McCormick, Daniel

Introduction: As importantly underscored by the SPRINT Trial, innovative blood pressure (BP) lowering and multiorgan protective drugs are needed for treating hypertension (HTN). We engineered a Mayo Clinic designer atrial natriuretic peptide (ANP)-based drug, ZD100, which possesses a unique 12 amino acid (AA) carboxy-terminus (CT) extension while maintaining the N-terminus (NT) of ANP. ZD100 is more potent than ANP in reducing BP, inducing natriuresis and inhibiting aldosterone via pGC-A and its second messenger cGMP. To date, the in vivo actions of ZD100 in a model of essential HTN are undefined. Further, in attempt to enhance and/or minimize pGC-A activation, the importance of specific AAs in the CT and NT of ZD100 is unknown and is critical to define. Hypothesis: We hypothesize that ZD100 is a potent anti-HTN drug activating pGC-A/cGMP system and that specific AA variations in the CT and/or NT of ZD100 will modify the ability of activating pGC-A. Methods: Spontaneously hypertensive rats (SHRs) received intravenous (IV) ZD100 or vehicle and BP, plasma and urinary cGMP and the natriuretic effects were determined. We then engineered 15 strategic ZD100 variants (Vs) consisting of mutated peptides in the CT or NT or both. We stimulated HEK293 cells overexpressing pGC-A with ZD100 and the 15 Vs (10-8 M) and measured intracellular cGMP generation. Results: In SHRs, acute IV ZD100 reduced BP, increased plasma and urinary cGMP generation and induced natriuresis demonstrating efficacy in HTN. In vitro, we found two more potent pGC-A activators (+100% cGMP activation) only among the CT-only mutated ZD100 Vs. Importantly, key mutations of either the CT or NT resulted in 6 loss-of-function Vs identifying importance of peptide-receptor dependency. Conclusions: We demonstrate for the first time in a genetic model of HTN that ZD100 reduces BP, activates systemic and renal pGC-A/cGMP and induces natriuresis. Enhanced or reduced pGC-A activation with targeted AA mutations in the CT and NT of ZD100 provides key structure function insights into ZD100 which may lead to next generation ZD100 therapeutic peptides. These findings support ZD100 as a novel peptide therapeutic strategy for HTN and will help guide the selection of best-in-class ZD100 analogues.

Abstract Experimental autoimmune thyroiditis (EAT) is a model for Hashimoto’s thyroiditis which is characterized by mononuclear cell infiltration and follicular destruction. We previously reported on an Eα-transgenic strain (H2A−E+) that is highly susceptible to human (h) thyroglobulin (Tg)-induced EAT, but strongly tolerant to mouse (m)Tg. After mTg and adjuvant, traditional, resistant strains do not develop thyroiditis, nor detectable primed T cell proliferation, but do make anti-mTg Abs. By contrast, this H2Eb+ strain does not even produce mTg Abs. To identify what epitopes in the mouse thyroid hTg-primed, thyroiditogenic T cells recognize to evoke EAT, we first confirmed that hTg-primed cells do not respond to mTg, nor to other thyroid Ags. We next selected the three immunogenic epitopes on hTg known to be processed and presented by H2Eb, as the basis for synthesizing potential mTg epitopes. One 15-mer peptide, mTg409, did prime T cells, elicit Ab, and generate moderate thyroiditis. Thyroiditis incidence was increased after adoptive transfer of in vitro activated, mTg409-primed cells. Moreover, cells primed to hTg410, the corresponding, pathogenic hTg peptide, cross-reacted with mTg409, and vice versa. This mTg autoepitope, identified by using thyroiditogenic hTg epitopes, helps to explain the severe thyroiditis seen in this novel H2E+ transgenic model. (Supported by NIH grant DK45960)

Abstract Pemphigus vulgaris (PV) is an Ab-mediated autoimmune blistering disease of mucotaneous surfaces. Over 95% of the patients with PV express DR4 or DRw6, and the disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. An appropriate animal model is required to understand immunoregulation and to address the role of immunogenetic components in the production of pathogenic Abs that are characteristic of PV. Therefore, we turned to the development of a mouse model. Four strains of female mice (BALB/c, DBA/1, SJL/J, and HRS/J) were screened for their ability to produce pathogenic anti-Dsg 3 Abs. We demonstrated that only BALB/c mice immunized with a full-length Dsg 3 can produce pathogenic Abs capable of causing acantholysis of human foreskin in culture and blistering in neonatal mice. This observation suggested that either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. No correlation was noted between a given isotype and the pathogenic potential of autoantibodies from different strains of mice. Similarly, the pattern of reactivity of Abs with a panel of 46 synthetic peptides that span the entire Dsg 3 failed to reveal any association between binding specificity and the pathogenic potential, and suggested that pathogenic Abs might recognize conformational epitopes. Moreover, our studies showed that the epitopes recognized by pathogenic Abs are contained within the extracellular Dsg 3.

Abstract We propagated from myasthenia gravis (MG) patients by stimulation in vitro with synthetic sequences (alpha 48-67, alpha 304-322, gamma 75-94 and gamma 321-340) of the human muscle acetylcholine receptor (AChR), CD4+ lines against four 20-residue sequence regions of the AChR alpha and gamma subunits that are recognized by Th cells of most MG patients. Most lines secreted IL-2 and not IL-4, suggesting that they comprise Th1 cells. For three lines we verified that, as reported previously, AChR epitopes are presented by DR molecules: their response to the relevant peptide was abolished by anti-DR Abs. The DR molecules presenting AChR epitopes were identified by testing the response of the lines to the relevant peptide, using APC from donors homozygous for the different DR alleles of the line. We tested the lines with single residue-substituted analogues of the epitope sequence. The results of these experiments indicated that the lines were polyclonal and recognized overlapping epitopes. Their response was abolished by some substitutions, identifying residues common to all epitopes within a given region, whereas other substitutions reduced but did not obliterate the response, indicating residues included in some but not all epitopes recognized by the line. Comparison of the residues involved in epitope formation for different lines supported the conclusion that within the 20-residue immunodominant regions investigated here, the same sequence segment is involved in formation of epitopes, even in DR-discordant patients.

Abstract We have identified in an extracellular segment of the alpha1 subunit of nicotinic acetylcholine receptor (AChR) of homologous muscle a 17-residue autoantigen that, without conjugation to a carrier, activates Th lymphocytes and induces production of autoantibodies that cause electrophysiologic signs of experimental autoimmune myasthenia gravis. A panel of overlapping synthetic peptides revealed two T cell epitopes, one encompassed by residues 121-136 and the other by 129-145. Residues 129 (glutamic acid) and 130 (isoleucine) were implicated in a pathogenic B cell epitope. Peptide 129-145 (Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu-Gln-Asn-Cys-Ser-Met-Lys, a conserved sequence in rat and human AChR) induced autoantibody production in 76% of rats. All seropositive rats had evidence of experimental autoimmune myasthenia gravis; five of five tested had electrophysiologic signs, and all had loss of immunochemically measured autologous muscle AChR. Analogues of 129-145, with single residues substituted by alanine, revealed phenylalanine 135, phenylalanine 137, and glutamic acid 139 as most important determinants of Ag/MHC-II/TCR interactions; phenylalanine 137 is critical for T cell activation. B cells were the major MHC-II-positive cell type to which the self-peptide 129-145 bound in a population of nonimmune splenic cells. More efficient processing and presentation of the Th cell epitope by an expanded population of immune B cells selected by specifically binding another epitope of the same peptide would greatly amplify the production of autoantibodies. Peptide autoantigens of this type could plausibly perpetuate the autoantibody response in myasthenia gravis, and are a rational target for strategies aimed at Ag-specific therapeutic intervention.

Abstract A series of 101 peptides each 20 amino acids in length (10-residue overlap) spanning the helical portion of the mature α-chain of human type II collagen (CII) was synthesized. DBA/1 (H-2q) mice were immunized with individual peptides, and draining lymph node cells were challenged in vitro. Strong responses were measured to three peptides: peptide I (residues 74-93), peptide 14 (residues 254-273), and peptide 81 (residues 924-943). B10.Q (H-2q) mice were responsive to peptides I and 81 but not to peptide 14. B10.RIII (H-2r) mice, which are resistant to arthritis induction following immunization with human CII, were unresponsive to peptides I, 14, and 81. Using single amino acid truncated peptides, we determined minimal immunostimulatory lengths for peptides I and 81. Residues critical to antigenicity were identified by introducing alanine and glycine substitutions into minimal length immunostimulatory peptides. The determinants within peptides I and 81 are 100% homologous to mouse CII and are autoantigens. Peptide 81 has homology to viral proteins. Peptide 14 is 90% homologous to mouse CII and has homology to heat shock proteins.

Abstract This study reports the synthesis of a disulfide-looped peptide corresponding to residues 125-147 (Cys 128-Cys 142) of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle, H alpha 125-147 (Lys-Ser-Tyr-Cys-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu-Gln- Asn-Cys-Ser-Nle-Lys Leu-Gly), and a nondisulfide-looped analogue, H alpha 125-147(S) (Lys-Ser-Tyr-Ser-Glu-Ile-Ile-Val-Thr-His-Phe-Pro-Phe-Asp-Glu- Gln-Asn-Cys-Ser-Nle-Lys-Leu-Gly), in which the amino acid Cys 128 was replaced with serine. Both peptides induced antigen-specific helper T cell responses, as evidenced in vitro by lymph node cell proliferation and in vivo by production of anti-AChR antibodies. Rats immunized with 100 micrograms of either synthetic peptide, without conjugation to a carrier, produced anti-peptide antibodies which bound to native AChR in immunoprecipitation assays and induced modulation of membrane-bound AChR from cultured human myotubes. Both peptides also induced electrophysiologic and biochemical signs of experimental autoimmune myasthenia gravis. Thus, region 125-147 of the AChR alpha-subunit is at least partly exposed extracellularly in human muscle and contains one or more autoantigenic sites capable of stimulating T cells and B cells. Disulfide-linkage between residues Cys 128 and Cys 142 is not essential for myasthenogenicity.

A key immunomodulatory cytokine, IL-10 (interleukin-10), has been shown to be dysregulated in preeclampsia, a pregnancy-specific hypertensive disorder, further characterized by multi-system involvement. However, studies have reported inconsistent findings about circulating IL-10 levels in preeclamptic versus normotensive pregnancies. The aim of the present systematic review and meta-analysis was to assess circulating IL-10 levels in preeclamptic and normotensive pregnancies at 2 time points: before, and at the time of preeclampsia diagnosis. PubMED, EMBASE, and Web of Science databases were searched to include all published studies examining circulating IL-10 levels in preeclamptic and normotensive pregnancies. Differences in IL-10 levels were evaluated by standardized mean differences. Of 876 abstracts screened, 56 studies were included in the meta-analysis. Circulating IL-10 levels were not different before the time of active disease (standardized mean differences, −0.01 [95% CI, −0.11 to 0.08]; P =0.76). At the time of active disease, women with preeclampsia (n=1599) had significantly lower IL-10 levels compared with normotensive controls (n=1998; standardized mean differences, −0.79 [95% CI, −1.22 to −0.35]; P =0.0004). IL-10 levels were lower in both early/severe and late/mild forms of preeclampsia. Subgroup analysis revealed that IL-10 measurement methodology (ELISA or multiplex bead array) and the sample type (plasma or serum) significantly influenced the observed differences, with the use of sera paired with ELISA technology providing the best distinction in IL-10 levels between preeclamptic and normotensive pregnancies. These findings support the role of decreased IL-10 levels in the pathophysiology of preeclampsia. Future studies should address the therapeutic potential of IL-10 in preeclampsia.