experimental groups of gnotobiotic newborn piglets was previously reported to cause severe, temporary small intestinal epithelium damage. We investigated archived intestinal samples for pro-inflammatory nuclear factor kappa B (NF-κB) activation, Interleukin (IL)-6 and IL-8 production and immune cell influx. Samples were collected 2, 4 and 7 days post-inoculation with C. suis strain S45/6 or mock inoculum (control). Increased nuclear localization of epithelial NF-κB, representative of activation, in the jejunum and ileum of C. suis-infected animals, compared to uninfected controls, began by 2 days post-infection (dpi) and persisted through 7 dpi. Infected animals showed increased production of IL-8, peaking at 2 dpi, compared to controls. Infection-mediated CD45-positive immune cell influx into the jejunal lamina propria peaked at 7 dpi, when epithelial damage was largely resolved. Activation of NF-κB appears to be a key early event in the innate response of the unprimed porcine immune system challenged with C. suis. This results in an acute phase, coinciding with the most severe clinical symptoms, diarrhea and weight loss. Immune cells recruited shortly after infection remain present in the lamina propria during the recovery phase, which is characterized by reduced chlamydial shedding and restored intestinal epithelium integrity.

Molecular Design, Optimization, and Genomic Integration of Chimeric B Cell Receptors in Murine B Cells.

Immune cell therapies based on the integration of synthetic antigen receptors comprise a powerful strategy for the treatment of diverse diseases, most notably T cells engineered to express chimeric antigen receptors (CAR) for targeted cancer therapy. In addition to T lymphocytes, B lymphocytes may also represent valuable immune cells that can be engineered for therapeutic purposes such as protein replacement therapy or recombinant antibody production. In this article, we report a promising concept for the molecular design, optimization, and genomic integration of a novel class of synthetic antigen receptors, chimeric B cell receptors (CBCR). We initially optimized CBCR expression and detection by modifying the extracellular surface tag, the transmembrane regions and intracellular signaling domains. For this purpose, we stably integrated a series of CBCR variants using CRISPR-Cas9 into immortalized B cell hybridomas. Subsequently, we developed a reliable and consistent pipeline to precisely introduce cassettes of several kb size into the genome of primary murine B cells also using CRISPR-Cas9 induced HDR. Finally, we were able to show the robust surface expression and antigen recognition of a synthetic CBCR in primary B cells. We anticipate CBCRs and our approach for engineering primary B cells will be a valuable tool for the advancement of future B cell- based immune cell therapies.

Survey on Chlamydiaceae in cloacal swabs from Swiss turkeys demonstrates absence of Chlamydia psittaci and low occurrence of Chlamydia gallinacean.

In Switzerland, domestic turkey meat is a niche product. Turkeys are fattened on mixed family-based farms scattered across the country, with most providing access to an uncovered outdoor pasture for the birds. Swiss fattening turkeys may therefore get infected with Chlamydiaceae via wild birds or their faeces, potentially shedding these bacteria at a later stage. The aim of the present study was to acquire baseline data about the shedding of Chlamydiaceae in clinically unremarkable Swiss fattening turkeys at slaughter, potentially exposing slaughterhouse workers to infection. In this large-scale study, 1008 cloacal swabs of Swiss turkeys out of 53 flocks from 28 different grow-out farms with uncovered outdoor pasture were collected over the course of 14 months and examined for the occurrence of Chlamydiaceae by a family-specific 23S-rRNA real-time PCR. Positive samples were further analyzed by Chlamydia psittaci (C. psittaci)-specific real-time PCR and the Arraymate DNA Microarray for species identification. All samples were negative for C. psittaci, but seven swabs out of one flock were tested positive for Chlamydia gallinacea (0.7%). Although turkeys with access to pasture may have contact with Chlamydiaceae-harbouring wild birds or their faeces, the infection rate in Swiss turkeys was shown to be low.

Antibody response in snakes with boid inclusion body disease.

Boid Inclusion Body Disease (BIBD) is a potentially fatal disease reported in captive boid snakes worldwide that is caused by reptarenavirus infection. Although the detection of intracytoplasmic inclusion bodies (IB) in blood cells serves as the gold standard for the ante mortem diagnosis of BIBD, the mechanisms underlying IB formation and the pathogenesis of BIBD are unknown. Knowledge on the reptile immune system is sparse compared to the mammalian counterpart, and in particular the response towards reptarenavirus infection is practically unknown. Herein, we investigated a breeding collection of 70 Boa constrictor snakes for BIBD, reptarenavirus viraemia, anti-reptarenavirus IgM and IgY antibodies, and population parameters. Using NGS and RT-PCR on pooled blood samples of snakes with and without BIBD, we could identify three different reptarenavirus S segments in the collection. The examination of individual samples by RT-PCR indicated that the presence of University of Giessen virus (UGV)-like S segment strongly correlates with IB formation. We could also demonstrate a negative correlation between BIBD and the presence of anti-UGV NP IgY antibodies. Further evidence of an association between antibody response and BIBD is the finding that the level of anti-reptarenavirus antibodies measured by ELISA was lower in snakes with BIBD. Furthermore, female snakes had a significantly lower body weight when they had BIBD. Taken together our findings suggest that the detection of the UGV-/S6-like S segment and the presence of anti-reptarenavirus IgY antibodies might serve as a prognostic tool for predicting the development of BIBD.

Bovine Fetal Placenta During Pregnancy and the Postpartum Period.

Bovine abortion is a worldwide problem, but despite extensive histopathologic and molecular investigations, the cause of abortion remains unclear in about 70% of cases. Cellular debris is a commonly observed histopathologic finding in the fetal placenta and is often interpreted as necrosis. In this study, the nature of this cellular debris was characterized, and histologic changes in the normal fetal placenta during pregnancy and after delivery were assessed. In addition, the presence of the most common abortifacient pathogens in Switzerland ( Chlamydiaceae, Coxiella burnetii, Neospora caninum) was tested by polymerase chain reaction. We collected 51 placentomes and 235 cotyledons from 41 and from 50 cows, respectively. In total, cellular debris was present in 48 of 51 (94%) placentomes and in 225 of 235 (96%) cotyledons, inflammation occurred in 1 of 51 (2%) placentomes and in 46 of 235 (20%) cotyledons, vasculitis was seen in 1 of 51 (2%) placentomes and 46 of 235 (20%) cotyledons, and 18 of 51 (35%) placentomes and 181 of 235 (77%) cotyledons had mineralization. The amount of cellular debris correlated with areas of positive signals for cleaved caspase 3 and lamin A. Therefore, this finding was interpreted as an apoptotic process. In total, 10 of 50 cotyledons (20%) were positive for C. burnetii DNA, most likely representing subclinical infections. The results of our study indicate that histologic features in the fetal placenta such as cellular debris, inflammation, vasculitis, and mineralization must be considered physiological processes during pregnancy and after delivery. Therefore, their presence in placentae of aborted fetuses must be interpreted with caution and might not be necessarily linked to an infectious cause of abortion.

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.

Chlamydiae in human intestinal biopsy samples.

Chlamydia trachomatis is frequently detected in anorectal specimens from men and women. A recent hypothesis suggests that C. trachomatis is a natural commensal organism asymptomatically colonizing the gastrointestinal tract. In this study, we investigated the presence of chlamydial DNA and antigen in intestinal biopsy samples taken during colonoscopy. Cases (n = 32) were patients whose histopathology reports included the term 'chlamydia', suggesting a possible history of infection. Control patients (n = 234) did not have chlamydia mentioned in their histopathology report and all tested negative for Chlamydiaceae DNA by 23S ribosomal RNA-based real-time PCR. Amongst the cases, C. trachomatis DNA was detected in the appendix and colon of two female and one male patients. Chlamydia abortus DNA was present in the colon of a fourth female patient. Thus, chlamydial DNA could be demonstrated in intestinal biopsy samples proximal to the anorectal site and inclusions were identified in rectum or appendix of two of these patients by immunohistochemistry. However, the findings in two cases were compatible with sexually acquired C. trachomatis. The identification of C. trachomatis DNA/antigen does not prove the presence of active infection with replicating bacteria. Larger prospective studies on fresh tissue samples are required to confirm the data obtained in this study.

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange.

The development of programmable nucleases has enabled the application of new genome engineering strategies for cellular immunotherapy. While targeted nucleases have mostly been used to knock-out or knock-in genes in immune cells, the scarless exchange of entire immunogenomic alleles would be of great interest. In particular, reprogramming the polymorphic MHC locus could enable the creation of matched donors for allogeneic cellular transplantation. Here we show a proof-of-concept for reprogramming MHC-specificity by performing CRISPR-Cas9-assisted cassette exchange. Using murine antigen presenting cell lines (RAW264.7 macrophages), we demonstrate that the generation of Cas9-induced double-stranded breaks flanking the native MHC-I H2-Kd locus led to exchange of an orthogonal H2-Kb allele. MHC surface expression allowed for easy selection of reprogrammed cells by flow cytometry, thus obviating the need for additional selection markers. MHC-reprogrammed cells were fully functional as they could present H2-Kd-restricted peptide and activate cognate T cells. Finally, we investigated the role of various donor template formats on exchange efficiency, discovering that templates that underwent in situ linearization resulted in the highest MHC-reprogramming efficiency. These findings highlight a potential new approach for the correcting of MHC mismatches in cellular transplantation.

Surveying the Delivery Methods of CRISPR/Cas9 for ex vivo Mammalian Cell Engineering.

The simplicity of the CRISPR/Cas9 technology has been transformative in making targeted genome editing accessible for laboratories around the world. However, due to the sheer volume of literature generated in the past five years, determining the best format and delivery method of CRISPR/Cas9 components can be challenging. Here, we provide a brief overview of the progress that has been made in the ex vivo genome editing of mammalian cells and summarize the key advances made for improving efficiency and delivery of CRISPR/Cas9 in DNA, RNA, and protein form. In particular, we highlight the delivery of Cas9 components to human cells for advanced genome editing applications such as large gene insertion.

Differential cytotoxicity induced by the Titanium(IV)Salan complex Tc52 in G2-phase independent of DNA damage.

BACKGROUND: Chemotherapy is one of the major treatment modalities for cancer. Metal-based compounds such as derivatives of cisplatin are in the front line of therapy against a subset of cancers, but their use is restricted by severe side-effects and the induction of resistance in treated tumors. Subsequent research focused on development of cytotoxic metal-complexes without cross-resistance to cisplatin and reduced side-effects. This led to the discovery of first-generation titanium(IV)salan complexes, which reached clinical trials but lacked efficacy. New-generation titanium (IV)salan-complexes show promising anti-tumor activity in mice, but their molecular mechanism of cytotoxicity is completely unknown. METHODS: Four different human cell lines were analyzed in their responses to a toxic (Tc52) and a structurally highly related but non-toxic (Tc53) titanium(IV)salan complex. Viability assays were used to reveal a suitable treatment range, flow-cytometry analysis was performed to monitor the impact of dosage and treatment time on cell-cycle distribution and cell death. Potential DNA strand break induction and crosslinking was investigated by immunostaining of damage markers as well as automated fluorometric analysis of DNA unwinding. Changes in nuclear morphology were analyzed by DAPI staining. Acidic beta-galactosidase activity together with morphological changes was monitored to detect cellular senescence. Western blotting was used to analyze induction of pro-apoptotic markers such as activated caspase7 and cleavage of PARP1, and general stress kinase p38. RESULTS: Here we show that the titanium(IV)salan Tc52 is effective in inducing cell death in the lower micromolar range. Surprisingly, Tc52 does not target DNA contrary to expectations deduced from the reported activity of other titanium complexes. Instead, Tc52 application interferes with progression from G2-phase into mitosis and induces apoptotic cell death in tested tumor cells. Contrarily, human fibroblasts undergo senescence in a time and dose-dependent manner. As deduced from fluorescence studies, the potential cellular target seems to be the cytoskeleton. CONCLUSIONS: In summary, we could demonstrate in four different human cell lines that tumor cells were specifically killed without induction of major cytotoxicity in non-tumorigenic cells. Absence of DNA damaging activity and the cell-cycle block in G2 instead of mitosis makes Tc52 an attractive compound for further investigations in cancer treatment.

Water-filtered infrared a irradiation in combination with visible light inhibits acute chlamydial infection.

New therapeutic strategies are needed to overcome drawbacks in treatment of infections with intracellular bacteria. Chlamydiaceae are Gram-negative bacteria implicated in acute and chronic diseases such as abortion in animals and trachoma in humans. Water-filtered infrared A (wIRA) is short wavelength infrared radiation with a spectrum ranging from 780 to 1400 nm. In clinical settings, wIRA alone and in combination with visible light (VIS) has proven its efficacy in acute and chronic wound healing processes. This is the first study to demonstrate that wIRA irradiation combined with VIS (wIRA/VIS) diminishes recovery of infectious elementary bodies (EBs) of both intra- and extracellular Chlamydia (C.) in two different cell lines (Vero, HeLa) regardless of the chlamydial strain (C. pecorum, C. trachomatis serovar E) as shown by indirect immunofluorescence and titration by subpassage. Moreover, a single exposure to wIRA/VIS at 40 hours post infection (hpi) led to a significant reduction of C. pecorum inclusion frequency in Vero cells and C. trachomatis in HeLa cells, respectively. A triple dose of irradiation (24, 36, 40 hpi) during the course of C. trachomatis infection further reduced chlamydial inclusion frequency in HeLa cells without inducing the chlamydial persistence/stress response, as ascertained by electron microscopy. Irradiation of host cells (HeLa, Vero) neither affected cell viability nor induced any molecular markers of cytotoxicity as investigated by Alamar blue assay and Western blot analysis. Chlamydial infection, irradiation, and the combination of both showed a similar release pattern of a subset of pro-inflammatory cytokines (MIF/GIF, Serpin E1, RANTES, IL-6, IL-8) and chemokines (IL-16, IP-10, ENA-78, MIG, MIP-1α/ß) from host cells. Initial investigation into the mechanism indicated possible thermal effects on Chlamydia due to irradiation. In summary, we demonstrate a non-chemical reduction of chlamydial infection using the combination of water-filtered infrared A and visible light.

A role for tumor necrosis factor and bacterial antigens in the pathogenesis of Crohn's disease-associated fistulae.

BACKGROUND: Intestinal fistulae represent a severe complication of Crohn's disease (CD). The authors have demonstrated that epithelial-to-mesenchymal transition plays a pivotal role in their pathogenesis. High levels of interleukin-13 and tumor necrosis factor (TNF) are detected in myofibroblast-like transitional cells covering the fistula tracts. Here, a functional role was investigated for the transcription factor Ets-1, TNF, and the bacterial wall component (muramyl dipeptide [MDP]) in the pathogenesis of CD-associated fistulae. METHODS: Perianal fistulae from CD patients were analyzed by immunohistochemistry. Primary colonic lamina propria fibroblasts (CLPFs) were isolated from CD patients with or without fistulizing disease. Messenger RNA (mRNA) levels were assessed by real-time polymerase chain reaction in CLPF or HT29 intestinal epithelial cells (IECs) grown as monolayers or spheroids. RESULTS: Strong expression of Ets-1 transcription factor was demonstrated in transitional cell covering the fistula tracts by immunohistochemistry. TNF induced mRNA expression of ETS-1 and ß6-integrin in HT29 IEC and in CLPF from fistulizing CD patients. These effects could be fully blocked by administration of anti-TNF antibodies. In HT29 cells, TNF further induced mRNA levels of TNF and transforming growth factor beta by treatment for 24 hours. In fistula CLPF derived from CD patients, TNF induced expression of ß6-integrin, TNF, and transforming growth factor beta. Of note, the bacterial wall component, MDP, induced mRNA levels of ETS-1, transforming growth factor beta, interleukin-13, SNAIL1, and ß6-integrin in HT29 IEC monolayers and fistula CLPF by treatment for 24 hours. CONCLUSIONS: TNF and MDP induce the expression of factors associated with epithelial-to-mesenchymal transition and invasion in IEC and fistula CLPF. Our findings indicate that TNF and MDP might synergize in the pathogenesis of CD-associated fistulae.

The role for dickkopf-homolog-1 in the pathogenesis of Crohn's disease-associated fistulae.

BACKGROUND: One of the most challenging conditions in Crohn's disease (CD) patients is the treatment of perianal fistulae. We have recently shown that epithelial-to-mesenchymal transition (EMT) plays a crucial role during CD-fistulae development. Dickkopf-homolog 1 (DKK-1) is known to play a key role during EMT. Here, we investigated a role for DKK-1 in the pathogenesis of CD-associated fistulae. METHODS: Dkk-1 protein expression in CD-fistula specimens were investigated by immunohistochemistry. Colonic lamina propria fibroblasts (CLPF) were obtained from either non-IBD control patients or patients with fistulizing CD. HT-29 intestinal epithelial cells (IEC) were either grown as monolayers or spheroids. Cells were treated with either TNF-α, TGF-ß or IL-13. Knock-down of DKK-1 or ß-Catenin was induced in HT-29-IEC by siRNA technique. mRNA expression was determined by real-time-PCR. RESULTS: Dkk-1 protein was specifically expressed in transitional cells lining the fistula tracts. TGF-ß induced DKK-1 mRNA expression in HT-29-IEC, but decreased it in fistula CLPF. On a functional level, DKK-1 knock-down prevented TGF-ß-induced IL-13 mRNA expression in HT-29-IEC. Further, loss of ß-Catenin was accompanied by reduced levels of DKK-1 and, again, IL-13 in IEC in response to TGF-ß. In turn, treatment of HT-29-IEC as well as fistula CLPF with IL-13 resulted in decreased levels of DKK-1 mRNA. Treatment with TNF-α or the bacterial wall component, muramyl-dipeptide, decreased DKK-1 mRNA levels in HT-29-IEC, but enhanced it in fistula CLPF. DISCUSSION: We demonstrate that DKK-1 is strongly expressed in cells lining the CD-fistula tracts and regulates factors involved in EMT initiation. These data provide evidence for a role of DKK-1 in the pathogenesis of CD-associated perianal fistulae.

Cytosolic Ca2+ shifts as early markers of cytotoxicity.

The determination of the cytotoxic potential of new and so far unknown compounds as well as their metabolites is fundamental in risk assessment. A variety of strategic endpoints have been defined to describe toxin-cell interactions, leading to prediction of cell fate. They involve measurement of metabolic endpoints, bio-energetic parameters or morphological cell modifications. Here, we evaluated alterations of the free cytosolic Ca2+ homeostasis using the Fluo-4 dye and compared results with the metabolic cell viability assay Alamar Blue. We investigated a panel of toxins (As2O3, gossypol, H2O2, staurosporine, and titanium(IV)-salane complexes) in four different mammalian cell lines covering three different species (human, mouse, and African green monkey). All tested compounds induced an increase in free cytosolic Ca2+ within the first 5 s after toxin application. Cytosolic Ca2+ shifts occurred independently of the chemical structure in all tested cell systems and were persistent up to 3 h. The linear increase of free cytosolic Ca2+ within the first 5 s of drug treatment correlates with the EC25 and EC75 values obtained in Alamar Blue assays one day after toxin exposure. Moreover, a rise of cytosolic Ca2+ was detectable independent of induced cell death mode as assessed by caspase and poly(ADP-ribose) polymerase (PARP) activity in HeLa versus MCF-7 cells at very low concentrations. In conclusion, a cytotoxicity assay based on Ca2+ shifts has a low limit of detection (LOD), is less time consuming (at least 24 times faster) compared to the cell viability assay Alamar Blue and is suitable for high-troughput-screening (HTS).