Preparation of pre-confluent retinal cells increases graft viability in vitro and in vivo: a mouse model

Purpose

Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure “transplant conditions” (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01.

Methods

Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts.

Results

Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001).

Conclusion

Pre-confluent cells should be used to maximize graft cell viability.     

Efficient removal of platelets from peripheral blood progenitor cell products using a novel micro-chip based acoustophoretic platform

Background

Excessive collection of platelets is an unwanted side effect in current centrifugation-based peripheral blood progenitor cell (PBPC) apheresis. We investigated a novel microchip-based acoustophoresis technique, utilizing ultrasonic standing wave forces for the removal of platelets from PBPC products. By applying an acoustic standing wave field onto a continuously flowing cell suspension in a micro channel, cells can be separated from the surrounding media depending on their physical properties.

Study Design and Methods

PBPC samples were obtained from patients (n = 15) and healthy donors (n = 6) and sorted on an acoustophoresis-chip. The acoustic force was set to separate leukocytes from platelets into a target fraction and a waste fraction, respectively. The PBPC samples, the target and the waste fractions were analysed for cell recovery, purity and functionality.

Results

The median separation efficiency of leukocytes to the target fraction was 98% whereas platelets were effectively depleted by 89%. PBPC samples and corresponding target fractions were similar in the percentage of CD34+ hematopoetic progenitor/stem cells as well as leukocyte/lymphocyte subset distributions. Median viability was 98%, 98% and 97% in the PBPC samples, the target and the waste fractions, respectively. Results from hematopoietic progenitor cell assays indicated a preserved colony-forming ability post-sorting. Evaluation of platelet activation by P-selectin (CD62P) expression revealed a significant increase of CD62P+ platelets in the target (19%) and waste fractions (20%), respectively, compared to the PBPC input samples (9%). However, activation was lower when compared to stored blood bank platelet concentrates (48%).

Conclusion

Acoustophoresis can be utilized to efficiently deplete PBPC samples of platelets, whilst preserving the target stem/progenitor cell and leukocyte cell populations, cell viability and progenitor cell colony-forming ability. Acoustophoresis is, thus, an interesting technology to improve current cell processing methods.     

14-3-3 σ expression effects G2/M response to oxygen and correlates with ovarian cancer metastasis

Background

In vitro cell culture experiments with primary cells have reported that cell proliferation is retarded in the presence of ambient compared to physiological O₂ levels. Cancer is primarily a disease of aberrant cell proliferation, therefore, studying cancer cells grown under ambient O₂ may be undesirable. To understand better the impact of O₂ on the propagation of cancer cells in vitro, we compared the growth potential of a panel of ovarian cancer cell lines under ambient (21%) or physiological (3%) O₂.

Principal Findings

Our observations demonstrate that similar to primary cells, many cancer cells maintain an inherent sensitivity to O₂, but some display insensitivity to changes in O₂ concentration. Further analysis revealed an association between defective G2/M cell cycle transition regulation and O₂ insensitivity resultant from overexpression of 14-3-3 σ. Targeting 14-3-3 σ overexpression with RNAi restored O₂ sensitivity in these cell lines. Additionally, we found that metastatic ovarian tumors frequently overexpress 14-3-3 σ, which in conjunction with phosphorylated RB, results in poor prognosis.

Conclusions

Cancer cells show differential proliferative sensitivity to changes in O₂ concentration. Although a direct link between O₂ insensitivity and metastasis was not determined, this investigation showed that an O₂ insensitive phenotype in cancer cells to correlate with metastatic tumor progression.     

Association of differentiation state of CD4+ T cells and disease progression in HIV-1 perinatally infected children

Background

In the USA, most HIV-1 infected children are on antiretroviral drug regimens, with many individuals surviving through adolescence and into adulthood. The course of HIV-1 infection in these children is variable, and understudied.

Methodology/Principal Findings

We determined whether qualitative differences in immune cell subsets could explain a slower disease course in long term survivors with no evidence of immune suppression (LTS-NS; CD4%≥25%) compared to those with severe immune suppression (LTS-SS; CD4%≤15%). Subjects in the LTS-NS group had significantly higher frequencies of naïve (CCR7+CD45RA+) and central memory (CCR7+CD45RA−) CD4+ T cells compared to LTS-SS subjects (p = 0.0005 and <0.0001, respectively). Subjects in the rapid progressing group had significantly higher levels of CD4+ T_EMRA (CCR7−CD45RA+) cells compared to slow progressing subjects (p<0.0001).

Conclusions/Significance

Rapid disease progression in vertical infection is associated with significantly higher levels of CD4+ T_EMRA (CCR7−CD45RA+) cells.     

Influence of short-term glucocorticoid therapy on regulatory T cells in vivo

Background

Pre- and early clinical studies on patients with autoimmune diseases suggested that induction of regulatory T(T_reg) cells may contribute to the immunosuppressive effects of glucocorticoids(GCs).

Objective

We readdressed the influence of GC therapy on T_reg cells in immunocompetent human subjects and naïve mice.

Methods

Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and T_reg cells in spleen and blood were analyzed by FACS. Sixteen patients with sudden hearing loss but without an inflammatory disease received high-dose intravenous prednisolone followed by stepwise dose reduction to low oral prednisolone. Peripheral blood T_reg cells were analyzed prior and after a 14 day GC therapy based on different markers.

Results

Repeated GC administration to mice for three days dose-dependently decreased the absolute numbers of T_reg cells in blood (100 mg dexamethasone/kg body weight: 2.8±1.8×10⁴ cells/ml vs. 33±11×10⁴ in control mice) and spleen (dexamethasone: 2.8±1.9×10⁵/spleen vs. 95±22×10⁵/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3⁺ T_reg cells amongst the CD4⁺ T cells also decreased in a dose dependent manner with the effect being more pronounced in blood than in spleen. The suppressive capacity of T_reg cells was unaltered by GC treatment in vitro. In immunocompetent humans, GCs induced mild T cell lymphocytosis. However, it did not change the relative frequency of circulating T_reg cells in a relevant manner, although there was some variation depending on the definition of the T_reg cells (FOXP3⁺: 4.0±1.5% vs 3.4±1.5%*; AITR⁺: 0.6±0.4 vs 0.5±0.3%, CD127^low: 4.0±1.3 vs 5.0±3.0%* and CTLA4+: 13.8±11.5 vs 15.6±12.5%; * p<0.05).

Conclusion

Short-term GC therapy does not induce the hitherto supposed increase in circulating T_reg cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating T_reg cell numbers.     

Impact of migration and acculturation on prevalence of type 2 diabetes and related eye complications in Indians living in a newly urbanised society

Background

Health of migrants is a major public health challenge faced by governments and policy makers. Asian Indians are among the fastest growing migration groups across Asia and the world, but the impact of migration and acculturation on diabetes and diabetes-related eye complications among Indians living in urban Asia remains unclear.

Methodologies/Principal Findings

We evaluated the influence of migration and acculturation (i.e., migration status and length of residence) on the prevalence of type-2 diabetes mellitus (T2DM) and diabetes-related eye complications (diabetic retinopathy (DR) and cataract), among first-generation (defined as participant born in India with both parents born in India, n = 781) and second-generation (participants born in Singapore with both parents born in India, n = 1,112) Indian immigrants from a population-based study of Adult Indians in Singapore. Diabetes was defined as HbA1c≥6.5%, use of diabetic medication or a physician diagnosis of diabetes. Retinal and lens photographs were graded for the presence of DR and cataract. Compared to first generation immigrants, second generation immigrants had a higher age- and gender-standardized prevalence of T2DM (34.4% versus 29.0%, p<0.001), and, in those with T2DM, higher age- and gender-standardized prevalence of DR (31.7% versus 24.8%, p<0.001), nuclear cataract (13.6% versus 11.6%, p<0.001), and posterior sub-capsular cataract (6.4% versus 4.6%, p<0.001). Among first generation migrants, longer length of residence was associated with significantly younger age of diagnosis of diabetes and greater likelihood of having T2DM and diabetes-related eye complications.

Conclusion

Second generation immigrant Indians and longer length of residence are associated with higher prevalence of diabetes and diabetes-related complications (i.e., DR and cataract) among migrant Indians living in Singapore. These data highlight potential worldwide impacts of migration patterns on the risk and burden of diabetes.     

Findings from a rapid assessment of avoidable blindness (RAAB) in Southern Malawi

Background

Data on prevalence and causes of avoidable blindness in Malawi are not readily available. The purpose of this study was to determine the prevalence and causes of blindness in persons aged 50 and above in southern Malawi to plan eye care services for the community.

Methodology

A population-based survey was conducted in 7 districts in southern Malawi. Villages were selected by probability proportionate to size within each district. Clusters were further subdivided into segments. A predetermined number of segments were selected randomly in each cluster. The survey team moved from house to house in each segment until they had examined 50 people over the age of 50. Examination consisted of visual acuity measurement with tumbling “E” chart and ocular examination by an ophthalmologist. Participants were categorized by visual acuity. Those who were visually impaired (VA<6/18 in the better eye with available correction) were assigned a main cause of visual loss. Further information was sought from anyone who had received cataract surgery.

Results

A total number of 3,583 persons aged 50 and above were sampled; among these 3,430 (95.7%) were examined. The prevalence of blindness (presenting visual acuity <3/60 in the better eye) among persons aged 50 and above was 3.3% (95% CI 2.5–4.1). Cataract was the most common cause of blindness contributing to 48.2% of all cases, followed by glaucoma (15.8%) and cornea scarring (12.3%). The cataract surgical coverage in blind persons was 44.6%.

Conclusion

The prevalence of blindness and visual impairment in persons aged 50 and above was lower than the WHO estimate for Malawi. The majority of the causes were avoidable, with cataract accounting for approximately half of all cases of blindness. The data suggests that expansion of eye care programs to address avoidable causes of blindness is necessary in this area of southern Malawi.     

A novel high-content flow cytometric method for assessing the viability and damage of rat retinal ganglion cells

Purpose

The aim of the study was to develop a high-content flow cytometric method for assessing the viability and damage of small, medium, and large retinal ganglion cells (RGCs) in N-methyl-D-aspartic acid (NMDA)-injury model.

Methods/Results

Retinal toxicity was induced in rats by intravitreal injection of NMDA and RGCs were retrogradely labeled with Fluoro-Gold (FG). Seven days post-NMDA injection, flatmount and flow cytometric methods were used to evaluate RGCs. In addition, the RGC area diameter (D_(a)) obtained from retinal flatmount imaging were plotted versus apparent volume diameter (D_(v)) obtained from flow cytometry for the same cumulative cell number (sequentially from small to large RGCs) percentile (Q) to establish their relationship for accurately determining RGC sizes. Good correlation (r = 0.9718) was found between D_(a) and apparent D_(v). Both flatmount and flow cytometric analyses of RGCs showed that 40 mM NMDA significantly reduced the numbers of small and medium RGCs but not large RGCs. Additionally, flow cytometry showed that the geometric means of FG and thy-1 intensities in three types of RGCs decreased to 90.96±2.24% (P<0.05) and 91.78±1.89% (P>0.05) for small, 69.62±2.11% (P<0.01) and 69.07±2.98% (P<0.01) for medium, and 69.68±6.48% (P<0.05) and 69.91±6.23% (P<0.05) for large as compared with the normal RGCs.

Conclusion

The established flow cytometric method provides high-content analysis for differential evaluation of RGC number and status and should be useful for the evaluation of various models of optic nerve injury and the effects of potential neuroprotective agents.     

Prevalence, causes and socio-economic determinants of vision loss in Cape Town, South Africa

Purpose

To estimate the prevalence and causes of blindness and visual impairment in Cape Town, South Africa and to explore socio-economic and demographic predictors of vision loss in this setting.

Methods

A cross sectional population-based survey was conducted in Cape Town. Eighty-two clusters were selected using probability proportionate to size sampling. Within each cluster 35 or 40 people aged 50 years and above were selected using compact segment sampling. Visual acuity of participants was assessed and eyes with a visual acuity less than 6/18 were examined by an ophthalmologist to determine the cause of vision loss. Demographic data (age, gender and education) were collected and a socio-economic status (SES) index was created using principal components analysis.

Results

Out of 3100 eligible people, 2750 (89%) were examined. The sample prevalence of bilateral blindness (presenting visual acuity <3/60) was 1.4% (95% CI 0.9–1.8). Posterior segment diseases accounted for 65% of blindness and cataract was responsible for 27%. The prevalence of vision loss was highest among people over 80 years (odds ratio (OR) 6.9 95% CI 4.6–10.6), those in the poorest SES group (OR 3.9 95% CI 2.2–6.7) and people with no formal education (OR 5.4 95% CI 1.7–16.6). Cataract surgical coverage was 68% in the poorest SES tertile (68%) compared to 93% in the medium and 100% in the highest tertile.

Conclusions

The prevalence of blindness among people ≥50 years in Cape Town was lower than expected and the contribution of posterior segment diseases higher than previously reported in South Africa and Sub Saharan Africa. There were clear socio-economic disparities in prevalence of vision loss and cataract surgical coverage in this setting which need to be addressed in blindness prevention programs.     

HER2-positive circulating tumor cells in breast cancer

Purpose

Circulating Tumor Cells (CTCs) detection and phenotyping are currently evaluated in Breast Cancer (BC). Tumor cell dissemination has been suggested to occur early in BC progression. To interrogate dissemination in BC, we studied CTCs and HER2 expression on CTCs across the spectrum of BC staging.

Methods

Spiking experiments with 6 BC cell lines were performed and blood samples from healthy women and women with BC were analyzed for HER2-positive CTCs using the CellSearch®.

Results

Based on BC cell lines experiments, HER2-positive CTCs were defined as CTCs with HER2 immunofluoresence intensity that was at least 2.5 times higher than the background. No HER2-positive CTC was detected in 42 women without BC (95% confidence interval (CI) 0–8.4%) whereas 4.1% (95%CI 1.4–11.4%) of 73 patients with ductal/lobular carcinoma in situ (DCIS/LCIS) had 1 HER2-positive CTC/22.5 mL, 7.9%, (95%CI 4.1–14.9%) of 101 women with non metastatic (M0) BC had ≥1 HER2-positive CTC/22.5 mL (median 1 cell, range 1–3 cells) and 35.9% (95%CI 22.7–51.9%) of 39 patients with metastatic BC had ≥1 HER2-positive CTC/7.5 mL (median 1.5 cells, range 1–42 cells). In CTC-positive women with DCIS/LCIS or M0 BC, HER2-positive CTCs were more commonly detected in HER2-positive (5 of 5 women) than HER2-negative BC (5 of 12 women) (p = 0.03).

Conclusion

HER2-positive CTCs were detected in DCIS/LCIS or M0 BC irrespective of the primary tumor HER2 status. Nevertheless, their presence was more common in women with HER2-positive disease. Monitoring of HER2 expression on CTCs might be useful in trials with anti-HER2 therapies.     

Resuscitation of newborn piglets. short-term influence of FiO2 on matrix metalloproteinases, caspase-3 and BDNF

Background

Perinatal hypoxia-ischemia is a major cause of mortality and cerebral morbidity, and using oxygen during newborn resuscitation may further harm the brain. The aim was to examine how supplementary oxygen used for newborn resuscitation would influence early brain tissue injury, cell death and repair processes and the regulation of genes related to apoptosis, neurodegeneration and neuroprotection.

Methods and Findings

Anesthetized newborn piglets were subjected to global hypoxia and then randomly assigned to resuscitation with 21%, 40% or 100% O₂ for 30 min and followed for 9 h. An additional group received 100% O₂ for 30 min without preceding hypoxia. The left hemisphere was used for histopathology and immunohistochemistry and the right hemisphere was used for in situ zymography in the corpus striatum; gene expression and the activity of various relevant biofactors were measured in the frontal cortex. There was an increase in the net matrix metalloproteinase gelatinolytic activity in the corpus striatum from piglets resuscitated with 100% oxygen vs. 21%. Hematoxylin-eosin (HE) staining revealed no significant changes. Nine hours after oxygen-assisted resuscitation, caspase-3 expression and activity was increased by 30–40% in the 100% O₂ group (n = 9/10) vs. the 21% O₂ group (n = 10; p<0.04), whereas brain-derived neurotrophic factor (BDNF) activity was decreased by 65% p<0.03.

Conclusions

The use of 100% oxygen for resuscitation resulted in increased potentially harmful proteolytic activities and attenuated BDNF activity when compared with 21%. Although there were no significant changes in short term cell loss, hyperoxia seems to cause an early imbalance between neuroprotective and neurotoxic mechanisms that might compromise the final pathological outcome.     

The dual PI3K/mTOR inhibitor NVP-BEZ235 induces tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer

Purpose

To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC).

Experimental Design

PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM) model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined.

Results

In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC₅₀ = 9.0–14.3 nM). Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01) vs. a 43% decrease (p = 0.008) in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003), no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013).

Conclusions

These studies provide the preclinical rationale for studies examining the efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type CRC.     

The association between systemic inflammatory cellular levels and lung function: a population-based study

Background

Lower lung function is associated with an elevated systemic white cell count in men. However, these observations have not been demonstrated in a representative population that includes females and may be susceptible to confounding by recent airway infections or recent cigarette smoking. We tested the hypothesis that lung function is inversely associated with systemic white cell count in a population-based study.

Methods

The study population consisted adults aged 17−90+ years who participated in the Third National Health and Nutrition Examination Survey who did not report a recent cough, cold or acute illness in a non-smoking and smoking population.

Results

In non-smoking adults with the highest quintile of the total white cell count had a FEV₁ 125.3 ml lower than those in the lowest quintile (95% confidence interval CI: −163.1 to –87.5). Adults with the highest quintile of the total white cell count had a FVC 151.1 ml lower than those in the lowest quintile (95% confidence interval CI: −195.0 to −107.2). Similar associations were observed for granulocytes, mononuclear cells and lymphocytes. In current smokers, similar smaller associations observed for total white cell count, granulocytes and mononuclear cells.

Conclusions

Systemic cellular inflammation levels are inversely associated with lung function in a population of both non-smokers and smokers without acute illnesses. This may contribute to the increased mortality observed in individuals with a higher baseline white cell count.     

Functional significance of vitamin D receptor FokI polymorphism in human breast cancer cells

Background

The FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids. Epidemiological studies have paradoxically implicated this polymorphism with increased breast cancer risk. 1α,25 (OH)₂D₃, the active metabolite of vitamin D, is known to inhibit cell proliferation, induce apoptosis and potentiate differentiation in human breast cancer cells. It is well documented that 1α,25 (OH)₂D₃ downregulates estrogen receptor α expression and inhibits estrogen mediated signaling in these cells. The functional significance of the VDR FokI polymorphism in vitamin D action is undefined.

Methods/Findings

To elucidate the functional role of FokI polymorphism in breast cancer, MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF stable cell lines were established from parental MCF-7 cells as single-cell clones. In response to 1α,25 (OH)₂D₃ treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells. The induction of the vitamin D target gene CYP24A1 mRNA was 1.8 fold higher in VDRFF cells than in VDRff cells. Estrogen receptor-α protein expression was downregulated by 62% in VDRFF cells compared to 25% in VDRff cells. VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide. PCR array analyses of VDRff and VDRFF cells revealed increased basal expression levels of pro-inflammatory genes Cyclooxygenase-2, Interleukin-8 and Chemokine (C-C Motif) Ligand 2 in MCF-7-VDRff cells by 14, 52.7 and 5 fold, respectively.

Conclusions/Significance

These results suggest that a VDRff genotype may play a role in amplifying aggressive breast cancer, paving the way for understanding why some breast cancer cells respond inefficiently to vitamin D treatment.     

H2S Donor,S-Propargyl-Cysteine,Increases CSE in SGC-7901 and Cancer-Induced Mice: Evidence for a Novel Anti-Cancer Effect of Endogenous H2S?

Background

S-propargyl-cysteine (SPRC), an H₂S donor, is a structural analogue of S-allycysteine (SAC). It was investigated for its potential anti-cancer effect on SGC-7901 gastric cancer cells and the possible mechanisms that may be involved.

Methods and Findings

SPRC treatment significantly decreased cell viability, suppressed the proliferation and migration of SPRC-7901 gastric cancer cells, was pro-apoptotic as well as caused cell cycle arrest at the G₁/S phase. In an in vivo study, intra-peritoneal injection of 50 mg/kg and 100 mg/kg of SPRC significantly reduced tumor weights and tumor volumes of gastric cancer implants in nude mice, with a tumor growth inhibition rate of 40–75%. SPRC also induced a pro-apoptotic effect in cancer tissues and elevated the expressions of p53 and Bax in tumors and cells. SPRC treatment also increased protein expression of cystathione-γ-lyase (CSE) in cells and tumors, and elevated H₂S levels in cell culture media, plasma and tumoral CSE activity of gastric cancer-induced nude mice by 2, 2.3 and 1.4 fold, respectively. Most of the anti-cancer functions of SPRC on cells and tumors were significantly suppressed by PAG, an inhibitor of CSE activity.

Conclusions

Taken together, the results of our study provide insights into a novel anti-cancer effect of H₂S as well as of SPRC on gastric cancer through inducing the activity of a new target, CSE.     

Switch of voltage-gated K+ channel expression in the plasma membrane of chondrogenic cells affects cytosolic Ca2+-oscillations and cartilage formation

Background

Understanding the key elements of signaling of chondroprogenitor cells at the earliest steps of differentiation may substantially improve our opportunities for the application of mesenchymal stem cells in cartilage tissue engineering, which is a promising approach of regenerative therapy of joint diseases. Ion channels, membrane potential and Ca²⁺-signaling are important regulators of cell proliferation and differentiation. Our aim was to identify such plasma membrane ion channels involved in signaling during chondrogenesis, which may serve as specific molecular targets for influencing chondrogenic differentiation and ultimately cartilage formation.

Methodology/Principal Findings

Using patch-clamp, RT-PCR and Western-blot experiments, we found that chondrogenic cells in primary micromass cell cultures obtained from embryonic chicken limb buds expressed voltage-gated Na_V1.4, K_V1.1, K_V1.3 and K_V4.1 channels, although K_V1.3 was not detectable in the plasma membrane. Tetrodotoxin (TTX), the inhibitor of Na_V1.4 channels, had no effect on cartilage formation. In contrast, presence of 20 mM of the K⁺ channel blocker tetraethyl-ammonium (TEA) during the time-window of the final commitment of chondrogenic cells reduced K_V currents (to 27±3% of control), cell proliferation (thymidine incorporation: to 39±4.4% of control), expression of cartilage-specific genes and consequently, cartilage formation (metachromasia: to 18.0±6.4% of control) and also depolarized the membrane potential (by 9.3±2.1 mV). High-frequency Ca²⁺-oscillations were also suppressed by 10 mM TEA (confocal microscopy: frequency to 8.5±2.6% of the control). Peak expression of TEA-sensitive K_V1.1 in the plasma membrane overlapped with this period. Application of TEA to differentiated chondrocytes, mainly expressing the TEA-insensitive K_V4.1 did not affect cartilage formation.

Conclusions/Significance

These data demonstrate that the differentiation and proliferation of chondrogenic cells depend on rapid Ca²⁺-oscillations, which are modulated by K_V-driven membrane potential changes. K_V1.1 function seems especially critical during the final commitment period. We show the critical role of voltage-gated cation channels in the differentiation of non-excitable cells with potential therapeutic use.     

αA Crystallin May Protect against Geographic Atrophy-Meta-Analysis of Cataract vs. Cataract Surgery for Geographic Atrophy and Experimental Studies

Background

Cataract and geographic atrophy (GA, also called advanced “dry” age-related macular degeneration) are the two major causes of visual impairment in the developed world. The association between cataract surgery and the development of GA was controversial in previous studies.

Methods/Principal Findings

We performed a meta-analysis by pooling the current evidence in literature and found that cataract is associated with an increased risk of geographic atrophy with a summary odds ratio (OR) of 3.75 (95% CI: 95% CI: 1.84–7.62). However, cataract surgery is not associated with the risk of geographic atrophy (polled OR = 3.23, 95% CI: 0.63–16.47). Further experiments were performed to analyze how the αA-crystallin, the major component of the lens, influences the development of GA in a mouse model. We found that theαA-crystallin mRNA and protein expression increased after oxidative stress induced by NaIO₃ in immunohistochemistry of retinal section and western blot of posterior eyecups. Both functional and histopathological evidence confirmed that GA is more severe in αA-crystallin knockout mice compared to wild-type mice.

Conclusions

Therefore, αA-crystallin may protect against geographic atrophy. This study provides a better understanding of the relationship between cataract, cataract surgery, and GA.     

Antiviral therapy and outcomes of patients with pneumonia caused by influenza A pandemic (H1N1) virus

Background

There is limited data on the clinical outcome of patients with pandemic H1N1 (pH1N1) pneumonia who received oseltamivir treatment, especially when the treatment was administered more than 48 hours after symptom onset.

Methods

During the pandemic in 2009, a cohort of pH1N1 influenza pneumonia was built in China, and their clinical information was collected systematically, and analyzed with Cox models.

Results

920 adults and 541 children with pneumonia who didn''t receive corticosteroids were analyzed. In-hospital mortality was higher in adults who did not receive antiviral therapy (18.2%) than those with who received oseltamivir ≤ 2days (2.9%), between 2–5 days (4.6%) and >5 days after illness onset (4.9%), p<0.01. A similar trend was observed in pediatric patients. Cox regression showed that at 60 days after symptoms onset, 11 patients (10.8%) who did not receive antivirals died versus 4 (1.8%), 18 (3.3%), and 23 (3.7%) patients whose oseltamivir treatment was started ≤ 2days, between 2–5days, and >5 days, respectively. For males patients, aged ≥ 14 years and baseline PaO₂/FiO₂<200, oseltamivir administration reduced the mortality risk by 92.1%, 88% and 83.5%, respectively. Higher doses of oseltamivir (>3.8 mg/kg/d) did not improve clinical outcome (mortality, higher dose 2.5% vs standard dose 2.8%, p>0.05).

Conclusions

Antiviral therapy might reduce mortality of patients with pH1N1 pneumonia, even when initiated more than 48 hours after onset of illness. Greater protective effects might be in males, patients aged 14–60 years, and patients with PaO₂/FiO₂<200.     

MRI of auto-transplantation of bone marrow-derived stem-progenitor cells for potential repair of injured arteries

Background

This study was to validate the feasibility of using clinical 3.0T MRI to monitor the migration of autotransplanted bone marrow (BM)-derived stem-progenitor cells (SPC) to the injured arteries of near-human sized swine for potential cell-based arterial repair.

Methodology

The study was divided into two phases. For in vitro evaluation, BM cells were extracted from the iliac crests of 13 domestic pigs and then labeled with a T2 contrast agent, Feridex, and/or a fluorescent tissue marker, PKH26. The viability, the proliferation efficiency and the efficacies of Feridex and/or PKH26 labeling were determined. For in vivo validation, the 13 pigs underwent endovascular balloon-mediated intimal damages of the iliofemoral arteries. The labeled or un-labeled BM cells were autotransplanted back to the same pig from which the BM cells were extracted. Approximately three weeks post-cell transplantation, 3.0T T2-weighted MRI was performed to detect Feridex-created signal voids of the transplanted BM cells in the injured iliofemoral arteries, which was confirmed by subsequent histologic correlation.

Principal Findings

Of the in vitro study, the viability of dual-labeled BM cells was 95–98%. The proliferation efficiencies of dual-labeled BM cells were not significantly different compared to those of non-labeled cells. The efficacies of Feridex- and PKH26 labeling were 90% and 100%, respectively. Of the in vivo study, 3.0T MRI detected the auto-transplanted BM cells migrated to the injured arteries, which was confirmed by histologic examinations.

Conclusion

This study demonstrates the capability of using clinical 3.0T MRI to monitor the auto-transplantation of BM cells that migrate to the injured arteries of large animals, which may provide a useful MRI technique to monitor cell-based arterial repair.     

IL-17A expression is localised to both mononuclear and polymorphonuclear synovial cell infiltrates

Introduction

This study examines the expression of IL-17A-secreting cells within the inflamed synovium and the relationship to in vivo joint hypoxia measurements.

Methods

IL-17A expression was quantified in synovial tissue (ST), serum and synovial fluid (SF) by immunohistochemistry and MSD-plex assays. IL-6 SF and serum levels were measured by MSD-plex assays. Dual immunofluorescence for IL-17A was quantified in ST CD15+ cells (neutrophils), Tryptase+ (mast cells) and CD4+ (T cells). Synovial tissue oxygen (tpO₂) levels were measured under direct visualisation at arthroscopy. Synovial infiltration was assessed using immunohistochemistry for cell specific markers. Peripheral blood mononuclear and polymorphonuclear cells were isolated and exposed to normoxic or 3% hypoxic conditions. IL-17A and IL-6 were quantified as above in culture supernatants.

Results

IL-17A expression was localised to mononuclear and polymorphonuclear (PMN) cells in inflamed ST. Dual immunoflourescent staining co-localised IL-17A expression with CD15+ neutrophils Tryptase+ mast cells and CD4+T cells. % IL-17A positivity was highest on CD15+ neutrophils, followed by mast cells and then CD4+T-cells. The number of IL-17A-secreting PMN cells significantly correlated with sublining CD68 expression (r = 0.618, p<0.01). IL-17A SF levels correlated with IL-6 SF levels (r = 0.675, p<0.01). Patients categorized according to tp0₂< or >20mmHg, showed those with low tp0₂<20mmHg had significantly higher IL-17A+ mononuclear cells with no difference observed for PMNs. Exposure of mononuclear and polymorphonuclear cells to 3% hypoxia, significantly induced IL-6 in mononuclear cells, but had no effect on IL-17A expression in mononuclear and polymorphonuclear cells.

Conclusion

This study demonstrates IL-17A expression is localised to several immune cell subtypes within the inflamed synovial tissue, further supporting the concept that IL-17A is a key mediator in inflammatory arthritis. The association of hypoxia with Il-17A expression appears to be indirect, probably through hypoxia-induced pro-inflammatory pathways and leukocyte influx within the joint microenvironment.