In vivo activation of langerhans cells and dendritic epidermal T cells in the elicitation phase of murine contact hypersensitivity

Langerhans cells (LCs) and dendritic epidermal T cells (DETCs) constitute the skin immune system. To demonstrate the kinetics of in vivo activation of murine LCs and DETCs in the elicitation phase of contact hypersensitivity, we measured the cell area positively stained for I-A and gammadeltaT-cell receptor (or Thy-1.2), respectively, under a fluorescence microscope at various time intervals after topical application of dinitrofluorobenzene. The fluorescence-positive area of LCs increased in parallel with that of DETCs at 1 h and 24 h, indicating the biphasic activation of LCs and DETCs. Early activation was hapten-specific and often exhibited close LC-to-DETC apposition. Experiments with in vivo administration of neutralizing anticytokine antibodies revealed that none of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta were involved in the induction of early activation of LCs and DETCs, while TNF-alpha and IL-1beta mediated late activation of LCs, and IFN-gamma and IL-1beta mediated that of DETCs. Our results indicate that LCs and DETCs are synchronously and biphasically activated in the epidermis during the elicitation phase of contact hypersensitivity and suggest that different mechanisms may control early and late activation.     

Suppressive effect of acupuncture stimulation to the sacral segment on the state of vigilance and the brainstem cholinergic neurons

The effects of acupuncture stimulation to the sacral segment on the electroencephalogram (EEG) and activity of the cholinergic neurons in the laterodorsal tegmental nucleus (LDT) were examined in urethane-anesthetized rats. When EEG was small amplitude and higher frequency, the stimulation to the sacral segment induced large amplitude and slow EEG with latencies ranged from 45 sec to 12 min, and durations from 48 sec to 56 min. The stimulus induced EEG is composed of significant increase in delta power and significant decrease in theta and beta powers. Firing rate of the cholinergic LDT neurons significantly decreased from 2.9+/-1.5 Hz to 1.1+/-0.8 Hz after the stimulus (n=12, p<0.05). The decrease of neuronal activity always preceded to the start of large and slow EEG, while the increase of the activity always preceded to the change of EEG from large slow wave to small faster wave. These results suggest that the acupuncture stimulation to the sacral segment changes the state of the animals from light anesthesia to deep anesthesia, and that the change is mediated by the suppression of the cholinergic neurons in the LDT.     

In vitro RNA interference against beta-catenin inhibits the proliferation of pediatric hepatic tumors

Mutations of beta-catenin have been identified in the majority of pediatric hepatic malignancies, including hepatoblastoma (HB) and hepatocellular carcinoma (HCC), suggesting its important contribution in hepatic tumorigenesis in this age group. However, the role of beta-catenin/canonical Wnt signaling pathway in the neoplastic growth of cancer cells has not been directly studied. To address beta-catenin's capability in maintaining the malignant phenotype in established pediatric HB and HCC cell lines, HuH-6 and HepG2, harboring mutated and overexpressed beta-catenin, we carried out a series of in vitro analyses through a transfection of short interfering RNAs (siRNAs) to generate a loss-of-function model. HuH-7, another HB cell line derived from a pediatric patient without a stabilizing mutation was used for comparison. RNA interference successfully manipulated the degradation of overexpressed beta-catenin. In all cell lines, beta-catenin mRNA was suppressed by 80-90% after 48 h of transfection, and a reduction of its protein expression was demonstrated. In HuH-6 and HepG2, the pre-existing beta-catenin nuclear accumulation disappeared and reductions of beta-catenin downstream target genes, c-myc and cyclinD1, were also evidenced after the treatment. The in vitro proliferation of both cell lines was transiently inhibited. In contrast, the suppression of beta-catenin in HuH-7 did not lead to a significant change in the expression of target genes or cellular proliferation. Our data indicate that beta-catenin can be considered a specific target for gene therapy in pediatric hepatic tumors with mutations and overexpression of this gene.     

Potential predictors of survival after surgery for colorectal cancer patients with synchronous unresectable liver metastases

Liver resection has been recognized as the best treatment for patients with colorectal liver metastases, but as a curative resection for multiple and bilobar colorectal liver metastases (MBCLM) it is definitely less effective. We clarify predictors of survival for unresectable MBCLM. Potential predictors of overall survival, and the correlation between tumor marker and survival were evaluated for patients with synchronous unresectable MBCLM, including 6 rectal and 17 colon cancers. In univariate analysis, survival in patients with the following parameters were longer than those without them: number of liver metastases (10) and a >1.0 ratio of postoperative CEA/preoperative CEA were factors of poor prognosis, and patients with two such factors had an even worse prognosis. There was a tendency for correlation between the ratio of postoperative CEA/pre-operative CEA and survival (R=-0.492, P=0.053; y=17.388-3.733x). Thus, we clarified some of the predictors of survival for MBCLM, and the usefulness of serum CEA.     

Heterogeneous CPA sensitivity of spontaneous excitation in smooth muscle of the rabbit urethra

1. To investigate the role of intracellular Ca stores in generating spontaneous excitation of the urethra, the effects of cyclopiazonic acid (CPA) on spontaneous contractions, transient increases in intracellular calcium concentration ([Ca2+]i; Ca transients) and depolarizations were examined in smooth muscles of the rabbit urethra. 2. In about 90% of circular smooth muscle (CSM) preparations, CPA (10 microM) increased the amplitude of spontaneous contractions by about 180% and reduced their frequency to some 25% of control values (CPA-resistant), while it readily abolished the contractions in the remaining preparations. 3. In about 70% of CSM preparations, CPA prevented the generation of spontaneous depolarizations termed slow waves, but increased their amplitude and duration in the remainder. CPA also prevented the generation of spontaneous Ca transients in about 40% of CSM preparations, while increasing their amplitude and duration in the remaining preparations. In CPA-resistant preparations that had been exposed to nicardipine (1 microM), subsequent CPA invariably abolished residual spontaneous depolarizations or Ca transients. CPA abolished caffeine-induced Ca transients in Ca-free solutions, suggesting that it effectively depleted intracellular Ca stores. 4. Longitudinal smooth muscles generated spontaneous action potentials, which had a shape distinct from that of slow waves in CSM. Spontaneous action potentials were abolished by nicardipine but not CPA. 5. Transmural nerve stimulation increased the frequency of Ca transients to give a sustained rise in [Ca2+]i, but inhibited their generation after blocking alpha-adrenoceptors with phentolamine (1 microM). These nerve-evoked responses were preserved in preparations that had been exposed to CPA. Similarly, both in control and CPA-treated CSM preparations, spontaneous Ca transients were accelerated by noradrenaline (NAd, 1 microM) and were suppressed by 3-morpholino-sydnonimine (SIN-1, 10 microM), a nitric oxide (NO) donor. 6. In conclusion, CSM of the urethra generates spontaneous activity, which depends on Ca release from intracellular Ca stores. However, after blocking this primary pacemaking mechanism, L-type Ca channel-dependent action potentials may drive CSM. Irrespective of the origin of pacemaking, neurally-released NAd and NO are capable of modulating spontaneous excitation.     

Induction of apoptosis by isoflavonoids from the leaves of Millettia taiwaniana in human leukemia HL-60 cells

We have isolated two new isoflavonoids, millewanin-F (1) and furowanin-A (2), together with five known isoflavonoids from the leaves of Millettia taiwaniana Hayata (Leguminosae) and examined their effects on the growth of human leukemia HL-60 cells. Among the isolated isoflavonoids, furowanin-A (2), warangalone (3), isoerysenegalensein-E (4), and euchrenone b10 (6) showed significant cytotoxicity against HL-60 cells. After treatment with three of the cytotoxic isoflavonoids, furowanin-A (2), warangalone (3), and isoerysenegalensein-E (4), fluorescence microscopy with Hoechst 33,342 staining revealed that the percentage of apoptotic cells with fragmented nuclei and condensed chromatin increased in a time-dependent manner. In addition, the activities of caspase-9 and caspase-3 were also enhanced in a time-dependent manner upon treatment with the isoflavonoids 2, 3, and 4. Caspase-9 and caspase-3 inhibitors suppressed apoptosis induced by isoflavonoids 2, 3, and 4. These results suggest that the isoflavonoids induced apoptosis in HL-60 cells through activation of the caspase-9/caspase-3 pathway, which is triggered by mitochondrial dysfunction.     

Effects of caffeine on the kinetics of fluvoxamine and its major metabolite in plasma after a single oral dose of the drug

The effects of caffeine on the kinetics of fluvoxamine (FLV) and its major metabolite fluvoxamino acid (FLA) in plasma, after a single oral dose of the drug, were studied in 12 healthy male volunteers. The subjects received caffeine 300 mg/d or placebo for 11 days in a double-blind randomized crossover manner, and on the eighth day they received a single oral 50-mg dose of FLV. Blood sampling and pharmacodynamic evaluation were conducted up to 72 hours after FLV dosing. Plasma concentrations of FLV and FLA were measured by high-performance liquid chromatography. Caffeine significantly decreased the plasma concentrations at 6 time points (P<0.05) and total area under the plasma concentration-time curve (156.5+/-51.7 vs. 118.9+/-38.2 ng/h/mL, P<0.01) of FLV. Plasma concentration and pharmacokinetic parameters of FLA were not affected by caffeine. Caffeine induced no significant change in the pharmacodynamic effects of FLV. The present study suggests that caffeine slightly induces the metabolism of FLV, probably mediated by CYP1A2.     

In vivo hepatotoxicity study of rats in comparison with in vitro hepatotoxicity screening system

For the establishment of a high throughput screening system using primary cell cultures, investigation of elucidated toxicities to assess the correlation between in vitro and in vivo hepatotoxicity is necessary in the safety evaluation of the compound. In the previous study, we reported the usability of rat primary cultured hepatocytes for establishment of high throughput screening system. To confirm the reliability of rat primary hepatocytes culture screening system, we conducted a single-dose in vivo study with relatively high dose of hepatotoxicant in rats using 4 reference compounds (acetaminophen, amiodarone, tetracycline, carbon tetrachloride), and investigated histopathological changes and expression of oxidative stress-related proteins by immunohistochemistry. We also carried out a proteomics analysis for estimating the reliable and sensitive biomarkers. Histopathologically, compound-specific hepatotoxicity was detected at 24 hr after administration in all compounds except amiodarone, which is known to induce phospholipidosis. Immunohistochemically, oxidative stress-related proteins were increased within 6 hr after administration in all treated groups. Proteomics analysis revealed several protein biomarkers related to oxidative stress and mitochondrial metabolism-regulation, which had been previously detected by proteomics analysis in in vitro screening system. Oxidative stress-related proteins were considered as useful biomarkers of hepatotoxicity; since they were detected by immunohistochemistry and proteomics analysis prior to appearance of compound-specific histopathological changes detected by light microscopy. Considering the relevance of in vitro system to in vivo system from the aspect of new biomarkers related to the toxicogenomics/toxicoproteomics, in vitro primary cell culture system would be sufficient to detect hepatotoxicity in the early stage of drug discovery.