Winston Chamberlain

Transcription of the FMR1 gene in individuals with fragile X syndrome

Fragile X syndrome generally arises as a consequence of a large expansion of a CGG trinucleotide repeat element that is located in the GC-rich promoter region of the fragile X mental retardation gene (FMR1). In the conventional model for fragile X, clinical involvement arises as a consequence of silencing of the FMR1 gene, with the attendant loss of FMR1 protein (FMRP). However, it has recently been demonstrated that most males with large premutation alleles (100-200 repeats), or with unmethylated full mutation alleles, have FMR1 mRNA levels that are higher than normal, despite reduced levels of FMRP. In the current work, we extend and confirm these observations using quantitative (fluorescent) reverse transcription polymerase chain reaction on larger sample populations, establishing that even for smaller premutation alleles (55-100 repeats) the mRNA levels are significantly elevated (mean 2.1-fold elevation; P = 3.9 x 10(-3)), relative to normal controls. Thus, an abnormal molecular phenotype is established close to the upper end of the normal range. We also demonstrate that the levels of FMR1 mRNA are elevated in females with premutation alleles; however, the mRNA levels are more varied than in the males, and are attenuated in a manner that is consistent with the fraction of normal alleles that are active in any given individual. Finally, we demonstrate that in lymphoblastoid cells derived from a patient with a severe form of fragile X caused by a point mutation in the second KH domain of the gene, but with a normal CGG element (25 repeats), the FMR1 mRNA level is normal. Thus, although models in which FMRP level (or level of function) modulates transcriptional activity remain viable, other explanations for the elevated message levels, including direct (cis) effects of the CGG element on transcription, must also be considered.

Comparison of Penetrating Keratoplasty Performed with a Femtosecond Laser Zig-Zag Incision versus Conventional Blade Trephination

PURPOSE To evaluate visual outcomes and astigmatism in patients who underwent penetrating keratoplasty (PK) with 2 different incision techniques. DESIGN Retrospective comparison of a consecutive surgical series. PARTICIPANTS Fifty-seven consecutive patients who underwent PK at the University of California, Irvine, academic referral practice. METHODS A comparison of 49 eyes of 43 patients that underwent femtosecond laser zig-zag incision pattern PK versus 17 eyes of 14 patients that underwent conventional Barron suction trephination PK performed contemporaneously. All PKs were closed with an identical, 24-bite running nylon suture technique. MAIN OUTCOME MEASURES Topographically determined astigmatism, best spectacle-corrected visual acuity (BSCVA), and recovery of full visual potential. RESULTS The postoperative follow-up ranged from 1 to 12 months. There was a significant difference in average astigmatism between the groups at postoperative month 1 (P = 0.013) and 3 (P = 0.018). By month 3, the average astigmatism was 3 diopters (D) in the zig-zag group and 4.46 D in the conventional group. Of the patients with normal macular and optic nerve function (n(ZZ) = 32; n(con) = 14), a significant difference in BSCVA was seen at month 1 (P = 0.0003) and month 3 (P = 0.006) with 81% of the zig-zag group versus 45% of the conventional group achieving BSCVA of > or =20/40 by month 3 (P = 0.03). CONCLUSIONS The femtosecond laser generated zig-zag-shaped incision results in a more rapid recovery of BSCVA and induces less astigmatism compared with conventional blade trephination PK. FINANCIAL DISCLOSURE(S) Proprietary commercial disclosure may be found after the references.

Comparison of Femtosecond Laser-assisted Keratoplasty versus Conventional Penetrating Keratoplasty

PURPOSE To compare postoperative outcomes for femtosecond laser-assisted keratoplasty (FLAK) with conventional penetrating keratoplasty (PK). DESIGN Retrospective, comparative surgical series. PARTICIPANTS Fifty consecutive patients who underwent FLAK and 50 case-controlled patients that had PK at the Casey Eye Institute (Oregon Health & Science University, Portland, OR). METHODS Data was collected for 50 consecutive cases that underwent zigzag incision FLAK and was compared with 50 subjects that had conventional blade trephine incision PK with similar age, diagnosis and concurrent ocular morbidities over a 2-year follow-up period. MAIN OUTCOME MEASURES Topographic astigmatism, best spectacle-corrected visual acuity, uncorrected visual acuity, pinhole visual acuity, and the timing of selective suture removal (or adjustment) over various follow-up intervals up to 2 years postoperatively. RESULTS Significantly lower topographic astigmatism was achieved in the FLAK group over the PK group in the 4- to 6-month follow-up period (P = 0.0324), which correlated well with significant earlier selective suture removal that occurred in that same group over both the 2- to 3-month (P = 0.0025) and 4- to 6-month (P = 0.0236) follow-up periods. This difference in astigmatism was no longer present at any other follow-up period up to 24 months postoperatively. The subset analysis of patients with keratoconus or post-LASIK ectasia did not show any difference in either astigmatism or visual acuity at any time. CONCLUSIONS Compared with PKP, FLAK had significant improvement in astigmatism before but not after the 6 month postoperative follow-up period. Earlier suture removal was noted in the FLAK group. No significant improvement in best spectacle-corrected visual acuity was noted at any time point. There were no complications or difficulties with trephination in the FLAK procedure across a wide range of corneal pathologies.

Genetic control of autoimmune myocarditis mediated by myosin-specific antibodies

Abstract Autoimmune disease involves both the development of autoreactivity and the expression of organ damage, and susceptibility is genetically complex. We recently reported that in autoimmune myocarditis susceptibility to antibody-mediated cardiac injury is strain specific. DBA/2 mice develop myocarditis following administration of myosin-specific antibody, while BALB/c mice do not. This susceptibility appears to be controlled by expression of myosin in the myocardial extracellular matrix. CByD2F1 mice are both resistant to induction of myocarditis and do not demonstrate extracellular myosin, indicating a recessive genetic component to these traits. A backcross analysis of susceptibility using DBA/2×CByD2F1 mice revealed a locus on chromosome 12 that is strongly linked with myocarditis. In male mice there was a second region on chromosome 1 that also contributes to disease susceptibility. However, genetic susceptibility in both female and male mice was genetically complex. This study demonstrates that the genetic basis of tissue injury can be analyzed separately from the genetic basis of autoreactivity. Future studies will determine whether the genetic factors identified in this study are also involved in susceptibility to rheumatic fever.

Topical/Mucosal Delivery of Sub-Unit Vaccines That Stimulate the Ocular Mucosal Immune System

Mucosal vaccination is proving to be one of the greatest challenges in modern vaccine development. Although ocular mucosal immunity is highly beneficial for achieving protective immunity, the induction of ocular mucosal immunity against ocular infectious pathogens, particularly herpes simplex virus type 1 (HSV-1), which is the leading cause of infectious corneal blindness, remains difficult. Recent developments in cellular and molecular immunology of the ocular mucosal immune system (OMIS) may help in the design of more effective and optimal immunization strategies against ocular pathogens. In this review, we highlight ocular mucosal immunoprophylactic and immunotherapeutic vaccine strategies that have been evaluated to control the many pathogens that attack the surface of the eye. Next, we describe the current understandings of the OMIS and elucidate the structure and the function of the humoral and cellular immune system that protects the surface of the eye. Results from our recent experiments using topical ocular delivery of peptides-CpG and lipopeptide-based vaccines against HSV-1 infection are presented. The future challenges and issues related to the ocular mucosal delivery of molecularly defined sub-unit vaccines are discussed.

Nasolacrimal Duct Closure Modulates Ocular Mucosal and Systemic CD4+ T-Cell Responses Induced following Topical Ocular or Intranasal Immunization

ABSTRACT Both topical ocular and topical intranasal immunizations have been reported to stimulate the ocular mucosal immune system (OMIS) and the systemic immune system. Nasolacrimal ducts (NLDs) are the connecting bridges between the OMIS and nasal cavity-associated lymphoid tissue (NALT). These ducts drain topical ocularly administrated solutions into the inferior meatus of the nose to reach the NALT. Inversely, NLDs also drain intranasally administrated solutions to the mucosal surface of the eye and thus the OMIS. This unique anatomical connection between the OMIS and NALT systems provoked us to test whether the OMIS and NALT are immunologically interdependent. In this report, we show that both topical ocular administration and topical intranasal administration of a mixture of immunodominant CD4+ T-cell epitope peptides from herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) emulsified with the CpG2007 mucosal adjuvant are capable of inducing local (in conjunctiva) as well as systemic (in spleen) HSV-peptide-specific CD4+ T-cell responses. Interestingly, surgical closure of NLDs did not significantly alter local ocular mucosal CD4+ T-cell responses induced following topical ocular immunization but did significantly enhance systemic CD4+ T-cell responses (as measured by both T-cell proliferation and gamma interferon (IFN-γ) production; P < 0.005). In contrast, NLD closure significantly decreased ocular mucosal, but not systemic, CD4+ T-cell responses following intranasal administration of the same vaccine solution (P < 0.001). The study suggests that NALT and the OMIS are immunologically interconnected.

Comparison of corneal surface higher-order aberrations after endothelial keratoplasty, femtosecond laser-assisted keratoplasty, and conventional penetrating keratoplasty.

Purpose: To evaluate and compare corneal higher-order aberrations (HOAs) after Descemet stripping automated endothelial keratoplasty (DSAEK), femtosecond laser–assisted penetrating keratoplasty (FLAK), and conventional penetrating keratoplasty (PKP). Methods: A retrospective comparison of consecutive surgical series of 67 eyes of 59 patients between 1.5 and 19 months after corneal transplant surgery (22, 34, and 11 corneas underwent DSAEK, FLAK, and PKP, respectively, by a single surgeon). The main outcome measures were anterior and posterior corneal surface HOAs (Zernike polynomials, third to eighth order) determined with Scheimpflug photography at 4.0- and 6.0-mm optical zones and best spectacle-corrected visual acuity (BSCVA) (logarithm of the minimum angle of resolution equivalents). Results: DSAEK had fewer total anterior HOAs compared with FLAK [P = 5.27 × 10−5 (4.0 mm) and P = 1.02 × 10−5 (6.0 mm)] and PKP [P = 1.82 × 10−4 (4.0 mm) and P = 1.56 × 10−4 (6.0 mm)] but greater total posterior HOAs than FLAK [P = 0.001 (4.0 mm) and P = 0.007 (6.0 mm)] and PKP [at 4.0-mm optical zone (P = 0.047)]. FLAK had fewer total anterior and posterior HOAs than PKP, but differences were not statistically significant. DSAEK grafts exhibited statistically significantly greater posterior HOAs than either type of PKP. The magnitude of anterior and posterior HOAs weakly correlated with BSCVA. Conclusions: DSAEK induces fewer anterior surface HOAs but greater posterior surface HOAs than FLAK or PKP. Differences between FLAK and PKP are not statistically significant. Anterior and posterior HOAs correlate weakly with poorer visual outcome and likely contribute to decreased BSCVA after keratoplasty.

Association of Central Corneal Thickness and 24-hour Intraocular Pressure Fluctuation

PurposeTo evaluate the association between office-hour central corneal thickness (CCT) and 24-hour intraocular pressure (IOP) fluctuation in patients with glaucoma. DesignObservational case-control study. MethodsMeasurements of IOP were obtained every 2 hours during a 24-hour period from 52 untreated glaucoma patients and 29 age-matched normal control subjects housed in a sleep laboratory. Habitual IOP measurements were obtained using a pneumatonometer in the sitting positions during the diurnal/wake period (7 AM to 11 PM) and in the supine position during the nocturnal/sleep period (11 PM to 7 AM). CCT was measured in all subjects using ultrasound pachymetry once during office hours. The association between IOP fluctuation (peak IOP-trough IOP) during the 24-hour period and the office-hour CCT was assessed in both glaucoma patients and healthy age-matched controls using Spearman rank order correlation. ResultsThere was no statistically significant correlation between IOP fluctuation and CCT in glaucomatous (P=0.405) and normal subjects (P=0.456). ConclusionsTwenty-four–hour IOP fluctuations were not correlated with single CCT measurements taken during office hours in glaucoma patients.

Imaging and quantification of endothelial cell loss in eye bank prepared DMEK grafts using trainable segmentation software

Abstract Purpose: To improve accuracy and efficiency in quantifying the endothelial cell loss (ECL) in eye bank preparation of corneal endothelial grafts. Methods: Eight cadaveric corneas were subjected to Descemet Membrane Endothelial Keratoplasty (DMEK) preparation. The endothelial surfaces were stained with a viability stain, calcein AM dye (CAM) and then captured by a digital camera. The ECL rates were quantified in these images by three separate readers using trainable segmentation, a plug-in feature from the imaging software, Fiji. Images were also analyzed by Adobe Photoshop for comparison. Mean times required to process the images were measured between the two modalities. Results: The mean ECL (with standard deviation) as analyzed by Fiji was 22.5% (6.5%) and Adobe was 18.7% (7.0%; p = 0.04). The mean time required to process the images through the two different imaging methods was 19.9 min (7.5) for Fiji and 23.4 min (12.9) for Adobe (p = 0.17). Conclusions: Establishing an accurate, efficient and reproducible means of quantifying ECL in graft preparation and surgical techniques can provide insight to the safety, long-term potential of the graft tissues as well as provide a quality control measure for eye banks and surgeons. Trainable segmentation in Fiji software using CAM is a novel approach to measuring ECL that captured a statistically significantly higher percentage of ECL comparable to Adobe and was more accurate in standardized testing. Interestingly, ECL as determined using both methods in eye bank-prepared DMEK grafts exceeded 18% on average.

Femtosecond-assisted deep anterior lamellar keratoplasty using big-bubble technique in a cornea with 16 radial keratotomy incisions

Purpose: To report on a patient with uncorrectable fluctuating astigmatism from multiple previous radial keratotomy (RK) procedures who successfully underwent a femtosecond laser-assisted deep anterior lamellar keratoplasty (DALK) with big-bubble technique. Methods: A 60-year-old woman with a history of multiple RK procedures with 16 radial incisions and 4 Ruiz-style cuts had fluctuating corneal astigmatism with preoperative best contact lens-corrected visual acuity of 20/80. Results: Intraoperatively, a femtosecond laser-assisted zigzag pattern DALK was performed. Descemet baring was successfully achieved using the big-bubble technique without air escaping from the radial incisions, likely related to precise depth of base of posterior side cut made by laser. Postoperative best-corrected visual acuity is 20/25 with 1.3 diopter of astigmatism at 5 months. Conclusions: To our knowledge, this is the first report to use this technique in a cornea with RK incisions. Femtosecond lasers may promote the success of DALK in challenging corneas.