Standardizing biomarker testing for Canadian patients with advanced lung cancer.

BACKGROUND: The development and approval of both targeted and immune therapies for patients with advanced non-small cell lung cancer (nsclc) has significantly improved patient survival rates and quality of life. Biomarker testing for patients newly diagnosed with nsclc, as well as for patients progressing after treatment with epidermal growth factor receptor (EGFR) inhibitors, is the standard of care in Canada and many parts of the world. METHODS: A group of thoracic oncology experts in the field of thoracic oncology met to describe the standard for biomarker testing for lung cancer in the Canadian context, focusing on evidence-based recommendations for standard-of-care testing for EGFR, anaplastic lymphoma kinase (ALK), ROS1, BRAF V600 and programmed death-ligand (PD-L1) at the time of diagnosis of advanced disease and EGFR T790M upon progression. As well, additional exploratory molecules and targets are likely to impact future patient care, including MET exon 14 skipping mutations and whole gene amplification, RET translocations, HER2 (ERBB2) mutations, NTRK, RAS (KRAS and NRAS), as well as TP53. RESULTS: The standard of care must include the incorporation of testing for novel biomarkers as they become available, as it will be difficult for national guidelines to keep pace with technological advances in this area. CONCLUSIONS: Canadian patients with nsclc should be treated equally; the minimum standard of care is defined in this paper.

Experiences with semi-routine production of riboflavin and UV-B pathogen-inactivated platelet concentrates in three blood centres.

BACKGROUND: For a clinical platelet (PLT) transfusion trial conducted in three countries, the production of PLT concentrates (PCs) that were pathogen inactivated with the Mirasol technology was set up and validated. While the Mirasol procedure is applied to an established PLT product, the PLT processing procedure still had to be modified to ensure a treated PC was of sufficient quality. Further, the effect of simulated transport conditions and the effect of ambient light on Mirasol-treated PCs was determined. STUDY DESIGN AND METHODS: Platelet concentrates in plasma were made from pooled buffy coats followed by Mirasol treatment. To mimic transport conditions, units were left unagitated for 6 h at room temperature. To mimic ambient light exposure, units were held unagitated for 4 h in direct fluorescent tube light. RESULTS: Measures had to be taken to allow 7-day storage of treated concentrates. In one site, PCs made from five buffy coats with >450 × 109 PLTs were removed from inventory. Another site went from five to four buffy coats per pool. Interruption of agitation for 6 h on day 3 did not induce meaningful changes in in vitro measures, even when stored up to 7 days. Exposure to ambient light for 4 h, either on day 3 or 6, had no effect on in vitro measures. CONCLUSION: The Mirasol pathogen inactivation process can be implemented in routine, but changes to current PLT processing methods might be needed. Transport conditions and 4-h-long ambient light exposure have no negative effect on the in vitro quality of Mirasol-treated PCs.

[The process of meaning-making in adjustment to multiple sclerosis]. / Le processus de construction de sens dans l'adaptation à la sclérose en plaques.

INTRODUCTION: Progressive neurological disorders require continual adaptation. People who maintain a better psychological balance in the face of the disease are those who find meaning in their experience. The ability to find a positive meaning in having multiple sclerosis (MS) is more common in people who are at the beginning of their disease or those who have few functional limitations (Pakenham, 2008). This qualitative study examines the process of meaning-making by people who are moderately to severely affected by MS. METHODS: Eight participants told the story of their experiences in individual semi-structured interviews. The verbatim transcripts were subjected to phenomenological analysis. RESULTS: The results allowed the identification of the essential elements that shape the experience of meaning-making. The adaptation happens in two main areas: (1) limiting the impact of specific symptoms, and (2) investing in activities that combine meaningful relationships and the feeling of being useful. Meaning is developed in the search for a new existential balance that reconciles creative tensions, particularly in the oscillation between letting go and determination. The process of meaning-making is distributed along a continuum of strategies that allows one to act as if nothing had happened, act within the limits of the disease, act despite the disease, act in new ways because of the disease, or act thanks to the disease. CONCLUSIONS: Understanding the process of meaning-making while adjusting to MS can allow the identification of possible interventions to better accompany people who are most severely affected by MS in their adaptation to the disease.

A first model of the fate of dietary calcium and phosphorus in broiler chickens.

To reduce P excretion and increase the sustainability of poultry farms, one needs to understand the mechanisms surrounding P metabolism and its close link with Ca metabolism to precisely predict the fate of dietary P and Ca and related requirements for birds. This study describes and evaluates a model developed to estimate the fate of Ca and P consumed by broilers. The Ca and P model relies on three modules: (1) digestion of Ca and P; (2) dynamics of Ca and P in soft tissue and feathers; and (3) dynamics of body ash. Exogenous phytase affects the availability of Ca and P; thus, to predict the absorption of those minerals, the model also accounts for the effect of phytase on Ca and P digestibility. We used a database to estimate the consequences of dietary Ca, P, and phytase over feed intake response. This study followed a four-step process: (1) Ca and P model development and its coupling with a growth broiler model; (2) model behavior assessment; (3) sensitivity analysis to identify the most influential parameters; and (4) external evaluation based on three databases. The proportion of P in body protein and the Ca to P ratio in bone are the most sensitive parameters of P deposition in soft tissue and bone, representing 91 and 99% of the total variation. The external evaluation results indicated that body water and protein had an overall mean square prediction error (rMSPE) of 7.22 and 12.3%, respectively. The prediction of body ash, Ca, and P had an rMSPE of 7.74, 11.0, and 6.56%, respectively, mostly errors of disturbances (72.5, 51.6, and 90.7%, respectively). The rMSPE for P balance was 13.3, 18.4, and 22.8%, respectively, for P retention, excretion, and retention coefficient, with respective errors due to disturbances of 69.1, 99.9, and 51.3%. We demonstrated a mechanistic model approach to predict the dietary effects of Ca and P on broiler chicken responses with low error, including detailed simulations to show the confidence level expected from the model outputs. Overall, this model predicts broilers' response to dietary Ca and P. The model could aid calculations to minimize P excretion and reduce the impact of broiler production on the environment. A model inversion is ongoing that will enable the calculation of Ca and P dietary quantities for a specific objective. This will simplify the use of the model and the feed formulation process.

Lipid-mediated inflammation and degeneration of bioprosthetic heart valves.

BACKGROUND: The durability of bioprosthetic valves is limited by structural valve degeneration (SVD) leading to bioprostheses (BPs) stenosis or regurgitation. We hypothesized that a lipid-mediated inflammatory mechanism is involved in the SVD of BPs. MATERIAL AND METHODS: Eighteen Freestyle stentless BP valves were explanted for SVD at a mean time of 5.9 +/- 3 years after implantation and were analysed by immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The mean age of the patients was 65 +/- 8 years and there were 11 male and seven female patients. Two of the 18 BPs had macroscopic calcification, whereas the other valves had minimal or no macroscopic calcification. Tears at the commissures leading to regurgitation was present in 16 BPs. Immunohistochemistry showed the presence of oxidized low-density lipoprotein (ox-LDL) and glycosaminoglycans in the fibrosa layer of 13 BPs. Areas with ox-LDL were infiltrated by macrophages (CD68(+)) co-expressing the scavenger receptor CD36 and metalloproteinase-9 (MMP-9). Zymogram showed the active form of MMP-9 within explanted BPs. EM studies revealed the presence of lipid-laden cells featuring foam cells and fragmented collagen. Nonimplanted control BPs obtained from the manufacturer (n = 4) had no evidence of lipid accumulation, inflammatory cell infiltration or expression of MMP9 within the leaflets. CONCLUSIONS: These results support the concept that lipid-mediated inflammatory mechanisms may contribute to the SVD of BPs. These findings suggest that modification of atherosclerotic risk factors with the use of behavioural or pharmacological interventions could help to reduce the incidence of SVD.

Temporal phases in apoptosis defined by the actions of Src homology 2 domains, ceramide, Bcl-2, interleukin-1beta converting enzyme family proteases, and a dense membrane fraction.

We have begun to explore the mechanisms of apoptosis using a cell-free system based on extracts from Xenopus eggs. Nuclei assembled or placed in these extracts undergo the morphological changes typical of apoptosis and eventually disintegrate. We used this system to investigate the potential involvement in apoptosis of proteins containing Src homology 2 (SH2) domains, which are known to interact with specific tyrosine-phosphorylated ligands. SH2 domains from a number of signaling proteins, including Lck, Src, and Abl, inhibited apoptosis when present at concentrations of 10-100 nM. The inhibition was dependent on specific interaction with endogenous tyrosine-phosphorylated ligands. A synthetic peptide ligand for Src family SH2 domains also inhibited apoptosis in a phosphotyrosine-dependent manner. Kinetic analysis defined three phases in the apoptotic process occurring in this cell-free system. SH2 domains and ceramide act throughout the first 60-90 min of the process (the "initiation" phase). Next, Bcl-2, interleukin-1beta converting enzyme family(CPP32-like) proteases, and the heavy membrane fraction act in a period occurring approximately 90-120 min after the start of incubation (the "sentencing" phase). In the final phase ("execution"), the process of active nuclear destruction ensues.

Crizotinib inhibition of ROS1-positive tumours in advanced non-small-cell lung cancer: a Canadian perspective.

The ros1 kinase is an oncogenic driver in non-small-cell lung cancer (nsclc). Fusion events involving the ROS1 gene are found in 1%-2% of nsclc patients and lead to deregulation of a tyrosine kinase-mediated multi-use intracellular signalling pathway, which then promotes the growth, proliferation, and progression of tumour cells. ROS1 fusion is a distinct molecular subtype of nsclc, found independently of other recognized driver mutations, and it is predominantly identified in younger patients (<50 years of age), women, never-smokers, and patients with adenocarcinoma histology. Targeted inhibition of the aberrant ros1 kinase with crizotinib is associated with increased progression-free survival (pfs) and improved quality-of-life measures. As the sole approved treatment for ROS1-rearranged nsclc, crizotinib has been demonstrated, through a variety of clinical trials and retrospective analyses, to be a safe, effective, well-tolerated, and appropriate treatment for patients having the ROS1 rearrangement. Canadian physicians endorse current guidelines which recommend that all patients with nonsquamous advanced nsclc, regardless of clinical characteristics, be tested for ROS1 rearrangement. Future integration of multigene testing panels into the standard of care could allow for efficient and cost-effective comprehensive testing of all patients with advanced nsclc. If a ROS1 rearrangement is found, treatment with crizotinib, preferably in the first-line setting, constitutes the standard of care, with other treatment options being investigated, as appropriate, should resistance to crizotinib develop.

Influence of smoking on airway inflammation and remodelling in asthma.

BACKGROUND: Although exposure to tobacco smoke has been associated with increased morbidity and mortality, cigarette smoking is still common in the asthmatic population. Induced sputum neutrophilia has been observed in asthmatic smokers, but the effects of regular smoking on their bronchial mucosa morphology remain to be defined. This study documents the inflammatory and remodelling features in bronchial biopsies of smoking compared with non-smoking asthmatics. METHODS: We analysed bronchial biopsies from 24 steroid-naïve young subjects with mild asthma: 12 non-smoking and 12 currently smoking subjects. In addition to airway morphology assessment, inflammation and remodelling were analysed by immunohistochemistry using antibodies against CD3, CD68, major basic protein, neutrophil elastase, and tryptase. Expression of the cytokines IL-4, IL-5, IL-8, IFN-gamma, transforming growth factor-beta, and TNF was determined by in situ hybridization. RESULTS: Compared with non-smoking asthmatic subjects, smoking asthmatics' bronchial mucosa showed squamous cell metaplasia, in addition to increased expression of subepithelial neutrophil elastase, IFN-gamma, and intraepithelial IL-8. CONCLUSIONS: Smoking status modifies morphological and inflammatory processes in young subjects with mild asthma. The changes may possibly affect asthma treatment responses and clinical outcomes.

Prenatal diagnosis of thrombosis of the dural sinuses: report of six cases, review of the literature and suggested management.

OBJECTIVES: To describe and assess the sonographic findings, evolution and clinical implications of thrombosis of the fetal dural sinuses. METHODS: We compiled a multicenter report of the outcomes of five cases with a prenatal diagnosis of thrombosis of the dural sinuses, and one case in which thrombosis of the dural sinus was diagnosed at necroscopy after termination of pregnancy. Prognostic factors are discussed, and suggestions made for prenatal and postnatal management. RESULTS: The mean (range) gestational age at diagnosis of thrombosis of the dural sinuses in the five cases in which it was made prenatally was 25.2 (22-31) weeks. In these five cases, diagnosis was made by sonography and confirmed by magnetic resonance imaging (MRI), which showed a blood clot in the region of the torcular herophili. Three of the six cases delivered vaginally with favorable sonographic findings, and normal clinical neurological development. Two pregnancies were terminated at the request of the parents. In one of these cases the prognosis was poor, with signs of fetal decompensation or cardiac failure; the pregnancy was terminated and necropsy revealed thrombosis of the occipital dural sinuses associated with a hemangioma. One infant, in whom the thrombosis developed in conjunction with a dural sinus malformation, died at 4 months of age. CONCLUSIONS: Thrombosis of the cerebral venous circulation can occur antenatally and is detectable by fetal real-time and color Doppler ultrasound examination. A review of the literature supports targeted evaluation of the fetus by serial ultrasound imaging and MRI to help guide the diagnosis, and to improve the counseling and management of such cases. Partial or total regression, isolated abnormality, absence of fetal decompensation or signs of cardiac failure and favorable clinical evolution are suggestive of favorable prognosis. In such cases, non-interventional neonatal management is recommended.

Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation.

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

Probing microtubules polarity in mitotic spindles in situ using Interferometric Second Harmonic Generation Microscopy.

The polarity of microtubules is thought to be involved in spindle assembly, cytokinesis or active molecular transport. However, its exact role remains poorly understood, mainly because of the challenge to measure microtubule polarity in intact cells. We report here the use of fast Interferometric Second Harmonic Generation microscopy to study the polarity of microtubules forming the mitotic spindles in a zebrafish embryo. This technique provides a powerful tool to study mitotic spindle formation and may be directly transferable for investigating the kinetics and function of microtubule polarity in other aspects of subcellular motility or in native tissues.

TCR/CD3-triggering causes increased activity of the p50csk tyrosine kinase and engagement of its SH2 domain.

The majority of known protein tyrosine kinases are integral membrane proteins or bound to cellular membranes via a covalently attached myristic acid. An exception is the newly described p50csk tyrosine kinase, which is believed to control the activity of the src-family of protein tyrosine kinases. This small kinase does not contain a myristylation signal or other known membrane attachment motifs, and indeed appears to be cytosolic. Recently, we found that p50csk specifically phosphorylates the negative regulatory Tyr-505 of the T cell-specific src-family kinase p56lck, and thereby suppresses its catalytic activity. Here we show that p50csk is activated in Jurkat T cells within one minute after stimulation of the cells with anti-CD3 MAbs. In parallel with this activation, p50csk formed a stable complex with one major 72 kDa tyrosine phosphorylated protein and minor polypeptides at 90 and 110 kDa. The isolated SH2 domain of p50csk specifically bound the 72 kDa protein in lysates from activated, but not resting, T or B cells. By several criteria, the 72 kDa protein was not a tyrosine kinase itself and it did not react with antibodies to a panel of known proteins of this molecular size. Our results suggest that p50csk is rapidly engaged via its SH2 domain and participates in the earliest events of T cell activation.

Two developmentally regulated mRNAs encoding actin-binding proteins in Physarum polycephalum.

A cDNA library from amoebae of Physarum polycephalum was screened by differential hybridization. Two clones contained inserts for mRNAs present in amoebae and absent in plasmodia. The LAV3-4 cDNA encodes a 402 aa protein (ABP-46) that shows sequence similarity to the actin binding site in the N-terminal region of the alpha-actinin family. The LAV3-5 cDNA is 76% identical to the Dictyostelium actin bundling protein, which cross-links and stabilizes actin filaments in amoebal filopodia.

Does histologic grade have a role in the management of head and neck cancers?

PURPOSE: High histologic grade is usually associated with a greater propensity to distant metastases (DM). Its role to predict DM in head and neck cancer is not yet defined. The aim of this study is to evaluate the role of histologic grade as an independent predictor of DM and to determine a subgroup of patients who may benefit from systemic chemotherapy. PATIENTS AND METHODS: This is a retrospective study of 1,266 consecutive patients treated by definitive or postoperative radiotherapy between 1989 and 1997. All patients received at least 50 Gy. All stages and subsites of head/neck were included. DM rates were evaluated by the Kaplan-Meier method with a subsequent Cox analysis. RESULTS: There is a strong correlation of grade with N stage (P <.000001). The metastases-free survival (MFS) was 98%, 90%, and 72% for grades 1, 2, and 3, respectively (P <.000001). In patients with N0 stage, MFS is always greater than 90%, whatever the grade. In the 222 N1 patients, MFS was more than 90% in grade 1 and 2 but dropped to 75% for grade 3 (P =.001). In patients with N2 and N3, MFS was 91%, 79%, and 59% for grades 1, 2, and 3, respectively (P =.008). The same conclusion is applicable when only patients with neck control are analyzed. In a Cox model, grade was an independent predictor of DM (P =.000001) as well as T stage (P =.003), N stage (P =.000001), and neck failure (P =.0003). Higher grade was also an independent predictor of survival (P =.02). CONCLUSION: Patients with histologic grade 1 and grade 2 (except N3) are at low risk of DM. Patients with grade 2 and N3 or patients with grade 3 and N1 to N3 have a higher risk of distant metastases and should be considered for systemic treatment.

Diminished TCR signaling in cutaneous T cell lymphoma is associated with decreased activities of Zap70, Syk and membrane-associated Csk.

Malignant cells of patients with cutaneous T cell lymphoma (CTCL) are of monoclonal origin and of the CD4+/CD45RO+ subset. Since unlike their normal counterparts, triggering of their TCR/CD3 in vitro elicits only a weak mitogenic response, we set out to determine which of the signal transduction molecules initiated by anti-CD3E antibodies are affected in neoplastic cells. The results obtained from analysis of tumor cells from four patients show a general reduction in basal and induced tyrosine phosphorylation of a wide range of signaling proteins. Furthermore, the function of members from distinct families of protein tyrosine kinases was altered in neoplastic cells. The enzymatic activity of the membrane-bound fraction of Csk was suppressed, and its association with other cellular proteins was altered. There was a decline in the amount and activity of Syk, and a slight decrease in the specific activity of Lck kinases. Zap70 tyrosyl phosphorylation was reduced or undetectable and the kinase associated weakly, or not at all, with the TCR zeta chain. We propose that dampened TCR-triggered responses in CTCL are caused by suppression of an array of effector molecules required for coupling cell surface receptors to early and late signaling events.

Adaptations to a diet-based weight-reducing programme in obese women resistant to weight loss.

The aim of this study was to assess energy intake, resting metabolic rate (RMR), appetite sensations, eating behaviours and sleep duration and quality in obese women resistant to body weight loss when subjected to a diet-based weight-reducing programme. A pooled cohort of obese women (n = 75; aged 39 ± 8 years; body mass index: 33 ± 4 kg m(-2)) participated in a 12-16-week diet-based weight loss programme targeting a daily energy deficit of 500-700 kcal d(-1). Women were classified in tertiles a posteriori based on the response of their body weight to dietary supervision (high, moderate and low responders). Post-intervention, mean weight loss was 3.3 ± 2.8 kg and explained by the 2.9 ± 2.6 kg reduction in fat mass. Mean weight loss was 6.2 ± 1.6, 3.4 ± 0.6 and 0.2 ± 1.4 kg in participants classified in the high, middle and low tertiles, respectively. Women in the low tertile reduced their daily energy intake and susceptibility to hunger during the programme to a lesser extent than those in the high tertile and had higher fasting hunger in response to the dietary intervention. Women in the high tertile maintained their RMR, which was in contrast to the significant decrease predicted by their weight loss. They also reported a significant improvement in sleep quality and an increase in sleep duration compared with other tertiles. The differences in the response of body weight to dietary supervision may be explained, in part, by variations in energy intake, eating behaviours, appetite sensations and sleep duration and quality.

Glucocorticoid-inducible retrovector for regulated transgene expression in genetically engineered bone marrow stromal cells.

Transplantable bone marrow stromal cells can be utilized for cell therapy of mesenchymal disorders. They can also be genetically engineered to express synthetic transgenes and subsequently serve as a platform for systemic delivery of therapeutic proteins in vivo. Inducible production of therapeutic proteins would markedly enhance the usefulness of stromal cells for cell therapy applications. We determined whether synthetic corticosteroid hormones can be used to tightly control transgene expression via the glucocorticoid response pathway in primary bone marrow stromal cells. This regulatory mechanism does not require the presence of potentially immunogenic prokaryotic or chimeric "Trans-activators." Further, synthetic corticosteroids are pharmaceutical agents that can be readily used in vivo. We designed a self-inactivating retroviral vector in which expression of the green fluorescent protein (GFP) reporter is controlled by a minimal synthetic promoter composed of five tandem glucocorticoid response elements upstream of a TATAA box. Vesicular stomatitis virus G-pseudotyped retroparticles were synthesized and utilized to transduce cultured cell lines and primary rat bone marrow stromal cells. We have shown that primary rat bone marrow stromal cells could be efficiently engineered with our GRE-containing retrovector, basal reporter expression was low in the absence of exogenous synthetic corticosteroids, and GFP expression was dexamethasone inducible and reversible. To summarize, this strategy allows dexamethasone-induced, "on-demand" transgene expression from transplantable genetically engineered bone marrow stromal cells.

An efficient expression, purification and immunodetection system for recombinant gene products.

We describe a modification of the mammalian expression vector pRc/CMV, which drives expression of inserted genes from either the human cytomegalovirus (CMV) immediate-early promoter or the bacteriophage T7 RNA polymerase promoter. The modification is designed to allow expression, simple purification and specific immunodetection of recombinant fusion proteins. The modified plasmid, termed pTag/CMV-neo, encodes a Kozak consensus ribosome-binding site (RBS) and a 30-amino acid fusion tag peptide. This peptide consists of a metal ion-binding site, (His)6, for single-step affinity purification using Ni(2+)-chelating resin and a multi-purpose HIV-1-derived peptide (p18HIV). This viral epitope can be used to identify, detect and characterize target fusion proteins in conjunction with a specific monoclonal antibody H902 that does not display cross-reactivity with cellular proteins. The H902 production hybridoma cell line is reagent #521 from the NIH AIDS Research and Reference Program.

Identification of a 16-kDa thymocyte membrane glycoprotein involved in the thymocyte/thymic medullary epithelial cell interaction.

We have previously described a type of lymphoepithelial interaction involving CD4+ CD8+ thymocytes and a medullary epithelial cell line (E-5). This interaction is mediated by the recently described gp23/45 epithelial adhesion molecule and an as yet unknown thymocyte receptor. The present work describes a thymocyte surface glycoprotein of 16 kDa which binds both to E-5 cells and to the purified gp23/45 adhesion molecule. In addition, a thymic lymphoma cell line (Ti-6), which interacts with the E-5 cells via the gp23/45 receptor, also present a 16-kDa glycoprotein on its surface. Taken together, the data suggest that the 16-kDa thymocyte surface glycoprotein participates in the binding between these cells and the thymic epithelium.