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Andrew J W Huang

Publications and source records attributed to Andrew J W Huang.

15 recordsLinked to original sources
PubMed2006

Identification of an amyloidogenic region on keratoepithelin via synthetic peptides.

Mutations of keratoepithelin (KE) gene in human chromosome 5q31 have been linked with corneal epithelial or stromal dystrophies characterized by the abnormal deposits of amyloid fibrils and/or non-amyloid aggregations in corneal tissue. We report herein that synthetic peptide containing amino acid (a.a.) residues of 515-532 of native KE protein can readily form beta-sheet-containing amyloid fibrils in vitro. Amyloid fibrils formed in various conditions from short synthetic peptides (containing a.a. 515-532 and 515-525, respectively) were characterized by thioflavin T (ThT) fluorescence assay, Congo red staining, electron microscopy (EM) and circular dichroism (CD). Triple-N-methylation of the synthetic peptides prevented the beta-sheet polymerization and related amyloid fibril formation. Comparison study with ThT fluorescence further demonstrated that synthetic peptides containing corneal dystrophy-related mutations within this region formed amyloid fibrils to various extents. Our results suggest that each individual dystrophy-related mutation by itself does not necessarily potentiate amyloid fibril formation of KE. Roles of these intrinsically amyloidogenic foci in abnormal KE aggregations and amyloid deposits of stromal corneal dystrophies await further investigation.

Amino Acid Sequence↗
PubMed2005

Pathways of corneal and ocular surface inflammation: a perspective from the cullen symposium.

The goal of this symposium was to coalesce information presented by 22 investigators in the field of corneal and ocular surface inflammation into common pathways of inflammation. The perspective elucidated in this article defines the components of the normal ocular surface immune architecture and describes the consensus reached on the mechanisms/pathways involved in 1) acute inflammation; 2) late-stage (chronic) response; and 3) allergic disease. Seven diagrams didactically illustrate mechanisms. This paper is the introductory article in a supplement containing 18 articles by the symposium participants.

Journal Article↗
PubMed2005

Modulation of corneal vascularization.

Avascularity of normal cornea is a result of homeostasis between anti-angiogenic and pro-angiogenic stimuli. Disruption of this delicate balance during corneal wound healing can lead to pathological corneal vascularization. Several unique characteristics in the ocular surface epithelia modulate corneal avascularity. Normal cornea contains heparan sulfate to prevent the release of potent angiogenic cytokines, such as basic fibroblast growth factor (bFGF) from the Bowman's layer. Potent angiostatic factors, such as endostatin and angiostatin, can be produced from degradation of corneal extracelluar matrix. In contrast, conjunctiva contains angiogenic cytokines, such as bFGF and vascular endothelial growth factor. In addition to regulating release of angiogenic and angiostatic cytokines, matrix metalloproteinases (MMPs) and other proteolytic enzymes can modulate corneal vascularization via matrix degradation to allow endothelial migration and tissue remodeling. When the cornea becomes vascularized, inflammatory cells and mediators gain uninhibited access to the cornea, thus rendering immune sensitization and increased risk of corneal graft rejection. Novel therapies targeting angiogenic cytokines or MMPs have been shown to suppress corneal vascularization effectively in animal models, and may have therapeutic potential for clinical use.

Journal Article↗
PubMed2004

Cytokeratin 12 in human ocular surface epithelia is the antigen reactive with a commercial anti-Galpha q antibody.

PURPOSE: In our initial attempt to identify differentiation markers for ocular surface epithelia, we observed a unique staining pattern by a commercial anti-Galphaq antibody. We further isolate and characterize the protein reactive with this anti-Galphaq antibody in human ocular surface epithelia. METHODS: Human donor corneoscleral buttons were sectioned and stained with a battery of commercial antibodies against Galpha proteins. Western blot analysis of cell lysates of corneal epithelial cells and HEK 293 cells transfected with Galphaq cDNA was used to determine the identity of the protein reactive with the anti-Galphaq antibody (E-17). Comparisons were made with another anti-Galphaq antibody (G4415) and an anti-cytokeratin 12C (J7) antibody. The isolated proteins reactive with E17 and J7 were then analyzed with two dimensional isoelectric focusing. Polypeptide sequences were identified using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) after in-gel protein digestion. RESULTS: The E-17 anti-Galphaq antibody preferentially stained the entire corneal epithelia and the suprabasal layers of the limbus with complete absence of staining in the basal limbus and conjunctiva. Western blot analysis of corneal epithelial cells showed that E-17 antibody identified a protein with a molecular weight of 55 kDa. However, the antibody did not react with the purported antigen, Galphaq protein (42 kDa) produced by Galphaq cDNA. Another anti-Galphaq antibody (G4415) did not react with the 55 kDa protein but did react with the 42 kDa Galphaq protein. Further comparison of the E-17 antibody with the J7 antibody revealed that both recognized the 55 kDa band in one and two dimensional analysis. MALDI-TOF MS analysis confirmed that the 55 kDa protein of interest was actually cytokeratin 12 (CK12), rather than Galphaq protein. CONCLUSIONS: The commercial E-17 anti-Galphaq antibody did not react with Galphaq protein, its purported antigen. Instead, it recognized a 55 kDa protein, which was characterized to be cytokeratin 12 by isoelectric focusing and peptide fingerprinting with mass spectrometry. Based on its reactivity with CK12, this commercial E-17 can be used as a differentiation marker to study ocular surface epithelia.

Autoantigens↗
PubMed2004

Identification of the promoter region of the human betaIGH3 gene.

PURPOSE: To isolate and characterize the promoter of the human betaIGH3 gene. METHODS: Primer extension and CapSite Hunting methods were used to determine the transcription start sites (TSS) of the human betaIGH3 gene. Putative transcription factor-binding sites and potential promoter regions were identified by online tools. Two clones containing 3 Kb and 1 Kb of the 5'-flanking region of the betaIGH3 gene were isolated and their respective promoter activities were characterized. Various fusion constructs of betaIGH3 promoter-luciferase reporter were made to transfect A549 cells. The responses of these fragments to TGF-beta1 were also measured after being treated with TGF-beta1 at different concentrations. Several human and nonhuman cell lines were also transfected with the 1 Kb betaIGH3 promoter-reporter construct to compare the activity of the betaIGH3 promoter in these cells. RESULTS: The transcription start site of human betaIGH3 mRNA was determined to be 65 bp upstream of the ATG start codon. Both the 3 Kb (-3011 to -1) and 1 Kb (-1000 to -1) fragments displayed strong and comparable promoter activity in transfected cells. Truncation analyses in A549 cells identified the nucleotide region from -336 to -1 as having high promoter activity (minimal promoter). The results also indicated that the nucleotide fragment from -1000 to -646 contained negative regulatory elements. Twenty ng/ml TGF-beta1 upregulated the activity of the 1 Kb construct, but did not upregulate the activity of the -336 to -1 construct, suggesting that TGF-beta1 responsive elements existed in the region from -1000 to -336. The 1 Kb construct universally demonstrated promoter activity in all cell lines tested. CONCLUSIONS: We identified the betaIGH3 gene promoter with a distinct regulatory pattern in the 1 Kb region upstream of the ATG start codon. Further elucidation of the functions of this promoter region may facilitate understanding of betaIGH3 and its related corneal dystrophies.

Animals↗
PubMed2004

In vivo confocal microscopy in patients with central cloudy dystrophy of François.

OBJECTIVE: To report in vivo corneal confocal microscopic findings of patients with central cloudy dystrophy of François. METHODS: Two unrelated patients, a 78-year-old man and a 75-year-old woman, with central cloudy dystrophy of François were examined using routine slitlamp biomicroscopy and confocal microscopy. RESULTS: In both cases, slitlamp biomicroscopy showed bilateral polygonal opacities separated by clear spaces. The corneal opacities were most prominent centrally and were located in the deeper stromal layer immediately anterior to the Descemet membrane. By confocal microscopy, normal superficial and basal epithelial layers, midstromal layers, and endothelial layers were noted in both cases. However, small highly refractile granules and deposits were observed in the anterior stromal layer in both cases. Also, multiple dark striae among the extracellular matrix with increased intensities were observed in the posterior stroma adjacent to the corneal endothelial layer in both cases. CONCLUSIONS: Abnormal stromal deposits and multiple dark striae were observed in central cloudy dystrophy of François using in vivo corneal confocal microscopy. Use of confocal microscopy to investigate these abnormal stromal opacities may be helpful in differentiating various corneal stromal pathologic features.

Aged↗
PubMed2004

Impression cytology in the diagnosis of acanthamoeba keratitis with surface involvement.

PURPOSE: To evaluate the use of impression cytology in the diagnosis of Acanthamoeba keratitis with surface involvement. DESIGN: Observational case series. METHODS: Three patients with culture-proven superficial Acanthamoeba keratitis were studied. Patients' medical records were reviewed for clinical information, treatment procedures and disease course. Impression cytology specimens taken from the cornea of those patients were processed and observed by bright field microscopy. The main outcome measure was the cytopathological examination and identification of amoebic organisms. RESULTS: In each of these three patients, impression cytology specimens revealed the amoeba cysts. Some specimens also showed pleomorphic trophozoites with typical amoeba nuclei. CONCLUSION: Corneal impression cytology successfully detected the cysts and trophozoites of Acanthamoeba from patients with superficial amoeba keratitis. This indicates that impression cytology can be a useful technique to facilitate early diagnosis of Acanthamoeba keratitis with surface involvement.

Acanthamoeba↗
PubMed2004

Treatment of recurrent corneal and conjunctival intraepithelial neoplasia with topical interferon alfa 2b.

OBJECTIVE: To evaluate topical interferon alfa 2b (IFNalpha2b) as a single therapeutic agent in the treatment of presumed recurrent corneal and conjunctival intraepithelial neoplasia. DESIGN: Noncomparative, retrospective case series. PARTICIPANTS: Seven consecutive patients with recurrent corneal and conjunctival intraepithelial neoplasia diagnosed at the University of Minnesota from July 2000 to November 2003 were studied retrospectively. All patients had a history of histologically proven primary corneal and conjunctival intraepithelial neoplasia and were treated by surgery, cryotherapy, radiation, and/or topical mitomycin C before recurrence. INTERVENTION: Patients with a clinical diagnosis of recurrent corneal and conjunctival intraepithelial neoplasia were treated with recombinant topical IFNalpha2b drops (1 million IU/ml) 4 times daily until lesion resolution was noted. MAIN OUTCOME MEASURES: A review of medical records was performed to assess the duration of and response to treatment with topical IFNalpha2b, defined by clinical resolution of corneal and conjunctival intraepithelial neoplasia. RESULTS: The average age of the 7 patients at the initiation of topical IFNalpha2b treatment for presumed recurrent corneal and conjunctival intraepithelial neoplasia was 68.7 years (range, 54-88). Six of 7 patients had successful treatment of recurrent corneal and conjunctival intraepithelial neoplasia lesions with topical IFNalpha2b treatment. The average length of IFNalpha2b treatment to resolution of recurrent corneal and conjunctival intraepithelial neoplasia was 14.5 weeks (range, 5-24). After treatment with topical IFNalpha2b for recurrent corneal and conjunctival intraepithelial neoplasia, 2 patients had another recurrence of corneal and conjunctival intraepithelial neoplasia, noted at 1 year and 2 months, respectively. The average post-treatment follow-up was 11.7 months (range, 8-17) after the resolution of recurrent corneal and conjunctival intraepithelial neoplasia. No side effects of treatment were noted in any patient. CONCLUSIONS: Topical IFNalpha2b as a single therapeutic agent is an effective treatment of presumed recurrent corneal and conjunctival intraepithelial neoplasia. It offers the benefits of topical therapy and avoids the risks of surgical or other interventions-specifically, ocular surface toxicity, cicatricial conjunctival changes, and limbal stem cell deficiency. Larger controlled studies with longer follow-up periods are recommended to confirm the long-term efficacy and safety of this topical treatment.

Administration, Topical↗
PubMed2004

Optimized expression and refolding of human keratoepithelin in BL21 (DE3).

Keratoepithelin (KE) is an extracellular protein participating in cell adhesion and differentiation. Mutations of the KE gene (on 5q31 in humans) cause deposition of abnormal proteins (amyloid and non-amyloid) in corneal stroma and lead to several corneal dystrophies in humans. However, further studies on the KE protein have been limited by the intrinsic difficulty of purifying this protein. A high-expression plasmid containing human KE gene was constructed to generate recombinant KE proteins in Escherichia coli. The plasmid was transformed into E. coli BL21 (DE3) and the recombinant protein was expressed as an insoluble His-tagged fusion protein and purified by nickel chelation affinity chromatography under denaturing conditions. On average, 12 mg of purified KE was routinely obtained from 1L of culture media. The recombinant KE was refolded in arginine-containing dialysis solutions and the recovery of bioactive KE typically was approximately 70%. The procedures developed in this report should enable reproducible production of KE and related mutant proteins in large quantities and facilitate future studies on biochemical and biophysical properties of KE and the pathogenesis of related corneal dystrophies.

Amino Acid Sequence↗
PubMed2003

Bilateral herpetic keratoconjunctivitis.

PURPOSE: To review the clinical characteristics and visual outcomes of patients with bilateral herpetic keratitis. DESIGN: Retrospective, noncomparative, observational case series. PATIENTS AND METHODS: A retrospective review of medical records of 544 patients with herpes simplex virus (HSV) eye disease treated between January 1996 and September 2001 was performed at the Department of Ophthalmology, University of Minnesota. Seven patients (1.3%) with bilateral herpetic keratoconjunctivitis were identified. RESULTS: In these seven patients, the age at the initial onset of corneal disease ranged from 7 weeks to 46 years, with a median of 18 years and a mean of 19.3 years. Five patients had systemic atopy, and two patients had severe ocular rosacea. Systemic immune disorders were noted in two patients. Recurrent blepharoconjunctivitis was noted in 8 eyes (57%), epithelial keratitis in 12 eyes (85.7%), stromal keratitis in 9 eyes (64.3%), necrotizing stromal keratitis in 5 eyes (35.7%), and progressive endotheliitis in 2 eyes (14.2%). Corneal complications included opacification, neovascularization, and corneal thinning or perforation. Penetrating keratoplasty was performed in 1 eye, in which endophthalmitis subsequently developed and which required enucleation. Four patients with continued use of oral antiviral prophylaxis (acyclovir 400 mg twice daily) since September 1999 showed significant decreases in recurrence. The average remission in these four patients was 1.7 years. The visual acuity at the last follow-up was 20/40 or worse in 6 eyes (42.8%). CONCLUSIONS: In contrast to unilateral HSV keratitis, our patients with bilateral herpetic corneal infections had underlying atopy or immune deviations and evinced more protracted clinical courses. Long-term prophylactic antiviral treatment has reduced the incidence of recurrence in this group of patients.

Acyclovir↗
PubMed2003

Detection by impression cytologic analysis of conjunctival intraepithelial invasion from eyelid sebaceous cell carcinoma.

OBJECTIVE: To demonstrate that conjunctival impression cytologic analysis can detect conjunctival intraepithelial invasion from sebaceous cell carcinoma of the eyelid. DESIGN: Observational case series with cytopathologic correlation. PARTICIPANTS: Four patients with unilateral blepharoconjunctivitis and biopsy-proven sebaceous cell carcinoma. METHODS: Impression cytologic analysis specimens were taken from the suspicious area of the bulbar conjunctiva of each patient. Staining of the specimens was performed with a modified Papanicolaou stain. MAIN OUTCOME MEASURE: Observation of the abnormal tumor cells in the collected specimens by bright field microscope. RESULTS: The technique of impression cytologic analysis allowed collection and identification of abnormal tumor cells with characteristic cytoplasmic vacuoles. CONCLUSIONS: Conjunctival impression cytologic analysis successfully detected the ocular surface sebaceous carcinoma cells from the eyelid. However, full-thickness biopsies are necessary to confirm the diagnosis. Judicious use of impression cytologic analysis may facilitate the detection and diagnosis of this invasive tumor.

Adenocarcinoma, Sebaceous↗
PubMed2003

Management of the late ocular sequelae of Stevens-Johnson syndrome.

The acute conjunctivitis seen initially in Stevens-Johnson syndrome is followed by a cicatricial phase, which often leads to severe ocular surface disease and visual morbidity. Manifestations include keratinization of the conjunctiva, lid margins, and lacrimal and meibomian ducts, resulting in an unstable tear film and mechanical trauma to the conjunctiva and cornea with blinking. Limbal stem cell deficiency is the most vision-threatening sequela of Stevens-Johnson syndrome, as it causes corneal neovascularization, chronic corneal inflammation, and an irregular corneal epithelium. Management of late sequelae often requires a multipronged approach, including strategies for ocular surface protection, ocular surface support, and ocular surface reconstruction. In this review, established therapies, as well as new experimental therapies, are discussed.

Journal Article↗