Inflammatory bone loss in experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in interleukin-1 receptor antagonist knockout mice.

The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up-regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin-1 receptor antagonist-deficient mice.

BACKGROUND AND OBJECTIVE: The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS). MATERIAL AND METHODS: Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10 µg/mL of Aggregatibacter actinomycetemcomitans LPS for 24 h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9 d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA. RESULTS: The levels of IL-1α, IL-1ß, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A. actinomycetemcomitans LPS were significantly increased by approximately 4- (p < 0.05), 5- (p < 0.05), 1.3- (p < 0.05) and 6- (p < 0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p < 0.05), 1.6- (p < 0.05), 2.5- (p < 0.05), 1.6- (p < 0.05) and 1.9- (p < 0.05) fold, respectively, in IL-1Ra KO mice stimulated with A. actinomycetemcomitans LPS compared with WT mice. CONCLUSION: IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.

Gene therapy for attenuating cardiac allograft arteriopathy using ex vivo E2F decoy transfection by HVJ-AVE-liposome method in mice and nonhuman primates.

Cardiac allograft arteriopathy, which limits the long-term survival of recipients, is characterized by diffuse intimal thickening composed of proliferative smooth muscle cells. The transcription factor E2F plays a pivotal role in the coordinated transcription of cell-cycle regulatory genes. To test the hypothesis that double-stranded DNA with specific affinity for E2F (E2F decoy) is effective in preventing intimal hyperplasia, we performed ex vivo single intraluminal delivery of E2F decoy into cardiac allografts of mice and Japanese monkeys using the hemagglutinating virus of Japan (HVJ) artificial viral envelope-liposome method. In murine models, antisense cyclin-dependent kinase 2 (cdk2) kinase oligodeoxynucleotide (ODN) and no transfers were performed to compare the effects. Severe intimal thickening was observed, and multiple cell-cycle regulatory genes were enhanced in untreated allografts. E2F decoy prevented neointimal formation and suppressed these genes for up to 8 weeks, whereas antisense cdk2 kinase ODN had limited effects. In primate models, E2F decoy dramatically prevented neointimal thickening and suppressed multiple cell-cycle regulatory genes, whereas intimal thickening developed in the nontransfected or mismatch decoy-transfected allografts. Gel mobility shift assay proved the specific effects of E2F decoy, and reverse transcriptase-polymerase chain reaction documented that neither complication nor dissemination of HVJ into other organs was observed. We demonstrate that ex vivo gene delivery to allografts is a potent strategy to modify allograft gene expression, resulting in prevention of graft arteriopathy without systemic adverse effects.

Functional hierarchy of c-kit and c-fms in intramarrow production of CFU-M.

Whereas the molecular natures of M-CSF/CSF-1 and its receptor c-fms are well characterized, its actual role in the intramarrow hematopoiesis remains obscure. This is because disruption of this signaling pathway results in the osteopetrosis mouse that lacks the bone cavity for hematopoiesis. To elucidate the role of c-fms in intramarrow hematopoiesis, we produced an antagonistic monoclonal antibody to murine c-fms and investigated its expression and function in the normal bone marrow. c-fms+ cells were detected both in mature and immature hematopoietic cells. Morphologically, c-kit+c-fms-, c-kit+c-fms+ and c-kit-c-fms+ cells were medium sized blasts, large promyelocytes with azurophilic granules and mature monocytes respectively. CFU-M was 10-fold more enriched in the c-kit+c-fms- than c-kit+c-fms+ fraction. Moreover, injection of the anti c-fms antibody had no effect on the production of CFU-M in the bone marrow, while anti-c-kit mAb could deplete them. As c-kit+c-fms+ cells were readily generated in the culture of c-kit+c-fms- cells, most of the CFU-M in the bone marrow are, in fact, c-fms- cells that differentiate into c-fms+ upon culture. These observations indicate a clear functional hierarchy of c-kit and c-fms in the bone marrow. Namely, c-kit plays the primary role in the production and maintenance of CFU-M, while c-fms, though it co-expressed with c-kit and functions as the growth receptor for M-CSF in the culture, has only a minimum role in the proliferation of c-fms+ cells in the bone marrow.

Tranilast inhibits cardiac allograft vasculopathy in association with p21(Waf1/Cip1) expression on neointimal cells in murine cardiac transplantation model.

Cardiac allograft vasculopathy is a major complication after cardiac transplantation, often limiting long-term recipient survival. N-(3,4-Dimethoxycinnamoyl)anthranilic acid (tranilast) inhibits cyclin-dependent kinase activity through p21(Waf1/Cip1) induction and arrests vascular smooth muscle cell proliferation in vitro. We tested a hypothesis that tranilast inhibits the vasculopathy characterized by diffuse intimal thickening in a murine heart transplantation model. Hearts from DBA/2 mice were heterotopically transplanted into B10.D2 mice as allografts. Oral administration of tranilast started 3 days before transplantation at doses of 550 or 1040 mg/kg per day until the animals were killed. Cardiac allograft vasculopathy was defined as luminal stenosis caused by neointimal formation. The percentage of luminal stenosis and cardiac rejection were analyzed 14 and 28 days after transplantation. Tranilast administration was associated with a marked reduction in luminal occlusion but with no significant effect on cardiac rejection. Immunohistochemical study of the tranilast-treated graft coronary arteries revealed enhancement of p21(Waf1/Cip1) and decreased expression of proliferating cell nuclear antigen in the neointima. The significant reduction in allograft vasculopathy concomitant with the enhancement of p21(Waf1/Cip1) indicates that tranilast has an antiproliferative effect that could be applicable to clinical treatment of cardiac allograft vasculopathy.

Studies on quinoline derivatives and related compounds. 5. Synthesis and antimicrobial activity of novel 1-alkoxy-1,4-dihydro-4-oxo-3-quinolinecarboxylic acids.

A series of novel 1-alkoxy-1,4-dihydro-4-oxo-3-quinolinecarboxylic acids was synthesized and screened as antimicrobial agents. The most active compounds in vitro against gram-negative microorganisms and Staphylococcus aureus were 1,4-dihydro-1-methoxy-6,7-methylenedioxy-4-oxo-3-quinolinecarboxylic acid (22), 1,2,6,9-tetrahydro-6-methoxy-9-oxofuro[3,2-f]quinoline-8-carboxylic acid (30, and 2,3,6,9-tetrahydro-6-methoxy-3-methyl -2,9-dioxothiazolo [5,4-f]quinoline-8-carboxylic acid (34). These compounds had antigram-negative activity comparable to that of the corresponding N-ethyl derivatives 1, 2, and 4. Their serum levels and urinary recovery rates in rats, however, were significantly improved relative to the latter compounds (1,2, and 4).

Effects of natural human interleukin-6 on thrombopoiesis and tumor progression in tumor-bearing mice.

The growth of inoculated colon 26 adenocarcinoma (colon 26) in mice gradually increased the platelet count owing to murine IL-6 secreted from the tumor, while Lewis lung carcinoma (LLC) decreased the platelet count in the hosts, depending on the tumor growth. Natural human IL-6 injections (hIL-6), 280 micrograms/kg/day, stimulated the platelet production in both types of carcinoma-bearing mice. When the administration of mitomycin C or cisplatin decreased the platelet number as a side reaction with a concomitant of suppressing the growth of colon 26 and LLC, respectively, hIL-6 could also increase the platelet count without the augmentation of tumor growth. However, loss of carcass weight was observed in colon 26-bearing mice treated with hIL-6, suggesting the development of cachexia is associated with hIL-6 administration. Despite the possibility of inducing cachexia in some types of tumors, our results suggest that IL-6 could be a useful means of restoring the decreased platelet number in cancer patients after intensive chemotherapy.

Differential Th1 and Th2 cell regulation of murine cardiac allograft acceptance by blocking cell adhesion of ICAM-1/LFA-1 and VCAM-1/VLA-4.

Administration of anti-intercellular adhesion molecule (ICAM)-1 monoclonal antibody (mAb) plus anti-lymphocyte function associated antigen (LFA)-1 mAb induces tolerance in murine cardiac transplantation, while anti-vascular cell adhesion molecule (VCAM)-1 mAb plus anti-very late antigen (VLA)-4 mAb administration prolongs graft survival, but leads to tolerance only in some cases. BALB/c mice hearts were transplanted into C3H/He recipients. Each combination of anti-VCAM-1 plus anti-VLA-4 mAbs (100 microg each/day, i.p.) or anti-ICAM-1 plus anti-LFA-1 mAbs (50 microg each/day, i.p.) was administered for 5 days. For control study, third group mice received daily with FK506 administration (1 mg/kg/day). The cardiac allografts and recipients' spleens were harvested on day 7; the expression of cytokines were detected using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and in situ RT-PCR. Th2 cytokines (IL-4 and IL-10) were markedly enhanced and Th1 cytokines (IFN-gamma and IL-2) were suppressed in recipients treated with anti-ICAM-1 mAb plus anti-LFA-1 mAb, while poor Th2 cytokine expression allowed persistent Th1 cytokine expression in recipient mice with anti-VCAM-1 mAb plus anti-VLA-4 mAb treatment. Both Th1 and Th2 cytokine expression was suppressed in FK506-treated mice. It is concluded that immunological tolerance and prolonged graft survival induced by blocking cell adhesion is regulated by different cytokine expression.

Dynamics of cerebral metabolism in patients with chronic subdural hematoma evaluated with phosphorous 31 MR spectroscopy before and after surgery.

PURPOSE: To determine whether the depression of cerebral bioenergetic metabolism caused by chronic subdural hematomas can account for neurologic dysfunction and whether the degree of metabolic depression may be useful for clinical assessment and therapy. METHODS: Sixteen patients who had chronic subdural hematomas with hemiparesis and/or mental disturbances underwent phosphorous 31 MR spectroscopy before and 10 to 14 days after surgery. Phosphorous 31 MR spectroscopy was also performed on 5 patients who had chronic subdural hematomas with only slight headaches who were treated by conservative therapy and on 10 healthy volunteers. RESULTS: The peroperative phosphocreatine-to-inorganic phosphate ratio (2.10 +/- 0.36) improved to normal values (2.69 +/- 0.44) after evacuation of hematomas. This improvement was accompanied by complete disappearance of hemiparesis and/or mental disturbance. Brain tissue pH also improved from 7.07 +/- 0.11 to 7.205 +/- 0.13 after surgery. On the other hand, patients who had chronic subdural hematomas with only slight headaches had the same phosphocreatine-to-inorganic phosphate ratio and brain intracellular pH as healthy volunteers. CONCLUSION: The phosphocreatine-to-inorganic phosphate ratio may be useful for determining when to operate on patients with chronic subdural hematomas and to assess the efficacy of treatment.

Hematopoietic progenitor cell counts performed by the Sysmex SE-9000 analyzer can guide timing of peripheral blood stem cell harvest.

BACKGROUND: We studied whether the hematopoietic progenitor cell (HPC) count in peripheral blood as evaluated by an automated counter, the Sysmex SE-9000, correlated with CD34 positive (+) cell count and therefore could guide the timing of peripheral blood stem cell (PBSC) harvest. MATERIALS AND METHODS: HPC count and flow cytometric CD34+ cell count were measured in 90 peripheral blood samples and 30PBSC samples. The correlation between HPC count and apheretic CD34+ cell yield was examined in 19 patients. RESULTS: HPC count showed good correlations with CD34+ cell count in peripheral blood (r = 0.699) and PBSC (r = 0.892). The correlation between peripheral blood HPC count and apheretic CD34+ cell yield also was good (r = 0.789). CONCLUSION: Automated HPC counting can be used as a screening test to guide the timing of PBSC harvest.

Assessment of T1 time course changes and tissue-blood ratios after Gd-DTPA administration in brain tumors.

Sequential T1 changes in brain tumor tissue after Gd-DTPA administration were investigated in 10 patients, including 4 meningiomas, 2 gliomas, 3 metastatic cerebral tumors and 1 brain abscess. T1 values were measured serially for 60 minutes following Gd-DTPA injection using a magnetic focusing technique. In vitro T1 of the whole blood samples was also comparatively examined. Time processes in the tissue-blood ratio (TBR) were calculated from two-point relaxation rates at 5 and 30 minutes. The obtained ratios of TBR were ranged from 1.0 to 3.0, probably depending on histological types of brain tumor (the value of 1.0 to 1.5 for meningioma and 1.5 to 3.0 for glioma and metastatic tumor). No significant changes in the T1 value were observed in the examined normal tissue and peritumoral edema. These results indicate that Gd-DTPA plays an important role not only as an image enhancer for tumor tissue but also as an indicator for estimating the blood-brain barrier function.

Spin-lattice relaxation times of bound water--its determination and implications for tissue discrimination.

Measurements were made of T1 of bound water (T1b) and bound water fraction (alpha) for gelatin solutions and human tissues (sera, brain tumor, cerebral white matter). Bound water fraction in each sample was measured by means of thermal analysis (differential scanning calorimetry: DSC). T1 values were measured by FONAR QED 80-alpha. T1b values were calculated by an equation derived from the fast-exchange two-state model. In the study of gelatin solutions, the relationship between T1 and water content differed depending on the sort of solutions. This was considered to be due to differences in T1b values. In each biological tissue the values of T1b and alpha had different distributions. These results indicate that values of T1b and alpha for biological tissues may be altered in correspondence to the changes in pathophysiological states in those tissues.

Thirty cases of bronchial asthma associated with exposure to pet hamsters.

BACKGROUND: The identification, isolation, and elimination of allergen(s) causing bronchial asthma are the most efficient form of treatment. The pet industry has diversified recently, increasing the risk of exposure of pet owners to many unknown antigens. We clinically studied the characteristics of asthma associated with exposure to pet hamsters. METHODS: The study group comprised 30 adults in whom the onset, recurrence, or exacerbation of asthma was triggered by contact with pet hamsters. Clinical characteristics such as sex, age, period required for symptom onset, species of hamster, treatment and disease course, smoking status, and hamster-specific IgE antibodies in serum were studied. RESULTS: The male: female ratio of the study group was 1:1.3, and mean age was 37.7 years. Patients with no previous history of asthma initially presented with cough, progressing to episodes of asthma. Asthmatic symptoms were associated with hamster contact and ranged in severity from mild to severe. Three patients required hospital admission for treatment. The mean period from the start of hamster exposure to the onset of asthmatic episodes was 15.7 months. Dwarf hamsters were responsible for most cases. The CAP-RAST score for hamster-specific IgE antibodies was 1 to 4 in 22 patients and 0 in 8 patients. Eight patients with a score of 1 or higher for hamster-specific IgE antibodies had a CAP-RAST score of 0 for mite antigen. In these patients, terminating hamster contact resulted in a rapid improvement in symptoms, with no need for further treatment. Twenty-three of the 30 subjects (76.7%) were smokers. CONCLUSION: Exposure to pet hamsters is an important risk factor for the onset, recurrence, or exacerbation of asthma. Smoking may also increase the risk of asthmatic symptoms in patients exposed to hamsters.

The antagonism of calcineurin inhibitors and costimulatory blockers: fact or fiction?

Blockade of T-cell costimulatory pathways has proven to be a useful approach in inhibiting allograft rejection and/or inducing tolerance in some experimental models of transplantation, and clinical development of B7 blockade by CTLA4-Ig in kidney transplant recipients is under way. Although calcineurin inhibitors are clinically effective immunosuppressive agents in their own right, conflicting findings have been reported as to whether concomitant use of these drugs with costimulatory blockade has a beneficial or adverse effect on preventing rejection and promoting long-term allograft survival or transplant tolerance. Here, we summarize the current understanding of the interaction between calcineurin inhibitors and T-cell costimulatory blockade strategies in transplantation.

[Analysis of pathological state of benign monoclonal gammopathy by beta 2-microglobulin and CEA].

In order to obtain the informations concerning benign monoclonal gammopathy (BMG)-related diseases, beta 2-microglobulin (beta 2-m) levels in sera from the patients with BMG were plotted against their serum carcinoembryonic antigen (CEA) levels, and then this figure was divided into the two regions, i.e., A-region (beta 2-m less than 7 mg/l & CEA less than 7 ng/ml) and B-region (beta 2-m greater than or equal to 7 mg/l & CEA greater than or equal to 7 ng/ml). Also, the normal region was expressed as C-region within A-region. In consequence, it was revealed that A-region and B-region mainly consisted of the inflammation and degeneration-related diseases and the tumor-related diseases, respectively.

[Time course of NMR images and T1 values associated with hypertensive intracerebral hematoma].

The present study describes time courses in tissue T1 values, as well as in NMR imagings, associated with hypertensive intracerebral hematoma (ICH). Non-operative 21 cases of ICH were examined by FONAR QED 80-alpha NMR system, which possessed dual modes of image display and focal T1 measurement (static magnetic field: 433 gauss). As the first step of examination SSFP images are displayed and then, at the regions of interest, absolute values of T1 are measured by field focusing technique. The extent of ICH was revealed as high density zone in NMR imaging, occasionally represented much wider extent of high density area than the finding on X-ray CT. Prolonged T1 values were obtained from such high density zone. This wide-spread high density area was regarded to reflect the spread of perifocal brain edema. T1 value of the hematoma itself was rather shortened in its initial phase within 2 weeks, thereafter followed by prolongation in the time lapse. This seemed to reflect the alterations in the properties of hematoma such as clot formation in earlier phase and resolution in later phase. On the contrary, T1 in the brain tissue surrounded to hematoma was apparently prolonged in the early phase within 2 weeks, representing the maximal values of 312 msec around 2 to 4 weeks after the onset, and then gradually normalized in the period over 1 month. This alteration in tissue T1 likely represents the processes of edema formation and its regression in perifocal zone. T1 values measured in perifocal region might be available for the evaluation of edema state in association with cerebrovascular accident.

[Clinical efficacy of leuprolide acetate and combined treatment with estramustine for advanced prostate cancer].

BACKGROUND/PURPOSE: Twenty-two institutes have organized Keio University Prostate Cancer Study Group to study clinical efficacy and safety of Leuprolide acetate (Leuplin) for the treatment of advanced prostate cancer (clinical stage D1 and D2). Cotreatment of Leuplin and Estramustine phosphate disodium (Estracyt) has been performed to investigate its clinical efficacy. MATERIALS AND METHODS: One hundred and two cases of advanced prostate cancer were treated either with Leuplin alone (group I), Leuplin and Estracyt (group II) or Estracyt alone (group III). After 12 weeks treatment, clinical effects against subjective symptoms (pain, voiding difficulty, performance status and body weight), serum testosterone level, tumor size and serum PSA level were examined to investigate short-term effect of each treatment. The treatment had been continued for 24 months and the treatment effects including progression free survival and overall survival were analyzed. RESULTS: Clinical efficacy after 12 weeks treatment were examined among 97 cases (group I; 35 cases, group II; 36 cases, group III; 26 cases). The background of those patients in each group was statistically equal. Treatment effects against subjective symptoms and serum testosterone level statistically revealed no significant difference among 3 groups. Treatment effects against primary tumor, bone metastatic lesion, lymphnode metastatic lesion and serum PSA level were investigated and anti-tumor effect was characterized by total efficacy rate (complete remission rate plus partial remission rate) of each treatment group. Treatment efficacy rates for each lesion and PSA demonstrated no statistical difference among 3 treatment groups. Total efficacy rate of group I, II and III were 88.2%, 84.0% and 78.3%, respectively, which statistically revealed no significant difference. Total efficacy rate of each group after completing 24 months treatment was; group I 80.0%, group II 55.6% and group III 83.3%, which statistically showed no significant difference among 3 treatment groups. The median day for progression free survival of group I, II and III were 661, 731 and 517, respectively. The overall survival rate of group I, II and III after completing 24 months treatment were 77.5%, 83.0% and 72.4%, respectively. Both progression free survival rates and overall survival rates revealed no significant difference among 3 groups. Side effects during 24 months treatment were seen in 8.6% of group I, 47.2% of group II and 26.9% of group III, and these occurrence rates were significantly different among the groups (p = 0.0013). CONCLUSION: Although number of the cases had not been able to continue the treatment for their side effects, the statistical characterization demonstrated that cotreatment of Leuplin and Estracyt had no greater treatment effect than monotreatment of each drug.

[Functional MRI in search of basal ganglia].

We investigated activation of the globus pallidus using conventional gradient echo sequence with a 1.5 tesla equipment. In accordance with complex finger tapping in the left hand, an increase in signal intensity from bilateral globus pallidus was attained, being grossly predominated on right side. On the other hand, activations in primary motor cortex were elicited mainly on right side, thereby being correctly accorded in attitude of on-off changes in signal intensity. fMRI in basal ganglia faces severer condition in detecting real activation, owing to influences to signal recording by ventricular pulsation, accumulation of iron by age, and differences in activation by the tasks applied. This study, however, suggests that fMRI of basal ganglia is a promising technique for mapping brain function in regard to correlations between cerebral cortex and deep structures.