Long-term immunosuppression after solitary islet transplantation is associated with preserved C-peptide secretion for more than a decade.

We report on two patients with type 1 diabetes (T1D) after solitary islet transplantation in 2001. They received steroid-sparing immunosuppression (daclizumab, sirolimus, and tacrolimus according to the Edmonton protocol). Both patients became insulin independent for 2 years: Patient A, a 42-year-old female with a 12-year history of T1D, received two islet infusions; patient B, a 53-year-old female with a 40-year T1D history, received one islet infusion. Pretransplant, both had undetectable C-peptide concentrations and frequent and severe hypoglycemia. Pretransplant, hemoglobin A1c (HbA1c) was 7.8% and 8.8% and insulin requirements were 0.47 and 0.33 units/kg/day, respectively. Posttransplant, C-peptide levels remained detectable while immunosuppression was continued, but decreased over time. Insulin was re-started 2 years posttransplant in both patients. Since patient A's glycemia and insulin requirements trended toward pretransplant levels, immunosuppression was discontinued after 13 years. This resulted in a sudden cessation of C-peptide secretion. Patient B continues on immunosuppression, has better HbA1c, and half the insulin requirement compared to pretransplant. Both patients no longer experience severe hypoglycemia. Herein, we document blood glucose concentrations over time (>30 000 measurements per patient) and ß cell function based on C-peptide secretion. Despite renewed insulin dependence, both patients express satisfaction with having undergone the procedure.

Effects of short-term sitagliptin treatment on immune parameters in healthy individuals, a randomized placebo-controlled study.

Sitagliptin, a dipeptidyl-peptidase 4 (DPP-4) inhibitor, improves blood glucose control in patients with type 2 diabetes by blocking cleavage of glucagon-like peptide 1 (GLP-1). In type 2 diabetes patients sitagliptin use is associated with an increase in minor infections, and in new-onset type 1 diabetes patients the ability of sitagliptin to dampen autoimmunity is currently being tested. DPP-4, also known as CD26, is expressed on leucocytes and can inactivate many chemokines important for leucocyte migration, as well as act as a co-stimulatory molecule on T cells. Therefore, this study was conducted to test whether sitagliptin is immunomodulatory. In this randomized, placebo-controlled trial, healthy volunteers were given sitagliptin or placebo daily for 28 days, and blood was drawn for immune assays. No significant differences were observed in the percentage of leucocyte subsets within peripheral blood mononuclear cells (PBMCs), plasma chemokine/cytokine levels or cytokines released by stimulation of PBMCs with either lipopolysaccharide (LPS) or anti-CD3. Individuals taking sitagliptin displayed increases in the percentage of cells expressing higher levels of CD26 at early time-points compared to placebo controls, but these differences resolved by day 28 of treatment. Therefore, in healthy volunteers, treatment with sitagliptin daily for 28 days does not overtly alter systemic immune function.

Beyond fast food and slow motion: weighty contributors to the obesity epidemic.

Decreased physical activity and marketing-driven increased consumption of "junk" food, dubbed "The Big Two", are generally regarded as the most important contributors to the obesity epidemic. However, the full picture contains many more pieces of the puzzle. We address several additional issues and review current clinical developments in obesity research. In spite of dramatic advancements in our understanding of the adipose organ and its endocrine and immune products, the ultimate causes of the obesity epidemic remain elusive. Treatment is plagued by poor adherence to life style modifications, and available pharmacological options are marginally effective, often also associated with major side effects. Surgical treatments, albeit effective in decreasing body weight, are invasive and expensive. Thus, our approaches to finding the causes, improving the existing treatments, and inventing novel therapies must be manifold.

Rising incidence and challenges of childhood diabetes. A mini review.

Approximately 215,000 people younger than 20 yr of age, or 1 in 500 children and adolescents, had diabetes in the United States in 2010--and the incidence is rising. We still have insufficient knowledge about the precise mechanisms leading to the autoimmune mediated ß-cell destruction in Type 1 diabetes, and the ß-cell failure associated with insulin resistance in Type 2 diabetes. Long-term complications are similar: micro- and macrovascular disease occurs prematurely and presents an enormous burden on affected individuals, often as early as in middle age. In Type 1 diabetes, technological advances have clearly improved blood glucose management, but chronic peripheral over-insulinization remains a problem even with the most advanced systems. Thus, in Type 1 diabetes our research must focus on 1) finding the stimulus that ignites the immune response and 2) developing treatments that avoid hyperinsulinemia. In Type 2 diabetes in youth, the challenges start much earlier: most young patients do not even benefit from existing therapies due to non-compliance. Therefore, prevention of Type 2 diabetes and improvement of compliance, especially with non-pharmacological interventions, are the greatest challenges.

Intrarenal complement fixation by cytotxic antibodies.

The reactivity of histocompatibility antigens with serum antibodies and complement was studied in a short term model of allotransplantation. Lewis and DA rat kidneys were perfused with cytotoxic antisera and tissue sections analyzed for adsorption of fluorescent antibodies against gamma-globulin and complement (beta(1C) protein). Both, the presumed cytotoxic antibody and C were localized at similar sites in the endothelial lining and in the walls of the arterial vessels and of the larger veins. The pattern of immune globulin deposition was distinct from that observed in glomerulonephritis. It may thus become helpful in the differential diagnosis between graft rejection and glomerulonephritis. At the sites of the deposits, the endothelial cells appeared enlarged and their nuclei protruded into the lumen. The findings are in line with the hypothesis that an antibody-mediated local activation of serum complement may be involved in the destruction of allografted tissue.

Allograft rejection in C'6 defective rabbits.

27 skin allografts were transplanted in duplicate to 13 rabbits defective in the sixth component (C'6) of complement (C'). 14 were rejected within the normal period of time and 9 only after a significant delay. In four grafts, no rejection was observed. Grafts exchanged between C' active and C' defective littermates tended to persist viable longer on the C' defective partners. It is concluded that in a species of higher vertebrates, the rabbit, allograft rejection can proceed in the absence of C'6 and the biological functions depending on it. This includes the cytotoxic function of complement. However, the prolonged survival in C'6 defective rabbits of some allografts strongly suggests that two pathways may be operative in the rejection reaction. The results are consistent with the view that a mechanism independent of C'6 was alone fully effective in the majority of the donor-host combinations. A C'6-dependent mechanism became influential only in some specific donor-host combinations.

Deficiency of the sixth component of complement in rabbits with an inherited complement defect.

A strain of rabbits with an inherited complement deficiency was shown to lack the sixth component of the hemolytic complement system. A method was elaborated for the partial purification of this component from normal rabbit serum. Upon injection of partially purified rabbit C'6 into C'6-deficient animals, an antibody was obtained which specifically inhibited the hemolytic activity of C'6. The data suggest that C'6-deficient serum either lacks the C'6 molecule or contains it in a chemically modified and inactive form.

Inter- and intra-individual variability of active glucagon-like peptide 1 among healthy adults.

Objective: To determine whether sex, age, and body mass index are correlated with active glucagon-like-peptide 1 concentrations and to investigate glucagon-like-peptide 1 reproducibility during repeated oral glucose tolerance tests. Methods: Sixty-one healthy volunteers underwent four 2-hour repeated oral glucose tolerance tests approximately 1 week apart. Because this randomized same-subject crossover trial was designed to investigate effects of non-nutritive sweeteners, participants received 355 mL (12 ounces) of water or a beverage containing non-nutritive sweeteners 10 minutes prior to each oral glucose tolerance test. Blood samples were collected 10 minutes before, and 0, 10, 20, 30, 60, 90, and 120 minutes following ingestion of 75 grams of glucose. Results: Basal active glucagon-like-peptide 1, peak glucagon-like-peptide 1, and glucagon-like-peptide 1 area-under-the-curve were higher in men than women (all p ≤0.04), adjusting for body mass index and age. Fasting and stimulated active glucagon-like-peptide 1 results were highly reproducible with little within-subject variability (between-subjects to within-subject variability ratio 4.2 and 3.5 for fasting glucagon-like-peptide 1 and glucagon-like-peptide 1 area-under-the-curve). Conclusion: Men had higher active glucagon-like-peptide 1 concentrations than women. In contrast to considerable inter-individual variability of basal and stimulated active glucagon-like-peptide 1 concentrations, intra-individual variability was low, consistent with tight physiological regulation.

Pancreatic beta cell function persists in many patients with chronic type 1 diabetes, but is not dramatically improved by prolonged immunosuppression and euglycaemia from a beta cell allograft.

AIMS/HYPOTHESIS: We measured serum C-peptide (at least 0.167 nmol/l) in 54 of 141 (38%) patients with chronic type 1 diabetes and sought factors that might differentiate those with detectable C-peptide from those without it. Finding no differences, and in view of the persistent anti-beta cell autoimmunity in such patients, we speculated that the immunosuppression (to weaken autoimmune attack) and euglycaemia accompanying transplant-based treatments of type 1 diabetes might promote recovery of native pancreatic beta cell function. METHODS: We performed arginine stimulation tests in three islet transplant and four whole-pancreas transplant recipients, and measured stimulated C-peptide in select venous sampling sites. On the basis of each sampling site's C-peptide concentration and kinetics, we differentiated insulin secreted from the individual's native pancreatic beta cells and that secreted from allografted beta cells. RESULTS: Selective venous sampling demonstrated that despite long-standing type 1 diabetes, all seven beta cell allograft recipients displayed evidence that their native pancreas secreted C-peptide. Yet even if chronic immunosuppression coupled with near normal glycaemia did improve native pancreatic C-peptide production, the magnitude of the effect was quite small. CONCLUSIONS/INTERPRETATION: Some native pancreatic beta cell function persists even years after disease onset in most type 1 diabetic patients. However, if prolonged euglycaemia plus anti-rejection immunosuppressive therapy improves native pancreatic insulin production, the effect in our participants was small. We may have underestimated pancreatic regenerative capacity by studying only a limited number of participants or by creating conditions (e.g. high circulating insulin concentrations or immunosuppressive agents toxic to beta cells) that impair beta cell function.

p53 downregulates expression of the G1/S cell cycle phosphatase Cdc25A.

Overexpression of Cdc25A phosphatase is often observed in cancer and results in poor prognosis. Cdc25A mainly dephosphorylates and thereby activates Cyclin-dependent kinase 2 and thus induces progression in the cell cycle from G(1) to S phase. Here, we demonstrate that the tumor suppressor p53 downregulates expression from the Cdc25A gene. In a p53-inducible cell system, Cdc25A expression on the mRNA and protein level is downregulated upon p53 expression. Promoter-reporter assays show that this regulation is dependent on the Cdc25A promoter. Mutant p53 fails to reduce Cdc25A transcription. In contrast to p53, neither p63 nor p73 can repress Cdc25A transcription. The Cdc25A promoter displays no p53 binding site, and p53 does not bind directly to the promoter DNA as shown by chromatin immunoprecipitation assays. Previously, the contribution of p53 to G(1)/S arrest has been mostly linked to activating the expression of the Cdk inhibitor p21(WAF1/CIP1). By downregulating Cdc25A expression, p53 may impair transition from G(1) to S phase independently of p21(WAF1/CIP1). Therefore, the data suggest that, as long as p53 is intact, Cdc25A transcriptional downregulation might play a role in cancer prevention.

Premature translation of oskar in oocytes lacking the RNA-binding protein bicaudal-C.

Bicaudal-C (Bic-C) is required during Drosophila melanogaster oogenesis for several processes, including anterior-posterior patterning. The gene encodes a protein with five copies of the KH domain, a motif found in a number of RNA-binding proteins. Using antibodies raised against the BIC-C protein, we show that multiple isoforms of the protein exist in ovaries and that the protein, like the RNA, accumulates in the developing oocyte early in oogenesis. BIC-C protein expressed in mammalian cells can bind RNA in vitro, and a point mutation in one of the KH domains that causes a strong Bic-C phenotype weakens this binding. In addition, oskar translation commences prior to posterior localization of oskar RNA in Bic-C- oocytes, indicating that Bic-C may regulate oskar translation during oogenesis.

Different in vivo and in vitro transformation of intestinal stem cells in mismatch repair deficiency.

Mutations in mismatch repair (MMR) genes result in microsatellite instability (MSI) and early onset of colorectal cancer. To get mechanistic insights into the time scale, sequence and frequency of intestinal stem cell (ISC) transformation, we quantified MSI and growth characteristics of organoids of Msh2-deficient and control mice from birth until tumor formation and related them to tissue gene expression. Although in Msh2-deficient organoids MSI continuously increased from birth, growth characteristics remained stable at first. Months before tumor onset, normal Msh2-deficient tissue contained tumor precursor cells forming organoids with higher MSI, cystic growth and growth rates resembling temporarily those of tumor organoids. Consistently, Msh2-deficient tissue exhibited a tumor-like gene signature. Normal Msh2-deficient organoids showed increased inheritable transient cyst-like growth, which became independent of R-spondin. ISC transformation proceeded faster in vitro than in vivo independent of the underlying genotype but more under MMR deficiency. Transient cyst-like growth but not MSI was suppressed by aspirin. In summary, as highlighted by organoids, molecular alterations continuously proceeded long before tumor onset in MMR-deficient intestine, thus increasing its susceptibility for ISC transformation.

Insulin and anti-insulin antibody interaction. Evidence for the formation of 7 S and 10 S structures.

The clearing of monoclonal and polyclonal and anti-insulin antibodies from homogeneous solutions at 100,000 X g was used to estimate the size of soluble insulin-antibody complexes at physiologic concentrations. Monoclonal antibodies cleared as a uniform population of 6.6 S independent of the insulin concentration. Polyclonal antibodies cleared as 6.6 S monomers at saturation and as 10 S particles when the amount of insulin bound decreased, suggesting that a soluble complex with two antibodies was formed. An increase of the affinity and a decrease of antibody valency can be related to the complex formation. The binding affinity of polyclonal sera depends on the composition of the affinities of the IgG monomers and on their ability to form 10 S complexes. The formation of insulin-antibody dimers precludes cross-linking and precipitation. Both types of insulin-antibody complexes have been found in the sera from patients treated with bovine insulin.

Conservation of substructures in proteins: interfaces of secondary structural elements in proteasomal subunits.

It is observed that during divergent evolution of two proteins with a common phylogenetic origin, the structural similarity of their backbones is often preserved even when the sequence similarity between them decreases to a virtually undetectable level. Here we analyzed, whether the conservation of structure along evolution involves also the local atomic structures in the interfaces between secondary structural elements. We have used as study case one protein family, the proteasomal subunits, for which 17 crystal structures are known. These include 14 different subunits of Saccharomyces cerevisiae, 2 subunits of Thermoplasma acidophilum and one subunit of Escherichia coli. The structural core of the 17 proteasomal subunits has 23 secondary structural elements. Any two adjacent secondary structural elements form a molecular interface consisting of two molecular patches. We found 61 interfaces that occurred in all 17 subunits. The 3D shape of equivalent molecular patches from different proteasomal subunits were compared by superposition. Our results demonstrate that pairs of equivalent molecular patches show an RMSD which is lower than that of randomly chosen patches from unrelated proteins. This is true even when patch comparisons with identical residues were excluded from the analysis. Furthermore it is known that the sequential dissimilarity is correlated to the RMSD between the backbones of the members of protein families. The question arises whether this is also true for local atomic structures. The results show that the correlation of individual patch RMSD values and local sequence dissimilarities is low and has a wide range from 0 to 0.41, however, it is surprising that there is a good correlation between the average RMSD of all corresponding patches and the global sequence dissimilarity. This average patch RMSD correlates slightly stronger than the C(alpha)-trace RMSD to the global sequence dissimilarity.

Inherited neuroaxonal dystrophy in C6 deficient rabbits.

We report the occurrence of a progressive neurological syndrome clinically characterized by subacute motor neuropathy in offspring of C6 deficient rabbits. On the basis of the pedigree analysis, the disease appears to be genetically transmitted, most probably with an autosomal recessive mode of inheritance. Pathological studies of affected animals revealed: transmitted, most probably with an autosomal recessive mode of inheritance. Pathological studies of affected animals revealed: 1) severe axonal degeneration in the sciatic nerve system involving mainly motor fibers; 2) occasional peripheral axonal enlargement closely associated with axonal degeneration; 3) presence of structured abnormal material in normal-size myelinated fibers of central nervous system (CNS) and peripheral nervous system (PNS); and 4) widespread occurrence of dystrophic axons and axonal spheroids in the gray matter of CNS. By ultrastructural examination, dystrophic axons are filled with tubulovesicular material, stalks of parallel membranes and dense bodies similar to what is described in human neuroaxonal dystrophies (NAD). The disease manifested by C6 deficient rabbits may represent an animal model of primary human NAD.

Islet amyloid polypeptide in pancreatic tissue of children with persistent hyperinsulinemic hypoglycemia caused by primary islet hyperplasia and nesidioblastosis.

Islet amyloid is a recognized characteristic finding in insulinoma and secondary islet hyperplasia resulting from severe insulin resistance. Little information is available about the presence of islet amyloid in primary islet hyperplasia (nesidioblastosis) of childhood. Here we report that islet amyloid was not present in 12 children with primary islet hyperplasia associated with hyperinsulinemic hypoglycemia.

Leptin secretion in Cushing's syndrome: preservation of diurnal rhythm and absent response to corticotropin-releasing hormone.

The normal inverse relationship between leptin and cortisol is lost in chronic hypercortisolism. We studied this apparent dysregulation in patients with Cushing's syndrome to investigate 1) the effect of chronic hypercortisolemia on the circadian rhythm of leptin secretion, 2) the response of leptin after administration of CRH, and 3) the short term effect of curative surgery on leptin. The preoperative morning leptin concentration was 54.2 +/- 8.1 ng/mL, and the nighttime value was 68.6 +/- 9.8 ng/mL, reflecting a mean rise of 32.8 +/- 7.6%, similar to the nocturnal increase observed in normal subjects. By contrast, cortisol's diurnal variation (21.8 +/- 1.7 vs. 16.9 +/- 1.1 mg/dL) was blunted. In women, but not men, body mass index correlated with leptin (P = 0.001). Preoperative ACTH and cortisol (both P < 0.0001), but not leptin levels increased after CRH. Ten days after surgery, basal cortisol values were subnormal (1.1 +/- 0.6 mg/dL), but leptin levels remained unchanged and did not increase after CRH. Body mass index and insulin also remained unchanged. Insulin, but not age, urinary free cortisol, or plasma cortisol correlated with leptin (P < 0.05). In summary, patients with Cushing's syndrome have moderately elevated leptin levels that maintain an intact circadian rhythm but do not respond to acute or subacute alterations of cortisol.

Complex demethylation patterns at Sp1 binding sites in F9 embryonal carcinoma cells.

The ubiquitous transcription factor Sp1 has been implicated in the mechanism which maintains CpG islands methylation-free. Plasmids containing GC boxes (Sp1 sites) were in vitro methylated at every CpG dinucleotide. After stable introduction into F9 embryonal carcinoma cells, we analysed the methylation of the sequence around the GC boxes with bisulphite sequencing. In agreement with restriction site analysis by other labs, we found preferential demethylation at GC box DNA versus control DNA. However, the bisulphite sequencing which permits the analysis of every CpG site on a given DNA molecule, revealed a complex pattern of methylated and unmethylated sites. Upon prolonged culture the pattern became simpler, with most sites demethylated but certain sites being consistently methylated.

Diabetes mellitus during adolescence.

Poorly controlled diabetes may affect the tempo and course of pubertal growth and development. Pubertal changes induce glucose metabolism or specific insulin resistance. Because puberty greatly increases the risk of diabetes complications, the management offered at this transitional age is critically important. An understanding of the environmental and developmental influences on diabetes control and the effect of the physiologic changes of puberty improves the approach to diabetes management in the adolescent with diabetes.

An unusual case of the nonketotic hyperglycemic syndrome during childhood.

The nonketotic hyperglycemic syndrome is rare during childhood and may occur as the initial manifestation of insulin-dependent diabetes mellitus or during an episode of gastroenteritis. In this article, we report an unusual case of this syndrome in a female infant who had atypically severe hyperglycemia in association with gastroenteritis. In addition, we provide a review of the literature and summarize the pathophysiologic mechanisms of the nonketotic hyperglycemic syndrome.