ABSTRACT
Androgen deprivation therapy (ADT) is recommended for the treatment of advanced prostate cancer. Inadequate suppression of testosterone while on ADT poses a clinical challenge and requires evaluation of multiple potential causes, including adrenal virilizing disorders. We present 2 cases of elderly patients with prostate cancer who had undiagnosed congenital adrenal hyperplasia (CAH) driving persistent testosterone elevation during ADT. The first patient is a 73-year-old man who underwent radical prostatectomy on initial diagnosis and was later started on ADT with leuprolide following tumor recurrence. He had a testosterone level of 294.4 ng/dL and prostate-specific antigen (PSA) level of 17.7 ng/mL despite leuprolide use. Additional workup revealed adrenal nodular hyperplasia, elevated 17-hydroxyprogesterone (19 910 ng/dL) and dehydroepiandrosterone sulfate (378 mcg/dL), and 2 mutations of the CYP21A2 gene consistent with simple virilizing CAH. The second patient is an 82-year-old man who received stereotactic radiation therapy at time of diagnosis. He had insufficient suppression of testosterone with evidence of metastatic disease despite treatment with leuprolide and subsequently degarelix. Laboratory workup revealed elevated 17-hydroxyprogesterone (4910 ng/dL) and dehydroepiandrosterone sulfate (312 mcg/dL). Based on clinical, radiographic and biochemical findings, the patient was diagnosed with nonclassic CAH. The first patient initiated glucocorticoid therapy, and the second patient was treated with the CYP17 inhibitor abiraterone in combination with glucocorticoids. Both patients experienced rapid decline in testosterone and PSA levels. Inadequate testosterone suppression during ADT should trigger evaluation for causes of persistent hyperandrogenemia. CAH can lead to hyperandrogenemia and pose challenges when treating patients with prostate cancer.
ABSTRACT
Importance: Although seroepidemiological studies indicate that greater than 50% of the population has been infected with John Cunningham virus (JCV), the sites of JCV persistence remain incompletely characterized. Objective: To determine sites of JCV persistence in immunologically healthy individuals. Design, Setting, and Participants: Tissue specimens from multiple sites including brain, renal, and nonrenal tissues were obtained at autopsy performed in the Department of Pathology at the University of Kentucky from 12 immunologically healthy patients between February 9, 2011, and November 27, 2012. Quantitative polymerase chain reaction was performed on the tissue specimens and urine. Serum JCV antibody status was determined by enzyme-linked immunosorbent assay. Main Outcomes and Measures: The detection and quantification of JCV from the tissues by quantitative polymerase chain reaction illuminated sites of viral persistence. These results were correlated with JCV antibody levels. Results: Autopsies were performed on 12 individuals, 10 men and 2 women, ranging in age from 25 to 75 years (mean, 55.3 years). Seven of 12 individuals were JCV antibody seropositive based on absorbance units. Serostatus was associated with amounts of JCV DNA in urine and its tissue distribution. John Cunningham virus DNA was found in 75% of genitourinary tissue samples from donors (18 of 24) with high JCV antibody levels, 13.3% of donors with low levels i(4 of 30), and 0% of seronegative persons (0 of 32). In nongenitourinary tissues, JCV DNA was detected in 45.1% of tissue samples of donors (32 of 71) with high JCV, 2.2% of donors with low JCV serostatus (2 of 93), and 0% of seronegative persons (0 of 43). Genitourinary tissues had higher copy numbers than other sites. John Cunningham virus DNA was detected in urine of seronegative individuals in a research-grade assay. Conclusions and Relevance: Persistent (latent or actively replicating) JCV infection mostly predominates in genitourinary tissues but distributes in other tissues at low copy number. The distribution and copy numbers of the virus appear to correlate with urinary JCV shedding and serostatus.
Subject(s)
Antibodies, Viral/blood , DNA, Viral/urine , JC Virus/genetics , JC Virus/immunology , Tumor Virus Infections , Adult , Aged , Autopsy , Female , Humans , Immunocompromised Host , Male , Middle Aged , Polyomavirus Infections/immunology , Seroepidemiologic Studies , Tissue Distribution , Tumor Virus Infections/genetics , Tumor Virus Infections/immunology , Tumor Virus Infections/virologyABSTRACT
Polyomavirus JC (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a rare and frequently fatal brain disease that afflicts a small fraction of the immune-compromised population, including those affected by AIDS and transplantation recipients on immunosuppressive drug therapy. Currently there is no specific therapy for PML. The major capsid viral protein 1 (VP1) involved in binding to sialic acid cell receptors is believed to be a key player in pathogenesis. PML-specific mutations in JCV VP1 sequences present at the binding pocket of sialic acid cell receptors, such as L55F and S269F, abolish sialic acid recognition and might favor PML onset. Early diagnosis of these PML-specific mutations may help identify patients at high risk of PML, thus reducing the risks associated with immunosuppressive therapy. As a first step in the development of such early diagnostic tools, we report identification and characterization of affinity reagents that specifically recognize PML-specific mutations in VP1 variants using phage display technology. We first identified 2 peptides targeting wild type VP1 with moderate specificity. Fine-tuning via selection of biased libraries designed based on 2 parental peptides yielded peptides with different, yet still moderate, bindinspecificities. In contrast, we had great success in identifying synthetic antibodies that recognize one of the PML-specific mutations (L55F) with high specificity from the phage-displayed libraries. These peptides and synthetic antibodies represent potential candidates for developing tailored immune-based assays for PML risk stratification in addition to complementing affinity reagents currently available for the study of PML and JCV.
Subject(s)
Capsid Proteins/immunology , JC Virus/immunology , Leukoencephalopathy, Progressive Multifocal/immunology , Mutation , Peptides/immunology , Single-Chain Antibodies/immunology , Capsid Proteins/genetics , Humans , JC Virus/genetics , Leukoencephalopathy, Progressive Multifocal/genetics , Peptides/genetics , Single-Chain Antibodies/geneticsABSTRACT
Antibodies are key components of the adaptive immune system and are well-established protein therapeutic agents. Typically high-affinity antibodies are obtained by immunization of rodent species that need to be humanized to reduce their immunogenicity. The complementarity-determining regions (CDRs) contain the residues in a defined loop structure that confer antigen binding, which must be retained in the humanized antibody. To design a humanized antibody, we graft the mature murine CDRs onto a germline human acceptor framework. Structural defects due to mismatches at the graft interface can be fixed by mutating some framework residues to murine, or by mutating some residues on the CDRs' backside to human or to a de novo designed sequence. The first approach, framework redesign, can yield an antibody with binding better than the CDR graft and one equivalent to the mature murine, and reduced immunogenicity. The second approach, CDR redesign, is presented here as a new approach, yielding an antibody with binding better than the CDR graft, and immunogenicity potentially less than that from framework redesign. Application of both approaches to the humanization of anti-α4 integrin antibody HP1/2 is presented and the concept of the hybrid humanization approach that retains "difficult to match" murine framework amino acids and uses de novo CDR design to minimize murine amino acid content and reduce cell-mediated cytotoxicity liabilities is discussed.
Subject(s)
Antibodies, Monoclonal, Humanized/biosynthesis , Complementarity Determining Regions/biosynthesis , Immunoglobulin Fab Fragments/biosynthesis , Amino Acid Sequence , Amino Acid Substitution , Animals , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/genetics , Antibody Affinity , Binding Sites , Cloning, Molecular , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Crystallography, X-Ray , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Hybridomas , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Jurkat Cells , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-DirectedABSTRACT
BACKGROUND: JC virus (JCV) infection is a prerequisite for development of progressive multifocal leukoencephalopathy (PML). We previously described the development of a novel, two-step enzyme-linked immunosorbent assay (ELISA) that detects anti-JCV antibodies in human serum or plasma, and the potential clinical utility of anti-JCV antibody status for PML risk stratification. OBJECTIVES: To validate the anti-JCV antibody ELISA at multiple clinical laboratories in order to demonstrate the robustness of the method. STUDY DESIGN: Analytical validation of the ELISA was performed at four laboratories by evaluation of intra- and inter-assay precision, analytical specificity and sensitivity, matrix interference, robustness, sample and reagent stability. RESULTS: Analytical validation demonstrated that the assay is sensitive, specific, and precise. The assay sensitivity was estimated at 1.7ng/mL using a humanized anti-JCV monoclonal antibody control. The sensitivity to detect JCV infection was estimated to be 97.5%. The specificity of the assay to discriminate JCV-specific antibodies from antibodies directed to BK virus, a related polyomavirus, was also demonstrated. The inter- and intra-assay precision was ≤6.0% and 9.8% for the screening step and 2.6% and 11.3% for the confirmation step. Results obtained for plasma and serum were highly congruent, and assay robustness was demonstrated by the highly concordant results generated by four laboratories testing a common panel of 100 blinded samples. CONCLUSIONS: The novel, two-step ELISA to detect anti-JCV antibodies in human serum and plasma is robust, and assay performance is consistent and reproducible in multiple laboratories, making it suitable to support testing for global clinical studies.
Subject(s)
Antibodies, Viral/blood , Enzyme-Linked Immunosorbent Assay/methods , JC Virus/immunology , Leukoencephalopathy, Progressive Multifocal/diagnosis , Antigens, Viral/immunology , Cross Reactions , Humans , Leukoencephalopathy, Progressive Multifocal/blood , Leukoencephalopathy, Progressive Multifocal/immunology , Leukoencephalopathy, Progressive Multifocal/virology , Protein Stability , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease caused by JC virus (JCV) infection of oligodendrocytes, may develop in patients with immune disorders following reactivation of chronic benign infection. Mutations of JCV capsid viral protein 1 (VP1), the capsid protein involved in binding to sialic acid cell receptors, might favor PML onset. Cerebrospinal fluid sequences from 37/40 PML patients contained one of several JCV VP1 amino acid mutations, which were also present in paired plasma but not urine sequences despite the same viral genetic background. VP1-derived virus-like particles (VLPs) carrying these mutations lost hemagglutination ability, showed different ganglioside specificity, and abolished binding to different peripheral cell types compared with wild-type VLPs. However, mutants still bound brain-derived cells, and binding was not affected by sialic acid removal by neuraminidase. JCV VP1 substitutions are acquired intrapatient and might favor JCV brain invasion through abrogation of sialic acid binding with peripheral cells, while maintaining sialic acid-independent binding with brain cells.
Subject(s)
Capsid Proteins/genetics , JC Virus/genetics , JC Virus/pathogenicity , Leukoencephalopathy, Progressive Multifocal/pathology , Mutation, Missense , Receptors, Virus/metabolism , Viral Tropism , Adult , Cerebrospinal Fluid/virology , Female , Human Development , Humans , JC Virus/isolation & purification , Male , Middle Aged , Nuclear Proteins , Promyelocytic Leukemia Protein , Transcription Factors , Tumor Suppressor Proteins , Virus AttachmentABSTRACT
BACKGROUND: Progressive multifocal leukoencephalopathy (PML) in natalizumab-treated MS patients is linked to JC virus (JCV) infection. JCV sequence variation and rearrangements influence viral pathogenicity and tropism. To better understand PML development, we analyzed viral DNA sequences in blood, CSF and/or urine of natalizumab-treated PML patients. METHODS: Using biofluid samples from 17 natalizumab-treated PML patients, we sequenced multiple isolates of the JCV noncoding control region (NCCR), VP1 capsid coding region, and the entire 5 kb viral genome. RESULTS: Analysis of JCV from multiple biofluids revealed that individuals were infected with a single genotype. Across our patient cohort, multiple PML-associated NCCR rearrangements and VP1 mutations were present in CSF and blood, but absent from urine-derived virus. NCCR rearrangements occurred in CSF of 100% of our cohort. VP1 mutations were observed in blood or CSF in 81% of patients. Sequencing of complete JCV genomes demonstrated that NCCR rearrangements could occur without VP1 mutations, but VP1 mutations were not observed without NCCR rearrangement. CONCLUSIONS: These data confirm that JCV in natalizumab-PML patients is similar to that observed in other PML patient groups, multiple genotypes are associated with PML, individual patients appear to be infected with a single genotype, and PML-associated mutations arise in patients during PML development.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Blood/virology , Immunologic Factors/administration & dosage , JC Virus/genetics , JC Virus/isolation & purification , Leukoencephalopathy, Progressive Multifocal/drug therapy , Leukoencephalopathy, Progressive Multifocal/virology , Amino Acid Substitution/genetics , Antibodies, Monoclonal, Humanized , Capsid Proteins/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , Humans , Mutation, Missense , Natalizumab , Sequence Analysis, DNAABSTRACT
OBJECTIVE: A study was undertaken to establish an enzyme-linked immunosorbent assay (ELISA) to detect JC virus (JCV)-specific antibodies in multiple sclerosis (MS) patients, and to evaluate its potential utility for identifying patients at higher or lower risk (ie, risk stratification) of developing progressive multifocal leukoencephalopathy (PML). METHODS: A 2-step assay for detecting and confirming the presence of anti-JCV antibodies in human serum and plasma was developed and demonstrated to be both sensitive and specific. ELISA cutpoints were statistically established using sera from >800 MS patients from natalizumab clinical studies. Subsequently, this assay was used to determine the presence of anti-JCV antibodies in natalizumab-treated PML patients where serum samples were collected 16-180 months prior to the diagnosis of PML. RESULTS: In our evaluation of natalizumab-treated MS patients, 53.6% tested positive for anti-JCV antibodies, with a 95% confidence interval of 49.9 to 57.3%. The false-negative rate of the ELISA was calculated to be approximately 2.5%, with an upper 1-sided confidence limit of 4.4%. Notably, we observed anti-JCV antibodies in all 17 available pre-PML sera samples, which was significantly different from the 53.6% seropositivity observed in the overall MS study population (p < 0.0001). INTERPRETATION: This 2-step assay provides a means to classify MS patients as having detectable or not detectable levels of anti-JCV antibodies. The finding that all 17 of the pre-PML samples that were available tested seropositive, and none tested seronegative, warrants further research on the clinical utility of the anti-JCV antibody assay as a potential tool for stratifying MS patients for higher or lower risk of developing PML.
Subject(s)
Antibodies, Anti-Idiotypic/therapeutic use , JC Virus/immunology , Leukoencephalopathy, Progressive Multifocal , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized , DNA, Viral/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Leukoencephalopathy, Progressive Multifocal/diagnosis , Leukoencephalopathy, Progressive Multifocal/immunology , Leukoencephalopathy, Progressive Multifocal/therapy , Natalizumab , Risk Factors , Viral Load/methodsABSTRACT
Progressive multifocal leukoencephalopathy (PML) is a rare but frequently fatal disease caused by the uncontrolled replication of JC virus (JCV), a polyomavirus, in the brains of some immunocompromised individuals. Currently, no effective antiviral treatment for this disease has been identified. As a first step in the identification of such therapy, we screened the Spectrum collection of 2,000 approved drugs and biologically active molecules for their anti-JCV activities in an in vitro infection assay. We identified a number of different drugs and compounds that had significant anti-JCV activities at micromolar concentrations and lacked cellular toxicity. Of the compounds with anti-JCV activities, only mefloquine, an antimalarial agent, has been reported to show sufficiently high penetration into the central nervous system such that it would be predicted to achieve efficacious concentrations in the brain. Additional in vitro experiments demonstrated that mefloquine inhibits the viral infection rates of three different JCV isolates, JCV(Mad1), JCV(Mad4), and JCV(M1/SVEDelta), and does so in three different cell types, transformed human glial (SVG-A) cells, primary human fetal glial cells, and primary human astrocytes. Using quantitative PCR to quantify the number of viral copies in cultured cells, we have also shown that mefloquine inhibits viral DNA replication. Finally, we demonstrated that mefloquine does not block viral cell entry; rather, it inhibits viral replication in cells after viral entry. Although no suitable animal model of PML or JCV infection is available for the testing of mefloquine in vivo, our in vitro results, combined with biodistribution data published in the literature, suggest that mefloquine could be an effective therapy for PML.
Subject(s)
Antiviral Agents/pharmacology , JC Virus/drug effects , Mefloquine/pharmacology , Neuroglia/virology , Virus Replication/drug effects , Antiviral Agents/chemistry , Astrocytes/virology , Cell Line, Transformed , Cells, Cultured , Humans , JC Virus/isolation & purification , JC Virus/physiology , Leukoencephalopathy, Progressive Multifocal/virology , Mefloquine/chemistry , Microbial Sensitivity Tests/methods , Models, Molecular , Neuroglia/cytology , Simian virus 40/pathogenicityABSTRACT
PML is a progressive and mostly fatal demyelinating disease caused by JC virus infection and destruction of infected oligodendrocytes in multiple brain foci of susceptible individuals. While JC virus is highly prevalent in the human population, PML is a rare disease that exclusively afflicts only a small percentage of immunocompromised individuals including those affected by HIV (AIDS) or immunosuppressive drugs. Viral- and/or host-specific factors, and not simply immune status, must be at play to account for the very large discrepancy between viral prevalence and low disease incidence. Here, we show that several amino acids on the surface of the JC virus capsid protein VP1 display accelerated evolution in viral sequences isolated from PML patients but not in sequences isolated from healthy subjects. We provide strong evidence that at least some of these mutations are involved in binding of sialic acid, a known receptor for the JC virus. Using statistical methods of molecular evolution, we performed a comprehensive analysis of JC virus VP1 sequences isolated from 55 PML patients and 253 sequences isolated from the urine of healthy individuals and found that a subset of amino acids found exclusively among PML VP1 sequences is acquired via adaptive evolution. By modeling of the 3-D structure of the JC virus capsid, we showed that these residues are located within the sialic acid binding site, a JC virus receptor for cell infection. Finally, we go on to demonstrate the involvement of some of these sites in receptor binding by demonstrating a profound reduction in hemagglutination properties of viral-like particles made of the VP1 protein carrying these mutations. Collectively, these results suggest that a more virulent PML causing phenotype of JC virus is acquired via adaptive evolution that changes viral specificity for its cellular receptor(s).
Subject(s)
Capsid Proteins/genetics , JC Virus/genetics , Leukoencephalopathy, Progressive Multifocal/virology , Mutation , Capsid Proteins/metabolism , Evolution, Molecular , Humans , JC Virus/isolation & purification , Leukoencephalopathy, Progressive Multifocal/metabolism , Phenotype , PhylogenyABSTRACT
BACKGROUND: WV Walks replicated the Wheeling Walks community-wide campaign methodology to promote physical activity. METHODS: A social marketing intervention promoted walking among insufficiently active 40- to 65-year-olds throughout the television media market in north-central West Virginia. The intervention included participatory planning, an 8-week mass media-based campaign, and policy and environmental activities. Pre and post random-digit-dial cohort telephone surveys were conducted at baseline and immediately postcampaign in intervention and comparison regions. RESULTS: The campaign resulted in maximal message awareness in north-central WV and demonstrated a significant increase in walking behavior represented by an absolute shift of 12% of the target population from insufficiently active to active (> or = 30 minutes, 5 days per week), versus the comparison community (adjusted odds ratio 1.82, CI: 1.05-3.17). Policy and environmental changes were also evident. CONCLUSIONS: This replication study increases our confidence that the initial effects observed in the Wheeling Walks intervention are generalizable to other similar rural communities.
Subject(s)
Health Promotion/organization & administration , Walking , Adult , Aged , Community Participation , Exercise , Female , Humans , Interviews as Topic , Male , Mass Media , Middle Aged , Social Marketing , West VirginiaABSTRACT
The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(6) expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(6) expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.
Subject(s)
Antibodies, Monoclonal/pharmacology , Carcinoma, Squamous Cell/pathology , Cell Proliferation/drug effects , Integrin alpha5/immunology , Pharyngeal Neoplasms/pathology , Transforming Growth Factor beta/physiology , Animals , Carcinoma, Squamous Cell/metabolism , Cells, Cultured , Disease Progression , Female , Humans , Immunoglobulin Fc Fragments/pharmacology , Integrin alpha5/metabolism , Integrin alpha5/physiology , Mice , Mice, Nude , Mink , Pharyngeal Neoplasms/metabolism , Protein Isoforms/immunology , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/pharmacology , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/chemistry , Recombinant Fusion Proteins/pharmacology , Signal Transduction/genetics , Smad Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
RATIONALE: Transforming growth factor (TGF)-beta has a central role in driving many of the pathological processes that characterize pulmonary fibrosis. Inhibition of the integrin alpha(v)beta6, a key activator of TGF-beta in lung, is an attractive therapeutic strategy, as it may be possible to inhibit TGF-beta at sites of alpha(v)beta6 up-regulation without affecting other homeostatic roles of TGF-beta. OBJECTIVES: To analyze the expression of alpha(v)beta6 in human pulmonary fibrosis, and to functionally test the efficacy of therapeutic inhibition of alpha(v)beta6-mediated TGF-beta activation in murine bleomycin-induced pulmonary fibrosis. METHODS: Lung biopsies from patients with a diagnosis of systemic sclerosis or idiopathic pulmonary fibrosis were stained for alpha(v)beta6 expression. A range of concentrations of a monoclonal antibody that blocks alpha(v)beta6-mediated TGF-beta activation was evaluated in murine bleomycin-induced lung fibrosis. MEASUREMENTS AND MAIN RESULTS: Alpha(v)beta6 is overexpressed in human lung fibrosis within pneumocytes lining the alveolar ducts and alveoli. In the bleomycin model, alpha(v)beta6 antibody was effective in blocking pulmonary fibrosis. At high doses, there was increased expression of markers of inflammation and macrophage activation, consistent with the effects of TGF-beta inhibition in the lung. Low doses of antibody attenuated collagen expression without increasing alveolar inflammatory cell populations or macrophage activation markers. CONCLUSIONS: Partial inhibition of TGF-beta using alpha(v)beta6 integrin antibodies is effective in blocking murine pulmonary fibrosis without exacerbating inflammation. In addition, the elevated expression of alpha(v)beta6, an activator of the fibrogenic cytokine, TGF-beta, in human pulmonary fibrosis suggests that alpha(v)beta6 monoclonal antibodies could represent a promising new therapeutic strategy for treating pulmonary fibrosis.
Subject(s)
Antibodies, Monoclonal/pharmacology , Disease Models, Animal , Integrins/antagonists & inhibitors , Pulmonary Fibrosis/immunology , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Antigens, Neoplasm/physiology , Collagen/metabolism , Dose-Response Relationship, Drug , Integrins/physiology , Mice , Mice, Inbred BALB C , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/immunology , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/therapy , Scleroderma, Systemic/immunology , Scleroderma, Systemic/pathology , Scleroderma, Systemic/therapy , Transforming Growth Factor beta/physiologyABSTRACT
The transforming growth factor (TGF)-beta-inducible integrin alpha v beta6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-beta. Herein, we show that alpha v beta6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of alpha v beta6 in renal disease, we studied the effects of function-blocking alpha v beta6 monoclonal antibodies (mAbs) and genetic ablation of the beta6 subunit on kidney fibrosis in Col4A3-/- mice, a mouse model of Alport syndrome. Expression of alpha v beta6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with alpha v beta6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in beta6-deficient Alport mice. Transcript profiling of kidney tissues showed that alpha v beta6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-beta RII treatment, suggesting shared regulatory functions of alpha v beta6 and TGF-beta. These findings demonstrate that alpha v beta6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target.
Subject(s)
Antigens, Neoplasm/biosynthesis , Integrins/biosynthesis , Nephritis, Hereditary/metabolism , Animals , Antibodies, Blocking/immunology , Antibodies, Blocking/pharmacology , Antigens, Neoplasm/immunology , Disease Models, Animal , Extracellular Matrix/drug effects , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Humans , Immunohistochemistry , Integrins/antagonists & inhibitors , Integrins/immunology , Kidney/metabolism , Kidney/pathology , Mice , Mice, Knockout , NIH 3T3 Cells , Nephritis, Hereditary/drug therapy , Nephritis, Hereditary/etiology , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/metabolism , Up-RegulationABSTRACT
Improving the affinity of a high-affinity protein-protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure-based computational methods to optimize the binding affinity of an antibody fragment to the I-domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd approximately 7 nM) and the moderate resolution (2.8 A) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high-affinity quadruple mutant complex at 2.2 A. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher-affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single-mutant success rate. Our results indicate that structure-based computational design can be successfully applied to further improve the binding of high-affinity antibodies.
Subject(s)
Antibodies/therapeutic use , Antibody Affinity , Binding Sites, Antibody , Computer-Aided Design , Drug Design , Amino Acid Substitution , Antigen-Antibody Complex/chemistry , Crystallography, X-Ray , Immunoglobulins , Integrin alpha1beta1/immunology , Models, Molecular , Structure-Activity RelationshipABSTRACT
PURPOSE: In a prior study, it was reported that both TGF-beta receptors type-I and -II are upregulated after wounding, suggesting that TGF-beta signaling may play a role in corneal epithelial repair. The Smad proteins, which translocate into the nucleus after activation of the TGF-beta receptors, are key factors in the major TGF-beta signaling pathway. The present study was undertaken to examine whether Smads 2 and 4 translocate into the nucleus during wound repair and whether the wound type affects the extent of translocation. METHODS: Either a 3-mm superficial keratectomy or epithelial debridement was performed on adult Sprague-Dawley rats. The eyes were allowed to heal from 4 hours to 2 weeks. Indirect immunofluorescence was performed with anti-Smads 2 and 4, anti-laminin, a marker of basement membrane, and anti-alphavbeta6 integrin, which has been implicated in TGF-beta activation. In addition, the effect of the p38MAPK inhibitor SB202190 on healing rates of debridement and keratectomy wounds was determined in organ culture. RESULTS: In unwounded tissue, Smad 2 was cytoplasmic. By 4 hours after keratectomy, nuclear localization was visible in a few epithelial basal cells at the leading edge of the wound. The number of basal cells expressing nuclear Smad 2 in the wound area increased with time, peaking at 48 hours (95%). However, in the debridement model, Smad 2 localization remained primarily cytoplasmic. Smad 4 showed similar localization. In both wound models, p38MAPK inhibitor slowed epithelial migration, and alphavbeta6 integrin appeared to be upregulated with localization primarily observed in the basal cells migrating over the wound area. CONCLUSIONS: The presence of the basement membrane appears to have an effect on the extent and duration of translocation of the Smad 2 and 4 proteins during corneal epithelial wound repair. The Smad pathway does not appear to be essential for migration; rather, it may play a role in resynthesis of the basement membrane.
Subject(s)
DNA-Binding Proteins/metabolism , Epithelium, Corneal/injuries , Trans-Activators/metabolism , Wound Healing , Animals , Antigens, Neoplasm/metabolism , Basement Membrane/physiology , Cell Culture Techniques , Cell Movement , Cytoplasm/metabolism , Debridement , Enzyme Inhibitors/pharmacology , Epithelium, Corneal/drug effects , Epithelium, Corneal/metabolism , Humans , Imidazoles/pharmacology , Integrins/metabolism , Male , Organ Culture Techniques , Protein Transport , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Smad2 Protein , Smad4 Protein , Up-Regulation , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
Mass media community-wide physical activity intervention to promote and sustain changes in walking was assessed using a 2-community longitudinal design. The intervention targeted sedentary 50- to 65-year-old residents of Wheeling, West Virginia. Telephone surveys of a probability sample followed cohorts at baseline and at 3-, 6-, and 12-month post-intervention with comparison communities. The intervention, consisting of paid advertisements, public relations, and community participatory planning, attained high levels of awareness and effected significant sustained population-wide changes among the most sedentary in Wheeling.
Subject(s)
Community Participation/methods , Health Promotion/methods , Mass Media , Walking , Cohort Studies , Female , Humans , Male , West VirginiaABSTRACT
A number of organizations have advised against the use of placebo substitution, including the American Pain Society, Agency for Healthcare Policy and Research, World Health Organization, Healthcare Facilities Accreditation Program, Joint Commission on Accreditation of Healthcare Organizations, Education for Physicians on End-of-Life Care Project (cosponsored by the American Medical Association and The Robert Wood Johnson Foundation), American Nursing Association, and the American Society of Pain Management Nurses. This white paper describes the literature and rationale in support of the American Osteopathic Association's (AOA's) position on the controversial subject of the use of placebos for pain management in terminally ill patients.
Subject(s)
Attitude of Health Personnel , Pain/drug therapy , Palliative Care/standards , Placebos/therapeutic use , Terminal Care/standards , Ethics, Clinical , Guideline Adherence , Humans , Narcotics/administration & dosage , Narcotics/adverse effects , Narcotics/therapeutic use , Opioid-Related Disorders/etiology , Opioid-Related Disorders/prevention & control , Osteopathic Medicine , Palliative Care/ethics , Palliative Care/legislation & jurisprudence , Patient Rights/legislation & jurisprudence , Placebos/adverse effects , Practice Guidelines as Topic , Terminal Care/legislation & jurisprudence , Terminal Care/methods , United StatesABSTRACT
We have generated a panel of potent, selective monoclonal antibodies that bind human and mouse alpha(v)beta(6) integrin with high affinity (up to 15 pm). A subset of these antibodies blocked the binding of alpha(v)beta(6) to the transforming growth factor-beta1 latency-associated peptide with IC(50) values as low as 18 pm, and prevented the subsequent alpha(v)beta(6)-mediated activation of transforming growth factor-beta1. The antibodies also inhibited alpha(v)beta(6) binding to fibronectin. The blocking antibodies form two biochemical classes. One class, exemplified by the ligand-mimetic antibody 6.8G6, bound to the integrin in a divalent cation-dependent manner, contained an RGD motif or a related sequence in CDR3 of the heavy chain, was blocked by RGD-containing peptides, and was internalized by alpha(v)beta(6)-expressing cells. Despite containing an RGD sequence, 6.8G6 was specific for alpha(v)beta(6) and showed no cross-reactivity with the RGD-binding integrins alpha(v)beta(3), alpha(v)beta(8),or alpha(IIb)beta(3). The nonligand-mimetic blocking antibodies, exemplified by 6.3G9, were cation-independent, were not blocked by RGD-containing peptides, were not internalized, and did not contain RGD or related sequences. These two classes of antibody were unable to bind simultaneously to alpha(v)beta(6), suggesting that they may bind overlapping epitopes. The "ligand-mimetic" antibodies are the first to be described for alpha(v)beta(6) and resemble those described for alpha(IIb)beta(3). We also report for the first time the relative abilities of divalent cations to promote alpha(v)beta(6) binding to latency-associated peptide and to the ligand-mimetic antibodies. These antibodies should provide valuable tools to study the ligand-receptor interactions of alpha(v)beta(6) as well as the role of alpha(v)beta(6) in vivo.