Iodine contrast volume reduction in preoperative transcatheter aortic valve implantation computed tomography: Comparison with 64- and 256-multidetector row computed tomography.

INTRODUCTION: This study aimed to compare the vascular enhancement and radiation dose in preoperative transcatheter aortic valve implantation (TAVI) computed tomography (CT) with a reduced contrast medium (CM) using volume scans in 256-multidetector row CT (MDCT) with a standard CM using 64-MDCT. METHODS: This study included 78 patients with preoperative TAVI CT with either 64- or 256-MDCT. The CM was injected at 1.5 mL/kg in the 64-MDCT group and 1.0 mL/kg in the 256-MDCT group. We compared vascular enhancement of the aortic root and access routes, image quality (IQ) scores, and radiation dose in both groups. RESULTS: Despite the reduced CM (by 33 %) in the 256-MDCT group, the mean vascular enhancement of the right and left subclavian arteries was significantly higher than that in the 64-MDCT group [284 and 267 Hounsfield units (HU) vs. 376 and 359 HU; p < 0.05]; however, no significant differences in the mean vascular enhancement in the ascending aorta, abdominal aorta at the celiac level, and bilateral common femoral arteries were observed between the two groups (p > 0.05 for all). The median IQ scores at the aortic root were higher in the 256-MDCT group than in the 64-MDCT group (3 vs. 4; p < 0.05), and those at the femoral access routes were comparable (4 vs. 4; p = 0.33). The mean effective dose was significantly reduced by 30 % in the 256-MDCT group (23.6 vs. 16.3 mSv; p < 0.05). CONCLUSION: In preoperative TAVI CT, volume scans using 256-MDCT provide comparable or better vascular enhancement and IQ with a 30 % reduction in CM and radiation dose than those using 64-MDCT. IMPLICATIONS FOR PRACTICE: Volume scan using 256-MDCT for preoperative TAVI CT may reduce CM and radiation dose in TAVI patients with renal dysfunction.

The combined application of the contrast-to-noise index and 80 kVp for cardiac CTA scanning before atrial fibrillation ablation reduces radiation dose exposure.

INTRODUCTION: To compare the radiation dose, diagnostic accuracy, and the resultant ablation procedures using 80 and 120-kVp cardiac computed tomography angiography (CCTA) protocols with the same contrast-to-noise ratio in patients scheduled for atrial fibrillation (AF) ablation. METHODS: This retrospective study was performed following institutional review board approval. We divided 140 consecutive patients who had undergone CCTA using a 64-MDCT scanner into two equal groups. Standard deviation (SD) of the CT number was set at 25 Hounsfield units (HU) for the 120-kVp protocol. To facilitate a reduction in radiation dose it was set at 40 HU for the 80 kVp protocol. We compared the two protocols with respect to the radiation dose, the diagnostic accuracy for detecting left atrial appendage (LAA) thrombi, matching for surface registration, and the resultant ablation procedures. RESULTS: At 120 kVp, the dose length product (DLP) was 2.2 times that at 80 kVp (1269.0 vs 559.0 mGy cm, p < 0.01). The diagnostic accuracy for thrombus detection was 100% using both protocols. There was no difference between the two protocols with respect to matching for surface registration. The protocols did not differ with respect to the subsequent time required for the ablation procedures and the ablation fluoroscopy time, and the radiation dose (p = 0.54, 0.33, and 0.32, respectively). CONCLUSION: For the same CNR, the DLP at 80 kVp (559.0 mGy cm) was 56% of that delivered at 120 kVp (1269.0 mGy cm). There was no reduction in diagnostic accuracy. IMPLICATIONS FOR PRACTICE: Maintaining CNR allows for a reduction in the radiation dose without reducing the image quality.

Result of neck clipping and coil embolization as a treatment for subarachnoid haemorrhage.

SUMMARY: The report of the International Subarachnoid Aneurysm Trial (ISAT) study showed that coil embolization was superior to neck clipping as a treatment for subarachnoid haemorrhage (SAH) (1). We compared the results of coil embolization and neck clipping in our institute. Generally better outcomes were obtained by endovascular surgery than neck clipping. Symptomatic vasospasm and symptomatic hydrocephalus occurred less frequently in coil embolization than neck clipping. Because not all cases of SAH can be treated by coil embolization due to the width of aneurysmal neck and relation of the aneurysm to parent arteries, we should also be able to perform neck clipping as another modality.

Comparison of digestibility and breath hydrogen gas excretion of fructo-oligosaccharide, galactosyl-sucrose, and isomalto-oligosaccharide in healthy human subjects.

OBJECTIVES: To clarify the difference of digestibility in the small intestine among fructo-oligosaccharide (FOS), galactosyl-sucrose (GS), and isomalto-oligosaccharide (IMO) using breath hydrogen test. DESIGN: The first step: screening test of breath hydrogen excretion and FOS tolerance test to select the subjects. The second step: breath hydrogen test of three kinds of oligosaccharides, carried out using precautionary regulations. The ingestion order was 10 g of FOS, GS, and IMO, with increases, at 1-week interval, up to 20 g, respectively. Breath gas was collected before, at 20 min intervals from 40 to 120 min after, and at 30 min intervals from 120 min to 7 h after ingestion of test substance. SETTING: Laboratory of Public Health Nutrition, Department of Nutrition and Health Sciences, Siebold University of Nagasaki, Nagasaki, Japan. SUBJECTS: A total of nine males (average: age 25.7+/-3.5 y, weight 61.9+/-8.8 kg, height 170.0+/-6.0 cm) and 29 females (average: 23.1+/-7.2 y, 52.9+/-5.3 kg, 157.5+/-5.1 cm) from the University of Tokyo and Siebold University of Nagasaki. MAIN OUTCOME MEASURES: Breath hydrogen excretion from end-expiratory gas. RESULT: : Breath hydrogen of FOS was more remarkably excreted than that of GS; that of IMO was slight; and that of AUC (10 g) was significantly different. FOS was 9768+/-3253 ppm, GS was 3662+/-2632 ppm, and IMO was 831+/-1154 ppm. A dose dependence was observed at doses between 10 and 20 g of FOS and GS, and the initial time of 20 g was earlier than that of 10 g. CONCLUSIONS: FOS was not hydrolyzed, GS was slightly hydrolyzed, and IMO was readily hydrolyzed by small intestinal enzymes. H(2) gas reflected fermentability in the large intestine. SPONSORSHIP: Siebold University of Nagasaki.

Argon plasma coagulation for successful treatment of early gastric cancer with intramucosal invasion.

BACKGROUND: In recent years, there has been an increasing number of cases of early gastric cancer (T1, NX) with intramucosal invasion, which are untreatable by surgical or endoscopic mucosal resection (EMR) because of their high risk. Currently, no adequate treatment is available for such patients. AIM: The main objective of this study was to evaluate whether argon plasma coagulation (APC) is an effective and safe modality for treating early gastric cancer untreatable by surgical resection or EMR. METHODS: The study group comprised 20 men and seven women diagnosed with gastric cancer with intramucosal invasion who were considered poor candidates for surgical resection or EMR due to risk factors such as severe cardiac failure or thrombocytopenia. Irradiation conditions for APC treatment were determined using swine gastric mucosa. We used an argon gas flow of 2 l/min at a power setting of 60 W and a maximum irradiation time of 15 s/cm(2). The follow up period of the 27 patients ranged from 18 to 49 months (median 30 months). RESULTS: All lesions were irradiated easily, including areas anatomically difficult for EMR such as the gastric cardia or the posterior wall of the upper gastric body. In 26 of 27 patients (96%) there was no evidence of recurrence during the follow up period (median 30 months). One patient showed recurrence six months after the treatment but was successfully retreated. No serious complications were found in any of the 27 patients but three patients (11%) experienced a feeling of abdominal fullness. INTERPRETATION: APC is a safe and effective modality for treatment of early gastric cancer with intramucosal invasion untreatable by surgical resection or EMR. However, further observations are necessary to determine the long term prognosis of patients undergoing this treatment.

Successful treatment of advanced peripheral T-cell lymphoma with an angiocentric growth pattern complicated with hemophagocytic syndrome by high-dose chemotherapy and autologous peripheral blood stem cell transplantation.

Peripheral T-cell lymphomas (PTCL) account for about 10% of all lymphomas in Western countries, respond poorly to therapy, and have short survival with no sustained remission. Furthermore, the complication of hemophagocytic syndrome (HPS) sometimes makes the prognosis of this disease extremely worse. We report here a case of PTCL with an angiocentric growth pattern complicated with HPS successfully treated by high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Our case suggests this approach is an excellent candidate for the treatment of this disease.

A case of true malignant histiocytosis: identification of histiocytic origin with use of immunohistochemical and immunocytogenetic methods.

We report here an autopsy case of true malignant histiocytosis. The patient was a 67-year-old woman who exhibited fever, wasting, hepatosplenomegaly, and progressive pancytopenia. The bone marrow aspiration disclosed hemophagocytosing cells, which resembled histiocytes. The molecular analysis did not show the clonal gene rearrangement of T-cell receptor or immunoglobulin heavy chain. Although the patient had been started on methylprednisolone pulse therapy and chemotherapy with etoposide, she died from cerebral hemorrhage. The autopsy specimens of spleen and liver showed extensive infiltration of atypical cells, for which histiocytic origin was identified with an immunohistochemical method using monoclonal antibodies against CD11c, CD68, macrophage colony-stimulating factor (M-CSF), M-CSF receptor, lysozyme, antitrypsin and alpha1-antichymotrypsin. Recent investigations have disclosed that in most cases diagnosed as malignant histiocytosis, hemophagocytosis was reactive and not evoked by histiocytic malignancy. True malignant histiocytosis, for which histiocytic origin is confirmed, is extremely rare.

Surgical treatment of a mixed pineocytoma/pineoblastoma in a 72-year-old patient.

BACKGROUND: Although pineal parenchymal tumours are very rare in elderly patients, we recently successfully treated a 72-year-old male patient. Interestingly, the histology of his pineal parenchymal tumour was mixed pineocytoma/pineoblastoma, which is reported to be extremely rare in aged patients. We present his clinical manifestations, follow-up MRI, surgical treatment, pathological findings, and review the literature. CLINICAL MATERIAL: This 72-year-old man had a mass in the pineal region detected 3 years previously on MRI in February 1996 following symptoms of headache and vertigo. Two years later, he experienced gait disturbance and disorientation. CT scans disclosed obstructive hydrocephalus, and ventriculo-peritoneal shunt placement was then performed. The tumour mass began to enlarge in July 1999 (at age 72). On October 13, 1999, total removal of the pineal region tumour was performed through an occipital transtentorial approach. The postoperative course was uneventful. The pathological diagnosis of the tumour was mixed pineocytoma/pineoblastoma. CONCLUSION: Pineal parenchymal tumours are uncommon in elderly patients, and mixed pineocytoma/pineoblastomas are particularly rare. We followed this patient closely for more than 3.5 years and finally performed total surgical removal of the tumour, with excellent outcome. The present case suggests that a mixed pineocytoma/pineoblastoma tumour is controllable even in elderly patients through careful evaluation and management.

F13459, a new derivative of mycophenolic acid. I. Taxonomy, isolation, and biological properties.

In the course of screening for inhibitors of intracellular trafficking of glycoprotein, a new inhibitor, F13459 was isolated from the culture broth of a Penicillium sp. It was purified using solvent extraction, silica gel, Sephadex LH-20 and ODS column chromatography. From structural analysis, F13459 was a derivative of mycophenolic acid, an inhibitor of inosine 5'-monophosphate dehydrogenase. F13459 inhibited hemagglutinin synthesis of NDV at concentrations more than 25 microg/ml. However, syncytium formation as a result of cell surface expression of F-glycoprotein of NDV was inhibited at concentrations of F13459 lower than those required for appreciable inhibition of glycoprotein synthesis.

A novel arterial infusion chemotherapy for the treatment of patients with advanced pancreatic carcinoma after vascular supply distribution via superselective embolization.

BACKGROUND: Patients with American Joint Committee on Cancer Stage IV advanced pancreatic carcinoma have been treated by systemic chemotherapy, intraarterial chemotherapy, radiation therapy, and multidisciplinary treatment using a combination of these. However, the outcome has not always been satisfactory. In the current study the authors describe the method and results of a new chemotherapy for advanced pancreatic carcinoma. METHODS: To restrict the blood flow into the pancreas (mainly to the great pancreatic artery and the caudal pancreatic artery), the peripancreatic blood vessels were embolized superselectively with microcoils. In 31 patients with advanced pancreatic carcinoma, the catheter tip for the arterial infusion chemotherapy was placed in the splenic artery just proximal to the branching of the great pancreatic artery when the treatment was given for primary tumors, and in the common hepatic artery when the treatment was given for a metastatic liver lesion. The other end of the catheter was connected to an implanted injection port embedded in the femoral region, and 5-fluorouracil and cisplatin were administered by continuous arterial infusion. RESULTS: Of the 31 patients with advanced pancreatic carcinoma, 23 (74%) underwent hemodynamic change and arterial infusion chemotherapy, with a response rate of 73.9% (complete response rate of 8.7% and a partial response rate of 65.2%) and a mean survival period of 18.26 +/- 10.06 months. The 1-year, 2-year, and 3-year survival rates were 90.9%, 42. 8%, and 18.3%, respectively, with a mean survival period of 19.0 months. Of these 23 patients, the 16 patients with liver metastases had a response rate of 68.8% and a mean survival period of 16.25 +/- 8.35 months, whereas the 7 patients without liver metastases had a response rate of 87.5% and a mean survival period of 22.86 +/- 12.69 months. CONCLUSIONS: In patients with Stage IV advanced pancreatic carcinoma, arterial infusion chemotherapy after hemodynamic change was found to be effective against both primary tumors and metastatic liver lesions. The authors believe that the treatment presented in the current study should be attempted, even in patients with advanced pancreatic carcinoma, as long as the blood vessels for vascular supply distribution exist.

Leukocyte integrin-dependent and antibody-independent cytotoxicity of macrophage against allografts.

Macrophages (Mphis), but not T cells, infiltrating into the rejection site of either i.p. allografted Meth A (H-2d) fibrosarcoma cells in C57BL/6 (B6) (H-2b) mice or BALB/c (H-2d) skin onto B6 mice are cytotoxic against allografts with H-2d specificity. To determine the mechanisms of specific killing of allografts by allograft-induced Mphi (AIM), we raised approximately 5,000 rat monoclonal antibodies (mAbs) against AIM and selected three of them (R1-73, R2-40 and R1-34), each of which inhibited cytotoxic activity against allografts in a dose-dependent manner. The antigens recognized by R1-73, R2-40 and R1-34 mAbs were defined by immunoprecipitation and Western blot analyses as CD11a, CD18 and CD11b, respectively; and the allografts expressed CD54, a ligand of CD11a or CD11b, suggesting leukocyte integrin-dependent killing. Although Ab-dependent cellular cytotoxicity has been recognized as a mechanism of specific killing by Mphis, the infiltration of AIM into the rejection site of allografts far (approximately 6 days) preceded the appearance of serum IgG Ab specific for the allograft. AIM exhibiting full cytotoxic activity against allografts was also induced in the transplantation site of Fcgamma receptor knockout [(B6x129) F1] mice as well as B10.D2 (H-2 compatible with allograft) and B6-xid (X-linked immunodeficiency with B cell-specific defect) strains of mice. In the latter two strains of mice, the levels of serum IgG Ab to the allograft were negligible. Moreover, the cytotoxic activity of AIM against allografts was not affected by pretreatment of the cells with anti-mouse IgG serum, suggesting Ab-independent cytotoxicity.

[Dissecting aneurysm of the middle cerebral artery: case report].

We present a surgical case of a dissecting aneurysm of the right middle cerebral artery associated with subarachnoid hemorrhage and intracranial hemorrhage. A 61-year-old woman with consciousness disturbance and left hemiplegia was referred to our hospital. She had suffered severe headache for a week. CT scan showed a subarachnoid hemorrhage in the right Sylvian fissure and intracranial hemorrhage in the right putamen. The right carotid angiogram revealed string sign in M1 portion and occlusion at M2 lower branch of the right middle cerebral artery. On the 12th day, we undertook surgery to confirm whether it was a dissecting aneurysm or not. In the operation, it was reddish in the M1 portion corresponding to the "string sign" and dark-purplish in the lower M2 portion corresponding to an "aneurysm-like lesion". To prevent bleeding, the arterial wall in the M1 portion was coated using muscle. Though the left hemiplegia was unchanged, the postoperative course was uneventful. The patient was transferred to another hospital and underwent rehabilitation. There has been no reccurrence during the four years since surgery. The middle cerebral artery dissecting aneurysm is extremely rare. We presented this case with review of the literature.

Nonpeptide mimic of bradykinin with long-acting properties.

Kinins, members of a family of peptides released from kininogens by the action of kallikreins, have been implicated in a variety of biological activities including vasodilation, increased vascular permeability, contraction of smooth muscle cells and activation of sensory neurons. However, investigation of the physiological actions of kinins have been greatly hampered because its effects are curtailed by rapid proteolytic degradation. We examined the pharmacological characteristics of the first nonpeptide bradykinin receptor agonist 8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl+ ++]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinolin e (FR190997). FR190997, whose structure is quite different from the natural peptide ligand, but is similar to the nonpeptide antagonists FR165649, FR167344 and FR173657, potently and selectively interacts with the human B2 receptor and markedly stimulates inositol phosphate formation in transfected Chinese hamster ovary (CHO) cells. FR190997 induces concentration-dependent contraction of isolated guinea pig ileum. In vivo, FR190997 mimics the biological action of bradykinin and induces hypotensive responses in rats with prolonged duration, presumably as a consequence of its resistance to proteolytic degradation. Therefore, FR190997 is a highly potent and subtype-selective nonpeptide agonist which displays high intrinsic activity at the bradykinin B2 receptor. This compound represents a powerful tool for further investigation of the physiology and pathophysiology of bradykinin receptors.

Rheumatoid arthritis-affected temporomandibular joint pain analgesia by linear polarized near infrared irradiation.

PURPOSE: To describe a new short-term treatment for pain in rheumatoid arthritis (RA)-affected temporomandibular joint (TMJ). CLINICAL FEATURES: We investigated four female patients (age 42.8+/-26.0 yr) with chronic rheumatoid arthritis affecting a single TMJ. Patients had received antirheumatic drugs such as sodium aurothiomalate, and as a result showed no symptoms in other body joints. Linear polarized near infrared radiation using Super Lizer was applied weekly with and/or without jaw movement to the unilateral skin areas overlying the mandibular fossa, anterior articular tubercle, masseter muscle and posterior margin of the ramus of the mandible. The duration of irradiation to each point was two seconds on and ten seconds off per cycle and the intensity at each point was approximately 138 J x cm(-2) at a wavelength of 830 nm. Interincisal distance was measured with maximal mouth opening in the absence and presence of pain before and after each treatment. Additionally, subjective TMJ pain scores assessed using a visual analog scale were performed for painful maximal mouth opening before and after each irradiation. TMJ pain disappeared after only four treatments. Moreover, painless maximal mouth opening without pain after irradiation in three patients was on average improved to 5.3+/-2.1 mm. However, one case was observed where the opening length prior to irradiation did not improve, despite the fact that the RA-affected TMJ pain had disappeared. CONCLUSION: Application of linear polarized near infrared irradiation to patients with RA-affected TMJ pain is an effective and non-invasive short-term treatment.

Imaging herpes virus thymidine kinase gene transfer and expression by positron emission tomography.

We report a series of studies that assess the feasibility and sensitivity of imaging of herpes virus type one thymidine kinase (HSV1-tk) gene transfer and expression with [124I]-5-iodo-2'-fluoro-1-beta-D-arabinofuranosyluracil ([124I]-FIAU) and positron emission tomography (PET) and the ability of [124I]-FIAU-PET imaging to discriminate different levels of HSV1-tk gene expression. Studies were performed in rats bearing multiple s.c. tumors derived from W256 rat carcinoma and RG2 rat glioma cells. In the first set, we tested the sensitivity of [124I]-FIAU-PET imaging to detect low levels of HSV1-tk gene expression after retroviral-mediated gene transfer. HSV1-tk gene transduction of one of preestablished wild-type W256 tumor in each animal was accomplished by direct intratumoral injection of retroviral vector-producer cells (W256-->W256TK* tumors). Tumors produced from W256 and W256TK+ cells served as the negative and positive control in each animal. Highly specific images of [124I]-FIAU-derived radioactivity were obtained in W256TK* tumors (that were transduced in vivo) and in W256TK+ tumors but not in nontransduced wild-type W256 tumors. The level of "specific" incorporated radioactivity in transduced portions of both W256TK* and W256TK+ tumors was relatively constant between 4 and 50 h. In the second set, we tested whether [124I]-FIAU and PET imaging can measure and discriminate between different levels of HSV1-tk gene expression. Multiple s.c. tumors were produced from wild-type RG2 cells and stably transduced RG2TK cell lines that express different levels of HSV1-tk. A highly significant relationship between the level of [124I]-FIAU accumulation [% injected dose/g and incorporation constant (Ki)] and an independent measure of HSV1-tk expression (sensitivity of the transduced tumor cells to ganciclovir, IC50) was demonstrated, and the slope of this relationship was defined as a sensitivity index. We have demonstrated for the first time that highly specific noninvasive images of HSV1-tk expression in experimental animal tumors can be obtained using radiolabeled 2'-fluoro-nucleoside [124I]-FIAU and a clinical PET system. The ability to image the location (distribution) of gene expression and the level of expression over time provides new and useful information for monitoring clinical gene therapy protocols in the future.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 2. Overcoming the species difference between guinea pig and man.

Recently we reported the identification of a series of 8-[[3-(N-acylglycyl-N-methylamino)-2, 6-dichlorobenzyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridines as the first orally active non-peptide bradykinin (BK) B2 receptor antagonists (1-3). These compounds inhibited the specific binding of [3H]BK to guinea pig ileum membrane preparations expressing B2 receptors with nanomolar IC50's and also displayed in vivo functional antagonistic activities against BK-induced bronchoconstriction in guinea pigs at 1 mg/kg by oral administration. However, it was found that their affinities for the B2 receptors in human A-431 cells (human epidermoid carcinoma) were much lower. Intensive modifications of the terminal substituents at the glycine moiety elucidated the structure-activity relationships (SAR) for human B2 receptors, leading to an extended basic framework which incorporated a novel key pharmacophore. Thus, we overcame the species difference and identified the first clinical candidate 18c (FR167344) with IC50's of 0.66 and 1.4 nM for guinea pig ileum and human A-431 cells, respectively. This compound displayed in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs with an ED50 value of 0.17 mg/kg by oral administration. This novel non-peptide B2 antagonist is extremely potent both in vitro and in vivo by oral administration and is expected to be the first member of a new class of drug for the treatment of various inflammatory diseases.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 3. Discovering bioisosteres of the imidazo[1,2-a] pyridine moiety.

Recently we reported on overcoming the species difference of our first orally active non-peptide bradykinin (BK) B2 receptor antagonists, incorporating an 8-[[3-(N-acylglycyl-N-methylamino)-2, 6-dichlorobenzyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridine skeleton, leading to identification of the first clinical candidate 4a (FR167344). With this potent new lead compound in hand, we then investigated further refinement of the basic framework by replacement of the imidazo[1,2-a]pyridine moiety and discovered several bioisosteric heterocycles. Extensive optimization of these new heteroaromatic derivatives revealed the detailed structure-activity relationships (SAR) around the imidazo[1, 2-a]pyridine ring and the 2,6-dichlorobenzyl moiety, leading to the discovery of our second clinical candidate 87b (FR173657) which inhibited the specific binding of [3H]BK to recombinant human B2 receptors expressed in Chinese hamster ovary (CHO) cells and guinea pig ileum membrane preparations expressing B2 receptors with IC50's of 1.4 and 0.46 nM, respectively. This compound also displayed excellent in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs with an ED50 value of 0.075 mg/kg by oral administration. Further modifications of the terminal substituents on the pyridine moiety led to a novel pharmacophore and resulted in the identification of 99 (FR184280), whose IC50 value for human B2 receptors (0.51 nM) was comparable to that of the second-generation peptide B2 antagonist Icatibant.

Tumor growth modulation by sense and antisense vascular endothelial growth factor gene expression: effects on angiogenesis, vascular permeability, blood volume, blood flow, fluorodeoxyglucose uptake, and proliferation of human melanoma intracerebral xenografts.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, has been investigated as a potent mediator of brain tumor angiogenesis and tumor growth. We evaluated the effect of VEGF expression on the pathophysiology of tumor growth in the brain. Human SK-MEL-2 melanoma cells, with minimal VEGF expression, were stably transfected with either sense or antisense mouse VEGF cDNA and used to produce intracerebral xenografts. Vascular permeability, blood volume, blood flow, and tumor fluorodeoxyglucose metabolism were assessed using tissue sampling and quantitative autoradiography. Tumor proliferation was assessed by measuring bromodeoxyuridine labeling indices. Tumor vascular density and morphological status of the blood-brain barrier were evaluated by immunohistochemistry. SK-MEL-2 cells transfected with sense VEGF (V+) expressed large amounts of mouse and human VEGF protein; V+ cells formed well-vascularized, rapidly growing tumors with minimal tumor necrosis. V+ tumors had substantial and significant increases in blood volume, blood flow, vascular permeability, and fluorodeoxyglucose metabolism compared to wild-type and/or V- (antisense VEGF) tumors. VEGF antisense transfected V- expressed no detectable VEGF protein and formed minimally vascularized tumors. V- tumors had a very low initial growth rate with central necrosis; blood volume, blood flow, vascular permeability, and glucose metabolism levels were low compared to wild-type and V+ tumors. A substantial inhibition of intracerebral tumor growth, as well as a decrease in tumor vascularity, blood flow, and vascular permeability may be achieved by down-regulation of endogenous VEGF expression in tumor tissue. VEGF-targeted antiangiogenic gene therapy could be an effective component of a combined strategy to treat VEGF-producing brain tumors.

Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase delta and replication factor C.

BACKGROUND: PCNA, an eukaryotic DNA sliding clamp interacts with replication factors and the cell cycle protein, p21(Cip1/Waf1) and functions as a molecular switch for DNA elongation. To understand how DNA replication is regulated through PCNA, elucidation of the precise mechanisms of these protein interactions is necessary. RESULTS: Loop-region mutants in which human PCNA sequences were substituted with the corresponding Saccharomyces cerevisiae PCNA regions were prepared. Analysis of their functions, along with previously prepared alanine scanning mutants, demonstrated that some loops interact with DNA polymerase delta (pol delta) and replication factor C (RFC). The p21 binding sites of PCNA, mapped by affinity measurement of the mutant forms, found to be located within a distinct structure of the PCNA monomer, overlap with RFC- and pol delta-interaction sites. Competition between p21 and pol delta or RFC for binding to PCNA results in efficient inhibition of its stimulation of pol delta DNA synthesis and RFC ATPase but not of PCNA loading on DNA by RFC. CONCLUSIONS: Semi-saturated amounts of p21 selectively block formation of the active pol delta complex but not the RFC-PCNA complex at 3'-ends of DNA primers. This differential effect may explain the specific inhibition of DNA replication by p21.