Jingsheng Tuo

The involvement of sequence variation and expression of CX3CR1 in the pathogenesis of age-related macular degeneration

This study examined the association between the sequence variation/expression of CX3CR1, a chemokine receptor, and age‐related macular degeneration (AMD). Peripheral blood from 85 AMD patients and 105 subjects without AMD (controls), as well as ocular tissue from 40 pathological sections with AMD and two normal eye sections, were screened for V249I and T280M, two single nucleotide polymorphisms (SNPs) in CX3CR1. An increased prevalence, with the highest odds ratio of 3.57, of the I249 and M280 carriers was found among the AMD cases as compared with the controls. When comparing CX3CR1 expression in the archived eye sections, CX3CR1 transcripts were not detectable in the maculae of AMD eyes bearing T/M280; however, transcripts were detected in the maculae of normal eyes bearing T/T280 or T/M280 as well as in the AMD maculae bearing T/T280. Furthermore, lower CX3CR1 protein expression was observed in the maculae of AMD eyes bearing T/M280 compared with the controls bearing T/T280. The I249 and M280 alleles result in a lowered number of receptor binding sites and a decreased ligand affinity. Our data suggest that a decrease, caused by sequence variation and/or lower CX3CR1 expression, in CX3CR1‐induced cellular activities could contribute to AMD development.

Polymerase Chain Reaction in the Diagnosis of Uveitis

Polymerase chain reaction (PCR) is a technique involving enzymatic amplification of nucleic acid sequences in repeated cycles of denaturation, oligonucleotide annealing, and DNA polymerase extension.1,2 The PCR uses in vitro enzymatic synthesis to amplify specific DNA sequences within a few hours. Since its inception in 1985, PCR has revolutionized research in the biologic sciences and medicine and has influenced criminology and law.3 The inventor of PCR, Kary Mullis, was awarded the Nobel Prize in Chemistry in 1993 in recognition of the extraordinary impact of PCR technology on scientific research.4,5 Polymerase chain reaction consists of repetitive cycles of specific DNA synthesis, defined by short stretches of preselected DNA. With each cycle, there is a doubling of the final, desired DNA product such that a million-fold amplification is possible.6 This powerful technique has numerous applications in diagnostic pathology, especially in the fields of microbiology, genetics, and oncology. Polymerase chain reaction has been used to diagnose uveitis, including viral uveitis, mycobacterial intraocular infections, infectious endophthalmitis, and protozoa eye diseases.7 However, the extremely high sensitivity of PCR can produce false-positive results, whereas its high specificity may produce false-negative results. These pitfalls can be minimized by techniques such as the use of both positive and negative controls, real-time PCR, and the performance of tests in an experienced laboratory. In any case, to ensure an accurate diagnosis, one must consider clinical data in the interpretation of a PCR result. We present diagnostic applications and examples of utilization of PCR in infectious and noninfectious uveitis, as well as in masquerade syndromes and other common ocular diseases with inflammatory components.

Macrophage polarization in the maculae of age-related macular degeneration: A pilot study

Macrophages can be polarized to exhibit either pro‐inflammatory M1 or pro‐angiogenic M2 phenotypes, but have high phenotypic plasticity. This pilot study investigated macrophage polarization in the macular retina and choroid of age‐related macular degeneration (AMD) and non‐AMD subjects, as well as in AMD choroidal neovascular membranes (CNVM). All specimens were evaluated for routine histopathology. Quantitative real‐time polymerase chain reaction for representative M1 (CXCL11) and M2 (CCL22) transcripts were performed on macular choroidal trephines (MCT) of 19 AMD and nine non‐AMD eye bank eyes, on the microdissected macular retinal cells from the archived slides of five geographic atrophic AMD, five exudative/neovascular AMD, and eight normal autopsied eyes, and on microdissected inflammatory cells from two surgically removed CNVM that did not respond to anti‐vascular endothelial growth factor (VEGF) therapy. High M2‐chemokine transcript and a low ratio of M1 to M2 chemokine transcript were found in aging non‐AMD MCT. Advanced AMD maculae had a higher M1 to M2 chemokine transcript ratio compared to normal autopsied eyes. Macrophages in the two CNVM of patients unresponsive to anti‐VEGF therapy were polarized toward either M1 or M2 phenotypes. The number of M2 macrophages was increased compared to M1 macrophages in normal aging eyes. A pathological shift of macrophage polarization may play a potential role in AMD pathogenesis.

Complement component C5a Promotes Expression of IL-22 and IL-17 from Human T cells and its Implication in Age-related Macular Degeneration

BackgroundAge related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly populations worldwide. Inflammation, among many factors, has been suggested to play an important role in AMD pathogenesis. Recent studies have demonstrated a strong genetic association between AMD and complement factor H (CFH), the down-regulatory factor of complement activation. Elevated levels of complement activating molecules including complement component 5a (C5a) have been found in the serum of AMD patients. Our aim is to study whether C5a can impact human T cells and its implication in AMD.MethodsHuman peripheral blood mononuclear cells (PBMCs) were isolated from the blood of exudative form of AMD patients using a Ficoll gradient centrifugation protocol. Intracellular staining and enzyme-linked immunosorbent assays were used to measure protein expression. Apoptotic cells were detected by staining of cells with the annexin-V and TUNEL technology and analyzed by a FACS Caliber flow cytometer. SNP genotyping was analyzed by TaqMan genotyping assay using the Real-time PCR system 7500.ResultsWe show that C5a promotes interleukin (IL)-22 and IL-17 expression by human CD4+ T cells. This effect is dependent on B7, IL-1β and IL-6 expression from monocytes. We have also found that C5a could protect human CD4+ cells from undergoing apoptosis. Importantly, consistent with a role of C5a in promoting IL-22 and IL-17 expression, significant elevation in IL-22 and IL-17 levels was found in AMD patients as compared to non-AMD controls.ConclusionsOur results support the notion that C5a may be one of the factors contributing to the elevated serum IL-22 and IL-17 levels in AMD patients. The possible involvement of IL-22 and IL-17 in the inflammation that contributes to AMD may herald a new approach to treat AMD.

Hypomethylation of the IL17RC promoter associates with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Although recent studies have demonstrated strong genetic associations between AMD and SNPs in a number of genes, other modes of regulation are also likely to play a role in the etiology of this disease. We identified a significantly decreased level of methylation on the IL17RC promoter in AMD patients. Furthermore, we showed that hypomethylation of the IL17RC promoter in AMD patients led to an elevated expression of its protein and messenger RNA in peripheral blood as well as in the affected retina and choroid, suggesting that the DNA methylation pattern and expression of IL17RC may potentially serve as a biomarker for the diagnosis of AMD and likely plays a role in disease pathogenesis.

Primary fibroblasts of Cockayne syndrome patients are defective in cellular repair of 8-hydroxyguanine and 8-hydroxyadenine resulting from oxidative stress

Cockayne syndrome (CS) is a genetic human disease with clinical symptoms that include neurodegeneration and premature aging. The disease is caused by the disruption of CSA, CSB, or some types of xeroderma pigmentosum genes. It is known that the CSB protein coded by the CS group B gene plays a role in the repair of 8‐hydroxyguanine (8‐OH‐Gua) in transcription‐coupled and non‐strand discriminating modes. Recently we reported a defect of CSB mutant cells in the repair of another oxidatively modified lesion 8‐hydroxyadenine (8‐OH‐Ade). We show here that primary fibroblasts from CS patients lack the ability to efficiently repair these particular types of oxidatively induced DNA damages. Primary fibroblasts of 11 CS patients and 6 control individuals were exposed to 2 Gy of ionizing radiation to induce oxidative DNA damage and allowed to repair the damage. DNA from cells was analyzed using liquid chromatography/isotope dilution mass spectrometry to measure the biologically important lesions 8‐OH‐Gua and 8‐OH‐Ade. After irradiation, no significant change in background levels of 8‐OH‐Gua and 8‐OH‐Ade was observed in control human cells, indicating their complete cellular repair. In contrast, cells from CS patients accumulated significant amounts of these lesions, providing evidence for a lack of DNA repair. This was supported by the observation that incision of 8‐OH‐Gua‐ or 8‐OH‐Ade‐containing oligodeoxynucleotides by whole cell extracts of fibroblasts from CS patients was deficient compared to control individuals. This study suggests that the cells from CS patients accumulate oxidatively induced specific DNA base lesions, especially after oxidative stress. A deficiency in cellular repair of oxidative DNA damage might contribute to developmental defects in CS patients.

Functional crosstalk between hOgg1 and the helicase domain of Cockayne syndrome group B protein.

We have previously reported that the Cockayne syndrome group B gene product (CSB) contributes to base excision repair (BER) of 8-hydroxyguanine (8-OH-Gua) and the importance of motifs V and VI of the putative helicase domains of CSB in BER of 8-OH-Gua. To further elucidate the function of CSB in BER, we investigated its role in the pathway involving human 8-OH-Gua glycosylase/apurinic lyase (hOgg1). Depletion of CSB protein with anti-CSB antibody reduced the 8-OH-Gua incision rate of wild type cell extracts but not of CSB null and motif VI mutant cell extracts, suggesting a direct contribution of CSB to the catalytic process of 8-OH-Gua incision and the importance of its motif VI in this pathway. Introduction of recombinant purified CSB partially complemented the depletion of CSB as shown by the recovery of the incision activity. This complementation could not fully recover the deficiency of the incision activity in WCE from CS-B null and mutant cell lines, suggesting that some additional factor(s) are necessary for the full activity. Electrophoretic mobility shift assays (EMSAs) showed a defect in binding of CSB null and motif VI mutant cell extracts to 8-OH-Gua-containing oligonucleotides. We detected less hOgg1 transcript and protein in the cell extracts from CS-B null and mutant cells, suggesting hOgg1 may be the missing component. Pull-down of hOgg1 by histidine-tagged CSB and co-localization of those two proteins after gamma-radiation indicated their co-existence in vivo, particularly under cellular stress. However, we did not detect any functional and physical interaction between purified CSB and hOgg1 by incision, gel shift and yeast two-hybrid assays, suggesting that even though hOgg1 and CSB might be in a common protein complex, they may not interact directly. We conclude that CSB functions in the catalysis of 8-OH-Gua BER and in the maintenance of efficient hOgg1 expression, and that motif VI of the putative helicase domain of CSB is crucial in these functions.

Ccl2/Cx3cr1-deficient mice: an animal model for age-related macular degeneration.

Background/Aims: Senescent Ccl2–/– mice develop cardinal features of human age-related macular degeneration (AMD). Loss-of-function single-nucleotide polymorphisms within CX3CR1 are associated with AMD. Methods: We generated Ccl2–/–/Cx3cr1–/– [double-knockout (DKO)] mice and evaluated the eyes using fundoscopy routine histology, immunochemistry, biochemistry and proteomics. Results: At 6 weeks old, all DKO mice developed AMD-like retinal lesions such as abnormal retinal pigment epithelium cells, drusen, photoreceptor atrophy and choroidal neovascularization, which progressed with age and reversed with high omega-3 long-chain polyunsaturated fatty acid diet. N-retinylidene-N-retinylethanolamine (A2E), a major lipofuscin fluorophore, illustrated by an emission peak at ∼600 nm, was significantly higher in DKO retinal pigment epithelium. Decreased ERp29 was found in the retina of DKO mice. Conclusion: A broad spectrum of AMD pathologies with early onset and high penetrance in these mice implicate certain chemokines, A2E and endoplasmic reticulum proteins in AMD pathogenesis.

Genetic factors of age-related macular degeneration

Age-related macular degeneration (AMD) is a leading cause of blindness in the United States and developed countries. Although the etiology and pathogenesis of AMD remain unknown, a complex interaction of genetic and environmental factors is thought to exist. The incidence and progression of all of the features of AMD are known to increase significantly with age. The tendency for familial aggregation and the findings of gene variation association studies implicate a significant genetic component in the development of AMD. This review summarizes in detail the AMD-related genes identified by studies on genetically engineered and spontaneously gene-mutated (naturally mutated) animals, AMD chromosomal loci identified by linkage studies, AMD-related genes identified through studies of monogenic degenerative retinal diseases, and AMD-related gene variation identified by association studies.

A High Omega-3 Fatty Acid Diet Reduces Retinal Lesions in a Murine Model of Macular Degeneration

Age-related macular degeneration (AMD) is one of the leading cause of blindness among the elderly; however, current therapy options are limited. Epidemiological studies have shown that a diet that is high in omega-3 polyunsaturated (n-3) fatty acids can slow disease progression in patients with advanced AMD. In this study, we evaluated the effect of such a diet on the retinas of Ccl2(-/-)/Cx3cr1(-/-) mice, a model that develops AMD-like retinal lesions that include focal deep retinal lesions, abnormal retinal pigment epithelium, photoreceptor degeneration, and A2E accumulation. Ccl2(-/-)/Cx3cr1(-/-) mice that ingested a high n-3 fatty acid diet showed a slower progression of retinal lesions compared with the low n-3 fatty acids group. Some mice that were given high levels of n-3 fatty acids had lesion reversion. We found a shunted arachidonic acid metabolism that resulted in decreased pro-inflammatory derivatives (prostaglandin E(2) and leukotriene B(4)) and an increased anti-inflammatory derivative (prostaglandin D(2)). We also measured lower ocular TNF-alpha and IL-6 transcript levels in the mice fed a diet of high n-3 fatty acids. Our findings in these mice are in line with human studies of AMD risk reduction by long-chain n-3 fatty acids. This murine model provides a useful tool to evaluate therapies that might delay the development of AMD.