Search

Abstract Background The definition of "clinical asthma remission" is based on absence of symptoms and use of medication. However, in the majority of these subjects airway inflammation is still present when measured. In the present study we investigated whether "complete asthma remission", additionally defined by the absence of bronchial hyperresponsiveness (BHR) and the presence of a normal lung function, is associated with the absence of airway inflammation. Methods Patients with a former diagnosis of asthma and a positive histamine provocation test were re-examined to identify subjects with complete asthma remission (no asthma symptoms or medication, PC20 histamine > 32 mg/ml, FEV1 > 90% predicted). Patients with PC20 histamine ≤ 32 mg/ml were defined as current asthmatics and were divided in two groups, i.e. asthmatics with and without BHR to adenosine 5'monophoshate (AMP). Sputum induction was performed 1 week before and 1 hour after AMP provocation. Sputum induction and AMP provocation were previously shown to be sensitive markers of airway inflammation. Results Seven patients met criteria for complete asthma remission. Twenty-three were current asthmatics, including twelve without hyperresponsiveness to AMP. Subjects with complete asthma remission showed no AMP-induced sputum eosinophilia (median (range) 0.2 (0 - 4.6)% at baseline and 0.2 (0 - 2.6)% after AMP). After AMP, current asthmatics had a significant increase in sputum eosinophils (0.5 (0 - 26.0)% at baseline and 2.6 (0 - 32.0) % after AMP), as had the subgroup of current asthmatics without hyperresponsiveness to AMP (0.2 (0 - 1.8)% at baseline and 1.3 (0 - 6.3)% after AMP). Conclusions Subjects with complete asthma remission, in contrast to subjects with current asthma, do not respond with eosinophilic inflammation in sputum after AMP provocations. These data lend support to the usefulness of the definition of complete asthma remission.

Summary: Background: As it is unknown whether complete asthma remission or progression of asthma is associated with airway inflammation and remodeling, we assessed these characteristics in bronchial biopsies of relevant subsets of asthma patients. Methods: Sputum and bronchial biopsies were obtained from asthma patients in remission (PC20 histamine>32mg/ml, PC20 AMP>320mg/ml) and from those with either a slow FEV1 decline (<30ml/year) or fast decline (>30ml/year). Inflammatory cells and mediators were determined in sputum, inflammatory cells and aspects of airway remodeling in bronchial biopsies. Results: Asthmatics in remission and asthma patients with a slow FEV1 decline had a similar extent of airway inflammation and remodeling in sputum and bronchial biopsies. Asthma patients with a fast FEV1 decline had high sputum eosinophil numbers. Moreover, FEV1 decline (ml/year) correlated with sputum eosinophil numbers (Rs=0.51, p=0.003) and ECP levels (Rs=0.57, p=0.001). Airway remodeling, i.e. basement membrane thickness, correlated with sputum eosinophils (Rs=0.69, p<0.001), sputum ECP (Rs=0.46, p=0.018) and airway wall eosinophil numbers (Rs=0.49, p=0.002). Conclusions: Asthma, even when in remission, is accompanied by airway inflammation and remodeling. Data suggest that eosinophils are important in a subset of asthma patients by association to accelerated FEV1 decline and change of basement membrane thickness. [ABSTRACT FROM AUTHOR]

Background: The definition of "clinical asthma remission" is based on absence of symptoms and use of medication. However, in the majority of these subjects airway inflammation is still present when measured. In the present study we investigated whether "complete asthma remission", additionally defined by the absence of bronchial hyperresponsiveness (BHR) and the presence of a normal lung function, is associated with the absence of airway inflammation. Methods: Patients with a former diagnosis of asthma and a positive histamine provocation test were re-examined to identify subjects with complete asthma remission (no asthma symptoms or medication, PC20 histamine > 32 mg/ml, FEV1 > 90% predicted). Patients with PC20 histamine ≥ 32 mg/ml were defined as current asthmatics and were divided in two groups, i.e. asthmatics with and without BHR to adenosine 5'monophoshate (AMP). Sputum induction was performed 1 week before and 1 hour after AMP provocation. Sputum induction and AMP provocation were previously shown to be sensitive markers of airway inflammation. Results: Seven patients met criteria for complete asthma remission. Twenty-three were current asthmatics, including twelve without hyperresponsiveness to AMP. Subjects with complete asthma remission showed no AMPinduced sputum eosinophilia (median (range) 0.2 (0 - 4.6)% at baseline and 0.2 (0 - 2.6)% after AMP). After AMP, current asthmatics had a significant increase in sputum eosinophils (0.5 (0 - 26.0)% at baseline and 2.6 (0 - 32.0) % after AMP), as had the subgroup of current asthmatics without hyperresponsiveness to AMP (0.2 (0 - 1.8)% at baseline and 1.3 (0 - 6.3)% after AMP). Conclusions: Subjects with complete asthma remission, in contrast to subjects with current asthma, do not respond with eosinophilic inflammation in sputum after AMP provocations. These data lend support to the usefulness of the definition of complete asthma remission. [ABSTRACT FROM AUTHOR]

Rationale: Smoking has detrimental effects on asthma outcome, such as increased cough, wheezing, sputum production, and frequency of asthma attacks. This results in accelerated lung function decline. The underlying pathological process of smoke-induced deterioration of asthma is unknown. Objectives: To compare bronchial inflammation and remodeling in never-smokers, ex-smokers, and current smokers with asthma. Methods: A total of 147 patients with asthma (66 never-smokers, 46 ex-smokers, and 35 current smokers) were investigated. Measurements and Main Results: Lung function, exhaled nitric oxide levels, and symptom questionnaires were assessed, and induced sputum and bronchial biopsies were obtained for determination of airway inflammation and remodeling. Smokers with asthma had lower FEV(1) and alveolar and bronchial nitric oxide levels than never-smokers. Smokers also had more goblet cells and mucus-positive epithelium, increased epithelial thickness, and a higher proliferation rate of intact and basal epithelium than ex-smokers and never-smokers. Smokers had higher numbers of mast cells and lower numbers of eosinophils than never-smokers. Ex-smokers had similar goblet cell numbers and mucus-positive epithelium, epithelial thickness, epithelial proliferation rate, and mast cell numbers as never-smokers. Conclusions: Smokers with asthma have epithelial changes that are associated with increased asthma symptoms, such as shortness of breath and phlegm production. The fact that epithelial characteristics in ex-smokers are similar to those in never-smokers suggests that the smoke-induced changes can be reversed by smoking cessation. [ABSTRACT FROM AUTHOR]

Purpose: DISP1 encodes a transmembrane protein that regulates the secretion of the morphogen, Sonic hedgehog, a deficiency of which is a major cause of holoprosencephaly (HPE). This disorder covers a spectrum of brain and midline craniofacial malformations. The objective of the present study was to better delineate the clinical phenotypes associated with division transporter dispatched-1 (DISP1) variants., Methods: This study was based on the identification of at least 1 pathogenic variant of the DISP1 gene in individuals for whom detailed clinical data were available., Results: A total of 23 DISP1 variants were identified in heterozygous, compound heterozygous or homozygous states in 25 individuals with midline craniofacial defects. Most cases were minor forms of HPE, with craniofacial features such as orofacial cleft, solitary median maxillary central incisor, and congenital nasal pyriform aperture stenosis. These individuals had either monoallelic loss-of-function variants or biallelic missense variants in DISP1. In individuals with severe HPE, the DISP1 variants were commonly found associated with a variant in another HPE-linked gene (ie, oligogenic inheritance)., Conclusion: The genetic findings we have acquired demonstrate a significant involvement of DISP1 variants in the phenotypic spectrum of midline defects. This underlines its importance as a crucial element in the efficient secretion of Sonic hedgehog. We also demonstrated that the very rare solitary median maxillary central incisor and congenital nasal pyriform aperture stenosis combination is part of the DISP1-related phenotype. The present study highlights the clinical risks to be flagged up during genetic counseling after the discovery of a pathogenic DISP1 variant., Competing Interests: Conflict of Interest All authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We have designed and developed novel and selective TLR7 agonists that exhibited potent receptor activity in a cell-based reporter assay. In vitro , these agonists significantly induced secretion of cytokines IL-6, IL-1β, IL-10, TNFa, IFNa, and IP-10 in human and mouse whole blood. Pharmacokinetic and pharmacodynamic studies in mice showed a significant secretion of IFNα and TNFα cytokines. When combined with aPD1 in a CT-26 tumor model, the lead compound showed strong synergistic antitumor activity with complete tumor regression in 8/10 mice dosed using the intravenous route. Structure-activity relationship studies enabled by structure-based designs of TLR7 agonists are disclosed., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Monogenic lipodystrophies are a heterogeneous group of rare disorders characterized by a lack of adipose tissue (AT), all of which predispose patients to the development of insulin resistance and its related metabolic sequelae. The extent of AT loss ranges from partial, as in familial partial lipodystrophy (FPLD), to a total absence of metabolically active AT in congenital generalized lipodystrophy (CGL) and is generally associated with the severity of metabolic complications. Significant genetic, allelic, phenotypic, and clinical heterogeneity exists among the lipodystrophies. Patients with FPLD3 due to mutations in the PPARG gene, which encodes a key transcriptional regulator of adipocyte development and function, provide a particularly striking example of this heterogeneity. We will present several gene-gene and gene-environment factors and mechanisms that are critical for adequate PPARγ expression and activity in AT and discuss how these interactions potentially contribute to the observed spectrum of FPLD3 phenotypes. Comparable mechanisms may play a role in other types of lipodystrophies too, and their elucidation may further improve our molecular understanding of AT dysfunction., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Several neurodegenerative diseases like Huntington's, a polyglutamine (PolyQ) disease, are initiated by protein aggregation in neurons. Furthermore, these diseases are also associated with a multitude of responses in non-neuronal cells in the brain, in particular glial cells, like astrocytes. These non-neuronal responses have repeatedly been suggested to play a disease-modulating role, but how these may be exploited to delay the progression of neurodegeneration has remained unclear. Interestingly, one of the molecular changes that astrocytes undergo includes the upregulation of certain Heat Shock Proteins (HSPs) that are classically considered to maintain protein homeostasis, thus resulting in cell autonomous protection. Previously, we discovered DNAJB6, a member of the human DNAJ family, as potent cell autonomous suppressor of PolyQ aggregation and related neurodegeneration. Using cell type specific expression systems in D. melanogaster, we show that exclusive expression of DNAJB6 in astrocytes (that do not express PolyQ protein) can delay neurodegeneration and expands lifespan when the PolyQ protein is exclusively expressed in neurons (that do not co-express DNAJB6 themselves). This provides direct evidence for a non-cell autonomous protective role of astrocytes in PolyQ diseases., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

BAG3 is a multi-domain hub that connects two classes of chaperones, small heat shock proteins (sHSPs) via two isoleucine-proline-valine (IPV) motifs and Hsp70 via a BAG domain. Mutations in either the IPV or BAG domain of BAG3 cause a dominant form of myopathy, characterized by protein aggregation in both skeletal and cardiac muscle tissues. Surprisingly, for both disease mutants, impaired chaperone binding is not sufficient to explain disease phenotypes. Recombinant mutants are correctly folded, show unaffected Hsp70 binding but are impaired in stimulating Hsp70-dependent client processing. As a consequence, the mutant BAG3 proteins become the node for a dominant gain of function causing aggregation of itself, Hsp70, Hsp70 clients and tiered interactors within the BAG3 interactome. Importantly, genetic and pharmaceutical interference with Hsp70 binding completely reverses stress-induced protein aggregation for both BAG3 mutations. Thus, the gain of function effects of BAG3 mutants act as Achilles heel of the HSP70 machinery.

Agonism of the 5-HT 2C receptor represents one of the most well-studied and clinically proven mechanisms for pharmacological weight reduction. Selectivity over the closely related 5-HT 2A and 5-HT 2B receptors is critical as their activation has been shown to lead to undesirable side effects and major safety concerns. In this communication, we report the development of a new screening paradigm that utilizes an active site mutant D134A (D3.32) 5-HT 2C receptor to identify atypical agonist structures. We additionally report the discovery and optimization of a novel class of nonbasic heterocyclic amide agonists of 5-HT 2C . SAR investigations around the screening hits provided a diverse set of potent agonists at 5-HT 2C with high selectivity over the related 5-HT 2A and 5-HT 2B receptor subtypes. Further optimization through replacement of the amide with a variety of five- and six-membered heterocycles led to the identification of 6-(1-ethyl-3-(quinolin-8-yl)-1H-pyrazol-5-yl)pyridazin-3-amine (69). Oral administration of 69 to rats reduced food intake in an ad libitum feeding model, which could be completely reversed by a selective 5-HT 2C antagonist.

G-protein-coupled receptor 119 (GPR119) is expressed predominantly in pancreatic β-cells and in enteroendocrine cells in the gastrointestinal tract. GPR119 agonists have been shown to stimulate glucose-dependent insulin release by direct action in the pancreas and to promote secretion of the incretin GLP-1 by action in the gastrointestinal tract. This dual mechanism of action has generated significant interest in the discovery of small molecule GPR119 agonists as a potential new treatment for type 2 diabetes. Herein, we describe the discovery and optimization of a new class of pyridone containing GPR119 agonists. The potent and selective BMS-903452 (42) was efficacious in both acute and chronic in vivo rodent models of diabetes. Dosing of 42 in a single ascending dose study in normal healthy humans showed a dose dependent increase in exposure and a trend toward increased total GLP-1 plasma levels.

There are numerous human diseases that are associated with protein misfolding and the formation of toxic protein aggregates. Activating the heat shock response (HSR)--and thus generally restoring the disturbed protein homeostasis associated with such diseases--has often been suggested as a therapeutic strategy. However, most data on activating the HSR or its downstream targets in mouse models of diseases associated with aggregate formation have been rather disappointing. The human chaperonome consists of many more heat shock proteins (HSPs) that are not regulated by the HSR, however, and researchers are now focusing on these as potential therapeutic targets. In this Review, we summarize the existing literature on a set of aggregation diseases and propose that each of them can be characterized or 'barcoded' by a different set of HSPs that can rescue specific types of aggregation. Some of these 'non-canonical' HSPs have demonstrated effectiveness in vivo, in mouse models of protein-aggregation disease. Interestingly, several of these HSPs also cause diseases when mutated--so-called chaperonopathies--which are also discussed in this Review.

Background: Control of asthma is the goal of asthma management worldwide. The Global Initiative for Asthma defined control by a composite measure of clinical findings and future risk but without using markers of airway inflammation, the hallmark of asthma. We investigated whether clinical asthma control reflects eosinophilic inflammation in a broad population., Methods: Control of asthma was assessed over a period of 4 weeks in 111 patients with asthma: 22 totally controlled, 47 well controlled and 42 uncontrolled. Lung function, quality of life, airway hyperresponsiveness to AMP, sputum and blood eosinophils, exhaled nitric oxide (NO) and bronchial biopsies were obtained., Results: The 69 subjects with controlled asthma (totally and well controlled combined) had lower median blood eosinophil numbers, slope of AMP hyperresponsiveness, and alveolar NO levels than the 42 subjects with uncontrolled asthma: 0.18 (range 0.01-0.54) versus 0.22 (0.06-1.16) × 10(9)/litre (p<0.05), 3.8 (-0.4-17 750) versus 39.7 (0.4-28 000) mg/ml (p<0.05) and 5.3 (1.5-14.9) versus 6.7 (2.6-51.7) ppb (p<0.05) respectively. Biopsies from subjects with controlled asthma contained fewer eosinophilic granules and more intact epithelium than uncontrolled subjects: 113 (6-1787) versus 219 (19-5313) (p<0.05) and 11.8% (0-65.3) versus 5.6% (0-47.6) (p<0.05) respectively. Controlled asthmatics had better Asthma Quality of Life Questionnaire scores than uncontrolled patients: 6.7 (5.0-7.0) versus 5.9 (3.7-7.0) (p<0.001)., Conclusions: The level of asthma control, based on a composite measure of clinical findings, is associated with inflammatory markers, particularly eosinophilic inflammation, with little difference between totally controlled and well controlled asthma.

Background: Heart failure has been associated with impaired cardiac sympathetic neuronal function. Cardiac imaging with radiolabeled agents that are substrates for the neuronal norepinephrine transporter (NET) has demonstrated the potential to identify individuals at risk of cardiac events. N-[3-Bromo-4-(3-[18F]fluoro-propoxy)-benzyl]-guanidine (LMI1195) is a newly developed 18F-labeled NET substrate designed to allow cardiac neuronal imaging with the high sensitivity, resolution, and quantification afforded by positron emission tomography (PET)., Methods and Results: LMI1195 was evaluated in comparison with norepinephrine (NE) in vitro and 123I-meta-iodobenzylguanidine (MIBG) in vivo. The affinity (Ki) of LMI1195 for NET was 5.16 ± 2.83 μmol/L, similar to that of NE (3.36 ± 2.77 μmol/L) in a cell membrane-binding assay. Similarly, LMI1195 uptake kinetics examined in a human neuroblastoma cell line had Km and Vmax values of 1.44 ± 0.76 μmol/L and 6.05 ± 3.09 pmol/million cells per minute, comparable to NE (2.01 ± 0.85 μmol/L and 6.23 ± 1.52 pmol/million cells per minute). In rats, LMI1195 heart uptake at 15 and 60 minutes after intravenous administration was 2.36 ± 0.38% and 2.16 ± 0.38% injected dose per gram of tissue (%ID/g), similar to 123I-MIBG (2.14 ± 0.30 and 2.19 ± 0.27%ID/g). However, the heart to liver and lung uptake ratios were significantly higher for LMI1195 than for 123I-MIBG. In rabbits, desipramine (1 mg/kg), a selective NET inhibitor, blocked LMI1195 heart uptake by 82%, which was more effective than 123I-MIBG (53%), at 1 hour after dosing. Sympathetic denervation with 6-hydroxydopamine, a neurotoxin, resulted in a marked (79%) decrease in LMI1195 heart uptake. Cardiac PET imaging with LMI1195 in rats, rabbits, and nonhuman primates revealed clear myocardium with low radioactivity levels in the blood, lung, and liver. Imaging in rabbits pretreated with desipramine showed reduced heart radioactivity levels in a dose-dependent manner. Additionally, imaging in sympathetically denervated rabbits resulted in low cardiac image intensity with LMI1195 but normal perfusion images with flurpiridaz F 18, a PET myocardial perfusion imaging agent. In nonhuman primates pretreated with desipramine (0.5 mg/kg), imaging with LMI1195 showed a 66% decrease in myocardial uptake. In a rat model of heart failure, the LMI1195 cardiac uptake decreased as heart failure progressed., Conclusions: LMI1195 is a novel (18)F imaging agent retained in the heart through the NET and allowing evaluation of the cardiac sympathetic neuronal function by PET imaging.

Rationale: Individuals with asthma may outgrow symptoms despite not using treatment, whereas others reach complete remission (CoR) with absence of airway obstruction and bronchial hyperresponsiveness. It is uncertain whether this associates with remission of all inflammatory and remodeling asthma features., Objectives: To compare the pathologic phenotype of individuals with asthma with CoR and clinical remission (ClinR) and those with active asthma, with and without the use of inhaled corticosteroids (ICS)., Methods: We investigated 165 individuals known with active asthma, on reexamination having CoR (n = 18), ClinR (n = 44), and current asthma (CuA, n = 103, 64 with and 39 without ICS). MEASUREMENTS MAIN RESULTS: Inflammatory cells were measured in blood, induced sputum, and bronchial biopsies; histamine and ECP in sputum; and eosinophilic peroxidase (EPX) immunopositivity and remodeling (epithelial changes, E-cadherin expression, basement membrane [BM] thickening, collagen deposition) in bronchial biopsies. Median (range) blood eosinophils from CoR were significantly lower than those from CuA (0.10 [0.04-0.24] vs. 0.18 [0.02-1.16] × 10⁹/L). Bronchial EPX immunopositivity was lower in CoR than in both ClinR and CuA (67 [0.5-462] vs. 95 [8-5329] and 172 [6-5313] pixels). Other inflammatory findings were comparable. BM thickness was lowest in CuA, caused by lower BM thickness in those using ICS (CoR, 6.3 [4.7-8.4]; ClinR, 6.5 [3.8-11.7]; CuA, 5.7 [2.8-12.6]; and ICS using CuA, 5.3 [2.8-8.2] μm)., Conclusions: CoR is still accompanied by airway abnormalities because BM thickness is similar in individuals with asthma with CoR, ClinR, and CuA without ICS. Airway eosinophilic activation best differentiates these three groups, signifying their importance in the clinical expression and severity of bronchial hyperresponsiveness in asthma.

The molecular basis of airway remodeling and loss of epithelial integrity in asthma is still undefined. We aimed to establish if exposure of human bronchial epithelium (16HBE cells) to asthma-related stimuli can induce epithelial-to-mesenchymal transition (EMT), a key process in tissue repair and remodeling associated with loss of intercellular contacts. We studied the effects of fibrogenic cytokine TGF-beta and protease-containing aeroallergen house dust mite (HDM) on mesenchymal and epithelial markers, cytoskeleton organization, and activation of beta-catenin-driven reporter TopFLASH. TGF-beta alone up-regulated vimentin and fibronectin, modestly down-regulated E-cadherin, but did not affect cytokeratin. HDM alone did not affect these markers, but promoted stress fibers. Importantly, when added to TGF-beta-primed epithelium, HDM induced E-cadherin internalization, enhanced beta-catenin-dependent transcription, and down-regulated cytokeratin. Regarding the underlying mechanisms, the stimuli together induced sustained myosin light chain phosphorylation, which was crucial for E-cadherin internalization and beta-catenin-dependent transcription. Previously, we showed that HDM signals through the epidermal growth factor receptor (EGFR). Accordingly, inhibition of EGFR prevented TGF-beta/HDM-induced mesenchymalization. TGF-beta facilitated uncoupling of EGFR from E-cadherin, its negative regulator, and prolonged EGFR signaling. Thus, we show that HDM promotes EMT in TGF-beta-primed epithelium. Analysis of primary epithelium appears consistent with this phenotypic change. We propose that TGF-beta secretion and dysregulated EGFR signaling may increase epithelial vulnerability to allergens and trigger the induction of EMT, a hitherto unrecognized contributor to airway remodeling in asthma.

Background: Ischemia/reperfusion injury (IRI) is a risk factor for the development of interstitial fibrosis. Previously we had shown that after renal IRI, bone marrow-derived cells (BMDC) can differentiate to interstitial myofibroblasts. Here we hypothesized that the immunosuppressant ciclosporin A (CsA), known for its profibrotic side effect, promotes myofibroblast differentiation of BMDC in the postischemic kidney., Methods: Using a model of unilateral renal IRI in rats reconstituted with R26-human placental alkaline phosphatase transgenic bone marrow, CsA was administered in a previously defined critical window for differentiation of BMDC to myofibroblasts. We evaluated fibrotic changes in the kidney and myofibroblast differentiation of BMDC on day 14 after CsA treatment., Results: CsA treatment for 14 days led to increased transforming growth factor-beta transcript levels and collagen III deposition in the postischemic kidney. However, neither the total number of alpha-smooth-muscle-actin-positive interstitial myofibroblasts, nor the bone marrow-derived fraction thereof was affected by CsA administration, irrespective of dosage and duration of treatment., Conclusions: In the critical postischemic window of BMDC differentiation to myofibroblasts, CsA did not promote BMDC differentiation to myofibroblasts, suggesting that, in the clinical setting, CsA is not involved in myofibroblastic differentiation of BMDC.

Background: In human renal allografts, recipient-derived cells engrafted in various kidney substructures, have been detected in the long term after transplantation. Here we investigated tubular engraftment and myofibroblast differentiation of recipient-derived cells at short term after experimental kidney transplantation, during a previously described window of regeneration and possible onset of renal interstitial fibrosis., Methods: Fisher (F344, syngeneic) and Dark Agouti (DA, allogeneic) kidneys were transplanted into F344-hPAP transgenic recipient rats, which allowed tracing of recipient-derived cells in nontransgenic donor kidneys. We evaluated tubular engraftment and myofibroblast differentiation of recipient-derived cells on day 14 after kidney transplantation., Results: Kidney transplantation resulted in tubular engraftment of recipient-derived cells. After allogeneic kidney transplantation, 9.7% of tubular cross-sections contained at least one recipient-derived cell, which represented a significant increase in comparison to syngeneic transplantation (4.0%, P<0.05). Moreover, recipient-derived myofibroblasts were present in the renal interstitium of the transplanted kidney. These cells contributed 39% of the total interstitial myofibroblast population in allografts, which was comparable to the syngeneic situation (28%, P=0.25)., Conclusions: In a defined early window of regeneration and possible onset of renal interstitial fibrosis after kidney transplantation, rejection-associated injury, superimposed on ischemic damage, increases tubular engraftment of recipient-derived cells, although it does not affect their relative contribution to the renal interstitial myofibroblast population.

Bone marrow-derived cells (BMDC) have been proposed to exert beneficial effects after renal ischemia/reperfusion injury (IRI) by engraftment in the tubular epithelium. However, BMDC can give rise to myofibroblasts and may contribute to fibrosis. BMDC contribution to the renal interstitial myofibroblast population in relation to fibrotic changes after IRI in rats was investigated. A model of unilateral renal IRI (45 min of ischemia) was used in F344 rats that were reconstituted with R26-human placental alkaline phosphatase transgenic BM to quantify BMDC contribution to the renal interstitial myofibroblast population over time. After IRI, transient increases in collagen III transcription and interstitial protein deposition were observed, peaking on days 7 and 28, respectively. Interstitial infiltrates of BMDC and myofibroblasts reached a maximum on day 7 and gradually decreased afterward. Over time, an average of 32% of all interstitial alpha-smooth muscle actin-positive myofibroblasts coexpressed R26-human placental alkaline phosphatase and, therefore, were derived from the BM. BMD myofibroblasts produced procollagen I protein and therefore were functional. The postischemic kidney environment was profibrotic, as demonstrated by increased transcription of TGF-beta and decreased transcription of bone morphogenic protein-7. TGF-beta protein was present predominantly in interstitial myofibroblasts but not in BMD myofibroblasts. In conclusion, functional BMD myofibroblasts infiltrate in the postischemic renal interstitium and are involved in extracellular matrix production.

Unlabelled: Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune responses. In our ongoing clinical trials, human leukocyte antigen (HLA)-A2.1+ melanoma patients are vaccinated with mature DC, presenting tumor-derived peptides in major histocompatibility complexes (MHC) to naive T cells. Previously, we have shown that both intradermally and intranodally injected (111)In-labeled mature DC migrate to draining lymph nodes. However, little is known about the fate of the MHC-peptide complex after injection of these peptide-loaded DC. The aim of the present study was to develop radiolabeled, tumor-derived peptides to monitor their binding to MHC Class I., Methods: The HLA-A2.1 binding peptide gp100:154-162mod (gp100:154m) was conjugated with diethylenetriamine pentaacetic acid (DTPA) either at the N-terminus (alpha-DTPA-gp100:154m) or at the epsilon amino group of the Lys(154) residue (epsilon-DTPA-gp100:154m) and labeled with (111)In., Results: The maximum specific activity for both peptides was 13 GBq/micromol. The IC50 of the alpha-[(111)In]DTPA-gp100:154m peptide was >75 microM. The IC50 of the (111)In-labeled epsilon-DTPA-gp100:154m was 3 microM, similar to the unconjugated peptide. MHC binding studies showed specific binding of the epsilon-[(111)In]DTPA-gp100:154m peptide to the JY cells at 4 degrees C. Interestingly, no specific binding was observed for the alpha-[(111)In]DTPA-gp100:154m peptide. In contrast to the alpha-[(111)In]DTPA-gp100:154m peptide, the epsilon-[(111)In]DTPA-gp100:154m peptide was recognized by cytotoxic T cells., Conclusion: When DTPA was conjugated to the epsilon NH2 group of the Lys(154) residue, MHC binding of the peptide was preserved and could still be recognized by cytotoxic T cells. These studies allow the noninvasive determination of the behavior of MHC-peptide complexes on DC in vivo.

Background: Ischemia/reperfusion (I/R) injury is a major cause of acute renal failure (ARF). ARF is reversible, due to an innate regenerative process, which is thought to depend partly on bone marrow-derived progenitor cells. The significance of these cells in the repair process has been questioned in view of their relatively low frequency. Here, we hypothesize that the severity of renal damage and the postischemic recovery time are determinants of tubular bone marrow-derived cell (BMDC) engraftment., Methods: We used a model of unilateral renal I/R in F344 rats reconstituted with R26-human placental alkaline phosphatase (hPAP) transgenic bone marrow, in which we quantified and characterized tubular BMDC engraftment with increasing severity of damage and in time., Results: After I/R injury, BMDC engrafted the tubular epithelium and acquired an epithelial phenotype. Tubular epithelial BMDC engraftment increased with longer ischemic time, indicating that tubular epithelial BMDC engraftment increases with the severity of damage. The number of circulating progenitor cells doubled early after I/R injury and was followed by a transient increase in tubular epithelial BMDC engraftment. The latter positively correlated with morphological recovery of the kidney over time. CONCLUSION. The extent of tubular BMDC engraftment depends on the severity of renal damage and follows a distinct time course after I/R injury. Therefore, the severity of damage and time course need to be taken into account when interpreting data on the role of tubular BMDC engraftment in renal repair after I/R injury.

In previous studies we demonstrated that lipophilic (99m)Tc-labeled LTB4 antagonist 1 (RP517) accumulated in infectious foci in rabbits, but hepatobiliary clearance hampered imaging of abdominal lesions. We now report the use of cysteic acid as a pharmacokinetic modifier to improve the water solubility and renal clearance of three hydrophilic analogues of 1. Divalent LTB4 antagonist 17 (DPC11870-11) is a DTPA conjugate for radiolabeling with In-111. Monovalent LTB4 antagonists 15 (BMS57868-88) and divalent LTB4 antagonist 18 (BMS57868-81) are conjugated to bifunctional chelator HYNIC for radiolabeling with (99m)Tc. The three compounds labeled efficiently with 111In or (99m)Tc with high radiochemical purity and specific activities. Scintigraphic images obtained in New Zealand White rabbits having acute intramuscular E. coli infection demonstrated that all agents were able to clearly visualize the abscess, and clearance was exclusively renal. The biodistribution of the (99m)Tc-labeled LTB4 antagonists was affected by the coligands used with the HYNIC chelator and by the monovalent or divalent nature of the receptor binding moiety. The best scintigraphic images were obtained with monovalent HYNIC conjugate 15 using tricine and isonicotinic acid as coligands with HYNIC for coordination with (99m)Tc.

Purpose: The aim of these studies was to develop a pretargeting strategy for CEA-expressing cancers using biologically produced bispecific monoclonal antibodies (bsMAb). The bsMAbs used in this system have affinity for the carcinoembryonic antigen on the one hand, and for indium-labeled diethylenetriaminepentaacetic acid (DTPA), on the other., Experimental Design: Stable quadroma clones producing bsMAb MN-14xDTIn-1 were isolated. LS174T tumor-bearing mice were injected with 1 to 100 microg of bsMAb followed by 1 to 60 ng of an (111)In-labeled bivalent peptide [Ac-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH2]. Mice were killed at 24 hours postinjection and the biodistribution of the radiolabel was determined. The biodistribution of diDTPA labeled with four different radionuclides ((111)In, 99mTc, nonresidualizing 125I, and residualizing 125I) was determined at various time points postinjection following pretargeting of LS174T tumors with bsMAb MN-14xDTIn-1., Results: Optimal tumor targeting was observed when tumors were pretargeted with 10 microg of bsMAb MN-14xDTIn-1 and when 6 ng of a radiolabeled peptide was given 72 hours later. The uptake of the four radiolabels in LS174T tumors at 4 hours postinjection was similar. However, at later time points, the (111)In-label and residualizing 125I-label were better retained in the tumor than the nonresidualizing 125I label. Although the absolute uptake in the tumor (in terms of percentage of injected dose per gram of tissue) was 5-fold lower than the uptake obtained with directly labeled MN-14, the pretargeting strategy revealed much higher tumor-to-blood ratios due to the rapid clearance of the radiolabel from the circulation as compared with (111)In-MN-14 (445 +/- 90 and 5.3 +/- 1.1, respectively, at 72 hours postinjection)., Conclusions: Effective targeting of carcinoembryonic antigen-expressing tumors was achieved with a newly produced bispecific antibody. The (111)In-labeled L-amino acid peptide and 125I-D-amino acid peptide were better retained in the tumor than the 99mTc- and 125I-L-amino acid peptide. Very high tumor-to-blood ratios were obtained due to rapid background clearance.

Unlabelled: Studies have demonstrated that the bivalent (111)In-labeled leukotriene B4 (LTB4) antagonist DPC11870 reveals infectious and inflammatory lesions in various rabbit models. The radioactive tracer accumulates quickly at the site of infection and clears rapidly from the circulation, resulting in high-quality images. In this study, 2 new hydrazinonicotinamide (HYNIC)-conjugated compounds that are structurally related to DPC11870 were studied to further improve image quality., Methods: A bivalent HYNIC-conjugated LTB4 antagonist (MB81) and a monovalent one (MB88) were labeled with (99m)Tc. The radiolabeled compounds were intravenously injected into New Zealand White rabbits with E. coli infection in the left thigh muscle. The imaging characteristics of both compounds were compared with those of the bivalent (111)In-labeled LTB4 antagonist., Results: Both (99m)Tc-labeled LTB4 antagonists revealed the abscess from 2 h after injection onward. Abscess uptake at 8 h after injection was similar for both compounds (0.22 +/- 0.08 percentage injected dose per gram [%ID/g] and 0.36 +/- 0.13%ID/g for the bivalent and monovalent compounds, respectively). However, visualization of the abscess and the quality of the images were better after injection of MB88 than after injection of either of the bivalent LTB4 antagonists. The excellent delineation of the abscess by MB88 was mainly due to the more rapid clearance of this compound from nontarget organs., Conclusion: The (99m)Tc-labeled HYNIC conjugated LTB4 antagonists MB88 and MB81 revealed infectious foci in rabbits within a few hours after injection. Imaging characteristics of monovalent (99m)Tc-MB88 were superior to those of the bivalent LTB4 antagonists DPC11870 and MB81. Therefore, of the 3 LTB4 antagonists, the monovalent LTB4 antagonist MB88 is the most potent and promising agent for visualizing and evaluating infection and inflammation in patients.

Unlabelled: Previous studies have shown that pretargeting allows rapid visualization of renal cell carcinomas (RCC) with an (111)In-labeled bivalent peptide. For radioimmunotherapy, a beta-emitting radionuclide labeled to a bivalent peptide is required. Therapeutic efficacy of these radionuclides depends on the E(max), physical half-life, and residence time of the radiolabel in the tumor. The (131)I radiolabel generally clears rapidly from the tumor after internalization and subsequent degradation of the bivalent l-amino acid peptide (l-a.a. peptide) in the tumor cells. To improve the residence time of the iodine label in the tumor, a new bivalent peptide was synthesized that is peptidase resistant and consists of 4 d-amino acids (d-a.a. peptide). Here we investigated the characteristics of the residualizing iodine label in SK-RC-52 RCC tumors., Methods: The d-a.a. peptide was manually synthesized according to standard solid-phase Fmoc/HBTU (2-[1H-benzotriazole-1-yl]-1,1,3,3-tetramethyluronium hexafluorophosphate) chemistry. The uptake and retention in the tumor of (111)In-/(125)I-labeled bivalent peptides (l-a.a. peptide and d-a.a. peptide) were studied in female BALB/c athymic mice with subcutaneous SK-RC-52 RCC tumors. Tumors were pretargeted with the bispecific monoclonal antibody (bs-mAb) G250xDTIn-1 and, 72 h later, mice were injected intravenously with one of both radiolabeled peptides. The effect of bs-mAb-diDTPA-bs-mAb (DTPA is diethylenetriaminepentaacetic acid) bridging at the tumor cell surface on the internalization of the bs-mAb-diDTPA complex was investigated in SK-RC-52 tumor-bearing mice., Results: The maximum uptake and retention of (125)I-labeled l-a.a. peptide in the tumor were significantly lower compared with that of the (111)In-labeled l-a.a. peptide. In contrast, the tumor uptake and retention of the (125)I-labeled d-a.a. peptide) were similar to that of the (111)In-labeled l-a.a. peptide but were superior at later time points. The biodistribution of the radioiodinated d-a.a. peptide was highly similar to that of the (111)In-labeled d-a.a. peptide, and both radiolabeled peptides were retained significantly better in the tumor than the (111)In-labeled l-a.a. peptide. bs-mAb-diDTPA-bs-mAb bridge formation did not affect internalization of the bs-mAb-diDTPA complex., Conclusion: Uptake and retention in the tumor of the iodinated peptide after pretargeting with a bs-mAb can be significantly improved using d-a.a. peptides. Accordingly, the radiation dose to the tumor, correlating with the therapeutic efficacy of pretargeted RCC, can be enhanced substantially.

Objectives: In the present study, we evaluated functional activity of monocytes in experimental preeclampsia induced by low-dose endotoxin infusion., Study Design: Pregnant (n = 12) and cyclic rats (n = 12) were equipped with a permanent jugular vein cannula and infused with either low-dose endotoxin or saline. One day before the infusion, and 4, 24, 72, and 168 hours after the infusion, blood samples (400 microL) were taken and white blood cell (WBC) and differential cell counts were measured. Samples were (re)stimulated with endotoxin, and the percentage of tumor necrosis factor-alpha (TNFalpha) producing monocytes was measured., Results: During experimental preeclampsia, monocyte TNFalpha production is persistently decreased, while total WBC and granulocyte counts are persistently increased compared with normal pregnant rats. No persistent effect of endotoxin was found in cyclic rats., Conclusion: Because decreased endotoxin-induced TNFalpha production is a feature of activated monocytes, the present results indicate that monocytes are persistently activated in experimental preeclampsia. Increased WBC counts and granulocyte numbers in these rats also point to an activated inflammatory response.

The need for effective community treatment for psychiatric clients has increased in recent years. The aftercare program descirbed in this paper provides a community treatment program for adult psychiatric clients that aims at facilitatin their adequate and independent adjustment to the community. The occupational therapist functions as a case manager of the treaatment team and is also program coordinator for the arts and crafts groups and the skill development groups. The arts and crafts groups are designed primarily to meet basic mental health needs; the skill development groups are aimed at teaching independent functioning in basic living skills. The behavior rehearsal model provides the theoretical framework for the skill development groups and is an effective approach in teaching skill acquisition to clients treated in the community.