Real-world visual acuity outcomes between ranibizumab and aflibercept in treatment of neovascular AMD in a large US data set.

PurposeTo examine 12-month real-world visual acuity outcomes and treatment patterns in neovascular age-related macular degeneration (nAMD) eyes, including those with pigment epithelial detachment (PED), receiving ranibizumab or aflibercept.Patients and methodsElectronic medical records were used to identify ranibizumab or aflibercept-treated nAMD eyes with 12 months follow-up from first prescription. The primary objective was to compare mean change in visual acuity (VA) between index and month 12, in eyes treated with ranibizumab and aflibercept to assess the non-inferiority of ranibizumab vs aflibercept using a -5 letter non-inferiority margin. The number of injections and non-injection visits during follow-up were key secondary objectives.ResultsA total of 3350 ranibizumab and 4300 aflibercept treatment-naive eyes were included. At month 12, mean change from index in VA letter score was -0.30 for ranibizumab and -0.19 for aflibercept (P=0.81). The adjusted difference of mean change was -0.14 (-0.79 to 0.51) (P=0.67) (generalized estimating equations method) confirming the non-inferiority of ranibizumab. Eyes received a similar number of injections during follow-up. The mean (±SD) number of ranibizumab and aflibercept injections were 6.70 (±2.54) and 7.00 (±2.40), respectively (P<0.0001). In PED eyes, the mean (±SD) change between baseline to month 12 was 1.25 (±11.3) for ranibizumab and -0.39 (±13.3) for aflibercept (adjusted between-group difference 1.80 (-0.71 to 4.30; P=0.16)) achieved with a mean (±SD) 7.85 (±2.68) ranibizumab and 7.47 (±2.45) aflibercept injections, (P=0.11).ConclusionsRanibizumab and aflibercept treatment yielded comparable VA outcomes in nAMD eyes, including those with PED, with similar treatment patterns over 12 months in real-world clinical practice.

Identification and characterization of a family of Rab11-interacting proteins.

Rab11a is a small GTP-binding protein enriched in the pericentriolar plasma membrane recycling systems. We hypothesized that Rab11a-binding proteins exist as downstream effectors of its action. Here we define a family of four Rab11-interacting proteins: Rab11-Family Interacting Protein 1 (Rab11-FIP1), Rab11-Family Interacting Protein 2 (Rab11-FIP2), Rab11-Family Interacting Protein 3 (Rab11-FIP3), and pp75/Rip11. All four interacting proteins associated with wild type Rab11a and dominant active Rab11a (Rab11aS20V) as well as Rab11b and Rab25. Rab11-FIP2 also interacted with dominant negative Rab11a (Rab11aS25N) and the tail of myosin Vb. The binding of Rab11-FIP1, Rab11-FIP2, and Rab11-FIP3 to Rab11a was dependent upon a conserved carboxyl-terminal amphipathic alpha-helix. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 colocalized with Rab11a in plasma membrane recycling systems in both non-polarized HeLa cells and polarized Madin-Darby canine kidney cells. GFP-Rab11-FIP3 also colocalized with Rab11a in HeLa cells. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 also coenriched with Rab11a and H(+)K(+)-ATPase on parietal cell tubulovesicles, and Rab11-FIP1 and Rab11-FIP2 translocated with Rab11a and the H(+)K(+)-ATPase upon stimulating parietal cells with histamine. The results suggest that the function of Rab11a in plasma membrane recycling systems is dependent upon a compendium of protein effectors.

8-Cl-adenosine induces growth arrest without differentiation of primary mouse epidermal keratinocytes.

In some cell systems, the antiproliferative effects of 8-Cl-cAMP, a site-selective cAMP analog specific for the type I cAMP-dependent protein kinase, are mediated by its metabolite, 8-Cl-adenosine. These effects were once thought to be specific to transformed cells. We investigated the ability of 8-Cl-adenosine to regulate growth and differentiation in primary cultures of mouse epidermal keratinocytes. A 24 h exposure of keratinocytes to 8-Cl-adenosine inhibited [3H]thymidine incorporation in a dose-dependent manner with an apparent IC(50) of 7.5 microM, and these effects were completely reversible. To determine the ability of 8-Cl-adenosine to induce differentiation of primary keratinocytes, we measured keratin-1 expression and transglutaminase activity, markers of early and later stages of keratinocyte differentiation, respectively. Interestingly, exposure of keratinocytes to 25 microM 8-Cl-adenosine for 24 h had no effect on keratin-1 expression or transglutaminase activity. The 8-Cl-adenosine-induced growth arrest of keratinocytes required uptake of the compound and was accompanied by an increase in protein expression of the cyclin-dependent protein kinase inhibitor p21(WAF1/Cip1). These results demonstrate that 8-Cl-adenosine inhibits growth in a non-transformed/non-immortalized cell system, possibly through an elevation in p21(WAF1/Cip1) protein levels, without inducing differentiation.

Inhibition of [(3)H]thymidine transport is a nonspecific effect of PDMP in primary cultures of mouse epidermal keratinocytes.

Inhibitors of sphingolipid metabolism are frequently used to investigate the role of ceramide and other sphingolipids as intracellular signaling molecules. For example, the inhibitor of glucosylceramide synthase D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) is commonly used to deplete glycosphingolipids and increase ceramide levels. Ceramide is known to induce growth arrest and differentiation of keratinocytes, and we hypothesized that PDMP would increase ceramide levels and induce growth arrest of primary cultures of mouse epidermal keratinocytes. As expected, PDMP increased ceramide levels and decreased the incorporation of [(3)H]thymidine into DNA, but surprisingly, PDMP was found to rapidly inhibit the intracellular transport of [(3)H]thymidine. This is likely due to a direct effect on nucleoside transport by PDMP and not due to elevations in ceramide levels because increasing ceramide levels by the addition of exogenous sphingomyelinase had no effect on [(3)H]thymidine transport. Furthermore, it is unlikely that the decreased [(3)H]thymidine transport is in response to growth arrest because PDMP had no effect on the cell cycle profile of keratinocytes. These results reveal that PDMP inhibits nucleoside transport independent of effects on ceramide levels or cell growth but probably by a direct effect on the nucleoside transport apparatus. Thus, this compound may be unsuitable for investigating the role of ceramide or other sphingolipids in some cellular processes.

Sustained phospholipase D activation is associated with keratinocyte differentiation.

Our previous results and data in the literature have suggested a potential role for phospholipase D (PLD) in the regulation of epidermal keratinocyte growth and differentiation. Therefore, we investigated the effect of agents reported to modulate keratinocyte growth and differentiation on PLD activation. The purported protein kinase C (PKC) 'inhibitor', staurosporine (Stsp), has been reported to activate PKC in keratinocytes, eliciting many of the same effects as active tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Stsp also induces a programmed pattern of differentiation similar to that seen in keratinocytes in vivo; TPA, on the other hand, appears to preferentially elicit markers consistent with late (granular) differentiation. In contrast, bradykinin is reported to stimulate keratinocyte proliferation. We found that these three agents had different effects on PLD activation in primary mouse epidermal keratinocytes. TPA increased PLD activity acutely and in a sustained fashion. In contrast, Stsp did not acutely activate PLD and inhibited acute TPA-induced activation of PLD. However, treatment of keratinocytes with Stsp for longer time periods (3-5 h) induced sustained PLD activation and this long-term effect was additive with that of TPA. Bradykinin activated PLD acutely but transiently. Both TPA and Stsp increased transglutaminase activity, a marker of late differentiation, whereas bradykinin had little or no effect on either cell proliferation or transglutaminase activity. These results suggest that a sustained activation of PLD is associated with the induction of keratinocyte differentiation. We hypothesize that PLD activity mediates late keratinocyte differentiation through generation of diacylglycerol and activation of specific PKC isoforms. Furthermore, we propose that the profound and immediate TPA-induced stimulation of PLD activity 'drives' the keratinocytes to late differentiation steps. However, the less efficacious (and more gradual) sustained activation of PLD by Stsp may allow a patterned differentiation more like that observed in skin.

1,25-dihydroxyvitamin D3 induces phospholipase D-1 expression in primary mouse epidermal keratinocytes.

The hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) elicits the programmed pattern of differentiation in epidermal keratinocytes. Based on data indicating a potential role of phospholipase D (PLD) in mediating keratinocyte differentiation, we investigated the effect of 1,25(OH)2D3 on PLD expression. A 24-h exposure to 1, 25(OH)2D3 stimulated PLD-1, but not PLD-2, mRNA expression. This 1, 25(OH)2D3-enhanced expression was accompanied by increased total PLD and PLD-1 activity. Time course studies indicated that 1,25(OH)2D3 induced PLD-1 expression by 8 h, with a maximal increase at 20-24 h. Exposure to 1,25(OH)2D3 inhibited proliferation over the same time period with similar kinetics. Expression of the early (spinous) differentiation marker keratin 1 decreased in response to 1, 25(OH)2D3 over 12-24 h. Treatment with 1,25(OH)2D3 enhanced the activity of transglutaminase, a late (granular) differentiation marker, by 12 h with a maximal increase after 24 h. In situ hybridization studies demonstrated that the highest levels of PLD-1 expression are in the more differentiated (spinous and granular) layers of the epidermis, with little expression in basal keratinocytes. Our results suggest a role for PLD expression/activity during keratinocyte differentiation.

A potential role for ceramide in the regulation of mouse epidermal keratinocyte proliferation and differentiation.

We have previously determined that sustained phospholipase D (PLD) activation is associated with differentiation induction in primary mouse epidermal keratinocytes. We therefore investigated the effect of two bacterial PLD on keratinocyte proliferation and differentiation. We found that Streptomyces sp. PLD was much less potent at inhibiting proliferation than S. chromofuscus PLD, with a half-maximal inhibitory concentration of 0.05 versus less than 0.001 IU per ml for S. chromofuscus PLD. Similarly, S. chromofuscus PLD stimulated transglutaminase activity more effectively and potently than S. sp. PLD. When we examined the formation of products by the two PLD, we found that the S. sp. PLD showed higher activity at all concentrations. Whereas the PLD from S. sp. is relatively inactive on sphingomyelin, S. chromofuscus PLD is known to hydrolyze both glycerophospholipids and sphingomyelin. Based on recent data indicating a role for ceramide in regulating cell growth and differentiation, we hypothesized that the ability of S. chromofuscus PLD to hydrolyze sphingomyelin might underlie its greater potency. Therefore, we examined the effect of exogenous sphingomyelinase and synthetic ceramides on DNA synthesis. We found that sphingomyelinase exhibited a potent concentration-dependent effect on [3H]thymidine incorporation, much like S. chromofuscus PLD. Synthetic cell-permeable ceramides (C6- and C2-ceramide) also concentration dependently inhibited DNA synthesis, with a half-maximal inhibitory concentration of approximately 12 microM. Finally, we obtained evidence suggesting that ceramide is generated in response to a physiologically relevant agent, because tumor necrosis factor-alpha, a known effector of sphingomyelin turnover in other systems and a cytokine that is produced and released by keratinocytes, increased ceramide levels in primary epidermal keratinocytes.

AANA journal course: update for nurse anesthetists--the laryngeal mask airway: attributes and inadequacies.

The laryngeal mask airway (LMA) is a relatively new and innovative means to control the airway during anesthesia or in emergent situations. It is simply a small infatable mask that forms a low pressure seal around the laryngeal inlet that can be used in spontaneous or controlled breathing in adult and pediatric patients. The LMA is less invasive than the endotracheal tube, does not require laryngoscopy or the use of muscle relaxants, and has been used in the management of the difficult airway and with fiberoptic facilitated intubation. It does not protect against aspiration and is contraindicated in patients at risk for this complication.

Probable succinylcholine-induced hyperkalemia in a trauma victim after recent benign anesthetics with succinylcholine.

Throughout its pharmacological history in anesthesia practice, succinylcholine has been notorious for its role in causing life-threatening hyperkalemia. Normally the serum potassium level will increase .5 to 1.0 mEq/L because of a sustained opening of the receptors in the neuromuscular junction and release of myoplasmic potassium. However, in certain patients the drug will result in a much higher level of serum potassium. The literature points out numerous conditions that predispose particular patients to this exaggerated intensification of potassium leakage from within the cell. Traumatized, neuromuscular diseased, infected, exsanguinated, acidotic, and closed head injury patients among this list. This case report describes a trauma patient requiring numerous orthopedic repairs in a relatively short period of time. He received succinylcholine on three separate occasions within a 20-day span without any untoward effects. However, during his sixth surgery and the fourth administration of succinylcholine, he developed ventricular fibrillation requiring defibrillation and cardiopulmonary resuscitation before converting to a sinus tachycardia. After ruling out other causes of the arrhythmia, the most probable one was hyperkalemia. A possible explanation of this probable succinylcholine-induced hyperkalemia may have been a combination of the trauma, tissue wasting, infection, immobility, or acute respiratory acidosis. The potassium elevation was treated successfully with hyperventilation, calcium chloride, sodium bicarbonate, glucose, and insulin. The patient recovered without complications and was later discharged to home. Succinylcholine definitely has its use in anesthesia, but it is imperative for the provider to be discriminatory in its administration. An all-encompassing history is paramount to discover any hidden rationale not to use succinylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreasing glycolysis increases sensitivity to mitochondrial inhibition in primary cultures of renal proximal tubule cells.

We have previously shown that shaking the culture plates (SHAKE) of rabbit renal proximal tubule cells (RPTC) to maintain adequate aeration increased aerobic metabolism and decreased the induction of glycolysis compared to RPTC cultured under standard conditions (STILL). However, glycolysis in SHAKE RPTC remained elevated compared to glycolysis in proximal tubules in vivo. In the present study the contribution of culture medium sugar composition and concentration to glycolytic metabolism was assessed in RPTC. SHAKE and STILL RPTC cultured in 5 mM glucose contained lactate levels equivalent to the respective SHAKE and STILL RPTC cultured in standard culture medium which contains 17.5 mM glucose. Similarly, the activity of lactate dehydrogenase was unchanged by lowering the medium glucose concentration. Substituting 5 mM galactose for 5 mM glucose in the culture medium significantly reduced the lactate content of both SHAKE and STILL RPTC but had no effect on lactate dehydrogenase activity. Cell growth was equivalent under all culture conditions. Sensitivity to mitochondrial inhibition was determined for each culture condition by measuring cell death after exposure to the respiratory inhibitor antimycin A. The results showed a hierarchy of sensitivity to antimycin A (5 mM galactose SHAKE > 5 mM glucose SHAKE > 17.5 mM glucose SHAKE = 17.5 mM glucose STILL), which was generally inversely correlated with the level of glycolysis as measured by lactate content (17.5 mM glucose STILL > 17.5 mM glucose SHAKE = 5 mM glucose SHAKE > 5 mM galactose SHAKE).

The role of short chain fatty acid substrates in aerobic and glycolytic metabolism in primary cultures of renal proximal tubule cells.

This study examined the role of odd and even short-chain fatty acid substrates on aerobic and glycolytic metabolism in well-aerated primary cultures of rabbit renal proximal tubule cells (RPTC). Increasing oxygen delivery to primary cultures of RPTC by shaking the dishes (SHAKE) reduced total lactate levels and lactate dehydrogenase (LDH) activity and reduced net glucose consumption compared to RPTC cultured under standard conditions (STILL). The addition of butyrate, valerate, heptanoate, or octanoate to SHAKE RPTC produced variable effects on glycolytic metabolism. Although butyrate and heptanoate further reduced total lactate levels and net glucose consumption during short-term culture (< 24 h), no fatty acid tested further reduced total lactate levels, net glucose consumption, or LDH activity during long-term culture (7 days). During the first 12 h of culture, maintenance of aerobic metabolism in SHAKE RPTC was dependent on medium supplementation with fatty acid substrates (2 mM). However, by 24 h, SHAKE RPTC did not require fatty acid substrates to maintain levels of aerobic metabolism equivalent to freshly isolated proximal tubules and greater than STILL RPTC. This suggests that SHAKE RPTC undergo adaptive changes between 12 and 24 h of culture, which give RPTC the ability to utilize other substrates for mitochondrial oxidation, therefore allowing greater expression of mitochondrial oxidative potential in SHAKE RPTC than in STILL RPTC.