Genetic evidence for serum amyloid P component as a drug target for treatment of neurodegenerative disorders.

The direct causes of neurodegeneration underlying Alzheimer's disease (AD) and many other dementias, are not known. Here we identify serum amyloid P component (SAP), a constitutive plasma protein normally excluded from the brain, as a potential drug target. After meta-analysis of three genome-wide association studies, comprising 44,288 participants, cis-Mendelian randomization showed that genes responsible for higher plasma SAP values are significantly associated with AD, Lewy body dementia and plasma tau concentration. These genetic findings are consistent with experimental evidence of SAP neurotoxicity and the strong, independent association of neocortex SAP content with dementia at death. Depletion of SAP from the blood and from the brain, as is provided by the safe, well tolerated, experimental drug, miridesap, may therefore contribute to treatment of neurodegeneration.

Plasmin activity promotes amyloid deposition in a transgenic model of human transthyretin amyloidosis.

Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTRS52P. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α2-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments.

Dementia in the older population is associated with neocortex content of serum amyloid P component.

Despite many reported associations, the direct cause of neurodegeneration responsible for cognitive loss in Alzheimer's disease and some other common dementias is not known. The normal human plasma protein, serum amyloid P component, a constituent of all human fibrillar amyloid deposits and present on most neurofibrillary tangles, is cytotoxic for cerebral neurones in vitro and in experimental animals in vivo. The neocortical content of serum amyloid P component was immunoassayed in 157 subjects aged 65 or more with known dementia status at death, in the large scale, population-representative, brain donor cohort of the Cognitive Function and Ageing Study, which avoids the biases inherent in studies of predefined clinico-pathological groups. The serum amyloid P component values were significantly higher in individuals with dementia, independent of serum albumin content measured as a control for plasma in the cortex samples. The odds ratio for dementia at death in the high serum amyloid P component tertile was 5.24 (95% confidence interval 1.79-15.29) and was independent of Braak tangle stages and Thal amyloid-ß phases of neuropathological severity. The strong and specific association of higher brain content of serum amyloid P component with dementia, independent of neuropathology, is consistent with a pathogenetic role in dementia.

Systemic Exosomal Delivery of shRNA Minicircles Prevents Parkinsonian Pathology.

The development of new therapies to slow down or halt the progression of Parkinson's disease is a health care priority. A key pathological feature is the presence of alpha-synuclein aggregates, and there is increasing evidence that alpha-synuclein propagation plays a central role in disease progression. Consequently, the downregulation of alpha-synuclein is a potential therapeutic target. As a chronic disease, the ideal treatment will be minimally invasive and effective in the long-term. Knockdown of gene expression has clear potential, and siRNAs specific to alpha-synuclein have been designed; however, the efficacy of siRNA treatment is limited by its short-term efficacy. To combat this, we designed shRNA minicircles (shRNA-MCs), with the potential for prolonged effectiveness, and used RVG-exosomes as the vehicle for specific delivery into the brain. We optimized this system using transgenic mice expressing GFP and demonstrated its ability to downregulate GFP protein expression in the brain for up to 6 weeks. RVG-exosomes were used to deliver anti-alpha-synuclein shRNA-MC therapy to the alpha-synuclein preformed-fibril-induced model of parkinsonism. This therapy decreased alpha-synuclein aggregation, reduced the loss of dopaminergic neurons, and improved the clinical symptoms. Our results confirm the therapeutic potential of shRNA-MCs delivered by RVG-exosomes for long-term treatment of neurodegenerative diseases.

A specific nanobody prevents amyloidogenesis of D76N ß2-microglobulin in vitro and modifies its tissue distribution in vivo.

Systemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein self-aggregation represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of ß2-microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed in vitro in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type ß2-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The ß2-microglobulin -binding nanobody, Nb24, more potently inhibits D76N ß2-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In ß2-microglobulin knock out mice, the D76N ß2-microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type ß2-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis.

Amyloid persistence in decellularized liver: biochemical and histopathological characterization.

Systemic amyloidoses are a group of debilitating and often fatal diseases in which fibrillar protein aggregates are deposited in the extracellular spaces of a range of tissues. The molecular basis of amyloid formation and tissue localization is still unclear. Although it is likely that the extracellular matrix (ECM) plays an important role in amyloid deposition, this interaction is largely unexplored, mostly because current analytical approaches may alter the delicate and complicated three-dimensional architecture of both ECM and amyloid. We describe here a decellularization procedure for the amyloidotic mouse liver which allows high-resolution visualization of the interactions between amyloid and the constitutive fibers of the extracellular matrix. The primary structure of the fibrillar proteins remains intact and the amyloid fibrils retain their amyloid enhancing factor activity.

Bifunctional crosslinking ligands for transthyretin.

Wild-type and variant forms of transthyretin (TTR), a normal plasma protein, are amyloidogenic and can be deposited in the tissues as amyloid fibrils causing acquired and hereditary systemic TTR amyloidosis, a debilitating and usually fatal disease. Reduction in the abundance of amyloid fibril precursor proteins arrests amyloid deposition and halts disease progression in all forms of amyloidosis including TTR type. Our previous demonstration that circulating serum amyloid P component (SAP) is efficiently depleted by administration of a specific small molecule ligand compound, that non-covalently crosslinks pairs of SAP molecules, suggested that TTR may be also amenable to this approach. We first confirmed that chemically crosslinked human TTR is rapidly cleared from the circulation in mice. In order to crosslink pairs of TTR molecules, promote their accelerated clearance and thus therapeutically deplete plasma TTR, we prepared a range of bivalent specific ligands for the thyroxine binding sites of TTR. Non-covalently bound human TTR-ligand complexes were formed that were stable in vitro and in vivo, but they were not cleared from the plasma of mice in vivo more rapidly than native uncomplexed TTR. Therapeutic depletion of circulating TTR will require additional mechanisms.

C-reactive protein is essential for innate resistance to pneumococcal infection.

No deficiency of human C-reactive protein (CRP), or even structural polymorphism of the protein, has yet been reported so its physiological role is not known. Here we show for the first time that CRP-deficient mice are remarkably susceptible to Streptococcus pneumoniae infection and are protected by reconstitution with isolated pure human CRP, or by anti-pneumococcal antibodies. Autologous mouse CRP is evidently essential for innate resistance to pneumococcal infection before antibodies are produced. Our findings are consistent with the significant association between clinical pneumococcal infection and non-coding human CRP gene polymorphisms which affect CRP expression. Deficiency or loss of function variation in CRP may therefore be lethal at the first early-life encounter with this ubiquitous virulent pathogen, explaining the invariant presence and structure of CRP in human adults.

Pathogenetic mechanisms of amyloid A amyloidosis.

Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid.

Monitoring systemic amyloidosis using MRI measurements of the extracellular volume fraction.

We report the in vivo evaluation, in a murine model, of MRI measurements of the extracellular volume fraction (ECV) for the detection and monitoring of systemic amyloidosis. A new inducible transgenic model was used, with increased production of mouse serum amyloid A protein controlled by oral administration of doxycycline, that causes both the usual hepatic and splenic amyloidosis and also cardiac deposits. ECV was measured in vivo by equilibrium contrast MRI in the heart and liver of 11 amyloidotic and 10 control mice. There was no difference in the cardiac function between groups, but ECV was significantly increased in the heart, mean (standard deviation) 0.20 (0.05) versus 0.14 (0.04), p < 0.005, and liver, 0.27 (0.04) versus 0.15 (0.04), p < 0.0005, of amyloidotic animals and was strongly correlated with the histological amyloid score, myocardium, ρ = 0.67, p < 0.01; liver, ρ = 0.87, p < 0.01. In a further four mice that received human serum amyloid P component (SAP) followed by anti-human SAP antibody, a treatment to eliminate visceral amyloid deposits, ECV in the liver and spleen returned to baseline after therapy (p < 0.01). MRI measurement of ECV is a sensitive marker of amyloid deposits with potential application for early detection and monitoring therapies promoting their clearance.

Exon skipping of hepatic APOB pre-mRNA with splice-switching oligonucleotides reduces LDL cholesterol in vivo.

Familial hypercholesterolemia (FH) is a genetic disorder characterized by extremely high levels of plasma low-density lipoprotein (LDL), due to defective LDL receptor-apolipoprotein B (APOB) binding. Current therapies such as statins or LDL apheresis for homozygous FH are insufficiently efficacious at lowering LDL cholesterol or are expensive. Treatments that target APOB100, the structural protein of LDL particles, are potential therapies for FH. We have developed a series of APOB-directed splice-switching oligonucleotides (SSOs) that cause the expression of APOB87, a truncated isoform of APOB100. APOB87, like similarly truncated isoforms expressed in patients with a different condition, familial hypobetalipoproteinemia, lowers LDL cholesterol by inhibiting very low-density lipoprotein (VLDL) assembly and increasing LDL clearance. We demonstrate that these "APO-skip " SSOs induce high levels of exon skipping and expression of the APOB87 isoform, but do not substantially inhibit APOB48 expression in cell lines. A single injection of an optimized APO-skip SSO into mice transgenic for human APOB resulted in abundant exon skipping that persists for >6 days. Weekly treatments generated a sustained reduction in LDL cholesterol levels of 34-51% in these mice, superior to pravastatin in a head-to-head comparison. These results validate APO-skip SSOs as a candidate therapy for FH.

Isolation and characterization of pharmaceutical grade human pentraxins, serum amyloid P component and C-reactive protein, for clinical use.

The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non-specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL-6 or IL-8, nor does SAP cause release of IL-1ß or IL-10. Furthermore neither of our preparations was pro-inflammatory in mice in vivo.

Protein engineered variants of hepatocyte growth factor/scatter factor promote proliferation of primary human hepatocytes and in rodent liver.

BACKGROUND & AIMS: Hepatocyte growth factor/scatter factor (HGF/SF) stimulates hepatocyte DNA synthesis and protects against apoptosis; in vivo it promotes liver regeneration and reduces fibrosis. However, its therapeutic value is limited by its complex domain structure, high cost of production, instability, and poor tissue penetration due to sequestration by heparin sulfate proteoglycans (HSPGs). METHODS: Using protein engineering techniques, we created a full-length form of HGF/SF (called HP21) and a form of the small, naturally occurring HGF/SF fragment, NK1 (called 1K1), which have reduced affinity for HSPG. We characterized the stability and proliferative and anti-apoptotic effects of these variants in primary human hepatocytes and in rodents. RESULTS: Analytical ultracentrifugation showed that 1K1 and NK1 were more stable than the native, full-length protein. All 4 forms of HGF/SF induced similar levels of DNA synthesis in human hepatocytes; 1K1 and NK1 required heparin, an HSPG analogue, for full agonistic activity. All the proteins reduced levels of Fas ligand-mediated apoptosis, reducing the activity of caspase-3/7 and cleavage of poly(adenosine diphosphate-ribose) polymerase. 1K1 was more active than NK1 in rodents; in healthy mice, 1K1 significantly increased hepatocyte DNA synthesis, and in mice receiving carbon tetrachloride, it reduced fibrosis. In rats, after 70% partial hepatectomy, daily administration of 1K1 for 5 days significantly increased liver mass and the bromodeoxyuridine labeling index compared with mice given NK1. CONCLUSIONS: 1K1, an engineered form of the small, naturally occurring HGF/SF fragment NK1, has reduced affinity for HSPG and exerts proliferative and antiapoptotic effects in cultured hepatocytes. In rodents, 1K1 has antifibrotic effects and promotes liver regeneration. The protein has better stability and is easier to produce than HGF/SF and might be developed as a therapeutic for acute and chronic liver disease.

Requirement for complement in antibody responses is not explained by the classic pathway activator IgM.

Animals lacking complement factors C1q, C2, C3, or C4 have severely impaired Ab responses, suggesting a major role for the classic pathway. The classic pathway is primarily initiated by antigen-Ab complexes. Therefore, its role for primary Ab responses seems paradoxical because only low amounts of specific Abs are present in naive animals. A possible explanation could be that the classic pathway is initiated by IgM from naive mice, binding with sufficient avidity to the antigen. To test this hypothesis, a knock-in mouse strain, Cµ13, with a point mutation in the gene encoding the third constant domain of the µ-heavy chain was constructed. These mice produce IgM in which proline in position 436 is substituted with serine, a mutation previously shown to abrogate the ability of mouse IgM to activate complement. Unexpectedly, the Ab response to sheep erythrocytes and keyhole limpet hemocyanin in Cµ13 mice was similar to that in WT mice. Thus, although secreted IgM and the classic pathway activation are both required for the normal primary Ab response, this does not require that IgM activate C. This led us to test Ab responses in animals lacking one of three other endogenous activators of the classic pathway: specific intracellular adhesion molecule-grabbing nonintegrin R1, serum amyloid P component, and C-reactive protein. Ab responses were also normal in these animals.

Antibodies to human serum amyloid P component eliminate visceral amyloid deposits.

Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one in a thousand deaths in developed countries. Localized amyloid can also have serious consequences; for example, cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present. There is therefore a major unmet need for therapy that safely promotes the clearance of established amyloid deposits. Over 20 different amyloid fibril proteins are responsible for different forms of clinically significant amyloidosis and treatments that substantially reduce the abundance of the respective amyloid fibril precursor proteins can arrest amyloid accumulation. Unfortunately, control of fibril-protein production is not possible in some forms of amyloidosis and in others it is often slow and hazardous. There is no therapy that directly targets amyloid deposits for enhanced clearance. However, all amyloid deposits contain the normal, non-fibrillar plasma glycoprotein, serum amyloid P component (SAP). Here we show that administration of anti-human-SAP antibodies to mice with amyloid deposits containing human SAP triggers a potent, complement-dependent, macrophage-derived giant cell reaction that swiftly removes massive visceral amyloid deposits without adverse effects. Anti-SAP-antibody treatment is clinically feasible because circulating human SAP can be depleted in patients by the bis-d-proline compound CPHPC, thereby enabling injected anti-SAP antibodies to reach residual SAP in the amyloid deposits. The unprecedented capacity of this novel combined therapy to eliminate amyloid deposits should be applicable to all forms of systemic and local amyloidosis.

Increased surface toll-like receptor 2 expression in superantigen shock.

OBJECTIVE: Examination of the interaction between gram-positive bacterial superantigens and toll-like receptor 2 (TLR2) in health and critical illness. DESIGN: Laboratory ex vivo model and prospective clinical, cohort study. SETTING: Two research laboratories in university hospitals and two intensive care units. SUBJECTS/PATIENTS: Laboratory study was performed in transfected HeLa cells and primary human monocytes from healthy volunteers. Clinical study used cells from 20 healthy controls and 45 critically ill patients with circulatory shock. INTERVENTIONS: HeLa cells and purified monocytes were exposed to purified superantigens or isogenic bacterial supernatants and readout obtained by cytokine enzyme-linked immunosorbent assay, flow cytometry, and quantitative real-time polymerase chain reaction. Peripheral blood mononuclear cells from patients with circulatory shock were compared with controls using flow cytometry and measurement of cytokines after ligand exposure. MEASUREMENTS AND MAIN RESULTS: Superantigens were unable to signal through ligation by TLR2. However, TLR2 was up-regulated on the surface of primary human monocytes, without detectable TLR2 messenger RNA neosynthesis, by a range of superantigens and superantigen-containing Streptococcus pyogenes supernatants, although not by isogenic superantigen-negative strains. Superantigen mutant constructs with disrupted major histocompatibility complex class II-binding sites did not support TLR2 up-regulation. TLR2 up-regulation was associated with an increase in the proinflammatory response to TLR2 ligands only at high ligand concentrations. TLR2 was up-regulated in a small subset of patients with severe S. pyogenes sepsis but not in patients with any other category of septic or circulatory shock; responses to TLR2 ligands were reduced in all categories of critically ill patient, however. CONCLUSIONS: Superantigens up-regulate monocyte surface TLR2 expression through major histocompatibility complex class II signaling. Enhanced surface TLR2 expression may be a specific feature of patients with S. pyogenes-induced shock. Importantly, intensity of TLR2 signaling is not necessarily coupled to TLR2 expression when ligand concentrations are low or after onset of critical illness.

Sexual dimorphism in superantigen shock involves elevated TNF-alpha and TNF-alpha induced hepatic apoptosis.

RATIONALE: There is conflicting evidence regarding sex differences in the outcome from severe sepsis and toxic shock. Superantigen-mediated toxic shock affects a higher proportion of female patients. OBJECTIVES: The objective of the current study was to investigate sexual dimorphism in superantigen-associated sepsis and in superantigen-mediated shock and to identify the key mechanisms responsible for this sex difference. METHODS: We measured mortality and serum cytokines after induction of sepsis with isogenic superantigen-positive and superantigen-negative Streptococcus pyogenes in HLA class II transgenics. During superantigen-mediated toxic shock, we measured mortality, T-cell responses, systemic tumor necrosis factor (TNF)-alpha and TNF receptors, TNF-alpha-induced hepatocyte apoptosis, and conditioning of these responses by tamoxifen treatment. MEASUREMENTS AND MAIN RESULTS: In both superantigen-associated sepsis and in superantigen-mediated shock, serum TNF-alpha was increased in females compared with males. This was not attributable to a detectable difference in splenic TNF-alpha transcription; rather, serum soluble TNF receptors were higher in males. Pretreatment of females with the estrogen receptor modulator tamoxifen increased serum soluble TNF receptors, reduced the early serum TNF-alpha response, and improved mortality in females challenged with staphylococcal enterotoxin B. Lethal superantigen shock was characterized by hepatocyte apoptosis, and was reproduced by injection of TNF-alpha. Females had enhanced susceptibility to TNF-alpha-mediated lethality. TNF-alpha-induced hepatocyte apoptosis was greater in females, and was reduced by tamoxifen pretreatment. CONCLUSIONS: Sexual dimorphism in experimental superantigen toxic shock results from increased systemic TNF-alpha in females, coupled with an increased susceptibility to TNF-alpha-induced hepatocyte apoptosis. Both processes are abrogated by estrogen receptor modulators.

High incidence of spontaneous disease in an HLA-DR15 and TCR transgenic multiple sclerosis model.

Multiple sclerosis (MS) is thought to involve CD4 T cell recognition of self myelin, many studies focusing on a pathogenic role for anti-myelin, HLA-DR15-restricted T cells. In experimental allergic encephalomyelitis, it is known which epitopes trigger disease and that disease is associated with determinant spread of T cell reactivity. Characterization of these events in human MS is critical for the development of peptide immunotherapies, but it has been difficult to define the role of determinant spread or define which epitopes might be involved. In this study, we report humanized transgenic mice, strongly expressing HLA-DR15 with an MS-derived TCR; even on a RAG-2 wild-type background, mice spontaneously develop paralysis. Disease, involving demyelination and axonal degeneration, correlates with inter- and intramolecular spread of the T cell response to HLA-DR15-restricted epitopes of myelin basic protein, myelin oligodendrocyte glycoprotein, and alphaB-crystallin. Spread is reproducible and progressive, with two of the epitopes commonly described in responses of HLA-DR15 patients. The fact that this pattern is reiterated as a consequence of CNS tissue damage in mice demonstrates the value of the transgenic model in supplying an in vivo disease context for the human responses. This model, encompassing pathologically relevant, spontaneous disease with the presentation of myelin epitopes in the context of HLA-DR15, should offer new insights and predictions about T cell responses during MS as well as a more stringent test bed for immunotherapies.

Modified amino acid copolymers suppress myelin basic protein 85-99-induced encephalomyelitis in humanized mice through different effects on T cells.

A humanized mouse bearing the HLA-DR2 (DRA/DRB1*1501) protein associated with multiple sclerosis (MS) and the myelin basic protein (MBP) 85-99-specific HLA-DR2-restricted T cell receptor from an MS patient has been used to examine the effectiveness of modified amino acid copolymers poly(F,Y,A,K)n and poly-(V,W,A,K)n in therapy of MBP 85-99-induced experimental autoimmune encephalomyelitis (EAE) in comparison to Copolymer 1 [Copaxone, poly(Y,E,A,K)n]. The copolymers were designed to optimize binding to HLA-DR2. Vaccination, prevention, and treatment of MBP-induced EAE in the humanized mice with copolymers FYAK and VWAK ameliorated EAE more effectively than Copolymer 1, reduced the number of pathological lesions, and prevented the up-regulation of human HLA-DR on CNS microglia. Moreover, VWAK inhibited MBP 85-99-specific T cell proliferation more efficiently than either FYAK or Copolymer 1 and induced anergy of HLA-DR2-restricted transgenic T cells as its principle mechanism. In contrast, FYAK induced proliferation and a pronounced production of the antiinflammatory T helper 2 cytokines IL-4 and IL-10 from nontransgenic T cells as its principle mechanism of immunosuppression. Thus, copolymers generated by using different amino acids inhibited disease using different mechanisms to regulate T cell responses.