Ascites production

Inoculation and Aseptic Transfer Monitoring of Growth Parameters and Control Containment and Containment Control Ascites Production Mouse Colony... 

The culture of hybridomas, thereby producing monoclonal antibodies, may be undertaken by ascites production or by direct animal cell culture. Ascites production entails injection of the hybridoma cells into the peritoneal cavity of mice (the mice essentially serve as a live fermentation chamber). The transplanted hybridoma cells produce antibody as they grow. Ascitic fluid collects in the cavity, which contains high concentrations (up to 15 mg/ml) of the desired antibody. On average, 5 ml of this fluid can be extracted per mouse. Most of the earlier monoclonal antibody preparations were produced in this manner, e.g. OKT-3, the first monoclonal antibody to be approved for therapeutic use by the FDA (see later), is produced using this strategy. 

Ascites production, however, suffers from a number of drawbacks. It is costly, and the product is contaminated by significant levels of various mouse proteins, rendering subsequent downstream processing more complex. As a result, monoclonal antibody production by standard animal cell culture techniques has become the method of choice for the production of pharmaceutical-grade monoclonal antibody preparations. 

Ascites Production. Anti-albumin clones HSA-1 and HSA-2 were thawed, subcloned, and reassayed by ELISA on human albumin, and a rapidly growing subclone was expanded for ascites production. Forty male BALB/c mice were primed with an intraperitoneal injection of 0.5 mL pristane on day 0. On day 14, 2 X 106 cells were injected into each mouse. Ascites fluid was collected over a 1-week period and pooled. Cells and other debris were removed by centrifugation. Approximately 180 mL of ascites fluid was obtained. 

The progression and growth of ovarian carcinoma are also dependent on chemokine-mediated angiogenesis. In one study, the in vitro production of CXCL8 by five human ovarian cancer lines correlated with tumor neovascularization and cancer-related death when implanted into the peritoneum of immunocompromised mice, whereas VEGF production correlated only with ascites production after implantation, but basic FGF did not correlate with the outcome [70]. This concept was further substantiated in patients with ovarian cancer where ascites fluid angiogenic activity directly correlated to CXCL8 [71]. 

Ascites production was induced by intraperitoneal injection of ATCC sarcoma TG180 (200 /rl, =10 cells). 

A. In Vivo (Ascites) Production of Monocionai Antibodies (Mabs)... 

The pathophysiologic mechanisms of portal hypertension and of cirrhosis itself are entwined with the mechanisms of ascites (Fig. 19-3). Cirrhotic changes and the subsequent decrease in synthetic function lead to a decrease in production of albumin (hypoalbuminemia). Albumin is the major intravascular protein involved in maintaining oncotic pressure in the vascular system low serum albumin levels and increased capillary permeability allow fluid to leak from the vascular space into body tissues. This can result in peripheral edema, ascites, and fluid in the pulmonary system. The obstruction of hepatic sinusoids and... 

Diuretics are often required in addition to the sodium restriction described previously. Spironolactone and jurosemide form the basis of pharmacologic therapy for ascites. Spironolactone is an aldosterone antagonist and counteracts the effects of activation of the renin-angiotensin-aldosterone system. In hepatic disease not only is aldosterone production increased, but its half-life is prolonged because it is hepatically metabolized. Spironolactone acts to conserve the potassium that would be otherwise excreted because of elevated aldosterone levels. 

The formation of 0-seryl or 0-prolyl esters (Figure 1) of certain N-hydroxy arylamines has been inferred from the observations that highly reactive intermediates can be generated in vitro by incubation with ATP, serine or proline, and the corresponding aminoacyl tRNA synthetases (11,12,119). For example, activation of N-hydroxy-4-aminoquinoline-l-oxide (119,120), N-hydroxy-4-aminoazobenzene (11) and N-hydroxy-Trp-P-2 (121) to nucleic acid-bound products was demonstrated using seryl-tRNA synthetase from yeast or rat ascites hepatoma cells. More recently, hepatic cytosolic prolyl-, but not seryl-, tRNA synthetase was shown to activate N-hydroxy-Trp-P-2 (12) however, no activation was detectable for the N-hydroxy metabolites of AF, 3,2 -dimethyl-4-aminobiphenyl, or N -acetylbenzidine (122). 

The development of ascites is related to systemic arterial vasodilation that leads to the activation of the baroreceptors in the kidney and an activation of the renin-angiotensin system, with sodium and water retention and vasoconstrictor production. 

Monoclonal antibodies can be produced not only in a cell culture but also in live animals. When injected into mice (in the peritoneal cavity, the gut), the hybridoma cells produce tumors containing an antibody-rich fluid called ascites fluid. Production in cell culture is usually preferred, as the ascites technique may be very painful to the animal and if replacement techniques exist, may be considered unethical. The process of producing monoclonal antibodies described above was invented by Georges Kohler. Cesar Milstein, and Niels Kaj Jeme in 1975 they shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery (http //en.wikipedia.org/ wiki/Antibody). 

The aim of the work was evaluation of the ability of photoexcited fullerene C60 and synthesized fullerene C60-containing composites to generate reactive oxygen species (ROS) and to perform comparative analysis of the state of the cells of two types (normal ones - thymocytes, and malignant ones - the cells of ascite Erhch carcinoma [EAC] and leucosis L1210) by such indexes as viability, content of LPO products, MTT test, and DNA fragmentation upon incubation in the presence of photoexcited fullerene C60. 

Literature data on cytotoxic effects of photoexcited fullerene C60 are controversial. In the studies on transformed B-lymphocytes of Raji fine, phototoxic action of water-soluble carboxy-C60 was not revealed even upon its concentration of 5 x 10 5 M (Irie et al., 1996). In the study (Kamat et al., 2000) damaging effect of fullerenes C60 in dependence on intensity of irradiation toward CHO cells has been demonstrated. Using microsomal fraction of rat liver that was treated with C -cyclodextrin complex, it was shown that already in 5-30 min after UV-irradiation the accumulation of LPO products occurs that is suppressed by antioxidants like ascorbic acid and a-tocopherol. Similar effect of fullerenes C60 has been revealed in microsomal fraction of the cells of ascitic sarcoma 180 (Kamat et al., 2000). 

Optimal fermentation parameters have been well established and air-lift, stirred tank, and hollow fibre systems have all been used. At commercial scale, fermentation volumes in excess of 1000 litres can be used, which can yield 100 g or more of final product. While hybridoma growth is straightforward, production levels of antibody can be quite low compared with ascites-based production systems. Typically, fermentation yields antibody concentrations of 0.1-0.5 mg/ml. Removal of cells from the antibody-containing media is achieved by centrifugation or filtration. An ultrafiltration step is then normally undertaken in order to concentrate the filtrate by up to 20-fold. 

OKT-3 was first approved for general medical use in the USA in 1986. Its indication was the treatment of acute kidney transplant rejection (Table 10.4). OKT-3 is produced via ascites grown in mice. The intact antibody is subsequently purified by a combination of ammonium sulphate fractionation and anion exchange chromatography. Despite its therapeutic effectiveness, the product does display some limitations. Its antigenicity in humans (the HAMA response) is one obvious factor which limits its prolonged use. 

Cirrhosis and other Uver diseases may result in the formation of excessive amounts of fluid in the abdomen ascites). The primary causes of ascites are usually elevation of pressure in the portal vein and a decreased amount of hepatic plasma protein production. Both factors tend to reduce the ability of the vascular compartment to retain fluid. The resultant ascites may contribute to decreased appetite and respiratory difficulties, among other symptoms. When these symptoms are present, careful reduction in the fluid volume through the use of diuretics is desirable. 

Antitoxic effect. Sesame oil, adiministered to male Wistar rats, ameliorated hepatic and renal damage in a dose-dependent manner and increased survival in lipopolysaccha-ride-treated rats. It decreased lipid peroxide concentration in serum but not in liver and kidney. Serum nitrite production was unaffected by sesame oil ingestion, and the activity of xanthine oxidase was reduced in the lipopolysaccharide-challenged rats k Anti-tumor activity. Water extract of the dried seed, administered intragastrically to mice at a dose of 50 mg/animal daily for 5 days, was active on CA-Ehrlich-ascites, 18% increase in life-span. Intraperitoneal administration was active on Dalton s lyphoma and CA-Ehrlich-ascites, 19 and 39% increase in life-span, respectively ". Seed oil, administered to rats intraperito-neally with 1,2,5,6-dibenzanthracene or re-tene, was active on sarcoma ". 

Affinity chromatography is a particularly powerful procedure that can be used to purify IgG, subpopulations of IgG, or the antigen-bmdmg fraction of IgG present in serum/ascitic fluid/hybridoma culture supernatant. This technique requires the production of a solid matrix to which a ligand that has either affinity for the relevant IgG or vice versa has been bound. Examples of ligands useful in this context are. 

Pannell, R. and Milstein, C. (1992) An oscillating bubble chamber for laboratory scale production of monoclonal antibodies as an alternative to ascitic tumors J Immunol Methods 146,43—48... 

For many producers the ideal chicken for organic production is one of the dual-purpose breeds, i.e. one of those developed for both egg and meat production (Fig. 6.1). These breeds fit into organic poultry systems more appropriately than breeds developed specifically for egg or meat production. Many are heritage breeds and have a much lower incidence of health problems such as ascites and SDS than found with commercial meat breeds. However,... 

W15. Wong, C. K., Fung, K. P., Lee, C. Y., and Choy, Y. M., In vivo production of tumor necrosis factor for the treatment of mice bearing Ehrlich ascites tumor. Cancer Lett. 63,7-13 (1992). 

Depending on the use of the mAbs, certain adaptations may be required for their preparation. When large quantities of mAbs are required, in vivo production of the antibody in ascitic fluid is not practical, because it will require the use of a large number of animals. Thus, it is often easier to cultivate the antibodies in an appropriate in vitro culture medium. However, given the strict nutritional requirements of the hybridomas and their fragility in the face of osmolality, pH variations, and the accumulation of metabolites, the production of large quantities of antibodies in vitro will necessitate special care. 

Unlike the production of other proteins from animal cells, mAbs can also be produced in vivo by inducing ascitic tumors in laboratory animals,... 

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