The effects of thalidomide on chemotactic migration of multiple myeloma cell lines.

We examined the effect of thalidomide and dexamethasone on the migration of multiple myeloma (MM) cell lines, U266, RPMI8226, and NCI-H929, using chemotaxis chamber plates. U266 underwent chemotactic migration in response to stromal-cell derived factor-1 alpha (SDF-1alpha), and other cell lines underwent random migration in response to SDF-1alpha or monocyte chemotactic protein-1 alpha. Following preincubation with 1 mug/ml thalidomide, the cell lines showed reduced migratory capacity in response to SDF-1alpha. Concerning the corresponding receptors, CXC chemokine receptor 4 was detected only on the surface of U266, by flow cytometry, whereas chemokine (C-C motif) receptor 2 was not detected on all three cell lines. Moreover, decreased migration by thalidomide was not accompanied by altered expression of the corresponding receptors of each cell line. This is the first report to show the effects of thalidomide on the migration of MM cell lines. The results suggest that the inhibition of chemotactic migration might be one of the mechanisms of the success of thalidomide in controlling MM.

Involvement of p38 MAP kinase in TGF-beta-stimulated VEGF synthesis in aortic smooth muscle cells.

Although it is known that transforming growth factor (TGF)-beta induces vascular endothelial growth factor (VEGF) synthesis in vascular smooth muscle cells, the underlying mechanisms are still poorly understood. In the present study, we examined whether the mitogen-activated protein (MAP) kinase superfamily is involved in TGF-beta-stimulated VEGF synthesis in aortic smooth muscle A10 cells. TGF-beta stimulated the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase, but not that of SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). The VEGF synthesis induced by TGF-beta was not affected by PD98059 or U0126, specific inhibitors of the upstream kinase that activates p42/p44 MAP kinase. We confirmed that PD98059 or U0126 did actually suppress the phosphorylation of p42/p44 MAP kinase by TGF-beta in our preparations. PD169316 and SB203580, specific inhibitors of p38 MAP kinase, significantly reduced the TGF-beta-stimulated synthesis of VEGF (each in a dose-dependent manner). PD169316 or SB203580 attenuated the TGF-beta-induced phosphorylation of p38 MAP kinase. These results strongly suggest that p38 MAP kinase plays a part in the pathway by which TGF-beta stimulates the synthesis of VEGF in aortic smooth muscle cells.

Trehalose suppresses lipopolysaccharide-induced osteoclastogenesis bone marrow in mice.

We have previously shown that trehalose suppresses bone loss in ovariectomized (OVX) mice by way of inhibiting osteoclast differentiation in bone marrow. Also, trehalose inhibits the secretion of interleukin-6 in bone marrow cell cultures, resulting in a decrease in osteoclast formation. In this study, we examined the effect of trehalose on osteoclastogenesis using another model of bone resorption, namely lipopolysaccharide (LPS)-stimulated osteoclast induction. Mice were given trehalose (1g/kg) by gastric intubation for 5 consecutive days, and 24 hours later, 14 mg/kg of LPS was injected intraperitoneally. Trehalose significantly suppressed LPS-induced tumor necrosis factor (TNF)-alpha production after 90 min and decreased the number of osteoclasts in the bone marrow 48 hours after LPS injection. These results indicate that trehalose suppresses excessive osteoclastogenesis not only in OVX mice but also in a LPS-induced bone resorption mouse model and further suggest that the latter finding may be mediated at least in part through a decrease in TNF-alpha production.

Magnitude of the regression to the mean within one-year intra-individual changes in serum lipid levels among Japanese male workers.

To investigate the magnitude of the 'regression to the mean' effect for the changes in serum total cholesterol (TC), triglycerides (TG), and high density lipoprotein cholesterol (HDLC) levels during one-year interval between annual health check-ups in occupational settings, the relationships between the baseline level and subsequent one-year change in TC, TG or HDLC were analyzed using paired health check-up data in 1998 and 1999 of 547 Japanese male clerical workers. After adjustment for age, body mass index (BMI), yearly change in BMI, drinking score and smoking score by the multivariate analyses, the yearly changes in each serum lipid (deltaTC, deltaLn(TG) or deltaHDLC) were clearly inversely associated with the lipid levels in 1998. For example, in the multiple linear regression analyses setting delta value in each serum lipid as a dependent variable, the partial regression coefficients for the baseline lipid levels (beta1) were - 0.21 (p<0.001) for the TC, -0.39 (p<0.001) for the Ln(TG) and -0.15 (p<0.001) for the HDLC, respectively. These results suggest that the observed yearly change in each serum lipid level may largely reflect the 'regression to the mean' effect in addition to the real yearly biological change.

Enhancement by sphingosine 1-phosphate in vasopressin-induced phosphoinositide hydrolysis in aortic smooth-muscle cells: involvement of p38 MAP kinase.

We previously reported that sphingosine 1-phosphate (S-1-P), a sphingomyelin metabolite, activates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in aortic smooth-muscle A10 cells. In the present study, we investigated the effect of sphingomyelin metabolites on phospholipase C-catalyzing phosphoinositide hydrolysis induced by arginine vasopressin (AVP) in A10 cells. C(2)-ceramide and sphingosine had little effect on inositol phosphate (IP) formation stimulated by AVP. S-1-P, which alone slightly stimulated the IPs formation, dose-dependently amplified the AVP-induced formation of IPs. Tumor necrosis factor-alpha enhanced the AVP-induced formation of IPs. However, S-1-P did not enhance the formation of IPs by NaF, a heterotrimeric GTP-binding protein activator. Pertussis toxin inhibited the effect of S-1-P. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the enhancement by S-1-P. SB203580, an inhibitor of p38 MAP kinase, suppressed the effect of S-1-P on the formation of IPs by AVP. SB203580 inhibited the AVP-induced phosphorylation of p38 MAP kinase. Pertussis toxin suppressed the phosphorylation of p38 MAP kinase by S-1-P. These results indicate that S-1-P amplifies AVP-induced phosphoinositide hydrolysis by phospholipase C through p38 MAP kinase in vascular smooth-muscle cells.

Interleukin-18 in combination with IL-2 enhances natural killer cell activity without inducing large amounts of IFN-gamma in vivo.

Interleukin-18 (IL-18) is known to synergistically enhance murine natural killer (NK) cell activity in vitro when combined with either IL-12 or IL-2. However, it has also been demonstrated that simultaneous administration of IL-18 and IL-12 to mice induces extraordinarily large amounts of interferon-gamma (IFN-gamma) in the serum. In this study, we examined the effects of a combination of IL-18 and IL-2 on in vivo NK cell activation in parallel with the induction of toxicity. In contrast to the IL-18 and IL-12 combination, the combined administration of IL-18 and IL-2 to BALB/c mice for 3 days induced neither high levels of IFN-gamma production nor other visible side effects. When compared with treatment with IL-18 or IL-2 alone, the combined treatment resulted in a significant increase in the number of DX-5 (pan-NK cells marker)-positive cells in spleens and a marked enhancement of splenic NK activity, as determined by standard cytotoxicity assays. Enhanced splenic cytotoxicity generated in the mice treated with both IL-18 and IL-2 was also observed against syngeneic Colon 26 adenocarcinoma cells. Consistent with this in vitro observation, combined treatment produced a significantly stronger inhibitory effect on the pulmonary metastases following i.v. injection of Colon 26 tumor cells than treatment with either cytokine alone. These results suggest that IL-18 combined with IL-2 potentiates in vivo NK cell activity and contributes to inhibition of tumor metastasis without inducing significant toxicity.

A new method for assaying adhesion of cancer cells to the greater omentum and its application for evaluating anti-adhesion activities of chemically synthesized oligosaccharides.

A new ex vivo method for assaying adhesion of cancer cells to the greater omentum has been developed using mouse greater omentum and [3H]labelled human gastric and mouse colorectal cancer cells. Since the adhesion rates were found to increase up to 18 h and labelled cells seemed to be stable during the period, the present method could be useful for investigating adhesion of cancer cells to the greater omentum, which must occur at the first step of the peritoneal dissemination. The adhesion of cancer cells to the greater omentum was inhibited by a series of chemically synthesized oligosaccharides and Gal beta1,3[3OMeGal beta1,4GlcNAc beta1,6]alphaBn was found to be the best inhibitor. The anti-tumor effect of this novel tetrasaccharide in vivo was shown in preliminary experiments using Balb/c mice and colon26 cells.

Molecular mechanisms of expression of Lewis b antigen and other type I Lewis antigens in human colorectal cancer.

Lewis b (Leb) antigens are gradiently expressed from the proximal to the distal colon, i.e., they are abundantly expressed in the proximal colon, but only faintly in the distal colon. In the distal colon, they begin to increase at the adenoma stage of cancer development and then increase with cancer progression. We aimed to clarify the molecular basis of Leb antigen expression in correlation with the expression of other type I Lewis antigens, such as Lewis a (Lea) and sialylated Lewis a (sLea), in colon cancer cells. Considering the Se genotype and the relative activities of the H and Se enzymes, the amounts of Leb antigens were proved to be determined by both the H and Se enzymes in noncancerous and cancerous colon tissues. But the Se enzyme made a much greater contribution to determining the Lebamounts than the H enzyme. In noncancerous colons, the Se enzyme were gradiently expressed in good correlation with the Leb expression, while the H enzyme was constantly expressed throughout the whole colon. In distal colon cancers, the H and Se enzymes were both significantly upregulated in comparison with in adjacent noncancerous tissues. In proximal colon cancers, expression of the H enzyme alone was highly augmented. The augmented expression of Leb antigens in distal colon cancers is caused mainly by upregulation of the Se enzyme and partly by the H enzymes, while it is caused by upregulation of the H enzyme alone in proximal colon cancers. The Se gene dosage profoundly influences the amounts of the Leb, Lea, and sLea antigens in whole colon tissues, regardless of whether they are noncancerous or cancerous tissues. It suggests that the Se enzyme competes with alpha2,3 sialyltransferase(s) and the Le enzyme for the type I acceptor substrates.

[The use of noninvasive positive pressure ventilation in the early postoperative period after cardiovascular surgery].

We used noninvasive positive pressure ventilation (NPPV) in 7 ICU patients after cardiovascular surgery. In 6 patients, we measured the variables of hemodynamics and arterial oxygenation by application of this nasal respiratory support (Companion 320 I/E, Puritan Bennett). Ventilator settings of expiratory positive airway pressure (EPAP) 3 cmH2O and inspiratory positive airway pressure (IPAP) 10 cmH2O were used and continued for 30 minutes. There were no significant changes in any hemodynamic variables during NPPV. Arterial oxygenation also remained unchanged at 30 min after discontinuation of NPPV. To conclude the efficacy of NPPV after cardiovascular surgery, higher level of IPAP and the combination with postural drainage should be studied further.

Allopurinol, an inhibitor of xanthine oxidase, improves the development of IVM/IVF bovine embryos (>4 cell) in vitro under certain culture conditions.

To determine the origin of free oxygen radicals in the culture medium of bovine embryos, the effect of allopurinol, an inhibitor of xanthine oxidase, on the development of embryos (>4 cell) in modified synthetic oviduct fluid (m-SOF) medium was examined. When embryos were cultured in the presence of 0.2 mM allopurinol under high oxygen tension (5% CO2 in air), the blastocyst rate significantly (P<0.05) increased compared with the absence of allopurinol (allopurinol (+) 42 vs. (-) 25%; Day 6, 63 vs. 51%; Day 7, 69 vs. 58%; Day 8). However, allopurinol had no effect on embryo development under low oxygen tension (5% CO2, 5% O2, 90% N2). Moreover, it was found that the developmental rate and the total cell number of blastocysts decreased (development rate: 60 vs. 28%, cell number: 132 vs. 74) when the embryos were cultured in medium containing 0.01 U/mL xanthine oxidase (XOD) and 0.1 mM hypoxanthine (HXT), and the damaging effect of XOD and HXT was removed by the addition of 0.2 mM allopurinol. The beneficial effect of allopurinol was also observed when the glucose concentration was increased to 4.5 mM from 1.5 mM (control: 22% vs. allopurinol: 34%; Day 8), but no beneficial effects were observed in the media without glucose (control: 55% vs. allopurinol: 59%). Taken together, these results suggested that a portion of the free oxygen radicals are generated from the XOD and HXT reactions under culture conditions, and this generation is enhanced by high oxygen tension in the gas atmosphere or by high glucose concentrations in the medium.

[Hemorrhagic shock during laparoscopic cholecystectomy detected by transesophageal echocardiography].

A 47-year-old man was scheduled for laparoscopic cholecystectomy under general anesthesia supplemented with epidural anesthesia. A direct arterial line and a transesophageal echocardiogram probe were inserted before surgery. Anesthesia was maintained with nitrous oxide and isoflurane but without epidural anesthesia. Severe hypotension occurred about 30 minutes after introducing pneumoperitoneum but surgeons denied massive bleeding in the operative field. Although this made us difficult to diagnose the incident as massive bleeding or pulmonary air embolism (PAE), a collapsed heart was detected by transesophageal echocardiography (TEE). Its end-diastolic diameter of the left ventricle was reduced to 20 mm and left ventricular end-systolic cavity obliteration was demonstrated. We could easily diagnose the decrease of blood volume due to PAE using TEE.

[Difficult tracheal intubation with von Recklinghausen disease].

We experienced a case of difficult tracheal intubation in a 15-year-old boy with von Recklinghausen disease scheduled for resection of a right neck tumor. His scoliosis made it difficult to intubate and to manage airway because he easily developed dyspnea. We tried nasotracheal intubation with the patient awake under sedation using a bronchofiberscope, but we found an unexpected tumor jeopardizing his airway patency near his vocal cord. Preoperative examination of a tumor in the airway is essential in the anesthetic management of the patients with von Recklinghausen disease.

Altered mRNA expression of specific molecular species of fucosyl- and sialyl-transferases in human colorectal cancer tissues.

Human colorectal cancers express various cancer-associated carbohydrate determinants such as Lewis Y or sialyl Lewis A, suggesting a considerable alteration in glycosyltransferase activities occurring upon malignant transformation. We investigated the mRNA amounts of fucosyltransferase (Fuc-T) and sialyltransferase (ST) isoenzymes, including Fuc-T III, IV, V, VI and VII and ST-3N, ST-30 and ST-4, in human colorectal cancer tissues by Northern blotting and RT-PCR. Regarding fucosyltransferases, mRNA of Fuc-T III and VI was not significantly altered, and only Fuc-T IV mRNA showed a moderate increase in cancer tissues when compared with adjacent non-malignant colonic epithelia taken from the same patient (273 +/- 96%; p < 0.001). The moderate increase of Fuc-T IV message may be related to an enhanced expression of Lewis Y in colon cancer tissues. In the ST isoenzymes, mRNA for ST-3N remained unchanged, whereas that for ST-4 decreased significantly in cancer tissues, to 32 +/- 29%, (p < 0.005). The most remarkable finding was that the message of ST-30 was prominently increased in cancer tissues compared with non-malignant colorectal mucosa. When further investigated by quantitative RT-PCR assays on a larger series of patients with colorectal cancers, the average increase in mRNA for ST-30 was 459 +/- 200% compared with that in adjacent non-malignant epithelium (significant at p < 0.0001). The increase of ST-30 message was more prominent in the cancer tissues strongly expressing sialyl Lewis A than in the cancer tissues expressing sialyl Lewis A only weakly or moderately (significant at p < 0.05). The marked increase in the message of ST-30 is suggested to be related to an enhanced expression of sialylated carbohydrate determinants in colon cancer tissues including sialyl Lewis A, since the enzyme exhibited a significant activity against the type 1 chain carbohydrate substrate and produced the precursors for sialyl Lewis A synthesis, when its cDNA was expressed in Cos-7 cells.

The usefulness of anti-fucosylated antigen antibody YB-2 for diagnosis of hepatocellular carcinoma.

Levels of fucosylated antigens in sera from patients with liver diseases were examined by a newly developed sandwich-type enzyme immuno assay with the aid of anti-fucosylated antigen antibody, YB-2 which reacts simultaneously with Y, Leb and H type 2 antigens. When the cut-off value was set arbitrarily at mean +3 SD values of normal, 30 (69.8%) of the 43 patients with HCC, 14 (53.8%) of the 26 patients with liver cirrhosis (LC) and 24 (45.3%) of the 53 patients with chronic hepatitis (CH) were found to be positive, whereas all of the 30 samples from healthy controls were negative. The levels of alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKA-II) in HCC were not correlated with those of YB-2 antigens. The positive rates of the combination YB-2 and AFP assay and YB-2 and PIVKA-II assay in HCC were significantly higher (83.7 and 86.0%, respectively) than that of the AFP and PIVKA-II combination (65.1%) which had been reported to be the best combination up to this time.

Elevation of an alpha(1,3)fucosyltransferase activity correlated with apoptosis in the human colon adenocarcinoma cell line, HT-29.

We studied changes in the carbohydrate expression following apoptotic cell death induced by treatment with interferon (IFN)-gamma and anti-Fas antibody using human colon adenocarcinoma HT-29 cells. An apoptotic cell death of HT-29 accompanied with typical DNA fragmentation was observed when the cells, were cultured sequentially with IFN-gamma and anti-Fas antibody. In flow cytometric analyses, the expression of Le(x) and Le(y) antigen was strongly and slightly enhanced, respectively, on the cell surface in accordance with the apoptosis. When the fucosyltransferase (Fuc-T) activities of the lysates from the treated cells were examined relative to those from untreated cells, a 2.5-fold increase of alpha(1,3)-Fuc-T activities and a slight increase of alpha(1,2)-Fuc-T activities were observed, but little or no increase of alpha(1,4)-Fuc-T activity was detected. In Northern blot analyses using probes for Fuc-T III, IV, V, VI and VII genes, strong RNA messages for Fuc-T III, V and/or VI and a weak RNA message for Fuc-T IV were detected in the untreated HT-29 cells. On the other hand, in the treated cells, the messages for Fuc-T III, V and/or VI were found to almost disappear and the 2.3 kb message for Fuc-T IV was observed to elevate 2.8-fold. Therefore, we suggest that the strongly increased expression of Le(x) antigen found on the HT-29 cell surface might be involved in the process of apoptosis, and that the enhancement of the antigen expression seems to result from the increased activity of alpha(1,3)-Fuc-T encoded mainly by the Fuc-T IV gene.

[Prostaglandin E1 at low-doses improved right ventricular ejection fraction in anesthetic management for CABG].

A 52-year-old male for CABG developed a severe right heart failure, because of the direct injury to the right ventricular wall, after cardiopulmonary bypass. The volume loading therapy could not improve the cardiac function, then we used an infusion of low-dose prostaglandin E1 (0.02-0.04 micrograms.kg-1.min-1) for the acute right heart failure with increased pulmonary vascular resistance. After continuous infusion of this dose, the pulmonary vascular resistance decreased quickly, the right ventricular ejection fraction increased, and the stroke volume index also improved. These hemodynamic changes are the result of the potent vasodilating effect of PGE1, that especially could decrease selectively the pulmonary vascular resistance, and increase the preload of the left ventricle. This dose of PGE1, did not cause a severe systemic hypertension that is a serious complication during vasodilating therapy with any vasoactive drugs. In the present case, we speculated that the low-dose PGE1, is effective to improve the right ventricular function during the acute right heart failure which resulted from the intrinsic right ventricular dysfunction.

[Anesthetic management of a patient with renal cell carcinoma extending into the right atrium].

A 60-year-old man with renal cell carcinoma extending through inferior vena cava into the right atrium was scheduled for the removal of the right kidney under general anesthesia and the cardiopulmonary bypass technique. In order to obtain a clear operative field and to minimize the risk for pulmonary embolism of necrotizing tumor, total circulatory arrest under profound hypothermia (20 degrees C) was performed. Anesthesia was maintained with high doses of fentanyl (62 micrograms.kg-1), midazolam and supplemented with enflurane. We attempted to prevent circulatory collapse due to acute pulmonary embolism by tumor fragments during operation. The body temperature of the patient was decreased down to 20 degrees C for protecting central nervous system with the minimal damage. No complications occurred during anesthesia and the post-operative period. For the safe anesthetic management of the patient such as our case, adequate monitoring of circulation and protection of central nervous system are essential.

[Perioperative managements of a mentally retarded child with obstructive sleep apnea syndrome for adeno-tonsillectomy].

We describe a mentally retarded child with obstructive sleep apnea syndrome (OSAS) in whom it was difficult to maintain upper airway in the perioperative period. The child underwent awake intubation, because the preanesthetic evaluation of the airway with a direct fiberoptic visualization revealed a very narrow airway. Also we considered that if we used an anesthetic agent, a perioperative airway management would be very troublesome. Postoperatively we continuously monitored for apnea and arrhythmias. When the child was sleeping, we found frequently that her thoratic movements were getting weak and percutaneous oxygen saturation went down to about 70 percent. The preoperative direct fiberoptic visualization of the upper airway is effective for the evaluation of the degree of airway obstruction in this child. We also recommend the continuous intensive postoperative monitorings including pulse oximetry, ECG, and apnea monitor which are very important to avoid life-threatening complications such as upper airway obstruction and serious arrhythmias in patients with obstructive sleep apnea syndrome.

Alpha 2-->3sialyltransferase associated with the synthesis of CA 19-9 in colorectal tumors.

BACKGROUND: A novel assay method specific for alpha 2-->3sialyltransferase that seems to be responsible for the synthesis of CA 19-9 antigen was developed and the levels of the enzyme in colorectal tumor tissues were measured and compared with the levels of alpha 1-->4fucosyltransferase and the CA 19-9 antigen. METHODS: Lacto-N-biose I (Gal beta 1-->3GlcNAc beta, Lewis(c) [Le(c)]) and 6-O-methyl-Le(c) (Gal beta 1-->3[6OMe]GlcNAc beta) were synthesized and covalently attached to bovine serum albumin (BSA). These two substrates were incubated with extracts from colorectal tissues in the presence of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) and their resulting products were detected by a sequential use of anti-BSA monoclonal antibody-coated beads and 125I-labelled anti-sialylated Le(c) antibody. Levels of alpha 2-->3sialyltransferase and alpha 1-->4fucosyltranferase activities and CA 19-9 antigen were measured in the extracts from colorectal tumors and their adjacent normal tissues. RESULTS: 6-O-Methyl-Le(c)-BSA showed a strong acceptor activity compared with Le(c)-BSA and was used as a specific acceptor for alpha 2-->3sialyltransferase. Similar elevation patterns alpha 2-->3sialyltransferase activities and CA 19-9 antigen levels were observed in tumor extracts but no clear correlation was present between the level of alpha 1-->4fucosyltransferase activities and CA 19-9 antigen levels were observed in tumor extracts but no clear correlation was present between the level of alpha 1-->4fucosyltransferase activities and CA 19-9 antigen levels in the same extracts. CONCLUSIONS: The accumulation of CA 19-9 antigen in colorectal tumors might be caused mainly by the activation of alpha 2-->3sialyltransferase but not by that of alpha 1-->fucosyltransferase.