Case report: CMV retinitis following local and systemic immunosuppression.

We report the case of a 71-year-old man who presented 2 years following renal transplantation with diffuse, unilateral cytomegalovirus retinitis five weeks after receiving an intravitreal dexamethasone implant device for the management of central retinal vein occlusion. Examination of the left eye showed diffuse retinal hemorrhages, attenuated and tortuous retinal vessels, and superior retinal whitening. The patient was successfully treated with serial intravitreal foscarnet injections and oral valganciclovir with disease regression observed by 12 weeks after presentation. The patient's visual acuity and examination remained stable at 9-months follow-up.

Exploring the Vitreoretinal Interface: A Key Instigator of Unique Retinal Hemorrhage Patterns in Pediatric Head Trauma.

PURPOSE: Various types of trauma can cause retinal hemorrhages in children, including accidental and nonaccidental head trauma. We used animal eyes and a finite element model of the eye to examine stress patterns produced during purely linear and angular accelerations, along with stresses attained during simulated repetitive shaking of an infant. METHODS: Using sheep and primate eyes, sclerotomy windows were created by removing the sclera, choroid, and retinal pigment epithelium to expose the retina. A nanofiber square was glued to a 5 mm2 area of retina. The square was pulled and separated from vitreous while force was measured. A finite element model of the pediatric eye was used to computationally measure tension stresses during shaking. RESULTS: In both sheep and primate eyes, tension stress required for separation of retina from vitreous range from 1 to 5 kPa. Tension stress generated at the vitreoretinal interface predicted by the computer simulation ranged from 3 to 16 kPa during a cycle of shaking. Linear acceleration generated lower tension stress than angular acceleration. Angular acceleration generated maximal tension stress along the retinal vasculature. Linear acceleration produced more diffuse force distribution centered at the poster pole. CONCLUSIONS: The finite element model predicted that tension stress attained at the retina during forcible shaking of an eye can exceed the minimum threshold needed to produce vitreoretinal separation as measured in animal eyes. Furthermore, the results show that movements that involve significant angular acceleration produce strong stresses localized along the vasculature, whereas linear acceleration produces weaker, more diffuse stress centered towards the posterior pole of the eye.

Determining the Tractional Forces on Vitreoretinal Interface Using a Computer Simulation Model in Abusive Head Trauma.

PURPOSE: Abusive head trauma (AHT) is the leading cause of infant death and long-term morbidity from injury. The ocular consequences of AHT are controversial, and the pathophysiology of retinal research findings is still not clearly understood. It has been postulated that vitreoretinal traction plays a major role in the retinal findings. A computer simulation model was developed to evaluate the vitreoretinal traction and determine whether the distribution of forces in different layers and locations of the retina can explain the patterns of retinal hemorrhage (RH) seen in AHT. DESIGN: Computer simulation model study. METHODS: A computer simulation model of the pediatric eye was developed to evaluate preretinal, intraretinal, and subretinal stresses during repetitive shaking. This model was also used to examine the forces applied to various segments along blood vessels. RESULTS: Calculated stress values from the computer simulation ranged from 3-16 kPa at the vitreoretinal interface through a cycle of shaking. Maximal stress was observed at the periphery of the retina, corresponding to areas of multiple vessel bifurcations, followed by the posterior pole of the retina. Stress values were similar throughout all 3 layers of the retina (preretinal, intraretinal, and subretinal layers). CONCLUSIONS: Ocular manifestations from AHT revealed unique retinal characteristics. The model predicted stress patterns consistent with the diffuse retinal hemorrhages (RH) typically found in the posterior pole and around the peripheral retina in AHT. This computer model demonstrated that similar stress forces were produced in different layers of the retina, consistent with the finding that retinal hemorrhages are often found in multiple layers of the retina. These data can help explain the RH patterns commonly found in AHT.

TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS.

Disrupted nucleocytoplasmic transport (NCT) has been implicated in neurodegenerative disease pathogenesis; however, the mechanisms by which disrupted NCT causes neurodegeneration remain unclear. In a Drosophila screen, we identified ref(2)P/p62, a key regulator of autophagy, as a potent suppressor of neurodegeneration caused by the GGGGCC hexanucleotide repeat expansion (G4C2 HRE) in C9orf72 that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We found that p62 is increased and forms ubiquitinated aggregates due to decreased autophagic cargo degradation. Immunofluorescence and electron microscopy of Drosophila tissues demonstrate an accumulation of lysosome-like organelles that precedes neurodegeneration. These phenotypes are partially caused by cytoplasmic mislocalization of Mitf/TFEB, a key transcriptional regulator of autophagolysosomal function. Additionally, TFEB is mislocalized and downregulated in human cells expressing GGGGCC repeats and in C9-ALS patient motor cortex. Our data suggest that the C9orf72-HRE impairs Mitf/TFEB nuclear import, thereby disrupting autophagy and exacerbating proteostasis defects in C9-ALS/FTD.

Retinopathy of prematurity: preferred practice patterns among pediatric ophthalmologists.

PURPOSE: The treatment of retinopathy of prematurity (ROP) is not standardized and can vary significantly between providers. This study aims to determine preferred practices in treating ROP by globally surveying pediatric ophthalmologists. METHODS: Between January and February 2017, an international pediatric ophthalmology interest group was invited to complete an anonymous survey of 18 questions. The main objectives were to determine the preferred first line of treatment for ROP, the preferred dosage of intravitreal bevacizumab (IVB) used, and the outcome and possible complications following bevacizumab injection. RESULTS: Out of 101 pediatric ophthalmologists, 72 (71.8%) stated that they had direct involvement in the treatment of ROP. When presented with type 1 ROP which requires treatment, 69 ophthalmologists (68.3%) stated that they prefer laser treatment over bevacizumab, and 33 ophthalmologists (32.7%) stated they would recommend bevacizumab as a first choice. Ninety-three ophthalmologists (92.1%) reported the success of 1 laser treatment between 75% and 100%, and 35 ophthalmologists (34.7%) perceive bevacizumab to be 75%-100% successful. Half dose of adult-prescribed bevacizumab at 0.625 mg/0.05 mL was preferred by 47 of the ophthalmologists (46.5%). No cases of endophthalmitis were reported with intravitreal injection. CONCLUSION: Laser photoablation remains the preferred mode of treatment for ROP among surveyed ophthalmologists across the world. Though bevacizumab is currently being used, this form of treatment is not as common, primarily due to the unknown safety profile and potential long-term ramifications of the drug.

Implementation of a Clinical Prediction Model Using Daily Postnatal Weight Gain, Birth Weight, and Gestational Age to Risk Stratify ROP.

PURPOSE: To develop a simple prognostic model using postnatal weight gain, birth weight, and gestational age to identify infants at risk for developing severe retinopathy of prematurity (ROP). METHODS: Medical records from two tertiary referral centers with the diagnosis code "Retinopathy of Prematurity" were evaluated. Those with a birth weight of 1,500 g or less, gestational age of 30 weeks or younger, and unstable clinical courses were included. Multivariate regression analysis was applied to transform three independent variables into a growth rate algorithm. RESULTS: Seventeen of 191 neonates had severe ROP. Weight gain of at least 23 g/d was determined as a protective cut-off value against development of severe ROP. This value maintained 100% sensitivity with 62% specificity to ensure all neonates who require treatment would be captured. Overall, the Omaha (OMA)-ROP model calculated a 58% reduction in eye examinations within the cohort. CONCLUSIONS: Inclusion of postnatal growth rate in risk stratification will minimize the number of eye examinations performed without increasing adverse visual outcomes. The OMA-ROP model predicts neonates who gain less than 23 g/d are at higher risk for developing severe ROP. Although promising, larger cohort studies may be necessary to validate and implement new screening practices among preterm infants. [J Pediatr Ophthalmol Strabismus. 2018;55(5):326-334.].

Endoscopic Ultrasound-Guided Tissue Acquisition Using Fork-Tip Needle Improves Histological Yield, Reduces Needle Passes, Without On-Site Cytopathological Evaluation.

Background and Aim: Endoscopic ultrasound (EUS)-guided fine needle biopsy (FNB) and fine needle aspiration (FNA) are established methods in tissue acquisition. A new fork-tip FNB needle has been used to obtain core tissue samples. We compared the performance of the FNB using fork-tip needles with that of the FNA using conventional needles in patients who had solid neoplastic lesions within and around the upper gastrointestinal (GI) tract. Methods: In this retrospective single-center study, patients who underwent EUS examinations for solid neoplastic lesions between October 2013 and February 2017 were included. The procedures were performed in the absence of an on-site cytologist. The main objectives were to compare the diagnostic yield and average number of passes of FNB using fork-tip needles versus those of FNA using conventional needles. Results: EUS/FNA and EUS/FNB were performed on 181 solid neoplastic lesions primarily in the pancreas and GI tract walls. There was no significant difference in patient's age, gender, tumor location, or tumor size. The mean number of needle passes was significantly lower in the fork-tip needle group than in the conventional needle group (3.8 vs. 5.9; p < 0.0001). There was a trend toward higher sensitivity (89.9% vs. 81%) using the fork-tip needles than when using the conventional needles (p = 0.119). No significant difference in rates of adverse events between two groups was found. Conclusions: Our study demonstrates that, compared with FNA using conventional needles, FNB using fork-tip needles required significantly fewer needle passes while achieving a relatively higher diagnostic yield due to its superior capacity in tissue acquisition from solid neoplastic lesions in and around GI tract walls without on-site cytological assessment.

New functions and signaling mechanisms for the class of adhesion G protein-coupled receptors.

The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region. In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis-inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF are associated noncovalently as a heterodimer at the plasma membrane. While the biological function of the GAIN domain-mediated autocleavage is not fully understood, mounting evidence suggests that the NTF and CTF possess distinct biological activities in addition to their function as a receptor unit. We discuss recent advances in understanding the biological functions, signaling mechanisms, and disease associations of the aGPCRs.

Persistence of the bacterial pathogen Granulibacter bethesdensis in chronic granulomatous disease monocytes and macrophages lacking a functional NADPH oxidase.

Granulibacter bethesdensis is a Gram-negative pathogen in patients with chronic granulomatous disease (CGD), a deficiency in the phagocyte NADPH oxidase. Repeated isolation of genetically identical strains from the same patient over years, and prolonged waxing and waning seropositivity in some subjects, raises the possibility of long-term persistence. G. bethesdensis resists killing by serum, CGD polymorphonuclear leukocytes (PMN), and antimicrobial peptides, indicating resistance to nonoxidative killing mechanisms. Although G. bethesdensis extends the survival of PMN, persistent intracellular bacterial survival might rely on longer-lived macrophages and their precursor monocytes. Therefore, we examined phagocytic killing by primary human monocytes and monocyte-derived macrophages (MDM). Cells from both normal and CGD subjects internalized G. bethesdensis similarly. G. bethesdensis stimulated superoxide production in normal monocytes, but to a lesser degree than in normal PMN. Normal but not CGD monocytes and MDM killed G. bethesdensis and required in vitro treatment with IFN-γ to maintain this killing effect. Although in vitro IFN-γ did not enhance G. bethesdensis killing in CGD monocytes, it restricted growth in proportion to CGD PMN residual superoxide production, providing a potential method to identify patients responsive to IFN-γ therapy. In IFN-γ-treated CGD MDM, G. bethesdensis persisted for the duration of the study (7 d) without decreasing viability of the host cells. These results indicate that G. bethesdensis is highly resistant to oxygen-independent microbicides of myeloid cells, requires an intact NADPH oxidase for clearance, and can persist long-term in CGD mononuclear phagocytes, most likely relating to the persistence of this microorganism in infected CGD patients.

Innate immunity against Granulibacter bethesdensis, an emerging gram-negative bacterial pathogen.

Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G. bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components (e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived in human serum concentrations of ≥90%, indicating a high degree of serum resistance. Consistent with the clinical host tropism of G. bethesdensis, CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, reduced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethesdensis was sensitive to H(2)O(2) but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance to O(2)-independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the relative inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms likely play an important role in G. bethesdensis pathogenesis.

Interaction of angiogenic microvessels with the extracellular matrix.

The extracellular matrix (ECM) plays a critical role in angiogenesis by providing biochemical and positional cues, as well as by mechanically influencing microvessel cell behavior. Considerable information is known concerning the biochemical cues relevant to angiogenesis, but less is known about the mechanical dynamics during active angiogenesis. The objective of this study was to characterize changes in the material properties of a simple angiogenic tissue before and during angiogenesis. During sprouting, there was an overall decrease in tissue stiffness followed by an increase during neovessel elongation. The fall in matrix stiffness coincided with peak matrix metalloproteinase mRNA expression and elevated proteolytic activity. An elevated expression of genes for ECM components and cell-ECM interaction molecules and a subsequent drop in proteolytic activity (although enzyme levels remained elevated) coincided with the subsequent stiffening. The results of this study show that the mechanical properties of a scaffold tissue may be actively modified during angiogenesis by the growing microvasculature.

Reactions in droplets in microfluidic channels.

Fundamental and applied research in chemistry and biology benefits from opportunities provided by droplet-based microfluidic systems. These systems enable the miniaturization of reactions by compartmentalizing reactions in droplets of femoliter to microliter volumes. Compartmentalization in droplets provides rapid mixing of reagents, control of the timing of reactions on timescales from milliseconds to months, control of interfacial properties, and the ability to synthesize and transport solid reagents and products. Droplet-based microfluidics can help to enhance and accelerate chemical and biochemical screening, protein crystallization, enzymatic kinetics, and assays. Moreover, the control provided by droplets in microfluidic devices can lead to new scientific methods and insights.

CYP2E1 induced by ethanol causes oxidative stress, proteasome inhibition and cytokeratin aggresome (Mallory body-like) formation.

The role of oxidative stress in alcoholic liver disease and cytokeratin aggresome formation is the focus of this in vitro study. HepG2 cells transduced to over express CYP2E1 (E47) and control HepG2 cells (C34) were first treated with arachidonic acid, then Fe-NAT, and finally with ethanol. In the E47 ethanol-treated cells, CYP2E1 was induced and a higher level of reactive oxygen species and carbonyl proteins were generated. The proteasome activity decreased significantly in the E47 ethanol-treated cells. This inhibition was prevented when CYP2E1 was inhibited by DAS. Microarray analysis showed gene expression down regulation of the proteasome subunit, as well as ubiquitin pathway proteins in the E47 ethanol-treated cells. 4-Hydroxynonenal (4-HNE) adducts were increased in the E47 cells treated with ethanol. Furthermore, the immunoprecipitated 4-HNE modified proteins from these cells stained positive with antibodies to the proteasome subunit alpha 6. These results indicate that the ethanol induced CYP2E1 generates oxidative stress that is responsible for the decrease in proteasome activity. Cytokeratin 8 and 18 were induced by ethanol treatment of E47 cells and polyubiquitinated forms of these proteins were found in the polyubiquitin smear upon Western blots analysis. Cytokeratin aggresomes and Mallory body-like inclusions formed in the ethanol-treated E47 cells, indicating that the ubiquitinated cytokeratins accumulated as a result of the inhibition of the proteasome by ethanol treatment when oxidation of ethanol induced oxidative stress. This is the first report where ethanol caused Mallory body-like cytokeratin inclusions in transformed human liver cells in vitro.

On-chip titration of an anticoagulant argatroban and determination of the clotting time within whole blood or plasma using a plug-based microfluidic system.

This paper describes extending plug-based microfluidics to handling complex biological fluids such as blood, solving the problem of injecting additional reagents into plugs, and applying this system to measuring of clotting time in small volumes of whole blood and plasma. Plugs are droplets transported through microchannels by fluorocarbon fluids. A plug-based microfluidic system was developed to titrate an anticoagulant (argatroban) into blood samples and to measure the clotting time using the activated partial thromboplastin time (APTT) test. To carry out these experiments, the following techniques were developed for a plug-based system: (i) using Teflon AF coating on the microchannel wall to enable formation of plugs containing blood and transport of the solid fibrin clots within plugs, (ii) using a hydrophilic glass capillary to enable reliable merging of a reagent from an aqueous stream into plugs, (iii) using bright-field microscopy to detect the formation of a fibrin clot within plugs and using fluorescent microscopy to detect the production of thrombin using a fluorogenic substrate, and (iv) titration of argatroban (0-1.5 microg/mL) into plugs and measurement of the resulting APTTs at room temperature (23 degrees C) and physiological temperature (37 degrees C). APTT measurements were conducted with normal pooled plasma (platelet-poor plasma) and with donor's blood samples (both whole blood and platelet-rich plasma). APTT values and APTT ratios measured by the plug-based microfluidic device were compared to the results from a clinical laboratory at 37 degrees C. APTT obtained from the on-chip assay were about double those from the clinical laboratory but the APTT ratios from these two methods agreed well with each other.

Controlling nonspecific protein adsorption in a plug-based microfluidic system by controlling interfacial chemistry using fluorous-phase surfactants.

Control of surface chemistry and protein adsorption is important for using microfluidic devices for biochemical analysis and high-throughput screening assays. This paper describes the control of protein adsorption at the liquid-liquid interface in a plug-based microfluidic system. The microfluidic system uses multiphase flows of immiscible fluorous and aqueous fluids to form plugs, which are aqueous droplets that are completely surrounded by fluorocarbon oil and do not come into direct contact with the hydrophobic surface of the microchannel. Protein adsorption at the aqueous-fluorous interface was controlled by using surfactants that were soluble in fluorocarbon oil but insoluble in aqueous solutions. Three perfluorinated alkane surfactants capped with different functional groups were used: a carboxylic acid, an alcohol, and a triethylene glycol group that was synthesized from commercially available materials. Using complementary methods of analysis, adsorption was characterized for several proteins (bovine serum albumin (BSA) and fibrinogen), including enzymes (ribonuclease A (RNase A) and alkaline phosphatase). These complementary methods involved characterizing adsorption in microliter-sized droplets by drop tensiometry and in nanoliter plugs by fluorescence microscopy and kinetic measurements of enzyme catalysis. The oligoethylene glycol-capped surfactant prevented protein adsorption in all cases. Adsorption of proteins to the carboxylic acid-capped surfactant in nanoliter plugs could be described by using the Langmuir model and tensiometry results for microliter drops. The microfluidic system was fabricated using rapid prototyping in poly(dimethylsiloxane) (PDMS). Black PDMS microfluidic devices, fabricated by curing a suspension of charcoal in PDMS, were used to measure the changes in fluorescence intensity more sensitively. This system will be useful for microfluidic bioassays, enzymatic kinetics, and protein crystallization, because it does not require surface modification during fabrication to control surface chemistry and protein adsorption.

Microfluidic systems for chemical kinetics that rely on chaotic mixing in droplets.

This paper reviews work on a microfluidic system that relies on chaotic advection to rapidly mix multiple reagents isolated in droplets (plugs). Using a combination of turns and straight sections, winding microfluidic channels create unsteady fluid flows that rapidly mix the multiple reagents contained within plugs. The scaling of mixing for a range of channel widths, flow velocities and diffusion coefficients has been investigated. Due to rapid mixing, low sample consumption and transport of reagents with no dispersion, the system is particularly appropriate for chemical kinetics and biochemical assays. The mixing occurs by chaotic advection and is rapid (sub-millisecond), allowing for an accurate description of fast reaction kinetics. In addition, mixing has been characterized and explicitly incorporated into the kinetic model.

Millisecond kinetics on a microfluidic chip using nanoliters of reagents.

This paper describes a microfluidic chip for performing kinetic measurements with better than millisecond resolution. Rapid kinetic measurements in microfluidic systems are complicated by two problems: mixing is slow and dispersion is large. These problems also complicate biochemical assays performed in microfluidic chips. We have recently shown (Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2003, 42, 768-772) how multiphase fluid flow in microchannels can be used to address both problems by transporting the reagents inside aqueous droplets (plugs) surrounded by an immiscible fluid. Here, this droplet-based microfluidic system was used to extract kinetic parameters of an enzymatic reaction. Rapid single-turnover kinetics of ribonuclease A (RNase A) was measured with better than millisecond resolution using sub-microliter volumes of solutions. To obtain the single-turnover rate constant (k = 1100 +/- 250 s(-1)), four new features for this microfluidics platform were demonstrated: (i) rapid on-chip dilution, (ii) multiple time range access, (iii) biocompatibility with RNase A, and (iv) explicit treatment of mixing for improving time resolution of the system. These features are discussed using kinetics of RNase A. From fluorescent images integrated for 2-4 s, each kinetic profile can be obtained using less than 150 nL of solutions of reagents because this system relies on chaotic advection inside moving droplets rather than on turbulence to achieve rapid mixing. Fabrication of these devices in PDMS is straightforward and no specialized equipment, except for a standard microscope with a CCD camera, is needed to run the experiments. This microfluidic platform could serve as an inexpensive and economical complement to stopped-flow methods for a broad range of time-resolved experiments and assays in chemistry and biochemistry.

Experimental test of scaling of mixing by chaotic advection in droplets moving through microfluidic channels.

This letter describes an experimental test of a simple argument that predicts the scaling of chaotic mixing in a droplet moving through a winding microfluidic channel. Previously, scaling arguments for chaotic mixing have been described for a flow that reduces striation length by stretching, folding, and reorienting the fluid in a manner similar to that of the baker's transformation. The experimentally observed flow patterns within droplets (or plugs) resembled the baker's transformation. Therefore, the ideas described in the literature could be applied to mixing in droplets to obtain the scaling argument for the dependence of the mixing time, t~(aw/U)log(Pe), where w [m] is the cross-sectional dimension of the microchannel, a is the dimensionless length of the plug measured relative to w, U [m s(-1)] is the flow velocity, Pe is the Péclet number (Pe=wU/D), and D [m(2)s(-1)] is the diffusion coefficient of the reagent being mixed. Experiments were performed to confirm the scaling argument by varying the parameters w, U, and D. Under favorable conditions, submillisecond mixing has been demonstrated in this system.

The human papillomavirus 16 E6 protein binds to tumor necrosis factor (TNF) R1 and protects cells from TNF-induced apoptosis.

High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that transfection of HPV 16 E6 into the tumor necrosis factor (TNF)-sensitive LM cell line protects expressing cells from TNF-induced apoptosis in a p53-independent manner, and the purpose of this study was to determine the molecular mechanism underlying this protection. Caspase 3 and caspase 8 activation were significantly reduced in E6-expressing cells, indicating that E6 acts early in the TNF apoptotic pathway. In fact, E6 binds directly to TNF R1, as shown both by co-immunoprecipitation and mammalian two-hybrid approaches. E6 requires the same C-terminal portion of TNF R1 for binding as does TNF R1-associated death domain, and TNF R1/TNF R1-associated death domain interactions are decreased in the presence of E6. HA-E6 also blocked cell death triggered by transfection of the death domain of TNF R1. Together, these results provide strong support for a model in which HPV E6 binding to TNF R1 interferes with formation of the death-inducing signaling complex and thus with transduction of proapoptotic signals. They also demonstrate that HPV, like several other viruses, has developed a method for evading the TNF-mediated host immune response.