Accrual and retention of diverse patients in psychosocial cancer clinical trials.

Background: Minority and older adult patients remain underrepresented in cancer clinical trials (CCTs). The current study sought to examine sociodemographic inequities in CCT interest, eligibility, enrollment, decline motivation, and attrition across two psychosocial CCTs for gynecologic, gastrointestinal, and thoracic cancers. Methods: Patients were approached for recruitment to one of two interventions: (1) a randomized control trial (RCT) examining effects of a cognitive-behavioral intervention targeting sleep, pain, mood, cytokines, and cortisol following surgery, or (2) a yoga intervention to determine its feasibility, acceptability, and effects on mitigating distress. Prospective RCT participants were queried about interest and screened for eligibility. All eligible patients across trials were offered enrollment. Patients who declined yoga intervention enrollment provided reasons for decline. Sociodemographic predictors of enrollment decisions and attrition were explored. Results: No sociodemographic differences in RCT interest were observed, and older patients were more likely to be ineligible. Eligible Hispanic patients across trials were significantly more likely to enroll than non-Hispanic patients. Sociodemographic factors predicted differences in decline motivation. In one trial, individuals originating from more urban areas were more likely to prematurely discontinue participation. Discussion: These results corroborate evidence of no significant differences in CCT interest across minority groups, with older adults less likely to fulfill eligibility criteria. While absolute Hispanic enrollment was modest, Hispanic patients were more likely to enroll relative to non-Hispanic patients. Additional sociodemographic trends were noted in decline motivation and geographical prediction of attrition. Further investigation is necessary to better understand inequities, barriers, and best recruitment practices for representative CCTs.

Are Semi-Quantitative Clinical Cultures Inadequate? Comparison to Quantitative Analysis of 1053 Bacterial Isolates from 350 Wounds.

Early awareness and management of bacterial burden and biofilm is essential to wound healing. Semi-quantitative analysis of swab or biopsy samples is a relatively simple method for measuring wound microbial load. The accuracy of semi-quantitative culture analysis was compared to 'gold standard' quantitative culture analysis using 428 tissue biopsies from 350 chronic wounds. Semi-quantitative results, obtained by serial dilution of biopsy homogenates streaked onto culture plates divided into 4 quadrants representing occasional, light, moderate, and heavy growth, were compared to total bacterial load quantified as colony-forming units per gram (CFU/g). Light growth, typically considered an insignificant finding, averaged a clinically significant 2.5 × 105 CFU/g (SE = 6.3 × 104 CFU/g). Occasional growth (range: 102-106 CFU/g) and light growth (103-107 CFU/g) corresponded to quantitative values that spanned a 5-log range; moderate and heavy growth corresponded to a range of 4-log and 6-log, respectively, with a high degree of overlap in range of CFU/g per category. Since tissue biopsy and quantitative culture cannot be widely practiced and semi-quantitative analysis is unreliable, other clinically relevant approaches are required to determine wound bioburden and guide best management practices. Fluorescence imaging is a point-of-care technology that offers great potential in this field.

Modulating Ocular Scarring in Glaucoma Filtration Surgery Using the Epigenetic Adjunct Suberoylanilide Hydroxamic Acid.

AIM: The aim of this study is to assess the effectiveness of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDI) with a broad spectrum epigenetic activity, in improving filtration bleb survival as an adjunct therapy to glaucoma filtration surgery (GFS) in the rabbit model. MATERIALS AND METHODS: Eighteen New Zealand White rabbits underwent GFS in the left eye and were randomized to receive either a subconjunctival (SC) injection of 0.1 mL SAHA (9.25 µg/mL) or balanced saline solution (BSS) at the end of surgery, or a 3-minute intraoperative topical application of 0.4 mg/mL mitomycin-C (MMC). Bleb survival and histology were compared. RESULTS: Blebs of rabbits receiving injections of SAHA survived an average (mean ± SD) of 23.2 ± 2.7 days. SAHA rabbits showed a nonsignificant improvement over rabbits that received an injection of BSS, which had a mean survival time of 19.7 ± 2.7 days (p = 0.38) according to a one-way analysis of variance (ANOVA). Eyes receiving intraoperative topical MMC survived an average of 32.5 ± 3.3 days, which is significantly longer than both the control group treated with BSS (p = 0.01) and the experimental group treated with the SAHA (p = 0.0495). SAHA was well tolerated and showed no significant avascularity, necrosis, or conjunctival thinning. CONCLUSION: Although it was well tolerated, a single intraoperative injection of SAHA did not significantly prolong bleb survival in the rabbit model. CLINICAL SIGNIFICANCE: Epigenetic adjuncts hold promise for improving GFS outcome; however, future studies must continue to examine different administration protocols and dosages to substantiate their efficacy. HOW TO CITE THIS ARTICLE: Rodgers CD, Lukowski ZL, et al. Modulating Ocular Scarring in Glaucoma Filtration Surgery Using the Epigenetic Adjunct Suberoylanilide Hydroxamic Acid. J Curr Glaucoma Pract 2019;13(1):37-41.

Interleukin-6 and body mass index, tobacco use, and sleep in gynecologic cancers.

OBJECTIVE: Elevated body mass index (BMI), tobacco use, and sleep disturbance are common health concerns among women with gynecologic cancers. The extent to which these factors are associated with systemic inflammation in gynecologic cancers is unknown. This is a significant literature gap given that (a) chronic, systemic inflammation may mediate relationships between behavioral health factors and cancer outcomes; and (b) elevated BMI, tobacco use, and sleep disturbances can be modified via behavioral interventions. This study examined Interleukin-6 (IL-6) relations with BMI, tobacco use history, and sleep disturbances in patients undergoing surgery for suspected gynecologic cancer. METHOD: Participants were 100 women (M age = 58.42 years, SD = 10.62 years) undergoing surgery for suspected gynecologic cancer. Smoking history was determined by participant self-report. Sleep quality/disturbance was assessed via the Pittsburgh Sleep Quality Index. BMI was abstracted from electronic health records. Presurgical serum IL-6 concentrations were determined using Enzyme-Linked Immunosorbent Assay. RESULTS: Controlling for the cancer type and stage, regression analyses revealed higher BMI, ß = 0.258, p = .007, and former/current smoking status, ß = 0.181, p = .046, were associated with higher IL-6. IL-6 did not differ between former and current smokers, ß = 0.008, p = .927. Global sleep quality, sleep latency, and sleep efficiency were not associated with IL-6. CONCLUSIONS: Higher BMI and any history of tobacco use predicted higher IL-6 among women undergoing surgery for suspected gynecologic cancers. Cognitive-behavioral interventions targeting primary and secondary obesity and tobacco use prevention may reduce systemic inflammation and optimize cancer outcomes in this population. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

In Vivo Knockdown of the Herpes Simplex Virus 1 Latency-Associated Transcript Reduces Reactivation from Latency.

During herpes simplex virus (HSV) latency, most viral genes are silenced, with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long noncoding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilized an adeno-associated virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition, these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness.IMPORTANCE Herpes simplex virus (HSV) establishes a lifelong infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency, with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This work shows that the LAT RNA is important for reactivation and suggests the potential of this treatment as a therapy for treating HSV infections.

Adeno-associated Virus Vectors Efficiently Transduce Mouse and Rabbit Sensory Neurons Coinfected with Herpes Simplex Virus 1 following Peripheral Inoculation.

UNLABELLED: Following infection of epithelial tissues, herpes simplex virus 1 (HSV-1) virions travel via axonal transport to sensory ganglia and establish a lifelong latent infection within neurons. Recent studies have revealed that, following intraganglionic or intrathecal injection, recombinant adeno-associated virus (rAAV) vectors can also infect sensory neurons and are capable of stable, long-term transgene expression. We sought to determine if application of rAAV to peripheral nerve termini at the epithelial surface would allow rAAV to traffic to sensory ganglia in a manner similar to that seen with HSV. We hypothesized that footpad or ocular inoculation with rAAV8 would result in transduction of dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. To test this, we inoculated the footpads of mice with various amounts of rAAV as well as rAAV capsid mutants. We demonstrated that this method of inoculation can achieve a transduction rate of >90% of the sensory neurons in the DRG that innervate the footpad. Similarly, we showed that corneal inoculation with rAAV vectors in the rabbit efficiently transduced >70% of the TG neurons in the optic tract. Finally, we demonstrated that coinfection of mouse footpads or rabbit eyes with rAAV vectors and HSV-1 resulted in colocalization in nearly all of the HSV-1-positive neurons. These results suggest that rAAV is a useful tool for the study of HSV-1 infection and may provide a means to deliver therapeutic cargos for the treatment of HSV infections or of dysfunctions of sensory ganglia. IMPORTANCE: Adeno-associated virus (AAV) has been shown to transduce dorsal root ganglion sensory neurons following direct intraganglionic sciatic nerve injection and intraperitoneal and intravenous injection as well as intrathecal injection. We sought to determine if rAAV vectors would be delivered to the same sensory neurons that herpes simplex virus (HSV-1) infects when applied peripherally at an epithelial surface that had been treated to expose the underlying sensory nerve termini. For this study, we chose two well-established HSV-1 infection models: mouse footpad infection and rabbit ocular infection. The results presented here provide the first description of AAV vectors transducing neurons following delivery at the skin/epithelium/eye. The ability of AAV to cotransduce HSV-1-infected neurons in both the mouse and the rabbit provides an opportunity to experimentally explore and disrupt host and viral proteins that are integral to the establishment of HSV-1 latency, to the maintenance of latency, and to reactivation from latency in vivo.

Connective tissue growth factor differentially binds to members of the cystine knot superfamily and potentiates platelet-derived growth factor-B signaling in rabbit corneal fibroblast cells.

AIM: To study the binding of connective tissue growth factor (CTGF) to cystine knot-containing ligands and how this impacts platelet-derived growth factor (PDGF)-B signaling. METHODS: The binding strengths of CTGF to cystine knot-containing growth factors including vascular endothelial growth factor (VEGF)-A, PDGF-B, bone morphogenetic protein (BMP)-4, and transforming growth factor (TGF)-ß1 were compared using the LexA-based yeast two-hybrid system. EYG48 reporter strain that carried a wild-type LEU2 gene under the control of LexA operators and a lacZ reporter plasmid (p80p-lacZ) containing eight high affinity LexA binding sites were used in the yeast two-hybrid analysis. Interactions between CTGF and the tested growth factors were evaluated based on growth of transformed yeast cells on selective media and colorimetric detection in a liquid ß-galactosidase activity assay. Dissociation constants of CTGF to VEGF-A isoform 165 or PDGF-BB homo-dimer were measured in surface plasma resonance (SPR) analysis. CTGF regulation in PDGF-B presentation to the PDGF receptor ß (PDGFRß) was also quantitatively assessed by the SPR analysis. Combinational effects of CTGF protein and PDGF-BB on activation of PDGFRß and downstream signaling molecules ERK1/2 and AKT were assessed in rabbit corneal fibroblast cells by Western analysis. RESULTS: In the LexA-based yeast two-hybrid system, cystine knot motifs of tested growth factors were fused to the activation domain of the transcriptional factor GAL4 while CTGF was fused to the DNA binding domain of the bacterial repressor protein LexA. Yeast co-transformants containing corresponding fusion proteins for CTGF and all four tested cystine knot motifs survived on selective medium containing galactose and raffinose but lacking histidine, tryptophan, and uracil. In liquid ß-galactosidase assays, CTGF expressing cells that were co-transformed with the cystine knot of VEGF-A had the highest activity, at 29.88 ± 0.91 fold above controls (P < 0.01). Cells containing the cystine knot of BMP-4 expressed the second most activity, with a 24.77 ± 0.47 fold increase (P < 0.01). Cells that contained the cystine knot of TGF-ß1 had a 3.80 ± 0.66 fold increase (P < 0.05) and the ones with the cystine knot of PDGF-B had a 2.64 ± 0.33 fold increase of ß-galactosidase activity (P < 0.01). Further SPR analysis showed that the association rate between VEGF-A 165 and CTGF was faster than PDGF-BB and CTGF. The calculated dissociation constant (KD) of CTGF to VEGF165 and PDGF-BB was 1.8 and 43 nmol/L respectively. PDGF-BB ligand and PDGFRß receptor formed a stable complex with a low dissociation constant 1.4 nmol/L. Increasing the concentration of CTGF up to 263.2 nmol/L significantly the ligand/receptor binding. In addition, CTGF potentiated phosphorylation of PDGFRß and AKT in rabbit corneal fibroblast cells stimulated by PDGF-BB in tissue culture condition. In contrast, CTGF did not affect PDGF-B induced phosphorylation of ERK1/2. CONCLUSION: CTGF has a differential binding affinity to VEGF-A, PDGF-B, BMP-4, and TGF-ß. Its weak association with PDGF-B may represent a novel mechanism to enhance PDGF-B signaling.

Sequential Therapy with Saratin, Bevacizumab and Ilomastat to Prolong Bleb Function following Glaucoma Filtration Surgery in a Rabbit Model.

To determine if sequential treatment with Bevacizumab (Avastin), a monoclonal, VEGF antibody that blocks angiogenesis; Saratin, a 12 kD polypeptide with anti-inflammatory and anti-thrombotic properties; and Ilomastat, a matrix metalloproteinase (MMP) inhibitor, prolongs bleb life following glaucoma filtration surgery (GFS) in a rabbit model. Thirty-two New Zealand White rabbits (eight rabbits per group) underwent GFS in the left eye. Group 1 received a perioperative injection of both Saratin and Bevacizumab, and later, subconjuctival injections of Ilomastat on days 8 and 15. Group 2 received only Saratin perioperatively, and also received Ilomastat injections on days 8 and 15. Group 3, the negative control, received a single perioperative injection of Balanced Saline Solution (BSS) along with post-operative BSS injections on days 8 and 15. Group 4, the positive control, received topical treatment with Mitomycin-C (MMC) at the time of surgery with no further treatment. Blebs were evaluated by an observer masked to treatment every third day. Histology was obtained on two eyes in each group on post-op day twelve as well as all eyes following bleb failure. Eyes in group 1 had a mean bleb survival time of 29 ± 2.7 days, whereas those in group 2 that received the experimental treatment without Bevacizumab had a mean survival time of 25.5 ± 2.7 days. An ANOVA test showed that the Saratin/Ilomastat/Bevacizumab group demonstrated a significant prolongation of bleb survival compared to the BSS control-mean survival time of 19.7 ±2.7 days-(p = 0.0252) and was not significantly different from the MMC positive control group (p = 0.4238)-mean survival time of 32.5 ± 3.3. From tissue histology at day 12, the four different groups showed marked differences in the cellularity and capsule fibrosis. The MMC eyes showed minimal cellularity, were avascular and had minimal fibrous tissue. BSS group showed high cellularity, moderate to high fibrosis, and thicker and more defined capsules than either of the treatment groups and the positive control. Both the Saratin/Ilomastat/Bevacizumab and Saratin/Ilomastat only eyes showed moderate cellularity with minimal fibrosis, with less cellularity and fibrosis present in the triple treatment group. Sequential therapy with multiple agents, including Bevacizumab, prolonged bleb function following GFS in the rabbit model and were significantly better than the negative BSS control. The experimental group did not show the same surface tissue histological thinning and side effects associated with MMC treatment.

Topical application of recombinant platelet-derived growth factor increases the rate of healing and the level of proteins that regulate this response.

A bipedicle ischaemic rat skin flap model was used to study the effects of daily topical applications of platelet-derived growth factor (PDGF) on the healing of ischaemic wounds. Levels of tumour necrosis factor-alpha (TNFA), interleukin 1-beta (IL1B) and both the latent and active forms of matrix metalloproteinase 2 (MMP2) and 9 (MMP9) were measured. Full-thickness wounds were made on a total of 72 adult male Sprague-Dawley rats. Each group of 18 rats with normal and ischaemic wounds received either vehicle or 0·01% recombinant PDGF-BB. Additional applications were made on the wounds on a daily basis. Wound areas were measured at 0, 1, 3, 5, 7 9 and 13 days after wounding. Ischaemia caused a delay in wound healing as well as an increase in TNFA, IL1B and both the pro and active forms of MMP2 and MMP9. PDGF accelerated the rate of wound healing in both normal and ischaemic wounds and negated the effect of ischaemia. PDGF reduced the TNFA concentration in both normal and ischaemic wounds, and the rate of wound healing closely resembled the pattern of TNFA protein expression. PDGF also reduced both the magnitude and duration of the increases in IL1B and both the pro and active forms of MMP2 and MMP9 induced by ischaemia.

Connective tissue growth factor and integrin αvß6: a new pair of regulators critical for ductular reaction and biliary fibrosis in mice.

UNLABELLED: Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell-matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvß6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvß6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter-driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvß6 during liver injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen-inducible Cre-loxP system down-regulated integrin αvß6 in DDC-damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvß6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis-related genes, smaller areas of alpha-smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvß6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)-ß1 activation in vitro. CONCLUSIONS: CTGF and integrin αvß6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF-ß1. CTGF and integrin αvß6 are potential therapeutic targets to control DRs and fibrosis in related liver disease.

A corneal scarring model.

Corneal opacification (i.e., haze) following a non-denaturing acute injury to the cornea is a process which takes about 5 days to manifest itself, indicating that it is the consequence of cellular and molecular biological processes. In order to obtain a better understanding of the haze development process, and to test candidate anti-haze therapies, we use a corneal scarring model whereby we create an excimer laser wound in the center of rabbit corneas. The primary data generated by this model are (1) changes in corneal thickness with time; (2) wound closure rates, or re-epithelialization; (3) changes in the location and density of corneal sub-epithelial haze; and (4) molecular and histological changes leading up to, during, and following the formation of haze. While the use of excimer lasers to generate consistent wounds in rabbit corneas is not a novel protocol for the study of corneal haze, the photographic technique presented here for the more objective recording and quantification of corneal haze is. At present, a qualitative, semiquantitative, grading system is employed whereby the amount of iris detail discernible through the scar is assigned a value between 0 and 4. Such a system makes direct comparisons amongst reported anti-haze trials nearly impossible. Furthermore, the additional "geographic" detail provided by the image provides a new layer of information about the formation of haze and the ability to troubleshoot dosing regimens. Altogether, with the information present herein, we believe that the study of corneal haze formation and the ability to compare and contrast candidate therapies are both greatly improved.

The progression of haze formation in rabbit corneas following phototherapeutic keratectomy.

PURPOSE: To determine the topographical location and time course of development of corneal haze in a phototherapeutic keratectomy model using slit lamp examination, macrophotography, quantitative image analysis, and immunofluorescence staining of corneal sections. METHODS: Rabbit corneas were ablated with an excimer laser and were observed and graded for haze via slit lamp, imaged, and graded by macrophotography. Corneal sections were stained for α-smooth muscle actin (α-SMA) and tenascin-C (TNC). The distribution of haze imaged in the macrophotographs and density of α-SMA and TNC staining were compared. A daily image time course of haze formation was generated using macrophotography. RESULTS: The first signs of corneal haze were apparent shortly after reepithelialization. The haze was distributed as a ring at the wound margin in all cases, while nearly all corneas also had some central islands of haze initiation. With time, the haze spread within the ablated zone and intensified. The pattern of immunofluorescent staining for α-SMA and TNC at the wound margin mirrored the haze distribution, spread, and intensification with time. CONCLUSIONS: The initiation and spread of subepithelial haze begins shortly after reepithelialization. The haze then spreads from the loci of initiation and becomes more dense with time, maturing as early as 14 days after wounding. The improved temporal and spatial resolution provided by these data improve the current model of light-scattering haze formation in wounded corneas, which will improve the design of studies aimed at maintaining corneal clarity following acute injury or surgery.

Prevention of ocular scarring after glaucoma filtering surgery using the monoclonal antibody LT1009 (Sonepcizumab) in a rabbit model.

PURPOSE: Excessive scarring leading to failure of the filtering bleb continues to be a major problem after glaucoma filtration surgery. This study examines the antifibrotic effects of the anti-S1P monoclonal antibody LT1009 (Sonepcizumab) in prolonging bleb survival in a rabbit model of glaucoma filtering surgery. METHODS: The frequency of LT1009 dosage was determined initially using an enzyme-linked immunosorbent assay assay measuring LT1009 eye tissue retention in 6 New Zealand White rabbits. A further 21 New Zealand White rabbits underwent glaucoma filtering surgery. Bleb tissues were observed and compared clinically and histologically. The duration of bleb elevation was compared among LT1009, balanced saline solution (BSS) negative control, and mitomycin-C (MMC)-positive control. RESULTS: The mean duration of bleb survival was 28.5±8.5 days for rabbits receiving injections of LT1009, 21.0±5.6 days for those receiving injections of BSS, and 33.8±5.6 days for rabbits receiving MMC. Analysis of variance with post hoc testing suggests a statistically significant trend of improvement in bleb duration for LT1009 when compared with BSS controls. Nonpainful, upper eyelid edema was noted after 5 injections of LT1009, which resolved over a 10-day period. MMC eyes developed avascular conjunctivas with areas of thinning and sparse cellularity, whereas the conjunctiva of LT1009 and BSS eyes remained relatively normal. CONCLUSIONS: The monoclonal antibody LT1009 demonstrated a longer duration of bleb elevation than BSS control without adverse conjunctival effects associated with MMC. However, after multiple doses LT1009 use was associated with short-term upper eyelid edema.

Ectopic epithelial implants following surface ablation of the cornea.

PURPOSE: To determine the direct contribution of the epithelium to the generation of complications using a phototherapeutic keratectomy model. METHODS: A mouse model with a genetically labeled epithelium was used to determine whether any epithelium-derived cells persist in the stroma up to 1 month after surgery. Also, gross histology and macrophotography of excimer-ablated rabbit corneas were analyzed for evidence epithelial ingrowths into the stroma. RESULTS: Epithelium-derived cells were present in the wounded stroma 1 month after surgery. Micrographs taken during the first 4 days during healing evidenced epithelial invasion of the stroma in one and sometimes more locations in the same cornea. Gross histology also revealed that the epithelial invasions can result in complete delamination of stromal tissue and subsequent inclusion of the stromal material in the epithelium. The epithelial inclusions ultimately created a highly irregular corneal surface. CONCLUSIONS: Ectopic epithelia are a known complication of LASIK and LASIK-like procedures. The data presented here indicate that ectopic epithelia are also a complication of surface ablation techniques. The knowledge that these complications are present following surface ablations provides a new understanding of the biological response to surface ablation techniques and suggests new avenues of study to improve clinical outcomes of those for whom LASIK-based techniques are not an option.

Comparison of single versus multiple injections of the protein saratin for prolonging bleb survival in a rabbit model.

PURPOSE: We compared the anti-fibrotic effects of single versus multiple postoperative injections of saratin following glaucoma filtration surgery (GFS) in the rabbit model. METHODS: The experiment was in two parts. To determine the optimal frequency for postoperative therapy, seven New Zealand White (NZW) rabbits received an injection of saratin under the superior conjunctiva bilaterally, and ocular tissue concentration was determined using Western blot and bicinchoninic acid (BCA) assay. Next, 32 additional NZW rabbits underwent filtration surgery and received either single or multiple-dose saratin treatments. Mitomycin-C (MMC) and balanced saline solution (BSS) treatment acted as positive and negative controls, respectively. RESULTS: Rabbits receiving only one perioperative saratin injection had a mean bleb survival time of 29.8 ± 5.3 days, while those receiving multiple (either 3 or 5+) injections of saratin had mean bleb survival times of 26.3 ± 8.1 and 26.4 ± 4.2 days, respectively. Analysis of variance with post-hoc testing showed the single injection group had a statistically favorable effect on bleb survival duration compared to BSS controls and was not significantly different from MMC. The conjunctivas of the saratin-treated rabbits did not show the thinning or avascularity that was seen in the MMC treatment group. Rabbits receiving more than three injections of saratin suffered temporary conjunctival redness and two rabbits had upper eyelid edema. CONCLUSIONS: A single postoperative injection of saratin was able to prolong the duration of bleb elevation when compared to BSS controls. Additional treatments of saratin seemed to reduce effectiveness and caused short-term eye inflammation.

Microbial barriers.

Barrier wound therapy is commonplace in the health care environment and functions to limit bacterial colonization and infection in both acute wounds and recalcitrant chronic wounds. This article reviews the nature of acute and chronic wounds and their available adjunctive barrier therapies.

CCN2/CTGF regulates neovessel formation via targeting structurally conserved cystine knot motifs in multiple angiogenic regulators.

Blood vessels are formed during development and tissue repair through a plethora of modifiers that coordinate efficient vessel assembly in various cellular settings. Here we used the yeast 2-hybrid approach and demonstrated a broad affinity of connective tissue growth factor (CCN2/CTGF) to C-terminal cystine knot motifs present in key angiogenic regulators Slit3, von Willebrand factor, platelet-derived growth factor-B, and VEGF-A. Biochemical characterization and histological analysis showed close association of CCN2/CTGF with these regulators in murine angiogenesis models: normal retinal development, oxygen-induced retinopathy (OIR), and Lewis lung carcinomas. CCN2/CTGF and Slit3 proteins worked in concert to promote in vitro angiogenesis and downstream Cdc42 activation. A fragment corresponding to the first three modules of CCN2/CTGF retained this broad binding ability and gained a dominant-negative function. Intravitreal injection of this mutant caused a significant reduction in vascular obliteration and retinal neovascularization vs. saline injection in the OIR model. Knocking down CCN2/CTGF expression by short-hairpin RNA or ectopic expression of this mutant greatly decreased tumorigenesis and angiogenesis. These results provided mechanistic insight into the angiogenic action of CCN2/CTGF and demonstrated the therapeutic potential of dominant-negative CCN2/CTGF mutants for antiangiogenesis.

Prevention of ocular scarring post glaucoma filtration surgery using the inflammatory cell and platelet binding modulator saratin in a rabbit model.

CLINICAL RELEVANCE: Late complications can occur with use of current antimetabolites to prevent scarring following glaucoma filtration surgery (GFS). Safer, more targeted, anti-fibrosis agents are sought. OBJECTIVES: The protein saratin has been shown to exhibit anti-fibrotic and anti-thrombotic properties in response to injury, but had not been used for glaucoma surgery. The goal of this study was to compare the efficacy of saratin with that of the widely accepted mitomycin-C (MMC) in prolonging bleb survival following GFS in the rabbit model. Two saratin delivery routes were compared; a single intraoperative topical application versus a combination of intraoperative topical application with two additional postoperative injections. METHODS: Twenty-four New Zealand White rabbits underwent GFS and received either intraoperative topical saratin, intraoperative topical saratin plus two injections on post-operative days 4 and 8, balanced saline solution (BSS), or MMC. The bleb tissues and their elevation durations were compared based on clinical and histological findings. RESULTS: Rabbits receiving topical+injections of saratin had a mean bleb survival of 33.6±8.5 days, significantly higher than the negative BSS controls, which averaged 17.4±6.0 days (p = 0.018). No improvement over BSS was seen for rabbits receiving topical saratin only (15.5±4.8 days, p = 0.749). Rabbits receiving saratin did not develop bleb avascularity and thinning associated with MMC treatment and there were no apparent clinical signs of toxicity. CONCLUSIONS: Treatment with a single intraoperative topical application plus two additional postoperative injections significantly prolonged bleb elevation comparable to MMC, but without toxicity; however, topical application alone was ineffective.

Vorinostat: a potent agent to prevent and treat laser-induced corneal haze.

PURPOSE: This study investigated the efficacy and safety of vorinostat, a deacetylase (HDAC) inhibitor, in the treatment of laser-induced corneal haze following photorefractive keratectomy (PRK) in rabbits in vivo and transforming growth factor beta 1 (TGFß1) -induced corneal fibrosis in vitro. METHODS: Corneal haze in rabbits was produced with -9.00 diopters (D) PRK. Fibrosis in cultured human and rabbit corneal fibroblasts was activated with TGFß1. Vorinostat (25 µm) was topically applied once for 5 minutes on rabbit cornea immediately after PRK for in vivo studies. Vorinostat (0 to 25 µm) was given to human/rabbit corneal fibroblasts for 5 minutes or 48 hours for in vitro studies. Slit-lamp microscopy, TUNEL assay, and trypan blue were used to determined vorinostat toxicity, whereas real-time polymerase chain reaction, immunocytochemistry, and immunoblotting were used to measure its efficacy. RESULTS: Single 5-minute vorinostat (25 µm) topical application on the cornea following PRK significantly reduced corneal haze (P<.008) and fibrotic marker proteins (α-smooth muscle actin and f-actin; P<.001) without showing redness, swelling, or inflammation in rabbit eyes in vivo screened 4 weeks after PRK. Vorinostat reduced TGFß1-induced fibrosis in human and rabbit corneas in vitro in a dose-dependent manner without altering cellular viability, phenotype, or proliferation. CONCLUSIONS: Vorinostat is non-cytotoxic and safe for the eye and has potential to prevent laser-induced corneal haze in patients undergoing PRK for high myopia.

Molecular Mechanisms of Biofilm Infection: Biofilm Virulence Factors.

BACKGROUND: Numerous planktonic virulence factors have been identified that enable bacteria to successfully produce acute infections in tissues. In contrast, very little is known about biofilm virulence factors that enhance the establishment and long-term survival of biofilms in tissues. THE PROBLEM: There is a need to identify the genes that encode biofilm virulence factors and understand how these factors function to enable bacteria to successfully establish chronic biofilm infections in tissues. BASIC SCIENCE ADVANCES: A methodology was developed to screen 6,912 Pseudomonas aeruginosa loss-of-function mutants in a murine model of airway infection to determine which genes were crucial for establishing chronic lung infections in mice. Genetic analysis of 16 bacterial mutants isolated using this methodology identified 15 separate biofilm virulence factor genes whose loss-of-function increased biofilm survival. Sequence analysis of clinical Isolates obtained from seven cystic fibrosis (CF) patients with chronic infections that were collected over 16.3 years identified loss-of-function mutants for 7 of these 15 genes. CLINICAL CARE RELEVANCE: Identifying biofilm virulence factor genes further defines the molecular mechanisms of establishing chronic biofilm infection and should lead to more effective and specific treatments to prevent biofilm formation and/or improve clearance of chronic biofilms in patients. CONCLUSION: Biofilm virulence factor genes were identified in P. aeruginosa using an animal model of chronic lung infection for mutant screening and from infected CF patient isolates. In many cases they were inactivated planktonic virulence factor genes. These results further demonstrate the opposing patterns of gene expression between acute planktonic bacterial infections and chronic biofilm infections.