Allelic resolution NGS HLA typing of Class I and Class II loci and haplotypes in Cape Town, South Africa.

The development of next-generation sequencing (NGS) methods for HLA genotyping has already had an impact on the scope and precision of HLA research. In this study, allelic resolution HLA typing was obtained for 402 individuals from Cape Town, South Africa. The data were produced by high-throughput NGS sequencing as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with the University of Cape Town and Stanford University. All samples were genotyped for 11 HLA loci, namely HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, and -DRB5. NGS HLA typing of samples from Cape Town inhabitants revealed a unique cohort, including unusual haplotypes, and 22 novel alleles not previously reported in the IPD-IMGT/HLA Database. Eight novel alleles were in Class I loci and 14 were in Class II. There were 62 different alleles of HLA-A, 72 of HLA-B, and 47 of HLA-C. Alleles A∗23:17, A∗43:01, A∗29:11, A∗68:27:01, A∗01:23, B∗14:01:01, B∗15:10:01, B∗39:10:01, B∗45:07, B∗82:02:01 and C∗08:04:01 were notably more frequent in Cape Town compared to other populations reported in the literature. Class II loci had 21 different alleles of DPA1, 46 of DPB1, 27 of DQA1, 26 of DQB1, 41 of DRB1, 5 of DRB3, 4 of DRB4 and 6 of DRB5. The Cape Town cohort exhibited high degrees of HLA diversity and relatively high heterozygosity at most loci. Genetic distances between Cape Town and five other sub-Saharan African populations were also calculated and compared to European Americans.

CD147 required for corneal endothelial lactate transport.

PURPOSE: CD147/basigin is a chaperone for lactate:H(+) cotransporters (monocarboxylate transporters) MCT1 and MCT4. We tested the hypothesis that MCT1 and -4 in corneal endothelium contribute to lactate efflux from stroma to anterior chamber and that silencing CD147 expression would cause corneal edema. METHODS: CD147 was silenced via small interfering ribonucleic acid (siRNA) transfection of rabbit corneas ex vivo and anterior chamber lenti-small hairpin RNA (shRNA) pseudovirus in vivo. CD147 and MCT expression was examined by Western blot, RT-PCR, and immunofluorescence. Functional effects were examined by measuring lactate-induced cell acidification, corneal lactate efflux, [lactate], central cornea thickness (CCT), and Azopt (a carbonic anhydrase inhibitor) sensitivity. RESULTS: In ex vivo corneas, 100 nM CD147 siRNA reduced CD147, MCT1, and MCT4 expression by 85%, 79%, and 73%, respectively, while MCT2 expression was unaffected. CD147 siRNA decreased lactate efflux from 3.9 ± 0.81 to 1.5 ± 0.37 nmol/min, increased corneal [lactate] from 19.28 ± 7.15 to 56.73 ± 8.97 nmol/mg, acidified endothelial cells (pHi = 6.83 ± 0.07 vs. 7.19 ± 0.09 in control), and slowed basolateral lactate-induced acidification from 0.0034 ± 0.0005 to 0.0012 ± 0.0005 pH/s, whereas apical acidification was unchanged. In vivo, CD147 shRNA increased CCT by 28.1 ± 0.9 µm at 28 days; Azopt increased CCT to 24.4 ± 3.12 vs. 12.0 ± 0.48 µm in control, and corneal [lactate] was 47.63 ± 6.29 nmol/mg in shCD147 corneas and 17.82 ± 4.93 nmol/mg in paired controls. CONCLUSIONS: CD147 is required for the expression of MCT1 and MCT4 in the corneal endothelium. Silencing CD147 slows lactate efflux, resulting in stromal lactate accumulation and corneal edema, consistent with lactate efflux as a significant component of the corneal endothelial pump.

Lactate-H⁺ transport is a significant component of the in vivo corneal endothelial pump.

PURPOSE: To confirm the expression of monocarboxylate transporters (MCT) 1, 2, and 4 in rabbit CE and to test the hypothesis that cellular buffering contributed by HCO3⁻, NBCe1, and carbonic anhydrase (CA) activity facilitates lactate-H⁺ efflux thereby controlling corneal hydration in vivo. METHODS: MCT1-4 expression of rabbit endothelium was examined by Western blotting and immunofluorescence staining. Lactate-induced acidification (LIA) was measured in perfused CE in the presence and absence of HCO3⁻ and acetazolamide (ACTZ) using tissue treated with siRNA specific to MCT1, 2, and 4. Corneal thickness and lactate concentration were measured in New Zealand White rabbits treated with the topical CA inhibitor Azopt, and from eyes that were injected intracamerally with ouabain, disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), and shRNA specific to the 1Na⁺:2HCO3⁻ cotransporter NBCe1. RESULTS: MCT1 and MCT4 are localized to the lateral membrane, while MCT2 is apical. Cell pH measurements showed LIA in response to 40 mM lactate in bicarbonate free (BF) Ringer's that was inhibited by niflumic acid and by MCT siRNA knockdown, and significantly reduced in the presence of HCO3⁻. Lactate-dependent proton flux in vitro was not significantly greater in the presence of HCO3⁻ or reduced by ACTZ. However, when active transport, NBCe1, or CA activity was disrupted in vivo, corneal edema ensued and was associated with significant corneal lactate accumulation. CONCLUSIONS: MCT1, 2, and 4 are expressed in rabbit CE on both the apical and basolateral surfaces and function to transport lactate-H⁺. Lactate-H⁺ flux is facilitated by active transport, HCO3⁻ transport and CA activity, disruption of which causes corneal edema in vivo and indicates that facilitation of lactate efflux is a component of the endothelial pump.

Telomerase immortalization of human corneal endothelial cells yields functional hexagonal monolayers.

Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells whose main function is to maintain corneal clarity by regulating corneal hydration. HCEnCs are derived from neural crest and are arrested in the post-mitotic state. Thus cell loss due to aging or corneal endothelial disorders leads to corneal edema and blindness-the leading indication for corneal transplantation. Here we show the existence of morphologically distinct subpopulations of HCEnCs that are interspersed among primary cells and exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. Colonies of these uniform and hexagonal HCEnCs (HCEnC-21) were selectively isolated and demonstrated high proliferative potential that was dependent on endogenous upregulation of telomerase and cyclin D/CDK4. Further transduction of HCEnC-21 with telomerase yielded a highly proliferative corneal endothelial cell line (HCEnT-21T) that was devoid of oncogenic transformation and retained critical corneal endothelial cell characteristics and functionality. This study will significantly impact the fields of corneal cell biology and regenerative medicine.

Bicarbonate, NBCe1, NHE, and carbonic anhydrase activity enhance lactate-H+ transport in bovine corneal endothelium.

PURPOSE: To identify and localize the monocarboxylate transporters (MCTs) expressed in bovine corneal endothelial cells (BCEC) and to test the hypothesis that buffering contributed by HCO(3)(-), sodium bicarbonate cotransporter (NBCe1), sodium hydrogen exchanger (NHE), and carbonic anhydrase (CA) activity facilitates lactate flux. METHODS: MCT1-4 expression was screened by RT-PCR, Western blot analysis, and immunofluorescence. Endogenous lactate efflux and/or pH(i) were measured in BCEC in HCO(3)(-)-free or HCO(3)(-)-rich Ringer, with and without niflumic acid (MCT inhibitor), acetazolamide (ACTZ, a CA inhibitor), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) (Na(+)/H(+) exchange blocker), disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; anion transport inhibitor), or with NBCe1-specific small interfering (si) RNA-treated cells. RESULTS: MCT1, 2, and 4 are expressed in BCEC. MCT1 was localized to the lateral membrane, MCT2 was lateral and apical, while MCT4 was apical. pH(i) measurements showed significant lactate-induced cell acidification (LIA) in response to 20-second pulses of lactate. Incubation with niflumic acid significantly reduced the rate of pHi change (dpH(i)/dt) and lactate-induced cell acidification. EIPA inhibited alkalinization after lactate removal. Lactate-dependent proton flux was significantly greater in the presence of HCO(3)(-) but was reduced by ACTZ. Efflux of endogenously produced lactate was significantly faster in the presence of HCO(3)(-), was greater on the apical surface, was reduced on the apical side by ACTZ, as well as on the apical and basolateral side by NBCe1-specific siRNA, DIDS, or EIPA. CONCLUSIONS: MCT1, 2, and 4 are expressed in BCEC on both the apical and basolateral membrane (BL) surfaces consistent with niflumic acid-sensitive lactate-H(+) transport. Lactate dependent proton flux can activate Na(+)/H(+) exchange and be facilitated by maximizing intracellular buffering capacity through the presence of HCO(3)(-), HCO(3)(-) transport, NHE and CA activity.

Overnight orthokeratology experience with XO material.

PURPOSE: To evaluate long-term safety and effectiveness of overnight orthokeratology in a large sample of myopes by using three different lens designs and the highly gas-permeable Boston XO material (Bausch & Lomb, Rochester, NY). METHODS: A total of three hundred forty-two subjects, 99 of whom were juveniles, were enrolled in the investigation at 26 clinical centers in the United States and Canada. Three different lens designs in Boston XO material were used. Subjects were limited to a measured visual need for correction of myopia less than or equal to 4.00 diopters and astigmatism less than or equal to 1.50 diopters with best-corrected Snellen visual acuity of 20/40 or better. Refractive error and unaided visual acuity were used to determine the effectiveness of the procedure at short-term (1 month) and long-term (12 months). The safety of the procedure was determined by the symptoms reported, and slitlamp findings suggested physiologic rejection of the procedure. RESULTS: One hundred thirty-three adults and 68 juveniles completed the 12-month study. More than 60% of the subjects reached 20/20 vision or better at the end of 1 month of overnight lens wear, and this number remained stable during the next 11 months. A reduction in myopia of 70% and of more than 90% was measured at the 1- and 12-month visits, respectively. Safety data indicate a decrease in symptoms and in general a decrease in positive slitlamp findings with time. CONCLUSIONS: The procedure with all three lenses in Boston XO material is effective. However, the safe use of orthokeratology lenses is predicated on the emphasis of clear patient direction on how to wear and take care of the lenses followed by regular examinations by an eye care practitioner.

Overnight orthokeratology: refractive and corneal recovery after discontinuation of reverse-geometry lenses.

PURPOSE: To determine the refractive and corneal topographic recovery after the use of reverse-geometry contact lenses for overnight orthokeratology. METHODS: Both eyes of 15 subjects were fitted with reverse-geometry contact lenses that were worn by the subjects for 1 month. Uncorrected visual acuity, refractive correction (sphere and spherical equivalent), corneal curvature, and corneal thickness were measured during this time and for 2 weeks after discontinuation of lens wear. RESULTS: Ten subjects completed the investigation. Uncorrected visual acuity, refractive correction, and corneal curvature had changed significantly (P = 0.01) after 1 month of lens wear. By the end of 1 month, central corneal thickness was significantly thinner than the baseline value (P = 0.01), but it recovered fully after one night of no lens wear. Recovery of corneal curvature was complete 1 week after lens wear was discontinued. Refractive correction and binocular uncorrected visual acuity recovered fully after 2 weeks. Monocular uncorrected visual acuity remained significantly (P = 0.01) different from baseline acuity 2 weeks after lens discontinuation. CONCLUSIONS: Full effect of overnight orthokeratology in low myopes is achieved within 1 week of initiating use of reverse-geometry lenses. Recovery after short-term use of reverse-geometry lenses is rapid for corneal thickness and corneal curvature. Refractive correction and binocular uncorrected visual acuity recovered fully after 2 weeks. Monocular uncorrected visual acuity was the slowest to recover and had not achieved full recovery after 2 weeks.

Overnight orthokeratology: visual and corneal changes.

PURPOSE: To achieve an optimal fit with reverse geometry Contex OK lenses and to determine a time course for and the stability of visual and corneal changes in achieving maximal refractive, corneal curvature, and corneal thickness changes after overnight wear of OK B and D series lenses. METHODS: This investigation was conducted under an Food and Drug Administration IDE G000059. Both eyes of 10 subjects were fitted with the lenses, and uncorrected visual acuity, refractive correction, contrast sensitivity, corneal curvature, and corneal thickness were measured at baseline and at 1 day, 1week, 1 month, and 3 months after lenses were worn. Except for baseline, data were collected at four different times during the day, immediately following lens removal and 4, 8, and 12 hours after lens removal. RESULTS: The results from eight subjects showed that uncorrected visual acuity, refractive correction, contrast sensitivity, and corneal curvature all changed significantly (P=0.01) overnight. By the end of 1 week, all corneal and visual changes had reached a maximal level and remained fairly stable during the day. These changes were sustained at 3 months. The epithelial thickness data from four subjects showed that the corneal epithelial thickness was reduced by approximately 19 microm after 3 months of lens wear. CONCLUSIONS: Successful fitting of OK B and D series lenses requires a thorough understanding of the lens-cornea relationship. Full effect of overnight orthokeratology is achieved by the end of 1 week. The visual and corneal changes remain stable for all waking hours of the day and allow patients to enjoy excellent device-free vision (20/20).