Effects of histatin-1 peptide on human corneal epithelial cells.

PURPOSE: Ocular surface and corneal epithelial wounds are common and potentially debilitating problems. Ideal treatments for these injuries would promote epithelial healing without inflammation, infection and scarring. In addition the best treatments would be cost-efficient, effective, non-toxic and easily applied. Histatin-1 peptides have been shown to be safe and effective enhancers of epithelial wound healing in other model systems. We sought to determine whether histatin-1 peptides could enhance human corneal epithelial wound healing in vitro. METHODS: Histatin-1 peptides were applied to human corneal epithelial cells and compared over useful dose ranges in scratch assays using time-lapse microscopy. In addition, path finding analysis, cell spreading assays, toxicity and proliferation assays were performed to further characterize the effects of histatin-1 peptide on human corneal limbal epithelial (HCLE). RESULTS: Histatin-1 enhanced human corneal epithelial wound healing in typical wound healing models. There was minimal toxicity and no significant enhancement of proliferation of corneal epithelium in response to histatin-1 application. Corneal epithelial spreading and pathfinding appeared to be enhanced by the application of histatin-1 peptides. CONCLUSIONS: Histatin -1 peptide may enhance migration of HCLE cells and wound healing in vitro. These peptides may have benefit in corneal epithelial wounds and need to be investigated further.

Tear fluid extracellular DNA: diagnostic and therapeutic implications in dry eye disease.

PURPOSE: To determine the abundance of extracellular DNA (eDNA) in tear fluid of patients with dry eye disease (DED) and to report clinical outcomes after DNase I eyedrops use to reduce excessive tear fluid eDNA. METHODS: Tear fluid was collected from healthy control subjects and patients with DED. The eDNA abundance was determined with the PicoGreen dye assay. The DED symptoms and clinical signs were recorded and correlated with eDNA abundance. Two patients with DED having excessive eDNA in tear fluid were treated with DNase I eyedrops. RESULTS: The PicoGreen dye assay measures tear fluid eDNA abundance after a 2-minute incubation time. With longer incubations, admixed cells also contribute to eDNA measurements. The mean (SE) eDNA abundance in healthy control subjects' tear fluid was 1.4 (0.2) µg/mL. The mean (SE) eDNA abundance in tear fluid of patients with nonautoimmune DED, autoimmune DED, and graft versus host disease was significantly higher: the values were 2.9 (0.6), 5.2 (1.2), and 9.1 (2.3) µg/mL, respectively (P < 0.05). In most of these patients, the PicoGreen dye kinetic assay of tear fluid showed an increase in fluorescence signal due to the presence of viable cells in tear fluid. Tear fluid eDNA had the best correlation with corneal Rose Bengal staining (r = 0.55). Treatment of patients having DED with DNase I eyedrops reduced eDNA abundance, abrogated signal increase, and improved comfort. CONCLUSIONS: Excessive eDNA is present in tear fluid of patients with dry eyes. A novel therapeutic approach for managing DED may be to measure eDNA abundance in tear fluid with the PicoGreen dye assay and reduce excessive amounts with DNase I eyedrops.

Case report of a small outbreak of epidemic dropsy.

Epidemic dropsy results from use of edible oils adulterated with Argemone mexicans oil. We report of the disease in a family caused due to sesame oil adulteration. Peculiarity to report this disease is that it occurred due to consumption of contaminated sesame seeds oil instead of mustard oiland secondly epidemic dropsy is also not very common in western Rajasthan. Five members of a family presented to the hospital with history of generalised body swelling, itching and marked cutaneous flush. Few of them also have shortness of breath, dry cough, palpitation, diarrhoea, vomiting and haemoptysis. On physical examination rashes, pallor and signs of cardiac failure were present in few patients. None of them had neurological symptoms and signs. After multiple interviews history of consumption of home made sesame seeds oil was explored in the family and the differential diagnosis of epidemic dropsy was made. Edible oil sample of the sesame seeds oil turned out to be positive for sanguinarine. Sanguinarine and dehydrosanguinarine are two major toxic alkaloids of Argemone oil, which cause widespread capillary dilatation, proliferation and increased capillary permeability. Leakage of the protein-rich plasma component into the extracellular compartment leads to the formation of oedema. There is no specific therapy. Removal of the adulterated oil and symptomatic treatment of congestive cardiac failure and respiratory symptoms, along with administration of anti-oxidants and multivitamins, remains the mainstay of treatment.

Ocular surface extracellular DNA and nuclease activity imbalance: a new paradigm for inflammation in dry eye disease.

PURPOSE: We determined whether nucleases are deficient in the tear fluid of dry eye disease (DED) patients, and whether this causes extracellular DNA (eDNA) and neutrophil extracellular trap (NET) accumulation in the precorneal tear film, thus causing ocular surface inflammation. METHODS: Exfoliated cells adhered to Schirmer test strips were collected on glass slides, and immunofluorescence confocal microscopy was used to evaluate neutrophils, eDNA, NETs, and their molecular components. Similar experiments were performed with mucoid films collected from the inferior conjunctival fornix or bulbar conjunctiva. We used quantitative PCR to evaluate eDNA signaling pathways and inflammatory cytokine expression. We also determined the amount of ocular surface eDNA and evaluated tear fluid nuclease activity. RESULTS: eDNA, NETs, and neutrophils were present on the ocular surface in DED patients and abundant in mucoid films. NETs consisted of eDNA, histones, cathelicidin, and neutrophil elastase. Tear fluid nuclease activity was decreased significantly in DED patients, whereas the amount of eDNA on the ocular surface was increased significantly. Expression of genes downstream of eDNA signaling, such as TLR9, MyD88, and type I interferon, as well as the inflammatory cytokines interleukin-6 and tumor necrosis factor-α, was significantly increased in DED patients. CONCLUSIONS: Extracellular DNA production and clearance mechanisms are dysregulated in DED. Nuclease deficiency in tear fluid allows eDNA and NETs to accumulate in precorneal tear film, and results in ocular surface inflammation. These findings point to novel therapeutic interventions in severe DED based on clearance of eDNA, NETs, and other molecular components from the ocular surface.

Fluoroscopic screening of asymptomatic patients implanted with the recalled Riata lead family.

BACKGROUND: The Food and Drug Administration recently issued a class I recall of the St. Jude Medical Riata implantable cardioverter-defibrillator lead presumably because of increased risk of electric failure and mechanical separation via inside-out abrasion. We sought to examine the incidence and time dependence of inside-out abrasion in asymptomatic patients implanted with the Riata lead. METHODS AND RESULTS: Asymptomatic patients implanted with the Riata lead at our institution were offered voluntary fluoroscopic screening in 3 views. Electric testing of the Riata lead with provocative isometric muscle contraction was performed at the time of fluoroscopic screening. Of the 245 patients undergoing fluoroscopic screening, 53 (21.6%) patients showed clear evidence of lead separation. Of these externalized leads, 0%, 13%, and 26% had a dwell time of <3 years, 3 to 5 years, and >5 years, respectively (P=0.037). Externalized leads had a significantly pronounced decrease in R-wave amplitude (-1.7±2.9 mV versus +0.35±2.5 mV; P<0.001), and more patients with externalized leads had ≥25% decrease in R-wave amplitude from baseline (28.0% versus 8.1%; P=0.018). One patient with externalization exhibited new noise on near-field electrogram. CONCLUSIONS: The Riata lead exhibits time-dependent high rates of cable externalization exceeding 20% at >5 years of dwell time. Externalized leads are associated with a more pronounced decrease in R-wave amplitude, which may be an early marker of future electric failure. The use of fluoroscopic and electric screening of asymptomatic patients with the Riata lead remains controversial in the management of patients affected by the recent Food and Drug Administration recall.

A novel manoeuvre for discerning supraventricular tachycardia mechanism.

AIMS: Discerning supraventricular tachycardia (SVT) mechanism during catheter ablation procedures can be difficult and time-consuming, which, when combined with diagnostic error, places patients at risk of unnecessary complications. Distinguishing atrial tachycardia (AT) from AV nodal re-entry tachycardia (AVNRT) may be particularly vexatious. Value-added techniques are thus always welcome, particularly if they are not time-consuming nor require complex intracardiac lead configurations. In this study, we assessed whether a new technique, simultaneous right atrial and right ventricular pacing (RA + RV) during ongoing SVT, met these criteria. METHODS AND RESULTS: Using a simple intracardiac lead configuration (right atrial appendage, His bundle, right ventricular apex), the response to RA + RV delivered at 80-90% of the SVT cycle length, was examined in 80 patients referred for catheter ablation. In each patient, the actual tachycardia mechanism was adjudicated by standard electrophysiologic criteria ± successful catheter ablation. Mechanisms of SVT included, non-exclusively, AVNRT (24 patients), accessory pathway-mediated (orthodromic) re-entry (AVRT; 23 patients), AT (10 patients), and sinus tachycardia (ST induced with isoproterenol; 49 patients). Immediately after cessation of RA + RV pacing during persistent SVT, the first intracardiac electrogram observed was right atrial in all AT whereas it was His bundle in all AVNRT. The response during AVRT was mixed. CONCLUSIONS: In this preliminary evaluation, RA + RV pacing appears to add value to the existing armamentarium of electrophysiologic indices to discern SVT mechanism, in particular with respect to discriminating between AVNRT and AT.

Correlation of electrical and mechanical reverse remodeling after cardiac resynchronization therapy.

BACKGROUND: Cardiac resynchronization therapy (CRT) improves clinical outcome in many patients with refractory heart failure (HF). This study examined whether CRT is associated with reverse electrical remodeling by surface electrocardiogram (ECG). METHODS: Consecutive CRT recipients at the University of Pittsburgh Medical Center with >90 days of follow-up were included in this analysis. ECG data were abstracted from medical records. Subjects with a relative increase of > or =15% in left ventricular ejection fraction (LVEF) after CRT were considered responders. RESULTS: A total of 113 patients (age 69 +/- 11 years, men 70%, white 92%) were followed for a mean duration of 407 +/- 290 (92-1439) days. Overall, LVEF increased after CRT (29 +/- 13% vs 24 +/- 9%; P < 0.01) and 50% of patients were responders. The mean native QRS interval among responders was higher than in nonresponders (163 +/- 32 ms vs 148 +/- 29 ms; P < 0.01). More than 3 months after CRT, there was no change in the paced QRS duration compared to baseline. Paced QRS duration, however, decreased among responders and increased among nonresponders and was significantly different by response status (P < 0.001). There was a significant correlation between increase in LVEF and decrease in paced QRS width in the overall population (r =-0.3; P < 0.01). CONCLUSIONS: Among responders to CRT, the paced QRS width decreases significantly, whereas it increases among nonresponders. Given the paced nature of the QRS, the improved conduction probably reflects enhanced cell-to-cell coupling after CRT as opposed to improved conduction within the His-Purkinje system. These findings have significant implications as to the mechanisms of benefit from CRT.

Biocatalysis of the anticancer sipholane triterpenoids.

The Red Sea sponge Callyspongia (= Siphonochalina) siphonella is a rich source of sipholane triterpenoids. Biocatalysis of the major sipholanes, sipholenol A (1) and sipholenone A (2), respectively, by Mucor ramannianus ATCC 9628 and Cunninghamella elegans ATCC 7929 afforded four new metabolites 3 - 6 along with sipholenol G and 28-hydroxysipholenol A. Major sipholanes along with their biocatalytic products were investigated for their antiproliferative activity against the highly malignant +SA mouse mammary epithelial cell line. Sipholenone A (2) was the most active sipholane inhibiting +SA cell proliferation with an IC(50) value of 20 - 30 microM. Sipholenone A, also, showed cytotoxicity against MCF-7 at a dose of 0.9 microM and antiangiogenic activity in the CAM (chorio-allantoic membrane) assay. This is the first report on anticancer activity of these triterpenoids.

Mechanisms of delayed electrical uncoupling induced by ischemic preconditioning.

Electrical uncoupling of cardiac myocytes during ischemia is delayed by ischemic preconditioning. This presumably adaptive response may limit development of arrhythmia substrates. To elucidate responsible mechanisms, we studied isolated, perfused rat hearts subjected to a standard preconditioning protocol of 3 cycles of 3 minutes of global no-flow ischemia each followed by 5 minutes of reperfusion before a 30-minute interval of ischemia. Changes in coupling were monitored by measuring whole-tissue resistance. Changes in phosphorylation and subcellular distribution of connexin43 (Cx43) were defined by quantitative immunoblotting and confocal microscopy. Preconditioning caused a 34% decrease in the maximal rate of uncoupling and delayed the time to plateau in uncoupling. Dephosphorylation of Cx43, known to occur during uncoupling induced by ischemia, was dramatically decreased in preconditioned hearts. Translocation of Cx43 from gap junctions to the cytosol, also known to occur during ischemia, was reduced by >5-fold in preconditioned hearts. The KATP channel blockers glybenclamide and 5-hydroxydecanoate prevented these effects in preconditioned hearts, whereas the KATP channel agonist diazoxide mimicked these effects in nonpreconditioned hearts. Intracellular translocation of Cx43 was blocked, but Cx43 dephosphorylation was not blocked during ischemia in preconditioned hearts treated with the PKC inhibitors chelerythrine and calphostin C. Uncoupling during ischemia was accelerated by PKC and KATP channel inhibition. Thus, delayed uncoupling in preconditioned hearts is likely related to diminished dephosphorylation and intracellular redistribution of Cx43 during prolonged ischemia. Both of these effects are regulated by activation of KATP channels, whereas PKC plays a role in internalization of Cx43.