Plasma levels of natriuretic peptides and year-by-year blood pressure variability: a population-based study.

Augmented blood pressure (BP) variability over various time periods has been recognized as a risk factor for cardiovascular diseases. Both atrial and B-type natriuretic peptides (ANP and BNP) are secreted in response to volume or pressure overload to the heart, exerting natriuretic and vasodilator actions. In this study, we examined the relationships between year-by-year BP variability and plasma levels of ANP and BNP in the general population. Study subjects were local residents receiving an annual heath checkup, who had an estimated glomerular filtration rate of >30 ml min-1 per 1.73 m2 and no history of heart disease. Of those, we selected 314 subjects that received checkups at least five times over the past 6 years. BP variability year-by-year was retrospectively evaluated by s.d., coefficient of variation, average real variability and variation independent of the mean of BP values of 6 or 7 time points. The four parameters of BP variability were each found to significantly correlate with logarithmically transformed ANP and BNP levels by simple regression. When classified by quartiles of s.d. of systolic BP, the highest quartile group showed significantly higher levels of the natriuretic peptides compared with other groups. Multivariate analyses revealed that BP variability was an independent determinant for the ANP and BNP levels. In conclusion, augmented year-by-year BP variability over the past 6 years was associated with elevation of plasma levels of ANP and BNP, suggesting a possible relationship between the BP variability and cardiac load, in the general population.

The validity and significance of substage IIIC by node involvement in epithelial ovarian cancer: impact of nodal metastasis on patient survival.

OBJECTIVE: The aim of this study was to examine whether there is a possible difference in patient survival (1) between node-positive and node-negative groups for Stage I, II, IIIA+B, and IIIC disease and (2) between node-positive Stage I through IIIB disease and Stage IIIC disease. METHODS: Medical records of 125 patients with Stage I, II, and III epithelial ovarian cancer were retrospectively analyzed for clinical and histological characteristics with special reference to node-positive or -negative status, and patient survival by lymph node status was compared for FIGO intraperitoneal stage. RESULTS: One hundred eleven of 125 patients were explored for retroperitoneal and inguinal nodes and divided into a node-positive group [n = 35 (31.5%)] and a node-negative group [n = 76 (68.5%)]. Stage III disease and histological grade 3 disease were significantly more frequent in the node-positive group in comparison with the corresponding figures in the node-negative group (P = 0.0007 and P = 0.0005). The 10-year survival was not significantly different between the node-positive and the node-negative group for each of Stage I, II, IIIA+B, and IIIC disease. However, survival was considerably worse in the node-positive group compared to that in the node-negative group for each of Stage I and II disease, and was significantly worse for Stage I+II disease (P = 0.0172). The survival was significantly worse in the node-positive group compared to that in the node-negative group also for Stage I through IIIB disease taken as a whole (P = 0.0212), although not for Stage IIIA+B disease alone. On the other hand, patient survival of node-positive Stage I through IIIB disease, taken as a whole, was significantly better than that in Stage IIIC disease (P < 0.0001). CONCLUSIONS: It might not be valid, on the basis of patient prognosis, to group node-positive Stage I, II, and IIIA+B disease into a criterion of Substage IIIC together with Stage III disease of abdominal implants greater than 2 cm in diameter outside the pelvis. For further evaluation of the prognostic significance of node involvement, survivals should be compared among patients with de novo node disease, with node disease removed, and otherwise similar patients without node disease, ideally in a prospective randomized trial with a large number of patients.

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones.

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min -1 at 100 g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 x 10(6) cells ml -1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process. (Himmelfarb et al. 1969; Feder and Tolbert 1983) usually have the problem of filter clogging, and perfusion culture processes with gravitational cell settling (Tokashiki and Arai 1989) are limited in scale-up because of low separation efficiency. Recently centrifugal separation of cells from culture medium has been used for perfusion culture processes (Hamamoto et al. 1989; Tokashiki et al. 1990; Apelman 1992; Jäger 1992). This process essentially has a high separation efficiency and can avoid further operational problems, such as filter clogging, by the type of filtration. To investigate the scale-up feasibility of the centrifuge culture system, we have developed a new type of centrifuge with four layers of spiral cell-settling zones to increase the separation area, and a novel cell separation and withdrawal method using a perfluorocarbon.

Perfusion culture apparatus for suspended mammalian cells.

A variety of processes have been proposed for mammalian cell culture in the commercial production of useful substances (e.g., monoclonal antibodies, therapeutic and diagnostic proteins). Among them, the perfusion culture of suspended non-immobilized cells is the most advantageous. Perfusion culture can be classified by the separation process of suspended cells from the culture mixture into three types, namely filtration, gravitational settling and centrifugation. From a commercial point of view, the present situation and technical problems of suspended cell perfusion culture will be reviewed based on the three types. The recent development of perfusion culture has been carried out mainly on the filtration separation process, but the centrifugation process seems to have a promising future because of operation stability and scale-up feasibility. The reasons will be explained in details.

Transferrin recycling perfusion culture of hybridoma cells.

A perfusion culture of hybridoma cells in serum-free medium recycling transferrin was carried out, which greatly reduced the level of transferrin that was needed. The culture was maintained even without supplying transferrin for nine days. IgG concentration reached 1.1 mg ml-1 in a month of recycling and its ratio to the total protein was 45.8%. The affinity of the antibody did not decrease and no degradation was observed after long recycling period. The cell density under recycling condition was 2-3 times higher than that without recycling. It was indicated that there was autocrine growth promoting activity in the culture supernatant.

Diagnostic hand anomalies in Smith-Magenis syndrome: four new patients with del (17)(p11.2p11.2)

We report clinical and cytogenetic findings of 4 children (2 boys and 2 girls) with the Smith-Magenis syndrome. All 4 patients had an interstitial deletion of 17p: del(17) (p11.2p11.2). Their clinical manifestations included brachycephaly, midface hypoplasia, prognathism, upper lip eversion, short and broad hands with short fingers, clinodactyly of the fifth fingers, fingertip pads, moderate mental retardation, and behavior problems. Analysis of the metacarpophalangeal pattern profiles in patient 2 showed progressive shortness from the metacarpals to the proximal, middle, and the distal phalanges. The fingerpads observed in all 4 patients have hitherto been noted in only one of 26 previously reported patients with the syndrome. These findings serve as a useful clue to the diagnosis of the syndrome.

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge.

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100-500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10(7) cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.

High density culture of mouse-human hybridoma cells using a perfusion culture apparatus with multi-settling zones to separate cells from the culture medium.

Mouse-human hybridoma X87X cells were cultivated using a novel perfusion culture apparatus provided with three-settling zones to separate the cells from the culture medium by gravitational settling. The maximum viable cell density in a serum-free culture medium attained 3.0×10(7) cells/ml, when the specific perfusion rate was set to 2.3 vol day(-1), and monoclonal antibody was continuously produced. These results were almost the same as those in the perfusion culture vessel with one settling zone and revealed that the process with a plurality of settling zones is a promising one for scale-up of a gravitation type of perfusion culture vessel.

Production of human-mouse chimeric antibody by high cell density perfusion culture.

Two mouse myeloma cell lines which were transfected with chimeric mouse variable-human constant immunoglobulin heavy and light chain genes have been cultured at high cell density in a settling perfusion culture vessel to produce chimeric antibody specific for human common acute lymphocytic leukemia antigen (cALLA).J558L transfectant proliferated well in a serum-free medium (ITES-eRDF) to a viable cell density of 3.7×10(7) cells/ml and produced chimeric antibody to a maximum value of 60 µg/ml in 120 ml scale vessel. X63Ag8.653 transfectant reached a density of 1.9×10(7) cells/ml in 1.2 I scale vessel in serum supplemented medium (10% FCS-eRDF) and produced chimeric antibody which consisted of chimeric gamma and chimeric kappa chains to a maximum value of 5.8 µg/ml.

High-Density culture of mouse-human hybridoma in serum-free defined medium.

Mouse-human hybridoma 4H11 cells producing anti-Pseudomonas sp. monoclonal antibody (IgA) grew in a serum-free medium supplemented with insulin, transferrin, ethanolamine, and selenite (ITES). The hybridoma could be applied to high-density culture in a serum-free medium supplemented with ITES, 0.5% BSA, egg yolk VLDL, and artificial blood FC-43 in a culture vessel equipped with hollow-fiber modules for medium exchange. Total cell density reached 1.1 x 10(7) cells/mL (viable cell density was 7.6 x 10(6) cells/mL), and the IgA productivity was around 20 mug/10(6) cells/day in the serum-free medium, which corresponded to the levels in serum-supplemented medium.

High cell density perfusion culture of hybridoma cells recycling high molecular weight components.

We have developed a high cell density and high product concentration culture system recycling high molecular weight components. The production of monoclonal antibodies in high concentration was performed by this culture system with mouse human hybridoma H2 and V6 cells in serum-free defined media.The concentration of IgG after 48 days culture of H2 cells in ITES-eRDF reached 2 mg/ml and the purity of IgG in culture fluid was 61%. In addition, high molecular weight components in serum-free media, such as transferrin or BSA, could be reduced to 5% of the original concentration.