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Biomedical subjects

David M Harlan

Publications and source records attributed to David M Harlan.

At least 19 recordsLinked to original sources
PubMed2007

Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation.

While insulin replacement remains the cornerstone treatment for type I diabetes mellitus (T1DM), the transplantation of pancreatic islets of Langerhans has the potential to become an important alternative. And yet, islet transplant therapy is limited by several factors, including far too few donor pancreases. Attempts to expand mature islets or to produce islets from stem cells are far from clinical application. The production and expansion of the insulin-producing cells within the islet (so called beta cells), or even creating cells that secrete insulin under appropriate physiological control, has proven difficult. The difficulty is explained, in part, because insulin synthesis and release is complex, unique, and not entirely characterized. Understanding beta-cell function at the molecular level will likely facilitate the development of techniques to manufacture beta-cells from stem cells. We will review islet transplantation, as well as the mechanisms underlying insulin transcription, translation and glucose stimulated insulin release.

Animals↗
PubMed2007

T cell-directed therapies: lessons learned and future prospects.

Agents interfering with T cell function are therapeutic mainstays for various autoimmune diseases and for transplant approaches to organ failure. The understanding of T cell biology has blossomed since the development of most agents now in use. Here we discuss T cell-specific agents now in use, others recently added to the therapeutic armamentarium and promising agents being investigated in clinical and preclinical studies. In addition, we reflect on the risks and benefits involved in the testing of such agents clinically, with examples of agents that have successfully been used in the clinic and agents that failed to reach therapeutic use.

Cell Adhesion↗
PubMed2006

Assessment of islet function following islet and pancreas transplantation.

Pancreas and islet transplant recipients are monitored using various metabolic and imaging methods. The inaccessibility of the transplanted whole pancreas and of the isolated islets poses specific problems (eg, all assessment techniques are indirect). Although successful pancreas transplantation typically restores normal glucose homeostasis, islet transplantation into the liver does not completely normalize islet hormone secretion and glucose metabolism. Development of better testing strategies, such as direct islet imaging, will significantly advance the field.

Diabetes Mellitus, Type 1↗
PubMed2006

Islet neogenesis associated protein transgenic mice are resistant to hyperglycemia induced by streptozotocin.

Islet neogenesis associated protein (INGAP) is a protein factor that can stimulate new islet mass from adult pancreatic progenitor cells. In models of islet neogenesis, INGAP expression is elevated in pancreatic acinar cells. Using a transgenic model to drive a sustained expression of INGAP in pancreatic acinar cells, we have identified a protection to chemical-induced hyperglycemia. A sustained expression of INGAP during development did not perturb islet development or basal blood glucose homeostasis, although beta-cell mass and pancreatic insulin content were significantly increased in the INGAP transgenic mice. When challenged with a diabetogenic dose of streptozotocin (STZ), mice carrying the INGAP transgene did not become hyperglycemic. In contrast, wild-type mice became and remained hyperglycemic, blood glucose > 550 mg/dl. The serum insulin levels and islet morphology were preserved in the transgenic mice after STZ treatment. These data suggest that the sustained expression of INGAP in the acinar pancreas confers resistance to a diabetogenic insult. The INGAP transgenic mouse provides a new model to uncover factors that are protective to diabetes onset and biomarkers to track beta-cell pathology.

Animals↗
PubMed2005

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.

Adoptive Transfer↗
PubMed2005

Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.

The recent success of pancreatic islet transplantation has generated considerable enthusiasm. To better understand the quality and characteristics of human islets used for transplantation, we performed detailed analysis of islet architecture and composition using confocal laser scanning microscopy. Human islets from six separate isolations provided by three different islet isolation centers were compared with isolated mouse and non-human primate islets. As expected from histological sections of murine pancreas, in isolated murine islets alpha and delta cells resided at the periphery of the beta-cell core. However, human islets were markedly different in that alpha, beta, and delta cells were dispersed throughout the islet. This pattern of cell distribution was present in all human islet preparations and islets of various sizes and was also seen in histological sections of human pancreas. The architecture of isolated non-human primate islets was very similar to that of human islets. Using an image analysis program, we calculated the volume of alpha, beta, and delta cells. In contrast to murine islets, we found that populations of islet cell types varied considerably in human islets. The results indicate that human islets not only are quite heterogeneous in terms of cell composition but also have a substantially different architecture from widely studied murine islets.

Animals↗
PubMed2005

Assessment of pancreatic islet mass after islet transplantation using in vivo bioluminescence imaging.

BACKGROUND: Pancreatic islet transplantation is an emerging therapy for type 1 diabetes, but it is difficult to assess islets after transplantation and thus to design interventions to improve islet survival. METHODS: To image and quantify islets, the authors transplanted luciferase-expressing murine or human islets (by adenovirus-mediated gene transfer) into the liver or beneath the renal capsule of immunodeficient mice and quantified the in vivo bioluminescence imaging (BLI) of mice using a cooled charge-coupled device camera and digital photon-counting image analysis. To account for variables that are independent of islet mass such as transplant site, animal positioning, and wound healing, the BLI of transplanted islets was calibrated against measurement of luminescence of an implanted bead emitting a constant light intensity. RESULTS: BLI of mice bearing islet transplants was seen in the expected anatomic location, was stable for more than 8 weeks after transplantation, and correlated with the number of islets transplanted into the liver or kidney. BLI of the luminescent bead and of transplanted islets in the kidney was approximately four times greater than when transplanted in the liver, indicating that photon emission is dependent on optical absorption of generated light and thus light source location. CONCLUSION: In vivo BLI allows for quantitative, serial measurements of pancreatic islet mass after transplantation and should be useful in assessing interventions to sustain or increase islet survival of transplanted islets.

Animals↗
PubMed2005

A single course of anti-CD3 monoclonal antibody hOKT3gamma1(Ala-Ala) results in improvement in C-peptide responses and clinical parameters for at least 2 years after onset of type 1 diabetes.

Despite advances in understanding autoimmune diabetes in animal models, there has been little progress in altering the natural course of the human disease, which involves progression to insulin deficiency. Studies with immunosuppressive agents have shown short-term effectiveness, but they have not induced tolerance, and continuous treatment is needed. We studied the effects of hOKT3gamma1(Ala-Ala), a humanized Fc mutated anti-CD3 monoclonal antibody, on the progression of type 1 diabetes in patients with recent-onset disease in a randomized controlled trial. In general, the drug was well tolerated. A single course of treatment, within the first 6 weeks after diagnosis, preserved C-peptide responses to a mixed meal for 1 year after diagnosis (97 +/- 9.6% of response at study entry in drug-treated patients vs. 53 +/- 7.6% in control subjects, P < 0.01), with significant improvement in C-peptide responses to a mixed meal even 2 years after treatment (P < 0.02). The improved C-peptide responses were accompanied by reduced HbA(1c) and insulin requirements. Clinical responses to drug treatment were predicted by an increase in the relative number of CD8(+) T-cells in the peripheral blood after the lymphocyte count recovered 2 weeks after the last dose of drug. We conclude that treatment with the anti-CD3 monoclonal antibody hOKT3gamma1(Ala-Ala) results in improved C-peptide responses and clinical parameters in type 1 diabetes for at least 2 years in the absence of continued immunosuppressive medications.

Adolescent↗
PubMed2004

Increased insulin translation from an insulin splice-variant overexpressed in diabetes, obesity, and insulin resistance.

Type 2 diabetes occurs when pancreatic beta-cells become unable to compensate for the underlying insulin resistance. Insulin secretion requires beta-cell insulin stores to be replenished by insulin biosynthesis, which is mainly regulated at the translational level. Such translational regulation often involves the 5'-untranslated region. Recently, we identified a human insulin splice-variant (SPV) altering only the 5'-untranslated region and conferring increased translation efficiency. We now describe a mouse SPV (mSPV) that is found in the cytoplasm and exhibits increased translation efficiency resulting in more normal (prepro)insulin protein per RNA. The RNA stability of mSPV is not increased, but the predicted secondary RNA structure is altered, which may facilitate translation. To determine the role of mSPV in insulin resistance and diabetes, mSPV expression was measured by quantitative real-time RT-PCR in islets from three diabetic and/or insulin-resistant, obese and nonobese, mouse models (BTBRob/ob, C57BL/6ob/ob, and C57BL/6azip). Interestingly, mSPV expression was significantly higher in all diabetic/insulin-resistant mice compared with wild-type littermates and was dramatically induced in primary mouse islets incubated at high glucose. This raises the possibility that the mSPV may represent a compensatory beta-cell mechanism to enhance insulin biosynthesis when insulin requirements are elevated by hyperglycemia/insulin resistance.

5' Untranslated Regions↗
PubMed2004

Pulmonary angiography for the diagnosis of thromboembolic events in the non-human primate.

BACKGROUND: Evaluating possible thromboembolic events in the non-human primate has traditionally required euthanasia, significantly limiting the ability to conduct longitudinal studies. We hypothesized that pulmonary angiography could offer a safe, reproducible, and non-lethal means to assess for pulmonary embolus in the non-human primate. METHODS: Eleven rhesus primates were studied using standard pulmonary angiography techniques. Five animals studied had previously received humanized anti-CD154 antibodies (associated with thromboembolism risk) in the context of skin transplantation 2 years before the angiography study. Four primates were studied after receiving mouse anti-human CD154 antibody following allogeneic islet or skin transplantation. RESULTS: Angiography was successful in all primates. We observed no complications, and all animals promptly recovered from the procedure. Angiographic findings consistent with thromboembolism were demonstrated in the three primates actively receiving anti-CD154 antibody and in one primate that last received anti-CD154 nearly 2 years before the study. The study was normal in both the streptozotocin-induced diabetic control animals. Histopathology of the lungs confirmed thrombus in two of the four primates, but no thromboembolus was identified in the other two. The first had limited pathologic evaluation without fine slices, and in the second (treated 2 years before with a humanized anti-CD154), ascariasis was found in the area identified as abnormal by the angiogram. CONCLUSIONS: Minimally invasive pulmonary angiography is a safe, reproducible, and inexpensive method to assess possible thromboembolic events in the non-human primate. This method may allow for the longitudinal assessment of non-human primates given novel agents that may promote thromboembolism.

Angiography, Digital Subtraction↗
PubMed2004

The contribution of Fc effector mechanisms in the efficacy of anti-CD154 immunotherapy depends on the nature of the immune challenge.

Blockade of the CD154-CD40 co-stimulatory pathway with anti-CD154 mAbs has shown impressive efficacy in models of autoimmunity and allotransplantation. Clinical benefit was also demonstrated in systemic lupus erythematosus (SLE) and idiopathic thrombocytopenia patients with the humanized anti-CD154 mAb, 5C8 (hu5C8). However, thromboembolic complications that occurred during the course of the hu5C8 clinical trials have proven to be a major setback to the field and safe alternative therapeutics targeting the CD154-CD40 pathway are of great interest. Recently, effector mechanisms have been shown to play a part in anti-CD154 mAb-induced transplant acceptance in murine models, while this issue remains unresolved for humoral-mediated models. Herein, aglycosyl anti-CD154 mAbs with reduced binding to FcgammaR and complement were used as a novel means to test the role of effector mechanisms in non-human primate and murine models not amenable to gene knockout technology. While aglycosyl hu5C8 mAb was relatively ineffective in rhesus renal and islet allotransplantation, it inhibited primary and secondary humoral responses to a protein immunogen in cynomolgus monkeys. Moreover, an aglycosyl, chimeric MR1 mAb (muMR1) prolonged survival and inhibited pathogenic auto-antibody production in a murine model of SLE. Thus, the mechanisms required for efficacy of anti-CD154 mAbs depend on the nature of the immune challenge.

Animals↗
PubMed2004

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.

Alternative Splicing↗
PubMed2004

Resistin serum levels in type 1 diabetes pre- and post-islet transplantation.

Resistin is a recently described secretory protein produced in adipocytes that is thought to be involved in insulin resistance, diabetes, and inflammation. While resistin can be detected in mouse and human serum, very little is known about the regulation of serum resistin levels, especially in humans. To test whether resistin levels are affected by type 1 diabetes mellitus (T1DM), we measured serum resistin levels in samples from 5 healthy volunteers and 6 patients with T1DM pre- and 3 months post-islet transplantation using a human resistin enzyme immunoassay (EIA). Interestingly, serum resistin levels were significantly higher in T1DM patients before transplantation compared to normal controls, but decreased to normal levels after islet transplantation. Thus, our results suggest that human resistin may be involved in the pathophysiology of T1DM and thereby reveal a heretofore unappreciated aspect of human resistin biology.

Diabetes Mellitus, Type 1↗
PubMed2004

Induction of diabetes in nonhuman primates by means of temporary arterial embolization and selective arterial injection of streptozotocin.

PURPOSE: To develop and assess a technique for induction of C peptide-negative diabetes in adult nonhuman primates in preparation for preclinical investigation of type 1 diabetes treatments. MATERIALS AND METHODS: First, temporary embolization of the hepatic and gastric arteries was performed in 14 adult nonhuman primates (six cynomolgus, five rhesus, and three pigtail macaques). After embolization was confirmed with angiography, streptozotocin was injected at a dose of 50-70 mg/kg into the celiac artery and branches supplying the pancreas. The macaques then were given intravenous injections of arginine and glucose, and blood levels of insulin and C peptide were measured with an enzyme-linked immunosorbent assay to determine whether diabetes had been induced. RESULTS: All but one of the macaques developed persistent long-term C peptide-negative diabetes after the streptozotocin injection. One macaque did not develop diabetes after the initial injection and was given a second dose of streoptozotocin, which did induce diabetes. None of the macaques showed any symptoms of hepatic or renal injury, and only one died (of gastric dilatation 5 days after the procedure). CONCLUSION: Streptozotocin injection after temporary embolization of the hepatic and gastric arteries is a safe and reproducible method for inducing C peptide-negative diabetes in adult nonhuman primates in preparation for preclinical investigation of type 1 diabetes treatments.

Animals↗
PubMed2004

Challenges facing islet transplantation for the treatment of type 1 diabetes mellitus.

Islet transplantation represents a most impressive recent advance in the search for a type 1 diabetes mellitus cure. While several hundred patients have achieved at least temporary insulin independence after receiving the islet "mini-organs" (containing insulin-producing beta cells), very few patients remain insulin independent beyond 4 years after transplantation. In this review, we describe historic as well as technical details about the procedure and provide insight into clinical and basic research efforts to overcome existing hurdles for this promising therapy.

Age Factors↗