Modeling Cell Energy Metabolism as Weighted Networks of Non-autonomous Oscillators.

Networks of oscillating processes are a common occurrence in living systems. This is as true as anywhere in the energy metabolism of individual cells. Exchanges of molecules and common regulation operate throughout the metabolic processes of glycolysis and oxidative phosphorylation, making the consideration of each of these as a network a natural step. Oscillations are similarly ubiquitous within these processes, and the frequencies of these oscillations are never truly constant. These features make this system an ideal example with which to discuss an alternative approach to modeling living systems, which focuses on their thermodynamically open, oscillating, non-linear and non-autonomous nature. We implement this approach in developing a model of non-autonomous Kuramoto oscillators in two all-to-all weighted networks coupled to one another, and themselves driven by non-autonomous oscillators. Each component represents a metabolic process, the networks acting as the glycolytic and oxidative phosphorylative processes, and the drivers as glucose and oxygen supply. We analyse the effect of these features on the synchronization dynamics within the model, and present a comparison between this model, experimental data on the glycolysis of HeLa cells, and a comparatively mainstream model of this experiment. In the former, we find that the introduction of oscillator networks significantly increases the proportion of the model's parameter space that features some form of synchronization, indicating a greater ability of the processes to resist external perturbations, a crucial behavior in biological settings. For the latter, we analyse the oscillations of the experiment, finding a characteristic frequency of 0.01-0.02 Hz. We further demonstrate that an output of the model comparable to the measurements of the experiment oscillates in a manner similar to the measured data, achieving this with fewer parameters and greater flexibility than the comparable model.

Clinical Imaging Findings of Vestibular Aqueduct Trauma in a Patient With Posttraumatic Meniere's Syndrome.

Posttraumatic Meniere's syndrome is a rare clinical entity. The pathomechanism by which temporal bone trauma leads to fluctuating audiovestibular symptoms, in some cases with a delay of onset many years after trauma, remains elusive. Here, a clinical case and the respective temporal bone imaging data were reviewed to investigate the underlying inner ear pathology. A 44-year-old patient presented with left-sided Meniere's syndrome 34 years after he suffered an ipsilateral temporal bone fracture caused by a car accident. Clinical imaging showed left cochleovestibular hydrops (gadolinium-enhanced MRI) and bony obliteration of the left VA (CT imaging), resulting in discontinuity of the ES. Our findings suggest that a temporal bone fracture with a "retrolabyrinthine" course, traversing the VA, caused intraaqueductal callus bone formation and progressive blockage of the VA. As a result, the extraosseous (distal) endolymphatic sac (eES) became separated from the cochleovestibular labyrinth, an event that presumably underlies endolymphatic hydrops formation and that precipitates the onset of clinical Meniere's symptoms in this case.

Vestibular Aqueduct Morphology Correlates With Endolymphatic Sac Pathologies in Menière's Disease-A Correlative Histology and Computed Tomography Study.

HYPOTHESIS: The vestibular aqueduct (VA) in Menière's disease (MD) exhibits different angular trajectories depending on the presenting endolymphatic sac (ES) pathology, i.e., 1) ES hypoplasia or 2) ES degeneration. BACKGROUND: Hypoplasia or degeneration of the ES was consistently found in inner ears affected by MD. The two etiologically distinct ES pathologies presumably represent two disease "endotypes," which may be associated with different clinical traits ("phenotypes") of MD. Recognizing these endotypes in the clinical setting requires a diagnostic tool. METHODS: 1) Defining the angular trajectory of the VA (ATVA) in the axial plane. 2) Measuring age-dependent normative data for the ATVA in postmortem temporal bone histology material from normal adults and fetuses. 3) Validating ATVA measurements from normative CT imaging data. 4) Correlating the ATVA with different ES pathologies in histological materials and CT imaging data from MD patients. RESULTS: 1) The ATVA differed significantly between normal adults and MD cases with ES degeneration, as well as between fetuses and MD cases with ES hypoplasia; 2) a strong correlation between ATVA measurements in histological sections and CT imaging data was found; 3) a correlation between the ATVA, in particular its axial trajectory in the opercular region (angle αexit), with degenerative (αexit < 120°) and hypoplastic ES pathology (αexit > 140°) was demonstrated. CONCLUSION: We established the ATVA as a radiographic surrogate marker for ES pathologies. CT-imaging-based determination of the ATVA enables endotyping of MD patients according to ES pathology. Future studies will apply this method to investigate whether ES endotypes distinguish clinically meaningful subgroups of MD patients.

Mechanical Compression of Coverslipped Tissue Sections During Heat-induced Antigen Retrieval Prevents Section Detachment and Preserves Tissue Morphology.

Heat-induced antigen retrieval (HIAR) is routinely employed on aldehyde-fixed tissue sections to enhance the reactivity of antibodies that exhibit weak or no specific interactions with tissue antigens when applied in conventional immunohistochemical protocols. A major drawback of HIAR protocols is, however, the heat-induced detachment of sections from the microscope slide with resultant impaired tissue morphology or loss of the section. We developed a method in which tissue sections mounted on glass slides are temporally coverslipped, and a clamp is used to compress the sections on the microscope slide during HIAR treatment. This "pressurized coverslipping" during HIAR was tested on various formalin-fixed tissues (murine kidneys and temporal bones, human tonsils and temporal bones) that were embedded in paraffin or celloidin. The method reliably kept the sections adherent to the slide, preserved the tissue morphology, and effectively retrieved tissue antigens for improved results in immunohistochemical labeling, even for exceptionally delicate, large, and poorly adhering sections, that is, decalcified human temporal bone sections. In summary, we present a simple method for improved slide adherence and morphological preservation of tissue sections during HIAR treatment that can be combined with all HIAR protocols and that requires only basic lab equipment.

Inner ear pathologies impair sodium-regulated ion transport in Meniere's disease.

Meniere's disease (MD), a syndromal inner ear disease, is commonly associated with a pathological accumulation of endolymphatic fluid in the inner ear, termed "idiopathic" endolymphatic hydrops (iEH). Although numerous precipitating/exacerbating factors have been proposed for MD, its etiology remains elusive. Here, using immunohistochemistry and in situ protein-protein interaction detection assays, we demonstrate mineralocorticoid-controlled sodium transport mechanisms in the epithelium of the extraosseous portion of the endolymphatic sac (eES) in the murine and human inner ears. Histological analysis of the eES in an extensive series of human temporal bones consistently revealed pathological changes in the eES in cases with iEH and a clinical history of MD, but no such changes were found in cases with "secondary" EH due to other otological diseases or in healthy controls. Notably, two etiologically different pathologies-degeneration and developmental hypoplasia-that selectively affect the eES in MD were distinguished. Clinical records from MD cases with degenerative and hypoplastic eES pathology revealed distinct intergroup differences in clinical disease presentation. Overall, we have identified for the first time two inner ear pathologies that are consistently present in MD and can be directly linked to the pathogenesis of EH, and which potentially affect the phenotypical presentation of MD.

An Aminocaprolactam Racemase from Ochrobactrum anthropi with Promiscuous Amino Acid Ester Racemase Activity.

The kinetic resolution of amino acid esters (AAEs) is a useful synthetic strategy for the preparation of single-enantiomer amino acids. The development of an enzymatic dynamic kinetic resolution (DKR) process for AAEs, which would give a theoretical yield of 100 % of the enantiopure product, would require an amino acid ester racemase (AAER); however, no such enzyme has been described. We have identified low AAER activity of 15 U mg-1 in a homologue of a PLP-dependent α-amino ϵ-caprolactam racemase (ACLR) from Ochrobactrum anthropi. We have determined the structure of this enzyme, OaACLR, to a resolution of 1.87 Šand, by using structure-guided saturation mutagenesis, in combination with a colorimetric screen for AAER activity, we have identified a mutant, L293C, in which the promiscuous AAER activity of this enzyme towards l-phenylalanine methyl ester is improved 3.7-fold.

Labyrinthitis Ossificans: On the Mechanism of Perilabyrinthine Bone Remodeling.

INTRODUCTION: It has been suggested that remodeling of the otic capsule is highly suppressed by the action of anti-resorptive signals emanating from structures of the inner ear space. Labyrinthitis ossificans (LO) is a severe complication to bacterial meningitis and is characterized by destruction of inner ear structures by the formation of new bone. The aim of this study was to explore the impact of LO on bone remodeling of the otic capsule. MATERIAL AND METHODS: In 11 human temporal bones with extensive LO and 10 control specimens, the degree of bone remodeling was explored indirectly by estimating the viability of osteocytes in perilabyrinthine bone and the mastoid. RESULTS: The viability of osteocytes was significantly lower in the perilabyrinthine bone compared to the mastoid in both groups. However, the loss of perilabyrinthine osteocytes was the same in the 2 groups, and the presence of cartilage remnants appeared to be the same. CONCLUSION: This study indicates that the factors affecting bone remodeling of the otic capsule and the degeneration of osteocytes are not altered by wholesale destruction of inner ear soft tissue and its replacement by bone. Therefore, alternative mechanisms may be implicated in the suppression of capsular bone remodeling.

Age as a factor in do not attempt cardiopulmonary resuscitation decisions: a multicentre blinded simulation-based study.

BACKGROUND: The European Resuscitation Council Guidelines recognise that there is a lack of direct evidence for the effect of age on outcome following cardiopulmonary resuscitation. AIM: To determine the role that advancing age plays in the decision by clinicians to complete a do not attempt cardiopulmonary resuscitation order based on perceived futility. DESIGN: A questionnaire-based trial. Clinicians were randomly assigned to receive one of two versions of a patient case, varying in age but otherwise identical (90 years vs 60 years). Participants were asked to decide whether a do not attempt cardiopulmonary resuscitation form should be completed based on perceived futility for a single patient case. Rates of do not attempt cardiopulmonary resuscitation order were compared between groups. PARTICIPANTS: Consultant physicians, surgeons and anaesthetists from 12 district general hospitals in England. RESULTS: In total, 291 questionnaires were returned. Overall, clinicians were significantly more likely to complete a do not attempt cardiopulmonary resuscitation form for a 90-year-old patient than a 60-year-old patient, when all other factors are equal (67.7% vs 7.4%, p < 0.001). This finding was consistent across speciality and experience level of the consultant. Surgeons were found to be significantly less likely to complete a do not attempt cardiopulmonary resuscitation order in the 90-year-old patient compared to other consultants (46.4% vs 74.1%, p < 0.001). Anaesthetists were more likely than other consultants to complete a do not attempt cardiopulmonary resuscitation order in the 60-year-old patient (17.8% vs 4.3%, p < 0.05). CONCLUSION: Age is a highly significant independent factor in a clinicians' decision to withhold cardiopulmonary resuscitation. We highlight a potential gap between current practice and supporting evidence base.

Temporal bone histopathology in a case of sensorineural hearing loss caused by superficial siderosis of the central nervous system and treated by cochlear implantation.

OBJECTIVE: To evaluate the histopathology of the temporal bones of a patient with documented superficial siderosis of the central nervous system who underwent right cochlear implantation six years before death. BACKGROUND: Superficial siderosis of the central nervous system is due to chronic or repeated subarachnoid hemorrhage and results in sensorineural deafness in 95% of affected individuals in addition to other neurologic findings. The deposition of hemosiderin in the meninges and around cranial nerves is thought to be causative. There have been no previous reports of temporal bone pathology in this disorder.This 57 year old man developed progressive, bilateral hearing loss starting in his 30's with loss of pure tone thresholds and word recognition. He underwent a right cochlear implant at age 51 with full insertion of the device. METHODS: The temporal bones and brainstem were fixed in formalin and prepared for histologic study by standard techniques. Special stains, including Gomori stain for iron were performed on sections of the temporal bones and cochlear nucleus. RESULTS: There was severe bilateral degeneration of the organ of Corti, spiral ligament, stria vascularis, and spiral ganglion cells. Gomori stain revealed iron deposits within the spiral ligament, stria vascularis and in the subepithelial mesenchymal tissue of the maculae of the vestibular system. Evaluation of the cochlear nucleus revealed iron deposits within glial cells and larger cells, probably macrophages, near the CSF surface. On the right side, the track created by the cochlear implant entered the scala tympani and continued to mm17, as measured from the round window. DISCUSSION AND CONCLUSION: This is the first known case of superficial siderosis with documented temporal bone histopathology. Hearing loss was likely caused by severe degeneration of spiral ganglion cells in both ears, despite the presence of remaining hair cells in the middle and apical turns. This was consistent with cochlear neuronal degeneration and retrograde degeneration of spiral ganglion cells within the inner ear, or alternatively, consistent with primary degeneration of hair cells and neural structures within the cochlea. Despite the presence of neural degeneration, the patient achieved a word recognition score of 28% six months following implantation.

TAK1 expression in the cochlea: a specific marker for adult supporting cells.

Transforming growth factor-ß-activated kinase-1 (TAK1) is a mitogen activated protein kinase kinase kinase that is involved in diverse biological roles across species. Functioning downstream of TGF-ß and BMP signaling, TAK1 mediates the activation of the c-Jun N-terminal kinase signaling pathway, serves as the target of pro-inflammatory cytokines, such as TNF-α, mediates NF-κß activation, and plays a role in Wnt/Fz signaling in mesenchymal stem cells. Expression of TAK1 in the cochlea has not been defined. Data mining of previously published murine cochlear gene expression databases indicated that TAK1, along with TAK1 interacting proteins 1 (TAB1), and 2 (TAB2), is expressed in the developing and adult cochlea. The expression of TAK1 in the developing cochlea was confirmed using RT-PCR and immunohistochemistry. Immunolabeling of TAK1 in embryonic, neonatal, and mature cochleas via DAB chromogenic and fluorescent immunohistochemistry indicated that TAK1 is broadly expressed in both the developing otocyst and periotic mesenchyme at E12.5 but becomes more restricted to specific types of supporting cells as the organ of Corti matures. By P1, TAK1 immunolabeling is found in cells of the stria vascularis, hair cells, supporting cells, and Kölliker's organ. By P16, TAK1 labeling is limited to cochlear supporting cells. In the adult cochlea, TAK1 immunostaining is only present in the cytoplasm of Deiters' cells, pillar cells, inner phalangeal cells, and inner border cells, with no expression in any other cochlear cell types. While the role of TAK1 in the inner ear is unclear, TAK1 expression may be used as a novel marker for specific sub-populations of supporting cells.

Extralabyrinthine manifestations of DFNA9.

DFNA9 is an autosomal dominant cause of non-syndromic adult-onset sensorineural hearing loss with associated variable vestibular dysfunction caused by mutations in the COCH gene. DFNA9 has previously been characterized by the presence of unique histopathologic features limited to the cochlear and vestibular labyrinth. This report describes newly discovered extralabyrinthine findings within the middle ear in DFNA9 and discusses their implications. The histopathologic anatomy of extralabyrinthine structures was reviewed in 12 temporal bones from seven individuals with DFNA9 and compared with age-matched controls. All temporal bones with DFNA9 had abnormal deposits within the tympanic membrane, incudomalleal joint, and incudostapedial joint. Hematoxylin and eosin stain and Movat's pentachrome stain both revealed different staining patterns of the extralabyrinthine deposits compared with the intralabyrinthine deposits suggesting that the composition of the deposits varies with location. The deposits within the tympanic membrane resembled cartilage morphologically and stained positively for aggrecan, an extracellular matrix protein found in cartilage. However, the cellular component of the tympanic membrane deposits did not stain with immunomarkers for chondrocytes (s100 and connective tissue growth factor). These novel findings in DFNA9 have implications for the phenotypic expression of the disorder and the clinical workup of adult-onset sensorineural hearing loss.

A method for MS(E) differential proteomic analysis of archival formalin-fixed celloidin-embedded human inner ear tissue.

Proteomic analysis of cadaveric formalin-fixed, celloidin-embedded (FFCE) temporal bone tissue has the potential to provide new insights into inner ear disorders. We have developed a liquid chromatography-mass spectrometry (LC-MS) method for tissue sections embedded with celloidin. Q-TOF (Quadrupole-time of flight mass spectrometry) MS(E) (mass spectrometry where E represents collision energy) and Identity(E)™ were used in conjunction with nano-UPLC (capillary ultrahigh pressure liquid chromatography) for robust identification and quantification of a large number of proteins. Formalin-fixed paraffin-embedded (FFPE) mouse liver sections were used to evaluate formalin de-cross-linking by five different methods. Unfixed fresh mouse liver tissue was used as a control. Five different methods for preparation of FFPE tissue for MS analysis were compared, as well as four methods for celloidin removal with FFCE mouse liver tissue. The methods judged best were applied to FFCE 20 µm sections of mouse inner ear samples, and FFCE 20 µm human inner ear and human otic capsule bone sections. Three of the five-tissue extraction methods worked equally in detecting peptides and proteins from FFPE mouse liver tissue. The modified Liquid Tissue kit protocol was chosen for further studies. Four different celloidin removal methods were compared and the acetone removal method was chosen for further analysis. These two methods were applied to the analysis of FFCE inner ear and otic capsule sections. Proteins from all major cellular components were detected in the FFCE archival human temporal bone sections. This newly developed technique enables the use of FFCE tissues for proteomic studies.

Differences in gene expression between the otic capsule and other bones.

Our long term goal is to understand the molecular pathology of otosclerosis and to develop better forms of therapy. Toward this goal, the current study focused on characterizing the molecular factors responsible for the unique biological features of the otic capsule: its minimal rate of remodeling, and lack of healing capacity when fractured. We compared expression levels of 62 genes involved in bone metabolism between the adult murine otic capsule and the tibia and parietal bones; the latter exemplify bones formed by endochondral and intramembranous ossification, respectively. Gene expression levels were measured using real-time quantitative RT-PCR and analyzed using tools of bioinformatics. Expression patterns of key genes were verified with in situ hybridization. The molecular profile of the otic capsule was distinctly different from that of the tibia and parietal bone. Genes found to be most characteristic of the otic capsule were: osteoprotegerin (opg), bone morphogenetic protein receptor 1b (bmpr1b) and bone morphogenetic protein 3 (bmp3). Expression levels were high for opg and bmpr1b, and minimal for bmp3 within the otic capsule. We concluded that opg and bmpr1b likely play important roles in inhibition of remodeling within the otic capsule.

Techniques of celloidin removal from temporal bone sections.

OBJECTIVES: We sought to determine whether the technique of celloidin removal influences the results of immunostaining in celloidin-embedded cochleae. METHODS: We compared four protocols of celloidin removal, including those using clove oil, acetone, ether-alcohol, and methanol saturated with sodium hydroxide. By optimally fixing our tissue (perfused mice), and keeping constant the fixative type (formalin plus acetic acid), fixation time (25 hours), and decalcification time (ethylenediaminetetraacetic acid for 7 days), we determined whether the technique of celloidin removal influenced the immunostaining results. Six antibodies were used with each removal method: prostaglandin D synthase, sodium, potassium adenosine triphosphatase (Na+,K(+)-ATPase), aquaporin 1, connective tissue growth factor, tubulin, and 200 kd neurofilament. RESULTS: Clove oil, acetone, and ether-alcohol resulted in incomplete removal of the celloidin, thereby negatively affecting the results of immunostaining. The methanol-sodium hydroxide method was effective in completely removing the celloidin; it produced the cleanest and most reproducible immunostaining for all six antibodies. CONCLUSIONS: Freshly prepared methanol saturated with sodium hydroxide and diluted 1:2 with methanol was the best solvent for removing celloidin from mouse temporal bone sections, resulting in consistent and reproducible immunostaining with the six antibodies tested.

Wolfram syndrome: a clinicopathologic correlation.

Wolfram syndrome or DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy and deafness) is a neurodegenerative disorder characterized by diabetes mellitus and optic atrophy as well as diabetes insipidus and deafness in many cases. We report the post-mortem neuropathologic findings of a patient with Wolfram syndrome and correlate them with his clinical presentation. In the hypothalamus, neurons in the paraventricular and supraoptic nuclei were markedly decreased and minimal neurohypophyseal tissue remained in the pituitary. The pontine base and inferior olivary nucleus showed gross shrinkage and neuron loss, while the cerebellum was relatively unaffected. The visual system had moderate to marked loss of retinal ganglion neurons, commensurate loss of myelinated axons in the optic nerve, chiasm and tract, and neuron loss in the lateral geniculate nucleus but preservation of the primary visual cortex. The patient's inner ear showed loss of the organ of Corti in the basal turn of the cochleae and mild focal atrophy of the stria vascularis. These findings correlated well with the patient's high-frequency hearing loss. The pathologic findings correlated closely with the patient's clinical symptoms and further support the concept of Wolfram syndrome as a neurodegenerative disorder. Our findings extend prior neuropathologic reports of Wolfram syndrome by providing contributions to our understanding of eye, inner ear and olivopontine pathology in this disease.

Immunocytochemical traits of type IV fibrocytes and their possible relations to cochlear function and pathology.

One of the more consistent and least understood changes in the aging human cochlea is the progressive loss of fibrocytes within the spiral ligament. This report presents an animal model for type IV fibrocyte loss, along with immunocytochemical evidence that noise-induced loss of these cells may account for previously unexplained hearing losses. The remarkably low threshold for noise-induced loss of type IV fibrocytes, approximately 24 dB less than the threshold for adjacent hair cell destruction, may account for the prevalence of missing fibrocytes in humans. In mice, changes in the spectrum of traumatizing noise had little effect upon the site of loss of the fibrocytes, suggesting that the primary site of damage that induced the loss was the basal-most cochlear turn, a site expected to be damaged by all three noise bands. Type IV fibrocytes were found to immunostain for connective tissue growth factor (CTGF) and for transforming growth factor beta receptor 3, a receptor that is known to activate CTGF expression. Type IV fibrocytes lack immunostaining for adenosine triphosphatase and connexins that are key players in potassium ion uptake and transmission, which suggests that they play little, if any, role in potassium recycling from perilymphatic space to the endolymphatic space. Consequently, their loss probably does not directly reduce this process. Immunostaining for a receptor for CTGF, low-density-lipoprotein-related protein 1, indicated that CTGF acts as an autocrine and a paracrine agent within the cochlea. The lack of CTGF paracrine effects following noise-induced loss of type IV fibrocytes may account for previously unexplained hearing losses.

Effects of fixative and embedding medium on morphology and immunostaining of the cochlea.

The localization of proteins by immunostaining is a powerful method to investigate otologic disorders. However, the use of fixatives and embedding media (necessary for the preservation of morphology) can obscure antigens, making it difficult to perform immunoassays. We performed a systematic investigation of the effects of fixative and embedding medium on morphology and immunostaining of the mouse cochlea. Three different fixative solutions [4% formaldehyde (F), 4% formaldehyde + 1% acetic acid (FA), and 4% formaldehyde + 1% acetic acid + 0.1% glutaraldehyde (FGA)] and 3 different embedding media (paraffin, polyester wax, and celloidin) were used. Morphology was assessed using light microscopy. Immunostaining was studied using a panel of 6 antibodies (to prostaglandin D synthase, aquaporin 1, connective tissue growth factor, 200-kDa neurofilament, tubulin and Na(+),K(+)-ATPase). Preservation of morphology was suboptimal with paraffin, adequate with polyester wax and superb with celloidin. Immunostaining was successful using all 6 antibodies in all 3 fixatives and all 3 embedding media. While there were differences in strength of signal and localization of antigen between the 3 fixatives, overall, FA and FGA gave the most uniform results. For a given fixative and antibody, there was surprisingly little difference in the quality of immunostaining between celloidin and paraffin, while results in polyester wax were not as good in some cases. These results suggest that celloidin may be the embedding medium of choice for both morphological and pathological studies, including immunostaining when morphology must be optimized.

CARMA3 mediates lysophosphatidic acid-stimulated cytokine secretion by bronchial epithelial cells.

NF-kappaB activation in bronchial epithelial cells is important for the development of allergic airway inflammation, and may control the expression of critical mediators of allergic inflammation such as thymic stromal lymphopoietin (TSLP) and the chemokine CCL20. Members of the caspase recruitment domain (CARD) family of proteins are differentially expressed in tissue and help mediate NF-kappaB activity in response to numerous stimuli. Here we demonstrate that CARMA3 (CARD10) is specifically expressed in human airway epithelial cells, and that expression of CARMA3 in these cells leads to activation of NF-kappaB. CARMA3 has recently been shown to mediate NF-kappaB activation in embryonic fibroblasts after stimulation with lysophosphatidic acid (LPA), a bioactive lipid-mediator that is elevated in the lungs of individuals with asthma. Consistent with this, we demonstrate that stimulation of airway epithelial cells with LPA leads to increased expression of TSLP and CCL20. We then show that inhibition of CARMA3 activity in airway epithelial cells reduces LPA-mediated NF-kappaB activity and the production of TSLP and CCL20. In conclusion, these data demonstrate that LPA stimulates TSLP and CCL20 expression in bronchial epithelial cells via CARMA3-mediated NF-kappaB activation.