Mast cells (MC)l are widely distributed throughout vascularized tissues and certain epithelia. They represent a source ofpotent mediators ofinflammation (reviewed in references 1-4). These mediators are released after sensitization with IgE immunoglobulins, which are bound to IgE receptors (FceRI) on the MC, and crosslinking with specific multivalent antigen (4). Such activation causes MC to degranulate releasing histamine, heparin, and other sulphated proteoglycans and certain neutral proteases. Activated MC also elaborate newly synthesized mediators such as products of the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism (reviewed in references 2-4). MC are widely regarded as critical effector cells in the inflammatory reactions underlying disorders of IgE-dependent immediate hypersensitivity and in the expression ofprotective immunity involving IgE (reviewed in references 1-4).Studies in mice indicate that MC are derived from multipotential bone marrowderived hematopoietic precursors which complete their program of differentiation and maturation in vascularized tissues, epithelia, and serosal cavities (reviewed in references 1, 5). This process results in the generation ofmast cell populations which vary in multiple aspects of their phenotype, including morphology, mediator content, and sensitivity to regulation by cytokines affecting proliferation and maturation (reviewed in reference 1). One such population, referred to as "mucosal" mast cells (MMC) because they occur in the mucosal layer of gastrointestinal tissues, appears to be exquisitely sensitive to regulation by the T cell-associated cytokines IL-3 and IL-4 (1). IL-3 probably represents the major cytokine regulating proliferation ofthis subset (6, 7), whereas in vitro studies indicate that IL-4 can act as a costimulant of proliferation (8). Thus, the mouse MMC population is regulated by products I Abbreviations used in this paper: AbMuLV, Abelson murine leukemia virus; Ag, antigen; BMCMC, bone marrow-derived cultured mast cell ; DNP3o-40-HSA 2,4-dinitrophenyl-human serum albumin; FceRI, cell surface receptor for the Fc portion of IgE; GM-CSF, granulocyte/macrophage colony-stimulating factor ; MC, mast cell; MIP, macrophage inflammatory protein; MMC, mucosal mast cell ; PKC, protein kinase C. J. Exp. MED.

Multiple sclerosis (MS) is a chronic debilitating disease of the central nervous system primarily mediated by T lymphocytes with specificity to neuronal antigens in genetically susceptible individuals. On the other hand, myasthenia gravis (MG) primarily involves destruction of the neuromuscular junction by antibodies specific to the acetylcholine receptor. Both autoimmune diseases are thought to result from loss of self-tolerance, which allows for the development and function of autoreactive lymphocytes. Although the mechanisms underlying compromised self-tolerance in these and other autoimmune diseases have not been fully elucidated, one possibility is numerical, functional, and/or migratory deficits in T regulatory cells (Tregs). Tregs are thought to play a critical role in the maintenance of peripheral immune tolerance. It is believed that Tregs function by suppressing the effector CD4+ T cell subsets that mediate autoimmune responses. Dysregulation of suppressive and migratory markers on Tregs have been linked to the pathogenesis of both MS and MG. For example, genetic abnormalities have been found in Treg suppressive markers CTLA-4 and CD25, while others have shown a decreased expression of FoxP3 and IL-10. Furthermore, elevated levels of pro-inflammatory cytokines such as IL-6, IL-17, and IFN-γ secreted by T effectors have been noted in MS and MG patients. This review provides several strategies of treatment which have been shown to be effective or are proposed as potential therapies to restore the function of various Treg subsets including Tr1, iTr35, nTregs, and iTregs. Strategies focusing on enhancing the Treg function find importance in cytokines TGF-β, IDO, interleukins 10, 27, and 35, and ligands Jagged-1 and OX40L. Likewise, strategies which affect Treg migration involve chemokines CCL17 and CXCL11. In pre-clinical animal models of experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune myasthenia gravis (EAMG), several strategies have been shown to ameliorate the disease and thus appear promising for treating patients with MS or MG.

High-density lipoproteins (HDLs) and their major protein, apoA-I, remove excess cellular cholesterol and protect against atherosclerosis. However, in acquired amyloidosis, non-variant full-length apoA-I deposits as fibrils in atherosclerotic plaques; in familial amyloidosis, N-terminal fragments of variant apoA-I deposit in vital organs damaging them. Recently, we used the crystal structure of Δ(185-243)apoA-I to propose that amyloidogenic mutations destabilize apoA-I and increase solvent exposure of the extended strand 44-55 that initiates β-aggregation. Here we test this hypothesis by exploring naturally occurring human amyloidogenic mutations, W50R and G26R, within or close to this strand. The mutations caused small changes in the protein’s α-helical content, stability, proteolytic pattern, and protein-lipid interactions. These changes alone were unlikely to account for amyloidosis, suggesting the importance of other factors. Sequence analysis predicted several amyloid-prone segments that can initiate apoA-I misfolding. Aggregation studies using N-terminal fragments experimentally verified this prediction. Three predicted N-terminal amyloid-prone segments, mapped on the crystal structure, formed an α-helical cluster. Structural analysis indicates that amyloidogenic mutations or Met86 oxidation perturb native packing in this cluster. Together, the results suggest that structural perturbations in the amyloid-prone segments trigger α-helix-to-β-sheet conversion in the N-terminal ~75 residues forming the amyloid core. Polypeptide outside this core can be proteolysed to form 9-11 kDa N-terminal fragments found in familial amyloidosis. Our results imply that apoA-I misfolding in familial and acquired amyloidosis follows a similar mechanism that does not require significant structural destabilization or proteolysis. This novel mechanism suggests potential therapeutic interventions for apoA-I amyloidosis.

High-density lipoproteins (HDL) mediate cholesterol removal and thereby protect against atherosclerosis. Mature spherical HDL contain the apolar lipid core and polar surface of proteins and phospholipids. Earlier, we showed that the structural integrity of HDL is modulated by kinetic barriers that prevent spontaneous protein dissociation and lipoprotein fusion and rupture. To determine the role of electrostatic interactions in the kinetic stability of mature HDL, here we analyze the effects of salt and pH on their thermal denaturation. In low-salt buffer at pH 5.7-7.7, HDL are highly thermostable. Increasing the salt concentration from 0 to 0.3 M NaCl causes low-temperature shifts in the calorimetric HDL transitions of up to -14 degrees C. This salt-induced destabilization leads to protein unfolding below 100 degrees C, facilitating the first Arrhenius analysis of HDL denaturation by circular dichroism spectroscopy. In 150 mM NaCl, two kinetic phases in HDL protein unfolding are observed: a faster phase with an activation energy E(a,fast) < or =15 kcal/mol and a slower phase with an E(a,slow) = 50 +/- 7 kcal/mol. Gel electrophoresis and electron microscopic data suggest that the faster phase involves partial protein unfolding but no significant protein dissociation or changes in HDL size, while the slower phase involves complete protein unfolding, partial protein dissociation, and HDL fusion. Hence, the slower phase may resemble HDL remodeling and fusion by plasma enzymes during metabolism. Analysis of the effects of various salts, sucrose, and pH suggests that HDL destabilization by salt results from ionic screening of favorable short-range electrostatic interactions such as salt bridges. Consequently, electrostatic interactions significantly contribute to the high thermostability of HDL in low-salt solutions.

Denaturation studies of high-density lipoproteins (HDL) containing human apolipoprotein A-2 (apoA-2) and dimyristoyl phosphatidylcholine indicate kinetic stabilization. Circular dichroism (CD) and light-scattering melting curves show hysteresis and scan rate dependence, indicating thermodynamically irreversible transition with high activation energy E(a). CD and light-scattering data suggest that protein unfolding triggers HDL fusion. Electron microscopy, gel electrophoresis, and differential scanning calorimetry show that such fusion involves lipid vesicle formation and dissociation of monomolecular lipid-poor protein. Arrhenius analysis reveals two kinetic phases, a slower phase with E(a,slow) = 60 kcal/mol and a faster phase with E(a,fast) = 22 kcal/mol. Only the fast phase is observed upon repetitive heating, suggesting that lipid-poor protein and protein-containing vesicles have lower kinetic stability than the disks. Comparison of the unfolding rates and the melting data recorded by differential scanning calorimetry, CD, and light scattering indicates the rank order for the kinetic disk stability, apoA-1 > apoA-2 > apoC-1, that correlates with protein size rather than hydrophobicity. This contrasts with the tighter association of apoA-2 than apoA-1 with mature HDL, suggesting different molecular determinants for stabilization of model discoidal and plasma spherical HDL. Different effects of apoA-2 and apoA-1 on HDL fusion and stability may reflect different metabolic properties of apoA-2 and/or apoA-1-containing HDL.

Background: Amyloids made of apolipoprotein A-I (apoA-I) contribute to the growth of the atherosclerotic plaques. Results: ApoA-I methionine oxidation by physiological levels of myeloperoxidase induces amyloid formation. Conclusion: Myeloperoxidase-mediated oxidation not only impairs the physiological functions of apoA-I but also promotes protein loss in form of amyloids. Significance: Our findings identify the physiological mechanism transforming wild-type apoA-I into an amyloidogenic protein.

Epigenetic alteration of the genome has been shown to provide palliative effects in mouse models of certain human autoimmune diseases. We have investigated whether chromatin remodeling could provide protection against autoimmune diabetes in NOD mice. Treatment of female mice during the transition from prediabetic to diabetic stage (18-24 weeks of age) with the well-characterized histone deacetylase inhibitor, trichostatin A effectively reduced the incidence of diabetes. However, similar treatment of overtly diabetic mice during the same time period failed to reverse the disease. Protection against diabetes was accompanied by histone hyperacetylation in pancreas and spleen, enhanced frequency of CD4 + CD62L + cells in the spleen, reduction in cellular infiltration of islets, restoration of normoglycemia and glucose-induced insulin release by beta cells. Activation of splenic T lymphocytes derived from protected mice in vitro with pharmacological agents that bypass the antigen receptor or immobilized anti-CD3 antibody resulted in enhanced expression of Ifng mRNA and protein without altering the expression of Il4, Il17, Il18, Inos and Tnfa genes nor the secretion of IL-2, IL-4, IL-17 and TNF-a proteins. Consistently, expression of the transcription factor involved in Ifng transcription, Tbet/Tbx21 but not Gata3 and Rorgt, respectively, required for the transcription of Il4 and Il17, was upregulated in activated splenocytes of protected mice. These results indicate that chromatin remodeling can lead to amelioration of diabetes by using multiple mechanisms including differential gene transcription. Thus, epigenetic modulation could be a novel therapeutic approach to block the transition from benign to frank diabetes.

The stability of human low-density lipoprotein (LDL), the major cholesterol carrier in plasma, was analyzed by heating samples of different concentrations at a rate from 11 to 90 K/h. Correlation of the calorimetric, circular dichroism, fluorescence, turbidity, and electron microscopic data shows that thermal disruption of LDL involves irreversible changes in the particle morphology and protein conformation but no global protein unfolding. Heating to 85 degrees C induces LDL conversion into smaller and larger particles and apparent partial dissociation, but not unfolding, of its sole protein, apoB. Further heating leads to partial unfolding of the beta-sheets in apoB and to fusion of the protein-depleted LDL into large aggregated lipid droplets, resulting in a previously unidentified high-temperature calorimetric peak. These lipid droplets resemble in size and morphology the extracellular lipid deposits formed in the arterial wall in early atherosclerosis. The strong concentration dependence of LDL fusion revealed by near-UV/visible CD, turbidity, and calorimetry indicates high reaction order, and the heating rate dependence suggests high activation energy that arises from transient disruption of lipid and/or protein packing interactions in the course of particle fusion and apparent apoB dissociation. Consequently, thermal stability of LDL is modulated by kinetic barriers. Similar barriers may confer structural integrity to LDL subclasses in vivo.