Relationship between cytokine single nucleotide polymorphisms and sarcoidosis among Japanese subjects.

Several susceptibility genes for sarcoidosis have been identified, but their relationship to the clinical state and prognosis remains to be elucidated. The aim of this study was to elucidate the relationship between sarcoidosis and five single nucleotide polymorphisms (SNPs) of three cytokines expected to play an important role in the inflammatory response. A case-control study was performed with 208 unrelated patients who met the diagnostic criteria for sarcoidosis used in Japan since 2006, and 328 control subjects. Five SNPs were analyzed: interleukin (IL)-10-819T/C (rs1800871), IL-10-592A/C(rs1800872), IL-6-634C/G (rs1800796), tumor necrosis factor-alpha (TNF-alpha)-857C/T (rs1799724), and TNF-alpha -1031T/C (rs1799964). No significant differences in SNPs were observed between the total sarcoidosis and control groups. However, the prevalence of rs1800871 and rs1800872 polymorphisms differed significantly in the sarcoidosis with eye involvement group compared with the control group [rs1800871 TT (vs. TC + CC): OR = 1.67, P = 0.034; rs1800872 AA (vs. AC + CC): OR = 1.66, P = 0.036]. Analyzing the cardiac involvement group, the prevalence of the rs1799724 polymorphism was significantly different from that of the control group [rs1799724 TT (vs. CC + CT): OR = 6.01. P = 0.006]. We concluded that the rs1799724 C/T polymorphism may affect susceptibility to cardiac sarcoidosis, while the rs1800871 T/C and rs1800872A/C polymorphisms may affect susceptibility to sarcoidosis with eye involvement.

Interleukin 6. A potential autocrine/paracrine factor in Paget's disease of bone.

Pagetic osteoclasts are greatly increased in number and size and have increased numbers of nuclei per cell compared to normal osteoclasts. The mechanisms responsible for enhanced osteoclast formation in Paget's disease are unknown. We have used our recently described model system for pagetic osteoclast formation to evaluate culture media conditioned by these atypical multinucleated cells (MNC) to determine if pagetic osteoclasts produce an autocrine or paracrine factor that enhances osteoclast formation. Conditioned media from long-term bone marrow cultures from patients with Paget's disease stimulated osteoclast-like MNC formation in normal marrow cultures. At least part of this activity could be ascribed to interleukin 6 (IL-6). In contrast, conditioned media from normal marrow cultures contained lower levels of IL-6 and did not stimulate formation of osteoclast-like MNC. 7 of 8 bone marrow plasma samples taken from involved bones and 18 of 27 peripheral blood serum samples from Paget's patients had high levels of IL-6. Normal marrow plasma and peripheral blood serum had no or very low levels of IL-6. These results suggest that IL-6 produced by marrow and/or bone cells in patients with Paget's disease may be an autocrine/paracrine factor for pagetic osteoclasts.

Antitumor activity of magainin analogues against human lung cancer cell lines.

Magainin 1 and magainin 2, originally isolated from African clawed frog Xenopus laevis skin, inhibit the growth of bacteria and fungi. Synthetic magainin A (MAG A) and magainin G (MAG G) are more potent against bacteria and protozoa. In order to determine the antitumor activity of these analogues, we have tested these two analogues against six small cell lung cancer (SCLC) cell lines NCI-H82, NCI-H526, NCI-H678, NCI-H735, NCI-H841, and NCI-H889, which were known to differ by more than 10-fold in their sensitivity to different chemotherapeutic agents, and four normal human fibroblast cell lines. Semiautomated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays of the six SCLC cell lines revealed average concentrations producing 50% inhibition (IC50) values of 2.6 microM (range, 0.49-9.30 microM) for cisplatin, 2.5 microM (range, 0.39-6.00 microM) for etoposide, and 138.8 nM (range, 55.0-450.0 nM) for doxorubicin. The average IC50 of MAG A was 8.64 microM (range, 6.23-11.7 microM) and that of MAG G was 8.82 microM (range, 4.44-12.5 microM) against the SCLC cell lines. Despite a 10-fold difference in sensitivity to standard chemotherapeutic agents, the IC50 of MAG A and MAG G differs by less than 3-fold. The average IC50 against four normal human fibroblast cell lines was 21.1 microM (range, 12.7-25.6 microM) for MAG A and 29.2 microM (range, 21.3-34.8 microM) for MAG G. Combined exposure to the IC50 concentration of MAG A or MAG G plus IC50 of etoposide or cisplatin decreased the percentage of surviving SCLC cells to 29.0% (range, 26.1-31.7%). MAG A or MAG G had an additive effect when used with standard chemotherapeutic agents. These data suggest that MAG A and MAG G have in vitro antitumor activity against SCLC cell lines.

Human small cell lung cancer cell lines express functional atrial natriuretic peptide receptors.

Small cell lung cancer cell (SCLC) lines, NCI-H82, NCI-H660, and NCI-H1284, and HeLa cells were analyzed for the presence of atrial natriuretic peptide (ANP) receptors. In these SCLC cell lines and HeLa cells, ANP A receptor mRNA was identified by Southern blot analyses of polymerase chain reaction products and RNase protection assays using poly(A)(+)-selected RNA. Saturable binding assays revealed that HeLa cells had 2000 to 5000 high affinity atrial natriuretic peptide receptors per cell with a dissociation constant of 140 pM. In the SCLC cell lines, the binding was saturable but too low to accurately estimate the number of binding sites. After addition of human ANP, radioimmunoassays revealed accumulation of cyclic GMP in SCLC cells as well as HeLa cells in a dose-dependent fashion. The half-maximal stimulation concentration of cyclic GMP accumulation in HeLa and these SCLC cell lines was approximately 2 nM. Tetrazolyl blue assays and tritiated thymidine incorporation did not show any remarkable growth inhibition or growth stimulation of SCLC cell lines after addition of human ANP up to 3.3 microM, more than 1000-fold greater than the half-maximal stimulation concentration of cyclic GMP accumulation. Our results indicate that human SCLC cells express functional ANP receptors but ANP addition produced no detectable change in their growth pattern.

Down-regulation of protein kinase C potentiates atrial natriuretic peptide-stimulated cGMP accumulation in vascular smooth-muscle cells.

It has been reported that atrial natriuretic peptide (ANP) produces inositol phosphates and diacylglycerol in vascular smooth muscle cells (VSMC). The purpose of this study is to investigate whether diacylglycerol produced by ANP affects ANP-induced cyclic GMP (cGMP) accumulation through the activation of protein kinase C. Short-term (15 min) treatment of rat aortic VSMC with protein kinase C activating phorbol 12-myristate 13-acetate (PMA, 100 nM) decreased ANP (100 nM)-induced cGMP accumulation by 34.7% in the presence of IBMX (0.5 mM). However, the long-term (24 h) treatment to decrease the activity of protein kinase C led to an enhancement of the cGMP accumulation by 69.6% compared with that of control VSMC. There were no significant differences in Bmax and Kd for ANP and ANP-dependent particular guanylyl cyclase activity between long-term PMA-treated and control VSMC. In the present study, we show that the activation of protein kinase C attenuates the cGMP accumulation induced by ANP and that down-regulation of protein kinase C results in an enhancement of the cGMP accumulation. These data are consistent with the role of protein kinase C as a negative regulator in ANP-receptor/guanylyl cyclase pathway.

Parathyroid hormone-related peptide is involved in protection against invasion of tooth germs by bone via promoting the differentiation of osteoclasts during tooth development.

In order to elucidate the role of parathyroid hormone-related peptide (PTHrP) in tooth development, we treated tooth germ explants of mouse molars with antisense phosphorothioate-oligodeoxynucleotide (ODN) against PTHrP. Antisense ODN-treatment of the explants resulted in the invasion of the tooth germs by bone. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells around the tooth germs in antisense ODN-treated explants was much lower than that of the control explants. Electron microscopic examination suggested that the antisense ODN-treatment inhibited differentiation of osteoclasts. Treatment of the explants with bisphosphonate or vitamin K2, inhibitors of the differentiation of osteoclasts, induced the invasion by bone into the tooth germs as observed in the antisense ODN-treated explants. The results obtained suggest that PTHrP is involved in the mechanism protecting tooth germs from bone invasion by promoting the differentiation of osteoclasts around them.

Increased serum and urinary levels of a parathyroid hormone-related protein COOH terminus in non-small cell lung cancer patients.

Parathyroid hormone-related protein (PTHrP) is expressed in a variety of human cancers including lung cancer. Three mature peptides with different COOH-terminal regions, PTHrP (1-139), PTHrP (1-173), and PTHrP (1-141), are translated from three different mRNAs through alternative splicing. In each, COOH-terminal fragment (C-PTHrP) is stable and measurable in the urine. In the present study, we measured concentrations of circulating and urinary C-PTHrP in 28 patients with primary lung cancer and normal serum calcium levels. We used PCR to evaluate PTHrP mRNA expression and its alternative splicing types in 16 lung cancer cell lines and 17 lung cancer tissues. The average serum C-PTHrP level was 38.95 +/- 19.41 pmol/l in 28 lung cancer patients, whereas that in 10 normal subjects was 26.53 +/- 9.43; the difference was statistically significant (P = 0.0065). Average urine C-PTHrP:urine creatinine ratio was 7.56 +/- 5.17 x 10(-1) pmol/mg creatinine in 28 lung cancer patients, whereas it was 4.91 +/- 1.77 in 10 normal subjects; the difference was statistically significant (P = 0.0287). C-PTHrP radioimmunoassays detected that 23% of non-small cell lung cancer patients had higher serum C-PTHrP levels, and 32% had higher urinary C-PTHrP:urine creatinine ratio than average + 2 SD of normal subjects. Reverse transcription-PCR detected PTHrP mRNA expression in 21 of 21 non-small cell lung cancer (NSCLC) samples and 3 of 12 small cell lung cancer samples. In the cancer cell lines and tissues that had detectable PTHrP mRNA, PTHrP (1-139) mRNA was found in 21 of 24, PTHrP (1-173) mRNA was found in 19 of 24, and PTHrP (1-141) mRNA was found in 23 of 24. Our results suggest that all PTHrP mRNA expression is common in lung cancers. We found that NSCLCs cancers had detectable PTHrP mRNA, and serum and urinary C-PTHrP levels in NSCLC patients were significantly higher than those in normal subjects. We concluded that NSCLC produced PTHrP more frequently, but there was no clear significance of C-PTHrP measurement in lung cancer patients for cancer detection using the present assay. We suggested that PTHrP probably plays a role similar to a growth factor or proliferation factor in lung cancer, especially NSCLC, at a level insufficient to cause humoral hypercalcemia of malignancy.

bcl-2 and p53 protein expression in non-small cell lung cancers: correlation with survival time.

Apoptosis-related genes have received increasing attention in carcinogenesis, drug sensitivity, radiation sensitivity, and patient survival. bcl-2 and mutated p53 genes have been reported to inhibit apoptosis. To determine bcl-2 and p53 protein expression and their impacts on survival time in lung cancers, we studied 99 surgically resected, paraffin-embedded non-small cell lung cancer (NSCLC) specimens by immunohistochemical staining. The bcl-2 protein was expressed in 19.2% of NSCLCs. bcl-2-positive cases were found in 30. 4% of stages I and II carcinomas in 36.8% of squamous cell carcinomas. Patients with bcl-2 expression survived longer than those without. p53 protein was found in 44.4%; there was no significant difference in survival time between patients with and without p53 expression. Patients who were both bcl-2 positive and p53 negative survived significantly longer than those who were bcl-2 negative or p53 positive. These results suggest that bcl-2 protein expression can be histologically specific and stage dependent, and that the bcl-2 protein expression is potentially valuable for prognosis in NSCLC, particularly in the early stages, when bcl-2 protein expression is considered with mutant p53 protein expression.

Evidence for an autocrine/paracrine role for interleukin-6 in bone resorption by giant cells from giant cell tumors of bone.

Interleukin-6 (IL-6) is a multifunctional cytokine whose role in osteoclastic bone resorption has not been clearly defined. Therefore, we have used giant cells, which express many features of osteoclasts, from giant cell tumors of bone as a model to examine the role that IL-6 may play in human osteoclastic bone resorption. We found that conditioned medium from 24-h cultures of highly purified giant cells (10(6)/ml) contained large amounts of IL-6 (37.9 +/- 8.8 ng/ml), similar to the amount of IL-6 produced by tumor stromal cells (29.8 +/- 11.5 ng/ml). Giant cells and stromal cells from giant cell tumors expressed IL-6 mRNA, as indicated by polymerase chain reaction analysis and in situ hybridization studies, and immunohistochemical techniques demonstrated that the giant cells expressed IL-6 receptors. The addition of a neutralizing antibody to IL-6 significantly decreased the area of dentine resorbed by purified giant cells in a dose-dependent manner, and the addition of IL-6 to cultures of purified giant cells pretreated with anti-IL-6 restored the resorbing capacity of the giant cells. These data suggest that IL-6 may act as both an autocrine and a paracrine factor for human osteoclasts and play an important role in the bone-resorbing capacity of these cells.

Induction of apoptosis in B lineage cells by activin A derived from macrophages.

A factor produced by P388D1 cell line murine macrophages showed a profound suppressive effect on the in vitro proliferation of B lineage cells. It was purified to homogeneity from conditioned media of P388D1 cells stimulated with phorbol 12-myristate 13-acetate for 48 h by a three-step procedure. The purified factor gave a single band of protein with a molecular mass of 16 kD on SDS-polyacrylamide gel electrophoresis. We show here that exposure of B lineage cells to this factor results in the induction of a cytotoxic effect and a significant increase in the proportion of fragmented DNA. DNA fragmentation was detected in B lineage cells after 3 h culture with the factor in the quantitative colorimetric determination. The mechanism of cell death was characterized by a ladder-like electrophoretic pattern of degraded chromosomal DNA, indicating that the factor induces apoptosis. The NH2-terminal amino acid sequence of this factor was identical with that of activin A over the 26 amino acid residues identified. We sought to determine whether apoptosis could be modulated by two kinds of inhibitor of protein kinases, H7 and HA1004, in concentrations that are below their toxicity limits. Apoptosis induced by the factor was suppressed by H7 but was relatively unaffected by HA1004. These findings suggest that the signals by protein kinases may regulate apoptotic B cell death by the factor activin A, derived from macrophages.

Multinucleated cells formed in vitro from Paget's bone marrow express viral antigens.

Paget's disease of bone is characterized by large numbers of osteoclasts that have viral-like nuclear and/or cytoplasmic inclusions. Pagetic osteoclasts express respiratory syncytial viral (RSV) and measles viral (MV) nucleocapsid antigens. The data suggest a possible viral etiology for Paget's disease. However, studies to characterize further the putative viral inclusions in Paget's osteoclasts have been severely hampered by the extreme difficulty in isolating large numbers of osteoclasts from pagetic bone. The recent demonstration that osteoclast-like multinucleated cells (MNC), that had certain characteristics of pagetic osteoclasts formed in marrow cultures from Paget's patients, may permit studies to describe this virus further. Therefore, we have cultured marrow samples from involved and uninvolved bones from Paget's patients and from normal subjects to determine if the MNC formed in these cultures express viral antigens. RSV and/or MV antigens were expressed in the mononuclear cells and/or the MNC formed in 12 of 12 marrow cultures from active lesions of patients with Paget's disease, with 40-50% of the cells expressing viral antigens. In contrast, less than 5% of cells isolated from cultures from normal subjects expressed RSV and/or MV. These results suggest that MNC formed in long-term marrow cultures from patients with Paget's disease frequently express paramyxoviral antigens and are very similar to pagetic osteoclasts. Thus, these marrow cultures may be useful for further characterizing the virus in Paget's disease.

Immunoelectron microscopic localization of fibronectin in the smooth muscle layer of mouse small intestine.

We studied the ultrastructural distribution of fibronectin in the smooth muscle layer of mouse small intestine with affinity-purified antibodies using the immunogold technique. Fibronectin was present over the pericellular area extending from the cell membrane to the extracellular matrix beyond the basal lamina. Distribution of the glycoprotein over the pericellular area was heterogeneous, i.e., it was localized more abundantly in the narrow space between smooth muscle cells, the gaps having a width of 60-80 nm where the two dense bands in adjacent cells matched each other. Such localization suggests that fibronectin contributes to cell adhesion. Within the basement membrane, gold label was localized both in lamina lucida and lamina densa, more densely in the latter than in the former. Fibronectin was also co-distributed with collagen fibers in the extracellular matrix. Within smooth muscle cells, gold particles were observed on rough endoplasmic reticulum and secretory vesicle-like structures. These results suggest that smooth muscle cells synthesize fibronectin and secrete it as a component of the basal lamina and extracellular matrix.

Inhibition of human small cell lung cancer cell growth by methylprednisolone.

Methylprednisolone (MP) inhibited cancer cell growth at concentrations between 0 and 2.0 mM. IC50 was 0.96 mM in H82, 0.44 mM in H345, 0.86 mM in H510A, 0.54 mM in N592 and 0.52 mM in H2081. The growth inhibition was not disturbed by addition of 1 microM RU38486. DNA fragmentation were observed in H510A and N592 at MP concentration of 0.2 mM. Glucocorticoid (GC) receptor mRNA expression was detectable only in H510A. We concluded that MP has potency as an anti-cancer agent at a high concentration. The effect was probably not through GC receptor binding, and growth inhibition was independent of apoptosis induction.

Frequent cyclin D1 expression in chromate-induced lung cancers.

Ex-chromate workers are frequently afflicted with lung cancers, especially central-type squamous cell carcinomas (SCCs) of the lung. However, little is known about the molecular and cellular biologic characteristics of chromate-induced lung cancers. We investigated expression of cyclin D1, bcl-2, and p53 proteins in chromate-induced lung cancers by immunohistochemistry, compared with those in lung cancers from nonexposed individuals and those in individuals with pneumoconiosis. Of 19 chromate-induced lung cancers, 16 tumors were SCCs, including 11 central and 5 peripheral types. Eleven (69%) of 16 chromate SCCs showed cyclin D1 expression. In contrast, cyclin D1 expression was observed in only 3 (12%) of 26 SCCs from nonexposed individuals and 6 (16%) of 37 SCCs that developed in patients with pneumoconiosis, respectively. The frequency of cyclin D1 expression proved to be significantly higher in chromate-induced SCCs than in SCCs from nonexposed individuals and from those with pneumoconiosis (P < .001). When comparisons were extended to all histologic types of lung cancer, cyclin D1 expression was observed significantly more often in chromate-induced lung cancers than in lung cancers from nonexposed subjects and those from patients with pneumoconiosis (11 [58%] of 19 v 5 [10%] of 52, P < .001, and 7 [11%] of 63, P < .001, respectively). Frequencies of bcl-2 and p53 expression were not significantly different among lung cancers from ex-chromate workers, nonexposed individuals and those with pneumoconiosis. The current study suggests that cyclin D1 expression may be involved in the development of chromate-induced lung cancers, although its underlying mechanism remains to be determined.

Gallium-67 scintigraphy, bronchoalveolar lavage, and pathologic changes in patients with pulmonary sarcoidosis.

The intensity of gallium-67 scintiscans, lymphocyte counts in bronchoalveolar lavage fluid, and pathologic changes were studied in 26 patients with untreated pulmonary sarcoidosis. Noncaseating granulomas were recognized with significantly greater frequency in stage 2 (80 percent; 8/10 cases) than in stage 1 (43 percent; 6/14 cases). Alveolitis showed little relation to the roentgenographic stage. There was a strong correlation between the intensity of gallium uptake in pulmonary parenchyma and the detection rate of granuloma; however, the detection rate of alveolitis was not statistically different from the intensity of gallium uptake. A highly significant correlation was revealed between the lymphocyte counts in bronchoalveolar lavage fluid and the intensity of alveolitis. These observations suggest that the gallium uptake reflects mainly the presence of granuloma, and the lymphocyte count in bronchoalveolar lavage fluid reflects the intensity of alveolitis in patients with pulmonary sarcoidosis.

Expression of syndecan-1 is common in human lung cancers independent of expression of epidermal growth factor receptor.

In order to determine syndecan-1 expression in lung cancer, we examined 115 lung cancer specimens and 17 lung cancer cell lines. Syndecan-1 was immunohistochemically stained with a polyclonal antibody in 115 paraffin-embedded specimens; 84 cases out of 97 non-small cell lung cancer (NSCLC) and eight cases out of 18 small cell lung cancer (SCLC) were positively stained. Simultaneously, epidermal growth-factor receptor (EGFR) was stained; 47 cases out of 97 NSCLC and one case of 18 SCLC were positively stained. No significant correlation was shown between EGFR and syndecan-1 expression (P=0.68). Syndecan-1 mRNA was detectable in 16 of 17 lung cancer cell lines and EGFR mRNA in nine of 17. Eight cell lines had syndecan-1 mRNA as well as EGFR mRNA. PR-39 (1 microM) and 80 pM transforming growth factor-beta(1) (TGF-beta(1)), did not increase expressions of syndecan-1 mRNA and EGFR in five lung cancer cell lines. We concluded that lung cancer had detectable syndecan-1; however, expression of syndecan-1 protein did not correlate with survival time of lung cancer patients.

Transforming growth factor-beta 1 proliferated vascular smooth muscle cells from spontaneously hypertensive rats.

To clarify whether the growth inhibitors, transforming growth factor-beta 1 (TGF-beta 1), heparin, and interferon-gamma (IFN-gamma) contribute to the development of vascular hypertrophy in spontaneously hypertensive rats (SHR), the growth of vascular smooth muscle cells (VSMC) was evaluated both for cell numbers over a period of 4 days, and [3H]thymidine incorporation over 24 h. Heparin and IFN-gamma inhibited the proliferation of VSMC from SHR and Wistar-Kyoto (WKY) rats. TGF-beta 1 enhanced SHR-VSMC proliferation by 16.6 +/- 8.9%; in contrast TGF-beta 1 inhibited WKY-VSMC proliferation by 60.5 +/- 7.4%. There was no difference in affinity, number of binding sites, or subtype expression of TGF-beta 1 receptor between SHR-VSMC and WKY-VSMC. This evidence suggests that the signal transduction system of TGF-beta 1 either the receptor itself or downstream signaling molecules, may be altered in SHR-VSMC versus WKY-VSMC. This abnormal responsiveness to TGF-beta 1 is involved in the proliferative characteristics of SHR-VSMC. Therefore, TGF-beta 1 could contribute to the development of hypertension or vascular hypertrophy in SHR.

Proximal gastric vagotomy with carbon dioxide laser: experimental studies in animals.

Proximal gastric vagotomy has been widely used as a surgical treatment for peptic ulcer disease. However, it is technically complex and time-consuming. Moreover, it may cause circulatory problems in the gastric mucosa. We have reported a new method of blood flow-preserving vagotomy with a carbon dioxide laser (CO2 laser vagotomy) developed in our laboratory. To assess its efficacy, we used cysteamine-induced ulcer and measured gastric mucosal blood flow in rats. The incidence of cysteamine-induced ulcer formation was reduced significantly in the group that underwent CO2 laser vagotomy compared with a group treated with proximal gastric vagotomy. Gastric mucosal blood flow was significantly better in the CO2 laser vagotomy group. Long-term follow-up of acid reduction was evaluated in dogs by the pentagastrin-stimulation test. Acid reduction in dogs was satisfactory during the 12 months of this study. CO2 laser vagotomy is a new, easy, time-saving, and circulatory-preserving technique for peptic ulcer disease.

Immunohistochemical studies on the distributions and age-related changes of types I and III collagen in the oral mucosa of mice.

The distribution of types I and III collagen in mouse oral mucosa and the age-related changes over 3 days to 2 years of age were examined by immuno-fluorescence and -electron microscopy, with use of affinity-purified polyclonal antibodies. Immunofluorescence microscopy revealed that types I and III collagen existed in all tissues and at all ages examined. The staining intensity for type I collagen was stronger and increased more markedly in the lamina propria of the hard palate (HPlp) and gingiva (G), compared with that in the submucosa of the hard palate (HPsm) and the buccal mucosa (BM). The staining intensity for type III collagen was strong and increased markedly with age in all connective tissues examined. Examination of immunogold-labeled tissues demonstrated that most of the collagen fibrils were labeled for both type I and type III collagen, which suggests that they were hybrid fibrils containing both types of collagen. The quantitative evaluation of the labeling densities of gold particles revealed that the labeling density of type III collagen in BM and HPsm was higher and increased more rapidly during growth than in HPlp and G, while the labeling density of type I collagen was higher in HPlp and G. The fibril diameters in HPlp were larger and increased more rapidly during growth than in BM and HPsm. These studies are the first to demonstrate the distribution of types I and III collagen and their age-related changes in mouse oral mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)