H-2-restricted cytotoxic effectors generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins

Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 μg of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.     

Effects of Tolcapone, a Catechol-O-Methyltransferase Inhibitor, and Sinemet on Intestinal Electrolyte and Fluid Transport in Conscious Dogs

Tolcapone (T) is a novelcatechol-O-methyltransferase (COMT) inhibitor recentlyintroduced for the treatment of Parkinson's disease. Inclinical efficacy studies, T has been associated with alow incidence of diarrhea. The objectives of the study wereto examine whether T and its adjunctive drug Sinemet (S)could influence intestinal fluid and electrolytetransport as a possible cause for the diarrhea. The studies were conducted in conscious dogssurgically prepared with Thiry-Vella loops constructedfrom a 40-cm jejunal segment. A physiologically bufferedtest solution was perfused into the orad stoma and collected from the caudad stoma. Secretionswere collected at 15-min intervals and analyzed forvolume, electrolytes, lipid phosphorus, and protein. Theacute oral administration of T (10 and 30 mg/kg doses) was well tolerated. Concurrent acuteadministration of S (25 mg/kg) with T (30 mg/kg) wasalso well tolerated. The acute oral administration of Tinduced a dose-dependent efflux of intestinal fluid and electrolytes (sodium, potassium, chloride, andbicarbonate) secretion (P < 0.05). The oralcoadministration of S (25 mg/kg) with T (30 mg/kg)accelerated the onset of the stimulation of intestinalsecretion. Despite the significant stimulation ofintestinal secretion, none of the dogs developeddiarrhea, indicating the importance of intestinalcompensatory mechanisms. Neither T nor T&S affectedcalcium, lipid, or protein efflux rates, suggesting thatthe stimulated secretion was not a consequence ofintestinal mucosal injury. The chronic (seven-day)administration of T and T&S was associated withreduced intestinal secretory responses when comparedwith the acute administration of the same drugs; Senhanced the T-induced tolerance development. The basisfor such tolerance is unknown. In conclusion, the stimulatory systemic actions of tolcapone onintestinal secretion may, under certain conditions,contribute to the induction of diarrhea in susceptiblepatients.     

Influence of dietary status and diabetes on aortic acyl-CoA hydrolase activity.

We have postulated that the accelerated snythesis of cholesteryl ester in atherosclerotic microsomes may result in part from decreased acyl-CoA hydrolase activity in arterial tissue, because acyl-CoA is a common substrate for both reactions. We have now investigated the influence of nutritional status, type of diet, and diabetes on the acyl-CoA hydrolase activity of otherwise normal aortic microsomes. Fasting rabbits for 16 hr diminished the acyl-CoA hydrolase activity approximately 30%. The activity of this aortic microsomal enzyme in rats maintained on a high-carbohydrate diet for 5 weeks was comparable to the activity observed on a high fat (olive oil) diet. The type of fat in the diet influences the acyl-CoA hydrolase activity: oils containing 77% oleic acid (high-oleic safflower oil) and containing 70% linoleic acid (conventional safflower oil) lowered the aortic microsomal acyl-CoA hydrolase activity in comparison to a more saturated fat (cocoa butter). Aortic preparations of rats made diabetic by streptozotocin exhibited higher acyl-CoA hydrolase activity than the normal. The results show that conditions associated with human atherogenesis (diabetes and saturated fat diet) increase rather than suppress the activity of this arterial enzyme in normal arterial tissues of the rat.