Detailed analysis of clinical test data on chemotherapy for colorectal cancer.

We reported previously that spline interpolation is effective as a pretreatment before analyzing clinical data by time series. However, further improvement is required to understand the detailed tendency of clinical data. In this study, the tendency of interpolated hematological data was investigated in the period between the most tolerated dose (MTD) and low-dose chemotherapy (LDC) for colorectal cancer. All patients were received both MTD and LDC. Hematological data, white blood cell count (WBC), red blood cell count (RBC) and mean corpuscular volume (MCV), were interpolated. The accuracy of interpolation was verified using leave-one-out cross-validation. The difference, Δ(i), was calculated from interpolated data and exhibited as a function of time. The predictions of RBC and MCV were accurate with high correlation coefficients, although the interpolation of WBC data was inaccurate. A marked difference was observed in the trend of Δ(i) between LDC and MTD periods. SD-RBC showed significant differences between LDC and MTD periods. The SD-MCV average in the LDC period was larger than in the MTD period. SD-MCV showed no significant difference. An attractor plot of Δ(i) in RBC clarified the tendency of the interpolated RBC data. There is a possibility that Δ(i) of RBC and/or SD-RBC may contribute to monitoring adverse reactions and decision of medication. Moreover, it is also useful to check on attractor plot of Δ(i) in RBC together with SD-RBC in order to find out untoward reactions and decision of medication.

[Five adult cases of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis].

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is more common in children, and is characterized by pancytopenia, liver dysfunction and coagulopathy caused by interactions between EBV-infected T cells and activated macrophages. We describe here five adults with EBV-HLH. The median age was 17 years (range 16 approximately 40). HLH developed in 4 patients within 2 months after the primary infection, and in the other one during the reactivation. All patients had a high EBV viral load in peripheral blood (2 x 10(2)-3 x 10(6) copies/ml) and monoclonal proliferation of EBV-infected T cells. All patients received immunosuppressive therapy with or without etoposide, and two patients required plasmapheresis due to the severity. Three patients are alive in complete remission (follow up periods; 13, 19, 30 months), while two patients became refractory to chemo-immunotherapy and died despite multidrug chemotherapy. EBV-HLH should be more widely recognized in adults in order to achieve early diagnosis and appropriate treatment.

Oleic acid-induced ADRP expression requires both AP-1 and PPAR response elements, and is reduced by Pycnogenol through mRNA degradation in NMuLi liver cells.

Fatty acids stimulate lipid accumulation in parallel with increased expression of adipose differentiation-related protein (ADRP) in liver cells. Although it is generally considered that the fatty acid effect on ADRP expression is mediated by peroxisome proliferator-activated receptors (PPARs), we identified here an additional molecular mechanism using the NMuLi mouse liver nonparenchymal cell line, which expresses PPARgamma and delta but not alpha. Oleic acid (OA) and specific ligands for PPARgamma and -delta stimulated ADRP expression as well as the -2,090-bp ADRP promoter activity which encompasses the PPAR response element (PPRE) adjacent to an Ets/activator protein (AP)-1 site. When the AP-1 site was mutated, OA failed to stimulate the activity despite the presence of the PPRE, whereas ligands for PPARgamma and -delta did stimulate it and so did a PPARalpha ligand under the coexpression of PPARalpha. DNA binding of AP-1 was stimulated by OA but not by PPAR ligands. Because we previously demonstrated that Pycnogenol (PYC), a French maritime pine bark extract, suppressed ADRP expression in macrophages partly by suppression of AP-1 activity, we tested the effect of PYC on NMuLi cells. PYC reduced the OA-induced ADRP expression along with suppression of lipid droplet formation. However, PYC neither suppressed the OA-stimulated ADRP promoter activity nor DNA binding of AP-1 but, instead, reduced the ADRP mRNA half-life. All these results indicate that the effect of OA on ADRP expression requires AP-1 as well as PPRE, and PYC suppresses the ADRP expression in part by facilitating mRNA degradation. PYC, a widely used dietary supplement, could be beneficial for the prevention of excessive lipid accumulation such as hepatic steatosis.

Adult patient with Epstein-Barr virus (EBV)-associated lymphoproliferative disorder: chronic active EBV infection or de novo extranodal natural killer (NK)/T-cell lymphoma, nasal type?

Chronic active Epstein-Barr virus (EBV) infection, which is considered to be a childhood disease, often develops into natural killer (NK) or T-cell lymphoma after recurrent infectious mononucleosis (IM)-like symptoms. We describe a 56-year-old woman who developed NK-cell lymphoma after 9 months of recurrent IM-like symptoms. The patient had an unusual anti-EBV antibody profile. Increased levels of EBV genome were detected in the liver and peripheral blood. Several chemotherapy regimens were ineffective, and the patient died of progression of lymphoma. Certain subtypes of NK-cell lymphoma showing a long-lasting prodrome related to chronic EBV infection may exist.

Sixty percent of patients with rheumatoid arthritis in Japan have used dietary supplements or health foods.

We conducted a survey on the use of dietary supplements and health foods (DS/HF) in definite rheumatoid arthritis (RA) patients treated by RA specialists. Among 296 patients (male 48, female 248), 179 patients (60.5%) had experience of DS/HF use. Prevalence of DS/HF use was significantly higher in female than in male patients (63.7% versus 43.8%). Overall, patients who have used DS/HF were significantly younger than those who have not used; it was particularly notable in female patients. The proportion of current users was significantly higher in those less than 5 years from diagnosis than those who had been diagnosed for 5 years or more. Products of herbs or algae (44.1%) and components of cartilage (40.8%) were the most popular DS/HF. Primary sources of product information were family members or friends (56.4%) and advertisements in the mass media (34.1%). Of the users, 73.7% did not disclose DS/HF use to their physicians. The users expected alleviation of the symptoms (35.2%) and improvement of health (34.6%). However, 59.2% of the users were unsure of the benefits. In conclusion, physicians should be aware of the high prevalence of DS/HF usage in patients with RA in Japan.

[Two young adult cases of Epstein-Barr virus associated-hemophagocytic lymphohistiocytosis with monoclonal proliferation of virus-infected cells within a short period after primary infection].

Hemophagocytic lymphohistiocytosis (HLH) is a rare but severe complication of Epstein-Barr virus (EBV) infection. Interactions between EBV-infected T cells and activated macrophages cause several conditions such as pancytopenia, liver dysfunction and coagulopathy. We describe here two young adults with EBV-associated HLH with monoclonal proliferation of EBV-infected T cells within a short period after infectious mononucleosis as a primary infection. One patient was a 16-year-old man who developed severe pancytopenia and liver dysfunction two months after infectious mononucleosis. Bone marrow examination showed hemophagocytosis, and laboratory data demonstrated monoclonal proliferation of EBV-infected T cells. Several treatments such as immunosuppressive therapy, chemotherapy and hematopoietic stem cell transplantation were not effective, and the patient died of progressive disease. The other patient was a 19-year-old woman who developed thrombocytopenia and liver dysfunction two months after infectious mononucleosis. Findings of hemophagocytosis and monoclonal proliferation of EBV-infected T cells were similar to those in the first case. Clinical signs and symptoms were resolved completely by immunosuppressive therapy containing methyl-prednisolone and cyclosporine. Since these two cases each demonstrated a distinct clinical course, an investigation of the prognostic factors and treatment strategies for EBV-HLH is warranted.

Pycnogenol, an extract from French maritime pine, suppresses Toll-like receptor 4-mediated expression of adipose differentiation-related protein in macrophages.

Adipose differentiation-related protein (ADRP) is highly expressed in macrophages and human atherosclerotic lesions. We demonstrated that Toll-like receptor (TLR) 4-mediated signals, which are involved in atherosclerosis formation, enhanced the expression of ADRP in macrophages. Lipopolysaccharide (LPS) enhanced the ADRP expression in RAW264.7 cells or peritoneal macrophages from wild-type mice, but not in macrophages from TLR4-deficient mice. Actinomycin D almost completely abolished the LPS effect, whereas cycloheximide decreased the expression at 12 h, indicating that the LPS-induced ADRP expression was stimulated at the transcriptional level and was also mediated by new protein synthesis. LPS enhanced the ADRP promoter activity, in part, by stimulating activator protein (AP)-1 binding to the Ets/AP-1 element. In addition, preceding the increase of the ADRP mRNA, LPS induced the expression of interleukin (IL)-6, IL-1alpha, and interferon-beta mRNAs, all of which stimulated the ADRP expression. Antibodies against these cytokines or inhibitors of c-Jun NH(2)-terminal kinase and nuclear factor (NF)-kappaB suppressed the ADRP mRNA level. Thus TLR4 signals stimulate the ADRP expression both in direct and indirect manners. Pycnogenol (PYC), an extract of French maritime pine, suppressed the expression of ADRP and the above-mentioned cytokines. PYC suppressed the ADRP promoter activity and enhancer activity of AP-1 and NF-kappaB, whereas it did not affect the LPS-induced DNA binding of these factors. In conclusion, TLR4-mediated signals stimulate the ADRP expression in macrophages while PYC antagonizes this process. PYC, a widely used dietary supplement, might be useful for prevention of atherosclerosis.

Long-term follow-up after nonmyeloablative allogeneic hematopoietic stem cell transplantation for systemic sclerosis.

Systemic sclerosis (SSc) characteristically consists of fibrotic changes in various organs, and immunological abnormality is the main cause of the disease. Although high-dose immunosuppressive therapies with autologous or allogeneic hematopoietic stem cell support can reverse the disease course, they have a high treatment-related mortality. We report the successful use of nonmyeloablative allogeneic hematopoietic stem cell transplantation (HSCT) for SSc. A 40-year-old woman with diffuse scleroderma and interstitial pneumonia underwent allogeneic peripheral blood stem cell transplantation from an HLA-identical sibling after conditioning with low-dose total-body irradiation and fludarabine. Prophylaxis for graft-versus-host disease (GVHD) consisted of cyclosporine and mycophenolate mofetil. No infection or acute GVHD developed. One year after transplantation, the patient developed membranous glomerulopathy caused by chronic GVHD that was successfully treated with prednisolone. The patient's skin score decreased dramatically, and her pulmonary function is stable 4 years after transplantation. Nonmyeloablative allogeneic HSCT may be more effective than conventional therapies for SSc.

[Graves' disease with splenomegaly and pancytopenia, mimicking B-cell lymphoproliferative disease].

A 48-year-old woman was referred to our hospital because of fever and general fatigue. Peripheral blood analysis showed a hemoglobin level of 82 g/l, a white blood cell count of 1.95 x 10(9)/l and a platelet count of 80 x 10(9)/l. There were 9% CD5-positive B-cells in peripheral blood and 35% CD10-positive B-cells in bone marrow. The patient had a high serum soluble interleukin-2 receptor (SIL-2R) level of 5,185 U/ml and splenomegaly. Lymphoproliferative disease was suspected, however monoclonal rearranged band of immunoglobulin heavy chain was not detected. She also showed hyperthyroidism, Graves' disease and then treatment with thiamazole started. However, the treatment was stopped because of agranulocytosis and she received subtotal thyroidectomy. After treatment for hyperthyroidism, serum SIL-2R level decreased to 504 U/ml and pancytopenia gradually improved. Fifteen months postoperatively, the percentage of CD5-positive B-cells in peripheral blood and CD10-positive B-cells in bone marrow decreased to 8% and 2%, respectively. This clinical course suggests that polyclonal B-cell proliferation was caused by hyperthyroidism.

Prevention of the hypercontractile response to thrombin by proteinase-activated receptor-1 antagonist in subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Cerebral vasospasm is one of the major complications of subarachnoid hemorrhage (SAH). Its pathogenesis still remains elusive, and effective therapeutic strategies are yet to be established. We investigated the role of proteinase-activated receptor-1 (PAR1) in the hypercontractile state in SAH. METHODS: Rabbit double hemorrhage model was used as a model of SAH. The contractile response to thrombin and the PAR1 expression were evaluated in the isolated rings of basilar artery. RESULTS: Thrombin exhibited only a minor contractile effect in the control, whereas it induced augmented contractions in SAH. The expression of PAR1 was upregulated in SAH. Intracisternal injection of PAR1 antagonist E5555 prevented the enhancement of the contractile responses to thrombin in SAH. The maximal prevention was obtained with 2 microg/kg weight/injection. The contractile responses to K(+) depolarization or endothelin-1 remained unaffected. The upregulation of PAR1 was also prevented by E5555 (2 microg/kg weight/injection) to a level similar to that seen in the control. Ex vivo treatment with E5555 (1 micromol/L) inhibited the contraction induced by thrombin, whereas it had little effect on the contraction induced by K(+) depolarization or endothelin-1, in the basilar artery of SAH. E5555 also inhibited the [Ca(2+)](i) elevation induced by thrombin, but not trypsin, in cultured smooth muscle cells. CONCLUSIONS: PAR1 plays a critical role in upregulating PAR1 itself, thereby enhancing the contractile response to thrombin in SAH. PAR1 could thus be a therapeutic target. However, the usefulness of PAR1 antagonist remains to be investigated in vivo.

Mortalin is a novel mediator of erythropoietin signaling.

Erythropoietin (EPO) stimulates erythroid growth by enhancing the proliferation, maturation and survival of late-stage erythroid progenitor cells. However, the entire process of EPO stimulation remains undetermined. To further clarify the intracellular mechanisms by which EPO affects the growth of erythroid progenitor cells, we analyzed proteins obtained from purified human erythroid colony-forming cells (ECFCs) cultured with or without EPO, and one of the proteins apparently related with EPO stimuli was identified as mortalin (mthsp70/PBP74/Grp75/mot-2), which is a member of the heat shock protein 70 family of chaperones. The amount of mortalin mRNA in ECFCs increased in an EPO dose-dependent manner, and ECFC growth was dependent on the amount of mortalin. Furthermore, expression of mortalin in ECFCs was suppressed by a phosphatidylinositol 3-kinase inhibitor. Finally, we analyzed gene expression patterns in ECFCs cultured with or without EPO after treatment with mortalin small interfering RNA (siRNA) using a DNA microarray. When ECFCs treated with mortalin siRNA were cultured with EPO, the expression of several genes overlapped with the profile seen in control ECFCs cultured without EPO. Our data suggest that mortalin is involved in the mediation of EPO signaling and plays an important role in stimulating the growth of erythroid progenitor cells.

RhoA/Rho kinase-mediated Ca2+ sensitization in the contraction of human prostate.

AIMS: The contractile mechanisms of prostatic smooth muscle have been extensively investigated at the receptor level. However, the intracellular mechanisms have not yet been fully elucidated, especially in human tissue. In the present study, we examined the functional role of RhoA/Rho kinase (ROCK), one of the major intracellular molecules involved in smooth muscle contraction, in the contraction of the human prostate. METHODS: Ring preparations made of cultured human prostatic stromal cells (CHPSCs) or fresh human prostatic tissue was used for an isometric tension study. Gene transfer using baculovirus vector and alpha-toxin permeabilized preparations were also used. RESULTS: RhoA, ROCK I and ROCK II proteins were all expressed in CHPSCs and fresh human prostatic tissue. In CHPSCs ring preparations, the contraction induced by endothelin (ET)-1 was enhanced by over-expression of RhoA and inhibited by ROCK inhibitor. In alpha-toxin permeabilized preparations, ET-1 or GTP-gammaS induced an additional contraction at a constant [Ca2+]i, that was inhibited by ROCK inhibitor. In fresh human prostatic tissue, norepinephrine (NE)-induced contraction was inhibited by ROCK inhibitor at a constant [Ca2+]i in alpha-toxin permeabilized preparations. CONCLUSIONS: These results suggested that RhoA/ROCK-mediated Ca2+ sensitization is likely involved in the contraction of the human prostate. The antagonisms of this pathway may thus be useful as an alternative target in the treatment of benign prostatic hyperplasia (BPH).

Plasmin induces endothelium-dependent nitric oxide-mediated relaxation in the porcine coronary artery.

OBJECTIVE: Plasmin is a key enzyme in fibrinolysis. We attempted to determine the possible role of plasmin in the regulation of vascular tone, while also investigating the mechanism of plasmin-induced vasorelaxation. METHODS AND RESULTS: In porcine coronary artery, plasmin induced an endothelium-dependent relaxation. This relaxing effect was mostly abolished by a proteinase inhibitor, a plasmin inhibitor, or a nitric oxide (NO) synthase inhibitor. The preceding stimulation with plasmin significantly inhibited the subsequent relaxation induced by thrombin but not that induced by proteinase-activated receptor-1-activating peptide. The relaxation induced by trypsin and substance P remained unaffected by the preceding plasmin stimulation. The pretreatment with plasmin, thrombin, or trypsin significantly attenuated the plasmin-induced relaxation. In porcine coronary artery endothelial cells (PCAECs) and human umbilical vein endothelial cells (HUVECs), plasmin induced a transient elevation in the cytosolic Ca2+ concentrations ([Ca2+]i). The preceding stimulation with plasmin inhibited the subsequent [Ca2+]i elevation induced by thrombin but not that induced by trypsin. In PCAECs, plasmin concentration-dependently induced NO production. CONCLUSIONS: The present study demonstrated, for the first time, that plasmin induced an endothelium-dependent NO-mediated relaxation in the porcine coronary artery, while also showing plasmin to specifically inactivate the thrombin receptor.

Prostaglandin F2alpha, but not latanoprost, increases the Ca2+ sensitivity of the pig iris sphincter muscle.

PURPOSE: To determine the mechanisms underlying prostaglandin (PG) F(2)alpha-(,) carbachol (CCh)-, or latanoprost (a PGF(2)alpha analogue)-induced contraction of the pig iris sphincter muscle. METHODS: Effects of these agents on myofilament Ca(2+) sensitivity were evaluated and compared with the use of receptor-coupled permeabilized preparations by alpha-toxin. The effects of PGF(2)alpha and CCh on the phosphorylation of myosin light chain (MLC) were also analyzed. RESULTS: In the intact strips, all three of these agents induced contractions. In permeabilized strips, PGF(2)alpha and CCh, but not latanoprost, caused an additional tension development at a fixed intracellular Ca(2+) concentration ([Ca(2+)](i)) and also shifted the [Ca(2+)](i)-tension curve to the left, thus indicating that PGF(2)alpha and CCh, but not latanoprost, induced increases in Ca(2+) sensitivity (Ca(2+) sensitization). This Ca(2+) sensitization could have been inhibited by Y27632, a rho kinase inhibitor, but not by GF109203X, a protein kinase C (PKC) inhibitor or by PD98059, a mitogen-activated protein (MAP) kinase inhibitor. PGF(2)alpha increased the level of MLC phosphorylation at a constant [Ca(2+)](i). CONCLUSIONS: PGF(2)alpha, but not latanoprost, induced Ca(2+) sensitization of the pig iris sphincter muscle in an MLC phosphorylation-dependent manner through the rho-rho kinase pathway. The effect of latanoprost on the Ca(2+) sensitization mechanism was different from that of PGF(2)alpha and was thought to play a beneficial role in glaucoma treatment.

Topics on the Na+/Ca2+ exchanger: involvement of Na+/Ca2+ exchanger in the vasodilator-induced vasorelaxation.

Many kinds of vasodilators induce relaxation of the vascular smooth muscle cells (VSMCs) through the production of cyclic AMP (cAMP) or cyclic GMP (cGMP). The relaxant effects mediated by these second messengers are thought to be mainly due to the decrease in intracellular Ca(2+) concentration ([Ca(2+)](i)), as well as the decrease in Ca(2+) sensitivity of the contractile apparatus of VSMCs. To explain the cAMP- or cGMP-mediated decrease in [Ca(2+)](i), several mechanisms have been proposed, including the inhibition of Ca(2+) influx due to a hyperpolarization, a stimulation of Ca(2+) uptake into the intracellular store, and an increase in Ca(2+) extrusion from VSMCs by stimulation of sarcolemmal Ca(2+)-pump. VSMCs have two major systems for Ca(2+) extrusion, namely, sarcolemmal Ca(2+)-pump and Na(+)/Ca(2+) exchanger (NCX). However, the involvement of NCX in the vasodilator-induced relaxation of VSMCs has not been well established. In this article, the possible involvement of NCX in the vasodilator-induced relaxation of VSMCs will be reviewed.

Pivotal role of Notch signaling in regulation of erythroid maturation and proliferation.

Notch signaling plays an important role in cell fate decisions in developmental systems. To clarify its role in committed hematopoietic progenitor cells, we investigated the effects of Notch signaling in erythroid colony forming cells (ECFCs) generated from peripheral blood. ECFCs express Notch receptors, Notch1 and Notch2, and Notch ligands Delta1, Delta4, and Jagged1. When we assayed the effects of Notch ligands on erythroid maturation by flow cytometry, we found that immobilized Delta1 and immobilized Delta4 in particular inhibited maturation, whereas Jagged1 had no effect. In addition, Delta4 inhibited proliferation without reducing cell viability. Increases in expression levels of the Notch target gene hairy enhancer of split (HES) -1 were evident by real-time PCR after stimulation with immobilized Delta4. The effect of soluble Delta4 on expression of HES-1 was less pronounced than that seen with the immobilized form, indicating that all surface-bound ligands are important for effective signal transduction. When ECFCs were cultured in the presence of soluble Delta4 at a low cell concentration, erythroid maturation was slightly inhibited, but at a high concentration, maturation was promoted via competition of soluble Delta4 with endogenous ligands. These results indicate a pivotal role of Notch signaling in regulating erythroid maturation and proliferation, and further suggest that cell-cell interactions modulate growth of erythroid progenitor cells via Notch system.

Upregulation of proteinase-activated receptors and hypercontractile responses precede development of arterial lesions after balloon injury.

Thrombin and other proteinases exert vascular effects by activating the proteinase-activated receptors (PARs). The expression of PARs has been shown to be upregulated after balloon injury and in human arteriosclerosis. However, the relationship between the receptor upregulation and the alteration of vasomotor function remains to be elucidated. We herein demonstrated that the contractile responses to the PAR-1 and PAR-2 agonist were markedly enhanced in the rabbit femoral arteries after balloon injury. Neointimal thickening was established 4 wk after the injury. No histological change was observed in the sham operation, where the saphenous artery was ligated without any balloon injury. The contractile response to K(+) depolarization was significantly attenuated 1 wk after the injury and then partly recovered after 4 wk. Thrombin, PAR-1-activating peptide, trypsin, and PAR-2-activating peptide induced no significant contraction in the control. All these stimulants induced enhanced responses 1 wk after balloon injury. Such enhanced responses were seen 4 wk after the injury, except for thrombin. There was no change in the Ca(2+) sensitivity of the contractile apparatus as evaluated in the permeabilized preparations. PAR-1-activating peptide (100 mumol/l), but no other stimulants, induced an enhanced contraction in the sham operation. The expression of PAR-1 and PAR-2 slightly increased after the sham operation, whereas it markedly and significantly increased after balloon injury. Our observations suggest that balloon injury induced the receptor upregulation, thereby enhancing the contractile response before the establishment of vascular lesions. The local inflammation associated with the sham operation may also contribute to the receptor upregulation.

Dependence of proliferating dedifferentiated vascular smooth muscle contraction on Rho-Rho kinase system.

Vascular smooth muscle cells (VSMCs) are not terminally differentiated and, owing to their remarkable plasticity, can change to a dedifferentiated state in response to vascular injury. Our understanding of the contractility of VSMCs is mainly based on the data obtained from normal adult animals. However, to obtain a better understanding of the abnormal contractility seen in the vascular diseases such as hypertension and vasospasm superimposed on atherosclerosis, it is important to also know the contractility of proliferating dedifferentiated VSMCs. To this end, we studied the contractility of cultured VSMCs that undergo dedifferentiation similar to that induced by vascular injury. There are only a few reports in which the contractility of cultured VSMCs has been extensively studied. We established a method to investigate the contractility of the cultured VSMCs and determined that their contraction is dramatically changed to be more dependent on the Rho-Rho kinase system but less dependent on the PKC-CPI-17 (protein kinase C-potentiated protein phosphatase 1 inhibitory protein)-mediated pathway. In this review, we focus on the contractility of the cultured VSMCs as a model of the proliferating dedifferentiated VSMCs.