Caitlyn Ngam

Delayed stress fiber formation mediates pulmonary myofibroblast differentiation in response to TGF-β.

Myofibroblast differentiation induced by transforming growth factor-β (TGF-β) and characterized by de novo expression of smooth muscle (SM)-specific proteins is a key process in wound healing and in the pathogenesis of fibrosis. We have previously shown that TGF-β-induced expression and activation of serum response factor (SRF) is required for this process. In this study, we examined the signaling mechanism for SRF activation by TGF-β as it relates to pulmonary myofibroblast differentiation. TGF-β stimulated a profound, but delayed (18-24 h), activation of Rho kinase and formation of actin stress fibers, which paralleled SM α-actin expression. The translational inhibitor cycloheximide blocked these processes without affecting Smad-dependent gene transcription. Inhibition of Rho kinase by Y-27632 or depolymerization of actin by latrunculin B resulted in inhibition TGF-β-induced SRF activation and SM α-actin expression, having no effect on Smad signaling. Conversely, stabilization of actin stress fibers by jasplakinolide was sufficient to drive these processes in the absence of TGF-β. TGF-β promoted a delayed nuclear accumulation of the SRF coactivator megakaryoblastic leukemia-1 (MKL1)/myocardin-related transcription factor-A, which was inhibited by latrunculin B. Furthermore, TGF-β also induced MKL1 expression, which was inhibited by latrunculin B, by SRF inhibitor CCG-1423, or by SRF knockdown. Together, these data suggest a triphasic model for myofibroblast differentiation in response to TGF-β that involves 1) initial Smad-dependent expression of intermediate signaling molecules driving Rho activation and stress fiber formation, 2) nuclear accumulation of MKL1 and activation of SRF as a result of actin polymerization, and 3) SRF-dependent expression of MKL1, driving further myofibroblast differentiation.

Control of Myofibroblast Differentiation by Microtubule Dynamics through a Regulated Localization of mDia2

Background: Myofibroblast differentiation plays a critical role in fibrosis. Results: Microtubule polymerization state inversely controls myofibroblast differentiation via Rho/SRF signaling. Dynamic localization of mDia2 to actin stress fibers is critical for myofibroblast differentiation and is regulated by the microtubule polymerization state. Conclusion: Microtubule polymerization state controls myofibroblast differentiation via regulation of mDia2 localization. Significance: This is a novel mechanism of myofibroblast differentiation and a therapeutic target. Myofibroblast differentiation plays a critical role in wound healing and in the pathogenesis of fibrosis. We have previously shown that myofibroblast differentiation is mediated by the activity of serum response factor (SRF), which is tightly controlled by the actin polymerization state. In this study, we investigated the role of the microtubule cytoskeleton in modulating myofibroblast phenotype. Treatment of human lung fibroblasts with the microtubule-destabilizing agent, colchicine, resulted in a formation of numerous stress fibers and expression of myofibroblast differentiation marker proteins. These effects of colchicine were independent of Smad signaling but were mediated by Rho signaling and SRF, as they were attenuated by the Rho kinase inhibitor, Y27632, or by the SRF inhibitor, CCG-1423. TGF-β-induced myofibroblast differentiation was not accompanied by gross changes in the microtubule polymerization state. However, microtubule stabilization by paclitaxel attenuated TGF-β-induced myofibroblast differentiation. Paclitaxel had no effect on TGF-β-induced Smad activation and Smad-dependent gene transcription but inhibited actin polymerization, nuclear accumulation of megakaryoblastic leukemia-1 protein, and SRF activation. The microtubule-associated formin, mDIA2, localized to actin stress fibers upon treatment with TGF-β, and paclitaxel prevented this localization. Treatment with the formin inhibitor, SMI formin homology 2 domain, inhibited stress fiber formation and myofibroblast differentiation induced by TGF-β, without affecting Smad-phosphorylation or microtubule polymerization. Together, these data suggest that (a) TGF-β promotes association of mDia2 with actin stress fibers, which further drives stress fiber formation and myofibroblast differentiation, and (b) microtubule polymerization state controls myofibroblast differentiation through the regulation of mDia2 localization.

Myofibroblasts exhibit enhanced fibronectin assembly that is intrinsic to their contractile phenotype

Background: Myofibroblasts have heightened expression of contractile genes and drive extracellular matrix formation during pulmonary fibrosis. Results: Enhanced fibronectin assembly by myofibroblasts requires smooth muscle α-actin expression. Conclusion: This study demonstrates a linkage between contractile gene expression and increased assembly of fibronectin fibrils by myofibroblasts. Significance: Targeting contractile gene expression in myofibroblasts may attenuate fibronectin matrix formation during fibrosis. Myofibroblasts have increased expression of contractile proteins and display augmented contractility. It is not known if the augmented contractile gene expression characterizing the myofibroblast phenotype impacts its intrinsic ability to assemble fibronectin (FN) and extracellular matrix. In this study we investigated whether myofibroblasts displayed increased rates of FN fibril assembly when compared with their undifferentiated counterparts. Freshly plated myofibroblasts assemble exogenous FN (488-FN) into a fibrillar matrix more rapidly than fibroblasts that have not undergone myofibroblast differentiation. The augmented rate of FN matrix formation by myofibroblasts was dependent on intact Rho/Rho kinase (ROCK) and myosin signals inasmuch as treatment with Y27632 or blebbistatin attenuated 488-FN assembly. Inhibiting contractile gene expression by pharmacologic disruption of the transcription factors megakaryoblastic leukemia-1 (MKL1)/serum response factor (SRF) during myofibroblast differentiation resulted in decreased contractile force generation and attenuated 488-FN incorporation although not FN expression. Furthermore, disruption of the MKL1/SRF target gene, smooth muscle α-actin (α-SMA) via siRNA knockdown resulted in attenuation of 488-FN assembly. In conclusion, this study demonstrates a linkage between increased contractile gene expression, most importantly α-SMA, and the intrinsic capacity of myofibroblasts to assemble exogenous FN into fibrillar extracellular matrix.

Tensin 1 Is Essential for Myofibroblast Differentiation and Extracellular Matrix Formation

&NA; Myofibroblasts, the primary effector cells that mediate matrix remodeling during pulmonary fibrosis, rapidly assemble an extracellular fibronectin matrix. Tensin (TNS) 1 is a key component of specialized cellular adhesions (fibrillar adhesions) that bind to extracellular fibronectin fibrils. We hypothesized that TNS1 may play a role in modulating myofibroblast‐mediated matrix formation. We found that TNS1 expression is increased in fibroblastic foci from lungs with idiopathic pulmonary fibrosis. Transforming growth factor (TGF)‐&bgr; profoundly up‐regulates TNS1 expression with kinetics that parallel the expression of the myofibroblast marker, smooth muscle &agr;‐actin. TGF‐&bgr;‐induced TNS1 expression is dependent on signaling through the TGF‐&bgr; receptor 1 and is Rho coiled‐coiled kinase/actin/megakaryoblastic leukemia‐1/serum response factor dependent. Small interfering RNA‐mediated knockdown of TNS1 disrupted TGF‐&bgr;‐induced myofibroblast differentiation, without affecting TGF‐&bgr;/Smad signaling. In contrast, loss of TNS1 resulted in disruption of focal adhesion kinase phosphorylation, focal adhesion formation, and actin stress fiber development. Finally, TNS1 was essential for the formation of fibrillar adhesions and the assembly of nascent fibronectin and collagen matrix in myofibroblasts. In summary, our data show that TNS1 is a novel megakaryoblastic leukemia‐1‐dependent gene that is induced during pulmonary fibrosis. TNS1 plays an essential role in TGF‐&bgr;‐induced myofibroblast differentiation and myofibroblast‐mediated formation of extracellular fibronectin and collagen matrix. Targeted disruption of TNS1 and associated signaling may provide an avenue to inhibit tissue fibrosis.

Assessment of Fidelity in Interventions to Improve Hand Hygiene of Healthcare Workers: A Systematic Review

OBJECTIVE Compliance with hand hygiene in healthcare workers is fundamental to infection prevention yet remains a challenge to sustain. We examined fidelity reporting in interventions to improve hand hygiene compliance, and we assessed 5 measures of intervention fidelity: (1) adherence, (2) exposure or dose, (3) quality of intervention delivery, (4) participant responsiveness, and (5) program differentiation. DESIGN Systematic review METHODS A librarian performed searches of the literature in PubMed, Cumulative Index to Nursing and Allied Health (CINAHL), Cochrane Library, and Web of Science of material published prior to June 19, 2015. The review protocol was registered in PROSPERO International Prospective Register of Systematic Reviews, and assessment of study quality was conducted for each study reviewed. RESULTS A total of 100 studies met the inclusion criteria. Only 8 of these 100 studies reported all 5 measures of intervention fidelity. In addition, 39 of 100 (39%) failed to include at least 3 fidelity measures; 20 of 100 (20%) failed to include 4 measures; 17 of 100 (17%) failed to include 2 measures, while 16 of 100 (16%) of the studies failed to include at least 1 measure of fidelity. Participant responsiveness and adherence to the intervention were the most frequently unreported fidelity measures, while quality of the delivery was the most frequently reported measure. CONCLUSIONS Almost all hand hygiene intervention studies failed to report at least 1 fidelity measurement. To facilitate replication and effective implementation, reporting fidelity should be standard practice when describing results of complex behavioral interventions such as hand hygiene.

Reducing Clostridium difficile in the Inpatient Setting: A Systematic Review of the Adherence to and Effectiveness of C. difficile Prevention Bundles

BACKGROUND Clostridium difficile infection (CDI) is the most common infectious cause of nosocomial diarrhea, and its prevention is an urgent public health priority. However, reduction of CDI is challenging because of its complex pathogenesis, large reservoirs of colonized patients, and the persistence of infectious spores. The literature lacks high-quality evidence for evaluating interventions, and many hospitals have implemented bundled interventions to reduce CDI with variable results. Thus, we conducted a systematic review to examine the components of CDI bundles, their implementation processes, and their impact on CDI rates. METHODS We conducted a comprehensive literature search of multiple computerized databases from their date of inception through April 30, 2016. The protocol was registered in PROSPERO, an international prospective register of systematic reviews. Bundle effectiveness, adherence, and study quality were assessed for each study meeting our criteria for inclusion. RESULTS In the 26 studies that met the inclusion criteria for this review, implementation and adherence factors to interventions were variably and incompletely reported, making study reproducibility and replicability challenging. Despite contextual differences and the variety of bundle components utilized, all 26 studies reported an improvement in CDI rates. However, given the lack of randomized controlled trials in the literature, assessing a causal relationship between bundled interventions and CDI rates is currently impossible. CONCLUSION Cluster randomized trials that include a rigorous assessment of the implementation of bundled interventions are urgently needed to causally test the effect of intervention bundles on CDI rates. Infect Control Hosp Epidemiol 2017;38:639-650.

Barriers and facilitators to Clostridium difficile infection prevention: A nursing perspective

Background: Clostridium difficile infection (CDI) is a critical patient safety issue. Consistent and regular performance of appropriate practices is effective in preventing CDI. Variation in adherence to these practices can impede their effective implementation and weaken CDI prevention. Methods: Using the Systems Engineering Initiative for Patient Safety (SEIPS) framework we convened a focus group of 10 nurses to identify barriers and facilitators to compliance with a CDI prevention bundle that includes (1) prompt diagnostic testing, (2) empirical isolation for patients with suspected CDI, (3) consistent and appropriate contact isolation, (4) hand hygiene, and (5) disinfection of the patient room and objects in the room. On completion of transcript coding, analyses were performed based on bundle intervention and the work system element of the SEIPS model. Results: A total of 58 excerpts were coded. Work system barriers or facilitators were associated with nearly every bundle intervention. The work system elements raised in over half of the excerpts were task (n = 31) (eg, amount of additional effort required to don and doff gloves and gowns) and organization (n = 30) (eg, recognition by all staff of the severity of CDI). Contact isolation was the most frequently discussed bundle intervention (n = 24). Conclusions: The SEIPS systems engineering framework is useful to evaluate infection prevention practices for CDI and identify opportunities for improvement. Addressing the work system barriers and facilitators identified in this study is essential to effective implementation of infection prevention interventions, specifically for CDI.

Work System Barriers and Facilitators to Compliance with Infection Prevention Intervention Initial Findings Regarding Hand Hygiene from Three Target Roles

In this paper we present preliminary findings that address the work system of physicians, nurses and environmental services workers and their ability to comply with one clinically accepted intervention associated with Clostridium difficile infection. Despite differences in their roles, responsibilities and focus related to management of patients with Clostridium difficile infection, numerous similarities exist in the type of work system considerations they must each face.

Compliance with PPE Requirements for C Difficile Isolation

a trial involving dual licensed personnel insertion and a “safety time out” was implemented in January 2015. The practice change involved review of the indications and proper steps for insertion per hospital policy by the two personnel involved in the procedure. The catheter was then insertedwith the second staff member being solely responsible to assure compliance with proper technique. The procedure was stopped if sterile technique was compromised. A data collection checklist was completed post-procedure indicating compliance with proper technique. The insertion-related CAUTI rate (number of CAUTIs occurring < 7 days after insertion/1000 catheters inserted) for January-June 2015 was compared with 2014 data using an independent t test. RESULTS: During the 6 month trial, 201 patients underwent dual personnel urinary catheter insertion in the ED. None of the patients with the dual personnel insertion developed a CAUTI. Comparison of the previous year’s insertion-related CAUTI rate yielded a statistical significance (P value) of .0078. CONCLUSIONS:Due to the success of the trial, dual personnel urinary catheter insertion has continued to remain as standard practice in the ED. In addition it may be considered hospital wide for specific cases with difficult insertion where technique can be jeopardized during insertion.