Using directed evolution to improve hydrogen production in chimeric hydrogenases from algal species.

Algae generate hydrogen from sunlight and water utilizing high-energy electrons generated during photosynthesis. The amount of hydrogen produced in heterologous expression of the wild-type hydrogenase is currently insufficient for industrial applications. One approach to improve hydrogen yields is through directed evolution of the DNA of the native hydrogenase. Here, we created 113 chimeric algal hydrogenase gene variants derived from combining segments of three parent hydrogenases, two from Chlamydomonas reinhardtii (CrHydA1 and CrHydA2) and one from Scenedesmus obliquus (HydA1). To generate chimeras, there were seven segments into which each of the parent hydrogenase genes was divided and recombined in a variety of combinations. The chimeric and parental hydrogenase sequences were cloned for heterologous expression in Escherichia coli, and 40 of the resultant enzymes expressed were assayed for H2 production. Chimeric clones that resulted in equal or greater production obtained with the cloned CrHydA1 parent hydrogenase were those comprised of CrHydA1 sequence in segments #1, 2, 3, and/or 4. These best-performing chimeras all contained one common region, segment #2, the part of the sequence known to contain important amino acids involved in proton transfer or hydrogen cluster coordination. The amino acid sequence distances among all chimeric clones to that of the CrHydA1 parent were determined, and the relationship between sequence distances and experimentally-derived H2 production was evaluated. An additional model determined the correlation between electrostatic potential energy surface area ratios and H2 production. The model yielded several algal mutants with predicted hydrogen productions in a range of two to three times that of the wild-type hydrogenase. The mutant data and the model can now be used to predict which specific mutant sequences may result in even higher hydrogen yields. Overall, results provide more precise details in planning future directed evolution to functionally improve algal hydrogenases.

COVID-19 Serology Control Panel Using the Dried-Tube Specimen Method.

The dried-tube specimen (DTS) procedure was used to develop the COVID-19 serology control panel (CSCP). The DTS offers the benefit of shipping materials without a cold chain, allowing for greater access without deterioration of material integrity. Samples in the panel were sourced from COVID-19 convalescent persons from March to May 2020. The immunoglobulin subtypes (total Ig, IgM, and IgG) and their respective reactivity to severe acute respiratory syndrome coronavirus 2 nucleocapsid, spike, and receptor-binding domain antigens of the samples were delineated and compared with the WHO International Standard to elucidate the exact binding antibody units of each CSCP sample and ensure the CSCP provides adequate reactivity for different types of serological test platforms. We distribute the CSCP as a kit with five coded tubes to laboratories around the world to be used to compare test kits for external quality assurance, for harmonizing laboratory testing, and for use as training materials for laboratory workers.

Evaluation of a multiplexed coronavirus antigen array for detection of SARS-CoV-2 specific IgG in COVID-19 convalescent plasma.

Mitigation of the COVID-19 pandemic requires an understanding of the antibody response to SARS-CoV-2. However, throughout the development of SARS-CoV-2 IgG antibody assays during the past year, cross-reactivity to other coronaviruses remained a question. To address these issues, we evaluated IgG in COVID-19 convalescent plasma samples for reactivity against three SARS-CoV-2 antigens including full-length spike, receptor binding domain, and the proximal extracellular fusion domain, and spike antigens from other coronaviruses (SARS-CoV, MERS-CoV, hCoV-HKU1, hCoV-OC43, hCoV-229E and hCoV-NL63) using the VaxArray Coronavirus SeroAssay which is a multiplexed antigen assay developed by InDevR Inc. These results were compared to two commercial SARS-CoV-2 IgG ELISAs targeting either the SARS-CoV-2 nucleocapsid or spike antigens and a live virus focus reduction neutralizing antibody test (FRNT). The VaxArray platform showed high specificity for detection of SARS-CoV-2 IgG, evident from lack of reactivity to SARS-CoV-2 antigens despite significant reactivity to endemic coronavirus antigens in pre-pandemic samples. SARS-CoV-2 IgG positive samples reacted weakly to SARS-CoV spike but not to MERS-CoV. While the VaxArray platform had overall comparable results to the spike and nucleocapsid IgG ELISAs, results were more similar to the spike antigen ELISA and the platform displayed a higher sensitivity and specificity than both ELISAs. Samples with FRNT titers below 1/23 reported negative on VaxArray, while positive samples on VaxArray had significantly higher neutralizing antibody titers. These results suggest that the VaxArray Coronavirus SeroAssay performs with high sensitivity and specificity for the detection of SARS-CoV-2 IgG, and positive results on the platform indicate SARS-CoV-2 neutralizing activity.

Presence and short-term persistence of SARS-CoV-2 neutralizing antibodies in COVID-19 convalescent plasma donors.

BACKGROUND: In March 2020, the Food and Drug Administration (FDA) approved use of COVID-19 convalescent plasma (CCP) as an investigational new drug for treatment of COVID-19. Since then, collection of CCP from COVID-19-recovered patients has been implemented in donor centers nationwide. Children's Hospital Colorado rapidly put into practice a CCP collection protocol, necessitating development and implementation of assays to evaluate SARS-CoV-2 antibodies in CCP units. STUDY DESIGN AND METHODS: We evaluated 87 units of CCP collected from 36 donors over two to four sequential donations using both antigen-binding assays for SARS-CoV-2 nucleoprotein and spike antigens and a live virus focus reduction neutralization test (FRNT50 ). RESULTS: Our data show that the majority of donors (83%) had a FRNT50 titer of at least 80, and 61% had a titer of at least 160, which met the FDA's criteria for acceptable CCP units. Additionally, our data indicate that analysis of antibodies to a single SARS-CoV-2 antigen is likely to miss a percentage of seroconverters; however, these individuals tend to have neutralizing antibody titers of less than 80. There was considerable variability in the short-term, sustained antibody response, measured by neutralizing antibody titers, among our donor population. CONCLUSION: The correlation of neutralizing activity and antigen-binding assays is necessary to qualify CCP for therapeutic use. Since SARS-CoV-2 antibody levels decline in a percentage of donors, and such a decline is not detectable by current qualitative assays implemented in many laboratories, robust, quantitative assays are necessary to evaluate CCP units best suited for therapeutic infusion in COVID-19 patients.

Analysis of COVID-19 convalescent plasma for SARS-CoV-2 IgG using two commercial immunoassays.

Coronavirus Disease 2019 (COVID-19) convalescent plasma (CCP) was approved by the FDA for use in severe cases of COVID-19 under an emergency Investigational New Drug (IND) protocol. Eligibility criteria for CCP donors includes documentation of evidence of COVID-19 either by viral RNA detection at the time of illness or positive SARS-CoV-2 IgG after recovery if diagnostic testing for COVID-19 was not performed at the time of illness. In addition to analysis of CCP, analysis of SARS-CoV-2 IgG provides information for possible past exposure and may support diagnosis when SARS-CoV-2 PCR is negative and clinical suspicion for COVID-19 is high. Furthermore, assays with high sensitivity and specificity for SARS-CoV-2 IgG are critical for understanding community exposure rates to SARS-CoV-2. Currently, there are several assays that test for antibodies to SARS-CoV-2 using a variety of methods, including point-of-care lateral flow-based devices, high throughput immunoassay analyzers, and manual methods such as ELISA. These assays target a number of SARS-CoV-2 antigens, including the nucleocapsid protein (N), full length spike protein (S), S1 subunit, or receptor binding domain (RBD) of the S protein. Given the heterogeneity among methods for, and antigenic targets used in SARS-CoV-2 antibody assays, it is necessary for careful evaluation of these assays prior to implementation for clinical use. We compared two assays that had received the CE mark of regulatory approval and that used either the N antigen or S1-RBD antigen as the target for analysis of a large set of CCP samples. Our data indicates that sensitivity and specificity vary between these assays and that more than one antigenic target may be required to improve the sensitivity and specificity of IgG detection to SARS-CoV-2.

Functional Analysis of Anti-cytokine Autoantibodies Using Flow Cytometry.

Autoantibodies to cytokines are increasingly being detected in association with immunodeficient, autoimmune and immune dysregulated states. Presence of these autoantibodies in an otherwise healthy individual may result in a unique phenotype characterized by predisposition to infection with specific organisms. The ability to detect these autoantibodies is of importance as it may direct treatment toward a combination of anti-microbial agents and immunomodulatory therapies that decrease autoantibody levels, thereby releasing the immune system from autoantibody-mediated inhibition. Ligand binding assays such as ELISA or bead multiplex assays have been used to detect these antibodies. However, not all anti-cytokine autoantibodies have demonstrable function in vitro and therefore their clinical significance is unclear. Assays that evaluate the functionality of anti-cytokine autoantibodies can supplement such ligand binding assays and add valuable functional information that, when viewed in the context of the clinical phenotype, may guide the use of adjunctive immunomodulatory therapy. This mini review provides an overview of anti-cytokine autoantibodies identified to date and their clinical associations. It also describes the use of flow cytometry for the functional analysis of anti-IFNγ and anti-GM-CSF autoantibodies.

Amotosalen/UVA treatment inactivates T cells more effectively than the recommended gamma dose for prevention of transfusion-associated graft-versus-host disease.

INTRODUCTION: Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare complication after transfusion of components containing viable donor T cells. Gamma irradiation with doses that stop T-cell proliferation is the predominant method to prevent TA-GVHD. Treatment with pathogen inactivation methodologies has been found to also be effective against proliferating white blood cells, including T cells. In this study, T-cell inactivation was compared, between amotosalen/ultraviolet A (UVA) treatment and gamma-irradiation (2500 cGy), using a sensitive limiting dilution assay (LDA) with an enhanced dynamic range. METHODS AND MATERIALS: Matched plasma units (N = 8), contaminated with 1 × 106 peripheral blood mononuclear cells (PBMCs) per mL, were either treated with amotosalen/UVA or gamma irradiation, or retained as untreated control. Posttreatment, cells were cultured under standardized conditions. T-cell proliferation was determined by the incorporation of 3 H-thymidine and correlated with microscopic detection. RESULTS: Range-finding experiments showed that after gamma irradiation (2500 cGy), significant T-cell proliferation could be observed at a 1 × 107 cell culture density, some proliferation at 1 × 106 , and none at 1 × 105 cells/well. Based on these facts, a quantitative comparison was carried out between amotosalen/UVA at the highest challenge of 1 × 107 PBMCs/well, and gamma irradiation at 1 × 106 and 1 × 105 PBMCs/well. Complete inactivation of the T cells after amotosalen/UVA treatment was observed, equivalent to greater than 6.2 log inactivation. Complete inactivation of the T cells was also observed after gamma irradiation when 1 × 105 PBMCs/well were cultured (>4.2 log inactivation). Proliferation was observed when 1 × 106 PBMCs/well were cultured (≤5.2 log inactivation) after gamma irradiation. CONCLUSION: Amotosalen/UVA treatment more effectively inactivates T cells than the current standard of gamma irradiation (2500 cGy) for the prevention of TA-GVHD.

Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species.

Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe-Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production. Two of the improved mutants have a logarithmic increase in hydrogen production in our in vitro assay. Furthermore, the model predicts hydrogenase sequences with hydrogen productions as high as 540-fold over the positive control. Taken together, these results demonstrate the potential of directed evolution to improve the native bacterial hydrogenases as a first step for improvement of hydrogenase activity, further in silico prediction, and finally, construction and demonstration of an improved algal hydrogenase in an in vivo assay of C. reinhardtii hydrogen production.

Bacterial Manipulation of NK Cell Regulatory Activity Increases Susceptibility to Listeria monocytogenes Infection.

Natural killer (NK) cells produce interferon (IFN)-γ and thus have been suggested to promote type I immunity during bacterial infections. Yet, Listeria monocytogenes (Lm) and some other pathogens encode proteins that cause increased NK cell activation. Here, we show that stimulation of NK cell activation increases susceptibility during Lm infection despite and independent from robust NK cell production of IFNγ. The increased susceptibility correlated with IL-10 production by responding NK cells. NK cells produced IL-10 as their IFNγ production waned and the Lm virulence protein p60 promoted induction of IL-10 production by mouse and human NK cells. NK cells consequently exerted regulatory effects to suppress accumulation and activation of inflammatory myeloid cells. Our results reveal new dimensions of the role played by NK cells during Lm infection and demonstrate the ability of this bacterial pathogen to exploit the induction of regulatory NK cell activity to increase host susceptibility.

Anticytokine Autoantibodies: Association with Infection and Immune Dysregulation.

The association of autoantibodies to cytokines with immune deficiency, autoimmunity and/or immune dysregulation is increasingly being recognized. For example, autoantibodies to interferon gamma have been found to be associated with chronic, treatment refractory infections with intracellular organisms such as mycobacteria, autoantibodies to interleukin 17 with chronic mucocutaneous candidiasis, and anti-interferon alpha autoantibodies with systemic lupus erythematosus. While low titer autoantibodies to these and other cytokines may be detected in normal individuals, patients with infectious or autoimmune manifestations tend to have high titer autoantibodies that may block or potentiate the function of the respective cytokine. Recognition of these autoantibodies is important because it may direct treatment toward a combination of adjunctive immunotherapy to modulate the autoantibody level while continuing with appropriate anti-microbial therapy. This review focuses on the anti-cytokine autoantibodies documented to date, their autoimmune, immune dysregulation and infectious disease associations, methods for detection of these antibodies and potential treatment options.

Rituximab as successful adjunct treatment in a patient with disseminated nontuberculous mycobacterial infection due to acquired anti-interferon-γ autoantibody.

An acquired immune deficiency due to interferon gamma (IFN-γ) autoantibodies was diagnosed in a 78-year-old Japanese man with treatment-refractory disseminated nontuberculous mycobacterial infection. In addition to standard antimycobacterial therapy, he was successfully treated with rituximab to eliminate B cells and thereby the autoantibody. Subsequently, he obtained a sustained remission from infection.

IL-4 confers resistance to IL-27-mediated suppression on CD4+ T cells by impairing signal transducer and activator of transcription 1 signaling.

BACKGROUND: TH2 cells play a critical role in the pathogenesis of allergic asthma. Established TH2 cells have been shown to resist reprogramming into TH1 cells. The inherent stability of TH2 cells poses a significant barrier to treating allergic diseases. OBJECTIVE: We sought to understand the mechanisms by which CD4(+) T cells from asthmatic patients resist the IL-27-mediated inhibition. METHODS: We isolated and cultured CD4(+) T cells from both healthy subjects and allergic asthmatic patients to test whether IL-27 can inhibit IL-4 production by the cultured CD4(+) T cells using ELISA. Culturing conditions that resulted in resistance to IL-27 were determined by using both murine and human CD4(+) T-cell culture systems. Signal transducer and activator of transcription (STAT) 1 phosphorylation was analyzed by means of Western blotting and flow cytometry. Suppressor of cytokine signaling (Socs) mRNA expression was measured by using quantitative PCR. The small interfering RNA method was used to knockdown the expression of Socs3 mRNA. RESULTS: We demonstrated that CD4(+) T cells from asthmatic patients resisted the suppression of IL-4 production mediated by IL-27. We observed that repeated exposure to TH2-inducing conditions rendered healthy human CD4(+) T cells resistant to IL-27-mediated inhibition. Using an in vitro murine culture system, we further demonstrated that repeated or higher doses of IL-4 stimulation, but not IL-2 stimulation, upregulated Socs3 mRNA expression and impaired IL-27-induced STAT1 phosphorylation. The knockdown of Socs3 mRNA expression restored IL-27-induced STAT1 phosphorylation and IL-27-mediated inhibition of IL-4 production. CONCLUSIONS: Our findings demonstrate that differentiated TH2 cells can resist IL-27-induced reprogramming toward TH1 cells by downregulating STAT1 phosphorylation and likely explain why the CD4(+) T cells of asthmatic patients are resistant to IL-27-mediated inhibition.

Patients with non-tuberculous mycobacterial lung disease have elevated transforming growth factor-beta following ex vivo stimulation of blood with live Mycobacterium intracellulare.

We previously found that a subset of patients with pulmonary non-tuberculous mycobacterial (pNTM) disease were taller, leaner, and had a higher prevalence of pectus excavatum and scoliosis than uninfected controls. Additionally, whole blood of pNTM patients stimulated ex vivo with live Mycobacterium intracellulare produced significantly less interferon-gamma (IFNγ) compared to that of uninfected controls. Since IFNγ production can be suppressed by transforming growth factor-beta (TGFß), an immunosuppressive cytokine, we measured basal and M. intracellulare-stimulated blood levels of TGFß in a group of 20 pNTM patients and 20 uninfected controls. In contrast to the IFNγ findings, we found that stimulated blood from pNTM patients produced significantly higher levels of TGFß compared to controls. Since pNTM patients frequently possess body features that overlap with Marfan syndrome (MFS), and increased TGFß expression is important in the pathogenesis of MFS, we posit that a yet-to-be-identified syndrome related to MFS predisposes certain individuals to develop pNTM disease.

Six-year outcome of thoracoscopic ventral spondylodesis after unstable incomplete cranial burst fractures of the thoracolumbar junction: ventral versus dorso-ventral strategy.

PURPOSE: The purpose of this study is to determine the long term-results after thoracoscopic spondylodesis particularly with respect to a ventral versus dorso-ventral treatment strategy. METHODS: In this prospective cohort study, a follow-up examination was performed in 19 patients (seven men, 12 women, average age: 37.7 years, follow-up rate: 79 %), six years after ventral thoracoscopic spondylodesis of unstable, incomplete burst fractures. Nine patients received a ventral monosegmental spondylodesis with iliac crest bone graft. The other ten cases were treated dorso-ventrally, five undergoing a ventral monosegmental treatment with iliac crest bone graft; the other five a ventral bisegmental treatment with expandable titanium cage. RESULTS: The complication rate was 15.7 %, the rate of revision of 10.5 %. No complication was related to the ventral thoracoscopic approach, whereas all of them were related to the iliac crest bone graft. The operative bisegmental kyphotic reduction was higher in the dorso-ventrally treated group. Afterwards, the loss of reduction was similar in both study groups. The mean VAS spine score summed up to more than 80 in both groups. The mean PCS scores were comparable to a normal healthy collective of the same age. CONCLUSIONS: The ventral thoracoscopic approach to the spine seems to be a safe therapeutic strategy. A dorso-ventral treatment concept goes along with a higher operative reduction potential.

6-Year follow-up of ventral monosegmental spondylodesis of incomplete burst fractures of the thoracolumbar spine using three cortical iliac crest bone grafts.

INTRODUCTION: Autologous bone graft is the gold standard for vertebral body replacement. Currently, after modern implants for vertebral body replacement are available, controversies exist regarding the optimal implant strategy. PATIENTS AND METHODS: Between 2002 and 2003, 17 patients were included in this study, all suffering from incomplete burst fractures of the thoracolumbar spine. All of them were treated by ventral monosegmental spondylodesis using iliac crest bone graft. The individual treatment strategy depended on the fracture situation and patient's condition. After an average of 74 months (range 66-84) a clinical and computer tomographic follow-up examination was performed in 14 patients (average age, 35.2 years) including VAS spine score and SF 36 score. Nine patients were treated ventral only five patients dorsoventrally. RESULTS: Complete osseous consolidation was visible in nine, partial consolidation (>30 %) in four, and lysis in one patient, without any significant differences between ventral only or dorsoventral approach. After removal of the fixateur interne the level of consolidation improved in all patients, treated dorsoventrally. There was no significant correlation between percentage of osseous consolidation and the clinical follow-up parameters. After 6 years, 71 % of the patients suffered from persistent pain associated with the approach to the iliac crest. Two revision surgeries have been necessary. CONCLUSION: High rates of osseous consolidation are visible 6 years after ventral spondylodesis by iliac crest bone grafts. A further improvement of consolidation can be expected after dorsal implant removal. But the surgical approach to the iliac crest is accompanied with a relevant complication rate.

The discovery and optimization of a novel class of potent, selective, and orally bioavailable anaplastic lymphoma kinase (ALK) inhibitors with potential utility for the treatment of cancer.

A class of 2-acyliminobenzimidazoles has been developed as potent and selective inhibitors of anaplastic lymphoma kinase (ALK). Structure based design facilitated the rapid development of structure-activity relationships (SAR) and the optimization of kinase selectivity. Introduction of an optimally placed polar substituent was key to solving issues of metabolic stability and led to the development of potent, selective, orally bioavailable ALK inhibitors. Compound 49 achieved substantial tumor regression in an NPM-ALK driven murine tumor xenograft model when dosed qd. Compounds 36 and 49 show favorable potency and PK characteristics in preclinical species indicative of suitability for further development.

Comparison of 2 cell-based phosphoprotein assays to support screening and development of an ALK inhibitor.

Anaplastic lymphoma kinase (ALK) when expressed as a fusion protein with nucleophosmin (NPM) has been implicated as a driving oncogene in a subset of lymphomas. Recent reports of ALK expression in a number of other cancers have raised the possibility that an ALK inhibitor may benefit patients with these diseases as well. In a campaign to identify and develop a selective ALK inhibitor, 2 assays were devised to measure the phosphorylation of tyrosine residue 1604 of ALK (pY(1604) ALK). Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen(®)) and phosflow platforms were used to detect modulation of pY(1604) ALK to determine the relative potency of a set of small-molecule inhibitors. Prior to making use of these assays in diverse settings, the authors attempted to ensure their equivalence with a direct comparison of their performance. The pY(1604) ALK assays correlated well both with each other and with assays of ALK enzyme activity or ALK-dependent cell proliferation. The AlphaScreen(®) assay was amenable to automation and enabled rapid, high-throughput compound assessment in an NPM-ALK-driven cell line, whereas the phosflow assay enabled the authors to characterize the activity of compounds with respect to their impact on targeted enzymes and pathways. Results show that both AlphaScreen(®) and phosflow ALK assays exhibited diverse characteristics that made them desirable for different applications but were determined to be equally sensitive and robust in the detection of inhibition of pY(1604) ALK.

Insulin and glucose regulate the expression of the DNA repair enzyme XPD.

Nucleotide excision repair (NER) of damaged DNA is operated by a complex network of DNA repair enzymes that include a protein termed xeroderma pigmentosum complementation group D (XPD). We have previously reported that the expression of XPD is regulated by activation of the insulin receptor and that mutations of the tyrosine kinase domain of the receptor inhibit the insulin-dependent increase in XPD messenger RNA (mRNA) and protein levels. In the present study, we characterize the insulin-dependent signaling pathway leading to the control of XPD expression. Using Chinese hamster ovary (CHO) cells transfected with the human insulin receptor, we demonstrated that the effect of insulin on XPD mRNA levels was mediated via the RAS-signaling and the p70 S6 kinase pathways. On the other hand, the intracellular level of XPD protein was under the exclusive control of the activation of the RAS-dependent cascade in response to insulin. We also studied the effect of acute and chronic exposures to different concentrations of glucose on the insulin-dependent regulation of intracellular XPD levels. A short-term exposure (48 h) to increasing concentrations of glucose potentiated the insulin-dependent regulation of XPD, and this was associated with an efficient protection against glucose-dependent damage to cellular DNA, as determined by the comet assay. Conversely, in cells that were grown for 3 weeks in the presence of glucose concentration greater than 10 mM, the capability of insulin to regulate the level of XPD was significantly reduced, and this promoted a glucose-dependent accumulation of products of DNA damage. In conclusion, glucose and insulin are important regulators of XPD, and prolonged exposure to toxic levels of glucose reduces the insulin-dependent regulation of DNA repair.

The short half-life of glucagon-like peptide-1 in plasma does not reflect its long-lasting beneficial effects.

The incretin hormone glucagon-like peptide-1 (GLP-1) is capable of ameliorating glucose-dependent insulin secretion in subjects with diabetes. However, its very short half-life (1.5-5 min) in plasma represents a major limitation for its use in the clinical setting. The present study was designed to characterize the duration of the effect of GLP-1 in the Zucker diabetic fatty (ZDF) rat. ZDF rats were subjected to a 48 h infusion of human GLP-1 (30 pmol/kg per min), followed by an i.p. glucose tolerance test (IPGTT) (1 g/kg body weight), 2 h after removing the infusion pump. At 15 min from the beginning of the test, GLP-1-treated animals had lower plasma glucose levels (442+/-38 mg/dl) than saline-infused controls (583+/-63 mg/dl, P<0.01). This was reflected in the higher insulin levels attained in the GLP-1-treated animals (1999+/-163 vs 1250+/-51 pmol/l, GLP-1 vs saline respectively, P<0.01). Repetition of the IPGTT on day 3, 9 and 16 from the removal of the infusion pump revealed a surprising lasting 'memory' of the exposure to GLP-1. Indeed, the best insulin secretory response was observed approximately 1 week after discontinuation of the GLP-1 infusion, and lasted up to 3 weeks from the early exposure to GLP-1. Detection of fasting plasma levels of GLP-1 during the 3 weeks of the experiment showed a very rapid decline, consistent with the data reported by others. Our findings provide evidence for a long-lasting beneficial effect of GLP-1 that persists for weeks even when the circulating levels of GLP-1 are back to normal.