Diagnostic accuracy of endoscopy in determining rectal tumor proximity to the peritoneal reflection.

PURPOSE: Treatment of invasive rectal adenocarcinoma is stratified into upfront surgery versus neoadjuvant chemoradiotherapy, in part, based on tumor distance from the anal verge (AV). This study examines the correlation between tumor distance measurements (endoscopic and MRI) and relationship to the anterior peritoneal reflection (aPR) on MRI. METHODS: A single-center retrospective study was performed at a tertiary center accredited by the National Accreditation Program for Rectal Cancer (NAPRC). 162 patients with invasive rectal cancer were seen between October of 2018 and April of 2022. Sensitivity and specificity were determined for MRI and endoscopic measurements in their ability to predict tumor location relative to the aPR. RESULTS: One hundred nineteen patients had tumors endoscopically and radiographically measured from the AV. Pelvic MRI characterized tumors as above (intraperitoneal) or at/straddles/below the aPR (extraperitoneal). True positives were defined as extraperitoneal tumors [Formula: see text] 10 cm. True negatives were defined as intraperitoneal tumors > 10 cm. Endoscopy was 81.9% sensitive and 64.3% specific in predicting tumor location with respect to the aPR. MRI was 86.7% sensitive and 92.9% specific. Utilizing a 12 cm cutoff, sensitivity of both modalities increased (94.3%, 91.4%) but specificity decreased (50%, 64.3%). CONCLUSION: For locally invasive rectal cancers, tumor position relative to the aPR is an important factor in determining the role of neoadjuvant therapy. These results suggest endoscopic tumor measurements do not accurately predict tumor location relative to the aPR, and may lead to incorrect treatment stratification recommendation. When the aPR is not identified, MRI-reported tumor distance may be a better predictor of this relationship.

Investigating transmission of SARS-CoV-2 using novel face mask sampling: a protocol for an observational prospective study of index cases and their contacts in a congregate setting.

INTRODUCTION: This study aims to measure how transmission of SARS-CoV-2 occurs in communities and to identify conditions that lend to increased transmission focusing on congregate situations. We will measure SARS-CoV-2 in exhaled breath of asymptomatic and symptomatic persons using face mask sampling-a non-invasive method for SARS-CoV-2 detection in exhaled air. We aim to detect transmission clusters and identify risk factors for SARS-CoV-2 transmission in presymptomatic, asymptomatic and symptomatic individuals. METHODS AND ANALYSIS: In this observational prospective study with daily follow-up, index cases and their respective contacts are identified at each participating institution. Contact definitions are based on Centers for Disease Control and Prevention and local health department guidelines. Participants will wear masks with polyvinyl alcohol test strips adhered to the inside for 2 hours daily. The strips are applied to all masks used over at least 7 days. In addition, self-administered nasal swabs and (optional) finger prick blood samples are performed by participants. Samples are tested by standard PCR protocols and by novel antigen tests. ETHICS AND DISSEMINATION: This study was approved by the Colorado Multiple Institutional Review Board and the WHO Ethics Review Committee. From the data generated, we will analyse transmission clusters and risk factors for transmission of SARS-CoV-2 in congregate settings. The kinetics of asymptomatic transmission and the evaluation of non-invasive tools for detection of transmissibility are of crucial importance for the development of more targeted control interventions-and ultimately to assist with keeping congregate settings open that are essential for our social fabric. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (#NCT05145803).

Factor H related proteins modulate complement activation on kidney cells.

Complement activation at a particular location is determined by the balance of activating and inhibitory proteins. Factor H is a key regulator of the alternative pathway of complement, and genetic or acquired impairments in Factor H are associated with glomerular injury. The human Factor H-related proteins (FHRs) comprise a family of five proteins that are structurally related to Factor H. Variations in the genes or expression levels of the FHRs are also associated with glomerular disease, although the mechanisms of glomerular protection/injury are incompletely understood. To explore the role of the FHRs on complement regulation/dysregulation in the kidney, we expressed and purified recombinant murine FHRs (FHRs A, B, C and E). These four distinct FHRs contain binding regions with high amino acid sequence homology to binding regions within Factor H, but we observed different interactions of the FHRs with Factor H binding ligands, including heparin and C3d. There was differential binding of the FHRs to the resident kidney cell types (mesangial, glomerular endothelial, podocytes, and tubular epithelial). All four FHRs caused complement dysregulation on kidney cell surfaces in vitro, although the magnitude of the effect differed among the FHRs and also varied among the different kidney cells. However, only FHR E caused glomerular complement dysregulation when injected in vivo but did not exacerbate injury when injected into mice with ischemic acute kidney injury, an alternative pathway-mediated model. Thus, our experiments demonstrate that the FHRs have unique, and likely context-dependent, effects on the different cell types within the kidney.

Inferior Vena Cava Obstruction Following Surgical Repair of a Secundum Atrial Septal Defect.

Obstruction of the inferior vena cava (IVC) following surgical repair of an atrial septal defect (ASD) is a rare complication. We present the case of a patient who developed IVC obstruction following surgical repair of a large secundum ASD. The diagnostic and management approaches used to care for this patient are discussed. (Level of Difficulty: Intermediate.).

Large incidental gastrointestinal stromal tumors in a patient presenting with acutely symptomatic nephrolithiasis: A case report.

Gastrointestinal stromal tumors (GISTs) are rare mesenchymal neoplasms, representing approximately 1%-2% of all primary gastrointestinal malignancies. Incidental GISTs are often less than 1 cm when discovered and have been reported predominantly in obese patients undergoing surgery for other medical indications. We present the rare case of a large incidental GIST in a nonobese patient with acutely symptomatic nephrolithiasis. Large GISTs may be treated with neoadjuvant imatinib mesylate to reduce tumor size prior to surgery, though some tumors may experience little change in size despite effective treatment. Treatment response for GISTs can be monitored via imaging studies, such as computed tomography or magnetic resonance imaging, but computed tomography is generally preferred over magnetic resonance imaging.

HIV-1 Neutralizing Antibody Signatures and Application to Epitope-Targeted Vaccine Design.

Eliciting HIV-1-specific broadly neutralizing antibodies (bNAbs) remains a challenge for vaccine development, and the potential of passively delivered bNAbs for prophylaxis and therapeutics is being explored. We used neutralization data from four large virus panels to comprehensively map viral signatures associated with bNAb sensitivity, including amino acids, hypervariable region characteristics, and clade effects across four different classes of bNAbs. The bNAb signatures defined for the variable loop 2 (V2) epitope region of HIV-1 Env were then employed to inform immunogen design in a proof-of-concept exploration of signature-based epitope targeted (SET) vaccines. V2 bNAb signature-guided mutations were introduced into Env 459C to create a trivalent vaccine, and immunization of guinea pigs with V2-SET vaccines resulted in increased breadth of NAb responses compared with Env 459C alone. These data demonstrate that bNAb signatures can be utilized to engineer HIV-1 Env vaccine immunogens capable of eliciting antibody responses with greater neutralization breadth.

Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs.

A vaccination regimen capable of eliciting potent and broadly neutralizing antibodies (bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here, we report the immunogenicity of longitudinal prime/boost vaccination regimens with a panel of HIV-1 envelope (Env) gp140 protein immunogens over a period of 200 weeks in guinea pigs. We assessed vaccine regimens that included a monovalent clade C gp140 (C97ZA012 [C97]), a tetravalent regimen consisting of four clade C gp140s (C97ZA012, 459C, 405C, and 939C [4C]), and a tetravalent regimen consisting of clade A, B, C, and mosaic gp140s (92UG037, PVO.4, C97ZA012, and Mosaic 3.1, respectively [ABCM]). We found that the 4C and ABCM prime/boost regimens were capable of eliciting greater magnitude and breadth of binding antibody responses targeting variable loop 2 (V2) over time than the monovalent C97-only regimen. The longitudinal boosting regimen conducted over more than 2 years increased the magnitude of certain tier 1 NAb responses but did not increase the magnitude or breadth of heterologous tier 2 NAb responses. These data suggest that additional immunogen design strategies are needed to induce broad, high-titer tier 2 NAb responses.IMPORTANCE The elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal for the HIV-1 vaccine field. In this study, we explored the use of a long-term vaccination regimen with different immunogens to determine if we could elicit bNAbs in guinea pigs. We found that longitudinal boosting over more than 2 years increased tier 1 NAb responses but did not increase the magnitude and breadth of tier 2 NAb responses. These data suggest that additional immunogen designs and vaccination strategies will be necessary to induce broad tier 2 NAb responses.

A Rare Case of Atrial Metastasis From a Rectal Adenocarcinoma.

Colorectal cancers typically metastasize to the lymph nodes, liver or lungs. Metastasis to the heart is rare and although a few cases of cardiac metastases from colon cancer are described in the literature, cases of metastatic rectal cancer to the heart are far fewer. A 69-year-old woman with a history of rectal adenocarcinoma treated with neo-adjuvant chemotherapy and radiation, followed by resection and adjuvant chemotherapy, presented with increasing dyspnea on exertion and lower extremity edema 5 years after oncology follow-up. Echocardiography revealed a mass within the right atrium, which was biopsied and found to be consistent with metastatic rectal adenocarcinoma and a thrombus. The patient was deemed to be a poor surgical candidate given her co-morbidities and overall prognosis. Chemotherapy was offered and refused by the patient. The medical literature has a paucity of similar cases of rectal adenocarcinoma metastasizing to the right atrium. Further studies are needed to help guide standardized treatment options.

HIV-1 ENVELOPE. Effect of the cytoplasmic domain on antigenic characteristics of HIV-1 envelope glycoprotein.

A major goal for HIV-1 vaccine development is the production of an immunogen to mimic native, functional HIV-1 envelope trimeric spikes (Env) on the virion surface. We lack a reliable description of a native, functional trimer, however, because of inherent instability and heterogeneity in most preparations. We describe here two conformationally homogeneous Envs derived from difficult-to-neutralize primary isolates. All their non-neutralizing epitopes are fully concealed and independent of their proteolytic processing. Most broadly neutralizing antibodies (bnAbs) recognize these native trimers. Truncation of their cytoplasmic tail has little effect on membrane fusion, but it diminishes binding to trimer-specific bnAbs while exposing non-neutralizing epitopes. These results yield a more accurate antigenic picture than hitherto possible of a genuinely untriggered and functional HIV-1 Env; they can guide effective vaccine development.

A multivalent clade C HIV-1 Env trimer cocktail elicits a higher magnitude of neutralizing antibodies than any individual component.

UNLABELLED: The sequence diversity of human immunodeficiency virus type 1 (HIV-1) presents a formidable challenge to the generation of an HIV-1 vaccine. One strategy to address such sequence diversity and to improve the magnitude of neutralizing antibodies (NAbs) is to utilize multivalent mixtures of HIV-1 envelope (Env) immunogens. Here we report the generation and characterization of three novel, acute clade C HIV-1 Env gp140 trimers (459C, 405C, and 939C), each with unique antigenic properties. Among the single trimers tested, 459C elicited the most potent NAb responses in vaccinated guinea pigs. We evaluated the immunogenicity of various mixtures of clade C Env trimers and found that a quadrivalent cocktail of clade C trimers elicited a greater magnitude of NAbs against a panel of tier 1A and 1B viruses than any single clade C trimer alone, demonstrating that the mixture had an advantage over all individual components of the cocktail. These data suggest that vaccination with a mixture of clade C Env trimers represents a promising strategy to augment vaccine-elicited NAb responses. IMPORTANCE: It is currently not known how to generate potent NAbs to the diverse circulating HIV-1 Envs by vaccination. One strategy to address this diversity is to utilize mixtures of different soluble HIV-1 envelope proteins. In this study, we generated and characterized three distinct, novel, acute clade C soluble trimers. We vaccinated guinea pigs with single trimers as well as mixtures of trimers, and we found that a mixture of four trimers elicited a greater magnitude of NAbs than any single trimer within the mixture. The results of this study suggest that further development of Env trimer cocktails is warranted.

Stable, uncleaved HIV-1 envelope glycoprotein gp140 forms a tightly folded trimer with a native-like structure.

The HIV-1 envelope spike [trimeric (gp160)3, cleaved to (gp120/gp41)3] is the mediator of viral entry and the principal target of humoral immune response to the virus. Production of a recombinant preparation that represents the functional spike poses a challenge for vaccine development, because the (gp120/gp41)3 complex is prone to dissociation. We have reported previously that stable HIV-1 gp140 trimers, the uncleaved ectodomains of (gp160)3, have nearly all of the antigenic properties expected for native viral spikes. Because of recent claims that uncleaved gp140 proteins may adopt a nonnative structure with three gp120 moieties "dangling" from a trimeric gp41 ectodomain in its postfusion conformation, we have inserted a long, flexible linker between gp120 and gp41 in our stable gp140 trimers to assess their stability and to analyze their conformation in solution. The modified trimer has biochemical and antigenic properties virtually identical to those of its unmodified counterpart. Both forms bind a single CD4 per trimer, suggesting that the trimeric conformation occludes two of the three CD4 sites even when a flexible linker has relieved the covalent constraint between gp120 and gp41. In contrast, an artificial trimer containing three gp120s flexibly tethered to a trimerization tag binds three CD4s and has antigenicity nearly identical to that of monomeric gp120. Moreover, the gp41 part of both modified and unmodified gp140 trimers has a structure very different from that of postfusion gp41. These results show that uncleaved gp140 trimers from suitable isolates have compact, native-like structures and support their use as candidate vaccine immunogens.

Design of lipid nanocapsule delivery vehicles for multivalent display of recombinant Env trimers in HIV vaccination.

Immunization strategies that elicit antibodies capable of neutralizing diverse virus strains will likely be an important part of a successful vaccine against HIV. However, strategies to promote robust humoral responses against the native intact HIV envelope trimer structure are lacking. We recently developed chemically cross-linked lipid nanocapsules as carriers of molecular adjuvants and encapsulated or surface-displayed antigens, which promoted follicular helper T-cell responses and elicited high-avidity, durable antibody responses to a candidate malaria antigen. To apply this system to the delivery of HIV antigens, Env gp140 trimers with terminal his-tags (gp140T-his) were anchored to the surface of lipid nanocapsules via Ni-NTA-functionalized lipids. Initial experiments revealed that the large (409 kDa), heavily glycosylated trimers were capable of extracting fluid phase lipids from the membranes of nanocapsules. Thus, liquid-ordered and/or gel-phase lipid compositions were required to stably anchor trimers to the particle membranes. Trimer-loaded nanocapsules combined with the clinically relevant adjuvant monophosphoryl lipid A primed high-titer antibody responses in mice at antigen doses ranging from 5 µg to as low as 100 ng, whereas titers dropped more than 50-fold over the same dose range when soluble trimer was mixed with a strong oil-in-water adjuvant comparator. Nanocapsule immunization also broadened the number of distinct epitopes on the HIV trimer recognized by the antibody response. These results suggest that nanocapsules displaying HIV trimers in an oriented, multivalent presentation can promote key aspects of the humoral response against Env immunogens.

Characterization and immunogenicity of a novel mosaic M HIV-1 gp140 trimer.

UNLABELLED: The extraordinary diversity of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein poses a major challenge for the development of an HIV-1 vaccine. One strategy to circumvent this problem utilizes bioinformatically optimized mosaic antigens. However, mosaic Env proteins expressed as trimers have not been previously evaluated for their stability, antigenicity, and immunogenicity. Here, we report the production and characterization of a stable HIV-1 mosaic M gp140 Env trimer. The mosaic M trimer bound CD4 as well as multiple broadly neutralizing monoclonal antibodies, and biophysical characterization suggested substantial stability. The mosaic M trimer elicited higher neutralizing antibody (nAb) titers against clade B viruses than a previously described clade C (C97ZA.012) gp140 trimer in guinea pigs, whereas the clade C trimer elicited higher nAb titers than the mosaic M trimer against clade A and C viruses. A mixture of the clade C and mosaic M trimers elicited nAb responses that were comparable to the better component of the mixture for each virus tested. These data suggest that combinations of relatively small numbers of immunologically complementary Env trimers may improve nAb responses. IMPORTANCE: The development of an HIV-1 vaccine remains a formidable challenge due to multiple circulating strains of HIV-1 worldwide. This study describes a candidate HIV-1 Env protein vaccine whose sequence has been designed by computational methods to address HIV-1 diversity. The characteristics and immunogenicity of this Env protein, both alone and mixed together with a clade C Env protein vaccine, are described.

Detection of complement activation using monoclonal antibodies against C3d.

During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially bind to the iC3b, C3dg, and C3d fragments in solution, but do not bind to intact C3 or C3b. The same 3 clones also bind to tissue-bound C3 activation fragments when injected systemically. Using mouse models of renal and ocular disease, we confirmed that, following systemic injection, the antibodies accumulated at sites of C3 fragment deposition within the glomerulus, the renal tubulointerstitium, and the posterior pole of the eye. To detect antibodies bound within the eye, we used optical imaging and observed accumulation of the antibodies within retinal lesions in a model of choroidal neovascularization (CNV). Our results demonstrate that imaging methods that use these antibodies may provide a sensitive means of detecting and monitoring complement activation-associated tissue inflammation.

HIV-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120.

HIV-1 envelope glycoprotein is the primary target for HIV-1-specific antibodies. The native HIV-1 envelope spike on the virion surface is a trimer, but trimeric gp140 and monomeric gp120 currently are believed to induce comparable immune responses. Indeed, most studies on the immunogenicity of HIV-1 envelope oligomers have revealed only marginal improvement over monomers. We report here that suitably prepared envelope trimers have nearly all the antigenic properties expected for native viral spikes. These stable, rigorously homogenous trimers have antigenic properties markedly different from those of monomeric gp120s derived from the same sequences, and they induce potent neutralizing antibody responses for a cross-clade set of tier 1 and tier 2 viruses with titers substantially higher than those elicited by the corresponding gp120 monomers. These results, which demonstrate that there are relevant immunologic differences between monomers and high-quality envelope trimers, have important implications for HIV-1 vaccine development.

Delineation of the complement receptor type 2-C3d complex by site-directed mutagenesis and molecular docking.

The interactions between the complement receptor type 2 (CR2) and the C3 complement fragments C3d, C3dg, and iC3b are essential for the initiation of a normal immune response. A crystal-derived structure of the two N-terminal short consensus repeat (SCR1-2) domains of CR2 in complex with C3d has previously been elucidated. However, a number of biochemical and biophysical studies targeting both CR2 and C3d appear to be in conflict with these structural data. Previous mutagenesis and heteronuclear NMR spectroscopy studies directed toward the C3d-binding site on CR2 have indicated that the CR2-C3d cocrystal structure may represent an encounter/intermediate or nonphysiological complex. With regard to the CR2-binding site on C3d, mutagenesis studies by Isenman and coworkers [Isenman, D. E., Leung, E., Mackay, J. D., Bagby, S. & van den Elsen, J. M. H. (2010). Mutational analyses reveal that the staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping contact residues on C3d: Implications for the controversy regarding the CR2/C3d cocrystal structure. J. Immunol. 184, 1946-1955] have implicated an electronegative "concave" surface on C3d in the binding process. This surface is discrete from the CR2-C3d interface identified in the crystal structure. We generated a total of 18 mutations targeting the two (X-ray crystallographic- and mutagenesis-based) proposed CR2 SCR1-2 binding sites on C3d. Using ELISA analyses, we were able to assess binding of mutant forms of C3d to CR2. Mutations directed toward the concave surface of C3d result in substantially compromised CR2 binding. By contrast, targeting the CR2-C3d interface identified in the cocrystal structure and the surrounding area results in significantly lower levels of disruption in binding. Molecular modeling approaches used to investigate disparities between the biochemical data and the X-ray structure of the CR2-C3d cocrystal result in highest-scoring solutions in which CR2 SCR1-2 is docked within the concave surface of C3d.

Biophysical investigations of complement receptor 2 (CD21 and CR2)-ligand interactions reveal amino acid contacts unique to each receptor-ligand pair.

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1-2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNalpha, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNalpha were titrated into (15)N-labeled SCR1-2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn(11), Arg(13), Ala(22), Arg(28), Ser(32), Arg(36), Lys(41), Lys(57), Tyr(64), Lys(67), Tyr(68), Arg(83), Gly(84), and Arg(89). With regard to IFNalpha, the binding is similar to the CR2-C3d interaction with specific residues being Arg(13), Tyr(16), Arg(28), Ser(42), Lys(48), Lys(50), Tyr(68), Arg(83), Gly(84), and Arg(89). We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K(d) values of 0.13 and 160 microm, whereas the CR2-gp350 and CR2-IFNalpha interactions were characterized as single site binding events with affinities of 0.014 and 0.035 microm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.

Intrinsic amino acid side-chain hydrophilicity/hydrophobicity coefficients determined by reversed-phase high-performance liquid chromatography of model peptides: comparison with other hydrophilicity/hydrophobicity scales.

An accurate determination of the intrinsic hydrophilicity/hydrophobicity of amino acid side-chains in peptides and proteins is fundamental in understanding many area of research, including protein folding and stability, peptide and protein function, protein-protein interactions and peptide/protein oligomerization, as well as the design of protocols for purification and characterization of peptides and proteins. Our definition of intrinsic hydrophilicity/hydrophobicity of side-chains is the maximum possible hydrophilicity/hydrophobicity of side-chains in the absence of any nearest-neighbor effects and/or any conformational effects of the polypeptide chain that prevent full expression of side-chain hydrophilicity/hydrophobicity. In this review, we have compared an experimentally derived intrinsic side-chain hydrophilicity/hydrophobicity scale generated from RP-HPLC retention behavior of de novo designed synthetic model peptides at pH 2 and pH 7 with other RP-HPLC-derived scales, as well as scales generated from classic experimental and calculation-based methods of octanol/water partitioning of Nalpha-acetyl-amino-acid amides or free energy of transfer of free amino acids. Generally poor correlation was found with previous RP-HPLC-derived scales, likely due to the random nature of the peptide mixtures in terms of varying peptide size, conformation and frequency of particular amino acids. In addition, generally poor correlation with the classical approaches served to underline the importance of the presence of a polypeptide backbone when generating intrinsic values. We have shown that the intrinsic scale determined here is in full agreement with the structural characteristics of amino acid side-chains.

Mapping of the C3d ligand binding site on complement receptor 2 (CR2/CD21) using nuclear magnetic resonance and chemical shift analysis.

Complement receptor 2 (CR2, CD21) is a cell membrane protein, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs (SCR1-2) mediate the interaction of CR2 with its four known ligands (C3d, Epstein-Barr virus gp350, interferon-alpha, and CD23). Inhibitory monoclonal antibodies against SCR1-2 block binding of all ligands. To develop ligand-specific inhibitors that would also assist in identifying residues unique to each receptor-ligand interaction, phage were selected from randomly generated libraries by panning with recombinant SCR1-2, followed by specific ligand-driven elution. Derived peptides were tested by competition ELISA. One peptide, C3dp1 (APQHLSSQYSRT) exhibited ligand-specific inhibition at midmicromolar IC(50). C3d was titrated into (15)N-labeled SCR1-2, which revealed chemical shift changes indicative of specific intermolecular interactions. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to C3d, the binding surface includes regions of SCR1, SCR2, and the inter-SCR linker, specifically residues Arg(13), Tyr(16), Arg(28), Tyr(29), Ser(32), Thr(34), Lys(48), Asp(56), Lys(57), Tyr(68), Arg(83), Gly(84), Asn(101), Asn(105), and Ser(109). SCR1 and SCR2 demonstrated distinct binding modes. The CR2 binding surface incorporating SCR1 is inconsistent with a previous x-ray CR2-C3d co-crystal analysis but consistent with mutagenesis, x-ray neutron scattering, and inhibitory monoclonal antibody epitope mapping. Titration with C3dp1 yielded chemical shift changes (Arg(13), Tyr(16), Thr(34), Lys(48), Asp(56), Lys(57), Tyr(68), Arg(83), Gly(84), Asn(105), and Ser(109)) overlapping with C3d, indicating that C3dp1 interacts at the same CR2 site as C3d.