Procedures are described for linking monomethoxypoly(ethylene glycol) (mPEG) to both epsilon and alpha amino groups of lysine. The lysine carboxyl group can then be activated as a succinimidyl ester to obtain a new mPEG derivative (mPEG2-COOSu) with improved properties for biotechnical applications. This branched reagent showed in some cases a lower reactivity toward protein amino groups than the linear mPEG from which it was derived. A comparison of mPEG- and mPEG2-modified enzymes (ribonuclease, catalase, asparaginase, trypsin) was carried out for activity, pH and temperature stability, Km and Kcat values, and protection to proteolytic digestion. Most of the adducts from mPEG and mPEG2 modification presented similar activity and stability toward temperature change and pH change, although in a few cases mPEG2 modification was found to increase temperature stability and to widen the range of pH stability of the adducts. On the other hand, all of the enzymes modified with the branched polymer presented greater stability to proteolytic digestion relative to those modified with the linear mPEG. A further advantage of this branched mPEG lies in the possibility of a precise evaluation of the number of polymer molecules bound to the proteins; upon acid hydrolysis, each molecule of mPEG2 releases a molecule of lysine which can be detected by amino acid analysis. Finally, dimerization of mPEG by coupling to lysine provides a needed route to monofunctional PEGs of high molecular weight.

427 3.3. Liposomes as Drug Carriers and Their in Vivo Fate 427 3.4. Long-Circulating Liposomes 428 3.4.1. GM1 and Glucoronide Liposomes 429 3.4.2. PEG Liposomes 430 3.4.3. Other Polymers Used in the Preparation of Long-Circulating Liposomes 430 3.4.4. Active Targeted, Thermosensitive, pH-Sensitive Long-Circulating Liposomes 431 3.4.5. Long-Circulating Liposomes for Tumor Diagnosis and Cancer Chemotherapy 432 3.5. General Considerations on Substance Entrapment in Liposomes 432 4. Particles 433 4.1. Introduction 433 4.2. Microparticles 433 4.3. Properties and Methods of Preparation of Nanoparticles 433 4.4. Nanoparticles as Drug Carriers 434 4.5. Long-Circulating Nanoparticles 434 4.6. General Considerations on Substance Entrapment in Particles 435 5. Additional Strategies 436 6. Conclusions 437

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-γ and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-γ and IL-12 knockout mutants (IFN-γ−/−, IL-12−/−) with Torpedo AChR (TAChR). WT and IFN-γ−/− mice developed EMG with similar frequency, IL-12−/−mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-γ−/− mice had significantly less IgG2c, and IL-12−/− mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-γ−/− mice had complement; WT mice had both IgG2b and IgG2c, IFN-γ−/− only IgG2b, and IL-12−/− neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-γ−/− mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12−/− mice secreted less IFN-γ, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12−/− mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-γ−/− mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.

No abstract

Ab to the acetylcholine receptor (AChR) cause experimental myasthenia gravis (EMG). Th1 cytokines facilitate EMG, whereas Th2 cytokines might be protective. IL-10 inhibits Th1 responses but facilitates B cell proliferation and Ig production. We examined the role of IL-10 in EMG by using wild-type (WT) C57BL/6 mice and transgenic (TG) C57BL/6 mice that express IL-10 under control of the IL-2 promoter. We immunized the mice with doses of AChR that cause EMG in WT mice or with low doses ineffective at causing EMG in WT mice. After low-dose AChR immunization, WT mice did not develop EMG and had very little anti-AChR serum Ab, which were mainly IgG1, whereas TG mice developed EMG and had higher levels of anti-AChR serum Ab, which were mainly IgG2, in addition to IgG1. At the higher doses, TG mice developed EMG earlier and more frequently than WT mice and had more serum anti-AChR Ab. Both strains had similar relative serum concentrations of anti-AChR IgG subclasses and IgG and complement at the muscle synapses. CD8+-depleted splenocytes from all AChR-immunized mice proliferated in the presence of AChR and recognized a similar epitope repertoire. CD8+-depleted splenocytes from AChR-immunized TG mice stimulated in vitro with AChR secreted significantly more IL-10, but less of the prototypic Th1 cytokine IFN-γ, than those from WT mice. They secreted comparable amounts of IL-4 and slightly but not significantly reduced amounts of IL-2. This suggests that TG mice had reduced activation of anti-Torpedo AChR Th1 cells, but increased anti-AChR Ab synthesis, that likely resulted from IL-10-mediated stimulation of anti-AChR B cells. Thus, EMG development is not strictly dependent on Th1 cell activity.