Reverse Engineering of Digital Measures: Inviting Patients to the Conversation.

Background: Digital measures offer an unparalleled opportunity to create a more holistic picture of how people who are patients behave in their real-world environments, thereby establishing a better connection between patients, caregivers, and the clinical evidence used to drive drug development and disease management. Reaching this vision will require achieving a new level of co-creation between the stakeholders who design, develop, use, and make decisions using evidence from digital measures. Summary: In September 2022, the second in a series of meetings hosted by the Swiss Federal Institute of Technology in Zürich, the Foundation for the National Institutes of Health Biomarkers Consortium, and sponsored by Wellcome Trust, entitled "Reverse Engineering of Digital Measures," was held in Zurich, Switzerland, with a broad range of stakeholders sharing their experience across four case studies to examine how patient centricity is essential in shaping development and validation of digital evidence generation tools. Key Messages: In this paper, we discuss progress and the remaining barriers to widespread use of digital measures for evidence generation in clinical development and care delivery. We also present key discussion points and takeaways in order to continue discourse and provide a basis for dissemination and outreach to the wider community and other stakeholders. The work presented here shows us a blueprint for how and why the patient voice can be thoughtfully integrated into digital measure development and that continued multistakeholder engagement is critical for further progress.

Biomarkers in osteoarthritis: current status and outlook - the FNIH Biomarkers Consortium PROGRESS OA study.

Currently, no disease-modifying therapies are approved for osteoarthritis (OA) use. One obstacle to trial success in this field has been our existing endpoints' limited validity and responsiveness. To overcome this impasse, the Foundation for the NIH OA Biomarkers Consortium is focused on investigating biomarkers for a prognostic context of use for subsequent qualification through regulatory agencies. This narrative review describes this activity and the work underway, focusing on the PROGRESS OA study.

Remote Cardiac Safety Monitoring through the Lens of the FDA Biomarker Qualification Evidentiary Criteria Framework: A Case Study Analysis.

Clinical safety findings remain one of the reasons for attrition of drug candidates during clinical development. Cardiovascular liabilities are not consistently detected in early-stage clinical trials and often become apparent when drugs are administered chronically for extended periods of time. Vital sign data collection outside of the clinic offers an opportunity for deeper physiological characterization of drug candidates and earlier safety signal detection. A working group representing expertise from biopharmaceutical and technology sectors, US Food and Drug Administration (FDA) public-private partnerships, academia, and regulators discussed and presented a remote cardiac monitoring case study at the FNIH Biomarkers Consortium Remote Digital Monitoring for Medical Product Development workshop to examine applicability of the biomarker qualification evidentiary framework by the FDA. This use case examined the components of the framework, including the statement of need, the context of use, the state of the evidence, and the benefit/risk profile. Examination of results from 2 clinical trials deploying 510(k)-cleared devices for remote cardiac data collection demonstrated the need for analytical and clinical validity irrespectively of the regulatory status of a device of interest, emphasizing the importance of data collection method assessment in the context of intended use. Additionally, collection of large amounts of ambulatory data also highlighted the need for new statistical methods and contextual information to enable data interpretation. A wider adoption of this approach for drug development purposes will require collaborations across industry, academia, and regulatory agencies to establish methodologies and supportive data sets to enable data interpretation and decision-making.

Biomarkers of Crohn's Disease to Support the Development of New Therapeutic Interventions.

BACKGROUND: Currently, 2 coprimary end points are used by health authorities to determine the effectiveness of therapeutic interventions in patients with Crohn's disease (CD): symptomatic remission (patient-reported outcome assessment) and endoscopic remission (ileocolonoscopy). However, there is lack of accepted biomarkers to facilitate regulatory decision-making in the development of novel therapeutics for the treatment of CD. METHODS: With support from the Helmsley Charitable Trust, Critical Path Institute formed the Crohn's Disease Biomarkers preconsortium (CDBpC) with members from the pharmaceutical industry, academia, and nonprofit organizations to evaluate the CD biomarker landscape. Biomarkers were evaluated based on biological relevance, availability of biomarker assays, and clinical validation data. RESULTS: The CDBpC identified the most critical need as pharmacodynamic/response biomarkers to monitor disease activity in response to therapeutic intervention. Fecal calprotectin (FC) and serum C-reactive protein (CRP) were identified as biomarkers ready for the regulatory qualification process. A number of exploratory biomarkers and potential panels of these biomarkers was also identified for additional development. Given the different factors involved in CD and disease progression, a combination of biomarkers, including inflammatory, tissue injury, genetic, and microbiome-associated biomarkers, will likely have the most utility. CONCLUSIONS: The primary focus of the Inflammatory Bowel Disease Regulatory Science Consortium will be development of exploratory biomarkers and the qualification of FC and CRP for IBD. The Inflammatory Bowel Disease Regulatory Science Consortium, focused on tools to support IBD drug development, will operate in the precompetitive space to share data, biological samples for biomarker testing, and assay information for novel biomarkers.

The Foundation for the National Institutes of Health Biomarkers Consortium: Past Accomplishments and New Strategic Direction.

The Foundation for the National Institutes of Health (FNIH) Biomarkers Consortium (BC) is a public-private partnership that aims to facilitate drug development with biomarkers across a range of therapeutic areas. The BC is organized to address specific precompetitive biomarker projects, giving participating stakeholders a role in the design and conduct of projects and making the results freely public. Ultimately, the goals of the BC are to accelerate the development of new medicines, inform regulatory decision making, and improve patient care. Here, we describe how the BC works and briefly highlight its accomplishments. The BC has had many notable successful biomarker projects in the past 12 years, including I-SPY2, which has improved clinical trials and biomarker use for breast cancer, and an evidentiary framework for biomarker qualification. Recently, the BC has undergone a strategic expansion of its scope to include related drug development tools along the lines of the Biomarkers, Endpoints, and other Tools (BEST) resource.

Predictive validity of biochemical biomarkers in knee osteoarthritis: data from the FNIH OA Biomarkers Consortium.

OBJECTIVE: To investigate a targeted set of biochemical biomarkers as predictors of clinically relevant osteoarthritis (OA) progression. METHODS: Eighteen biomarkers were measured at baseline, 12 months (M) and 24 M in serum (s) and/or urine (u) of cases (n=194) from the OA initiative cohort with knee OA and radiographic and persistent pain worsening from 24 to 48 M and controls (n=406) not meeting both end point criteria. Primary analyses used multivariable regression models to evaluate the association between biomarkers (baseline and time-integrated concentrations (TICs) over 12 and 24 M, transposed to z values) and case status, adjusted for age, sex, body mass index, race, baseline radiographic joint space width, Kellgren-Lawrence grade, pain and pain medication use. For biomarkers with adjusted p<0.1, the c-statistic (area under the curve (AUC)), net reclassification index and the integrated discrimination improvement index were used to further select for hierarchical multivariable discriminative analysis and to determine the most predictive and parsimonious model. RESULTS: The 24 M TIC of eight biomarkers significantly predicted case status (ORs per 1 SD change in biomarker): sCTXI 1.28, sHA 1.22, sNTXI 1.25, uC2C-HUSA 1.27, uCTXII, 1.37, uNTXI 1.29, uCTXIα 1.32, uCTXIß 1.27. 24 M TIC of uCTXII (1.47-1.72) and uC2C-Human Urine Sandwich Assay (HUSA) (1.36-1.50) both predicted individual group status (pain worsening, joint space loss and their combination). The most predictive and parsimonious combinatorial model for case status consisted of 24 M TIC uCTXII, sHA and sNTXI (AUC 0.667 adjusted). Baseline uCTXII and uCTXIα both significantly predicted case status (OR 1.29 and 1.20, respectively). CONCLUSIONS: Several systemic candidate biomarkers hold promise as predictors of pain and structural worsening of OA.

Developing Outcomes Assessments as Endpoints for Registrational Clinical Trials of Antibacterial Drugs: 2015 Update From the Biomarkers Consortium of the Foundation for the National Institutes of Health.

One important component in determining the benefits and harms of medical interventions is the use of well-defined and reliable outcome assessments as endpoints in clinical trials. Improving endpoints can better define patient benefits, allowing more accurate assessment of drug efficacy and more informed benefit-vs-risk decisions; another potential plus is facilitating efficient trial design. Since our first report in 2012, 2 Foundation for the National Institutes of Health Biomarkers Consortium Project Teams have continued to develop outcome assessments for potential uses as endpoints in registrational clinical trials of community-acquired bacterial pneumonia and acute bacterial skin and skin structure infections. In addition, the teams have initiated similar work in the indications of hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia. This report provides an update on progress to date in these 4 diseases.

Renal epithelial cell-derived monocyte colony stimulating factor as a local informant of renal injury and means of monocyte activation.

Monocyte accumulation in renal allografts is associated with allograft dysfunction. As monocyte influx occurs acutely following reperfusion, we investigated the effect of ischemia-reperfusion injury (IRI) on monocyte colony stimulating factor (m-CSF), a key cytokine in monocyte recruitment. We hypothesized that renal tubule epithelial cells (RTECs) could produce m-CSF in response to IRI, which could in turn promote monocyte activation. Real time PCR was used to measure levels of intragraft m-CSF transcripts in patients during IRI and clinical rejection. Also, m-CSF production by RTECs following IRI simulation in vitro was measured using ELISA. Monocyte expression of CD40 and CD80 was then analyzed using flow cytometry following co-culture with supernatants of RTECs after IRI. Monocyte expression of CD40, CD80 and HLA-DR was then examined following treatment with rh-m-CSF (10, 36, and 100 ng/ml), as was monocyte size and granularity. We found that intragraft m-CSF transcription was significantly increased postreperfusion (P = 0.002) and during clinical rejection (P = 0.002). We also found that RTECs produced m-CSF in response to IRI in vitro (P = 0.036). Monocytes co-cultured with the supernatants of postischemic RTECs became activated as evidenced by increased expression of CD40 and CD80. Also, monocytes treated with recombinant m-CSF assumed an activated phenotype exhibiting increased size, granularity and expression of CD40, CD80, CD86, and HLA-DR, and demonstrating enhanced phagocytic activity. Taken together, we suggest that renal tubular cell derived m-CSF is a stimulus for monocyte activation and may be an important target for control of IRI-associated immune activation.

Neonatal-onset multisystem inflammatory disease responsive to interleukin-1beta inhibition.

BACKGROUND: Neonatal-onset multisystem inflammatory disease is characterized by fever, urticarial rash, aseptic meningitis, deforming arthropathy, hearing loss, and mental retardation. Many patients have mutations in the cold-induced autoinflammatory syndrome 1 (CIAS1) gene, encoding cryopyrin, a protein that regulates inflammation. METHODS: We selected 18 patients with neonatal-onset multisystem inflammatory disease (12 with identifiable CIAS1 mutations) to receive anakinra, an interleukin-1-receptor antagonist (1 to 2 mg per kilogram of body weight per day subcutaneously). In 11 patients, anakinra was withdrawn at three months until a flare occurred. The primary end points included changes in scores in a daily diary of symptoms, serum levels of amyloid A and C-reactive protein, and the erythrocyte sedimentation rate from baseline to month 3 and from month 3 until a disease flare. RESULTS: All 18 patients had a rapid response to anakinra, with disappearance of rash. Diary scores improved (P<0.001) and serum amyloid A (from a median of 174 mg to 8 mg per liter), C-reactive protein (from a median of 5.29 mg to 0.34 mg per deciliter), and the erythrocyte sedimentation rate decreased at month 3 (all P<0.001), and remained low at month 6. Magnetic resonance imaging showed improvement in cochlear and leptomeningeal lesions as compared with baseline. Withdrawal of anakinra uniformly resulted in relapse within days; retreatment led to rapid improvement. There were no drug-related serious adverse events. CONCLUSIONS: Daily injections of anakinra markedly improved clinical and laboratory manifestations in patients with neonatal-onset multisystem inflammatory disease, with or without CIAS1 mutations. (ClinicalTrials.gov number, NCT00069329 [ClinicalTrials.gov].).

Nitroglycerin reperfusion reduces ischemia-reperfusion injury in non-heart-beating donor lungs.

BACKGROUND: Lung transplantation is severely limited by an inadequate supply of lungs from brain-dead donors. A potential solution is use of lungs from non-heart-beating donors (NHBDs) with retrieval at intervals after circulatory arrest and death. A warm ischemic period with concomitant reperfusion injury is a major limiting factor in the transplantation of lungs retrieved from NHBDs. We hypothesized that the administration of the nitric oxide-donor nitroglycerin to lungs from NHBDs would reduce ischemia-reperfusion injury by activation of guanylate cyclase to form guanosine 3',5'-cyclic monophosphate (cGMP). METHODS: An in situ isolated perfused rat lung model was used. Lungs were retrieved from rats at varying intervals after circulatory arrest and death. Lungs were either ventilated with O(2) in situ or not ventilated. Lungs were reperfused at intervals after death with Earle's solution with or without nitroglycerin (0.1 mg/ml). Lung ischemia-reperfusion injury was assessed by capillary filtration coefficient, wet-to-dry lung weight ratio, and pulmonary hemodynamics. Tissue levels of adenine nucleotides and cGMP concentrations were measured by high-performance liquid chromatography and enzyme immunoassay, respectively. RESULTS: Reperfusion with nitroglycerin decreased capillary filtration coefficient compared with reperfusion without nitroglycerin at all post-mortem ischemic times, irrespective of pre-harvest ventilation. cGMP levels increased significantly with nitroglycerin-reperfusion and attenuated decreases in high-energy adenine nucleotides. CONCLUSIONS: Reperfusion of lungs with nitroglycerin may facilitate safe lung transplantation from NHBDs by reducing capillary leak after reperfusion.

Results from a human renal allograft tolerance trial evaluating T-cell depletion with alemtuzumab combined with deoxyspergualin.

BACKGROUND: Perioperative lymphocyte depletion induces allograft tolerance in some animal models, but in humans has only been shown to reduce immunosuppressive requirements. Without maintenance immunosuppression, depleted human renal allograft recipients experience rejection characterized by infiltration of the allograft with monocytes and macrophages. T-cell depletion combined with a brief course of deoxyspergualin (DSG), a drug with inhibitory effects on monocytes and macrophages, induces tolerance in nonhuman primates. We therefore performed a trial to determine if lymphocyte depletion with alemtuzumab combined with DSG would induce tolerance in humans. METHODS: Five recipients of live donor kidneys were treated perioperatively with alemtuzumab and DSG and followed postoperatively without maintenance immunosuppression. Patients were evaluated clinically, by flow cytometry, and by protocol biopsies analyzed immunohistochemically and with real-time polymerase chain reaction. Results were compared to previously studied patients receiving alemtuzumab alone or standard immunosuppression. RESULTS: Despite profound T-cell depletion and therapeutic DSG dosing, all alemtuzumab/DSG patients developed reversible rejection that was similar in timing, histology, and transcriptional profile to that seen in patients treated with alemtuzumab alone. Chemokine expression was marked prior to and during rejections. CONCLUSIONS: We conclude that treatment with alemtuzumab and DSG does not induce tolerance in humans. Chemokine production may not be adequately suppressed using this approach.

Disruption of CD40/CD40-ligand interactions in a retinal autoimmunity model results in protection without tolerance.

We examined the role of CD40/CD40L interactions on the development of experimental autoimmune uveoretinitis (EAU), a cell-mediated, Th1-driven autoimmune disease that serves as a model for autoimmune uveitis in humans. EAU-susceptible B10.RIII mice immunized with the retinal autoantigen interphotoreceptor retinoid binding protein in CFA and treated with anti-CD40L Ab (MR1) had reduced incidence and severity of disease. Real-time PCR analysis revealed that the innate and adaptive responses of protected mice were reduced, without an obvious shift toward a Th2 cytokine profile. In contrast to some other reports, no evidence was found for regulatory cells in adoptive transfer experiments. To determine whether CD40L blockade resulted in long-term tolerance, mice protected by treatment with MR1 Ab were rechallenged for uveitis after circulating MR1 Ab levels dropped below the detection limit of ELISA. MR1-treated mice developed severe EAU and strong cellular responses to interphotoreceptor retinoid binding protein, comparable to those of control mice. These responses were higher than in mice that had not received the primary immunization concurrently with anti-CD40L treatment. We conclude that 1) CD40/CD40L interaction is required for EAU and its disruption prevents disease development; 2) CD40L blockade inhibits the innate response to immunization and reduces priming, but does not result in immune deviation; and 3) protection is dependent on persistence of anti-CD40L Abs, and long-term tolerance is not induced. Furthermore, immunological memory develops under cover of CD40L blockade causing enhanced responses upon rechallenge. Taken together, our data suggest that ongoing CD40/CD40L blockade might be required to maintain a therapeutic effect against uveitis.

Immunocompetent T-cells with a memory-like phenotype are the dominant cell type following antibody-mediated T-cell depletion.

T-cell depletion facilitates reduced immunosuppression following organ transplantation and has been suggested to be pro-tolerant. However, the characteristics of post-depletional T cells have not been evaluated as they relate to tolerance induction. We therefore studied patients undergoing profound T-cell depletion with alemtuzumab or rabbit anti-thymocyte globulin following renal transplantation, evaluating the phenotype and functional characteristics of their residual cells. Naive T cells and T cells with potential regulatory function (CD4+CD25+) were not prevalent following aggressive depletion. Rather, post-depletion T cells were of a single phenotype (CD3+CD4+CD45RA-CD62L-CCR7-) consistent with depletion-resistant effector memory T cells that expanded in the first month and were uniquely prevalent at the time of rejection. These cells were resistant to steroids, deoxyspergualin or sirolimus in vitro, but were calcineurin-inhibitor sensitive. These data demonstrate that therapeutic depletion begets a limited population of functional memory-like T cells that are easily suppressed with certain immunosuppressants, but cannot be considered uniquely pro-tolerant.

Functionally significant renal allograft rejection is defined by transcriptional criteria.

Renal allograft acute cellular rejection (ACR) is a T-cell mediated disease that is diagnosed histologically. However, many normally functioning allografts have T-cell infiltrates and histological ACR, and many nonimmune processes cause allograft dysfunction. Thus, neither histological nor functional criteria are sufficient to establish a significant rejection, and the fundamental features of clinical rejection remain undefined. To differentiate allograft lymphocyte infiltration from clinically significant ACR, we compared renal biopsies from patients with ACR to patients with: sub-clinical rejection (SCR, stable function with histological rejection); no rejection; and nontransplanted kidneys. Biopsies were compared histologically and transcriptionally by RT-PCR for 72 relevant immune function genes. Neither the degree nor the composition of the infiltrate defined ACR. However, transcripts up-regulated during effector T(H)1 T-cell activation, most significantly the transcription factor T-bet, the effector receptor Fas ligand and the costimulation molecule CD152 clearly (p = 0.001) distinguished the patient categories. Transcripts from other genes were equivalently elevated in SCR and ACR, indicating their association with infiltration, not dysfunction. Clinically significant ACR is not defined solely by the magnitude nor composition of the infiltrate, but rather by the transcriptional activity of the infiltrating cells. Quantitative analysis of selected gene transcripts may enhance the clinical assessment of allografts.

Insulinomas and expression of an insulin splice variant.

BACKGROUND: Insulinomas are beta-cell tumours characterised by uncontrolled insulin secretion even in the presence of hypoglycaemia. However, the mechanisms allowing such excessive insulin secretion are not known. Insulin secretion can occur only when the beta-cell insulin stores have been replenished by insulin biosynthesis, which is mainly controlled by translation. Such specific translational regulation often involves the 5' untranslated region. We have identified an insulin splice variant in isolated human pancreatic islets of non-diabetic donors that retains 26 bp of intron 1 and thereby changes the 5' untranslated region, but leaves the coding region unchanged. This splice variant has increased translation efficiency in vitro and in vivo compared with native insulin mRNA. However, splice variant expression is less than 1% of native insulin mRNA in normal islets. METHODS: To test whether this splice variant is involved in insulin production by human insulinomas, we extracted RNA from nine laser-captured surgical insulinoma samples and from isolated islets of nine donors who did not have diabetes. We then determined the ratio of splice variant to native insulin mRNA by quantitative real-time RT-PCR. FINDINGS: The mean ratio of the splice variant to native insulin mRNA was increased more than 50-fold in insulinomas compared with normal islets, and this difference was present in all nine human insulinomas. Overexpression of the splice variant therefore seems to be a general characteristic of insulinomas and is estimated to contribute about 90% to insulin synthesis by these tumours. INTERPRETATION: Overexpression of the insulin splice variant with increased translation efficiency in insulinomas might explain how these tumours maintain high levels of insulin synthesis and secretion leading to hyperinsulinaemia-the hallmark of this disease.

Resistin is expressed in pancreatic islets.

Resistin, a recently described adipocyte factor, is regulated by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. While resistin has been proposed to mediate insulin resistance in rodents, little is known about human resistin and its expression in pancreatic islets has not been tested. The goal of the present study was therefore to analyze whether resistin, like PPARgamma, is expressed in islets. Human islets from seven donors were analyzed by quantitative RT-PCR revealing resistin expression in all samples. Immunohistochemistry using a resistin-specific antibody on human pancreatic sections localized resistin protein to the islets. Mouse resistin was also detected in the Min6 beta cell line. Interestingly, we found a 4-fold increase in islet resistin expression in insulin resistant A-ZIP transgenic compared to wild-type mice. Our results demonstrate that resistin is expressed in islets and up-regulated in insulin resistance and thereby shed new light on the role of resistin in mice and humans.

Immune profiling: molecular monitoring in renal transplantation.

Molecular techniques have become a mainstay for most biomedical research. In particular, sensitive methods for gene transcript detection and advanced flow cytometry have been crucial in fostering our understanding of the basic mechanisms promoting allosensitization and adaptive immune regulation. These technologies have been validated in vitro, and in pre-clinical settings, and as such their clinical application is now clearly appropriate. It is becoming increasingly clear that these robust techniques hold much promise to better elucidate human transplant biology, and more importantly, guide clinical decision making with mechanistically-based information. This article will discuss our laboratory's use of several novel technologies, including gene polymorphism analysis, real-time polymerase chain reaction transcript quantification, and multi-color flow cytometry in clinical human renal transplantation. Specific technical methodology will be presented outlining keys for effective clinical application. Clinical correlations will be presented as examples of how these techniques may have clinical relevance. Suggestions for the adaptation of these methods for therapeutic intervention will be given. We propose that clinical transplantation should proceed in close step with modern molecular diagnostics.

Results from a human renal allograft tolerance trial evaluating the humanized CD52-specific monoclonal antibody alemtuzumab (CAMPATH-1H).

BACKGROUND: Profound T-cell depletion before allotransplantation with gradual posttransplant T-cell repopulation induces a state of donor-specific immune hyporesponsiveness or tolerance in some animal models. Alemtuzumab (Campath-1H, Millennium Pharmaceuticals, Cambridge, MA) is a humanized CD52-specific monoclonal antibody that produces profound T-cell depletion in humans and reduces the need for maintenance immunosuppression after renal transplantation. We therefore performed a study to determine if pretransplant T-cell depletion with alemtuzumab would induce tolerance in human renal allografts and to evaluate the nature of the alloimmune response in the setting of T-cell depletion. METHODS: Seven nonsensitized recipients of living-donor kidneys were treated perioperatively with alemtuzumab and followed postoperatively without maintenance immunosuppression. Patients were evaluated clinically by peripheral flow cytometry, protocol biopsies evaluated immunohistochemically, and real-time polymerase chain reaction-based transcriptional analysis. RESULTS: Lymphocyte depletion was profound in the periphery and secondary lymphoid tissues. All patients developed reversible rejection episodes within the first month that were characterized by predominantly monocytic (not lymphocytic) infiltrates with only rare T cells in the peripheral blood or allograft. These episodes were responsive to treatment with steroids or sirolimus or both. After therapy, patients remained rejection-free on reduced immunosuppression, generally monotherapy sirolimus, despite the recovery of lymphocytes to normal levels. CONCLUSIONS: T-cell depletion alone does not induce tolerance in humans. These data underscore a prominent role for early responding monocytes in human allograft rejection.

Pulmonary preservation studies: effects on endothelial function and pulmonary adenine nucleotides.

BACKGROUND: Lung transplantation is an effective therapy plagued by a high incidence of early graft dysfunction, in part because of reperfusion injury. The optimal preservation solution for lung transplantation is unknown. We performed experiments using an isolated perfused rat lung model to test the effect of lung preservation with three solutions commonly used in clinical practice. METHODS: Lungs were retrieved from Sprague-Dawley rats and flushed with one of three solutions: modified Euro-Collins (MEC), University of Wisconsin (UW), or low potassium dextran and glucose (LPDG), then stored cold for varying periods before reperfusion with Earle's balanced salt solution using the isolated perfused rat lung model. Outcome measures were capillary filtration coefficient (Kfc), wet-to-dry weight ratio, and lung tissue levels of adenine nucleotides and cyclic AMP. RESULTS: All lungs functioned well after 4 hr of storage. By 6 hr, UW-flushed lungs had a lower Kfc than LPDG-flushed lungs. After 8 hr of storage, only UW-flushed lungs had a measurable Kfc. Adenine nucleotide levels were higher in UW-flushed lungs after prolonged storage. Cyclic AMP levels correlated with Kfc in all groups. CONCLUSIONS: Early changes in endothelial permeability seemed to be better attenuated in lungs flushed with UW compared with LPDG or MEC; this was associated with higher amounts of adenine nucleotides. MEC-flushed lungs failed earlier than LPDG-flushed or UW-flushed lungs. The content of the solution may be more important for lung preservation than whether the ionic composition is intracellular or extracellular.