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Low denfity lipoprot¢ins (LDL) oxidati~ly modified by maeropha~s have been shown to tm atherogeni¢ in ~ vivo studio, W= studied the potential role of nitric oxide (NO), a free r-adi~l produt:ed by maerophag=;, in LDL modifi~tion, Human LDL (I mg/ml) were incubated with mouse Ixritom:al ma¢rophag~ in Ham's F-IO medium. The ¢¢1ls w=r¢ then stimulat~ by interferon.y and tumor n¢¢rosi= fa=or-¢ to inerea~ their production of NO from t.3 to 12.2 tam in 24 h. us mcatsured by nitrite. Lipid l~roxidation of LDL, as m~sured by thio~rbitudc acid.tractive materials (";'BARS). was reduexd in stimulated c¢{l~ in a time.dependent manner. At 24 h, the d=rcar¢ ~s about 2"/%. In tl;¢ preu:n¢¢ of an NO syntha~ inhibitor (N".aminophomoarginin¢). the Ilcncration of NO was diminished and tim proration a~init LDL lipid pcroaidation wa~ reverted, The extent of LDL protein modifiemtion was al~ assessed by examining its electropho~tic mobility. It wall found that macrophage NO reduced the change in LDL ¢lectromobility, These data ladS=ate that the production of NO may inlfibit the oxidative modification of LDL with cytokine,stimulatcd macrophag¢*, We suggest that NO plays a protective role in limiting macrophage.indutu=d LDL modifl=ation, [15,16], In the presence of an antioxidant, such as probucol, the formation of foam ~lls is inhibited both in vitro and in vivo [17][18][19][20][21]. However, the sources and species of those free radicals responsible for the oxidation of LDL are not well established, The superoxid¢ anion (Of) produced by macrophages is known to be involve, sin~ the pretreatment of macrophages with superoxid¢ dismuta~e (SOD) attenuates LDL lipid peroxidation [22]. Macrophages have been shown to produce the nitric oxide (NO) radical via nitric oxide synthase. This enzyme converts arginine into citrulline and is indued by cytokin, treatment [23,24], Although NO can neutrali~ Oi-to form a stable peroxynitrit¢ anion (ONOO') [25], under some conditions th¢ peroxynitrit¢ can also rapidly decompose to form a strong oxidant with reactivity similar to the hydroxyl radical In addition. NO generation in vivo may lead to the mobilization of iron with a subsequent increase in the level of reactive oxygen slzt¢ics [2?]. These observation= could ¢~plain the cytotoxi¢ action of NO that has been demonstrated in a numL~r of studies [28,29]. The purpose of the present study was to dctermin, the ,ff~t of NO production by macrophagcs on LDL lipid [mroxi. datioa. To this end we incubated LDL with macrophages in which the synthesis of NO was induced with interf¢ron-7 and tumor necrosis factor-~, and followed the extent of oxidative modification of LDL. ,i, Nitric oxide .¢k.tztha~'e inhibitor (MDL lO0.24tt) $-Mcthylisothio~emi~ar "b~tde hydroio,Jid¢ (17,8 8. 0.0?fi tool) was added to L-ly£n¢ monohydrate (9,3 $, 0,076 tool) in water (t00 ml) and adjusted to pH 9.5 with I N NaOH, The r~gtion mi~tu~ wa= h~t~ and stirred at 40"C for 30 h. The solution was then cooled to room teml~rature and acidified to pH 4.0 with acetic acid. F~vianic acid (...

The modification of the terminal ionizable charges of an immunogenic peptide, HEL (46-61), was found to greatly increase the immunogenicity of the peptide. The modified peptide had 100- to 1000-fold enhanced activity in both in vitro and in vivo T cell assays. The mechanism of the enhancement was investigated by determining the binding affinities to I-Ak as well as circular dichroism (CD) studies. The native and enhanced peptides had indistinguishable binding affinities, as well as similar kinetics. The CD studies revealed that in aqueous solution, neither peptide had any detectable helicity; however, the addition of trifluoroethanol did result in significant helicity; with the two peptides being indistinguishable. These same modifications were also shown to enhance other immunogenic peptides if they contained a basic carboxy-terminal amino acid residue. Thus, by modifying the termini of T cell epitopes, their immunogenicity can be dramatically increased, but the molecular basis for this enhancement is still unclear.

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