Rescue of abasic hammerhead ribozymes by exogenous addition of specific bases.

We have synthesized 13 hammerhead ribozyme variants, each containing an abasic residue at a specific position of the catalytic core. The activity of each of the variants is significantly reduced. In four cases, however, activity can be rescued by exogenous addition of the missing base. For one variant, the rescue is 300-fold; for another, the rescue is to the wild-type level. This latter abasic variant (G10.1X) has been characterized in detail. Activation is specific for guanine, the base initially removed. In addition, the specificity for guanine versus adenine is substantially altered by replacing C with U in the opposite strand of the ribozyme. These results show that a binding site for a small, noncharged ligand can be created in a preexisting ribozyme structure. This has implications for structure-function analysis of RNA, and leads to speculations about evolution in an "RNA world" and about the potential therapeutic use of ribozymes.     

The physical state of a meal affects hormone release and postprandial thermogenesis

There is evidence that food consistency may influence postprandial physiological responses. Recently we found that homogenization of a vegetable-rich meal significantly delayed the gastric emptying rate and was more satiating than the same meal in solid-liquid form. In this present study we investigated whether homogenization also influences endocrine and metabolic responses to the meal. Eight healthy men, aged 21-28 (mean 24.5) years, were given the meal (cooked vegetables 250 g, cheese 35 g, croutons 50 g and olive oil 25 g, with water 300 ml; total energy 2.6 MJ) in both solid-liquid (SM) and homogenized (HM) form, in random order, at 1-week intervals. Variables assayed were plasma glucose, insulin and glucose-dependent insulinotropic peptide (GIP) levels for 2 h and diet-induced thermogenesis (DIT) for 5 h. Plasma glucose pattern was similar after both meals. However, HM induced significantly greater insulin, GIP and DIT responses than SM. Mean integrated areas under the curves (AUC) were 1.7 (SEM 0.38) v. 1.2 (SEM 0.33) U/l per 120 min (P = 0.005) for insulin, 19.9 (SEM 2.44) v. 16 (SEM 1.92) nmol/l per 120 min (P = 0.042) for GIP, and 237.7 (SEM 16.32) v. 126.4 (SEM 23.48) kJ/300 min (P = 0.0029) for DIT respectively. Differences between GIP-AUC after HM and SM correlated significantly with differences between insulin-AUC after HM and SM (r2 0.62, P = 0.021). These findings demonstrate that homogenization of a meal results in a coordinated series of changes of physiological gastroentero-pancreatic functions and confirm that the physical state of the meal plays an important role in modulating endocrine and metabolic responses to food.     

Plasma cyclic nucleotide levels in acute leukemia patients

To verify the clinical usefulness of extracellular cyclic nucleotide determination as a tumor marker, plasma cyclic AMP (cAMP) and cyclic GMP (cGMP) levels were measured in 70 normal subjects and 173 acute leukemia patients studied in different stages of their disease. Mean plasma cAMP levels were similar in leukemic and normal subjects, although in 48 patients in the active stage of the disease, first diagnosis, or relapse, the cAMP values were below the normal range, and most of these patients failed to respond to chemotherapy. Plasma cGMP levels were markedly elevated in untreated patients, normalized in all patients who attained complete remission, and increased promptly to pretreatment values in patients who relapsed, suggesting that their determination may be useful to monitor the patients' response to treatment.     

Comparison of Cytomegalovirus (CMV) Enzyme-Linked Immunosorbent Spot and CMV Quantiferon Gamma Interferon-Releasing Assays in Assessing Risk of CMV Infection in Kidney Transplant Recipients

Assessing cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) represents an appealing strategy for identifying transplant recipients at risk of infection. In this study, we compared two gamma interferon-releasing assays (IGRAs), Quantiferon-CMV and CMV enzyme-linked immunosorbent spot (ELISPOT), to determine the ability of each test to predict protective CMV-specific T-cell responses. Two hundred twenty-one Quantiferon-CMV and ELISPOT tests were conducted on 120 adult kidney transplant recipients (KTRs), including 100 CMV-seropositive transplant recipients (R⁺) and 20 CMV-seronegative transplant recipients of a CMV-positive donor (D⁺/R⁻). As a control cohort, 39 healthy adult subjects (including 33 CMV-seropositive and 6 CMV-seronegative subjects) were enrolled. CMV IgG serology was used as a reference for both tests. In the CMV-seropositive individuals, the ELISPOT and Quantiferon-CMV assays provided 46% concordance with the serology, 12% discordance, 18% disagreement between ELISPOT or Quantiferon-CMV and the serology, and 24% gray areas when one or both tests resulted in weak positives. None of the CMV-seronegative subjects showed detectable responses in the ELISPOT or the Quantiferon-CMV test. In transplant recipients, both the ELISPOT and Quantiferon-CMV assays positively correlated with each other and negatively correlated with CMV DNAemia in a significant way (P < 0.05). During the antiviral prophylaxis, all 20 D⁺/R⁻ KTRs we examined displayed undetectable Quantiferon-CMV and ELISPOT results, and there was no evidence of CMV seroconversion. The receiving operator curve (ROC) statistical analysis revealed similar specificities and sensitivities in predicting detectable viremia (areas under the curve [AUC], 0.66 and 0.62 for Quantiferon-CMV and ELISPOT, respectively). ELISPOT and Quantiferon-CMV values of >150 spots/200,000 peripheral blood mononuclear cells (PBMCs) and >1 to 6 IU gamma interferon (IFN-γ) were associated with protection from CMV infection (odds ratios [OR], 5 and 8.75, respectively). In transplant recipients, the two tests displayed similar abilities for predicting CMV infection. Both the ELISPOT and Quantiferon-CMV assays require several ameliorations to avoid false-negative results.     

Stimulatory effects of ghrelin on circulating somatostatin and pancreatic polypeptide levels

Ghrelin, the recently identified endogenous ligand of the GH secretagogue receptor, is a gut-brain peptide with endocrine, orexigenic, and gastrointestinal effects. In rodents it increases circulating gastrin and insulin levels, whereas in man it appears to decrease insulin secretion despite a rise in blood glucose levels. The aim of the present study was to evaluate the effects of ghrelin administration on total circulating somatostatin (SS), pancreatic polypeptide (PP), and gastrin levels compared with those elicited on insulin, glucose, and GH. Eight healthy volunteers of normal weight (four women and four men) were injected with 3.3 microg/kg ghrelin or saline after an overnight fast on 2 different days. Blood was taken every 15 min for 1 h and then every 30 min for 2 h. As expected, ghrelin injection elicited a prompt GH and glucose increase with a peak at 30 min and an insulin decrease with a nadir at 60 min. Gastrin concentrations were not modified, whereas significant rises were observed in both SS (in a biphasic pattern with peaks at 15 and 120 min) and PP (which increased promptly with a peak at 15 min). A significant negative correlation was found between SS (first peak) and insulin changes (r = -0.86; P < 0.01). In conclusion, this study clearly demonstrates that ghrelin stimulates SS and PP release in man. Although the underlying mechanisms and biological significance of these pharmacological effects remain to be elucidated, a causal relationship between the SS increase and the insulin changes may be hypothesized. Finally, these findings strongly support ghrelin's postulated role in linking the endocrine control of energy balance and growth with the regulation of gastrointestinal functions.     

Influence of caloric intake on gastric emptying of solids assessed by 13C-octanoic acid breath test

BACKGROUND: The 13C-octanoic breath test (13C-OBT), a recently developed technique to evaluate gastric emptying of solids, has been validated in comparison to scintigraphy with low caloric meals (250 kcal). However, there is consensus that for clinical studies total caloric load should be in excess of 300 kcal, but studies comparing 13C-OBT results after low and medium caloric meals are lacking. METHODS: Ten healthy subjects were given a 250-kcal and a 550-kcal meal in randomized order. Gastric emptying was assessed simultaneously by ultrasonography and 13C-OBT. Breath samples were taken according to both classic (21 samples over 5 h) and simplified (11 samples) schedules. RESULTS: Increasing the meal energy content resulted in significantly longer half emptying time (T(1/2)) estimates by both ultrasonography (P < 0.01, Wilcoxon test) and 13C-OBT (P < 0.05). T(1/2) estimates by the two methods significantly correlated for both the 250 (r(s) = 0.733, P = 0.018) and the 550 (r(s) = 0.637, P = 0.035) kcal meal. However, differences between T(1/2) estimates by 13C-OBT and ultrasonography were greater after the 550- than the 250-kcal meal (median 172.5 versus 76.5 min, P < 0.05). Interindividual variability was also 2-fold greater for indexes estimated by 13C-OBT with the 550-kcal meal compared with the 250-kcal meal. For both meals 13C-OBT yielded similar results with the classic and simplified schedules. CONCLUSIONS: In healthy subjects caloric intake is a major determinant of gastric emptying rate. However, after a medium caloric meal 13C-OBT shows some inaccuracy, which raises questions about its routine clinical application. Nevertheless, when using 13C-OBT one must take into account that the simplified schedule is just as effective as the classic one, and is far lower in cost.     

Cerebellar degeneration and hearing loss in a patient with idiopathic myenteric ganglionitis

A 35-year-old male with an 11-year history of intestinal pseudo-obstruction associated with an idiopathic inflammatory insult of the myenteric plexus and the presence of circulating anti-Hu antibodies developed a neurological syndrome characterized by bilateral hearing loss, deteriorating balance, an unsteady gait and difficulty in estimating distances. A similar neurological syndrome has previously been described in older patients among the paraneoplasic syndromes associated with small-cell lung carcinoma and the presence of circulating anti-Hu antibodies, but never in the rare cancer-free patients with anti-Hu-associated chronic idiopathic intestinal pseudo-obstruction. The patient underwent a steroid treatment. No further episodes of functional intestinal obstruction were observed and, after an initial improvement, the neurological symptoms stabilized, leaving a permanent reduction in hearing function and an unsteady gait. The case shows that an idiopathic inflammatory insult of the myenteric plexus may precede (and perhaps lead to) central nervous system impairment in patients with anti-Hu-associated chronic idiopathic intestinal pseudo-obstruction.     

Plasma chromogranin A in patients with autoimmune chronic atrophic gastritis, enterochromaffin-like cell lesions and gastric carcinoids

OBJECTIVE: In atrophic body gastritis (ABG) chronic hypergastrinaemia stimulates enterochromaffin-like (ECL) cell proliferation with development of cell hyperplasia, dysplasia and possibly type-1 gastric carcinoids. As circulating chromogranin A (CgA) levels are a marker of neuroendocrine tumours, we evaluated the clinical usefulness of CgA assay in ABG patients to detect those with carcinoids. DESIGN AND METHODS: Plasma CgA levels were measured using a commercial ELISA in 45 healthy volunteers, nine patients with type-1 gastric carcinoids and 43 consecutive ABG patients (21 without and 22 with ECL cell hyperplasia/dysplasia). RESULTS: CgA levels were significantly higher in ABG patients with and without gastric carcinoids than in healthy subjects (P < 0.001). The highest values occurred in patients with carcinoids (median (interquartile range): 58.1 (44.5-65.3) U/l) and with ECL cell hyperplasia/dysplasia (35.5 (31.8-48.65) U/l) but there were no significant differences in CgA among the various subgroups of ABG patients classified according to ECL cell status. Nevertheless, in ABG patients without carcinoids CgA values correlated with the presence and severity of ECL cell lesions (r(s) = 0.428, P < 0.01). The sensitivity and specificity of the CgA assay in identifying patients with carcinoids were 100 and 23% respectively. CONCLUSIONS: CgA plasma levels reflect the histological degree of ECL cell lesions in patients with ABG but the assay specificity is too low to detect among these patients those with gastric carcinoids.     

High frequency of anti-endomysial reactivity in candidates to heart transplant

BACKGROUND: A possible link between coeliac disease and dilated cardiomyopathy has recently been suggested. AIMS:. To assess the frequency of anti-endomysial antibodies, the marker for coeliac disease, in patients with different forms of heart failure, and to establish the clinical features of those endomysial antibody positive. SUBJECTS AND METHODS:. A total of 642 consecutive patients entering the waiting list for heart transplantation from 1995 through 1997 were studied. The prevalence of endomysial IgA antibodies, determined by indirect immunofluorescence, was compared to that observed in three surveys conducted in the Italian general population. RESULTS: Of the 642 patients, 12 (1.9%; 95% confidence interval 0.97-3.2) resulted endomysial antibody positive, versus 34/9,720 healthy controls (0.35%; 95% confidence interval, 0.23-0.47), accounting for a relative risk of 5.3 (95% confidence interval, 2.8-10.3). Anti-endomysial antibodies were found in 6/275 patients with dilated cardiomyopathy and 6/367 with other forms of heart failure (2.2% versus 1.6%; 95% confidence interval 0.8-4.7 and 0.6-3.5), with no statistical difference. The 12 endomysial antibody positive patients were leaner (body mass index, 22.0 +/- 1.9 vs 24.2 +/- 3. 1, p<0. 05) than 36 seronegative patients matched for baseline demographics and aetiology of cardiomyopathy No differences were observed as regards clinical, biochemical and echocardiographic features, mortality in waiting list and 2-year post-transplant survival. CONCLUSIONS: Patients with end-stage heart failure are at increased risk for coeliac disease as compared to the general population.