Pilot study on circulating miRNA signature in children with obesity born small for gestational age and appropriate for gestational age.

BACKGROUND: Children born small for gestational age (SGA) are at increased risk of metabolic dysfunction. Dysregulation of specific microRNAs (miRNAs) contributes to aberrant gene expression patterns underlying metabolic dysfunction. OBJECTIVE: We aimed to determine and compare circulating miRNA (c-miRNA) profile of SGA and appropriate for gestational age (AGA) children with obesity and with normal weight, in order to identify biomarkers for early detection of increased risk of developing metabolic dysfunction in SGA and AGA children with obesity. METHODS: Small non-coding RNAs from serum of 15 SGA children with obesity (OB-SGA), 10 SGA children with normal weight (NW-SGA), 17 AGA children with obesity (OB-AGA) and 12 AGA children with normal weight (NW-AGA) (mean age 11.2 ± 2.6) have been extracted and sequenced in order to detect and quantify miRNA expression profiles. RESULTS: RNA-seq analyses showed 28 miRNAs dysregulated in OB-SGA vs. NW-SGA and 19 miRNAs dysregulated in OB-AGA vs. NW-AGA. Among these, miR-92a-3p, miR-122-5p, miR-423-5p, miR-484, miR-486-3p and miR-532-5p were up regulated, and miR-181b-5p was down regulated in both OB-SGA and OB-AGA compared with normal weight counterparts. Pathway analysis and miRNA target prediction suggested that these miRNAs were particularly involved in insulin signalling, glucose transport, insulin resistance, cholesterol and lipid metabolism. CONCLUSION: We identified a specific profile of c-miRNAs in SGA and AGA children with obesity compared with SGA and AGA children with normal weight. These c-miRNAs could represent specific biomarkers for early detection of increased risk of developing metabolic dysfunction in SGA and AGA children with obesity.

Gamma rays induce a p53-independent mitochondrial biogenesis that is counter-regulated by HIF1α.

Mitochondrial biogenesis is an orchestrated process that presides to the regulation of the organelles homeostasis within a cell. We show that γ-rays, at doses commonly used in the radiation therapy for cancer treatment, induce an increase in mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence, in the presence of a functional p53. Although the main effector of the response to γ-rays is the p53-p21 axis, we demonstrated that mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a murine double minute 2 (MDM2)-mediated hypoxia-inducible factor 1α (HIF1α) degradation, leading to the release of peroxisome-proliferator activated receptor gamma co-activator 1ß inhibition by HIF1α, thus promoting mitochondrial biogenesis. Mimicking hypoxia by HIF1α stabilization, in fact, blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally, we also show in vivo that post-radiotherapy mitochondrial DNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of cell senescence.

Regulation of IGFBP3 gene expression in short children born small for gestational age.

OBJECTIVE: Approximately 6% of newborns at term are small for gestational age (SGA) and present a birth weight and/or length less than -2SD from the mean. SGA infants are at increased risk for perinatal morbidity, associated psychological and/or mental problems, persistent short stature (about 15% of subjects) and metabolic alterations. Insulin-like growth factors (IGFs), their common receptor (IGF1R) and their binding proteins (IGFBPs) play a critical role in fetal and postnatal growth. In these genes common polymorphisms, such as single nucleotide polymorphisms and variable number of tandem repeats, have been investigated with conflicting results with respect to SGA-related outcomes, and the functional role of these gene variants remains to be elucidated. DESIGN: The study group consisted of 100 pre-pubertal short children born SGA and 94 healthy controls, matched for sex and age, recruited at the Department of Biomedicine of Development Age of the Bari University and at the Paediatric Department of the Messina Hospital. In the present study we analyzed the allelic frequency of the polymorphisms -795 G/A, -667 G/A, -396 C/T in the IGFBP3 in SGA children and their influence on the basal and insulin-stimulated transcriptional activity of the gene. RESULTS: We found that the polymorphisms -667 G/A and -396 C/T in the IGFBP3 promoter region are capable of having an effect on the transcriptional activity of the gene, although with opposing effects. Interestingly, the -667 G/A polymorphism has a negative impact on the IGFBP3 transcription, while the -396 C/T polymorphism determines an increase of the transcriptional activity of the IGFBP3 gene promoter. Interestingly, we found that the -396 C/T polymorphism correlates with lower birth length in SGA children. Most importantly, while the diminished IGFBP3 transcriptional activity induced by the -667A polymorphism was significantly recovered after insulin administration (p-value<0.05), the increased transcriptional activity caused by the -396T polymorphism was not restored to baseline levels by insulin. CONCLUSIONS: Altogether our results demonstrated that the -667 G/A and the -396 C/T polymorphisms in IGFBP3 promoter region influence the basal transcriptional activity of the gene.

The influence of donor and recipient factors in allograft rejection of the human cornea.

UNLABELLED: The health of the corneal endothelium is essential in maintaining the clarity of the transplanted human cornea. Immune-mediated endothelial rejection is a complex series of events, which may culminate in the decompensation of the donor button. It is the commonest instigator of failure in penetrating corneal transplantation. METHODS: This retrospective case note review of 203 penetrating keratoplasties with adequate follow-up data during a 5-year study period from 1 January 2000 to 31 December 2003 at Manchester Royal Eye Hospital, were used for analysis. RESULTS: Forty-three of the 203 donor grafts underwent at least one single episode of immune-mediated endothelial rejection, an incidence of 21% over a 5-year follow-up. Recipient's age was inversely associated with the risk of rejection. The average age for the cohort of 58.7 years and average age for rejecting patients of 47.6 years were strongly significantly different (P=0.009). Rejection in keratoconic patients accounted for 30% of cases. Death to enucleation time (P=0.03) was also associated with an increased risk of rejection. CONCLUSION: Although penetrating keratoplasty is an effective long-term treatment option for improving visual function, the endothelial rejection rate in our study was 21% over a mean follow-up of over 5 years. Host vascularisation, regrafts, younger recipient age group, and donor factors were found to be significantly associated with a risk of rejection. Rejection in keratoconic recipients was more common than expected.

Risk of Creutzfeldt-Jakob disease transmission by ocular surgery and tissue transplantation.

Creutzfeldt-Jakob disease (CJD) is a rare, fatal neurodegenerative disease that occurs in sporadic, genetic, variant, and iatrogenic forms. The transformation of normal prion protein (PrP(C)) to the abnormal form (PrP(Sc)) is a key step in the pathogenesis of CJD and leads to the accumulation of amyloid and spongiform changes in the brain. The presence of PrP(Sc) in tissue is a surrogate marker for CJD infectivity. Sporadic CJD, whose cause is unknown, is by far the most frequent form with 1-2 cases per million population occurring every year-the genetic forms of CJD are rather rarer. The majority of variant CJD cases have occurred in the United Kingdom, where there have been four reports of transmission of vCJD by blood transfusion. The great majority of iatrogenic transmissions of CJD have resulted from the use of pituitary-derived hormones or dura mater with only a very few cases attributable to neurosurgical instruments or corneal transplants. In the absence of a validated test for CJD infectivity in eye donors, the application of appropriate donor selection criteria and the use of single-use instruments in eye banks are currently the most effective means of reducing the risk of CJD transmission. Onward transmission by reusable ophthalmic surgical instruments has not been reported, but the risk cannot be excluded. Use of appropriate cleaning and disinfection protocols and the ability to identify and quarantine instruments that may have been used on an infected patient are important safeguards.

Fungal keratitis in the United Kingdom 2003-2005.

PURPOSE: To describe the incidence and current management of fungal keratitis in the United Kingdom. METHODS: Cases were identified prospectively through the British Ophthalmologic Surveillance Unit (BOSU) from December 2003 to November 2005. Questionnaire data were requested at diagnosis and at 6 months follow-up. Inclusion criteria were a positive culture or microsopic proof from a scraping or biopsy, and a normal residence in the United Kingdom. RESULTS: Data were available on 39 confirmed cases at diagnosis and 34 cases at follow-up. The minimum average annualised incidence was 0.32 (95% CI, 0.24-0.44) cases per million individuals. In 22 cases (56%), only Candida was isolated and 14 of these (63%) had prior ocular surface disease treated with topical steroid. A filamentary fungus infection was more common in male patients (P=0.02), often following trauma, and the differences in risk factors between types of fungal infection was statistically significant (P<0.001). One case had a mixed yeast and filamentary fungus infection. The most frequent initial topical therapies were amphotericin B (38%) or econazole (28%). In addition, oral fluconazole was used in 11 (31%) patients and oral itraconazole in six (15%). At follow-up, the vision in 15 eyes (44%) was <6/60 including three eyes eviscerated. CONCLUSIONS: This study provides data on the minimum incidence of fungal keratitis in the United Kingdom. It provides evidence of frequent delay in diagnosis after presentation to eye departments, inconsistent management, and poor outcome. Issues that can now be addressed.

Penetrating keratoplasty: indications, outcomes, and complications.

PURPOSE: Corneal transplantation is the most common form of transplantation with approximately 2500 grafts undertaken annually in the United Kingdom. The modern day success of transplantation is attributed to eye bank storage techniques, ocular pharmacology, and improved surgical techniques. METHODS: This retrospective case note review identified 203 penetrating keratoplasties (PKs) performed during a period from 1 January 2000 to 31 December 2003 at Manchester Royal Eye Hospital. Preoperative risk factors, surgical technique, postoperative complications, and Snellen acuity were analysed. RESULTS: The mean age of the recipient group was 56.7 years, with 107 right eyes and 96 left eyes. The mean follow-up was 61 months. The overall 5-year survival was 82%, with keratoconus and corneal dystrophies at 93 and 89%, respectively. Visual acuity had improved to 6/12 or better in 48% of patients postoperatively, compared with 8% preoperatively. Forty-three donor grafts (21%) underwent at least a single episode of endothelial rejection. Glaucoma was a finding in 37 (18%) of patients following PK. In all, 16 grafts of 15 patients were noted to have suffered microbial keratitis (MK), an incidence of 8%. CONCLUSIONS: PK is currently an effective long-term treatment option for improving visual function. An overall survival rate of 82% over 5 years is comparable with other published studies and is largely dependent on recipient factors. This report emphasises the significant complications of immunological rejection, glaucoma, and microbial keratitis, which continue to limit success.

Increased Rac2 mRNA expression in peripheral blood during human corneal graft rejection.

PURPOSE: Allograft rejection is the main cause of graft failure in human corneal transplantation, for which underlying pathomechanism is not yet clear. We compared gene expression in the peripheral blood of patients who after undergoing corneal transplantation experienced graft rejection with those patients who accepted grafts. METHODS: Sixty-six patients who underwent corneal transplantation were studied including 18 patients who suffered subsequent graft rejection. cDNA array technology was used to survey and quantify transcript expression. A semiquantitative reverse transcriptase-PCR (RT-PCR) was used to confirm the gene expression pattern measured by a cDNA array of selected genes. RESULTS: Among 265 genes present on the array, eight genes were found to be differentially expressed. Four genes (Rac 2, RhoA, paxillin, and CD18) were further analysed by semiquantitative RT-PCR, and significant differences in mRNA expression levels in the rejection group were confirmed. CONCLUSIONS: Our study demonstrated that the expression of Rac2 mRNA was upregulated in the peripheral blood of patients experiencing corneal transplantation rejection compared to those patients who had no rejection episodes. In addition, three genes, RhoA, paxillin, and CD18, showed decreased expression in rejecting patients. cDNA array technology provides a potentially useful approach to identify novel genes that might participate in pathogenic pathways during corneal graft rejection.

A comparison of clinical performance between disposable and Goldmann tonometers.

PURPOSE: Applanation tonometry as performed in routine clinical practice is a significant potential vehicle for cross-infection particularly in an emergency eye care setting. The aim of this study is to evaluate the accuracy and reliability of three single-use devices (Tonoshield, Tonosafe, Tonojet) as an alternative to standard Goldmann prisms in an emergency eye department. METHODS: All patients attending the eye casualty at the Manchester Royal Eye Hospital for a period of 4 months who required intraocular pressure measurement were eligible for this prospective study. Exclusion criteria were: age below 18 years, corneal anomalies that might affect measurement, and refusal to participate. After taking informed consent, the patient was examined by one experienced nurse practitioner, who measured the intraocular pressure three times. In the first part of the study, we compared the standard Goldmann prism vsTonoshield and Tonosafe prisms, while for the second part of the study we used standard Goldmann, Tonosafe, and Tonojet prisms. Agreement and repeatability tests were carried out on separate samples. RESULTS: Tonosafe and Tonojet correlated well with standard Goldmann tonometry (P<0.001), while the measurements obtained with Tonoshield were higher, especially for raised intraocular pressure measurements. Tonojet and Tonosafe measurements were more reproducible than Tonoshield measurements. CONCLUSIONS: This study shows that Tonosafe and Tonojet are accurate and reliable alternatives to standard Goldmann tonometry.

Epithelial proliferative potential of organ cultured corneoscleral rims; implications for allo-limbal transplantation and eye banking.

AIMS: To determine the epithelial proliferative capacity of organ cultured limbal tissue and correlate this with various donor and eye banking factors. METHODS: 24 corneoscleral limbal (CSL) rims left over from penetrating keratoplasty were split in half and set up as in vitro explant cultures. Corneal epithelial proliferative potential (CEPP) was assessed by the number of "cycles" of growth achieved before explants underwent exhaustion and failure to generate an epithelium to subconfluence. The dependence of CEPP on the age of the donor, time of death to enucleation, time of enucleation to organ culture, and time in organ culture in the eye bank was determined. RESULTS: CSL rims were capable of up to four cycles of culture with a wide variation between tissue samples. Of the various factors examined, death to enucleation time was the only statistically significant factor affecting the CEPP (regression coefficient: -0.062 (cycles/hour), CI -0.119 to -0.004, p = 0.037). Time in organ culture had little effect on CEPP. CONCLUSIONS: Preselected organ cultured CSL rims from eye banks may offer a viable alternative tissue source for use in allo-limbal transplantation.

Abnormal prion protein in the retina of the most commonly occurring subtype of sporadic Creutzfeldt-Jakob disease.

BACKGROUND: Involvement of the eye has been reported in patients with variant Creutzfeldt-Jakob disease (vCJD), but there is disagreement on whether retinal involvement occurs in sporadic Creutzfeldt-Jakob disease (sCJD). METHODS: Western blotting, paraffin embedded tissue blotting, and immunohistochemistry were used to test whether the abnormal form of the prion protein (PrPSc) accumulates to detectable levels in the eye in a case of the most common subtype of sCJD (MM1). RESULTS: Low levels of PrPSc were detectable in the retina, localised to the plexiform layers of the central retina. PrPSc was not detectable in other ocular tissues. CONCLUSIONS: The abnormal form of the prion protein is present in the retina in the most common sCJD subtype (MM1), albeit at levels lower than those found previously in vCJD and in sCJD of the VV2 subtype.

Incidence of keratitis of varying severity among contact lens wearers.

AIM: To determine the incidence of non-severe keratitis (NSK) and severe keratitis (SK) among wearers of current generation contact lenses. METHODS: A 12 month, prospective, hospital based epidemiological study was conducted by examining all contact lens wearers presenting with a corneal infiltrate/ulcer to a hospital centre in Manchester. A clinical severity matrix was used to differentiate between NSK and SK, based on the severity of signs and symptoms. The size of the hospital catchment population and the wearing modalities (daily wear (DW) or extended wear (EW)) and lens types being used were estimated from relevant demographic and market data. RESULTS: During the survey period, 80 and 38 patients presented with NSK and SK, respectively. The annual incidences (cases per 10,000 wearers) for each wearing modality and lens type were: DW rigid--NSK 5.7, SK 2.9; DW hydrogel daily disposable--NSK 9.1, SK 4.9; DW hydrogel (excluding daily disposable)--NSK 14.1, SK 6.4; DW silicone hydrogel--NSK 55.9, SK 0.0; EW rigid--NSK 0.0, SK 0.0; EW hydrogel--NSK 48.2, SK 96.4; EW silicone hydrogel--NSK 98.8, SK 19.8. The difference in SK between EW hydrogel and EW silicone hydrogel was significant (p = 0.04). CONCLUSIONS: A clinical severity matrix has considerable utility in assessing contact lens related keratitis. There is a significantly higher incidence of SK in wearers who sleep in contact lenses compared with those who only use lenses during the waking hours. Those who choose to sleep in lenses should be advised to wear silicone hydrogel lenses, which carry a five times decreased risk of SK for extended wear compared with hydrogel lenses.

Ocular findings in patients with solid tumours treated with the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib ('Iressa', ZD1839) in Phase I and II clinical trials.

PURPOSE: To describe the strategy used for large-scale ophthalmological monitoring in the clinical development of the novel anticancer agent gefitinib ('Iressa', ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, which had demonstrated ocular effects in preclinical animal models. METHODS: In this extensive clinical trial programme, patients in Phase I and II trials underwent frequent and intensive ophthalmological monitoring at baseline and during the trials. Data were reviewed by an external independent Ophthalmology Advisory Board. RESULTS: Ophthalmological data for 221 patients in Phase I trials of gefitinib and 425 patients in Phase II trials revealed no evidence of any consistent or drug-related ophthalmological toxicity. Interestingly, the baseline data revealed that, in an asymptomatic population, transient ophthalmological events are identified during monitoring. CONCLUSIONS: This study reports the methodology and normative data in an ophthalmological screening programme that should prove useful for future studies.

External ocular infections due to methicillin-resistant Staphylococcus aureus (MRSA).

PURPOSE: To determine the prevalence and clinical characteristics of external ocular infections caused by methicillin-resistant Staphylococcus aureus (MRSA) in an ophthalmic hospital in the UK. METHODS: A retrospective analysis of the case notes of patients who had culture proven external ocular Staphylococcal infections during a 44-month period was undertaken. RESULTS: There were a total of 548 external eye infections caused by Staphylococcus aureus. Of these, 17 (3%) were MRSA positive. The most common presentation was conjunctivitis seen in six patients. All MRSA isolates were sensitive to chloramphenicol. Ofloxacin resistance was observed in all isolates from patients over the age of 50 years. All patients had an underlying history of either an ocular surface disease, malignancy, or a debilitating medical illness. CONCLUSIONS: MRSA is as yet an infrequent cause of external ocular infections. Patients typically have underlying ocular risk factors and/or are medically debilitated. Different strains infect young and old age groups with characteristic antimicrobial sensitivity. This study highlights the need for more work to establish the role of MRSA commensals and ocular infections.