Local and retrograde gene transfer into primate neuronal pathways via adeno-associated virus serotype 8 and 9.

Viral vector-mediated gene transfer has become increasingly valuable for primate brain research, in particular for application of genetic methods (e.g. optogenetics) to study neuronal circuit functions. Neuronal cell tropisms and infection patterns are viable options for obtaining viral vector-mediated transgene delivery that is selective for particular neuronal pathways. For example, several types of viral vectors can infect axon terminals (retrograde infections), which enables targeted transgene delivery to neurons that directly project to a particular viral injection region. Although recent studies in rodents have demonstrated that adeno-associated virus serotype 8 (AAV8) and 9 (AAV9) efficiently transduce neurons, the tropisms and infection patterns remain poorly understood in primate brains. Here, we constructed recombinant AAV8 or AAV9, which expressed an enhanced green fluorescent protein (EGFP) gene driven by a ubiquitous promoter (AAV8-EGFP and AAV9-EGFP, respectively), and stereotaxically injected it into several brain regions in marmosets and macaque monkeys. Immunohistochemical analyses revealed almost exclusive colocalization of EGFP fluorescence via AAV9-mediated gene transfer with a neuron-specific marker, indicating endogenous neuronal tropism of AAV9, which was consistent with our previous results utilizing AAV8. Injections of either AAV8-EGFP or AAV9-EGFP into the marmoset striatum resulted in EGFP expression in local striatal neurons as a result of local infection, as well as expression in dopaminergic neurons of the substantia nigra via retrograde transport along nigrostriatal axonal projections. Retrograde infections were also observed in the frontal cortex and thalamus, which are known to have direct projections to the striatum. These local and retrograde gene transfers were further demonstrated in the geniculocortical pathway of the marmoset visual system. These findings indicate promising capabilities of AAV8 and AAV9 to deliver molecular tools into a range of primate neural systems in pathway-specific manners through their neuronal tropisms and infection patterns.

A case of adenocarcinoma with mixed subtypes displaying ground-glass opacity on high-resolution CT.

We report a case of adenocarcinoma with mixed subtypes with pleural dissemination and lymphatic permeation, although the CT results showed ground-glass opacity that led to the diagnosis of bronchioloalveolar carcinoma without foci of active fibroblastic proliferation.

Bronchopulmonary arterial anastomosis at the precapillary level in human lung. Visualization using CT angiography compared with microangiography of autopsied lung.

PURPOSE: To investigate the interrelationships between the bronchial and pulmonary circulations including the existence of precapillary bronchopulmonary arterial anastomoses. MATERIAL AND METHODS: CT of bronchial arteriography (BAG-CT) was performed in 10 patients and BAG-CT during a pulmonary artery block test (PA-block) in 5 patients with lung cancer. Bronchial and pulmonary circulations were evaluated in 5 autopsied normal lungs by injecting silicone rubber with different colors into the bronchial and pulmonary arteries. RESULTS: BAG-CT correlated well with the findings at silicone rubber injection into lung autopsy samples. BAG-CT demonstrated inflow of contrast medium into the pulmonary artery during PA-block in all cases, while no inflow was observed before and following reversal of PA-block. Mixed silicone rubber was observed in the lobar to subsubsegmental bronchial arteries in all cases and in the subsubsegmental pulmonary artery in one case. CONCLUSION: Precapillary bronchopulmonary arterial anastomoses may exist at the level of the lobar bronchi to the periphery. If either the pulmonary or bronchial circulation is disturbed, flow occurs inside the anastomoses to supplement the other flow, especially flow from the bronchial to the pulmonary arteries via the anastomoses, which occurs within 30 min.

Effect of troglitazone on endocrine and ovulatory performance in women with insulin resistance-related polycystic ovary syndrome.

OBJECTIVE: To investigate the effect of troglitazone, a new antidiabetic agent that improves insulin resistance, on endocrine, metabolic, and ovulatory performance in women with insulin resistance-related polycystic ovary syndrome (PCOS). DESIGN: Prospective clinical study. SETTING: Infertility outpatient clinic, Niigata University Hospital, Niigata, Japan. PATIENT(S): Thirteen women with PCOS and insulin resistance. INTERVENTION(S): Troglitazone (400 mg/d) was administered for 12 weeks. MAIN OUTCOME MEASURE(S): Insulin and other hormone (gonadotropins, androgens) levels; various parameters relating to glucose and lipid metabolism before, during, and after troglitazone administration; and ovulation rate. RESULT(S): The mean (+/-SD) fasting insulin concentration was significantly reduced, from 18.3+/-8.9 to 10.5+/-7.1 microU/mL. The LH level was reduced from 9.7+/-3.4 to 4.8+/-3.9 mIU/mL and the testosterone level was reduced from 0.9+/-0.5 to 0.5+/-0.3 ng/mL in accordance. Atherosclerotic lipid levels also were normalized. Before troglitazone administration, the ovulation rate during clomiphene citrate therapy was 34.9% per cycle (15/43). This increased significantly to 72.7% (8/11) during troglitazone coadministration. Further, an ovulation rate of 42.3% (11/26) was achieved with troglitazone alone. CONCLUSION(S): In women with PCOS and insulin resistance, the reduction of hyperinsulinemia that is produced by troglitazone improves the hyperandrogenism that characterizes PCOS, restoring ovulation.

Polycystic ovary syndrome. Insulin resistance and ovulatory responses to clomiphene citrate.

OBJECTIVE: To evaluate insulin secretion detected on oral glucose tolerance testing in relation to clomiphene citrate (CC) responses in women with polycystic ovary syndrome (PCOS). STUDY DESIGN: A total of 37 PCOS women were enrolled. Plasma baseline levels of gonadotropins, sex hormone binding globulin, androstenedione, dehydroepiandrosterone sulfate and testosterone (T) were determined. Insulin and glucose serum concentrations were analyzed in all samples after a 75-g oral glucose tolerance test. A dose of 100 mg/d of CC was given for ovulation induction. Ovulation was detected by ultrasonographic observation in conjunction with a serum progesterone level. RESULTS: During three consecutive cycles of CC administration, two or more anovulatory cycles were observed in 12 of 37 PCOS women (defined as CC nonresponders). The serum T level and the area under the curve (AUC) of insulin were significantly higher in CC nonresponders. The receiver operating characteristic curve of the AUC of insulin provided the most appropriate cutoff point for the prediction of CC resistance. CONCLUSION: We found higher androgen secretion and insulin resistance in CC nonresponders. The AUC of insulin was the most reliable method for the prediction of CC resistance associated with insulin resistance.

[Combination of transcatheter arterial infusion of SMANCS and embolization (SMANCS-TAE) for hepatocellular carcinoma--second report].

Patients with unresectable hepatocellular carcinoma (hepatoma) with hypervascularity were treated by SMANCS-TAE. A superselective catheterization technique was used to inject gelatin sponge particles after administration of SMANCS. In 30 patients of first hepatoma treated by SMANCS-TAE. Grade 4 was obtained after 1.7(1-3) courses. The 2-year survival rate was 22%. Some of the 24 patients of second hepatoma treated by SMANCS-TAE have survived over 2 years. Sixteen patients with advanced hepatoma (Vp2-3 or T4) were treated only by SMANCS injection, but none survived over 1 year. SMANCS-TAE appears to have the same potential and safety as L-TAE, when used selectively. Moreover, we can reduce the course of treatment and obtain good QOL for hepatoma patients except in advanced cases.

Mutational analysis of BRCA1 gene in ovarian and breast-ovarian cancer families in Japan.

We analyzed the alteration of BRCA1 in DNA obtained from 83 individuals of 13 Japanese site-specific ovarian cancer families and 6 breast-ovarian cancer families. Six germline mutations were detected in 7 families, which consisted of 4 breast-ovarian cancer and 3 site-specific ovarian cancer families, by single-strand conformation polymorphism analysis, followed by direct sequence determination. The mutations included three frameshifts, two nonsense mutations, and one missense mutation causing loss of a zinc-binding motif. The frequency of loss of heterozygosity at the microsatellite markers on the BRCA1 gene was 57% (8 of 14 cases) in site-specific ovarian cancer families, and 100% (6 of 6 cases) in breast-ovarian cancer families. All tumors of the patients carrying a mutation of BRCA1 showed deletion of wild-type alleles, implicating BRCA1 as a tumor suppressor gene. These results suggest that germline mutations of the BRCA1 gene play an important role in the carcinogenesis of breast and/or ovarian cancer in a majority of breast-ovarian cancer families and in some site-specific ovarian cancer families.

Direct intratumoral gene transfer of the herpes simplex virus thymidine kinase gene with DNA-liposome complexes: growth inhibition of tumors and lack of localization in normal tissues.

To constitute the site-specific expression of the herpes simplex virus thymidine-kinase (HSV-TK) gene in tumor cells, we have assessed the promoter function of the simian virus 40 (SV40) promoter and the 5'flanking region of c-erbB-2 gene using a luciferase-expressing reporter plasmid. After the transfection of the luciferase plasmid directed by the promoter region of c-erbB-2 gene, a large amount of luciferase activity was observed in c-erbB-2-expressing cells (Colo201, MCF-7, and HEC1-A), while none was detected in cells with no expression of c-erbB-2 protein (HRA and KF cells). On the other hand, a high level of luciferase activity was detected in all tumor cell lines tested, when the transfection was performed with SV40 promoter. The repeated transfection of the liposome-conjugated HSV-TK gene regulated by the SV40 promoter or by the promoter region of c-erbB-2 gene with cultivation in 100 micrograms/ml of aciclovir for 5 days in vitro resulted in growth inhibition for all four cell lines examined or for only c-erbB-2-expressing cells in the presence of SV40 promoter or c-erbB-2 promoter, respectively. Finally, direct injection of the DNA-liposome complex into established tumors in the presence of 50 mg/kg of aciclovir led to significant tumor volume reduction in all three tumors tested when SV40 promoter was employed. However, this anti-tumor effect was noted only in c-erbB-2-positive cells (Colo201 cells) upon intratumoral injection of HSV-TK gene regulated by c-erbB-2 promoter. In the case of intratumoral gene transfer, foreign DNA was detected in only one of seven mice by polymerase chain reaction (PCR) analysis performed 7 days following injection. When PCR analysis was carried out at 14 or 21 days following injection, no DNA signal was found at all. However, DNA was detected in several normal tissues at all three times tested in the case of intravenous injection. No abnormalities were seen in histologic examinations of normal tissues or in serum biochemical parameters following DNA liposome delivery. These results suggest that the direct gene transfer of HSV-TK gene regulated by tumor-specific transcriptional units may be one of the most clinically promising of the selective genetic strategies against cancer.

[Genetic testing of BRCA1 for familial ovarian cancers].

Ovarian cancer is the most lethal kind of gynecological malignancy. Though combination therapy has improved, the prognosis is still poor. It is a risk factor to have a member affected with breast or ovarian cancer in one's family or to have been affected with these diseases. Genetic testing will more effectively enable earlier diagnosis and cure of such diseases. Including the selected results of familial ovarian cancers, we discuss the effectiveness and difficulty of genetic testing of BRCA1 in Japan.

Accumulation of Tc-99m HMDP in hepatic metastasis from colon carcinoma without detectable calcification.

A 51-year-old man was found to have hepatic metastasis from colon carcinoma. Bone scintigraphy with 99mTc-hydroxymethylene diphosphonate showed a ringlike accumulation corresponding to the hepatic mass, which was without calcification on computed tomography. Treatment with chemotherapeutic agents was started. After 4 months, computed tomography showed that the mass was smaller than before and that the margin was calcified. The extraosseous accumulation of the radionuclide may be evidence supporting earlier speculation on calcification that could not yet be detected by computed tomography.

Studies on the cytogenetic and endocrinologic background of spontaneous abortion.

OBJECTIVE: To determine whether chronic menstrual irregularity with elevated LH levels has a role in spontaneous abortion. DESIGN: In 41 consecutively examined women with spontaneous abortions occurring within 11 complete gestational weeks, fetal chromosomal analyses were performed. The differences of endocrinologic background in relation to the result of fetal chromosomal analyses were investigated. MAIN OUTCOME MEASURES: Endocrinologic profiles, including menstrual history, basal hormone levels (LH, FSH, and PRL), ovarian morphology on ultrasound, and fetal chromosomal karyotypes. RESULTS: The women with normal fetal karyotypes compared with those with abnormal fetal karyotypes showed higher frequency of menstrual irregularity (47.1% versus 8.3%), elevated basal LH levels (7.42 +/- 5.12 versus 4.41 +/- 1.39 mIU/mL; conversion factor to SI unit, 1.00), and higher frequency of polycystic ovary (41.2% versus 8.3%). CONCLUSION: Elevated LH-polycystic ovary may be involved in the genesis of spontaneous abortions that are not explainable by fetal chromosomal abnormality.

Establishment of two distinct anti-cardiolipin antibody-producing cell lines from the same individual by Epstein-Barr virus transformation.

Two anti-cardiolipin antibody (ACA)-producing cell lines were established, using Epstein-Barr virus transformation followed by a repeated cluster-picking from the same individual with antiphospholipid syndrome who had a history of 8 consecutive fetal losses and deep venous thrombosis. Characterization of the two ACAs derived from these cell lines revealed that one (Ab-019, subclass IgM, kappa) reacted exclusively with cardiolipin and showed strong lupus anticoagulant activity, while the other (Ab-226, subclass IgM, lambda) reacted with negatively charged phospholipids such as phosphatidylserine and phosphatidylglycerol, as well as cardiolipin. Furthermore, Ab-226 showed reactivity with human umbilical vein endothelial cells whereas Ab-019 did not. It is suggested that ACA is heterogeneous even in the same individual, and that reactivity against negatively charged phospholipids corresponds to reactivity against endothelial cell.

Serum erythropoietin levels in gynecologic cancer patients during cisplatin combination chemotherapy.

To investigate the participation of erythropoietin (Epo) in anemias induced by cisplatin combined chemotherapy, serum Epo levels were measured by radioimmunoassay on a serial basis in eight patients with gynecologic cancer undergoing this chemotherapy. The data demonstrated that serum Epo levels, the mean level before chemotherapy being 20.1 +/- 6.6 mU/ml, were significantly elevated after the first course of the chemotherapy (52.1 +/- 32.2 mU/ml; P less than 0.05) when anemia was not evident. The serum Epo level continued to increase as the course of chemotherapy advanced, and furthermore, patients with normocytic anemia after a multiple course of this chemotherapy still showed high Epo levels (115.2 +/- 53.5 mU/ml) that were appropriate for given degrees of anemia. It is suggested that cisplatin combined chemotherapy caused the elevated serum Epo levels through an unknown mechanism other than anemia, and that in anemias induced by cisplatin, Epo deficiency is not evident.

Influence of immunotherapy on the cellular immunity of unexplained recurrent aborters.

Changes in lymphocyte subsets in whole blood were analyzed sequentially by flow cytometry with an automated leukocyte differential system in 15 patients with unexplained recurrent spontaneous abortions, each of whom underwent vaccination(s) with her husband's lymphocytes. Mitogen responses of peripheral blood lymphocytes (PBL) were also examined in these patients. The reactivity of PBL against mitogens revealed no significant change in each patient before and after vaccination(s) with her husband's lymphocytes. The CD4:8 ratio was observed to decrease significantly during 22 and 28 days after the first vaccination with a significant increase in the percentage of T suppressor-cytotoxic (CD8) cells. This change was also observed after the second vaccination. The percentages of other subsets did not change significantly after vaccination(s). In 11 patients out of 15, the pregnancy continued successfully and correlated with a predominance of Ts/c (CD8) over TH/I (CD4) cells in the first trimester. These changes in lymphocyte subsets may indicate the induction of immune enhancing mechanisms and it is suggested that continuation of the predominance of Ts/c cells induced by immunotherapy might be important for the successful maintenance of pregnancy.

[Rapid etiologic diagnosis of epidemic keratoconjunctivitis].

We evaluated enzyme-linked immunosorbent assay (Adenoclone) in 253 cases of epidemic keratoconjunctivitis (EKC), pharyngeal conjunctival fever (PCF) and acute conjunctivitis. The clinical diagnosis due to adenoviral ocular infection was 86/114 (75%) in EKC, 26/50 (52%) in PCF and 25/89 (28%) in acute conjunctivitis, Adenoclone was positive only within 7 days after the disease onset. The specificity (87%) and sensitivity (74%) of Adenoclone were confirmed by isolate cell culture. Cases with corneal complication due to adenoviral infection show higher a value than the OD value in Adenoclone and the bulbar conjunctiva is a suitable site to detect adenoviral antigen. In this survey, isolated adnovirus type 4 was recognized in 53%, Ad8 in 14%, Ad3 in 13%, Ad37 in 5%, Ad19 in 2% and non typed Ad in 12%. Clinical findings due to Ad4 showed EKC in 50%, PCF in 30% and acute conjunctivitis in 20% respectively.

Cytotoxic antibody against trophoblast and lymphocytes present in pregnancy with intrauterine fetal growth retardation and its relation to anti-phospholipid antibody.

To elucidate immunological mechanisms involved in the genesis of intra-uterine fetal growth retardation (IUGR), an in vitro cytotoxicity assay against normal trophoblast and lymphocytes was performed. The data demonstrated the existence of cytotoxic antibody directed against trophoblast exclusively in the IgG fraction of the sera of 9 out of 15 mothers with IUGR, but in none of the sera from normal pregnant women. This antibody showed differential reactivity patterns that may be indicative women. This antibody showed differential reactivity patterns that may be trophoblast in common. Out of 9 IUGR mothers with this cytotoxic antibody, in 6 cases (66.7%) chronic villitis was evident upon microscopic examination of the placenta, the frequency being significantly higher than that in IUGR mothers without cytotoxicity or in control mothers (P less than 0.02). It is suggested that in situ inflammatory change triggered by this antibody might lead to IUGR through chronic villitis. This antibody showed cross-reactivity with anti-negatively charged phospholipid antibody, as confirmed by an absorption experiment, indicating that the trophoblast antigenic stimuli in pregnancy can induce the production of various autoantibodies including anti-phospholipid antibodies. These results are of interest in relation to the pathogenesis of autoimmune diseases.

[Effects of anti-toxic agents in septic shock].

From 1975 to 1987, we had 56 patients of septic shock in the Department of Surgery. Multiple organ failure occurred in many septic patients. Glucocorticoids inhibited the secretion of chemical mediators (histamine, serotonin and bradykinin) and then prevented septic shock. Intravenous administration of dexamethasone showed no change in amounts of leukotrienes (LTC4, LTD4, LTE4) in venous blood in peritonitis rats. Dexamethasone treatment of septic rats corrected FDP and nearly normalized PEP values. When glucocorticoid was given intravenously at the time of cecal incision, PFKase, PKase, G6Pase and PEPCK were stimulated, respectively. Protease inhibitor FUT-175 was infused in 5% dextrose (0.1mg/ml/hr) in septic rats. Survival time was 12.1 +/- 2.3 hour in FUT-175 group and 6.6 +/- 1.1 hr without FUT-175. In FUT-175 injected rats G6P decreased by 20%, FDP increased 50% and lactate doubled. PEP levels increased 30% above peritonitis values. The amounts of leukotrienes (LTC4, LTD4, LTE4) in venous blood were gradually decreased by pretreatment with the specific 5-lipoxygenase inhibitor AA-861 after peritonitis. Specific treatments in septic shock should be instituted administration of glucocorticoid, antibiotics, protease inhibitor and lipoxygenase inhibitor. The importance of septic shock as a factor contributing to organ failure must be acknowledge. We believe that the prompt and efficacious treatment of septic shock is the best therapy.

[Mitral valve prolapse in patients with surgically-closed atrial septal defect].

To evaluate the prevalence, causes and clinical significance of mitral valve prolapse (MVP) associated with surgically-closed atrial septal defect (ASD), 90 patients (M: 41, F: 49) were studied using two-dimensional and color-coded Doppler echocardiography. Among the 90 patients, preoperative echocardiograms were available in 27. MVP was found in 21 of the 27 patients (78%) preoperatively, but it was found in 59% (16/27) postoperatively. In total, MVP was detected in 50 of the 90 patients (56%) postoperatively. The postoperative MVP group had higher pulmonary-to-systemic flow ratios (3.6 +/- 1.9 vs 2.8 +/- 1.1, p less than 0.05) and higher mean pulmonary arterial pressures (21 +/- 11 vs 13 +/- 5 mmHg, p less than 0.01) at the time of surgery. Between the two groups with or without MVP postoperatively, there was no difference (p less than 0.05) in age at surgery, the postoperative duration and left ventricular (LV) deformity index both in pre- and postoperative states. A mitral regurgitant (MR) murmur was recorded in seven patients postoperatively. However, only two had clinically severe MR. It was concluded that MVP is frequently detected in patients with closure of ASD and it is related neither to degree of the LV deformity nor to age at operation; rather, it is related to the severity of the preoperative hemodynamic state. Clinically significant MR is rare in the postoperative period.

A new polyvinylchloride blood bag plasticized with less-leachable phthalate ester analogue, di-n-decyl phthalate, for storage of platelets.

To compare changes in platelets stored in the new di-n-decyl phthalate (DnDP)-plasticized polyvinyl chloride (PVC) bag with those in a di-(2-ethylhexyl) phthalate (DEHP)-plasticized PVC bag, single-donor apheresis platelet concentrates (PCs), 133 +/- 11 x 10(7) platelets per ml (n = 7), were stored with 94 +/- 3 ml of plasma in a new 1-liter bag with a surface area of 44 +/- 7.1 cm2 per 10(10) platelets. Oxygen and carbon dioxide gas diffusion properties of PVC-DnDP films were respectively, 1.6 and 2 times those of standard PVC-DEHP films. The amounts of DnDP leaked into the plasma of PCs were low at 0.58 +/- 0.06 mg per bag after 5-day storage, which is about one-eightieth the amount of DEHP leaked. The pH of PCs in PVC-DnDP bags amounted to 6.99 +/- 0.03 after 5-day storage, with glycolysis accelerated somewhat in the new bags. However, the platelet oxygen consumption was no different from that in the PVC-DEHP bags. Platelet aggregation and responses to hypotonic shock were significantly better in the new bags at the end of storage. Shape changes of platelets into spherical forms with dendrites were more frequently observed in PVC-DnDP bags than in PVC-DEHP bags. The study indicated that platelets stored in the new DnDP-plasticized PVC bags have retained aggregation and responses to hypotonic shock more than platelets in the PVC-DEHP bags, but spherical forms and anaerobic metabolism increased in the new bags.