Efforts to develop methods for in vivo evaluation of the native beta-cell mass

Visualization and quantification of the native beta-cell mass in vivo in humans appear to be important in the study of the natural course of diabetes, and in ongoing trials aimed at preserving beta-cell mass in patients with diabetes. This cannot be done by biopsy sampling, and therefore there is a great need for development of a non-invasive method. This article discusses the principle theoretical requirements for reaching this goal. In addition, it provides an overview of tracer probes, which have been examined as potential beta-cell mass imaging agents in the past. Finally, some future perspectives are discussed.     

Regional pre- and postsynaptic sympathetic system in the failing human heart--regulation of beta ARK-1

OBJECTIVES: Regional presynaptic sympathetic innervation varies considerably in the cardiomyopathic human heart, as shown in previous studies in vivo and in vitro. The goal of the present study was to correlate markers of presynaptic sympathetic innervation with local measurement of the postsynaptic beta-adrenergic system in failing human hearts. METHODS AND RESULTS: In nine left ventricular regions of hearts explanted from patients suffering from dilated cardiomyopathy, we measured the density of uptake(1) carriers ([3H]mazindol binding) as a marker of presynaptic function as well as beta-receptor density ([3H]CGP 12177 binding) and beta ARK-1 levels as the pivotal compounds of postsynaptic adrenergic signal transduction. Additionally, a subgroup of the patients was examined in vivo by HED-PET prior to heart transplantation. The density of uptake(1) was related to local hydroxyephedrine (HED) retention (as determined by pre-operative PET, r=0.65), whereas it was inversely correlated to regional beta ARK-1 levels (r=-0.61, P=0.04). In contrast, beta-adrenergic receptor density was not significantly correlated either to uptake(1) density or to local HED retention (r=0.15 and r=0.21). CONCLUSIONS: Regional beta ARK-1 levels rather than beta-adrenergic receptor density were correlated with presynaptic alterations in cardiomyopathic human left ventricles. It can be assumed that in the cardiomyopathic human heart, regional beta-adrenergic desensitization might be determined by differences in local beta ARK levels rather than by changes in beta-receptor density.     

Ile164 variant of beta2-adrenoceptor does not influence outcome in heart failure but may interact with beta blocker treatment

BACKGROUND: The Ile164 variant of the beta2-adrenoceptor has been shown to alter cardiovascular phenotypes and adversely affect survival in heart failure patients. AIMS: We aimed to replicate this observation by genotyping a cohort of 451 heart failure patients for the Ile164 polymorphism. METHODS: Patient outcome was recorded over a median follow-up period of 3.09 years, and genotypes were derived by multiplex amplification refractory mutation system PCR. RESULTS: Genotypes were obtained for 443 patients, and 3.2% of these (14 patients) were heterozygous for the Ile164 SNP. Demographic data, cardiac function and neurohormonal profiles did not differ between genotype groups. Ile164 genotype did not significantly affect survival in this cohort (Thr164 homozygotes 48.9%, Ile164 heterozygous 42.9%, p=0.66), although multivariate analysis suggested that beta-blocker treatment may negatively impact survival in the heterozygote group. CONCLUSION: This study suggests that the Ile164 polymorphism of the beta2-adrenoceptor does not have a major impact on outcome in individuals with heart failure, although it's potential interaction with beta-blockers requires further examination.     

Effects of low glucose degradation products peritoneal dialysis fluid on the peritoneal fibrosis and vascularization in a chronic rat model

In the present study, we examined the effects of a new peritoneal dialysis fluid (PDF) with a low level of low glucose degradation products (GDP) on the functional and structural stability of the peritoneal membrane (PM). Male Sprague-Dawley rats were divided into three groups: group C (n = 8), without dialysate infusion; group P (n = 12), infused with low-level GDP solution (4.25% Physioneal, pH 7.0-7.4); and group D (n = 12), infused with conventional solution (4.25% Dianeal, pH 5.2, adjusted to pH 7.0). In groups D and P, animals were infused through a permanent catheter with 25 mL of PDF, twice daily for 8 weeks. Lipopolysaccharide was added into the PDF immediately before infusion on days 8, 9 and 10 in the two dialysis groups. When compared with group P, group D showed a higher glucose mass transfer at weeks 6 and 8, D/P urea at week 8, TGF-beta1 at weeks 4 and 8, and VEGF level at week 8. The submesothelial matrix layer of the parietal peritoneum was significantly thickened in group D and the lectin-stained blood vessels in this layer were well-visualized in group D compared with group P. There were significantly more peritoneal blood vessels in group D than group P. The transforming growth factor-beta induced gene-h3 (betaig-h3) and TGF-beta1 levels in the peritoneal effluent correlated with the submesothelial thickness, which correlated with the dialysate-to-plasma ratio (D/P) of protein and, inversely, with the rate of glucose transport (D/D(0) glucose, where D is glucose concentration in the dialysate and D(0) is glucose concentration in the dialysis solution before it is infused into the peritoneal cavity). The present study showed that low-GDP PDF effectively attenuated the peritoneal vascularization and fibrosis related to conventional solution.     

Relationship between polymorphisms in the 5' leader cistron, positions 16 and 27 of the adrenergic β2 receptor gene and asthma in a Han population from southwest China

Background and objective: Adrenergic β2 receptors (ADRB2) play an important role in regulating pulmonary function. Many previous studies have investigated possible associations between polymorphisms in the ADRB2 gene and asthma, but have yielded conflicting results. Furthermore, little is known regarding the possible role of the Arg19Cys polymorphism in susceptibility to asthma among Chinese. Methods: This case–control association study involved 238 patients with asthma and 265 healthy subjects from a Han population in southwest China. For all subjects, the 5′ leader cistron Arg19Cys, Arg16Gly and Gln27Glu polymorphisms in the ADRB2 gene were characterized by direct sequencing. Genotype, allele and haplotype frequencies were determined. In addition, to evaluate the association between the ADRB2 polymorphisms and lung function, bronchodilator response to inhaled β2 agonists (400 µg of albuterol) was assessed in the asthmatic patients. Results: There were no significant differences in genotype or allele frequencies for the three ADRB2 polymorphisms between the two cohorts. The Arg19/Arg16/Gln27 haplotype was more frequent among asthmatic patients than control subjects (odds ratio 2.24, 95% confidence interval (CI): 1.05–4.73, P = 0.04). Moreover, the Arg19/Cys19 genotype was associated with a lower FEV₁% (mean difference ?4.5, 95% CI: ?12.5 to 3.6, P = 0.02) and FEV₁/FVC (mean difference 8.9, 95% CI: 8.5–9.4, P = 0.01). The bronchodilator response to albuterol was also marginally lower in individuals who were homozygous for the Arg19 genotype (mean difference 4.2, 95% CI: 3.7–4.8, P = 0.03). Conclusions: The Arg19/Cys19 genotype was an independent risk factor for lower FEV₁% and FEV₁/FVC. Asthmatic patients with the Arg19/Arg19 genotype showed decreased responsiveness to albuterol. Furthermore, the Arg19/Arg16/Gln27 haplotype may contribute to increased susceptibility to asthma in the Chinese population.     

Compound heterozygote state for GgammaAgamma(deltabeta) degrees -thalassemia and hereditary persistence of fetal hemoglobin

We report a hitherto undescribed interaction of a deletional (deltabeta) degrees -thalassemia and a deletional hereditary persistence of fetal hemoglobin (HPFH) in an adult Thai individual. He was a 40-year-old Thai male who had the following hematologic data: Hb 13.9 g/dL, Hct 43.8%, MCV 78.0 fL, MCH 24.7 pg, MCHC 31.6 g/dL, and RDW 17.1%. Hemoglobin analysis revealed 97% Hb F with Ggamma-globin chain predominant. Globin gene analyses demonstrated that he carried the GgammaAgamma(deltabeta) degrees -thalassemia deletion in trans to the HPFH-6. Hematologic data of the patient were compared to those of the heterozygotes for these high-Hb F determinants found in his parents and an unrelated Thai patient with a compound HPFH-6/deletion-inversion Ggamma(Agammadeltabeta) degrees -thalassemia previously described.     

Concordant fetal hemoglobin response to hydroxyurea in siblings with sickle cell disease

Fetal hemoglobin (HbF) level and the HbF responses to hydroxyurea (HU) vary among patients with sickle cell disease and are, at least in part, genetically regulated. We hypothesized that siblings with sickle cell disease are likely to share the same parental beta-like globin gene clusters with their cis-acting regulatory sequences and therefore, if regulation of this response is linked to the beta-globin gene cluster, might have concordant HbF responses to HU. Accordingly, we studied 26 families (30 sib pairings), 20 with sickle cell anemia (three families had three siblings) and 6 families with HbS-beta-thalassemia (one family had three siblings, and one family consisted of monozygotic twins), to see if siblings with sickle cell disease had discordant or concordant changes in HbF during HU treatment. Intraclass correlation coefficients (r) showed a high, positive correlation between sibs for HbF levels before and during HU treatment and a concordant change in HbF response from baseline to treatment-associated levels. Changes in mean corpuscular volume (MCV) paralleled HbF levels, while the expected correlations between treatment-associated fall in leukocyte count and increase in MCV were also present. Our results provide additional evidence that some elements that regulate HbF expression are linked to the beta-globin gene cluster.     

XmnI Ggamma-polymorphism in six unrelated Pakistani families with Inv/Del Ggamma(Agammadeltabeta) degrees deltabeta-thalassemia

The XmnI Ggamma-polymorphism (C-T polymorphism at position -158 to the Ggamma-globin gene) was studied in 13 individuals from six unrelated Pakistani families with deltabeta-thalassemia. All of the subjects had the Asian-Indian Inv/Del Ggamma(Agammadeltabeta) degrees that included six heterozygotes, six homozygotes, and one compound heterozygote of deltabeta- and beta-thalassemia. All seven deltabeta-thalassemia heterozygotes (including one compound heterozygote) had the -/+ genotype, whereas all six of the homozygotes had the +/+ genotype. The results strongly suggest a tight linkage between the XmnI Ggamma-polymorphism and the Asian-Indian Inv/Del Ggamma(Agammadeltabeta) degrees . The finding could explain the unusually well-preserved capacity to produce fetal hemoglobin in deltabeta-thalassemia.     

Role of beta1- and alpha2c-adrenergic receptor polymorphisms and their combination in heart failure: a case-control study

BACKGROUND: Adrenergic activation has a central role in the development of HF. The function of the beta1- and the alpha2C-adrenergic receptors is influenced by gene polymorphisms: the beta1Arg389 variant is associated with increased beta1-receptor sensitivity and the alpha2C-receptor Del322-325 variant is associated with decreased alpha2C receptor function and increased norepinephrine release. We hypothesised that these polymorphisms could influence the prevalence of heart failure. METHODS: The role of the beta1- and alpha2C-adrenergic receptor gene polymorphisms as risk factors for heart failure (HF) was assessed in an Italian white Caucasian population using a case-control study design. Genomic DNA was analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RLFP). RESULTS: We compared 260 Caucasian patients with HF and 230 normal subjects. The beta1Arg389 allele was frequent both in the patients with HF (69%) and in the normal subjects (73%). The alpha2CDel322-325 variant was rare in both groups (9% and 8%, respectively). Patients homozygotes for either the beta1Arg389 or the alpha(2C)Del322-325 alleles had no increased risk of HF (odds ratio [OR], 0.8; 95%CI: 0.5-1.2 and OR, 0.8; 95% CI: 0.4-1.8, respectively). Patients homozygotes for both the beta1Arg389 and the alpha(2C)Del322-325 alleles had no increased risk of HF as well (OR: 0.6; 95% CI: 0.2-2.1). CONCLUSIONS: Beta1-ARs and alpha2C-ARs polymorphisms are not associated with an increased risk of HF in an Italian white Caucasian population.     

Hb Florida: a novel elongated C-terminal beta-globin variant causing dominant beta-thalassemia phenotype

We report here a new frameshift mutation in exon 3 of the beta-globin gene, a single nucleotide deletion (-C) in between codons 140/141 (GCC/CTG-->GCC/TG), found in an 8-year-old Argentinean girl with clinical picture of thalassemia intermedia. It leads to a beta-chain that is elongated to 156 amino acids [(141)Trp-Pro-Thr-Ser-Ile-Thr-Lys-Leu-Ala-Phe-Leu-Leu-Ser-Asn-Phe-(156)Tyr-COOH]. The resulting hemoglobin, which we named Hb Florida, was not detected in peripheral blood; however, erythroid hyperplasia and dyserythropoiesis with large inclusion bodies on methyl violet staining were observed in bone marrow, suggesting that this is a hyperunstable variant producing a dominant beta-thalassemia phenotype, since the other beta-allele was completely normal.     

Molecular characteristics of pediatric patients with sickle cell anemia and stroke

Cerebrovascular accidents (CVA) are serious complications of sickle cell anemia (SS) in children. Factors that predispose children to this complication are not well established. In an effort to elucidate the risk factors associated with CVA in SS, we have determined the alpha-globin genotype and the beta(S) haplotype of children with this complication. Among 700 children with SS followed at Children's Hospital of Michigan, 41 (6%) are on chronic transfusions because of stroke due to cerebral infarction. The mean age of patients with CVA at the time of stroke was 5.6 +/- 3.2 years (mean +/- SD). The male/female ratio was 2/3. Only 8 of 41 patients (19.5%) had one alpha-gene deletion, compared to the reported prevalence of 30% in African-Americans. None of the patients had two alpha-gene deletions, and two (5%) had five alpha-genes. These findings are different than those in our adult patients with SS, where the prevalence of -alpha/-alpha and alphaalphaalpha/alphaalpha is 4% and <2%, respectively. Ten different beta(S)-haplotypes were detected in the patients studied. The majority of the patients (31%) were doubly heterozygous for the Ben/CAR haplotypes followed by Ben/Ben, Ben/Sen, and CAR/CAR haplotypes, respectively. The prevalence of these haplotypes, with the exception of the CAR/CAR haplotype, was higher in females than males. All the patients with CAR/CAR haplotype were males, had four alpha-genes, and ranked third in prevalence. Three patients were heterozygous for the Cameron haplotype. The Cameron and atypical haplotypes were more prevalent than reported in patients with SS at large. The data suggest that CVA in children seems to occur more frequently in females and in patients with certain beta(S) haplotype. alpha-Gene deletion seems to offer a protective effect against this complication. Neonates with four or more alpha-genes whose beta(S) haplotype is Ben/CAR, atypical, or CAR/CAR seem to be at a higher risk for CAV than other patients. A prospective study on a larger group of patients with or without CVA may clarify this issue.     

Characterization of a novel deletion causing (deltabeta)0-thalassemia in a Thai family

A novel deletion of the human beta-globin gene cluster associated with the increased level of fetal hemoglobin (Hb F) in adult life has been demonstrated in a Thai family. A Thai girl who was mistakenly diagnosed as beta-thalassemia/HbE is found to be the compound heterozygote of this mutation and Hb E. The heterozygous father had mild hypochromic and microcytic red blood cells and a high level of Hb F (23.2%). Polymorphic restriction sites in the beta-globin gene cluster identified the homozygous alleles, which localized the deletion region between the psibeta-globin and the 3' beta-globin genes. DNA polymerase that can amplify a long DNA template was employed to examine DNA fragment encompassing this deletion. A 11.3 kilobases (kb) of DNA deletion, beginning approximately 3.1 kb 5' to the delta-globin gene and end in the intron 2 of the beta-globin gene was detected. DNA analysis revealed that this is a case of (deltabeta)(0)-thalassemia with a novel mutation, which can lead to a mild form of beta-thalassemia upon interaction with Hb E.     

Similar effects of recombinant interferon-alpha-2b and natural interferon-alpha when combined with intra-arterial 5-fluorouracil for the treatment of advanced hepatocellular carcinoma.

AIM: Intra-arterial 5-fluorouracil (5-FU) plus interferon (IFN) combination therapy is effective against advanced hepatocellular carcinoma (HCC) with portal vein tumour thrombosis. In this study, we compared the efficiency and safety of recombinant IFN-alpha-2b with natural IFN-alpha as components of the combination therapy. METHODS: Consecutive HCC patients (n=31) with portal vein tumour thrombosis were enrolled in this prospective study. They received combination therapy of 5-FU and either recombinant IFN-alpha-2b (R group, n=15) or natural IFN-alpha (N group, n=16). We compared the two groups for the early response rate, adverse reactions, time to progression (TTP) and survival rates. In addition, we assessed the cost-effectiveness of each protocol. RESULTS: The early response rate (R: 26.7%, N: 31.2%), median TTP (R: 5.8 months, N: 5.6 months) and median survival time (R: 7.5 months, N: 6.5 months) were not significantly different between the R and N groups. There were no differences in adverse reactions between the two groups. The estimated cost-effectiveness ratio of recombinant IFN-alpha-2b was better than natural IFN-alpha. CONCLUSIONS: In our protocol of combination therapy, there were no significant differences between recombinant IFN-alpha-2b and natural IFN-alpha with regard to early response to therapy, adverse effects, TTP and survival rates. 5-FU could be combined with either recombinant IFN-alpha-2b or natural IFN-alpha, although the cost-effectiveness of the former warrants its use clinically.     

Inhibitory effect of human interferon-beta-1a on activated rat and human hepatic stellate cells

Background and Aims: Hepatic stellate cells (HSC) are the primary cell type mediating hepatic fibrosis. Although known for its antiviral effects, the inhibitory effects of interferon‐beta (IFN‐β) on HSC treatment have not yet been established. Methods: Both human and rat activated HSC cell lines were incubated with increasing concentrations of recombinant human IFN‐β1a (rhIFN‐β1a) for 24, 48 or 72 h. The effects of rhIFN‐β1a on α‐smooth muscle actin (α‐SMA), collagen types I and III, transforming growth factor‐β1 (TGF‐β1), platelet‐derived growth factor‐BB (PDGF‐BB), and mothers against decapentaplegic homolog (Smad4, Smad7) expression in HSC were examined using Western blotting and immunocytochemistry. Proliferation of HSC was evaluated via bromodeoxyuridine assay. Results: rhIFN‐β1a treatment had a dose‐dependent, inhibitory effect on α‐SMA and collagen type I protein expression. In addition, rhIFN‐β1a decreased the expression of collagen type III, TGF‐β1, PDGF‐BB and Smad4 protein expression in HSC compared with untreated cells. We also observed increased Smad7 protein expression and decreased proliferation in rhIFN‐β1a‐treated HSC. Conclusions: Our data suggest that rhIFN‐β1a treatment decreased α‐SMA and collagen expression and inhibited the activation of HSC through the inhibition of the TGF‐β and PDGF pathways.     

Distribution of melatonin receptors in murine pancreatic islets

Melatonin has multiple receptor-dependent and receptor-independent functions. At the cell membrane, melatonin interacts with its receptors MT1 and MT2, which are expressed in numerous tissues. Genome-wide association studies have recently shown that the MTNR1B/MT2 receptor may be involved in the pathogenesis of type 2 diabetes mellitus. In line with these findings, expression of melatonin receptors has been shown in mouse, rat, and human pancreatic islets. MT1 and MT2 are G-protein-coupled receptors and are proposed to exert inhibitory effects on insulin secretion. Here, we show by immunocytochemistry that these membrane melatonin receptors have distinct locations in the mouse islet. MT1 is expressed in α-cells while MT2 is located to the β-cells. These findings help to unravel the complex machinery underlying melatonin's role in the regulation of islet function.     

Anti-interferon-α neutralizing antibody is associated with nonresponse to pegylated interferon-α plus ribavirin in chronic hepatitis C

Summary. Pegylated interferon-α (PEG-IFN-α) plus ribavirin (RBV) treatment fails to achieve a sustained virological response (SVR) in approximately 20-50% of patients with chronic hepatitis C virus (HCV) infection. We assessed the contribution of an anti-IFN-α neutralizing antibody (NAb) on the nonresponse to treatment. NAbs were detected using an antiviral assay that assessed the neutralizing effects of serum samples against IFN. Serum samples were obtained at the end of the treatment and evaluated for the presence of NAbs using recombinant IFN-α as a standard. We studied 129 PEG-IFN-α/RBV-treated patients. In the 82 end-of-treatment responders, no NAbs were detected. Of the 47 patients who did not respond, seven (15%) were positive for NAbs. We also examined an additional 83 patients who had not responded to PEG-IFN-α treatment, and detected 12 with NAbs. Patients with good IFN-responsive characteristics, including HCV genotype 2/3 and major allele homozygotes for interleukin-28B, were included in the 19 patients with NAbs. No NAbs interfered with the antiviral activity of natural human IFN-β (nIFN-β) and re-treatement of patients with NAbs with nIFN-β/RBV achieved SVR. Our analyses revealed that the emergence of anti-IFN-α NAbs was a candidate causal factor of PEG-IFN-α-treatment failure. Therefore, these antibodies should be assayed in patients who do not respond to PEG-IFN-α therapy, and if detected, other effective treatments, i.e., medications that are not neutralized by anti-IFN-α NAbs, should be considered.     

FR167653, a potent suppressant of interleukin-1 and tumor necrosis factor-alpha production, ameliorates colonic lesions in experimentally induced acute colitis.

BACKGROUND: Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are believed to play a significant role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-1 and TNF-alpha possess overlapping and synergetic activities inducing the production in cascade of other cytokines, adhesion molecules, arachidonic acid metabolites, as well as activating immune and non-immune cells. FR167653 (C24H18FN5O2-H2SO4-H2O) is a newly synthesized organic compound with a potent inhibitory effect on IL-1beta and TNF-alpha production. We hypothesized that the suppression of IL-1 and TNF-alpha induced by FR167653 could effectively attenuate experimentally induced colonic damage. METHODS: Colonic lesions were induced in male Sprague-Dawley rats (250-300 g) by intrarectal instillation of 4% acetic acid. The effect of FR167653 administration at 1.0, 1.5, 2.5 mg/kg per 6 h subcutaneously on acetic acid-induced colonic damage was assessed. The lesion area, microscopic findings, colonic and serum levels of TNF-alpha and IL-1beta were also evaluated. RESULTS: Treatment with FR167653 at 1.5 and 2.5 mg/kg per 6 h was able to ameliorate the gross macroscopic appearance of colonic lesions significantly, as well as ameliorate the lesion area induced by acetic acid. Colonic mucosal TNF-alpha and IL-1beta levels of rats treated with FR167653 showed significant decrease in a dose-dependent fashion compared with the control group. In the same manner, serum TNF-alpha of rats treated with FR167653 was significantly lower than that of respective controls. CONCLUSIONS: Subcutaneous administration of FR167653 was able to ameliorate the acute changes induced by acetic acid instillation in a dose-dependent manner. This is the first report to evaluate the dual inhibition of the production of IL-1 and TNF-alpha, offered by FR167653, in acute experimental colitis. Further studies are necessary to evaluate FR167653's efficacy and safety on long-term conditions.