Expression of the macrophage colony-stimulating factor and c-fms genes in human acute myeloblastic leukemia cells.

Macrophage colony-stimulating factor (CSF-1; M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocytes. The effects of CSF-1 are mediated through binding to specific, high-affinity surface receptors encoded by the c-fms gene. CSF-1 and c-fms gene expression was investigated in fresh human acute myeloblastic leukemic cells by Northern blot hybridization using cDNA probes. 4.0-kb CSF-1 transcripts were detected in 10 of 17 cases of acute myeloblastic leukemia (AML), while c-fms transcripts were detected in 7 of 15. Coexpression of CSF-1 and c-fms was observed in five cases, and in five other cases neither gene was expressed. In situ hybridization demonstrated that transcripts for CSF-1 were present in 70-90% of cells in each of three cases studied while c-fms mRNA was detected in 40-70% of cells. The constitutive expression of CSF-1 transcripts was associated with production of CSF-1 protein, although detectable amounts of CSF-1 were not secreted unless the cells were exposed to phorbol ester. These results demonstrate that leukemic myeloblasts from a subset of patients with AML express transcripts for both the CSF-1 and CSF-1 receptor genes, often in the same leukemic cells in vitro.

Transcriptional and posttranscriptional regulation of CSF-1 gene expression in human monocytes.

Regulation of CSF-1 gene expression was investigated in human monocytes. CSF-1 transcripts were at low or undetectable levels in resting monocytes. However, in monocytes treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), CSF-1 mRNA was increased by 3 h and reached maximal levels by 12 h of drug exposure. When nuclear run-on assays were used, CSF-1 gene transcription was also at low or undetectable levels in resting monocytes but was activated after TPA exposure. TPA-treated monocytes exposed to actinomycin D further demonstrated that the half-life of the CSF-1 mRNA is 0.9 h. The results also demonstrated that the protein synthesis inhibitor, cycloheximide (CHX), increases CSF-1 mRNA levels in both resting and TPA-treated monocytes. These effects of CHX occurred in the absence of detectable increases in CSF-1 gene transcription. Moreover, treatment of monocytes with CHX and actinomycin D demonstrated that inhibition of protein synthesis is associated with stabilization of the CSF-1 transcript. Taken together, these findings indicated that CSF-1 gene expression is controlled at both transcriptional and posttranscriptional levels in human monocytes.

Posttranscriptional stabilization of c-fms mRNA by a labile protein during human monocytic differentiation.

The c-fms proto-oncogene encodes a transmembrane glycoprotein that is closely related or identical to the receptor for the monocyte colony-stimulating factor CSF-1. The present studies examined the mechanisms responsible for the regulation of c-fms gene expression during human monocytic differentiation. Levels of c-fms mRNA were undetectable in HL-60 promyelocytic leukemia cells, while 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of these cells was associated with the appearance of these transcripts. Run-on transcription assays demonstrated that the c-fms gene was transcriptionally active in uninduced HL-60 cells and that the rate of transcription was unchanged after TPA treatment. These findings suggested that c-fms mRNA levels in HL-60 cells are controlled by posttranscriptional mechanisms. The half-life of c-fms transcripts in TPA-induced HL-60 cells was found to be at least 6 h, while inhibition of protein synthesis with cycloheximide (CHX) decreased this half-life to 4 h. Moreover, inhibition of protein synthesis was associated with decreases in c-fms mRNA levels and a block in the induction of c-fms transcripts by TPA. These findings indicated that the c-fms transcript is stabilized by a labile protein. In contrast to HL-60 cells, c-fms mRNA is constitutively expressed in resting human monocytes and is down-regulated by treatment of these cells with TPA. Run-on assays demonstrated that TPA-induced downregulation of c-fms mRNA levels in monocytes occurred at the posttranscriptional level. Moreover, the results demonstrate that levels of c-fms mRNA are regulated posttranscriptionally by a labile protein. In this regard, the half-life of the c-fms transcript was 6.1 h in monocytes, while treatment of these cells with CHX decreased the half-life to 30 min. Furthermore, this effect of CHX occurred in the absence of changes in the rate of c-fms gene transcription. Together, these findings indicate that c-fms gene expression is regulated at a posttranscriptional level both in HL-60 cells induced to differentiate along the monocytic lineage and in human monocytes. The findings also indicate that levels of c-fms mRNA are regulated by the synthesis of a labile protein which is involved in stabilization of the c-fms transcript.

Role of arachidonic acid metabolism in transcriptional induction of tumor necrosis factor gene expression by phorbol ester.

The treatment of human HL-60 promyelocytic leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with induction of tumor necrosis factor (TNF) transcript. The study reported here has examined TPA-induced signaling mechanisms responsible for the regulation of TNF gene expression in these cells. Run-on assays demonstrated that TPA increases TNF mRNA levels by transcriptional activation of this gene. The induction of TNF transcripts by TPA was inhibited by the isoquinolinesulfonamide derivative H7 but not by HA1004, suggesting that this effect of TPA is mediated by activation of protein kinase C. TPA treatment also resulted in increased arachidonic acid release. Moreover, inhibitors of phospholipase A2 blocked both the increase in arachidonic acid release and the induction of TNF transcripts. These findings suggest that TPA induces TNF gene expression through the formation of arachidonic acid metabolites. Although indomethacin had no detectable effect on this induction of TNF transcripts, ketoconazole, an inhibitor of 5-lipoxygenase, blocked TPA-induced increases in TNF mRNA levels. Moreover, TNF mRNA levels were increased by the 5-lipoxygenase metabolite leukotriene B4. In contrast, the cyclooxygenase metabolite prostaglandin E2 inhibited the induction of TNF transcripts by TPA. Taken together, these results suggest that TPA induces TNF gene expression through the arachidonic acid cascade and that the level of TNF transcripts is regulated by metabolites of the pathway, leukotriene B4 and prostaglandin E2.

Detection of c-fms and CSF-1 RNA by in situ hybridization.

Recent studies have demonstrated by Northern blot analysis that both the c-fms proto-oncogene and the CSF-1 gene are expressed during human monocytic differentiation. In order to examine c-fms and CSF-1 expression at the cellular level, we have applied alkaline phosphatase detection of biotinylated v-fms and CSF-1 cDNA probes in situ. Using this approach, we demonstrate that c-fms and CSF-1 transcripts are detectable in HL 60 cells induced along the monocytic lineage but not in uninduced cells. The specific detection of these transcripts is further supported by the absence of histochemical staining in RNase-treated cells and when using pBR322 plasmid without insert as the biotinylated probe. Finally, the results indicate that most of the induced HL-60 cells have detectable levels of both c-fms and CSF-1 RNA. This approach should be useful for studying expression of these genes in populations of leukemic blasts and normal hematopoietic cells.

Highest microvessel count as a long-term prognostic factor in Japanese breast cancer patients.

The aim of this study was to determine whether microvessel density (MVD) could add useful information in predicting the prognosis of breast cancer patients. In our study, MVD was calculated by counting microvessels per x200 field in the highest neovascularized area of the tumor (highest microvessel count, HMC). HMC significantly increased according to the increased number of positive nodes. Higher HMC significantly correlated with worse relapse-free survival (RFS) of patients with negative node, one to three positive nodes in the axilla or with stage I and II tumors. HMC, however, was not predictive for RFS of patients with four or more positive nodes or with stage III tumors. Multivariate analysis revealed that HMC was second only to nodal status and tumor size as being predictive for RFS. These results suggest that HMC could be used in selection of patients with early-stage breast cancer who are at high risk for having occult metastasis.

Serum concentration of hepatocyte growth factor in patients with metastatic breast cancer.

The serum concentration of hepatocyte growth factor (HGF) was examined in 34 patients with metastatic breast cancer. Although no significant difference was observed between HGF concentration and the site of metastasis, serum HGF levels were slightly higher in patients with liver metastasis and in patients with multiple metastatic sites than in patients with other lesions. Significantly higher levels of serum HGF were observed in patients with progressive metastasis of breast cancer compared with those with stable metastasis. The patients with high HGF levels exhibited a significantly shorter survival rate than those with low HGF levels. Circulating HGF levels may be a useful indicator for the progression of metastatic lesions and the prognosis of patients with metastatic breast cancer.

A pilot study of the effects of short-term tamoxifen therapy on Ki-67 labelling index in women with primary breast cancer.

In this study we demonstrate the change in estrogen receptor (ER) level and cell proliferation in human breast cancer after a short-term tamoxifen therapy. Ten pre- and post-treatment breast tumor samples were examined immunohistochemically using ER and Ki-67 antibodies. Before tamoxifen treatment, six (60%) of ten patients were positive for ER. Tamoxifen increased the ER level in one patient and decreased the level in 4 patients. There was no significant change in ER level by tamoxifen therapy. On the other hand, Ki-67 labelling index (LI) significantly decreased after tamoxifen treatment. When Ki-67 LI was analyzed according to ER level, there was no difference between pre- and post-tamoxifen treatment in ER-negative patients, however, a significant decrease of Ki-67 LI by tamoxifen treatment was seen in ER-positive patients. Patients who showed down-regulation of ER expression tended to show a decrease of Ki-67 LI after tamoxifen therapy. In conclusion, short-term tamoxifen therapy decreased the proliferation of breast cancer, in ER-positive breast tumor samples.

Angiogenesis and stromal fibronectin expression in invasive breast carcinoma.

Angiogenesis and stromal fibronectin (SFN) expression were immunohistochemically analyzed in 83 breast cancer specimens. Microvessel count (MVC), which correlated with lymph node metastasis, TNM stage, recurrence, and mortality, was relatively low in SFN-positive tumors. SFN expression did not correlate with lymph node metastases or tumor size. However, SFN positivity was less likely in patients with recurrent disease than in those without recurrence, and relapse-free survival was significantly better in patients with SFN-positive tumors than in those with SFN-negative tumors. MVC and SFN positivity were significant independent predictors of relapse-free survival as well as tumor size and axillary nodal status (Cox's proportional hazards regression analysis). Angiogenesis and SFN expression, both of which are inversely related, can be used prognostically in patients with breast cancer.

c-erbB-2 status is an independent predictor of survival after first recurrence.

We studied retrospectively the interaction between c-erbB-2 overexpression and the prognosis in 239 invasive breast cancer patients who underwent radical operations between January 1984 and April 1991. The c-erbB-2 protein was overexpressed in 42 (17.6%) of 239 patients. There was no correlation between c-erbB-2 overexpression and age at operation, tumor size, lymph node involvement, or clinical stage. Only an inverse correlation was found between c-erbB-2 overexpression and hormone receptor levels. Patients with c-erbB-2 overexpression had a significantly worse overall survival than those without c-erbB-2 overexpression. In relation to lymph node involvement or estrogen receptor status, a significant difference in overall survival between the c-erbB-2-positive and -negative groups was found in patients with lymph node metastasis or in those with estrogen receptor-negative tumors. Out of 237 patients (two were lost to follow-up), 42 recurred and 25 died of breast cancer. The c-erbB-2-negative patients survived significantly longer after the time of first recurrence than the c-erbB-2-positive patients. In a multivariate analysis using Cox proportional-hazard regression model, c-erbB-2 status and disease-free interval were independent predictors of survival after first recurrence. In conclusion, c-erbB-2 status is an independent prognostic indicator of survival after first recurrence.

Inhibitory effects of an antiestrogen, toremifene, on the phorbol ester-induced adhesive capacity of breast carcinoma cells.

Previous studies have revealed that protein kinase C (PKC) is responsible for malignant progression. In the present study, we investigated the potent inhibitory effects of an antiestrogen, toremifene, on PKC-mediated cellular adhesion. A phorbol ester, phorbol 12-myristate 13-acetate (PMA), significantly enhanced alpha2beta1 integrin-dependent adhesion of MCF-7 breast carcinoma cells. This PMA-induced adhesion was partially inhibited by incubating cells with toremifene prior to PMA exposure in a time- and dose-dependent manner. FACS analysis demonstrated that the PMA-induced alpha2beta1-dependent cellular adhesion was accompanied with elevated expression of alpha2beta1+integrin subunit on the cell surface. However, toremifene did not affect the elevated expression levels of these integrins but rather the avidity of alpha2beta1 integrin. We concluded that toremifene inhibited cellular adhesion activated by PMA, probably through mechanism which inhibits PKC.

Schizophrenia-associated idiopathic unconjugated hyperbilirubinemia (Gilbert's syndrome).

BACKGROUND: Idiopathic unconjugated hyperbilirubinemia (Gilbert's syndrome) is a benign hyperbilirubinemia found in the general population. There has been only 1 previous report of Gilbert's syndrome occurring in schizophrenic patients. The present study was conducted to determine the frequency of Gilbert's syndrome in schizophrenic patients relative to patients with other psychiatric disorders. METHOD: Plasma bilirubin concentrations of every patient admitted to the psychiatric hospital during a 3-year period were collected, and patients were examined to exclude all other causes of hyperbilirubinemia. In addition, the psychiatric symptoms of schizophrenic patients (ICD-10 criteria) with hyperbilirubinemia were evaluated by the Positive and Negative Syndrome Scale (PANSS). RESULTS: Schizophrenic patients showed a significantly higher incidence of hyperbilirubinemia (p < .05) relative to patients suffering from other psychiatric disorders, and schizophrenic patients with hyperbilirubinemia showed significantly higher scores on the positive and general psychiatric subscales of the PANSS (p < .0001) than patients without hyperbilirubinemia. CONCLUSION: The apparently higher frequency of Gilbert's syndrome in schizophrenic patients may reflect a relationship between hyperbilirubinemia and schizophrenic psychosis. Hypothetical explanations, such as a possible genetic disposition for Gilbert's syndrome, an increased vulnerability of red cell membranes, and the role of estrogens in schizophrenic patients, are discussed.

The relationship of the platelet 5-HT-induced calcium response to clinical symptoms in eating disorders.

Clinical observations indicate that persons with eating disorders exhibit many psychopathologic symptoms such as difficulty with impulse control and depressed mood associated with impaired regulation of serotonin (5-HT) synaptic function in the central nervous system. In this study, we focused on the relationship between the 5-HT-induced calcium response in platelets and the clinical symptoms. While age, body weight, and severity of depressive symptoms were not correlated with 5-HT-induced response, there was an enhanced response in patients with bulimic symptoms or other impulsive behaviors. Moreover, patients with multi-impulsive behaviors had a significantly higher maximal increase than patients with uni-impulsive behavior, i.e., those who had only bulimic symptoms, as well as non-impulsive patients, and controls. Considering these results, the 5-HT-induced response may be related to difficulty with impulse control in general rather than bulimic eating attitudes specifically.

Involvement of the alpha2-adrenergic system in polydipsia in schizophrenic patients: a pilot study.

Animal studies have suggested the involvement of the adrenergic system in drinking behavior. The present study investigated the involvement of the alpha2-adrenergic system in the polydipsia of patients with chronic schizophrenia by use of an alpha2 agonist and an antagonist. Four patients with schizophrenic disorders accompanied by intermittent hyponatremia and polydipsia were the subjects of, and completed, this study. Drinking behavior was assessed by calculating the percent of maximum weight gain [PMWG: (maximum diurnal weight - standard weight) x 100/standard weight]. Standard weight was defined as body weight after 8 h of water restriction. Clonidine (75, 150, and 225 mg/day) increased the PMWG in a dose-dependent manner in the four subjects. In contrast, in three of the subjects, mianserin (30, 60, and 90 mg/day) decreased PMWG, and the severe polydipsia disappeared almost completely. These findings indicate clearly that the alpha2-adrenergic system is involved in the drinking behavior of schizophrenic patients. Mianserin appears to be clinically useful in treating such patients with polydipsia.

Estrogen receptor alpha mediated induction of the transforming growth factor alpha gene by estradiol and 4-hydroxytamoxifen in MDA-MB-231 breast cancer cells.

The selective estrogen receptor modulator, 4-hydroxytamoxifen (4-OHT) is a full agonist at the transforming growth factor (TGF) alpha gene in ER negative breast cancer cells stably transfected with ER alpha cDNA (Levenson et al., Br. J. Cancer 77 (1998) 1812-1819). E(2) and 4-OHT increase TGF alpha mRNA and protein in a concentration dependent manner. The responses to E(2) and 4-OHT are blocked by the pure antiestrogen ICI 182,780, which does not induce TGF alpha. Transfected MDA-MB-231 cells contain functional ER alpha but no ER beta function was detected. Neo transfected cells that did not express ER alpha or cells stably transfected with the DNA binding domain mutant C202R/E203V which prevents gene activation did not induce TGF alpha mRNA after either E(2) or 4-OHT treatment. An examination of the time course for either 10 nM E(2) or 1 microM 4-OHT for MDA-MB-231 cells stably transfected with cDNA for ER alpha showed increases in TGF alpha mRNA within 2 or 3 h respectively. Cells pretreated with cycloheximide (1 microg/ml) showed induced TGF alpha mRNA in response to E(2) or 4-OHT but TGF alpha mRNA induction was blocked by actinomycin D (1 microg/ml). We conclude that both E(2) and 4-OHT induce TGF alpha by direct interaction of ER alpha with DNA and that ER beta is not involved in the estrogen-like response to 4-OHT in the MDA-MB-231 cells.

Differential regulation by pregnenolone sulfate of intracellular Ca2+ increase by amino acids in primary cultured rat cortical neurons.

We investigated the effects of pregnenolone sulfate (PS) on the [Ca2+]i increase induced by gamma-aminobutyric acid (GABA) and N-methyl-D-aspartate (NMDA) using fluorescence imaging. PS inhibited the 50 microM GABA-induced increase in [Ca2+]i in a dose-dependent manner with an IC50 of 30 microM. The inhibitory effect of PS was apparent within 5 min and was in a non-competitive manner, suggesting that PS may act directly to the membrane level but indirectly to the GABA binding sites. Our previous study has already shown that the GABA-induced Ca2+ increase involves GABAA receptors and the similar pathway to a high K(+)-induced Ca2+ response (Takebayashi et al., 1996). Because 50 microM of PS could not inhibit a 25 mM K(+)-induced Ca2+ increase, it seems likely that the site of the inhibitory action of PS on the GABA-induced Ca2+ increase may be independent of the pathway of the high K(+)-induced Ca2+ response, but rather at GABAA receptor complex. In contrast, PS potentiated the 50 microM NMDA-induced increase in [Ca2+]i in a dose-dependent manner. The magnitude of the NMDA response was approximately doubled in the presence of 100 microM of PS. However, PS did not affect the acetylcholine(Ach)-induced increase in [Ca2+]i. Furthermore, corticosterone had little effect on the GABA- and NMDA-induced Ca2+ increases, indicating that the alteration of the Ca2+ response is specific for PS. In conclusion, it is suggested that PS modulates differentially [Ca2+]i increase induced by GABA and NMDA.

A new triphenylethylene derivative, TAT-59; hormone receptors; insulin-like growth factor 1; and growth suppression of hormone-dependent MCF-7 tumors in athymic mice.

TAT-59 ((E)-4-[1-[4-[2-(dimethylamino)ethoxy]-phenyl]-2-(4- isopropyl)phenyl-1-butenyl]-phenyl-monophosphate) treatment was performed on hormone-dependent MCF-7 tumors in athymic mice. TAT-59 given at 1, 5, and 20 mg/kg inhibited the estrogen-stimulated growth of MCF-7 tumors in athymic mice in a dose-dependent fashion. The most clear decrease in tumor growth was shown in the TAT-59 alone group, although it was not dramatic. Average serum concentrations of DP-TAT-59((Z)-[1-[4-[2-(dimethylamino)- ethoxy]phenyl]-2-(4-isopropyl)phenyl-1-butenyl]-4-hydroxybenzene) and DM-DP-TAT-59(desmethyl-DP-TAT-59), metabolites of TAT-59, increased in a dose-dependent manner. Much higher levels of DP-TAT-59 and DM-DP-TAT-59 were shown in tumors (target tissues of estrogen) as compared with muscles (nontarget tissues of estrogen) or serum. A serum concentration of DP-TAT-59 or DM-DP-TAT-59 corresponding to the physiologic levels of serum estradiol in premenopausal women was sufficient to inhibit the estrogen-stimulated growth of MCF-7 tumors in mice. TAT-59 induced a dose-dependent increase in estrogen receptor levels in the MCF-7 tumors. In contrast, it prevented the estradiol (E2)-induced increase in progesterone receptor levels in a dose-dependent manner. Insulin-like growth factor 1 levels measured in the MCF-7 tumors significantly decreased in the TAT-59 alone group and in the no treatment group as compared with the E2 alone group. These results show the pronounced antiestrogenic action of TAT-59 on hormone-dependent MCF-7 tumors in athymic mice.

A combination chemoendocrine therapy of mitoxantrone, doxifluridine, and medroxyprogesterone acetate for anthracycline-resistant advanced breast cancer.

Between January 1993 and October 1995, 34 patients with anthracycline-resistant advanced breast cancer were treated with a combination chemoendocrine therapy of mitoxantrone (MIT), doxifluridine (5'-DFUR) and medroxyprogesterone acetate (MPA). Of 34 patients, 28 were evaluable for efficacy of this combination therapy, and 30 including 2 for whom data were incomplete were assessed for adverse drug reactions. Adriamycin (ADM) was used for pretreatment in 12 patients, 4'-epi-ADM in 6, and THP-ADM in 12. In the eligible patients, 8.0 mg/m2 MIT was administered intravenously every 4 weeks, and 600 mg MPA and 600 mg 5'-DFUR were given orally every day. The median follow-up period was 25 weeks (range 2-90 weeks). The median cumulative dose of mitoxantrone was 66 mg (range 12-121 mg). Of the 28 patients, 11 (39.3%) responded to this combination therapy. As for response in relation to predominant site of lesion, 1 of 5 soft tissue lesions (20%) and 8 of 12 bone metastases (66.7%) showed a partial response, and one complete response and one partial response (25.0%) were seen in eight lung lesions. None of three pleural lesions responded to this therapy. The median duration of response was 31 +/- weeks (range 12-82 weeks). Adverse drug reactions were controllable or tolerable. Combined chemoendocrine therapy with a low dose of MIT is a well-tolerated and moderately effective regimen for the treatment of anthracycline-resistant advanced breast cancer.

Lithium chloride inhibits thrombin-induced intracellular calcium mobilization in C6 rat glioma cells.

In this study, the authors have demonstrated the effect of lithium, a typical mood stabilizer, on thrombin-evoked Ca2+ mobilization in C6 cells to elucidate the action mechanisms of the drug. Thrombin-induced Ca2 mobilization was reduced 24 hr after 1 or 10 mM lithium chloride (LiCl) pretreatment. The Ca2+ rise was reduced in a time-dependent manner, and the significant inhibition was observed 9 hr pretreatment with 10 mM LiCl. On the other hand, pretreatment of the cells with 10 mM LiCl for 24 hr did not alter the amount of Galphaq/11 significantly. Pretreatment with 10 mM LiCl for 24 hr failed to reduce the 5-HT-induced Ca2+ mobilization or to affect the desensitization of the 5-HT signal. Finally, thrombin-elicited Ca2+ rise was markedly inhibited in the presence of 0.05 U/ml plasmin, however, the Ca2+ rise was not further attenuated in the presence of plasmin in C6 cells pretreated with LiCl for 24 hr. These results indicate that pretreatment with LiCl attenuated thrombin-evoked intracellular Ca2+ mobilization in plasmin sensitive manner in C6 rat glioma cells. Thus, it is important to investigate the effect of lithium on thrombin-induced cellular responses to clarify the action mechanism of lithium in relation to some abnormality in thrombin-evoked Ca2+ rise observed in bipolar disorders.

Parkinson's disease associated with argyrophilic grains clinically resembling progressive supranuclear palsy: an autopsy case.

A 70-year-old male began to show akinesia, rigidity of extremities, finger tremor, disturbed vertical external ocular movement, and nuchal dystonia, which progressed slowly. Brain CT scan and magnetic resonance images showed slight atrophy of the frontal lobe and slight enlargement of the lateral ventricles. Hasegawa's dementia rating scale-revised version gave a moderate score of 11/30 points. He died of pneumonia at the age of 76. The clinical diagnosis was progressive supranuclear palsy (PSP). However, there were no neuropathological characteristics of PSP. Neuropathologically, Parkinson's disease was diagnosed. In addition, many argyrophilic grains (ArGs) in the gray matter were stained, especially in the insula, amygdala, hippocampus, parahippocampal gyrus, lateral occipitotemporal gyrus, and substantia nigra, by the Gallyas-Braak method. We consider that ArGs could modify the symptoms of Parkinson's disease and that Parkinson's disease with ArGs may show a PSP-like clinical course.