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The toxic effects of acrylamide on cytoskeletal integrity and ion channel balance is well-established in many cell types, but there has been little examination regarding the effects of acrylamide on primary cardiomyocytes, despite the importance of such components in their function. Furthermore, acrylamide toxicity is generally examined using concentrations higher than those found in vivo under starch-rich diets. Accordingly, we sought to characterize the dose-dependent effects of acrylamide on various properties, including cell morphology, contraction patterns, and junctional connexin 43 staining, in primary cardiomyocytes. We show that several days exposure to 1-100 μM acrylamide resulted in altered morphology, irregular contraction patterns, and an increase in the amount of immunoreactive signal for connexin 43 at cell junctions. We conclude that dietary levels of acrylamide may alter cellular function with prolonged exposure, in primary cardiomyocytes.

Tetraspanin CD151 is highly expressed in endothelial cells and regulates pathologic angiogenesis. However, the mechanism by which CD151 promotes vascular morphogenesis and whether CD151 engages other vascular functions are unclear. Here we report that CD151 is required for maintaining endothelial capillary-like structures formed in vitro and the integrity of endothelial cell-cell and cell-matrix contacts in vivo. In addition, vascular permeability is markedly enhanced in the absence of CD151. As a global regulator of endothelial cell-cell and cell-matrix adhesions, CD151 is needed for the optimal functions of various cell adhesion proteins. The loss of CD151 elevates actin cytoskeletal traction by up-regulating RhoA signaling and diminishes actin cortical meshwork by down-regulating Rac1 activity. The inhibition of RhoA or activation of cAMP signaling stabilizes CD151-silenced or -null endothelial structure in vascular morphogenesis. Together, our data demonstrate that CD151 maintains vascular stability by promoting endothelial cell adhesions, especially cell-cell adhesion, and confining cytoskeletal tension.

Arrhythmogenic right ventricular cardiomyopathy is a primary heart muscle disorder characterized by the early occurrence of arrhythmias often out of proportion to the extent of structural remodeling and contractile derangement. Approximately 40% of patients with arrhythmogenic right ventricular cardiomyopathy have one or more mutations in genes encoding proteins in desmosomes, intercellular adhesion junctions which, in cardiac myocytes, reside within intercalated disks. Some desmosomal proteins fulfill roles both as structural proteins in cell–cell adhesion junctions and as signaling molecules in pathways mediated by Wnt ligands. Evidence is increasing that mutations in desmosomal proteins can perturb the normal balance of critical proteins in junctions and the cytosol which, in turn, could alter gene expression by circumventing normal Wnt signaling pathways. This review highlights recent advances in understanding the pathogenesis of arrhythmogenic right ventricular cardiomyopathy and presents evidence suggesting that the disease is caused by a combination of altered cellular biomechanical behavior and altered signaling.

Growth factor signaling can affect tissue remodeling through autocrine/paracrine mechanisms. Recent evidence indicates that EGF receptor transactivation by heparin-binding EGF (HB-EGF) contributes to hypertrophic signaling in cardiomyocytes. Here, we show that HB-EGF operates in a spatially restricted circuit in the extracellular space within the myocardium, revealing the critical nature of the local microenvironment in intercellular signaling. This highly localized microenvironment of HB-EGF signaling was demonstrated with 3D morphology, consistent with predictions from a computational model of EGF signaling. HB-EGF secretion by a given cardiomyocyte in mouse left ventricles led to cellular hypertrophy and reduced expression of connexin43 in the overexpressing cell and in immediately adjacent cells but not in cells farther away. Thus, HB-EGF acts as an autocrine and local paracrine cardiac growth factor that leads to loss of gap junction proteins within a spatially confined microenvironment. These findings demonstrate how cells can coordinate remodeling with their immediate neighboring cells with highly localized extracellular EGF signaling.

Viscoelastic models of cells often treat cells as homogeneous objects. However, studies have demonstrated that cellular properties are local and can change dramatically on the basis of the location probed. Because membrane receptors are linked in various ways to the intracellular space, with some receptors linking to the cytoskeleton and others diffusing freely without apparent linkages, the cellular physical response to mechanical stresses is expected to depend on the receptor engaged. In this study, we tested the hypothesis that cellular mechanical stiffness as measured via cytoskeletally linked receptors is greater than stiffness measured via receptors that are not cytoskeletally linked. We used a magnetic micromanipulator to apply linear stresses to magnetic beads attached to living cells via selected receptors. One of the receptor classes probed, the dystroglycan receptors, is linked to the cytoskeleton, while the other, the transferrin receptors, is not. Fibronectin-coated beads were used to test cellular mechanical properties of the cytoskeleton without membrane dependence by allowing the beads to endocytose. For epithelial cells, transferrin-dependent stiffness and endocytosed bead-dependent stiffness were similar, while dystroglycan-dependent stiffness was significantly lower. For smooth muscle cells, dystroglycan-dependent stiffness was similar to the endocytosed bead-dependent stiffness, while the transferrin-dependent stiffness was lower. The conclusion of this study is that the measured cellular stiffness is critically influenced by specific receptor linkage and by cell type and raises the intriguing possibility of the existence of separate cytoskeletal networks with distinct mechanical properties that link different classes of receptors.

Cells face not only a complex biochemical environment but also a diverse biomechanical environment. How cells respond to variations in mechanical forces is critical in homeostasis and many diseases. The mechanisms by which mechanical forces lead to eventual biochemical and molecular responses remain undefined, and unraveling this mystery will undoubtedly provide new insight into strengthening bone, growing cartilage, improving cardiac contractility, and constructing tissues for artificial organs. In this article we review the physical bases underlying the mechanotransduction process, techniques used to apply controlled mechanical stresses on living cells and tissues to probe mechanotransduction, and some of the important lessons that we are learning from mechanical stimulation of cells with precisely controlled forces.

The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including α6β1. To probe strength of α6β1-dependent adhesion to laminin-1, defined forces (0–1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-α6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening α6β1 integrin-mediated adhesion to laminin-1.

Aims The majority of patients diagnosed with arrhythmogenic right ventricular cardiomyopathy (ARVC) have mutations in genes encoding desmosomal proteins, raising the possibility that abnormal intercellular adhesion plays an important role in disease pathogenesis. We characterize cell mechanical properties and molecular responses to oscillatory shear stress in cardiac myocytes expressing mutant forms of the desmosomal proteins plakoglobin and plakophilin, which are linked to ARVC in patients. Methods and Results Cells expressing mutant plakoglobin or plakophilin showed no differences in cell-cell adhesion relative to controls, while knocking down these proteins weakened cell-cell adhesion. However, cells expressing mutant plakoglobin failed to increase the amount of immunoreactive signal for plakoglobin or N-cadherin at cell-cell junctions in response to shear stress, as seen in control cells. Cells expressing mutant plakophilin exhibited a similar attenuation in the shear-induced increase in junctional plakoglobin immunoreactive signal in response to shear stress, suggesting the phenotype is independent of the type of mutant protein being expressed. Cells expressing mutant plakoglobin also showed greater myocyte apoptosis compared to controls. Apoptosis rates increased greatly in response to shear stress in cells expressing mutant plakoglobin, but not in controls. Abnormal responses to shear stress in cells expressing either mutant plakoglobin or plakophilin could be reversed by SB216763, a GSK3β inhibitor. Conclusions Desmosomal mutations linked to ARVC do not significantly affect cell mechanical properties, but cause myocytes to respond abnormally to mechanical stress through a mechanism involving GSK3β. These results may help explain why patients with ARVC experience disease exacerbations following strenuous exercise.

There is a need to characterize biomechanical cell–cell interactions, but due to a lack of suitable experimental methods, relevant in vitro experimental data are often masked by cell–substrate interactions. This study describes a novel method to generate partially lifted substrate-free cell sheets that engage primarily in cell–cell interactions, yet are amenable to biological and chemical perturbations and, importantly, mechanical conditioning and characterization. A polydimethylsiloxane (PDMS) mold is used to isolate a patch of cells, and the patch is then enzymatically lifted. The cells outside the mold remain attached, creating a partially lifted cell sheet. This simple yet powerful tool enables the simultaneous examination of lifted and adherent cells. This tool was then deployed to test the hypothesis that the lifted cells would exhibit substantial reinforcement of key cytoskeletal and junctional components at cell–cell contacts, and that such reinforcement would be enhanced by mechanical conditioning. Results demonstrate that the mechanical strength and cohesion of the substrate-free cell sheets strongly depend on the integrity of the actomyosin cytoskeleton and the cell–cell junctional protein plakoglobin. Both actin and plakoglobin are significantly reinforced at junctions with mechanical conditioning. However, total cellular actin is significantly diminished on dissociation from a substrate and does not recover with mechanical conditioning. These results represent a first systematic examination of mechanical conditioning on cells with primarily intercellular interactions.

Connections between cell-cell junctions and cytoskeleton profoundly influence cell shape, motility and gene expression. However, cell-cell interactions are easily masked by cell-substrate interactions thus remains poorly characterized. A cell monolayer can be considered the simplest multicellular tissue structure, yet they fulfill critical roles in human physiology and tissue engineering. A systematic characterization of intercellular adhesion, cytoskeletal organization and signaling in a substrate-free cell monolayer is crucial for unlocking the potential for cell-sheet tissue engineering. To test our hypothesis that intercellular junctions and cytoskeleton components likely play significant roles in establishing cell sheet cohesion, we have developed a unique partial-lifting technique that allows generation and investigation of the biological and mechanical properties of substrate-freehuman keratinocytecell monolayer.Results demonstrated that through alterations of spatial organization and expression of their cytoskeleton and specialized intercellular adhesions, substrate-free cells create a monolayer tissue that has a much stronger mechanical strength than their substrate-adhering controls. To determine the contribution of intercellular junctions and cytoskeleton to cell sheet mechanics, we altered the junctional protein plakoglobin by siRNA interference, altered actin-myosin interactions by ROCK kinase inhibitor and actin disruption agent. Knockdown of plakoglobin significantly diminished its participation in intercellular junctions and resulted in weakened cell sheet strength. Similarly, actin disruption and actin-myosin contraction inhibition also diminished cell sheet strength. Next, we demonstrated that external mechanical stimuli further boosted the sheet's mechanical strength via increased junctional localizations and elevated expression levels, suggesting possible involvement of plakoglobin-related Wnt/β-catenin signaling and actin-related RhoA/ROCK signaling.Given the near-ubiquity of plakoglobin and actin in multicellular organisms, these findings could have significant implications for understanding the fundamental mechanisms that regulating cell adhesion and biomechanics, and improving the mechanical properties of tissue engineering constructs.

Rheological characterization of cells using passive particle tracking techniques can yield substantial information regarding local cellular material properties. However, limited work has been done to establish the changes in material properties of mechanically-responsive cells that experience external stimuli. In this study, cardiac fibroblasts plated on either fibronectin or collagen were treated with cytochalasin, mechanically stretched, or both, and their trajectories and complex moduli were extracted. Results demonstrate that both solid and fluid components were altered by such treatments in a receptor-dependent manner, and that, interestingly, cells treated with cytochalasin were still capable of stiffening in response to mechanical stimuli despite gross stress fiber disruption. These results suggest that the material properties of cells are dependent on a variety of environmental cues and can provide insight into physiological and disease processes.

Immortalized cells are commonly used as analogs for primary cells in many cell mechanics, tissue engineering, and biochemical assays. However, it is not well-established whether immortal cell lines can mimic the behavior of primary cells in more physiological (three-dimensional) environments. For this project, we investigate the mechanical properties of primary cardiac fibroblasts (CFs) and 3T3 transformed fibroblasts when cultured in cell bi-layers by comparing the cells’ viscoelastic properties.Copyright © 2012 by ASME

Cell–cell junctional proteins play important structural and functional roles in several physiological systems. Recent studies have illuminated key aspects in the relationship of junctional proteins with normal cell and tissue function as well as various pathologies. In this review article, the roles of cell–cell junctional proteins will be presented in four classes: adherens junctions, desmosomes, gap junctions, and tight junctions, and discussed primarily in the context of cardiovascular cell and tissue physiology and pathophysiology. The functions of the proteins are described from the perspective of mechanotransductive regulation of physiological and disease processes, with focus being laid on more biomechanical aspects, such as cell adhesion, migration, and mechanosignaling.

Mutations in genes encoding desmosomal proteins have been implicated in the pathogenesis of heart and skin diseases. This has led to the hypothesis that defective cell-cell adhesion is the underlying cause of injury in tissues that repeatedly bear high mechanical loads. In this study, we examined the effects of two different mutations in plakoglobin on cell migration, stiffness, and adhesion. One is a C-terminal mutation causing Naxos disease, a recessive syndrome of arrhythmogenic right ventricular cardiomyopathy (ARVC) and abnormal skin and hair. The other is an N-terminal mutation causing dominant inheritance of ARVC without cutaneous abnormalities. To assess the effects of plakoglobin mutations on a broad range of cell mechanical behavior, we characterized a model system consisting of stably transfected HEK cells which are particularly well suited for analyses of cell migration and adhesion. Both mutations increased the speed of wound healing which appeared to be related to increased cell motility rather than increased cell proliferation. However, the C-terminal mutation led to dramatically decreased cell-cell adhesion, whereas the N-terminal mutation caused a decrease in cell stiffness. These results indicate that different mutations in plakoglobin have markedly disparate effects on cell mechanical behavior, suggesting complex biomechanical roles for this protein.

Background: Ex vivo T-cell depletion (TCD) by CD34+ selection has been used successfully for graft-versus-host disease prevention alleviating the need for additional pharmacologic immunosuppressants. TCD has been associated with higher rates of viral infections and cytomegalovirus (CMV) disease in particular compared to conventional (CONV) HCT. Modern methods of TCD achieve up to 5 log reduction of T-cells. At MSK, TCD HCT recipients (R) are routinely monitored for CMV, adenovirus (ADV), Epstein-Barr virus (EBV), and human herpes virus 6 (HHV6) in the blood by quantitative polymerase chain reaction (qPCR) assays. We report the incidence of viremia and end organ disease (EOD) by these viruses in TCD HCT recipients. Methods: Retrospective review of adults with acute leukemia (AL) and myelodysplastic syndrome (MDS) patients (pts) who received TCD HCT at MSK from June 2010 -December 2014. TCD was performed by the FDA approved CliniMACS CD34+ reagent system (Miltenyi Biotec, Gladbach, Germany). CMV was monitored at least weekly from day 14-100 and at least every 14 days until day 180. EBV was monitored through day 180. Monitoring for CMV and EBV was performed by the Clinical Microbiology Laboratory at MSK using Roche analyte specific reagents, and since March 2013, the Cobas Ampliprep/Cobas Taqman (CAP/CTM) CMV qPCR in plasma. Monitoring for ADV/HHV6 was performed by Viracor-IBT laboratories (Lenexa, KS). Preemptive treatment for CMV was started for ≥2 consecutive PCR >500copies/ml in whole blood or >300 IU/ml in plasma. Treatment of ADV/HHV6 viremia was at clinician's discretion. EBV post-transplant lymphoproliferative disorder (PTLD) was treated with rituximab as primary therapy. During the study, 11 pts were enrolled in CMV prevention trials of brincidofovir (BCV), and 5 pts received BCV for treatment of ADV. EOD was scored by standard criteria. Follow up was through June 2015. The cumulative incidence (CI) was estimated for viremia at 180 days and EOD at 365 days for CMV, ADV, HHV6, and EBV. Kaplan-Meier method and log-rank test were used to compare CI between groups. P Results: Of239 pts (median age 56.3 years, range 19.6-73.3), 161 (67%) pts had AL and 78 (33%) pts had MDS. Donors were matched-related 76 (32%), matched unrelated 116 (48%), or mismatched (related or unrelated) 47(20%); CMV R+ 148 (62%) or R- 91 (38%). CMV infection had the earliest onset post HCT at a median of 28 days (d) (IQR: 25-33), followed by HHV6 33d (25-52), ADV 71d (44-89), and EBV 85d (58-107). 188 (79%) pts had ≥1 dsDNA viral infection with 182 (76%) pts experiencing infection prior to d100. The CIs of viremia were: CMV 42% (68% in R+ vs 0% in R-, P Conclusions: 1) Viremias by dsDNA viruses occurred frequently and early post TCD HCT: 189 (79%) pts had at least 1 dsDNA viral infection, with onset of first infection before d100 in 183 (77%) pts. Around 58% of pts with CMV had coinfections with additional dsDNA virus(es). 2) Two thirds (67%) of CMV R+ and 41% of total pts received CMV antiviral treatment. 3) Overall, 18% of patients developed end organ disease by dsDNA viruses. 4) Viremias were managed effectively with intense monitoring and antiviral treatment, and rates of EOD were comparable to those of CONV HCT. Our data highlights the need for effective strategies to reduce the total burden of dsDNA viruses after TCD HCT. Figure 1. CIs of dsDNA viremia. Figure 1. CIs of dsDNA viremia. Figure 2. dsDNA viral co-infections Figure 2. dsDNA viral co-infections Figure 3. Coinfections among pts with CMV viremia Figure 3. Coinfections among pts with CMV viremia Disclosures Perales: Merck: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Giralt:TAKEDA: Consultancy, Honoraria, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; AMGEN: Consultancy, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding.

Background: Streptococcus pneumoniae is a major cause of sepsis, pneumonia and meningitis leading to significant morbidity and mortality. Invasive pneumococcal disease (IPD) and mortality rates in cancer patients (pts) are higher than in the general population. Since the introduction of routine childhood immunization with the 7-valent pneumococcal conjugate vaccine (PREVNAR) in 2000, the overall rate of invasive pneumococcal disease (IPD) decreased in adults in the US. The impact of PREVNAR on rates of IPD in cancer pts is not well studied. Methods: Retrospective review of pts treated at Memorial Sloan-Kettering Cancer Center from 1991-2012. Unique patient visits per year were obtained from the hospital database. Prevalence was calculated as number of cases per 1000 patient-visits per year. Pts with positive culture(s) for S. pneumoniae were identified from microbiology records. Serotype data is available through 2001. IPD was defined as a positive S. pneumoniae culture from a sterile site such as blood, CSF, or pleural fluid culture. Pneumococcal pneumonia was defined as a positive S. pneumoniae culture from thelower respiratory tract or a positive upper respiratory culture with either radiographic evidence for pneumonia and/or ICD-9 code "pneumococcal pneumonia" or "pneumonia" associated with the positive culture for S. pneumoniae. Colonization was defined as positive culture for S. pneumoniae without IPD or pneumonia. We compared prevalence between "early" (prior to PREVNAR, 1992-2001) and "late" (2002-2012) periods, in those with diagnoses of solid tumors and hematologic malignancies, and between ages ≤21 and >21 years. Pneumococcal vaccination rates were calculated as the number of patients who received any pneumococcal vaccine divided by the number of patient-visits per year. Results: The mean number of patient-visits per year was 46,394 and 96,954, for the early and late period, respectively. Of 1,033 patients with ≥1 culture positive for S. pneumoniae during the study, 329 (32%) had IPD, 394 (38%) had pneumococcal pneumonia and 310 (30%) had colonization. S. pneumoniae type 6 accounted for 39 (22%) isolates where serotype data was available. The annual prevalence of IPD, pneumonia and colonization gradually declined over the course of the study (Figure 1). Compared to the early period, the prevalence of IPD, pneumonia and colonization were lower in the late period (Table 1). The highest prevalence of IPD was observed in pts ≤21 years of age (2.8 and 1.3 for early and late periods, respectively). The prevalence of IPD in pts with hematologic malignancies was 5-fold higher than pts with solid tumors in the early period (1.8 vs 0.39 respectively; P Conclusions: 1) Over a 20 year period we observed a decline in the prevalence of IPD, pneumococcal pneumonia and colonization in cancer patients at a tertiary cancer center in NYC. 2) The prevalence of IPD, pneumococcal pneumonia and colonization was significantly lower (P Table 1. Prevalence for IPD, Pneumonia and Colonization by Period Prevalence Period IPD PNA Colonization 1992-2001 0.36 0.48 0.48 2002-2012 0.16 0.16 0.08 Prevalence ratio (Early/Late) 2.25 3.0 6.0 95% CI 2.0-2.7 2.6-3.4 4.8-7.7 P value Figure 1. Annual prevalence of IPD, pneumonia and colonization Figure 1. Annual prevalence of IPD, pneumonia and colonization Disclosures Kaltsas: Pfizer: Research Funding.

Background: Hematopoietic cell transplant (HCT) patients are at risk for serious infections by Herpes simplex1/2 (HSV) and Varicella zoster(VZV). Current guidelines recommend prophylaxis (PPX) with acyclovir (ACV) during the peri-transplant period and continuing throughout the period of immunosuppression. Brincidofovir (BCV, CMX001) is a broad-spectrum nucleotide analog with in vitro activity against double stranded DNA (dsDNA) viruses including HSV and VZV. BCV is in Phase 3 development for the prevention of cytomegalovirus (CMV) infection post-HCT. At present, there is no clinical data to support the efficacy of BCV PPX for HSV/VZV post-HCT. At Memorial Sloan-Kettering Cancer Center (MSK) from 2010 to June 2015, approximately 70 alloHCT recipients were treated with BCV for various indications. We examined rates of breakthrough HSV and VZV infections during administration of BCV. Methods: Retrospective review of alloHCT patients (pts) treated for ≥14 days with BCV, on protocols or emergency investigational new drug application (EIND) at MSK. Administration of concomitant (CON) ACV for HSV PPX during BCV treatment was at clinician discretion. CMX001-350 was an expanded access study of BCV for treatment of subjects with dsDNA viruses and limited or no therapeutic options. CMX001-202 was a placebo-controlled study of BCV as preemption of adenovirus (ADV) post-HCT. CMX001-304 is a currently ongoing open label study of BCV for preemption of ADV disease. After June 2011, all pts received BCV 200mg total weekly dose [100mg twice weekly (BIW) or 200mg weekly (QW)] for adult/adolescent pts and 4mg/kg total weekly dose (2mg/kg BIW or 4mg/kg QW) for pediatric pts. Patients enrolled on CMX-304 through June 16, 2015 are included in this analysis. Based on CON ACV PPX during BCV treatment, 3 patient groups were identified: 1) No CON ACV PPX (group A); 2) Partial CON ACV PPX (group B); and 3) CON ACV PPX (group 3). Diagnosis of HSV or VZV infection was based on viral culture or PCR and clinical symptoms. Data was extracted from medical, pharmacy, and microbiology records. Results: Of 55 alloHCT pts who received BCV ≥14 days, 27 (49%) were treated on CMX001-350, 1 (2%) on CMX001-202, 12 (22%) on CMX001-304 and 15 (27%) under EIND. The median age was 53 years (range, 3-69). Thirty nine (71%) pts received T-cell depleted allografts, 9 (16%) cord blood, and 7 (13%) conventional allografts. BCV started a median of 85 days [Interquartile range (IQR), 59.5-209.5] post-HCT. The primary indication for BCV was: ACV resistant HSV in 2 (4%) pts, disseminated VZV in 1 (2%) pts, CMV in 28 (51%) pts, ADV in 21 (38%) pts and other dsDNA viruses (BK polyoma virus, Human herpesvirus 6, John Cunningham virus) in 3 (5%) pts. Thirty one (56%) pts received BCV 100mg BIW. Among 35 pts in group A, duration of BCV was a median of 39 days (IQR, 29.5-78). One pt in group A received concomitant anti-CMV antivirals for 12 days. Among 13 pts in group B, the median duration of BCV monotherapy was 23 days (IQR, 13-40). Two pts in group B had brief concomitant exposure to anti-CMV antivirals. Among 7 patients in group C, the median duration of BCV treatment was 71 days (IQR, 40-132) (Table 1). Two pts developed HSV infections during BCV treatment. For one pt, the primary indication for BCV was ACV resistant HSV. The pt initially responded to BCV with resolution of oral lesions. During his treatment with BCV, he had intermittent oral ulcers and oral cultures positive for HSV. The second pt developed breakthrough HSV infection during interruption of BCV for severe diarrhea related to graft versus-host disease. There were no breakthrough VZV infections. For one pt, the primary indication for BCV was disseminated VZV. The pt had complete resolution of VZV and no further VZV reactivation during BCV treatment. Conclusions: 1) During 2,806 patient-days of BCV monotherapy in highly immunosuppressed HCT pts, there were no VZV infections and only 2 breakthrough HSV infections. 2) BCV treatment interruptions and impaired gastrointestinal absorption likely explain the breakthrough HSV infections. 3) Our retrospective analysis in this heterogeneous cohort of sick alloHCT recipients supports that BCV administration at doses currently studied for CMV prevention is likely effective for prophylaxis of HSV/VZV in HCT. Disclosures Off Label Use: Brincidofovir (CMX001) for the efficacy of prophylaxis for Herpes simples 1/2 and Varicella zoster..

Junctional adhesion molecule-A (JAM-A) is an adhesive protein expressed in endothelial cells, epithelial cells, platelets, and some leukocytes. JAM-A localizes to the tight junctions between contacting endothelial and epithelial cells, where it contributes to cell-cell adhesion and to the control of paracellular permeability. JAM-A also regulates cell motility, even though the quantitative biophysical features have not been characterized. In this study, we evaluated the role of JAM-A in the regulation of cell motility using JAM-A-expressing and JAM-A-deficient murine endothelial cells. We report that, in the absence of shear stress, JAM-A absence increases cell motility by increasing directional persistence but not cell speed. In addition, in the presence of shear stress, JAM-A absence increases protrusion extension in the direction of flow and increased downstream cellular displacement (while, conversely, decreasing upstream displacement). All these effects of JAM-A absence are mitigated by the microtubule-stabilizing compound taxol. A motility- and microtubule-related function, integrin-mediated adhesiveness, was only slightly reduced in JAM-A-deficient cells compared with JAM-A-expressing cells. However, overexpression of JAM-A in the JAM-A-deficient cells increased integrin adhesiveness to the same levels as those observed in taxol-treated JAM-A-deficient cells. Taken together, these data indicate that JAM-A regulates cell motility by cooperating with microtubule-stabilizing pathways.

Abstract —Vascular smooth muscle cells must monitor and respond to their mechanical environment; however, the molecular response of these cells to mechanical stimuli remains incompletely defined. By applying a highly uniform biaxial cyclic strain to cultured cells, we used DNA microarray technology to describe the transcriptional profile of mechanically induced genes in human aortic smooth muscle cells. We first identified vascular endothelial growth factor (VEGF) as a mechanically induced gene in these cells; VEGF served as a positive control for these experiments. We then used a DNA microarray with 5000 genes with putative functions to identify additional mechanically induced genes. Surprisingly, relatively few genes are mechanically induced in human aortic smooth muscle cells. Only 3 transcripts of 5000 were induced >2.5-fold: cyclooxygenase-1, tenascin-C, and plasminogen activator inhibitor-1. Downregulated transcripts included matrix metalloproteinase-1 and thrombomodulin. The transcriptional profile of mechanically induced genes in human aortic smooth muscle cells suggests a response of defense against excessive deformation. These data also demonstrate that in addition to identifying large clusters of genes that respond to a given stimulus, DNA microarray technology may be used to identify a small subset of genes that comprise a highly specific molecular response.

Introduction: Mechanotransduction is the process by which cells couple their external and internal environments by transducing mechanical stimuli into a biochemical response. While this process is responsible for regulating cell fate through gene expression (i.e. - c-myc, c-fos, etc.), it remains incompletely characterized in cardiac myocytes. Our previous work demonstrated that primary neonatal rat ventricular myocytes (NRVM) respond to mechanical shear stress via junctional remodeling of plakoglobin (JUP) and N-cadherin (NC). Here, we study myocyte mechanotransduction by investigating ERK phosphorylation, c-myc & c-fos gene regulation; we also investigate the effects of inhibiting primary cilia in NRVM, one potential mechanism for transduction of mechanical stimuli.Methods: NRVM were transfected with adenoviruses to express either of two different mutations in JUP (2057del2 & S39_K40insS) which cause arrhythmogenic right ventricular cardiomyopathy (ARVC). Primary cilia were inhibited by transfecting NRVM with siRNA against intraflagellar transport protein 88 (IFT88). NRVM were sheared under oscillatory flow in a parallel-plate shear chamber at physiologically-relevant shear stresses (forces in the plane of the cell layer that mimic those in contracting myocardium; 0.06 Pascal). Samples were subsequently stained and imaged on an Olympus IX-81 confocal microscope using quantitative confocal microscopy. Immunoblotting was performed on NRVM lysates separated by PAGE.Conclusions: We demonstrate that ERK phosphorylation, and c-myc & c-fos regulation are unaffected by mutant JUP expression. Our previous results indicate that cardiac myocytes are responsive to shear stimuli, and undergo junctional remodeling of both NC and JUP under oscillatory shear, a phenomenon absent in cells expressing ARVC-causing mutant JUP. Interestingly, knockdown of IFT88 does not have a dramatic effect on junctional remodeling; these results suggest that the role of primary cilia in cardiomyocyte shear sensing is varied and does not affect all shear-dependent pathways.

Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhd(flox/flox) mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.

Control over cell viability is a fundamental property underlying numerous physiological processes. Cell spreading on a substrate was previously demonstrated to be a major factor in determining the viability of individual cells. In multicellular organisms, cell-cell contact is likely to play a significant role in regulating cell vitality, but its function is easily masked by cell-substrate interactions, thus remains incompletely characterized. In this study, we show that suspended immortalized human keratinocyte sheets with persisting intercellular contacts exhibited significant contraction, junctional actin localization, and reinforcement of cell-cell adhesion strength. Further, cells within these sheets remain viable, in contrast to trypsinized cells suspended without either cell-cell or cell-substrate contact, which underwent apoptosis at high rates. Suppression of plakoglobin weakened cell-cell adhesion in cell sheets and suppressed apoptosis in suspended, trypsinized cells. These results demonstrate that cell-cell contact may be a fundamental control mechanism governing cell viability and that the junctional protein plakoglobin is a key regulator of this process. Given the near-ubiquity of plakoglobin in multicellular organisms, these findings could have significant implications for understanding cell adhesion, modeling disease progression, developing therapeutics and improving the viability of tissue engineering protocols.

Background There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking. Results In this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion. Conclusions All components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network.