Fibronectin plays a major role in hypoxia-induced lenvatinib resistance in hepatocellular carcinoma PLC/PRF/5 cells.

Resistance to lenvatinib mesylate (LEN), a systemic chemotherapy that can be administered orally, has been a major issue for treatment of hepatocellular carcinoma (HCC). Although HCC is the tumor that most exhibits intratumoral hypoxia, which has been shown to be involved in the development of treatment resistance, there are no reports of LEN resistance in HCC treatment under hypoxia. The purpose of our study was to elucidate the mechanism of treatment resistance to LEN under hypoxia using HCC cell lines. We confirmed LEN resistance under hypoxic conditions in HCC cell lines. There was a significant increase in the IC50 value of PLC/PRF/5 cells from 13.0±0.8 µM in normoxia to 21.3±1.1 µM in hypoxia, but in HepG2 cells, the increase was not significant. To elucidate the LEN resistance mechanism of PLC/PRF/5 cells under hypoxia, we performed microarray analysis and extracted genes that are thought to be related to this mechanism. Furthermore, in-silico analysis confirmed significant changes in the extracellular matrix, and among them, FN1 encoding fibronectin was determined as the hub of the gene cluster. The expression of fibronectin in PLC/PRF/5 cells examined with immunofluorescence staining was significantly elevated in and outside of cells under hypoxia, and tended to decrease when cells were exposed to LEN under normoxia. Furthermore, the fibronectin concentration in the culture solution of PLC/PRF/5 cells examined by ELISA was 2.3 times higher under hypoxia than under normoxia under LEN(-) conditions, and 1.6 times higher under hypoxia than under normoxia under LEN(+) conditions. It is assumed that in PLC/PRF/5 cells, fibronectin is probably suppressed as an indirect effect of LEN under normoxia, but transcription factors such as HIF-1α are induced under hypoxia, thus enhancing the production of fibronectin and attenuating the effect of LEN, resulting in drug resistance. This behavior of fibronectin with LEN exposure under hypoxia is probably specific to PLC/PRF/5 cells. Further studies should verify the combined effective inhibition of fibronectin and the MAPK pathway as a promising therapeutic strategy to enhance the value of LEN in HCC treatment.

Partial loss of function of colony-stimulating factor 1 receptor in a patient with white matter abnormalities.

BACKGROUND AND PURPOSE: Mutations in colony-stimulating factor 1 receptor (CSF1R) cause adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Patients with ALSP can be misdiagnosed as having acute ischemic stroke due to hyperintensity lesions on diffusion-weighted magnetic resonance imaging. Mutant CSF1R proteins identified in ALSP show a complete loss of autophosphorylation of CSF1R. METHODS: We conducted mutation screening of CSF1R in 123 patients with definite acute ischemic cerebrovascular syndrome and positive family history of stroke. The pathogenicity of identified variants was evaluated using functional analyses. The levels of autophosphorylation of CSF1R in response to treatment with ligands of CSF1R were examined in cells transfected with wild-type and mutant CSF1R. RESULTS: We identified eight CSF1R variants, six were known non-pathogenic polymorphisms, whereas the other two were missense variants inducing substitution of amino acid residues (p.Glu573Lys and p.Gly747Arg). Functional assay showed that the levels of autophosphorylation of p.Gly747Arg were similar to those of wild-type when treated with ligands. The autophosphorylation of p.Glu573Lys was detectable, but significantly decreased compared with those of wild-type CSF1R (P < 0.001, two-way anova with Bonferroni). The clinical presentation of the patient with p.Glu573Lys was consistent with cerebral embolism. The patient did not have typical clinical findings of ALSP. However, periventricular white matter abnormalities, unrelated to the recent infarct, were evident on brain magnetic resonance imaging. CONCLUSIONS: In contrast to ALSP-associated missense mutations, CSF1R p.Glu573Lys variant in a patient with acute ischemic cerebrovascular syndrome showed a partial loss of autophosphorylation of CSF1R; its clinical significance warrants further investigation.

Diagnostic Value of Brain Calcifications in Adult-Onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia.

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia is a rare neurodegenerative disease resulting from mutations in the colony stimulating factor 1 receptor gene. Accurate diagnosis can be difficult because the associated clinical and MR imaging findings are nonspecific. We present 9 cases with intracranial calcifications distributed in 2 brain regions: the frontal white matter adjacent to the anterior horns of the lateral ventricles and the parietal subcortical white matter. Thin-section (1-mm) CT scans are particularly helpful in detection due to the small size of the calcifications. These calcifications had a symmetric "stepping stone appearance" in the frontal pericallosal regions, which was clearly visible on reconstructed sagittal CT images. Intrafamilial variability was seen in 2 of the families, and calcifications were seen at birth in a single individual. These characteristic calcification patterns may assist in making a correct diagnosis and may contribute to understanding of the pathogenesis of leukoencephalopathy.

Adenosine A2A receptor mediated protective effect of 2-(6-cyano-1-hexyn-1-yl)adenosine on retinal ischaemia/reperfusion damage in rats.

AIMS: To determine the effect of 2-(6-cyano-1-hexyn-1-yl)adenosine (2-CN-Ado), an adenosine A2A receptor agonist, on retinal ischaemia/reperfusion damage in rats. METHODS: Retinal ischaemia/reperfusion damage was induced by elevating the intraocular pressure of one eye to 130 mm Hg for 60 minutes and returning it to normal. 7 days later, retinal ischaemia/reperfusion damage was histologically quantified by measuring the thickness of retinal layers. Intraocular pressure was measured by pressure transducer. RESULTS: Retinal ischaemia/reperfusion caused cell loss in the ganglion cell layer and thinning of the inner plexiform and nuclear layer. Both ocular topical and intravenous administration of 2-CN-Ado caused a reduction of retinal ischaemia/reperfusion damage. A selective A2A receptor antagonist, 1,3,7-trimethyl-8-(3-chlorostyryl) xanthine (CSC), but not a selective A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), or a selective A2B receptor antagonist, alloxazine, reduced the protective effect of 2-CN-Ado. While ocular topical administration of 2-CN-Ado caused a sustained reduction of intraocular pressure, intravenous administration of 2-CN-Ado showed a transient ocular hypotensive effect. CONCLUSIONS: These results suggest that 2-CN-Ado attenuates retinal ischaemia/reperfusion damage, and at least some of this protective effect of 2-CN-Ado might be mediated via activation of the adenosine A2A receptor.

Gene expressions of canine angiopoietin-1 and -2 in normal tissues and spontaneous tumours.

The angiopoietin (Ang) family of proteins are central to the regulation of angiogenesis. The purposes of this study were to determine cDNA sequences of canine Ang-1 and Ang-2 and investigate their expressions in normal tissues and spontaneous tumours. The cDNA sequences of canine Ang-1 and Ang-2 were 1,494 and 1,488 bp, and the deduced amino acid sequences were 497 and 495 residues, respectively. The cDNA sequences of canine Ang-1 and Ang-2 showed high homology with those of the other mammalian species. Canine Ang-1 and Ang-2 mRNA were detectable in all 22 normal tissues and spontaneous tumours. Higher mRNA expression level of canine Ang-2 was demonstrated in mammary simple carcinomas, haemangiosarcoma and hepatocellular carcinoma in comparison with normal tissues.

Application of dye-enhanced laser ablation for liver resection. Production of protein sealant on the cut surface of the liver by enhanced thermal energy of low power diode laser.

BACKGROUND: Dye-enhanced laser ablation (DLA) using a low-power diode laser for indocyanine green (ICG)-stained tissue has proven its effectiveness in dye-enhanced laser photocoagulation of retinal vessels or endoscopic surgical mucosectomy. We have applied DLA in hepatectomy and described its histological distinction in comparison with the cavitron ultrasonic surgical aspirator (CUSA). METHODS: A diode laser (UDL-60 Laser unit, Olympus, Tokyo, Japan) with 810 +/- 20 nm wavelength was employed for this study. The ICG dye (Diagnogreen, Daiichi Pharmaceutical, Tokyo, Japan) with a peak absorption wavelength at 800-810 nm was injected topically into the resection plane of the liver. The liver tissue was divided by touching the tip of the diode laser. Three different concentrations of ICG solution such as 2.0, 1.0 and 0.5 mg/ml were tested in the preliminary animal experiment. The use of a low-power diode laser at 10 W with an ICG concentration of 0.5 mg/ml was the appropriate combination for liver resection. In the clinical series, 27 hepatectomies were performed by DLA, and 10 with CUSA. RESULTS: DLA demonstrated smooth cutting and good hemostasis in liver resection. Among the hepatectomy cases given DLA, no postoperative hemorrhage or bile leakage was noted. The postoperative hospital stay was significantly shorter in the DLA than the CUSA group. The cut surface of the liver was sealed microscopically with a layer of protein coagulum. CONCLUSIONS: A layer of protein sealant on the cut surface of the liver contributes to the short postoperative hospital stay when using DLA.

Roles of aquaporin-3 water channels in volume-regulatory water flow in a human epithelial cell line.

Membrane water transport is an essential event not only in the osmotic cell volume change but also in the subsequent cell volume regulation. Here we investigated the route of water transport involved in the regulatory volume decrease (RVD) that occurs after osmotic swelling in human epithelial Intestine 407 cells. The diffusion water permeability coefficient (Pd) measured by NMR under isotonic conditions was much smaller than the osmotic water permeability coefficient (Pf) measured under an osmotic gradient. Temperature dependence of Pf showed the Arrhenius activation energy (Ea) of a low value (1.6 kcal/mol). These results indicate an involvement of a facilitated diffusion mechanism in osmotic water transport. A mercurial water channel blocker (HgCl(2)) diminished the Pf value. A non-mercurial sulfhydryl reagent (MMTS) was also effective. These blockers of water channels suppressed the RVD. RT-PCR and immunocytochemistry demonstrated predominant expression of AQP3 water channel in this cell line. Downregulation of AQP3 expression induced by treatment with antisense oligodeoxynucleotides was found to suppress the RVD response. Thus, it is concluded that AQP3 water channels serve as an essential pathway for volume-regulatory water transport in, human epithelial cells.

Multistep nucleus formation and a separate subunit contribution of the amyloidgenesis of heat-denatured monellin.

Monellin (MN) is a sweet-tasting plant protein known to form fibrous aggregates in the heat-denatured state. Here the amyloid-type aggregation process of MN is extensively characterized. The amyloidgenesis was initiated in a highly denatured state of MN. A seeding effect of skipping a lag phase of the amyloid formation kinetics established a nucleation-dependent aggregation mechanism. A finely controlled experimental protocol revealed an additional prenucleus stage preceding the maturation of the nucleus, indicating that the initial lag phase is composed of multiple conformational events. The results obtained for the aggregation properties of the separate A and B subunit chains of MN and a recombinant single-chain MN suggest that the B chain exclusively contributed to the amyloid-type aggregation. These findings suggest a scheme for the amyloidgenesis of MN and their subunits, and provide a unique model of amyloidgenesis that is regulated by the subunit composition of protein.

Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS.

This review article describes three aspects of polymeric drugs. The general mechanism of the EPR (enhanced permeability and retention) effect and factors involved in the effect are discussed, in view of the advantages of macromolecular therapeutics for cancer treatment, which are based on the highly selective EPR-related delivery of drug to tumor. Also described are advantages of more general water-soluble polymeric drugs as primary anticancer agents, using SMANCS as an example. Last, SMANCS/Lipiodol is discussed with reference to the type of formulation for arterial injection with most pronounced tumor selective delivery, as well as its advantages, precautions, and side effects from the clinical standpoint.

Phosphinic acid pseudopeptides analogous to glutamyl-gamma-glutamate: synthesis and coupling to pteroyl azides leads to potent inhibitors of folylpoly-gamma-glutamate synthetase.

Several routes to a complex phosphinate phosphapeptide analogous to the gamma-glutamyl peptide Glu-gamma-Glu have been investigated. Formation of gamma-phosphono glutamate derivatives via addition of a phosphorus-based radical to protected vinylglycine was found to be of limited value because of the elevated temperatures required. Alkylation and conjugate addition reactions of trivalent phosphorus (P(III)) species were investigated. In situ generation of bis-trimethylsilyl esters of phosphinous acids proved to be an effective route to phosphinates of modest structural complexity. However, this chemistry could not be extended to the incorporation of an amino acid moiety at the N-terminal side of the desired phosphinate. A successful synthesis of the target phosphinate phosphapeptide was effected using P(III) chemistry and dehydrohalogenation to yield an alpha,beta-unsaturated phosphinic acid ester, following which conjugate addition of diethylacetamido malonate and acid-mediated hydrolysis afforded the desired phosphinate phosphapeptide. Coupling of the unprotected phosphinate phosphapeptide with two acyl azides derived from folic acid and methotrexate led to the corresponding pteroylphosphapeptides of interest as possible mimics of tetrahedral intermediates in the reaction catalyzed by folylpolyglutamate synthetase.

K-ras gene mutations in non-small cell lung cancer in Japanese.

To evaluate the association of mutations in the K-ras gene with the incidence of non-small cell lung cancer (NSCLC) in Japanese patients, 410 patients treated surgically were studied. DNA was extracted from frozen specimens, and polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) hybridization assay was used to examine K-ras mutations in codons 12, 13 and 61. K-ras mutations were found in 33 patients (8.0%), and all were smokers or ex-smokers. There were no significant differences in clinical or pathological stage between wild-type cases and mutant cases. The most frequently identified mutation was a G-->T transversion (25/33, 75.8%) that resulted in the substitution of a glycine for a cysteine or a valine. Previous studies have shown that the majority of K-ras mutations among smokers are G-->T transversion. In our study, K-ras mutations were found only in smokers, and there was a high frequency of G-->T transversions. A clear correlation exists between smoking and K-ras gene mutations.

Urinary metabolites of DX-8951, a novel camptothecin analog, in rats and humans.

Urinary metabolites of DX-8951 ((1S,9S)-1-amino-9-ethyl-5-fluoro- 1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione, CAS 171335-80-1, exatecan) in rats and humans were identified. Rats were dosed with the drug, and two major metabolites (UM-1 and UM-2) in the urine were isolated and purified by using ion-exchange column and HPLC. From NMR and mass spectra, they are suggested to be 4-hydroxymethyl metabolite (UM-1) and 3-hydroxy metabolite (UM-2) of the drug. Their chemical structures were confirmed by comparing their NMR spectra with those of chemically synthesized metabolites. Two major metabolites were found in human urine obtained in phase I trial. They were also confirmed to be UM-1 and UM-2 by LC/MS/MS by comparing their mass fragment patterns with those of synthetic metabolites.

Validation study of assay method for DX-8951 and its metabolite in human plasma and urine by high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry.

A new liquid chromatographic/mass spectrometric assay has been developed for the determination of DX-8951, a new anti-tumor drug, and its 4-hydroxymethyl metabolite (UM-1) in human plasma and urine. Solid-phase extractions were used for sample preparation. A gradient reverse-phase HPLC separation was developed with mobile phases consisting of trifluoroacetic acid and methanol. The detection was conducted using atmospheric pressure chemical ionization tandem mass spectrometry in the selected reaction monitoring mode. A structural analog, camptothecin (CPT), was used as the internal standard. The assay was validated for the determination of DX-8951 and UM-1 in human plasma and urine. The lower limits of quantitation of DX-8951 and UM-1 were 0.1 ng/mL in plasma and 1 ng/mL in urine. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity.

Treatment of VX2 carcinoma implanted in the liver with arterial and intraperitoneal administration of oily anticancer agents.

PURPOSE: Long-term survival and cure cannot be achieved in patients with unresectable, advanced abdominal cancer, because no chemotherapeutic treatment has definite antitumor activity for malignant solid tumor and its dissemination. In this study, arterial and intraperitoneal administration of oily anticancer agents, which have properties that permit targeted chemotherapy for VX2 carcinoma implanted in the liver, was attempted to achieve long-term survival. MATERIALS AND METHODS: Rabbits bearing VX2 tumors in the liver measuring 1-2 cm in diameter received an arterial injection of 0.2 ml of nitrogen mustard N-Oxide (HN2-O) dissolved in Lipiodol (7.5 mg/ml), a newly developed oily anticancer agent, for the tumor and an intraperitoneal injection of a cocktail of oily anticancer agents for the prevention of intraperitoneal dissemination. RESULTS: Twelve out of thirteen rabbits survived and VX2 cancer was not observed in these 12 rabbits. The controls received a sham operation, an intraperitoneal injection of the cocktail of oily anticancer agents alone, or an arterial injection of HN2-O/Lipiodol alone. In these control groups, 27 out of 29 rabbits died of cancer. To examine the dose form for arterial injection, 14 rabbits received an arterial injection of the simple mixture of HN2-O dissolved in physiological saline and Lipiodol, with an additional intraperitoneal injection of the cocktail. Eight of these 14 rabbits died of enlargement of the hepatic tumor and peritoneal dissemination. CONCLUSION: Long-term survival and cure was achieved in almost all rabbits bearing VX2 tumor in the liver by simultaneous arterial and intraperitoneal injection of oily anticancer agents.

High-Performance liquid chromatographic analysis of lactone and hydroxy acid of new antitumor drug, DX-8951 (exatecan), in mouse plasma.

A sensitive high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of lactone and total drug (lactone plus hydroxy-acid) of DX-8951 in mouse plasma. Solid-phase extraction by C18 cartridge separated lactone from total drug of DX-8951. Analysis was performed using a reverse-phase ODS column with a mobile phase consisting of acetonitrile/0.05 M potassium dihydrogen phosphate (pH 3) (18: 82, v/v) at a flow rate of 1 ml/min. The limits of quantitation of lactone and total drug were 3 ng/ml in plasma and a linear range of determination were observed over the concentration of 3 to 500 ng/ml. This method was applied to pharmacokinetic study in male mice treated with a single intravenous administration of either lactone or hydroxy-acid of DX-8951. The plasma concentrations of lactone from 2 to 6 h after dosing were similar regardless of the form of DX-8951 administered.

Improved survival of children with advanced neuroblastoma treated by intensified therapy including myeloablative chemotherapy with stem cell transplantation: a retrospective analysis from the Tohoku Neuroblastoma Study Group.

In the hospitals of the Tohoku Neuroblastoma Study Group (TNBSG), treatment for children with advanced neuroblastoma (NB) was intensified in the mid-1990's with the introduction of myeloablative therapy (MT) with stem cell transplantation (SCT) including the use of autologous peripheral blood stem cells (PBSC) and bone marrow transplantation (BMT). In this report, we examined whether the intensified therapy improved the outcome of children with advanced NB (age> 12 months) who were diagnosed between 1991 and 1997. Patients were 36 children (23 boys and 13 girls) with an average age of 3.4 years (range; 1 to 14 years). Six of them had stage III disease, and the other 30 had stage IV. They were treated initially with induction chemotherapy, surgery, and post-operative chemoradiotherapy, after which 17 of them continued further chemotherapy and the other 19 received MT/SCT (18 with PBSCT and 1 with BMT). Progression-free survival (PFS) rate at seven years from diagnosis was 43.5% for all patients, 66.7% for stage III patients and 38.2% for stage IV patients. The difference between stage III and IV patients was not significant. Among the 30 patients with stage IV disease, PFS at seven years was significantly higher in the 19 patients who received MT/SCT (55.6%) than in the 11 patients who did not receive it (12.5%). There was no difference in clinical and biological risk factors between these two groups, except for the proportion of patients with favorable response to initial therapy (36% and 80% for patients without and with MT/SCT, respectively). Furthermore, the proportion of patients with N-myc amplification was significantly higher in patients with progressive disease (PD) after MT/SCT than in those in CR after MT/SCT. The results of this retrospective study of children with advanced NB suggest that therapy intensification involving MT/SCT might result in lengthened survival time for patients with stage IV disease, and that post-transplant PD remains a risk for patients with high levels of N-myc amplification.

A new alternative method to quantify residual structure in 'unfolded' proteins.

Pig (pCSD1) and human (hCSD1) calpastatin domain 1 proteins were studied to characterize common features of the denatured state of proteins. These proteins were chosen for the present investigation, because pCSD1 was suggested previously to be unstructured in water even at 25 degrees C (1) [T. Konno et al., Biochim. Biophys. Acta 1342 (1997) 73-82]. hCSD1 could be expected to exhibit similar features on the basis of preliminary spectroscopic studies. In the present study, the experimental grounds for the estimate of residual structure in the unfolded state were differential scanning calorimetry heat capacity and circular dichroism (CD) measurements over the temperature range 10-80 degrees C. At selected temperatures, we studied also the effect of guanidinium hydrochloride (GdnHCl) which is known to promote further unfolding of the polypeptide chain. All other measurements were performed at pH 6 in pure water. The present results support the conclusion that the comparison of the experimentally obtained heat capacity data with theoretical heat capacity values calculated on the basis of a newly established increment system gives insight into the degree of hydration of the unfolded polypeptide chain. The percentage by which the experimental heat capacity of the unfolded polypeptide chain differs from the calculated heat capacity permits a quantitative estimate of the residual structure. This estimate is in good agreement with that based on CD absorption. The heat capacity approach has the advantage of comparing fully hydrated and partially hydrated residues in the same aqueous environment, whereas for example spectroscopic measurements, such as CD, are generally referred to the fully unfolded chain in concentrated urea or GdnHCl solutions. As the unfolded chains of pCSD1 and hCSD1 exhibit a smaller heat capacity than that calculated on the new peptide-based increment system [M. Häckel et al., J. Mol. Biol. 291 (1999) 197-213], we conclude that the residues in the unfolded polypeptide chain are less hydrated than the same residues in oligopeptides. This suboptimal hydration is the result of residual structure in the chain as observed in both CD and heat capacity measurements.

Columnar epidermal necrosis: a unique manifestation of transfusion-associated cutaneous graft-vs-host disease.

BACKGROUND: In 1978, the first case of columnar epidermal necrosis was reported in a 6-year-old boy. There were scaly, partially vesicular or crusty, erythematous lesions mainly involving the extremities that histopathologically showed peculiar features of focal, total epidermal necrosis accompanied by a lichenoid tissue reaction. He developed the skin eruption after receiving a blood transfusion from his mother when he showed debility induced by vaccination with an alternated live measles virus vaccine. The lesions rapidly regressed after sun exposure. To our knowledge, there has been no report of a similar case despite such unique features. OBSERVATION: We encountered a similar case of columnar epidermal necrosis in a 15-year-old Japanese girl with chronic graft-vs-host disease; the lesions occurred 3 months after the transfusion of peripheral blood stem cells from her HLA antigen-matched brother. However, there was no exacerbation of liver dysfunction, diarrhea, or bone marrow aplasia. The peculiar cutaneous lesions responded well to topical phototherapy. CONCLUSION: These 2 patients shared a similarity in their lesions and circumstances under which the blood transfusion was performed to a debilitated patient from a close family member. We believe that focal epidermal necrosis observed in patients with this condition represents a variant of blood transfusion-associated lichenoid graft-vs-host disease that occurs uniquely in a skin-targeted fashion.

Tumor-targeted chemotherapy with SMANCS in lipiodol for renal cell carcinoma: longer survival with larger size tumors.

OBJECTIVES: To evaluate the anticancer effects of a lipophilic macromolecular anticancer agent, poly(styrene-co-maleic acid)-conjugated neocarzinostatin (SMANCS), dissolved in a lipid contrast medium (Lipiodol) given via the renal artery to patients with renal cell carcinoma. METHODS: Among 467 patients with renal cell carcinoma treated between April 1984 and March 1993, 191 were treated with SMANCS dissolved in a lipid contrast medium (a 3:2 mixture of Lipiodol F and Lipiodol Ultrafluid; Lpd). Selective arterial infusion of SMANCS/Lpd was performed at a dose of 1.0 or 1. 5 mg/mL. The infusion was repeated at intervals of about 2 weeks or longer, but the doses and the total number of infusions varied among patients, according to results of computed tomography analysis. RESULTS: Statistical analysis was performed for 415 patients who met the criteria of this study. Twenty-six surgical patients with metastases who underwent infusion therapy of SMANCS/Lpd for primary lesions showed 3 and 5-year survival rates of 23.0% and 12.8%, respectively; the rates were 19.3% and 9.7% in 31 patients who did not receive SMANCS infusion therapy. In 125 surgical patients without metastases who underwent SMANCS/Lpd infusion, the 5 and 10-year survival rates were 83.0% and 75.2%, respectively, whereas rates of 84.6% and 78.9% were observed in 199 surgical patients whose median tumor size was significantly smaller, however, than the SMANCS/Lpd infusion group. The maximal tumor diameter at the beginning of treatment was significantly larger (mean diameter 70.8 mm) in the SMANCS/Lpd infusion group than in the noninfusion group (59.1 mm). The survival rate was statistically better for patients with tumors of 100 mm diameter or larger in the SMANCS/Lpd infusion group (P <0.05): 5 and 10-year survival rates were 70.4% and 61.6%, respectively, for the infusion group and 64.6% and 50.9% for the group receiving no drug. In patients with larger tumor (greater than 110 mm), the survival rate at 13 years was 75% in the SMANCS/Lpd infusion group and 0% in the surgery group. CONCLUSIONS: Arterial infusion therapy with SMANCS/Lpd appears to be effective for large renal cell carcinoma without metastases in conjunction with surgery.

PCR-RFLP analysis of cytomegalovirus infections associated with bone marrow transplantation in Japanese children.

In order to investigate the longitudinal molecular epidemiology of cytomegalovirus (CMV) infections associated with bone marrow transplantation (BMT) in Japanese children, we analyzed 36 CMV strains from 11 cases. Three regions (DNA polymerase, glycoprotein H, and immediate-early regions) of CMV DNA were amplified by polymerase chain reaction (PCR), and amplified products were each digested with two restriction enzymes, followed by electrophoresis. These restriction fragment length polymorphism (RFLP) analyses allowed the differentiation of 36 strains into 13 genotypes. Each patient excreted his or her own CMV with distinct genotype over the study period of up to one year. CMVs of two different genotypes were recovered during a one-month study from one recipient, who received a peripheral blood stem cell transplantation. Although the majority of patients and donors were CMV-seropositive before BMT, multiple CMV infections might not be common and the reactivation of latently infected CMV might be prominent in Japanese children receiving transplants.