Time course localization of immunoglobulin M monoclonal antibody and its fragments in leukemic tumor-bearing mice

Summary In vivo localization of a mouse monoclonal antibody (F₂-10.23 IgM) binding leukemic L 1210 cells was studied in DBA/2 mice bearing an L 1210 tumor. F(ab)₂ fragments were prepared and their specific binding to L 1210 cells was analyzed by flow cytofluorometry. Radiolocalization studies were performed by using ¹²⁵I- or ¹³¹I-labeled IgM monoclonal antibody or its F(ab')₂ fragments to ascertain their capacity to visualize the L 1210 tumor. F(ab)₂ fragments were cleared more rapidly than the whole IgM; the clearance was as fast in healthy as in tumor-bearing mice. The tumor-to-muscle ratio observed 24 h after injection of ¹²⁵I-radiolabeled F(ab)₂ fragments and ¹²⁵I-radiolabeled IgM was 10; the radioactivity level in the blood with F(ab)₂ fragments was lower than with IgM, and so -camera imaging was workable with F(ab)₂ fragments without background substraction. The tumor localization was studied over a period of 5 days by recording the distribution of the iodinated fragments in the tumor-bearing leg compared with that in the normal leg, and by computer analysis of the region of interest. F(ab)₂ fragments gave better results than intact IgM in tumor visualization. Nevertheless, the rapid clearance of this antibody or its F(ab)₂ fragments make them hardly suitable as carriers of toxic drugs. Abbreviations used are: MEM Minimum essential medium; SDS sodium dodecylsulfate; PAGE polyacrylamide gel electrophoresis     

Lymphokine regulation of human lymphocyte proliferation: Formation of resting G0 cells by removal of interteukin 2 in cultures of proliferating T lymphocytes

The question of whether lymphocytes which have once been activated and have completed one or several cell cycle(s) can return to the G₀ phase and stay ready for a new activation (G₀-G₁ transition), rather than simply die, was investigated. To do so interleukin 2 (IL-2) was removed from cultures of continuously proliferating human T lymphocytes and the formation of resting (G₀) cells was measured. Kinetic analyses in freshly prepared peripheral blood lymphocytes (PBL) revealed that the onset of detectable RNA synthesis and the appearance of structures binding the anti-Tac antibody occurred simultaneously. This allowed the expansion of the definition of G₀ T lymphocytes as cells having a low RNA (and DNA) content, and no Tac antigen. When cultured human T cells proliferating continuously by means of IL-2 were characterized in terms of their distribution in the cell cycle, 7 days after the initial PHA stimulation, it could be demonstrated that very few cells were in the G₀ phase, supporting the concept of direct S/G₂/M-G₁ transition. However, when IL-2 was removed from the cultures, the [³H]thymidine incorporation per 10⁴ cells and correspondingly the number of cells in the S/G₂/M and G₁ phases were reduced drastically and during the following 72-hr period, the number of G₀ cells increased markedly. Restimulation of such in vitro formed G₀ cells, under conditions permitting observation of their shift from the G₀ to G₀ phase, demonstrated that most cells could respond normally. Based on these observations, it was concluded that IL-2 not only ensures T-lymphocyte survival and proliferation, but IL-2 starvation induces many continuously proliferating T lymphocytes to stop cycling and to return to the G₀ phase of the cell cycle where they remain functional.     

Human autologous rosettes. IV. Their relation with interleukin 2 activity production and natural killer cells in cancer patients

Peripheral blood lymphocytes (PBL) of solid-tumor-bearing cancer patients produced a lower interleukin 2 (IL-2) activity after lectin stimulation than did those from normal subjects. Moreover natural killer (NK) cell activity and autologous rosette forming (ARF) cell rate are found significantly correlated with IL-2 production in these patients. No direct relation is observed between ARF cell ratio and NK cell activity in a given patient. A central role for IL-2 in cancer patient immune dysfunctions is suggested. Two lines of pathogenetic mechanisms are documented. First, PBL exhibited cellular function defects, namely, autologous receptor expression, IL-2 production, and NK activity. Second, these dysfunctions involved, at least partly, plasma factors. The possibility of specific deficiency, (e.g., thymic factors) is not documented. Conversely it is demonstrated that patient plasma contain immunosuppressive factor(s) that block(s) IL-2 production and ARF cell expression. Involvement of ARF cell receptor in T-cell activation is discussed.     

Stimulation par la lumière rouge lointain de la synthèse de la δ-aminolévulinate déshydratase des cotylédons de radis

Stimulation of de novo synthesis of δ-aminolevulinate dehydralasc of radishes grown under far-red light .
Density labelling studies of δ-aminolevulinate dehydratase (ALAD) in cotyledons of radish ( Raphanus sativus L. cv. Longue Rave Saumonée) seedlings demonstrate that far-red light stimulates de novo synthesis of ALAD and that the turn-over of this enzyme is very poor. Cycloheximide reduces considerably both the increase of ALAD activity and the incorporation of deuterium in ALAD, which indicates that ALAD synthesis depends upon cytoplasmic ribosomes.     

Effects of nitrogen source, calcium concentration and osmotic stress on protoplasts and protoplast-derived cell cultures of Pinus Pinaster cotyledons

The experiments described emphasize the effects of several factors crucial to the maintenance of cell divisions leading to increased cell numbers in suspension and colony formation from cotyledon protoplasts of Pinus Pinaster Ait. Osmotic potential of the incubation and culture media are critical. Reducing the osmolality from 680 mOsm kg H₂O⁻¹ during protoplast isolation to 610 mOsm kg H₂O⁻¹ during washing and culture was essential to achieve a high frequency of cell division. Survival of the cells beyond 3 weeks of culture occurs only if the calcium concentration is decreased from 5.6 m M to 1.5 m M . Glutamine as sole source of nitrogen shortens the lag phase of response of the protoplasts and increases their plating efficiency. After 6 weeks of culture, a combination of low osmolality (225 mOsm kg H₂O⁻¹) and high level of glutamine (40 m M ) is a prerequisite for obtaining actively growing cell suspensions.     

Use of microcalorimetry for the characterization of marine metabolic activity at the water-sediment interface

Metabolic processes occurring at the sea-water-sediment interface were studied using a circulation flow microcalorimeter. A methodology was developed to characterize rapid and global changes in metabolism and energy flow, not easily detectable with reductionist approaches. Sea water was pumped continuously, 5–10 mm above the sediment, in experimental microcosms; a 100-μm filter prevented passage of meiofauna. This “circulating interface” was taken through the microcalorimeter and from there to an oxygen electrode, and was returned to the microcosm. The microcosms were experimentally eutrophicated using peptone (4 mg·ml ^?1). The relationship between heat production and oxygen tension in the circulating interface has been compared with ATP production, ¹⁴CO₂ and [¹⁴C]particulate matter turnovers. Initial heat steady-state production rises to a peak of 130 to 180 μW·ml^?1 in 6 to 8 h after peptone treatment. The microcalorimetric peak is closely correlated with ¹⁴CO₂ turnover and partially correlated with micro-events on the pO₂ curve. ATP concentration and particulate-¹⁴C turnover increase constantly and then stabilize, with the establishment of a new heat production steady state. The approach provides an indication of the temporal behaviour of complex mixtures of microorganisms and ciliates at the water-sediment interface, and gives holistic measurements of energy flow after induced perturbation (eutrophication) of the ecosystem. Although many problems remain to be solved in this field, it is shown here that flow microcalorimetric measurements can be used to monitor the effects of addition of reagents like pollutants and nutrients.     

Ovarian and haemolymph titres of ecdysteroid during the gonadotrophic cycle in Diploptera punctata

The free (non-conjugated) ecdysteroid in the ovaries during the first gonadotrophic cycle of Diploptera punctata was identified as 20-hydroxyecdysone. The hormone, quantified by radioimmunoassay and by ultraviolet absorbance, was detectable in the ovary toward the end of vitellogenesis; the quantity increased rapidly during chorion formation. Ovaries with chorionated eggs contained 67 μg of 20-hydroxyecdysone per g fresh weight. The haemolymph free-ecdysteroid, not identified physicochemically, was quantified by radioimmunoassays. The highest concentration was observed at adult emergence; the titre declined between days 1–3 and then remained at a relatively constant level through oviposition (which occurs between day 7 and 8); titres in pregnant females were higher. Ovariectomized females exhibited the same pattern of ecdysteroid titres in the haemolymph as the sham operated controls throughout the period corresponding to the first gonadotrophic cycle. Thus the ovary may not be the only source of haemolymph ecdysteroid related to reproduction in adult females.     

Species variations amongst proteinases in liver lysosomes

Cathepsin B, H, L and D activities in liver lysosomes were compared between species. Although cathepsin B and D were detected in bovine, pig, chicken and rat liver, striking species differences were evident for cathepsin H and L. Cathepsin L activity was particularly high in chicken lysosomal extracts, but could not be detected in bovine and pig extracts. Whereas there was no significant cathepsin H activity in bovine extracts, rat liver lysosomal extracts contained large amounts of cathepsin H activity.