CT135 mediates the resistance of Chlamydia trachomatis to primate interferon gamma stimulated immune defenses.

Evading host innate immune defenses is a critical feature of Chlamydia trachomatis infections, and the mechanisms used by C. trachomatis to subvert these pathways are incompletely understood. We screened a library of chimeric C. trachomatis mutants for genetic factors important for interference with cell-autonomous immune defenses. Mutant strains with predicted truncations of the inclusion membrane protein CT135 were susceptible to interferon gamma-activated immunity in human cells. CT135 functions to prevent host-driven recruitment of ubiquitin and p62/SQSTM to the inclusion membrane. In a nonhuman primate model of C. trachomatis infection, a CT135-deficient strain was rapidly cleared, highlighting the importance of this virulence factor for C. trachomatis pathogenesis. Analysis of CT135 phenotypes in primary macaque cells revealed that cell-autonomous immune defenses against C. trachomatis are conserved between humans and nonhuman primates and connects mechanistic findings with in vivo infection outcomes.

Immunization with a tri-antigen syphilis vaccine significantly attenuates chancre development, reduces bacterial load, and inhibits dissemination of Treponema pallidum.

Syphilis continues to be a significant public health concern worldwide. The disease is endemic in many low- and middle-income countries, and rates have risen sharply in high-income countries over the last decade. The continued prevalence of infectious and congenital syphilis worldwide highlights the need for the development of an effective syphilis vaccine to complement public health measures for syphilis control. The complex, multi-stage course of syphilis infection necessitates a holistic approach to the development of an effective vaccine, in which immunization prevents both the localized stage of infection (typified by the highly infectious chancre) and the disseminated stages of infection (typified by the secondary rash, neurosyphilis, and destructive tertiary lesions, as well as congenital syphilis). Inhibiting development of the infectious chancre would reduce transmission thus providing community- level protection, while preventing dissemination would provide individual-level protection by reducing serious sequelae and may also provide community level protection by reducing shedding during secondary syphilis. In the current study we build upon prior investigations which demonstrated that immunizations with individual, well characterized T. pallidum TprK, TprC, and Tp0751 peptides elicits partial protection against infection in the animal model. Specifically, we show here that immunization with a TprC/TprK/Tp0751 tri-antigen cocktail protects animals from progressive syphilis lesions and substantially inhibits dissemination of the infection.

B-Cell Epitope Mapping of TprC and TprD Variants of Treponema pallidum Subspecies Informs Vaccine Development for Human Treponematoses.

Several recent studies have focused on the identification, functional analysis, and structural characterization of outer membrane proteins (OMPs) of Treponema pallidum (Tp). The Tp species encompasses the highly related pallidum, pertenue, and endemicum subspecies of this pathogen, known to be the causative agents of syphilis, yaws, and bejel, respectively. These studies highlighted the importance of identifying surface-exposed OMP regions and the identification of B-cell epitopes that could be protective and used in vaccine development efforts. We previously reported that the TprC and TprD OMPs of Tp are predicted to contain external loops scattered throughout the entire length of the proteins, several of which show a low degree of sequence variability among strains and subspecies. In this study, these models were corroborated using AlphaFold2, a state-of-the-art protein structure modeling software. Here, we identified B-cell epitopes across the full-length TprC and TprD variants using the Geysan pepscan mapping approach with antisera from rabbits infected with syphilis, yaws, and bejel strains and from animals immunized with refolded recombinant TprC proteins from three syphilis strains. Our results show that the humoral response is primarily directed to sequences predicted to be on surface-exposed loops of TprC and TprD proteins, and that the magnitude of the humoral response to individual epitopes differs among animals infected with various syphilis strains and Tp subspecies. Rather than exhibiting strain-specificity, antisera showed various degrees of cross-reactivity with variant sequences from other strains. The data support the further exploration of TprC and TprD as vaccine candidates.

An Evaluation of Fish Tissue Monitoring Alternatives for Mercury and Selenium: Fish Muscle Biopsy Samples Versus Homogenized Whole Fillets.

Fish contaminant studies with human health protection objectives typically focus on muscle tissue, recognizing that fillets are the commonly consumed tissue fraction. Muscle biopsy punch sampling for mercury analysis has recently been used as an alternative to harvesting fish for fillets; however, there is limited information comparing fillet plug results to whole fillet results. This study was conducted to address that data gap and to test the applicability of plugs for monitoring associated with United States Environmental Protection Agency's fish tissue-based mercury and selenium water quality criteria. The mercury phase included 300 fillet homogenates and 300 field-extracted plug samples from 60 fish, and the selenium phase included 120 fillet homogenates and 120 plugs from 30 fish. Both phases showed that there were no statistically significant differences between fillet plug and homogenized fillet results at the community level; however, a selenium plug monitoring alternative must employ a sufficiently sensitive analytical method and consider total solids. Plug and fillet sampling alternatives have inherent advantages and disadvantages. Fillet sampling provides sufficient mass to consider multiple contaminants but requires fish to be harvested. Plug sampling only provides adequate mass for a single analyte but may allow fish survival, although additional research is needed on survival following plug removal.

Transcriptional and immunological analysis of the putative outer membrane protein and vaccine candidate TprL of Treponema pallidum.

BACKGROUND: An effective syphilis vaccine should elicit antibodies to Treponema pallidum subsp. pallidum (T. p. pallidum) surface antigens to induce pathogen clearance through opsonophagocytosis. Although the combination of bioinformatics, structural, and functional analyses of T. p. pallidum genes to identify putative outer membrane proteins (OMPs) resulted in a list of potential vaccine candidates, still very little is known about whether and how transcription of these genes is regulated during infection. This knowledge gap is a limitation to vaccine design, as immunity generated to an antigen that can be down-regulated or even silenced at the transcriptional level without affecting virulence would not induce clearance of the pathogen, hence allowing disease progression. PRINCIPAL FINDINGS: We report here that tp1031, the T. p. pallidum gene encoding the putative OMP and vaccine candidate TprL is differentially expressed in several T. p. pallidum strains, suggesting transcriptional regulation. Experimental identification of the tprL transcriptional start site revealed that a homopolymeric G sequence of varying length resides within the tprL promoter and that its length affects promoter activity compatible with phase variation. Conversely, in the closely related pathogen T. p. subsp. pertenue, the agent of yaws, where a naturally-occurring deletion has eliminated the tprL promoter region, elements necessary for protein synthesis, and part of the gene ORF, tprL transcription level are negligible compared to T. p. pallidum strains. Accordingly, the humoral response to TprL is absent in yaws-infected laboratory animals and patients compared to syphilis-infected subjects. CONCLUSION: The ability of T. p. pallidum to stochastically vary tprL expression should be considered in any vaccine development effort that includes this antigen. The role of phase variation in contributing to T. p. pallidum antigenic diversity should be further studied.

Adequacy of sample size for estimating a value from field observational data.

In 2011, the U.S. EPA Office of Research and Development released a field-based method for deriving aquatic life benchmarks for conductivity. Since its release, it has been verified, validated, and corroborated by the authors, reviewers, and independent researchers. However, the method and published results have been recently challenged as being artifacts of small sample sizes, prompting this re-evaluation. This paper supplements prior causal analyses by weighing evidence that specifically addresses the hypothesis that the benchmark is a statistical artifact. Four types of evidence are presented: (1) Permutation analyses show that the data sets are able to reliably estimate the extirpation of 5% of genera. (2) Analyses show that 25 occurrences of a genus are sufficient to estimate extirpation. (3) Coherent ecological explanations show that the claimed influence of sample size is actually a result of community ecology. (4) A review of relevant independent studies supports the benchmark. The permutation test is a useful test of the adequacy of field data sets. Furthermore, this weight-of-evidence approach and the individual types of evidence can be a model for analysis of other field-based benchmark values.

Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates.

BACKGROUND: Syphilis is resurgent in many developed countries and still prevalent in developing nations. Current and future control campaigns would benefit from the development of a vaccine, but although promising vaccine candidates were identified among the putative surface-exposed integral outer membrane proteins of the syphilis spirochete, immunization experiments in the rabbit model using recombinant antigens have failed to fully protect animals upon infectious challenge. We speculated that such recombinant immunogens, purified under denaturing conditions from Escherichia coli prior to immunization might not necessarily harbor their original structure, and hypothesized that enhanced protection would result from performing similar immunization/challenge experiments with native antigens. METHODS: To test our hypothesis, we engineered non-infectious Borrelia burgdorferi strains to express the tp0897 (tprK) and tp0435 genes of Treponema pallidum subsp. pallidum and immunized two groups of rabbits by injecting recombinant strains intramuscularly with no adjuvant. TprK is a putative integral outer membrane protein of the syphilis agent, while tp0435 encodes the highly immunogenic T. pallidum 17-kDa lipoprotein, a periplasmic antigen that was also shown on the pathogen surface. Following development of a specific host immune response to these antigens as the result of immunization, animals were challenged by intradermal inoculation of T. pallidum. Cutaneous lesion development was monitored and treponemal burden within lesions were assessed by dark-field microscopy and RT-qPCR, in comparison to control rabbits. RESULTS: Partial protection was observed in rabbits immunized with B. burgdorferi expressing TprK while immunity to Tp0435 was not protective. Analysis of the humoral response to TprK antigen suggested reactivity to conformational epitopes. CONCLUSIONS: Immunization with native antigens might not be sufficient to obtain complete protection to infection. Nonetheless we showed that non-infectious B. burgdorferi can be an effective carrier to deliver and elicit a specific host response to T. pallidum antigens to assess the efficacy of syphilis vaccine candidates.

Macrolide Resistance in Treponema pallidum Correlates With 23S rDNA Mutations in Recently Isolated Clinical Strains.

BACKGROUND: High rates of 23S rDNA mutations implicated in macrolide resistance have been identified in Treponema pallidum samples from syphilis patients in many countries. Nonetheless, some clinicians have been reluctant to abandon azithromycin as a treatment for syphilis, citing the lack of a causal association between these mutations and clinical evidence of drug resistance. Although azithromycin resistance has been demonstrated in vivo for the historical Street 14 strain, no recent T. pallidum isolates have been tested. We used the well-established rabbit model of syphilis to determine the in vivo efficacy of azithromycin against 23S rDNA mutant strains collected in 2004 to 2005 from patients with syphilis in Seattle, Wash. METHODS: Groups of 9 rabbits were each infected with a strain containing 23S rDNA mutation A2058G (strains UW074B, UW189B, UW391B) or A2059G (strains UW228B, UW254B, and UW330B), or with 1 wild type strain (Chicago, Bal 3, and Mexico A). After documentation of infection, 3 animals per strain were treated with azithromycin, 3 were treated with benzathine penicillin G, and 3 served as untreated control groups. Treatment efficacy was documented by darkfield microscopic evidence of T. pallidum, serological response, and rabbit infectivity test. RESULTS: Azithromycin uniformly failed to cure rabbits infected with strains harboring either 23S rDNA mutation, although benzathine penicillin G was effective. Infections caused by wild type strains were successfully treated by either azithromycin or benzathine penicillin G. CONCLUSIONS: A macrolide resistant phenotype was demonstrated for all strains harboring a 23S rDNA mutation, demonstrating that either A2058G or A2059G mutation confers in vivo drug resistance.

Microbiological profiles of fungal keratitis: a 10-year study at a tertiary referral center.

BACKGROUND: Given the rise in cases of fungal keratitis in recent years, this study was performed to better elucidate the microbiological profile, risk factors, and surgical intervention rates of fungal keratitis at a tertiary referral center in the Southeastern USA. FINDINGS: This is a retrospective case series of fungal keratitis infections treated at Duke University Eye Center from January 1, 1998, to October 6, 2008. Of the 4651 culture-proven corneal ulcers identified, 63 (1.4 %) were positive for fungal keratitis with a total of 69 fungal organisms isolated. The majority of isolates were filamentous species (44 of 69, 64 %), and the most commonly isolated organism was Curvularia (11 of 69, 16 %). Bacterial coinfections were found in 24 of the 63 cases (38 %). The most commonly associated risk factors were contact lens wear (n = 15, 24 %) and prior penetrating keratoplasty (PKP) (n = 15, 24 %). Twenty-three cases (37 %) required surgical intervention. The rate of surgical intervention was highest in patients with prior PKP (7/15, 47 %). CONCLUSIONS: In this study, the leading risk factors for fungal keratitis were contact lens wear and prior PKP. Filamentous species were the most common causative pathogens. A relatively high rate of mixed bacterial-fungal infections was found. Patients with prior PKP were more likely to require surgery than patients without history of keratoplasties.

Antigenic variation of TprK facilitates development of secondary syphilis.

Although primary syphilis lesions heal spontaneously, the infection is chronic, with subsequent clinical stages. Healing of the primary chancre occurs as antibodies against outer membrane antigens facilitate opsonophagocytosis of the bacteria by activated macrophages. TprK is an outer membrane protein that undergoes antigenic variation at 7 variable regions, and variants are selected by immune pressure. We hypothesized that individual TprK variants escape immune clearance and seed new disseminated lesions to cause secondary syphilis. As in human syphilis, infected rabbits may develop disseminated secondary skin lesions. This study explores the nature of secondary syphilis, specifically, the contribution of antigenic variation to the development of secondary lesions. Our data from the rabbit model show that the odds of secondary lesions containing predominately TprK variant treponemes is 3.3 times higher than the odds of finding TprK variants in disseminated primary lesions (odds ratio [OR] = 3.3 [95% confidence interval {CI}, 0.98 to 11.0]; P = 0.055) and that 96% of TprK variant secondary lesions are likely seeded by single treponemes. Analysis of antibody responses demonstrates significantly higher antibody titers to tprK variable region sequences found in the inoculum compared to reactivity to tprK variant sequences found in newly arising secondary lesions. This suggests that tprK variants escape the initial immune response raised against the V regions expressed in the inoculum. These data further support a role for TprK in immune evasion and suggest that the ability of TprK variants to persist despite a robust immune response is instrumental in the development of later stages of syphilis.

DWI findings of optic nerve ischemia in the setting of central retinal artery occlusion.

A 67-year-old African-American male with untreated hypertension, hyperlipidemia, and diabetes mellitus presented with sudden, staggering, progressive loss of vision in his left eye over the course of 8 days. Ophthalmologic and fluorescein angiography exams confirmed central retinal artery conclusion, but revealed no embolus. Magnetic resonance imaging of the brain serendipitously revealed restricted diffusion within the distal left optic nerve, illustrating a more proximal occlusion, which matched the fluorescein angiographic findings. Extensive workup revealed no embolic source, postulating primary hypertension as the underlying etiology.

Clinical outcomes of xeno-free allogeneic cultivated limbal epithelial transplantation for bilateral limbal stem cell deficiency.

PURPOSE: To report the clinical outcomes of allogeneic cell-based therapy for bilateral corneal blindness due to limbal stem cell deficiency (LSCD). METHODS: This retrospective study included 28 eyes of 21 patients, at least 8 years of age, with bilateral and total LSCD, treated between 2001 and 2010. A limbal biopsy was obtained from the eye of an adult living related donor. The limbal epithelial cells were cultivated in the laboratory using a xeno-free explant culture technique and transplanted onto the recipient eye after 10-14 days. All transplant recipients received topical and systemic immunosuppressants. RESULTS: At a mean follow-up of 4.8 ± 2.8 years, 20 (71.4%) eyes maintained a completely epithelised, avascular and stable corneal surface, and among them 13 (46.4%) eyes subsequently underwent a penetrating keratoplasty (PK). The Kaplan-Meier survival rate of the PK allograft was 76.9 ± 11.7% at 1 year with a median survival of 3.3 years. Visual acuity improved to 20/60 or better in 19 (67.8%) eyes. No donor or recipient eyes developed serious ocular complications. CONCLUSIONS: Allogeneic cultivated limbal epithelial transplantation, followed by PK when needed, can successfully restore the ocular surface and improve vision in patients with corneal blindness due to bilateral LSCD.

Dislocation of the donor graft to the posterior segment in descemet stripping automated endothelial keratoplasty.

PURPOSE: To report a series of dislocations of the donor graft into the posterior segment associated with Descemet stripping endothelial keratoplasty (DSAEK) and to identify possible risk factors for dislocation and clinical outcomes. DESIGN: Retrospective case series. METHODS: Cases of donor graft dislocation into the posterior segment associated with endothelial keratoplasty were identified from the clinical experience of 7 surgeons. Observations included the preoperative surgical history of each eye, preoperative and postoperative visual acuity, management of the complication, and the postoperative clinical course. No identified cases were excluded from this series. RESULTS: Eight posterior graft dislocations were associated with DSAEK surgery. Each eye had a history of vitrectomy. Five eyes had sutured posterior chamber intraocular lenses, 1 eye had a sulcus intraocular lens, and 2 eyes were aphakic. Each eye required repeat grafting, and in 6 of 8 eyes, pars plana vitrectomy was used to remove the dislocated graft. Final visual acuities ranged from 20/30 to no light perception. CONCLUSIONS: Graft dislocation into the posterior segment is a rare complication of DSAEK surgery that can lead to permanent vision loss. It has occurred in eyes that have undergone previous vitrectomy and complicated intraocular lens placement or were aphakic. As is the case with a dropped lens nucleus during cataract extraction, visual acuities after a dropped DSAEK graft range from very good to no light perception. Better postoperative results seem to be associated with prompt removal of the posteriorly dislocated graft.

Cataracts: we have perfected the surgery, but is it time for prevention?

PURPOSE OF REVIEW: This review presents a discussion of cataract prevention, with a focus on the societal burden of untreated cataracts and pathophysiologic mechanisms of prevention. RECENT FINDINGS: Multiple studies have implicated cataract surgery and vision loss due to cataract as a major cause of disability and lost productivity. Long-term use of antioxidants such as vitamin C, lutein, and zeaxanthin have been associated with lower incidence and progression of cataracts, but prospective studies of vitamin supplementation have shown little effect. There are currently over 400 cataract-related clinical trials; one trial of a topical medication for the treatment of cataract is currently in progress. SUMMARY: Vision loss due to cataract, disability associated with cataract blindness, and the surgical treatment of this disease present a significant public health burden. Useful strategies for prevention may include public health campaigns targeted at young adults, with a focus on making healthy choices to prevent this disease.

Endothelial Keratoplasty: From DLEK to DMEK.

The last decade has heralded a revolutionary shift in the treatment of corneal endothelial disease. Only 15 years ago, the only surgical treatment for pseudophakic bullous keratopathy and Fuchs dystrophy was penetrating keratoplasty (PK). Although used successfully for over a century, PK requires many months of refractive adjustments before the eye achieves visual stability. Starting with the advent of posterior lamellar keratoplasty in the late 1990s, a number of procedures have been developed, refined, and widely adopted, which have given patients faster recoveries and improved globe stability in comparison to traditional corneal transplantation. Each iteration of endothelial keratoplasty (EK) has involved the increasingly selective transplantation of corneal endothelial cells. Preliminary results of the most recent form of EK, Descemet's membrane EK, suggest that pure endothelial cell transplantation is on the horizon.

Descemet stripping automated endothelial keratoplasty in a child.

We present the case of a 9-year-old patient with corneal decompensation that was treated with Descemet stripping automated endothelial keratoplasty (DSAEK) and followed for 18 months. Although the procedure has been used successfully in adult populations, to our knowledge, this is the first report of DSAEK with long-term follow-up in a child. The outcome suggests that endothelial keratoplasty may be a good alternative to penetrating keratoplasty in children. Endothelial keratoplasty is a new form of corneal transplantation that promises faster visual recovery compared with penetrating keratoplasty. With endothelial keratoplasty, the corneal architecture is preserved; therefore, large spherical and astigmatic errors are not induced, and stable refraction is achieved earlier in the course of therapy.

Nutrition and the prevention of cataracts.

PURPOSE OF REVIEW: Oxidative stress is a major cause of cataract development. Numerous studies have been published regarding the effects of nutritional supplementation on cataract progression. RECENT FINDINGS: Basic science research has demonstrated a protective effect of antioxidants on lens tissue, and supplementation with vitamin C and lutein/zeaxanthin has been associated with a decreased risk of cataract formation in multiple observational studies. One large interventional trial demonstrated a significant difference in participants treated with high-dose vitamin C versus placebo, but a more recent interventional study did not replicate these findings. In a review of the carotenoids lutein and zeaxanthin, the Food and Drug Administration concluded there is insufficient evidence to suggest that supplementation with these carotenoids lowers the risk of cataract formation. While high doses of multivitamins, antioxidants, or lutein and zeaxanthin are unlikely to be of significant ophthalmic benefit to the general public, these nutrients may help individuals exposed to high oxidative stress, such as heavy smokers, and those with poor nutrition. SUMMARY: Supplementation with vitamin C, lutein, zeaxanthin, or a multivitamin may help certain populations, but is unlikely to affect the progression of cataracts in most patients.

Evaluation of a new CPC-to-gauge bonding technique with the use of in vitro fluid flow.

Strain gauging enables the measurement of bone deformation during physical activity, leading to a better understanding of the physiological effects of loading on bone growth and remodeling. Development of a technology that will withstand long-term in vivo exposure and bond securely to bone is imperative for accurate, consistent measurement collection. Polysulfone is currently used to attach calcium-phosphate ceramic (CPC) particles, which promote bone-to-gauge bonding, to polyimide-backed strain gauges. This study evaluated the use of an implant-grade epoxy as an alternative CPC-polyimide adhesive. Polyimide-epoxy-CPC interfaces were loaded to failure and shear strengths calculated. In vitro studies providing a constant flow of medium over test specimens were designed, and long-term in vitro fluid exposure studies of the epoxy's shear strength were conducted. Average shear strength of polysulfone-polyimide interfaces were reported to be 7 MPa. The average shear strength of the epoxy-polyimide interface before long-term in vitro exposure was 17 MPa, which is stronger than the shear strength of the bone-CPC interface. The strength of the epoxy-polyimide interface decreased to 6.8 MPa after 24 weeks in vitro and 3 MPa after 24 weeks in vivo.