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Background: Kidney biopsy is the gold-standard for diagnosing medical renal diseases, but the accuracy of the diagnosis greatly depends on the quality of the biopsy specimen, particularly the amount of renal cortex obtained. Inadequate biopsies, characterized by insufficient cortex or predominant medulla, can lead to inconclusive or incorrect diagnoses, and repeat biopsy. Unfortunately, there has been a concerning increase in the rate of inadequate kidney biopsies, and not all medical centers have access to trained professionals who can assess biopsy adequacy in real time. In response to this challenge, we aimed to develop a machine learning model capable of assessing the percentage cortex of each biopsy pass using smartphone images of the kidney biopsy tissue at the time of biopsy., Methods: 747 kidney biopsy cores and corresponding smartphone macro images were collected from five unused deceased donor kidneys. Each core was imaged, formalin-fixed, sectioned, and stained with Periodic acid-Schiff (PAS) to determine cortex percentage. The fresh unfixed core images were captured using the macro camera on an iPhone 13 Pro. Two experienced renal pathologists independently reviewed the PAS-stained sections to determine the cortex percentage. For the purpose of this study, the biopsies with less than 30% cortex were labeled as inadequate, while those with 30% or more cortex were classified as adequate. The dataset was divided into training ( n =643), validation ( n =30), and test ( n =74) sets. Preprocessing steps involved converting High-Efficiency Image Container iPhone format images to JPEG, normalization, and renal tissue segmentation using a U-Net deep learning model. Subsequently, a classification deep learning model was trained on the renal tissue region of interest and corresponding class label., Results: The deep learning model achieved an accuracy of 85% on the training data. On the independent test dataset, the model exhibited an accuracy of 81%. For inadequate samples in the test dataset, the model showed a sensitivity of 71%, suggesting its capability to identify cases with inadequate cortical representation. The area under the receiver-operating curve (AUC-ROC) on the test dataset was 0.80., Conclusion: We successfully developed and tested a machine learning model for classifying smartphone images of kidney biopsies as either adequate or inadequate, based on the amount of cortex determined by expert renal pathologists. The model's promising results suggest its potential as a smartphone application to assist real-time assessment of kidney biopsy tissue, particularly in settings with limited access to trained personnel. Further refinements and validations are warranted to optimize the model's performance., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shree Sharma reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Multiple autoantigens have been identified in membranous nephropathy (MN) by tissue-based proteomics. However, antigenic targets of disease are unknown for over 10% of patients with MN and over half of those with membranous lupus nephritis (MLN). Here, we identified multiple new targets in PLA2R-/THSD7A-/EXT-/NELL1-quadruple negative MN biopsies through mass spectrometry of immune complexes recovered from biopsy tissue of patients with MN. Patients with MN negative for these four antigens were identified from Arkana Laboratories case archives. Protein G immunoprecipitation recovered immune complexes from frozen biopsy tissue from 142 quadruple-negative cases and 278 cases of known antigen type, followed by interrogation by mass spectrometry. Potential putative antigens were confirmed through paraffin immunofluorescence and co-localization with IgG within immune deposits. Consecutive series of 165 cases of PLA2R-negative MN and 142 MLN biopsies were screened to determine the frequency for each potential antigen. Seven putative antigens were discovered within immune complexes from biopsies of patients with MN including FCN3, CD206, EEA1, SEZ6L2, NPR3, MST1, and VASN. Peptides from these proteins were not enriched in the 278 cases of known antigen type. Between three to 30 unique peptides were detected for each new target. Frequencies of each biomarker, determined by staining consecutive case series, ranged from under 1 to 4.9%. NPR3 and CD206 were only positive in index cases. All cases showed co-localization of IgG within the immune deposits. Thus, seven putative antigens were newly identified in MN and MLN. Due to the number of antigens identified, it is becoming impractical to type PLA2R-negative MN or MLN cases through immunostaining alone. A multiplex approach is needed for subtyping of these diseases., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Membranous lupus nephritis is a frequent cause of nephrotic syndrome in patients with systemic lupus erythematosus. It has been shown in phospholipase A2 receptor positive membranous nephropathy that known antibodies can be detected within sera, determination of the target autoantigen can have diagnostic significance, inform prognosis, and enable non-invasive monitoring of disease activity. Here we utilized mass spectrometry for antigen discovery in laser captured microdissected glomeruli from formalin-fixed paraffin embedded tissue and tissue protein G immunoprecipitation studies to interrogate immune complexes from frozen kidney biopsy tissue. We identified neural cell adhesion molecule 1 (NCAM1) to be a target antigen in some cases of membranous lupus nephritis and within rare cases of primary membranous nephropathy. The prevalence of NCAM1 association was 6.6% of cases of membranous lupus nephritis and in 2.0% of primary membranous nephropathy cases. NCAM1 was found to colocalize with IgG within glomerular immune deposits by confocal microscopy. Additionally, serum from patients with NCAM1-associated membranous nephropathy showed reactivity to NCAM1 recombinant protein on Western blotting and by indirect immunofluorescence assay, demonstrating the presence of circulating antibodies. Thus, we propose that NCAM1 is a target autoantigen in a subset of patients with membranous lupus nephritis. Future studies are needed to determine whether anti-NCAM1 antibody levels correlate with disease activity or response to therapy., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Membranous lupus nephritis (MLN) comprises 10%-15% of lupus nephritis and increases morbidity and mortality of patients with SLE through complications of nephrotic syndrome and chronic kidney failure. Identification of the target antigens in MLN may enable noninvasive monitoring of disease activity, inform treatment decisions, and aid in prognostication, as is now possible for idiopathic MN caused by antibodies against the phospholipase A2 receptor. Here, we show evidence for type III TGF- β receptor (TGFBR3) as a novel biomarker expressed in a subset of patients with MLN., Methods: Mass spectrometry was used for protein discovery through enrichment of glomerular proteins by laser capture microdissection and through elution of immune complexes within MLN biopsy specimens. Colocalization with IgG within glomerular immune deposits from patients and disease controls was evaluated by confocal microscopy. Immunostaining of consecutive case series was used to determine the overall frequency in MN and MLN., Results: TGFBR3 was found to be enriched in glomeruli and coimmunoprecipitated with IgG within a subset of MLN biopsy specimens by mass spectrometry. Staining of consecutive MN cases without clinical evidence of SLE did not show TGFBR3 expression (zero of 104), but showed a 6% prevalence in MLN (11 of 199 cases). TGFBR3 colocalized with IgG along the glomerular basement membranes in TGFBR3-associated MN, but not in controls., Conclusions: Positive staining for TGFBR3 within glomerular immune deposits represents a distinct form of MN, substantially enriched in MLN. A diagnosis of TGFBR3-associated MN can alert the clinician to search for an underlying autoimmune disease., Competing Interests: J. M. Arthur reports serving as a scientific advisor for, or member of, Kidney360; and receiving honoraria from Travere Pharmaceuticals. L. H. Beck reports serving on the advisory boards of Alexion, Genentech, Ionis, and Visterra; serving on the editorial boards of Kidney International Reports and Kidney Medicine; receiving research funding from Pfizer and Sanofi-Genzyme; receiving honoraria from UpToDate, Inc.; and being a coinventor on, and receive royalties related to, the US patent “Diagnostics for Membranous Nephropathy.” D. J. Kenan reports having patents and inventions with EMD Millipore. All remaining authors have nothing to disclose., (Copyright © 2021 by the American Society of Nephrology.)

Patients with membranous nephropathy have an increased risk of malignancy compared to the general population, but the target antigen for malignancy-associated membranous nephropathy is unknown. To explore this, we utilized mass spectrometry for antigen discovery in malignancy-associated membranous nephropathy examining immune complexes eluted from frozen kidney biopsy tissue using protein G bead immunoglobulin capture. Antigen discovery was performed comparing cases of membranous nephropathy of unknown and known type. Mass spectrophotometric analysis revealed that nerve epidermal growth factor-like 1 (NELL1) immune complexes were uniquely present within the biopsy tissue in membranous nephropathy. Additional NELL1-positive cases were subsequently identified by immunofluorescence. In a consecutive series, 3.8% of PLA2R- and THSD7A-negative cases were NELL1-positive. These NELL1-positive cases had segmental to incomplete IgG capillary loop staining (93.4%) and dominant or co-dominant IgG1-subclass staining (95.5%). The mean age of patients with NELL1-positive membranous nephropathy was 66.8 years, with a slight male predominance (58.2%) and 33% had concurrent malignancy. Compared with PLA2R- and THSD7A-positive cases of membranous nephropathy, there was a greater proportion of cases with malignancies in the NELL1-associated group. Thus, NELL1-associated membranous nephropathy has a unique histopathology characterized by incomplete capillary loop staining, IgG1-predominance, and is more often associated with malignancy than other known types of membranous nephropathy., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Coronavirus Disease-19 (COVID-19), caused by the coronavirus SARS-CoV-2, was initially recognized in Wuhan, China and subsequently spread to all continents. The disease primarily affects the lower respiratory system, but may involve other organs and systems. Histopathologic evaluation of tissue from affected patients is crucial for diagnostic purposes, but also for advancing our understanding of the disease. For that reason, we developed immunohistochemical (IHC) and in situ hybridization (ISH) assays for detection of the. virus. A total of eight autopsy lungs, one placenta, and ten kidney biopsies from COVID-19 patients were stained with a panel of commercially available antibodies for IHC and commercially available RNA probes for ISH. Similarly, autopsy lungs, placentas and renal biopsies from non-COVID-19 patients were stained with the same antibodies and probes. All eight lungs and the placenta from COVID-19 patients stained positive by IHC and ISH, while the kidney biopsies stained negative by both methodologies. As expected, all specimens from non-COVID-19 patients were IHC and ISH negative. These two assays represent a sensitive and specific method for detecting the virus in tissue samples. We provide the protocols and the list of commercially available antibodies and probes for these assays, so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes.

Membranous-like glomerulopathy with masked IgG kappa deposits (MGMID) is a recently described pattern of glomerulonephritis with a unique histopathology. The pattern is characterized by subepithelial and/or mesangial immune deposits that are "masked", to immunoglobulin staining by routine immunofluorescence but strongly stain for IgG and kappa light chain after protease digestion. Patients with this pattern of glomerulonephritis are most commonly young females presenting with proteinuria and a vague history of autoimmune disease such as low titer antinuclear antibodies. Here we compared the mass spectrometry profile of laser capture microdissected glomeruli from nine MGMID renal biopsies with eight biopsies showing other patterns of membranous glomerulopathy. The protein most significantly increased in MGMID was serum amyloid P. Immunostaining showed serum amyloid P colocalized with IgG in the glomeruli of MGMID but not with PLA2R-associated membranous glomerulopathy. Serum amyloid P was positive in the glomeruli of all 32 MGMID biopsies but negative in biopsies of other types of membranous glomerulopathies such as those associated with PLA2R and THSD7A. There were four biopsies with glomerular serum amyloid P staining among the 173 biopsies that did not fulfill criteria for MGMID or amyloidosis. All four of these biopsies with positive serum amyloid P staining had a membranous pattern of glomerulopathy with IgG kappa deposits that only differed from MGMID by the lack of "masking". Thus, positive staining within glomerular deposits for serum amyloid P identifies a unique form of glomerulonephritis likely sharing a common pathophysiologic mechanism of disease., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

In immunocompromised patients, reactivation of latent BK polyomavirus (BKPyV) can cause disease with lytic infections of the kidneys and the lower urinary tract. Emerging evidence also links BKPyV to oncogenesis and high-grade intrarenal and transitional cell carcinomas. These neoplasms strongly express polyomavirus large-T antigen as a defining feature; that is, they are 'large-T-positive carcinomas'. Such neoplasms arise in immunocompromised patients, typically in renal allograft recipients, and preferentially in tissues harbouring latent BKPyV. In recent articles in this journal, it was shown that tumour cells harbour replication-incompetent clonal BKPyV. The virus can be truncated and randomly integrated into the genome, and/or it can be mutated in an episomal state. Truncation and/or deletions in the BKPyV non-coding control region can hamper late viral gene expression, replication, and cell lysis, while facilitating overexpression of early genes, including that encoding large-T. Biologically active fusion proteins or alterations in human tumour suppressor or promoter function have not been described so far, making uncontrolled large-T gene expression in non-lytically infected cells a prime suspect for neoplastic transformation. Current concepts of BKPyV-induced disease, including recent reports from this journal, are discussed, and evolving paradigms of BKPyV-associated oncogenesis are highlighted. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

It has been suggested that BK-polyomavirus is linked to oncogenesis via high expression levels of large T-antigen in some urothelial neoplasms arising following kidney transplantation. However, a causal association between BK-polyomavirus, large T-antigen expression and oncogenesis has never been demonstrated in humans. Here we describe an investigation using high-throughput sequencing of tumour DNA obtained from an urothelial carcinoma arising in a renal allograft. We show that a novel BK-polyomavirus strain, named CH-1, is integrated into exon 26 of the myosin-binding protein C1 gene (MYBPC1) on chromosome 12 in tumour cells but not in normal renal cells. Integration of the BK-polyomavirus results in a number of discrete alterations in viral gene expression, including: (a) disruption of VP1 protein expression and robust expression of large T-antigen; (b) preclusion of viral replication; and (c) deletions in the non-coding control region (NCCR), with presumed alterations in promoter feedback loops. Viral integration disrupts one MYBPC1 gene copy and likely alters its expression. Circular episomal BK-polyomavirus gene sequences are not found, and the renal allograft shows no productive polyomavirus infection or polyomavirus nephropathy. These findings support the hypothesis that integration of polyomaviruses is essential to tumourigenesis. It is likely that dysregulation of large T-antigen, with persistent over-expression in non-lytic cells, promotes cell growth, genetic instability and neoplastic transformation., (© 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Cowpox virus infection induces interleukin-10 (IL-10) production from mouse bone marrow-derived dendritic cells (BMDCs) or cells of the mouse macrophage line (RAW264.7) at about 1800 pg/ml, whereas infections with vaccinia virus (strains WR or MVA) induced much less IL-10. Similarly, in vivo, IL-10 levels in bronchoalveolar lavage fluids of mice infected with cowpox virus were significantly higher than those after vaccinia virus infection. However, after intranasal cowpox virus infection, although dendritic and T-cell accumulations in the lungs of IL-10 deficient mice were greater than those in wild-type mice, weight-loss and viral burdens were not significantly different. IL-10 deficient mice were more susceptible than wild-type mice to re-infection with cowpox virus even though titers of neutralizing antibodies and virus-specific CD8 T cells were similar between IL-10 deficient and wild-type mice. Greater bronchopneumonia in IL-10 deficient mice than wild-type mice suggests that IL-10 contributes to the suppression of immunopathology in the lungs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A bifunctional peptide coating was designed, synthesized, and evaluated as a potential pro-healing stent coating. The bifunctional peptide consisted of a short 28-mer sequence that on the N-terminus has a motif with affinity for polystyrene binding and at the C-terminus has a motif that was shown to bind selectively human endothelial cells but not platelets. Results showed that the selective coating, a polystyrene-binding peptide terminated in RRETAWA (FFSFFFPASAWGSSGSSGK(biotin)CRRETAWAC), bound endothelial cells quantitatively as well as the common RGD motif, but unlike RGD, it did not show any preference for platelet adherence. Follow-up work examining the difference in cell line selectivity between endothelial cells, whose binding should be encouraged, and smooth muscle cells, whose binding should be deprecated in a stenting application, did identify a temporal preference of the RRETAWA-terminated peptide coating for endothelial cells. However, the in vivo implications of this apparent selectivity need to be examined in more detail before definitive conclusions can be drawn. The positive in vitro results encourage the continued development of other novel coatings that mimic biological structures, signaling capabilities, or both to direct cellular processes on the surface of synthetic materials.

Bacterial infections can have adverse effects on the efficacy, lifetime and safety of an implanted device and are the second most commonly attributed cause of orthopedic implant failure. We have previously shown the assembly of PEGylated titanium-binding peptides (TBPs) on Ti to obtain a bacteriophobic surface coating that can effectively resist protein adsorption and Staphylococcus aureus (S. aureus) adhesion. In the present study, we examine the effect of multiple TBP repeats on coating performance in vitro. Mono, di, and tetravalent peptides were synthesized and assessed for binding affinity and serum stability. PEGylated analogs were prepared and evaluated for their effect on S. aureus attachment and biofilm formation. Coating performance improved with the number of TBP repeats, with the tetravalent coating, TBP(4)-PEG, showing the best performance in all assays. In particular, TBP(4)-PEG forms a serum-resistant surface coating capable of preventing S. aureus colonization and subsequent biofilm formation. These results further support the role that multivalency can play in the development of improved surface coatings with enhanced stabilities and efficacy for in vivo clinical use., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Resorbable scaffolds such as polyglycolic acid (PGA) are employed in a number of clinical and tissue engineering applications owing to their desirable property of allowing remodeling to form native tissue over time. However, native PGA does not promote endothelial cell adhesion. Here we describe a novel treatment with hetero-bifunctional peptide linkers, termed "interfacial biomaterials" (IFBMs), which are used to alter the surface of PGA to provide appropriate biological cues. IFBMs couple an affinity peptide for the material with a biologically active peptide that promotes desired cellular responses. One such PGA affinity peptide was coupled to the integrin binding domain, Arg-Gly-Asp (RGD), to build a chemically synthesized bimodular 27 amino acid peptide that mediated interactions between PGA and integrin receptors on endothelial cells. Quartz crystal microbalance with dissipation monitoring (QCMD) was used to determine the association constant (K (A) 1 x 10(7) M(-1)) and surface thickness (~3.5 nm). Cell binding studies indicated that IFBM efficiently mediated adhesion, spreading, and cytoskeletal organization of endothelial cells on PGA in an integrin-dependent manner. We show that the IFBM peptide promotes a 200% increase in endothelial cell binding to PGA as well as 70-120% increase in cell spreading from 30 to 60 minutes after plating.

Appropriate surface chemistry between a material and its surrounding biological environment is crucial to the eventual integration and performance of any implant, whether metal, plastic, or ceramic. A robust peptide-based coating technology capable of easily modifying the surface of titanium (Ti) metal through noncovalent binding is described. A short peptide possessing affinity for Ti was identified using a phage display screening process and subjected to an amino acid substitution exercise using solid-phase chemical synthesis. Through these studies, the HKH tripeptide motif was elucidated as an important contributor to Ti binding within the Ti-binding peptide. This peptide spontaneously and selectively adsorbs onto a Ti surface from dilute aqueous solution with submicromolar binding affinities as determined by ELISA and quartz crystal microbalance with dissipation monitoring (QCM-D), through a process largely dominated by electrostatic interactions. Atomic force microscopy (AFM) reveals a densely packed peptide adlayer with an average height of approximately 0.5 nm. Subsequently, a PEGylated analogue of the peptide was shown to rapidly coat Ti to afford a nonfouling surface that efficiently blocked the adsorption of fibronectin and significantly reduced the extent of Staphylococcus aureus attachment and biofilm formation in vitro. These PEGylated-peptide coatings show promise in terms of resolving two major hurdles common to implanted metals: (i) nonspecific protein adsorption and (ii) bacterial colonization. At the same time, the facile one-step modification process will facilitate the point-of-care application of these coatings in the surgical suite.

The generation of recombinant single-chain antibodies from either non-immune or immune phage display antibody libraries is an effective means to obtain high affinity antibodies against a specific target. Non-immune libraries contain a wide variety of antibodies but these are often low affinity. Immune libraries contain a high frequency of high affinity antibodies, but are typically limited to a single antigen. Due to the V(H) and V(L) recombination that occurs during antibody library construction, we hypothesized that an immune antibody library produced against one member of a protein family would contain antibodies specific for other members of the same protein family. Here, we tested this hypothesis by mining an existing anti-human Toll-like receptor-2 (hTLR2) antibody library for antibodies specific for other members of the TLR family. This procedure, referred to as homolog mining, proved to be effective. Using a cell-based system to pan and screen the anti-TLR2 library, we identified single chain antibodies specific for three of the four hTLR2 homologs we targeted. The antibodies identified, anti-murine TLR2, anti-hTLR5, and anti-hTLR6, bind specifically to their target, with no cross-reactivity to hTLR2 or other TLRs tested. These results demonstrate that combinatorial re-assortment of V(H) and V(L) fragments from multiple sources during Ab library construction increases Ab repertoire complexity, allowing antibody libraries produced by immunization with one antigen to be used to obtain antibodies specific to related antigens. The principle of homolog mining may be extended to other protein families and will facilitate and accelerate antibody production processes.

Biomaterials used in implants have traditionally been selected based on their mechanical properties, chemical stability, and biocompatibility. However, the durability and clinical efficacy of implantable biomedical devices remain limited in part due to the absence of appropriate biological interactions at the implant interface and the lack of integration into adjacent tissues. Herein, we describe a robust peptide-based coating technology capable of modifying the surface of existing biomaterials and medical devices through the non-covalent binding of modular biofunctional peptides. These peptides contain at least one material binding sequence and at least one biologically active sequence and thus are termed, "Interfacial Biomaterials" (IFBMs). IFBMs can simultaneously bind the biomaterial surface while endowing it with desired biological functionalities at the interface between the material and biological realms. We demonstrate the capabilities of model IFBMs to convert native polystyrene, a bioinert surface, into a bioactive surface that can support a range of cell activities. We further distinguish between simple cell attachment with insufficient integrin interactions, which in some cases can adversely impact downstream biology, versus biologically appropriate adhesion, cell spreading, and cell survival mediated by IFBMs. Moreover, we show that we can use the coating technology to create spatially resolved patterns of fluorophores and cells on substrates and that these patterns retain their borders in culture.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.

The antiangiogenic protein angiostatin inhibits ATP synthase on the endothelial cell surface, blocking cellular proliferation. To examine the specificity of this interaction, we generated monoclonal antibodies (mAb) directed against ATP synthase. mAb directed against the beta-catalytic subunit of ATP synthase (MAb3D5AB1) inhibits the activity of the F(1) domain of ATP synthase and recognizes the catalytic beta-subunit of ATP synthase. We located the antibody recognition site of MAb3D5AB1 in domains containing the active site of the beta-subunit. MAb3D5AB1 also binds to purified Escherichia coli F(1) with an affinity 25-fold higher than the affinity of angiostatin for this protein. MAb3D5AB1 inhibits the hydrolytic activity of F(1) ATP synthase at lower concentrations than angiostatin. Like angiostatin, MAb3D5AB1 inhibits ATP generation by ATP synthase on the endothelial cell surface in acidic conditions, the typical tumor microenvironment where cell surface ATP synthase exhibits greater activity. MAb3D5AB1 disrupts tube formation and decreases intracellular pH in endothelial cells exposed to low extracellular pH. Neither angiostatin nor MAb3D5AB1 showed an antiangiogenic effect in the corneal neovascularization assay; however, both were effective in the low-pH environment of the chicken chorioallantoic membrane assay. Thus, MAb3D5AB1 shows angiostatin-like properties superior to angiostatin and may be exploited in cancer chemotherapy.

Complex DNA nanostructures have been developed as structural components for the construction of nanoscale objects. Recent advances have enabled self-assembly of organized DNA nanolattices and their use in patterning functional bio-macromolecules and other nanomaterials. Adapter molecules that bind specifically to both DNA lattices and nanomaterials would be useful components in a molecular construction kit for patterned nanodevices. Herein we describe the selection from phage display libraries of single-chain antibodies (scFv) for binding to a specific DNA aptamer and their development as adapter molecules for nanoscale construction. We demonstrate the decoration of various DNA tile structures with aptamers and show binding of the selected single-chain antibody as well as the self-assembly of mixed DNA-protein biomolecular lattices.

Amphiphilic macromolecules containing a polystyrene-adherent peptide domain and a cell-repellent poly(ethylene glycol) domain were designed, synthesized, and evaluated as a cytophobic surface coating. Such cytophobic, or cell-repellent, coatings are of interest for varied medical and biotechnological applications. The composition of the polystyrene binding peptide domain was identified using an M13 phage display library. ELISA and atomic force spectroscopy were used to evaluate the binding affinity of the amphiphile peptide domain to polystyrene. When coated onto polystyrene, the amphiphile reduced cell adhesion of two distinct mammalian cell lines and pathogenic Staphylococcus aureus strains.

Proteins with expanded polyglutamine domains cause nine dominantly inherited, neurodegenerative diseases, including Huntington's disease. There are no therapies that inhibit disease onset or progression. To identify a novel therapeutic, we screened phage displayed peptide libraries for phage that bind preferentially to expanded polyglutamine repeats. We identified a peptide motif that inhibits polyglutamine aggregation in vitro and inhibits death in cellular and Drosophila models of the polyglutamine repeat diseases. In this chapter, we describe in detail how to screen a peptide phage display library and highlight results demonstrating the success of this approach. A similar experimental approach could be used for other diseases caused by conformational change in disease proteins, including prion, Alzheimer's, and Parkinson's diseases.

A receptor for angiostatin was identified on the surface of endothelial cells as F(1)-F(0) ATP synthase (Moser et al., 1999). Proc. Natl. Acad. Sci. U.S.A. 96, 2811-2816. This ectopic ATP synthase catalyzes ATP synthesis and is inhibited by angiostatin over a wide pH range. Endothelial cells grown at normal pH suffer no ill effects from this angiostatin-mediated inhibition of ATP synthase, whereas endothelial cells grown at low, tumor-like extracellular pH cannot maintain a normal intracellular pH and die. Angiostatin inhibits both ATP synthesis and ATP hydrolysis (Moser et al., 2001) and interferes with intracellular pH regulation (Wahl and Grant, 2002; Wahl et al., 2002). Although angiostatin administered intravenously is cleared from the circulation in a matter of minutes, angiostatin-mimetics that are more stable have potential for clinical application. An angiostatin-mimetic activity has recently been observed using a polyclonal antibody against the beta catalytic subunit of ATP synthase. In order to explore the mechanism of action of angiostatin and its mimetics, further work needs to be done to evaluate clinical applicability, specificity, and contraindications for this class of therapeutics.

Tumor growth requires the development of new vessels that sprout from pre-existing normal vessels in a process known as "angiogenesis" [Folkman (1971) N Engl J Med 285:1182-1186]. These new vessels arise from local capillaries, arteries, and veins in response to the release of soluble growth factors from the tumor mass, enabling these tumors to grow beyond the diffusion-limited size of approximately 2 mm diameter. Angiostatin, a naturally occurring inhibitor of angiogenesis, was discovered based on its ability to block tumor growth in vivo by inhibiting the formation of new tumor blood vessels [O'Reilly et al. (1994a) Cold Spring Harb Symp Quant Biol 59:471-482]. Angiostatin is a proteolytically derived internal fragment of plasminogen and may contain various members of the five plasminogen "kringle" domains, depending on the exact sites of proteolysis. Different forms of angiostatin have measurably different activities, suggesting that much remains to be elucidated about angiostatin biology. A number of groups have sought to identify the native cell surface binding site(s) for angiostatin, resulting in at least five different binding sites proposed for angiostatin on the surface of endothelial cells (EC). This review will consider the data supporting all of the various reported angiostatin binding sites and will focus particular attention on the angiostatin binding protein identified by our group: F(1)F(O) ATP synthase. There have been several developments in the quest to elucidate the mechanism of action of angiostatin and the regulation of its receptor. The purpose of this review is to describe the highlights of research on the mechanism of action of angiostatin, its' interaction with ATP synthase on the EC surface, modulators of its activity, and issues that should be explored in future research related to angiostatin and other anti-angiogenic agents., (Copyright (c) 2005 Wiley-Liss, Inc.)

Angiostatin binds to endothelial cell (EC) surface F(1)-F(0) ATP synthase, leading to inhibition of EC migration and proliferation during tumor angiogenesis. This has led to a search for angiostatin mimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F(1) ATP synthase and inhibited F(1)-dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. Although exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared with angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis.

The unliganded thyroid hormone receptor beta (TRbeta) represses the basal transcriptional activity of target genes, in part through interactions with the nuclear receptor corepressor (N-CoR). In this study we have identified a rather unexpected interaction between N-CoR and the nuclear receptor coactivator ACTR. We have demonstrated in vitro and in intact cells that N-CoR directly associates with ACTR and that the interaction surfaces on N-CoR and ACTR are distinct from those required for TR binding. The significance of this finding was demonstrated by showing that N-CoR facilitates an interaction between unliganded-TRbeta and ACTR. One possible consequence of the formation of the trimeric complex of N-CoR/ACTR/unliganded-TR is that N-CoR may raise the local concentration of ACTR at target gene promoters. In support of this hypothesis it was demonstrated that the presence of N-CoR can enhance TRbeta-mediated transcriptional activation. It is proposed, therefore, that TRbeta- mediated activation and repression are integrally linked in a manner that is not predicted by the current models of nuclear receptor action.

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F(1)-F(O) ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F(1) ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F(1) ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the alpha- and beta-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C terminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experiment in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of translation of the HCV RNA genome.

Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.

Recruitment of transcriptional coactivators following ligand activation is a critical step in nuclear receptor-mediated target gene expression. Upon binding an agonist, the receptor undergoes a conformational change which facilitates the formation of a specific coactivator binding pocket within the carboxyl terminus of the receptor. This permits the alpha-helical LXXLL motif within some coactivators to interact with the nuclear receptors. Until recently, the LXXLL motif was thought to function solely as a docking module; however, it now appears that sequences flanking the core motif may play a role in determining receptor selectivity. To address this issue, we used a combinatorial phage display approach to evaluate the role of flanking sequences in influencing these interactions. We sampled more than 10(8) variations of the core LXXLL motif with estradiol-activated estrogen receptor alpha (ERalpha) as a target and found three different classes of peptides. All of these peptides interacted with ERalpha in an agonist-dependent manner and disrupted ERalpha-mediated transcriptional activity when introduced into target cells. Using a series of ERalpha-mutants, we found that these three classes of peptides showed different interaction patterns from each other, suggesting that not all LXXLL motifs are the same and that receptor binding selectivity can be achieved by altering sequences flanking the LXXLL core motif. Most notable in this regard was the discovery of a peptide which, when overexpressed in cells, selectively disrupted ERbeta- but not ERalpha-mediated reporter gene expression. This novel ERbeta-specific antagonist may be useful in identifying and characterizing the ERbeta-regulated process in estradiol-responsive cells. In conclusion, using a combinatorial approach to define cofactor-receptor interactions, we have clearly been able to demonstrate that not all LXXLL motifs are functionally equivalent, a finding which suggests that it may be possible to target receptor-LXXLL interactions to develop receptor-specific antagonists.

Whipple's disease (WD) is an uncommonly diagnosed infection caused by the recently characterized bacillus, Tropheryma whippelii. The association of WD with pericarditis and endocarditis is widely recognized, although less attention has been paid to the myocardium as a site of disease. Although the disease was uniformly fatal before antibiotic therapy, current treatment usually results in cure. We report two patients whose deaths were directly related to cardiac involvement by WD and whose underlying disease escaped diagnosis for years. The first, a 60-year-old white woman, suffered a cardiovascular collapse, and lymphocytic myocarditis was demonstrated at autopsy. The second, a 48-year-old black man, had a lengthy history of progressive cardiac failure that terminated in arrhythmia. Extensive myocardial fibrosis, with lymphocytic and granulomatous inflammation, was demonstrated at autopsy. The presence of T. whippelii was confirmed by electron microscopic examination in both cases and by polymerase chain reaction in one. Patients with WD might harbor an undiagnosed lymphocytic or granulomatous myocarditis, and this diagnosis should be considered in the evaluation of cardiac failure.

A correlative microscopy method for the ultrastructural analysis of focal viral tissue infections is presented. Using a confocal scanning laser microscope, foci of infection are identified in tissue sections prior to embedment; a variety of techniques can be employed for viral detection, including staining with standard histochemical reagents and fluorescently labeled antibodies. Areas of infection identified using confocal microscopy are excised from the tissue sections, embedded, and examined by transmission electron microscopy. Applications of this technique in both diagnostic and basic research settings are described.

We recently reported purification, determination of the nucleotide sequence, and cloning of a 60-nucleotide RNA (I-RNA) from the yeast Saccharomyces cerevisiae which preferentially blocked cap-independent, internal ribosome entry site (IRES)-mediated translation programmed by the poliovirus (PV) 5' untranslated region (UTR). The I-RNA appeared to inhibit IRES-mediated translation by virtue of its ability to bind a 52-kDa polypeptide which interacts with the 5' UTR of viral RNA. We demonstrate here that the HeLa 52-kDa I-RNA-binding protein is immunologically identical to human La autoantigen. Moreover, I-RNA-mediated purified La protein. By using I-RNAs with defined deletions, we have identified sequences of I-RNA required for inhibition of internal initiation of translation. Two smaller fragments of I-RNA (16 and 25 nucleotides) inhibited PV UTR-mediated translation from both monocistronic and bicistronic RNAs. When transfected into HeLa cells, these derivatives of I-RNA inhibited translation of PV RNA. A comparison of protein binding by active and inactive I-RNA mutants demonstrates that in addition to the La protein, three other polypeptides with apparent molecular masses of 80, 70, and 37 kDa may influence the translation-inhibitory activity of I-RNA.

We have characterized the in vivo and in vitro binding of human La protein to the human immunodeficiency virus type 1 (HIV-1) leader RNA, the trans-activation response element (TAR). In immunoprecipitation studies using anti-La serum, La-TAR ribonucleoproteins were recovered from HIV-1-infected lymphocytes. Further characterization of this interaction revealed that La has preference for the TAR stem. However, TAR RNA recognition tolerated changes in the primary sequence of the stem as long as the secondary structure was conserved. This structural aspect of La-TAR recognition was confirmed in competition studies in which certain homopolymers influenced complex formation while other single-stranded and double-stranded RNAs had no effect. Deletion mutants of recombinant La protein were used to demonstrate that the residues responsible for binding to polymerase III precursor transcripts overlapped the binding domain for the TAR leader RNA. This finding of a direct interaction between La and TAR has functional implications for translational regulation of HIV-1 mRNAs as demonstrated in the accompanying report (Y. V. Svitkin, A. Pause, and N. Sonenberg, J. Virol. 68:7001-7007, 1994).

Ample evidence indicates that Alu family interspersed elements retrotranspose via primary transcripts synthesized by RNA polymerase III (pol III) and that this transposition sometimes results in genetic disorders in humans. However, Alu primary transcripts can be processed posttranscriptionally, diverting them away from the transposition pathway. The pol III termination signal of a well-characterized murine B1 (Alu-equivalent) element inhibits RNA 3' processing, thereby stabilizing the putative transposition intermediary. We used an immobilized template-based assay to examine transcription termination by VA1, 7SL, and Alu class III templates and the role of transcript release in the pol III terminator-dependent inhibition of processing of B1-Alu transcripts. We found that the RNA-binding protein La confers this terminator-dependent 3' processing inhibition on transcripts released from the B1-Alu template. Using pure recombinant La protein and affinity-purified transcription complexes, we also demonstrate that La facilitates multiple rounds of transcription reinitiation by pol III. These results illustrate an important role for La in RNA production by demonstrating its ability to clear the termination sites of class III templates, thereby promoting efficient use of transcription complexes by pol III. The role of La as a potential regulatory factor in transcript maturation and how this might apply to Alu interspersed elements is discussed.

Initiation of poliovirus RNA translation by internal entry of ribosomes is believed to require the participation of trans-acting factors. The mechanism of action of these factors is poorly defined. The limiting amount of one of these factors, La protein, in rabbit reticulocyte lysates (RRL) has been postulated to partially explain the inefficient translation of poliovirus RNA in this system. To further characterize La activity in translation and to identify other potential limiting factors, we assayed the ability of La protein as well as purified initiation factors, eIF-2, guanine nucleotide exchange factor (GEF), eIF-4A, eIF-4B, eIF-4F, and eIF-3, to stimulate the synthesis of P1, the capsid precursor protein, in poliovirus type 1 (Mahoney) RNA-programmed RRL. Of the proteins tested, only La, GEF, and to some extent eIF-2 stimulated the synthesis of P1. The enhanced translation of P1 in response to La occurred concomitantly with the inhibition of synthesis of most aberrant polypeptides, resulting from initiation in the middle of the genome. Deletion of the carboxy-terminal half (214 amino acids) of La did not decrease its binding to the poliovirus 5' untranslated region but abrogated the stimulatory and correcting activity in translation. In contrast to La, GEF and eIF-2 stimulated the overall translation and increased the synthesis of aberrant products as well as P1. Neither La, GEF, nor any other factor stimulated translation of encephalomyocarditis virus RNA in RRL. The implications of these findings for the mechanism of internal translation initiation on picornavirus RNAs are discussed.

Surface technologies based upon selection of ligands from combinatorial libraries herald a revolution in molecular research and drug discovery. Molecular diversity is generated by random combinations of monomeric building blocks to form polymeric conformers that constitute 'shape libraries'. The media for exploring surfaces of target molecules include synthetic or biological polymers consisting of natural or modified amino acids, nucleotides, carbohydrates and other organic materials. Targets can be any biological surface, including enzymes, antibodies, receptors and other regulatory molecules. The power of combinatorial selection is in finding conceptual leads for designing high-affinity ligands and effector molecules for the analysis and manipulation of biochemical interactions.

Translation initiation on poliovirus RNA occurs by internal binding of ribosomes to a sequence within the 5' untranslated region. We have previously characterized a HeLa cell protein, p52, that binds to a fragment of the poliovirus 5' untranslated region (K. Meerovitch, J. Pelletier, and N. Sonenberg, Genes Dev. 3:1026-1034, 1989). Here we report the purification of the HeLa p52. Protein microsequencing identified p52 as La autoantigen. The La protein is a human antigen that is recognized by antibodies from patients with autoimmune disorders such as systemic lupus erythematosus and Sjögren's syndrome. We show that the La protein stimulates translation of poliovirus RNA, but not brome mosaic virus, tobacco mosaic virus, and alfalfa mosaic virus 4 RNA, translation in a reticulocyte lysate. In addition, La corrects aberrant translation of poliovirus RNA in a reticulocyte lysate. Subcellular immunolocalization showed that La protein is mainly nuclear, but after poliovirus infection, La is redistributed to the cytoplasm. Our results suggest that La protein is involved in poliovirus internal initiation of translation and might function through a similar mechanism in the translation of cellular mRNAs.

An antiserum raised against a peptide was used to select a unique RNA species from a degenerate pool of RNAs designed to resemble an autoantibody recognition site in U1 RNA. The peptide and the selected RNA epitope could compete for antibody binding, suggesting that both RNA and peptide epitopes occupy the same or overlapping antigen-combining sites. Thus, the RNA epitope functioned as a specific inhibitor of the antibody-antigen interaction. We demonstrate that the RNA epitope can be used to tag unrelated RNA molecules and also to detect the presence of the antibody. We propose that sequence-specific recognition of RNA by antibodies may involve protein-RNA contacts similar to those occurring in other nucleic acid-binding proteins. In addition, these findings are compatible with the suggestion that nucleic acid-binding autoantibodies may arise through immunological cross-reactivity between proteins and nucleic acids.

Members of a family of proteins containing a conserved approximately 80-amino acid RNA recognition motif (RRM) bind specifically to a wide variety of RNA molecules. Structural studies, in combination with sequence alignments, indicate the structural context of both conserved and non-conserved elements in the motif. These analyses suggest that all RRM proteins share a common fold and a similar protein-RNA interface, and that non-conserved residues contribute additional contacts for sequence-specific RNA recognition.

The DNA sequence of a cloned segment of the Escherichia coli chromosome containing ftsQ, ftsA, and part of the ftsZ gene was determined and interpreted for genetic complementation and promoter fusion data for the region. The contiguous genes ftsQ, ftsA, and ftsZ were transcribed in the same direction (clockwise on the genetic map) and each had at least one associated promoter which allowed it to be transcribed independently of neighboring genes. ftsA and ftsZ possessed promoters within the coding sequences of the juxtaposed upstream structural genes, and a promoter element for ftsA was surrounded by a region of twofold symmetry which corresponded closely to a symmetrical element in the region of a putative ftsZ promoter. The structural gene of ftsQ consisted of 838 nucleotides, encoding a 276-residue amino acid polypeptide of molecular weight 31,400; the structural gene of ftsA consisted of 1,260 nucleotides, encoding a 420-residue amino acid polypeptide of molecular weight 45,400. The observation that the termination codon of ftsQ overlaps with a potential initiation codon for ftsA suggested that these two genes may be translationally coupled when transcription is initiated upstream of the ftsQ coding sequence.

A 1.2-kilobase-pair BamHI fragment from a cell envelope-cell division gene cluster of Escherichia coli containing ddl and part of ftsQ was cloned and sequenced, and the sequence was interpreted with the aid of genetic complementation and promoter fusion data for the region. Both ddl and ftsQ were transcribed in the same direction (clockwise on the genetic map). ddl was shown to be capable of independent expression from a promoter of its own, and a promoter was identified within the ddl structural gene. The structural gene of ddl consisted of 918 nucleotides, encoding a 306-residue polypeptide of molecular weight 32,840; the synthesis of a protein of this molecular weight was shown to be directed from the 1.2-kilobase-pair BamHI fragment in minicells. Analysis of the DNA sequence further showed that the termination codon of ddl is separated from the initiation codon of ftsQ by one base, which suggests that these two genes may be translationally coupled when transcription is initiated upstream of ddl. This represents a second instance of potential translational coupling within this gene cluster and also indicates that the ddl and ftsQ transcriptional units must overlap (as has been reported earlier for ftsQ and ftsA and for ftsA and ftsZ).