Membrane-bound form of monocyte chemoattractant protein-1 enhances antitumor effects of suicide gene therapy in a model of hepatocellular carcinoma.

Suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system combined with monocyte chemoattractant protein-1 (MCP-1) provides significant antitumor efficacy. The current study was designed to evaluate the antitumor immunity of a newly developed membrane-bound form of MCP-1 (mMCP-1) in an immunocompetent mouse model of hepatocellular carcinoma (HCC). A recombinant adenovirus vector (rAd) harboring the human MCP-1 gene and the membrane-spanning domain of the CX3CL1 gene was used. Large amounts of MCP-1 protein were expressed and accumulated on the tumor cell surface. The growth of subcutaneous tumors was markedly suppressed when tumors were treated with mMCP-1, as compared with soluble MCP-1, in combination with the HSV-tk/GCV system (P<0.01). The numbers of Mac-1-, CD4- and CD8a-positive cells were significantly higher in tumor tissues (P<0.05), and tumor necrosis factor (TNF) mRNA expression levels with mMCP-1 were almost five-fold higher than those with soluble MCP-1. These results indicate that the delivery of the mMCP-1 gene greatly enhanced antitumor effects following the apoptotic stimuli by promoting the recruitment and activation of macrophages and T lymphocytes, suggesting a novel strategy of immune-based gene therapy in the treatment of patients with HCC.

Monocyte chemoattractant protein-1 gene delivery enhances antitumor effects of herpes simplex virus thymidine kinase/ganciclovir system in a model of colon cancer.

Suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system is a well-characterized tool for cancer gene therapy; however, it does not yet exhibit sufficient efficacy to cure patients of malignancies. We have reported that adenovirally delivered monocyte chemoattractant protein (MCP)-1 augmented the antitumor effects of the HSV-tk/GCV system in an athymic nude mouse model. The current study, which uses an immunocompetent mouse model of colon cancer, was designed to evaluate the antitumor effects of MCP-1 gene delivery in conjunction with this suicide gene therapy system. Subcutaneous tumor foci were directly transduced with both recombinant adenoviruses (rAds) expressing an HSV-tk gene and either of the MCP-1, CD80 and LacZ genes, followed by GCV administration. The growth of tumors was markedly suppressed by codelivery of HSV-tk and MCP-1 genes, which was exclusively associated with the recruitment of monocytes/macrophages, T helper 1 (Th1) cytokine gene expression and cytotoxic activity of the splenocytes. Furthermore, the antitumor effects were more efficient than that obtained by the combination of HSV-tk and CD80 genes. These results suggest an immunomodulatory effect of MCP-1 in the context of suicide gene therapy of colon cancer via orchestration of innate and acquired immune responses.

Enhanced anti-tumor effects of herpes simplex virus thymidine kinase/ganciclovir system by codelivering monocyte chemoattractant protein-1 in hepatocellular carcinoma.

The therapeutic efficacy of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system in many types of tumors is unsatisfactory due to the insufficient spread of gene transfer and insufficient cell killing. In the current study, we investigated whether adenovirally delivered monocyte chemoattractant protein (MCP)-1 potentiates the antitumor effects of the HSV-tk/GCV system in hepatocellular carcinoma (HCC) cells. Subcutaneous tumor foci of the human HCC cell line, HuH7, established in athymic mice were directly transduced with a recombinant adenovirus (rAd) harboring an HSV-tk gene driven by a human alpha-fetoprotein promoter, followed by GCV administration. Subsequently, another rAd expressing MCP-1 under the universal CAG promoter was injected. The growth of tumors was markedly suppressed by codelivering HSV-tk and MCP-1 genes compared to that by either HSV-tk/GCV or MCP-1 delivery. In the tumor tissues, monocyte/macrophage infiltration was detected immunohistochemically. The antitumor effects of the rAd expressing MCP-1 were markedly reduced by the administration of carrageenan, a compound known to inactivate macrophage. These results indicate that adenovirally delivered MCP-1 enhanced the antitumor effects of the HSV-tk/GCV system synergistically by recruitment/activation of macrophages in tumor tissues, suggesting an effective immunotherapy for HCC and other lineages of tumors when used adjuvantly with a suicide gene.

Laparoscopic cholecystectomy via two ports, using the "Twin-Port" system.

We developed a "Twin-Port" system that allows a 5-mm camera and a forceps to be inserted through a single port for the laparoscopic cholecystectomy procedure. An infraumbilical incision of approximately 10mm is made to insert the "Twin-Port". After pneumoperitoneum is performed, a 5-mm camera and grasper are inserted to expose the gallbladder. A 5-mm trocar is inserted approximately 1 cm below the xiphoid process, and laparoscopic cholecystectomy is performed via two ports. The gallbladder is removed through the opened "Twin-Port". The operation was performed in 40 patients without acute inflammatory gallbladder disease. None of the patients required open abdominal surgery. In 3 patients, an additional 5-mm trocar was inserted because of difficulty in removing the gallbladder from the gallbladder fossa. Mean operation time was 49min. The size of the infraumbilical wound was almost the same as that with the conventional procedure using a 10-mm trocar. The "Twin-Port" system was devised to make laparoscopic cholecystectomy possible through two ports in the clinical setting. It may be less invasive than other LC procedures, and also has cosmetic and cost advantages. This procedure appears promising as a practical surgical treatment for cholecystolithiasis and gallbladder polyps.

Functional disorders of the sympathetic nervous system in mice lacking the alpha 1B subunit (Cav 2.2) of N-type calcium channels.

N-type voltage-dependent Ca(2+) channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, we have generated mice genetically deficient in the alpha(1B) subunit (Ca(v) 2.2). The alpha(1B)-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to omega-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, was markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation were dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice was nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction.

[Day surgery for cholecyst lithiasis].

Since 1997, laparoscopic cholecystectomy has been performed as one-day surgery (LC/DS) at our institution. Among the 122 patients enrolled in this program, 97 (80%) were successfully discharged within 24 hours after admission. Discharge was delayed for the other 25 patients, although 12 (48%) of them were discharged on postoperative day (POD) 2 or 3. This study not only verified the efficacy of LC/DS in shortening convalescence and allowing an early resumption of work but also confirmed the safety of LC/DS except in one patient with hemophilia A who required laparotomy for intraabdominal bleeding on POD 13. LC/DS is now the first choice of treatment for cholelithiasis regardless of symptoms. Discharge can be expected within 24 hours after admission in most cases, although the preference of patients should be considered when determining the timing of discharge.

Competitive NMDA receptor antagonists disrupt prepulse inhibition without reduction of startle amplitude in a dopamine receptor-independent manner in mice.

Prepulse inhibition is thought to reflect the operation of the sensorimotor gating system in the brain, and is reduced in schizophrenic patients and in animals treated with non-competitive NMDA receptor antagonists such as phencyclidine and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ((+)-MK-801). Previously, we reported that a competitive NMDA receptor antagonist, cis-4-phosphonomethyl-2-piperidine-carboxylate hydrochloride (CGS 19755), also disrupts prepulse inhibition concomitantly with a marked reduction of startle amplitude elicited by pulse alone in rats. In the present study, the effect of NMDA receptor antagonists on prepulse inhibition was tested in mice. In addition, involvement of the dopaminergic system in CGS 19755-induced disruption of prepulse inhibition was examined. When CGS 19755 was subcutaneously administered at 40 and 80 mg/kg, prepulse inhibition was disrupted without any change in the startle amplitude elicited by pulse alone. Intracerebroventricularly administered CGS 19755 disrupted prepulse inhibition at dosages of 0.1 and 0.2 microg/mouse. The same dosages of R-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP), another competitive NMDA receptor antagonist, also decreased prepulse inhibition, while its less active enantiomer, S-CPP, did not affect prepulse inhibition at 0.2 microg/mouse (i.c.v.). A typical neuroleptic, haloperidol, did not significantly improve CGS 19755 (40 mg/kg s.c.)-induced disruption of prepulse inhibition. These results suggest that the disruption of prepulse inhibition by CGS 19755 and R-CPP is NMDA receptor-mediated and dopamine receptor-independent.

Differential effects of the strychnine-insensitive glycine site antagonist (+)-HA-966 on the hyperactivity and the disruption of prepulse inhibition induced by phencyclidine in rats.

The amplitude of the acoustic startle response is reduced by a preceding weak stimulation which by itself does not elicit the startle response. This phenomenon is named prepulse inhibition (PPI) and is thought to reflect the operation of the sensorimotor gating system, which is deficient in schizophrenic patients. It has been reported that an antagonist at the strychnine-insensitive glycine site has atypical neuroleptic properties in experimental animals. To evaluate the effect of an antagonist at the site on disrupted PPI, we examined whether (+)-HA-966 antagonizes phencyclidine-induced (3 mg/kg s.c.) and apomorphine-induced (1 mg/kg s.c.) disruption of PPI in rats. In addition, its effect on phencyclidine-induced hyperactivity was tested. The effects of (+)-HA-966 were compared with those of haloperidol, a typical neuroleptic. (+)-HA-966 antagonized phencyclidine-induced hyperactivity, but not phencyclidine-induced disruption of PPI, which is thought to be a model of refractory symptoms in schizophrenia. Furthermore, (+)-HA-966 did not improve the deficit in PPI induced by apomorphine. On the other hand, haloperidol antagonized phencyclidine-induced hyperactivity and the disruption of PPI by apomorphine, but not by phencyclidine. The results of this study might mean that (+)-HA-966 antagonizes the behavioral change induced by excessive dopamine release (the increment of locomotor activity due to phencyclidine), but not the effect induced by a direct dopamine agonist or the dopamine-independent effect of phencyclidine (the disruption of PPI). Thus, as regards antagonism of phencyclidine-induced disruption of PPI, (+)-HA-966 does not appear to have an atypical neuroleptic-like effect.

[Changes in serum anti-Helicobacter pylori IgG antibody, pepsinogen I, and pepsinogen II after eradication therapy of Helicobacter pylori].

To investigate the changes in serum anti-Helicobacter pylori IgG antibody (HP Ab), pepsinogen I (PG I), pepsinogen II (PG II), and pepsinogen I/II ratio (PG I/II) after eradication therapy of Helicobacter pylori (HP), we studied 78 patients with HP-positive peptic diseases. They received combination therapy (proton pump inhibitor + amoxicillin: n = 17, proton pump inhibitor + amoxicillin + clarithromycin: n = 61). In the 68 patients in whom HP was eradicated, HP Ab, PG I, and PG II decreased and PG I/II increased significantly after eradication. Especially, the decrease in PG II and the increase in PG I/II were rapid and remarkable, found 2 months after the beginning of eradication therapy, and then continued. On the other hand, in the patients in whom HP was not eradicated, HP Ab and PG I/II did not change significantly, while PG I and PG II temporarily increased at the end of administration of proton pump inhibitor. In conclusion, it seems that the measurement of PG II and PG I/II is useful for the early detection of HP eradication.

Hyponatremia with consciousness disturbance caused by omeprazole administration. A case report and literature review.

A 68-year-old man developed severe consciousness disturbance after daily administration of 20 mg omeprazole for four days for the treatment of bleeding gastric ulcer. Systemic investigation revealed severe hyponatremia (111 meq/liter). Consciousness did not become clear until his sodium intake was increased to 480 meq/day, and his serum sodium concentrations reached 130 meq/liter. After the discontinuation of omeprazole, his serum sodium levels returned to the normal range with only minimum supplementation of sodium in the form of dietary sodium chloride intake of 10 g/day. Although the mechanism of hyponatremia induced by omeprazole is not clear, an excessive loss of urinary sodium appears to be more likely than water retention with an increase in fluid intake. The literature was also reviewed.

E2011 a novel, selective and reversible inhibitor of monoamine oxidase type A.

E2011, (5R)-3-[2-((1S)-3-cyano-1hydroxypropyl)benzothiazol- 6-yl]-5-methoxymethyl-2-oxazolidine, is a novel inhibitor of monoamine oxidase type A (MAO-A). We have characterized the neurochemical and pharmacological profiles of E2011 and compared them with those of known inhibitors of MAO-A. E2011 potently inhibited MAO-A with more than 30,000 times higher selectivity for MAO-A relative to MAO-B in rat brain homogenate. E2011 did not affect putative neural receptors or reuptake of biogenic amines into synaptosomes of rat brain, which suggests that it is specific to monoaminergic systems. In vivo, E2011 at a dose of 0.3 mg/kg p.o. exhibited potent MAO-A inhibitory activity, whereas MAO-B inhibition was not observed even at 100 mg/kg p.o. E2011 inhibited monoamine metabolism in the rat brain, but the effect disappeared 24 h after administration. Like other reversible MAO-A inhibitors, E2011 did not show a cumulative inhibitory effect during repeated administration for 7 days. However, inhibition of MAO-A by E2011 in ex vivo experiments appeared to be less potent than that by moclobemide. The MAO-A inhibition by E2011 was partially but significantly reversed by dialysis at 4 degrees C for 24 h, which indicates that E2011 could be dissociated from the enzyme. These findings suggest that E2011 is a reversible and highly selective inhibitor of MAO-A. The potency of inhibition by highly reversible MAO-A inhibitors such as E2011 is likely to be underestimated in ex vivo studies because of dilution of the homogenate in the assay system.

Dopamine D3 agonists disrupt social behavior in rats.

Behavioral studies were conducted in rats administered a selective D3 agonist, 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) or 4aR, 10bR-(+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1] benzopyrano[4,3-b]-1,4-oxazin-9-ol (PD 128907). Both drugs induced disruption of huddling behavior in rats at doses that did not produce hyperlocomotion. The effects of the D3 agonists were dependent upon dosage and time after administration. These results suggest that D3 receptors are concerned with social interaction in rats.

Involvement of P-type calcium channels in high potassium-elicited release of neurotransmitters from rat brain slices.

Several types of voltage-dependent calcium channels appear to occur in neurons, although coupling of the particular subtype of calcium channels to the release of neurotransmitter has not been clearly understood. We have examined the effects of subtype-specific inhibitors of the calcium channels on depolarization-induced release of endogenous neurotransmitters from brain slices. High potassium-induced release of glutamate and aspartate from hippocampal and striatal slices was almost completely inhibited by a P-type channel blocker, omega-agatoxin IVA. omega-Agatoxin IVA also completely inhibited the release of serotonin from the hippocampal slices with almost the same potency as in the case of glutamate, whereas the potency in blocking the release of serotonin and dopamine from striatal slices was lower than that from the hippocampal slices. Another calcium channel blocker, omega-agatoxin TK, that was recently found to block P-type channels with very similar selectivity and potency to omega-agatoxin IVA, also inhibited the release of amino acid transmitters and monoamines, though its potency was lower than that of omega-agatoxin IVA. An N-type channel blocker, omega-conotoxin GVIA, partially inhibited the neurotransmitter release, but an L-type channel blocker, nifedipine was ineffective. We propose that the activation of P-type calcium channels makes a major contribution to depolarization-elicited neurotransmitter release in the CNS and that multiple P-type channels sensitive to omega-agatoxin IVA and omega-agatoxin TK modulate the neurotransmitter release.

Application of pharmacokinetic studies to a novel antidepressant, E2011.

1. The original drug tested here, (5R)-3-[2-(3-cyanopropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxaz olidinone (ER-4539), exhibited strong MAO-A inhibitory activity in vitro, but its bioavailability in rat was very low. After ER-4539 was administered orally to dog, a metabolite was found in plasma. 2. The metabolite was isolated by hplc after incubation with dog liver microsomal preparations. Its structure, determined by ms and nmr analysis, was alpha-hydroxy-ER-4539. The configuration of the alpha-hydroxy metabolite was (S), determined in comparison with the authentic sample of (R) and (S) by hplc. The isolated metabolite had potent MAO-A inhibitory action in vitro, indicating that it would have antidepressant action. 3. (5R)-3-[2-((1S)-3-Cyano-1-hydroxypropyl)benzothiazol-6-yl]-5- methoxymethyl-2-oxazolidinone (E2011), the synthesized metabolite, has been improved in regard to biopharmaceutical characteristics in rat and dog.

[Ulcerative colitis complicated by early carcinoma of the rectum and cholecystolithiasis].

Reported is a case of a 73-year-old male with a history of ulcerative colitis that had started at the age of 57. In 1985, on receiving a barium enema, a polypoid lesion was found in his rectum. In 1986, the results of a colonoscopy showed that the polypoid lesion had reached an IIa-aggregated elevation, and biopsies of this lesion were diagnosed as an adenoma or a hyperplastic polyp. A year later, in 1987, another biopsy specimen was taken and was histologically diagnosed as being an adenomatous cancer. Thus, a pull-through operation and a cholecystectomy were performed. The lesion was 4.5 x 3.0 cm in diameter, and the histological findings showed it to be a well-differentiated adenocarcinoma with a submucosal invasion. Accordingly, physicians should be advised that patients with a longstanding ulcerative colitis ought to undergo periodic examination that includes a colonoscopy and a biopsy of any suspicious growth.

Effects of caffeine on gluconeogenesis and urea synthesis induced by alpha-adrenergic stimulation in suspensions of rat hepatocytes.

Effects of caffeine on gluconeogenesis and urea synthesis of rat isolated hepatocytes were investigated in the presence of hormonal agonists. Phenylephrine at 10 microM stimulated 1.7-fold gluconeogenesis and 1.9-fold (compared to control) urea synthesis from 4 mM glutamine. Stimulative effects of caffeine in the range from 0.1 to 10 mM were biphasic depending on its concentration, and it showed maxima at about 1 mM. Caffeine at 1 mM stimulated 2.1-fold gluconeogenesis and 2.4-fold urea synthesis. Caffeine without phenylephrine did not stimulate both syntheses. These effects of caffeine and phenylephrine diminished in the absence of extracellular Ca2+. Results on uptake of 45Ca2+ into hepatocytes and change in quin-2 fluorescence indicated that phenylephrine induced Ca2+ influx into the cell and consequently increased the intracellular Ca2+ concentration, [Ca2+], and that the addition of caffeine did not further stimulate the effect of phenylephrine on [Ca2+]. Therefore, we suggest that stimulation of gluconeogenesis and urea synthesis by phenylephrine is due to increase in [Ca2+]. Since caffeine is known to inhibit phosphodiesterase, the additional stimulation of both syntheses by caffeine plus phenylephrine may be due to the synergistic effect of increases in cAMP and [Ca2+]. The increase in the rates of gluconeogenesis and urea synthesis similarly depended on the caffeine concentration. Furthermore, the ratio of [acetoacetate]/[3-OH-butyrate] which shows intramitochondrial redox state, also depended on the caffeine concentration, indicating a possible coupling of the redox function of mitochondria with [Ca2+].

Studies on complications of diverticular disease of the colon.

Out of 1,124 cases of diverticular disease of the colon seen during the past 15 years, 27 cases (2.4%) of diverticulitis and 44 cases (3.9%) of diverticular hemorrhage were found. The incidence of diverticulitis was more frequent in the both-sides colon type, and also in the multiple form having 10 or more diverticula. The average age was higher in diverticulitis of the sigmoid colon (57.8 years) than in diverticulitis of the right-side colon (47.9 years). Twenty-two cases (81%) of these patients were recovered by medical treatment, and in 5 cases (19%) of these, elective colectomy was carried out. On the other hand, the incidence of diverticular hemorrhage was more frequent in patients over 70 years old, and also in the multiple form having 10 or more diverticula. Though anemia was seen in 11 (25%) of 44 cases, all patients were recovered by medical management. Namely, diverticulitis of the right-side colon is more frequent in middle age, and both diverticulitis of the sigmoid colon and diverticular hemorrhage are more frequent in old age.

[The effect of oral synthetic protease inhibitor (FOY 305) on endocrine pancreas and carbohydrate and lipid metabolism in normal and streptozotocin-induced diabetic rats].

The effect of long-term oral synthetic protease inhibitor (FOY 305) administration on fasting blood sugar (FBS), body weight, glucose tolerance, plasma insulin and glucagon levels, pancreatic insulin and glucagon contents, hepatic enzyme activities, and plasma lipids in normal and streptozotocin (STZ)-induced diabetic rats was studied. Normal rats treated with oral FOY 305 for 9 weeks were found to have pancreatic hypertrophy and decreased body weight gain as compared with the untreated normal controls. FBS, glucose tolerance, plasma insulin and glucagon levels, pancreatic insulin and glucagon contents, and plasma lipids were uninfluenced in FOY 305 treated normal rats. STZ-induced diabetic rats treated with oral FOY 305 were found to have decreased FBS for 5 weeks after the beginning of FOY 305 administration as compared with the untreated diabetic controls, whereas at the 7th and 9th week after treatment there was no difference in FBS between FOY 305 treated and untreated diabetic rats. In the metabolic balance observed at the 4th week after treatment, a slight improvement of the diabetic state was found in FOY 305 treated diabetic rats. There was no apparent difference in the blood sugar curve and insulin response following oral glucose load between diabetic rats treated for 7 weeks and untreated diabetic rats. All the rats were sacrificed after 9 weeks of treatment. Diabetic rats treated with oral FOY 305 for 9 weeks showed pancreatic hypertrophy and decreased plasma glucagon level and decreased pancreatic glucagon content as compared with the untreated diabetic controls, whereas there was no difference in body weight, plasma insulin level and pancreatic insulin content between FOY 305 treated and untreated diabetic rats. Furthermore, oral FOY 305 treatment improved hyperlipidemia in STZ-induced diabetic rats and also significantly improved the hepatic pyruvate kinase and phosphoenlpyruvate carboxykinase activities of diabetic rats. These improvements might partly be due to a decreased pancreatic content and secretion of glucagon and/or a direct action of the synthetic PI, FOY 305 to tissues.