The scale-up from 0.1 to 100 liter of a unit process system based on 3-mm-diameter glass spheres for the production of four strains of FMDV from BHK monolayer cells

This paper describes the scale-up from 0.1 to 100 liter of the unit process based on 3-mm-diameter glass spheres for the growth of BHK monolayer cells. The production of four strains of FMD virus at the 0.1-, 10-, and 100-liter scales was examined. Cell growth was estimated from measurements of the concentration of glucose in the growth medium, while the release of virus was inferred from measuring the concentration of LDH in the culture supernatant fluid. The yields of virus at 0.1-, 1-, and 10-liter scales were similar but that from the 100-liter version was somewhat lower. The reason for this lower yield and the method used to overcome it over outlined.     

Persistently low natural killer cell activity in normal adults: immunological, hormonal and mood correlates

This current study is concerned with the identification of the incidence of persistently low natural killer (NK) cell activity in an essentially healthy population of young adults, and the characterization of the psychological profile associated with this pattern of NK activity. Eighty-eight individuals, between the ages of 18 and 44 years, were accrued to this project. Subjects were tested serially, at baseline induction into the study, and at 2 and 4 weeks follow-up. Baseline assessment included a complete physical examination and laboratory work-up in order to exclude anyone ill at the time of enrollment, and to have complete physical status information on all subjects at the time of accrual. NK activity, urinary catecholamine levels and psychological status were also assessed at baseline, and at the specified follow-up intervals. Individuals with low NK activity were operationally defined as those having NK function either below the group mean, or below the group lowest quartile, at baseline and at two follow-up assessments. Results showed that in both univariate analyses, as well as in logistic regression models, age and the perception of environmental stressors or 'hassles' predicted persistently low NK activity. Younger subjects, who perceived environmental events to which they were exposed as more serious in nature, were more likely to exhibit a persistently low NK profile over time than older individuals who perceived daily events as less important to them.     

Effects of cytokines on in vitro growth of tumor-infiltrating lymphocytes obtained from human primary and metastatic liver tumors

Summary Tumor-infiltrating lymphocytes (TIL) were isolated from 22 human primary and metastatic liver tumors, and expanded in vitro in the presence of either interleukin-2 (IL-2, 100 U/ml) plus tumor necrosis factor (TNF, 1000 U/ml), IL-2 (1000 U/ml) plus IL-4 (1000 U/ml) or IL-2 (1000 U/ml) alone. TIL proliferated in culture in 20/22 cases. Among different cytokine combinations, TNF and IL-2 were most effective in promoting the outgrowth of CD3⁺ CD8⁺ T lymphocytes (mean ± SEM: 90%±5) in the cultures of TIL from primary liver tumors. Cytotoxicity against autologous tumor cells was demonstrated in all early cultures of TIL from primary liver cancers in the presence of IL-2 plus TNF. In contrast, cultures of TIL derived from colon cancer metastatic to liver had significantly lower levels of autotumor cytotoxicity and proportions of CD3⁺ CD8⁺ cells (40%±13) than those of TIL from primary liver tumors. The addition on day 0 of interferons ( or ) to TIL cultured in the presence of TNF and IL-2, significantly augmented cytotoxicity against autologous tumor. In contrast, incubation of TIL in the presence of IL-4 and IL-2 did not result in increased autotumor responses in the cultures of TIL from primary liver tumors. The expansion (-fold) of TIL (day 30) cultured in the presence of IL-2 alone compared to that in the presence of TNF and IL-2 was significantly greater for hepatocellular carcinoma (median, 280 vs 260) than for autologous peripheral blood lymphocytes (36 vs 27), cholangiocarcinoma (42 vs 51) or TIL from metastatic colon cancer (39 vs 30). Outgrowth of TIL in IL-2 plus TNF offers an opportunity for in vitro enrichment in cells with autotumor cytotoxicity in primary liver tumors. However, this cytokine combination was unable to promote and sustain growth of autotumor effectors from TIL in metastatic liver cancer.Supported by ACS grants IM27 077 and IM588 A (TLW) and Organ Transplant Program Project 1P01-CA-4744501 AZ     

Reflex cardiovascular responses caused by stimulation of pulmonary C-fibers with capsaicin in dogs

The purpose of these studies was to determine quantitatively the reflex cardiovascular responses to stimulation of the pulmonary C-fibers in dogs. We used a preparation in which the airway, pulmonary artery, and the pulmonary veins to the left lung were cannulated in situ. Ventilation and perfusion of the right lung maintained the animal in relatively normal homeostasis. Capsaicin, a decylenic acid amide of vanillylamine that selectively stimulates nerve endings of unmyelinated fibers (C-fibers), was injected into the left pulmonary artery in 5-ml boluses. Maximal reflex responses were obtained with concentrations as low as 0.8-1.6 X micrograms-1 X kg-1. Heart rate, hindlimb resistance, and left ventricular contractility were lowered transiently (the maximal responses showing declines of 40, 13, and 15.2%, respectively). As a result of these changes, combined with vasodilation in other resistance vessels, cardiac output fell 28% and blood pressure fell 35%. Interrupting the afferent neural pathway by severing the ipsilateral cervical vagus nerve eliminated these responses, confirming the distribution of their reflex origin. Although the role of these reflexes in homeostasis has not been decided, the results of this study suggest that the lungs of dogs, if appropriately stimulated, potentially can exert a major inhibitory influence on the neural regulation of cardiovascular function.     

Development of heart cells in culture: studies using an affinity purified antibody to a myosin light chain

Cultured neonatal rat heart cells can be used to study the factors that regulate cardiac contractility and myocyte development in vitro. An antibody to the 26,000 dalton light chain of myosin (MLC1), has been produced and purified on a Sepharose 4B affinity column prepared with rat heart myosin. When primary cultures of myocytes are studied by indirect immunofluorescence using this antibody a predictable pattern of myofibrillar structure is observed to develop over 72 h. This myosin cytoskeleton is highly organized and the myosin fibrils exhibit cross striations. The antibody does not stain non-muscle heart cells and there is no evidence for myocyte division in culture. The qualitative immunofluorescent pattern of myosin organization is the same in both spontaneously beating and in non-contracting cells.     

The degradation of canavanine by jack bean cotyledons

Summary Germinating jack bean cotyledons liberated ¹⁴CO₂ when fed ¹⁴C-guanidoxy-canavanine but did not accumulate any ¹⁴C-compounds other than the applied canavanine. This suggested that the canavanine was being degraded by the action of canavanase to canaline and urea, the urea then being converted to ammonia and carbon dioxide by the action of urease. Hydroxyurea and acetohydroxamic acid (both inhibitors of urease activity) strongly inhibited the liberation of ¹⁴CO₂ from ¹⁴C-guanidoxy-canavanine by the cotyledons but neither compound induced the accumulation of ¹⁴C-urea within the tissues. This inhibitory action of hydroxyurea on ¹⁴CO₂ output was thought to be due at least in part, to this inhibition of canavanase activity.     

Effect of Enzymes on the Composition and Structure of Chromobacterium violaceum Cell Envelopes

Cell envelopes of Chromobacterium violaceum were isolated and treated under controlled conditions with trypsin, Pronase, lipase, phospholipase C, lysozyme, and a mixture of enzymes produced by a bacteriolytic Pseudomonas sp. After each enzyme treatment, losses in dry weight, protein, lipid, carbohydrate, 2,6-diaminopimelic acid, and total phosphorus were determined. Electron-microscopic examination of the enzyme-treated envelopes indicated complete or partial loss of envelope rigidity or some envelope fragmentation, or both. Each enzyme hydrolyzed at least one envelope component and liberated several others into the supernatant fluid, where they appeared as nondialyzable particulate components, identified by means of electron microscopy. Unlike the other enzymes, the Pseudomonas sp. enzyme mixture partially liberated all major envelope components except phosphorus, heptose, and 2-keto-3-deoxy octonic acid. In spite of these large losses, the envelopes preserved some features of their integrity and elongated shape.