Acanthamoeba griffini. Molecular characterization of a new corneal pathogen

Purpose. Acanthamoeba was isolated from the cornea of a soft contact lense wearer who had keratitis. The protozoan was also isolated from the contact lens storage case and the domestic water supply used to clean the case. Using morphologic features, all three isolates were identified tentatively as A. griffini, a species not previously associated with keratitis. Complete small subunit ribosomal RNA gene (18S rDNA) sequence analysis was used to characterize further the three isolates. Methods. 18S rDNA was polymerase chain reaction-amplified from whole cell DNA derived from amoebal lysates. The genes were cloned and sequenced. Complete sequences of approximately 2800 base pairs were obtained from each culture and compared with those stored in a data base of homologous Acanthamoeba sequences. Results. The isolates were unequivocally identified as A. griffini both by comparison of the gene sequence available for the type strain of the species and the presence of a unique group I intron located within the small subunit rDNA. Sequences obtained for the three isolates were identical, indicating that they were the same strain. Conclusions. The first direct connection between human disease and A. griffini is reported from a case of Acanthamoeba keratitis. The type strain of this species was isolated from a marine environment, but the disease<ausing strain was isolated from a domestic water supply. The DNA sequences obtained confirmed unequivocally the epidemiologic association between a keratitis-causing strain of Acanthamoeba, the contact lens storage case, and the domestic water supply

Sex alters thyroid hormone’s effect on protein O-GlcNAcylation in the aged mouse heart

Abstract Background The aging heart undergoes physiological changes, many of which are sex dependent and encompass differential responses to cardiac stress. However, much about the molecular changes that occur within the aging heart is still unknown. Thyroid hormone (TH) and the posttranslational modification O-GlcNAcylation (O-GlcNAc) are independently known to regulate cardiac function; therefore, we tested the hypothesis that TH disorders affect cardiac protein O-GlcNAcylation in aged hearts. Results We treated male and female 18–22 month-old aged C57BL/6 mice to create euthyroid, hypothyroid, or hyperthyroid states. Western blots and RT-qPCR from cardiac tissue were used to determine changes in global O-GlcNAc levels along with key regulatory proteins in the O-GlcNAcylation process. Immunoprecipitation and western blotting compared global O-GlcNAc changes to differences on an individual protein. We found increased total O-GlcNAc levels for female hypo- and hyperthyroid mice and male hyperthyroid mice compared to sex-matched euthyroid hearts, with no change for male hypothyroid mice. TH’s O-GlcNAc effect on female mice appears heart specific as liver O-GlcNAc levels were unchanged. The proteins regulating O-GlcNAcylation also demonstrated sex differences. Female hyperthyroid mice had increased protein expression of the O-GlcNAc regulatory proteins GFAT 1, GFAT 2, and OGT, whereas the hyperthyroid male mice showed decreased expression for the regulatory protein OGA. The hypothyroid female mice had increased protein expression for OGT and NAGK, whereas the hypothyroid male mice showed increased protein expression for NAGK alone. Interestingly, the directional changes in these protein levels did not match RNA transcription. We further found O-GlcNAc levels of the mitochondrial thiolase protein ACAA2 diverged from global O-GlcNAc changes. ACAA2 was hyper O-GlcNAcylated in the female hypothyroid group and hypo O-GlcNAcylated in the male hyperthyroid group whereas there was no change in female hyperthyroid or male hypothyroid. Conclusion Protein O-GlcNAcylation is potentially an important mechanism whereby TH perturbations affect the aged heart. We found sex influences O-GlcNAc regulation, global O-GlcNAc levels, and O-GlcNAc protein specificity in response to thyroid hormone perturbations. Our results also suggest the changes in cardiac O-GlcNAc levels are not solely due to TH transcriptional regulation of key O-GlcNAc regulatory enzymes

Sex impacts cardiac function and the proteome response to thyroid hormone in aged mice

AbstractBackgroundSex and age have substantial influence on thyroid function. Sex influences the risk and clinical expression of thyroid disorders (TDs), with age a proposed trigger for the development of TDs. Cardiac function is affected by thyroid hormone levels with gender differences. Accordingly, we investigated the proteomic changes involved in sex based cardiac responses to thyroid dysfunction in elderly mice.MethodsAged (18–20 months) male and female C57BL/6 mice were fed diets to create euthyroid, hypothyroid, or hyperthyroid states. Serial echocardiographs were performed to assess heart function. Proteomic changes in cardiac protein profiles were assessed by 2-D DIGE and LC-MS/MS, and a subset confirmed by immunoblotting.ResultsSerial echocardiographs showed ventricular function remained unchanged regardless of treatment. Heart rate and size increased (hyperthyroid) or decreased (hypothyroid) independent of sex. Pairwise comparison between the six groups identified 55 proteins (≥ 1.5-fold difference andp &lt; 0.1). Compared to same-sex controls 26/55 protein changes were in the female hypothyroid heart, whereas 15/55 protein changes were identified in the male hypothyroid, and male and female hyperthyroid heart. The proteins mapped to oxidative phosphorylation, tissue remodeling and inflammatory response pathways.ConclusionWe identified both predicted and novel proteins with gender specific differential expression in response to thyroid hormone status, providing a catalogue of proteins associated with thyroid dysfunction. Pursuit of these proteins and their involvement in cardiac function will expand our understanding of mechanisms involved in sex-based cardiac response to thyroid dysfunction.</jats:sec

Sex Impacts Cardiac Function and the Proteome Response to Thyroid Hormone in Aged Mice

Abstract Background Sex and age have substantial influence on thyroid function. Sex influences the risk and clinical expression of thyroid disorders (TDs), with age a proposed trigger for the development of TDs. Cardiac function is affected by thyroid hormone levels with gender differences. Accordingly, we investigated the proteomic changes involved in sex based cardiac responses to thyroid dysfunction in elderly mice. Methods Aged (18–20 months) male and female C57BL/6 mice were fed diets to create euthyroid, hypothyroid, or hyperthyroid states. Serial echocardiographs were performed to assess heart function. Proteomic changes in cardiac protein profiles were assessed by 2-D DIGE and LC-MS/MS, and a subset confirmed by immunoblotting. Results Serial echocardiographs showed ventricular function remained unchanged regardless of treatment. Heart rate and size increased (hyperthyroid) or decreased (hypothyroid) independent of sex. Pairwise comparison between the six groups identified 55 proteins (≥ 1.5-fold difference and p &lt; 0.1). Compared to same-sex controls 26/55 protein changes were in the female hypothyroid heart, whereas 15/55 protein changes were identified in the male hypothyroid, and male and female hyperthyroid heart. The proteins mapped to oxidative phosphorylation, tissue remodeling and inflammatory response pathways. Conclusion We identified both predicted and novel proteins with gender specific differential expression in response to thyroid hormone status, providing a catalogue of proteins associated with thyroid dysfunction. Pursuit of these proteins and their involvement in cardiac function will expand our understanding of mechanisms involved in sex-based cardiac response to thyroid dysfunction.</jats:p

Detection of Bacterial Endosymbionts in Clinical Acanthamoeba Isolates

PURPOSE: To determine the presence of four clinically relevant bacterial endosymbionts in Acanthamoeba isolates obtained from patients with Acanthamoeba keratitis (AK) and the possible contribution of endosymbionts to the pathogenesis of AK. DESIGN: Experimental study PARTICIPANTS: Acanthamoeba isolates (N=37) recovered from cornea and contact lens paraphernalia of 23 patients with culture proven AK and 1 environmental isolate. METHODS: Acanthamoeba isolates were evaluated for the presence of microbial endosymbionts belonging to the bacterial genera Legionella, Pseudomonas, Mycobacteria and Chlamydia using molecular techniques (Polymerase chain reaction and sequence analysis, fluorescent in situ hybridization) and transmission electron microscopy. Corneal toxicity and virulence of Acanthamoeba isolates with and without endosymbionts were compared using a cytopathic effect (CPE) assay of human corneal epithelial cells in vitro. Initial visual acuity (VA), location and characteristics of the infiltrate, time to detection of the infection and symptoms duration at presentation were evaluated in all patients. MAIN OUTCOME MEASURES: Prevalence and potential pathobiology of bacterial endosymbionts detected in Acanthamoeba isolates recovered from AK. RESULTS: Twenty-two of the 38 (59.4%) cultures examined contained at least one bacterial endosymbiont. One isolate contained two endosymbionts, Legionella and Chlamydia, confirmed by fluorescence in situ hybridization. Corneal toxicity (CPE) was significantly higher for Acanthamoebae hosting endosymbionts compared to isolates without endosymbionts (p<0.05). Corneal pathogenic endosymbionts such as Pseudomonas and Mycobacterium enhanced Acanthamoeba CPE significantly more than Legionella (p<0.05). In the presence of bacterial endosymbionts, there was a trend toward worse initial VA (p>0.05), central location (p<0.05), absence of radial perineuritis (p<0.05), delayed time to detection (p>0.05) and longer symptoms duration at presentation (p>0.05). CONCLUSION: The majority of Acanthamoeba isolates responsible for AK harbors one or more bacterial endosymbionts. The presence of endosymbionts enhances the corneal pathogenicity of Acanthamoeba isolates and might impact detection time and clinical features of AK

Cysticidal Activity of Antifungals against Different Genotypes of Acanthamoeba

Antifungal drugs have been proposed as a novel treatment for Acanthamoeba keratitis. The cysticidal activity of several antifungal compounds was tested against different genotypes of culture collection and clinical isolates of Acanthamoeba. Only voriconazole and posaconazole were found to be cysticidal, with no differences in activity observed between clinical and culture collection isolates

Abstract 68: Echocardiographic Evaluation of the Kawasaki Disease Mouse Model

Background: Mouse echocardiography is an established, non-invasive method to evaluate experimental cardiovascular disease. The murine Kawasaki disease (KD) model demonstrates aortic and coronary artery inflammation by histology. The clinical relevance of these pathological changes has not been confirmed. In children, KD involves coronary artery abnormalities visualized by echocardiography. However, echocardiography has not been previously applied to the mouse KD model. We hypothesized that coronary vasculitis caused the lactobacillus casei cell wall extract (LCE) causes abnormalities detectable by echo. Methods: Male mice (ages 3-4 months) were injected with LCE or vehicle. Sedated echos were performed with a VisualSonics Vevo 2100 system. Twelve mice (8 LCE and 4 vehicle) had pre-injection echos followed by serial studies for 6-10 weeks. Evaluations were blinded to treatment group. 2-D measurements were taken at consistent locations in the arteries. Aortic regurgitation was rated based upon clinical criteria. Histology was evaluated at 6 weeks post-LCE. Results: Mild aortic regurgitation (AR) was present in 6 of 8 mice injected with LCE starting at 1 week post-injection. No AR was present at baseline or vehicle injected mice. LCE mice showed increased aortic root diameter at 6 weeks compared to baseline (1.78mm±0.01 vs 1.52±0.02, p&lt;0.05). Overall left coronary artery dimensions were not changed from baseline at 6 weeks post-LCE, but coronary imaging was difficult. One mouse had a diffusely enlarged left coronary system and severely diminished left ventricular function at 10 weeks post-LCE. Histology findings were present in all LCE-injected mice including aortic valvulitis, myointimal proliferation and a single case of infarction. Conclusion: The most prominent echo findings in the mouse KD model are severe aortitis with dilation and valvular regurgitation. These features could serve as non-invasive experimental measurements. Coronary dilation was a rare finding; however the coronaries are difficult to evaluate in mice. Overall, the mouse KD model demonstrates greater aortic pathology than found in human KD. Further studies are underway to evaluate additional non-invasive measurements such as vascular strain. </jats:p