Protein overproduction alters exosome secretion in Chinese hamster ovary cells.

Despite the abundance of available cell lines, nearly 70% of all recombinant therapeutic proteins today are produced in Chinese hamster ovary (CHO) cells. The impact of protein overproduction on the secretion of exosomes by CHO cells has been investigated here. Increased secretion of extracellular vesicles (EVs) by protein overexpressing CHO cells was demonstrated with protein content assay, nanoparticle tracking analysis, and capillary electrophoresis. Our results revealed that a protein overproduction might induce EVs secretion, which might be accompanied by the sequestration and loading of overexpressed proteins into the exosomes. These findings are of vital importance for the manufacturing of therapeutics in CHO expression systems due to the risk of product loss during downstream processing of culture medium as well as the application of exosomes as nanocarriers of therapeutic proteins. The study indicates also the importance of culturing process control.

PARS PLANA VITRECTOMY IN ADVANCED CASES OF VON HIPPEL-LINDAU EYE DISEASE.

PURPOSE: To investigate spectrum of patients with Von Hippel-Lindau disease (VHL) that required pars plana vitrectomy and evaluate anatomical and functional outcomes of surgery. METHODS: Twenty-three patients who underwent surgery for advanced VHL eye disease were assessed by genetic tests, diagnostic tests for systemic lesions, and clinical eye examination. The vitrectomized eyes were divided into two groups: with or without retinotomy (group R vs. NR). Functional and anatomical outcome was analyzed and compared between the groups. RESULTS: All patients had central nervous system hemangioblastomas and 57% had other systemic tumors. Point germline mutations, large partial deletions, and complete vhl gene deletions were found in 64%, 27%, and 9% of patients, accordingly. Destruction of hemangioblastomas by retinotomy, laser, or cryotherapy and anatomical attachment of the retina were achieved in all eyes. Preoperative mean distance best-corrected visual acuity was logarithm of the minimum angle of resolution 2.66 (20/9,140) in group R and 1.76 (20/1,150) in group NR (P < 0.05). At 6 months postoperatively, distance best-corrected visual acuity improved in 20 eyes (83%). After over 24 months postoperatively, distance best-corrected visual acuity remained better than preoperatively in 36% in the R group and in 70% in the NR group of eyes. During 24 months postoperatively in 17 eyes, new retinal capillary hemangiomas developed. The mean number of new retinal capillary hemangiomas per eye was higher in group R than in group NR (3.14 vs. 0.70; P < 0.01). In group R, number of new retinal capillary hemangioblastoma was higher in retinal segments where retinotomy was performed (n = 29) than in other areas (n = 13) (P < 0.01). CONCLUSION: Advanced VHL eye disease correlates with occurrence of central nervous system and systemic lesions. Spectrum of vhl gene mutation in the patients corresponds to that of the general VHL population. Pars plana vitrectomy in advanced VHL eye disease can improve or preserve visual function, but postoperative progression of ocular VHL disease can be accelerated in cases where retinotomy is performed.

Large deletion causing von Hippel-Lindau disease and hereditary breast cancer syndrome.

Patients with intragenic mutations of the VHL gene have a typical disease presentation. However in cases of large VHL gene deletions which involve other genes in the proximity of the VHL gene a presentation of the disease can be different. To investigate whether large VHL deletions that remove the FANCD2 gene have an effect on the disease phenotype, we studied a family with a 50 kb large deletion encompassing these two genes. Four patients in this family were affected by VHL-related lesions. However one carrier of the deletion also had bilateral ductal breast cancer at age 46 and 49. Both tumors were of ~2 cm in diameter. On one side lymph nodes were affected. One tumor was ER- and PR-negative (HER2 s unknown) and the second was ER- and PR-positive, and HER2-negative. Our study suggests that a deletion of FANCD2 gene, an important gene in the DNA repair pathway, may be associated with an increased risk of breast cancer, but further studies are needed in this regard.

Pattern electroretinogram (PERG) in the early diagnosis of normal-tension preperimetric glaucoma: a case report.

INTRODUCTION: The purpose of this case is to present the use of pattern electroretinogram (PERG) in the early diagnosis of normal-tension preperimetric glaucoma in 56 years old woman. METHODS AND RESULTS: At baseline the results were as follows: distance-corrected visual acuity in the right eye (RE) and left eye (LE) 1.0 and 0.7, respectively (Snellen table), normal anterior segments in both eyes, normal fundus in the RE and abnormal cup to disc ratio (0.6) in the LE. Intraocular pressure (IOP) was within normal limits in both eyes: RE-14 mmHg, LE-18 mmHg (Goldmann tonometer). Results of standard automated perimetry (SAP), short wavelength automated perimetry (SWAP) and nerve fiber analyzer (GDx) were normal in both eyes. PERG result was normal in the RE but in the LE reduced amplitudes of P50 and N95 waves were observed. After topical treatment (Xalacom to the LE), a reduction of IOP to 13 mmHg was achieved and was accompanied by amplitudes increase of PERG waves. After discontinuation of the therapy, IOP increased to 18 mmHg and P50 and N95 amplitudes decreased to the values before treatment, suggesting the influence of IOP lowering therapy on electrical function of retinal ganglion cells. After 4 years from the baseline, static perimetry results were still normal, but abnormalities in retinal nerve fiber layer thickness were detected in GDx. CONCLUSIONS: PERG was a useful test not only for the early diagnosis of normal-tension preperimetric glaucoma, but also in evaluating the effectiveness of antiglaucomatous treatment.

[Combined photodynamic therapy and intravitreal injection of triamcinolone acetonide in patients with wet form of AMD. Introductory report]. / Zastosowanie terapii laczonej PDT z iniekcjami doszklistkowymi octanu triamcinolonu u pacjentów z wysiekowa postacia AMD. Doniesienie wstepne.

PURPOSE: To evaluate the efficacy of combined PDT and 4 mg intravitreal triamcinolone acetonide injection, performed 48-72 hours after PDT, in patients with wet form of AMD. MATERIAL AND METHODS: Nonrandomised, interventional case series, 13 eyes of 13 patients with subfoveal CNV due to AMD that did not respond to PDT monotherapy - 7 females, 6 males - at the age of 65-85 (average age 76.6 +/- 6.7 years); standard PDT was performed in all patients followed by a 4 mg intravitreal injection of triamcinolone acetonide given 48-72 hours after PDT. Follow up visits were scheduled 1 and 7 days after the injection and then every 3 months afterwards and included: BVCA (Snellen chart), IOP measurements, FA, OCT, slit lamp and eye fundus examination. Lesions with active CNV leakage in FA were retreated every 3 months. RESULTS: Average observation time was 10.8 +/- 3.5 months. Baseline visual acuity before PDT monotherapy was applied (Vo) was 0.17 +/- 0.12 (0.06-0.5), and after the therapy decreased to (V1) 0.14 +/- 0.13 (0.05-0.2). After combined PDT and Tc treatment BVCA increased to (V2) 0.21 +/- 0.13 (0.06-0.5), p<0,03. 76,9% of patients gained or maintained visual acuity after combined therapy in the observation time. In 70% of eyes no signs of active CNV was observed in AF and OCT after 1 session of combined PDT and Tc treatment. Only 4 patients required 1 repeated treatment session. CONCLUSIONS: 1. Combination of PDT and IVTA may be effective in patients with wet AMD with no response to PDT alone and significantly reduces the repeated treatment rate. 2. Intravitreal Tc injection performed 48-72 hours after PDT may improve the final functional effects in treated eyes as compared with PDT monotherapy. Our results need further investigation.

[Eye's risk factors in age-related macular degeneration (AMD). Part I]. / Oczne czynniki ryzyka zwyrodnienia plamki zwiazanego z wiekiem (AMD). Czesc I.

PURPOSE: To evaluate the relationship between cataract, past cataract surgery and age-related macular degeneration (AMD). MATERIAL AND METHODS: 281 eyes with AMD diagnosed with fundus fluorescein angiography were included to the study and divided into two groups: with dry (208) and exudative (73) type of AMD. 149 eyes without AMD were controls. We performed ophthalmological examination and questionnaire. We collected data regarding past cataract surgery, exposition to the sun, using of sunglasses and other. RESULTS: Eyes with AMD were more likely to have cataract and nuclear sclerosis than controls. History of cataract surgery was significantly more frequent in group with exudative AMD compared to controls. Cataract (OR = 1.93), nuclear sclerosis (OR = 3.84) and past cataract surgery (OR = 2.32) were associated with increased risk of age-related macular degeneration in multivariate analyses adjusted for age. CONCLUSIONS: The concomitance of cataract (especially nuclear sclerosis) and age-related macular degeneration may support the theory of common pathogenesis of both pathologies. There is increased risk of exudative type of AMD in eyes after cataract surgery.

[Eye's risk factors in age-related macular degeneration (AMD). Part II]. / Oczne czynniki ryzyka zwyrodnienia plamki zwiazanego z wiekiem (AMD). Czesc II.

PURPOSE: To evaluate the relationship between different ocular factors and age-related macular degeneration (AMD). MATERIAL AND METHODS: 281 eyes with AMD diagnosed with fundus fluorescein angiography were included to the study and divided into two groups: with dry and exudative type of AMD. 149 eyes without AMD consisted of control group. We performed ophthalmic examination and questionnaire. We collected data concerning exposition to the sun, using of sunglasses and other factors. RESULTS: Arterial sclerosis in the retina was associated with twice 2-fold increased risk (OR = 1.96) of exudative AMD. There was not association of iris color, using of sunglasses, work conditions and age-related macular degeneration. CONCLUSIONS: The coexistance of arterial sclerosis in the retina suggests that vascular disorders take part in pathogenesis of exudative type of AMD. Factors like iris color, using of sunglasses and work are not associated with the risk of AMD.

[Use of statins as a form of protection against age-related macular degeneration (AMD)]. / Leczenie statynami jako forma ochrony przed zwyrodnieniem plamki zwiazanym z wiekiem (AMD).

PURPOSE: To evauate the association between use of lipid-lowering medications and occurrence of age-related macular degeneration (AMD). MATERIAL AND METHODS: 91 patients with dry and 55 with exudative type AMD diagnosed with fundus fluorescein angiography were included to the study. Control group for the study Consisted of 68 participants without AMD. Data regarding use of cholesterol-lowering medications were collected with details. RESULTS: In control group we noted significantly higher percentage of statin users compared to patients with AMD. CONCLUSIONS: Use of statins may be a protective factor against age-related macular degeneration.

A 43-year-old man with reduced visual acuity and normal fundus: occult macular dystrophy--case report.

PURPOSE: Occult macular dystrophy (OMD) is an unusual, inherited macular dystrophy characterized by a slowly progressive decline of visual acuity with normal fundus and fluorescein angiography (FA). The authors present a 43-year-old man who was diagnosed as having OMD because of the results of electrophysiological, psychophysical, optical coherence tomography (OCT) tests. METHODS: Routine ophthalmological evaluation, FA, visual field tests, electroretinographic examinations (EOG, ERG, PERG and mfERG recordings according to ISCEV standards) and foveal thickness measurements (OCT) were performed. RESULTS: Funduscopic examinations, FA, full field ERG as well as PERG results were all normal. In both eyes, the abnormalities were observed in static perimetry (relative central scotomas), mfERG (significant reduction of P1 amplitude in the central retinas) and OCT (significantly thinner foveal thickness). CONCLUSIONS: A new case with OMD is added to preceding reports. The mfERG and OCT tests are important in detection of OMD patients. It can help in differential diagnosis of amblyopia, optic nerve diseases and non-organic visual disorders.

[The potential role of oxidative stress in the pathogenesis of the age-related macular degeneration (AMD)]. / Potencjalny udzial stresu oksydacyjnego w patogenezie zwyrodnienia plamki zwiazanego z wiekiem (AMD).

Age-related macular degeneration (AMD) is one of the most important causes of blindness among the elderly. Although the disease presents a serious social problem, its pathogenesis is still unclear. AMD involves the posterior pole of the retina, the place responsible for acute vision. Retinal factors (intensive oxygen metabolism, continual exposure to light, a high concentration of polyunsaturated fatty acids, the presence of photosensitizers) increase the production of reactive oxygen species. Oxidative stress is aggravated by the presence of lipofuscin. The pigment accumulates with age, especially in the eyes of those with AMD. The most important risk factors for AMD, beside genetic predisposition, are factors leading to oxidative stress in the retina, e.g. age above 65 years, cigarettes smoking, obesity, exposition to blue light, and bright irises. Macular pigment is a natural barrier protecting the central retina against oxidative damage. It is formed by two dihydroxycarotenoids, lutein and zeaxanthin. The prereceptoral location of the macular pigment permits it to act as an optical filter that absorbs short-wavelength visible light. Carotenoids also demonstrate antioxidant activity. Eyes with a predisposition to develop AMD or which already have developed the disease have considerably less macular pigment and a greater risk of oxidative damage compared with healthy eyes. Investigations have shown that diet poor in antioxidant micronutrients (vitamin C, E, carotenoids, zinc) and low plasma levels of antioxidants may favor the development of the age-related macular degeneration. The findings demonstrated that micronutrient supplementation enhances antioxidant defense and might prevent or retard AMD or modify the course of the disease.