Immune response to a syngeneic mammary adenocarcinoma. II. In vitro generation of cytotoxic lymphocytes.

A method is described for the consistent in vitro generation cytotoxic cells by incubating Fischer 344 rat spleen cells on monolayers of a syngeneic mammary adenocarcinoma. Significant cytotoxicity by in vitro culture is generated as early as 3 days after initiation and effector cells are cytolytic only toward target cells of the sensitizing monolayer. Reciprocal sensitization with allogeneic fibroblasts as the immunizing monolayer yielded effector cells cytolytic for the fibroblasts but without effect on the mammary tumor. The consistency in the generation of cytotoxic cells by in vitro culture should permit its standardized use in following other related immune phenomena such as blocking by serologic factors and suppression, recritment of memory for cytotoxic function.     

Immune response to a syngeneic mammary adenocarcinoma. III. Development of memory and suppressor functions modulating cellular cytotoxicity.

Measurement of the development of cytolytic activity by mammary tumor primed or unprimed syngeneic spleen cells on in vitro monolayers of the 13762 rat mammary tumor operationally defined several subpopulations of lymphoid cells involved in the cytotoxic response. In vitro sensitization of cells from Fischer 344 animals injected 2 to 10 days earlier with 2 x 10(7) viable tumor cells always resulted in a higher and earlier lytic response than cells from non-inoculated animals. Adoptive transfer of the same in vivo primed cells for 5 days in irradiated syngeneic hosts removed any cytotoxic cells originally present but subsequent in vitro sensitization still resulted in a higher and earlier cytolytic response. We defined such cells as "memory" cells for cytotoxicity. Memory cells were radiosensitive and specific for the immunizing target cell. In contrast to cells from animals inoculated for 3 to 10 days, cells obtained 11 and 12 days after immunization had a lower response than unprimed cells on vitro sensitization. The anamnestic response could be restored either by culturing 12-day primed cells in vitro for 2 days without antigen or by adoptive transfer for 5 days into irradiated syngeneic rats. This suggests that another population of cells is present in spleen and suppresses the conversion of memory to cytotoxic cells. A more direct measurement of suppressor cell function was obtained by coincubating tumor-primed and unprimed cells on monolayers during in vitro sensitization. Cells from animals bearing tumors for 5 to 10 days always caused an increase in the response of the mixed lymphocyte groups, whereas 11- to 13-day tumor primed cells always caused a marked decrease in the cytolytic response. These results suggest the following interpretation of the kinetics of cell-mediated cytotoxicity to syngeneic tumor inoculation. Cytotoxic cells appear about 6 days after immunization, reach peak levels 2 days later, and then decrease rapidly. Memory cells are generated at a faster rate, reach peak levels before maximum cytolytic activity, but are then functionally inhibited from converting into differentiated cytotoxic cells by a new population of suppressor cells which reach peak activity about 12 days after immunization.     

Calmodulin and ATP dependence of the high and low calcium affinity p-nitrophenylphosphatase from human erythrocyte membranes

In calmodulin depleted membranes from human erythrocytes, the Ca2+-dependent phosphatase showed different sensitivity to calmodulin and ATP with variable affinity towards free calcium concentrations: a calmodulin-dependent activity with high calcium affinity, K1/2 = 1.2 X 10(-7) mol/l calcium, that was fully activated at submicromolar calcium concentrations, higher concentrations being rather inhibitory; an ATP-dependent activity with lower calcium affinity, K1/2 = 10(-6) mol/l calcium, that was fully activated at 10(-5) mol/l calcium in the presence of 50-200 mumol/l ATP and was insensitive to calmodulin, and a calcium dependent phosphatase that was active at a wider ranger of free calcium, 10(-8)-10(-5) mol/l, and required the presence of both calmodulin and ATP.     

Evaluation of populations of fluorescent pseudomonads related to decline of take-all patch on turfgrass

Take-all on turfgrass caused by Gaeumannomyces graminis var. avenae (Gga) occurs as patches of yellowish plants. On some patches the central zone was recolonized by the same grass species, Festuca sp., previously damaged by the fungus despite the centrifugal extension of the disease. This disease remission was assimilated to decline. Rhizosphere bacterial counts showed that total population of bacteria was nearly the same in all zones across the patches. However, the ratio of fluorescent Pseudomonas spp./ total bacteria was 1/22, 1/15.4, 1/3.5 and 1/2.9 in the disease free area, the front margin of the patch, in the damaged part of the patch, and in the recolonized central part respectively. Furthermore, in this last mentioned zone, 44 to 82% of the fluorescent Pseudomonas spp. were antagonistic in vitro to Gga, whereas only 12 to 34% from the disease free area were antagonistic. So the development of take-all on turf induced quantitative and qualitative changes in populations of fluorescent pseudomonads. The remission of the disease in the center was correlated to higher amount of antagonistic fluorescent pseudomonads in this part of the patches. This typical patch with the well defined zones can provide a good model for the study of changes in bacterial populations related to the build up of take-all decline.     

Anti-inflammatory effect of l-cysteine (a semi-essential amino acid) on 5-FU-induced oral mucositis in hamsters

Oral mucositis is an inflammation of the oral mucosa mainly resulting from the cytotoxic effect of 5-fluorouracil (5-FU). The literature shows anti-inflammatory action of l-cysteine (l-cys) involving hydrogen sulfide (H₂S). In view of these properties, we investigate the effect of l-cys in oral mucositis induced by 5-FU in hamsters. The animals were divided into the following groups: saline 0.9%, mechanical trauma, 5-FU 60–40 mg/kg, l-cys 10/40 mg and NaHS 27 µg/kg. 5-FU was administered on days 1st to 2nd; 4th day excoriations were made on the mucosa; 5th–6th received l-cys and NaHS. For data analysis, histological analyses, mast cell count, inflammatory and antioxidants markers, and immunohistochemistry (cyclooxygenase-2(COX-2)/inducible nitric oxide synthase (iNOs)/H₂S) were performed. Results showed that l-cys decreased levels of inflammatory markers, mast cells, levels of COX-2, iNOS and increased levels of antioxidants markers and H₂S when compared to the group 5-FU (p < 0.005). It is suggested that l-cys increases the H₂S production with anti-inflammatory action in the 5-FU lesion.

     

Comparison and Analysis of Zinc and Cobalt-Based Systems as Catalytic Entities for the Hydration of Carbon Dioxide

In nature, the zinc metalloenzyme carbonic anhydrase II (CAII) efficiently catalyzes the conversion of carbon dioxide (CO₂) to bicarbonate under physiological conditions. Many research efforts have been directed towards the development of small molecule mimetics that can facilitate this process and thus have a beneficial environmental impact, but these efforts have met very limited success. Herein, we undertook quantum mechanical calculations of four mimetics, 1,5,9-triazacyclododedacane, 1,4,7,10-tetraazacyclododedacane, tris(4,5-dimethyl-2-imidazolyl)phosphine, and tris(2-benzimidazolylmethyl)amine, in their complexed form either with the Zn²⁺ or the Co²⁺ ion and studied their reaction coordinate for CO₂ hydration. These calculations demonstrated that the ability of the complex to maintain a tetrahedral geometry and bind bicarbonate in a unidentate manner were vital for the hydration reaction to proceed favorably. Furthermore, these calculations show that the catalytic activity of the examined zinc complexes was insensitive to coordination states for zinc, while coordination states above four were found to have an unfavorable effect on product release for the cobalt counterparts.