Modulation of mucin secretion using combined polyethylene glycol–propylene glycol topical formulation in a hyperosmotic stress-based explant model

Purpose: Ocular surface discomfort and dry eye disease are caused by a dysfunctional tear film. The efficacy of lubricating eye drops on the human eye is known, but the compositions may show differential effects on rescuing the tear film. Mucins form a critical layer of the tear film, a reduction of which may be causative for ocular surface conditions. Therefore, it is essential to develop relevant human?derived models to test mucin production. Methods: Human corneoscleral rims were obtained from a healthy donor (n = 8) post?corneal keratoplasty and cultured in DMEM/F12 media. Hyperosmolar stress mimicking dry eye disease was induced by exposing the corneoscleral rim tissues to +200 mOsml NaCl?containing media. The corneoscleral rims were treated with polyethylene glycol–propylene glycol (PEG–PG)?based topical formulation. Gene expression analysis was performed for NFAT5, MUC5AC, and MUC16. Secreted mucins were measured by enzyme?linked immunosorbent assay (ELISA) (Elabscience, Houston, TX, USA) for MUC5AC and MUC16. Results: The corneoscleral rims responded to hyperosmolar stress by upregulating NFAT5, a marker for increased osmolarity, as observed in the case of dry eye disease. The expression of MUC5AC and MUC16 was reduced upon an increase in hyperosmotic stress. The corneoscleral rim tissues showed induction of MUC5AC and MUC16 expression upon treatment with PEG–PG topical formulation but did not show significant changes in the presence of hyperosmolar treatments. Conclusion: Our findings showed that PEG–PG?based topical formulation slightly alleviated hyperosmolar stress?induced decrease in MUC5AC and MUC16 gene expression that is encountered in DED

Type 1–type 2 interferon imbalance in dry eye disease

Purpose: Dry eye disease (DED) is characterized by altered ocular surface proinflammatory and antiinflammatory factors. Interferons (IFNs) are a class of pleiotropic cytokines well known for their antimicrobial, inflammatory, and immunomodulatory roles. Hence, this study investigates the ocular surface expression of different types of IFNs in patients with DED. Methods: The cross?sectional, observational study included patients with DED and normal subjects. Conjunctival impression cytology (CIC) samples were obtained from the study subjects (controls, n = 7; DED, n = 8). The mRNA expression levels of type 1 IFN (IFN?, IFN?), type 2 IFN (IFN?), and type 3 IFN (IFN?1, IFN?2, IFN?3) were measured by quantitative PCR (polymerase chain reaction) in CIC samples. IFN? and IFN? expression under hyperosmotic stress was also studied in human corneal epithelial cells (HCECs) in vitro. Results: The mRNA expression levels of IFN? and IFN? were significantly lower and that of IFN? was significantly higher in DED patients compared to healthy controls. The mRNA levels of IFN?, IFN?, and IFN? were significantly lower compared to IFN? in DED patients. An inverse association between tonicity?responsive enhancer?binding protein (TonEBP; hyperosmotic stress maker) and IFN? or IFN? expression and a positive association between TonEBP and IFN? expression was observed in CIC samples. The expression of IFN? was lower than IFN? in HCECs undergoing hyperosmotic stress compared to HCECs without the stress. Conclusion: The presence of an imbalance between type 1 and type 2 IFNs in DED patients suggests newer pathogenic processes in DED, plausible ocular surface infection susceptibility in DED patients, and potential therapeutic targets in the management of DED

Hyperosmotic stress and perfusion culture strategies increase the yield of recombinant adenoviral vector produced by HEK 293 cells / 生物工程学报

With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×1010 IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.

Choline-betaine pathway contributes to hyperosmotic stress and subsequent lethal stress resistance in Pseudomonas protegens SN15-2

Pseudomonas protegens SN15-2, a typical non-spore-forming rhizosphere bacterium, has excellent biocontrolcapabilities; thus, it is necessary to explore the stress resistance of SN15-2. The choline–glycine betainepathway is considered as an important mechanism by which bacteria adapt to stressful environments. In thiswork, we demonstrated that the expression of the betA and betB genes, which are involved in the choline–glycine betaine pathway in SN15-2, was highly increased by 12-fold and 26-fold, respectively, by hyperosmotic stress and choline treatment. The accumulation of betaine in SN15-2 (5.54 g/L) was significantly higherthan that in the mutants D betA (3.44 g/L) and D betB (2.68 g/L) under hyperosmotic stress and cholinetreatment. Moreover, choline enhanced the growth of SN15-2 greatly, but it did not enhance the growth of DbetB under hyperosmotic stress. Choline combined with hyperosmotic adaptation significantly increased thelethal stress resistance of SN15-2 while the resistance of D betA and D betB was significantly decreased. Thisresearch illuminated a strategy underlying the adaptation to osmotic stress in P. protegens and provided aneffective method to improve the stress resistance of this species, thus provided a theoretical basis for thepractical application of P. protegens SN15-2.

Protective effect of Dexamethasone combined with hypertonic solution on corneal endothelium in rabbit eyes / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To explore the protective effect of dexamethasone combined with hypertonic solution on corneal endothelial cells of rabbit eyes. <p>METHODS: We made four groups of rabbits for different treatments with dexamethasone and hypertonic solution. All groups were treated with low-permeability solution for 10min to maintain anterior chamber perfusion. Group A was treated with 0.2mL dexamethasone subconjunctival injection, and hypertonic glucose eye drop immediately after surgery. Group B(control group)was subjected to 0.2mL of 0.9% saline subconjunctival injection, and balanced salt solution eye drop immediately after surgery. In group C, 0.2mL dexamethasone subconjunctival injection, and hypertonic glucose eye drop were given on the second day of surgery and in group D(control group), 0.2mL of 0.9% saline through subconjunctival injection and balanced salt solution eye drop were given on the second day of the surgery. The edema degree of cornea was observed with slit lamp before the operation and on the 1, 3, 5 and 7d after operation. The cornea were also examined by anterior segment OCT, and the corneal thickness was measured by A-scan ultrasonography.<p>RESULTS: In group A, during the entire experimental observation period, the cornea had no edema or only mild edema, the thickness of the central cornea hardly increased, and the number of corneal endothelial cells did not change significantly. There was no significant difference compared with(control group B)before modeling(<i>P</i>>0.05). In groups B, C, and D, corneal edema and corneal thickness increased after the rabbit eyes were modeled. There was a significant difference compared with group A(<i>P</i><0.05). The number of corneal endothelial cells in the rabbit eyes of groups B and D could not be measured during the observation period due to corneal edema. The number of corneal endothelial cells can be measured in group C up to the 7d after modeling. The number of corneal endothelial cells in group C was significantly reduced as compared with group A(<i>P</i><0.05), which before modeling and the 7d after modeling.<p>CONCLUSION: Dexamethasone combined with hypertonic glucose solution has a good protective effect on rabbit corneal endothelial cells. And early application can effectively prevent rabbit corneal edema and this method can also prevent the progression of corneal endothelial decompensation.

Efficacy of Anterior Stromal Puncture Using 5% NaCl Eye Drops for Prolonged Time in Recurrent Corneal Erosion Syndrome

PURPOSE: The purpose of this study is to evaluate the efficacy of the combined treatment of anterior stromal puncture with the concurrent use of 5% NaCl eye drops for 6 months for recurrent corneal erosion syndrome. METHODS: A retrospective chart review was conducted for 21 patients (21 eyes) who underwent the combined treatment of anterior stromal puncture with 5% NaCl eye drops qid for 6 months to treat recurrent corneal erosion syndromes. The patients' age, gender, history of corneal trauma, history of ocular surgery, recurrence rate, need for additional treatments after recurrence and complication were analyzed. RESULTS: The mean age was 42.05 ± 13.83 years (19-68 years) with no significant difference in gender, and the mean follow up period was 8.48 ± 2.76 months (6-15 months). Among the 21 eyes 14 eyes (66.7%) had a previous history of corneal trauma, 2 eyes (9.52%) had previous refractive surgery, 1 eye (4.8%) had corneal dystrophy, 2 eyes (9.5%) had diabetic mellitus and 5 eyes (28.81%) had an unknown origin. Furthermore, 3 eyes (14.3%) developed recurrences and 2 eyes were resolved with conservative treatment while 1 eye needed additional anterior stroma puncture. There was no recurrence after retreatment with anterior stroma puncture. No significant complications such as infection or inflammation developed with the exception of corneal opacity in which one patient developed glare due to corneal opacity. CONCLUSIONS: The combined treatment of anterior stromal puncture with 5% NaCl eye drops for a prolonged time might be effective at preventing the recurrence of refractory recurrent corneal erosion syndrome with conservative treatments.

Efficacy of hyperosmotic glucose in treatment of corneal edema after cataract surgery / 国际眼科杂志(Guoji Yanke Zazhi)

AlM: To explore clinical efficacy of hyperosmotic glucose after cataract surgery.METHODS: Eighty eyes ( 80 cases ) with ll level and above grades corneal edema after small incision cataract operation were enrolled. They were randomly divided into treatment group and control group. Control group used TobraDex eye drops and other conventional treatment and the treatment group received hyperosmotic glucose(500g/L) with the bases of the control group. The regression of corneal edema was observed by slit lamp microscope. SPSS 17. 0 statistical software was used for statistical analysis.RESULTS: The level of corneal edema at the first day after surgery, age, sexuality and nuclear hardness in the treated group were compared with those in the control group, the differences were without significances ( P>0. 05). Disappearance time of corneal edema was 4. 95 ± 3. 62d of the treatment group, and was 6. 80±3. 33d of the control group, the treatment group was better than control group (t=8. 55, P=0. 00).CONCLUSlON: Hyperosmotic glucose has exact and obvious efficacy for early corneal edema after cataract surgery.

Comparison of Nd:YAG Laser versus Conservative Management in the Treatment of Recurrent Corneal Erosion

PURPOSE: To compare the treatment effects of neodymium:yttrium-aluminum-garnet (Nd:YAG) laser and conservative management in treatment of recurrent corneal erosion. METHODS: Twenty-three eyes that received Nd:YAG laser treatment and 24 eyes that underwent conservative management including hyperosmotic agent were retrospectively reviewed for the rate and frequency of recurrence, presentation, time to recurrence, final visual acuity and complications. RESULTS: The rate of recurrence in eyes treated with Nd:YAG laser was 56.5% and that in eyes with conservative management was 50.0%. The difference was not statistically significant (p = 0.654). However, 10 of 12 eyes that recurred after conservative treatment presented with macroform erosion at the time of recurrence, whereas 5 of 13 eyes that recurred after Nd:YAG laser had an accompanying epithelial defect (p = 0.041). After the Nd:YAG laser treatment or conservative treatment, 64.1% and 60.5% of the eyes, respectively, remained free from recurrence 1 year after treatment, however the success rate decreased over time (p = 0.649). The final visual acuity in the group that received Nd;YAG laser treatment was 0.05 +/- 0.06 log MAR and 0.09 +/- 0.08 log MAR (p = 0.649) in the group that received conservative management. None of the patients in either group experienced complications of corneal scarring. CONCLUSIONS: The difference in the recurrence rates between Nd:YAG laser treatment and conservative treatment were not statistically different, however, the severity of recurred episodes were milder in the eyes that received Nd:YAG laser treatment.

Hyperosmotic Stimulus Down-regulates 1alpha, 25-dihydroxyvitamin D3-induced Osteoclastogenesis by Suppressing the RANKL Expression in a Co-culture System

The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on 1alpha,25(OH)2D3-induced osteoclastogenesis was investigated in an osteoblast-preosteoclast co-culture system. Serial doses of sucrose were applied as a mechanical force. These hyperosmotic stimuli significantly evoked a reduced number of 1alpha,25(OH)2D3-induced tartrate-resistant acid phosphatase-positive multinucleated cells and 1alpha,25(OH)2D3-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor kappaB ligand (RANKL) and Runx2 expressions were down-regulated in response to 1alpha,25(OH)2D3. Knockdown of Runx2 inhibited 1alpha,25(OH)2D3-induced RANKL expression in osteoblastic cells. Finally, the hyperosmotic stimulus induced the overexpression of TonEBP in osteoblastic cells. These results suggest that hyperosmolarity leads to the down-regulation of 1alpha,25(OH)2D3-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.

Changes of Gene Expression in NIH3T3 Cells Exposed to Osmotic and Oxidative Stresses

Cells consistently face stressful conditions, which cause them to modulate a variety of intracellular processes and adapt to these environmental changes via regulation of gene expression. Hyperosmotic and oxidative stresses are significant stressors that induce cellular damage, and finally cell death. In this study, oligonucleotide microarrays were employed to investigate mRNA level changes in cells exposed to hyperosmotic or oxidative conditions. In addition, since heat shock protein 70 (HSP70) is one of the most inducible stress proteins and plays pivotal role to protect cells against stressful condition, we performed microarray analysis in HSP70 overexpressing cells to identify the genes expressed in a HSP70 dependent manner. Under hyperosmotic or oxidative stress conditions, a variety of genes showed altered expression. Down regulation of protein phosphatase1 beta (PP1 beta) and sphingosine 1 phosphate phosphatase 1 (SPPase1) was detected in both stress conditions. Microarray analysis of HSP70 overexpressing cells demonstrated that diverse mRNA species depend on the level of cellular HSP70. Genes encoding lysyl oxidase, thrombospondin 1, and procollagen displayed altered expression in all tested conditions. The results of this study will be useful to construct networks of stress response genes.

Side effects of inhalation of hyperosmotic saline on sputum induction in asthmatic patients / 第三军医大学学报

0 05). Excessive bronchoconstriction occurred in one patient (1/41, 2.44%). Nausea and vomiting, pharyngeal dryness and pharyngalgia, dizziness and headache, discomfort of chest, and so on were found in other 43 9% patients, but the side effects were mild and endurable. Conclusion Sputum induction after inhalation of short acting ? 2 agonist is safe for asthmatic patients in the remission stage.

Effects of hyperoxic-hyperosmotic sodium chloride solution on blood lactate and arterial blood gas in rabbits with hemorrhagic shock / 中国普通外科杂志

Objective To investigate the effects of hyperoxic-hyperosmotic sodium chloride solution(HHSCS) on arterial blood gas and blood lactate in rabbits with hemorrhagic shock, and evaluate the HHSCS effects on hemorrhagic shock resuscitation.Methods Hemorrhagic shock models were induced in 30 male rabbits.The median arterial pressure(MAP) was declined to 40mmHg within 10 minutes,then maintained for 60(minutes).The animals were then randomly divided into 3 groups:normal saline hyperoxic solution(NSO)group,hyperosmotic sodium chloride solution(HS)group and HHSCS group.Sixty minutes later,NSO,HS or HHSCS 6ml/kg was infused intravenously in 5 minutes.Heart rate(HR) 、respiratory rate(RR)、 MAP and urine drops(UD) before shock and after fluid infusion were recorded;blood lactate(BL) and blood gas were measured before shock,60min after shock,and 30min,60min,120min postinfusion.Finally,the(rabbits) were killed by bleeding,the lungs were observed in autopsy,and the lung coefficient was measured.Results HS and HHSCS more effectively raised MAP,enhanced heart function,and increased UD than NSO.HHSCS group significantly reduced BL.At 60,120min after infusion,SaO_2 in HHSCS group was(significantly) higher than that in HS group and NSO group(P