Regulation of human erythrocyte metabolism by insulin: cellular distribution of 6-phosphofructo-1-kinase and its implication for red blood cell function

Human erythrocytes are highly specialized cells whose function is oxygen transport. These cells' sole metabolic source of energy is the fermentation of glucose via glycolysis. They contain an active insulin receptor and respond to insulin by increasing phosphorylation of tyrosine residues in several proteins. However, no metabolic effects have yet been associated with activation of this receptor in human erythrocytes. Here, we show that insulin increases the rate of glycolysis in human erythrocytes. Lactate production increased 56 and 173% in the presence of 10 and 100 nM insulin, respectively. A higher insulin concentration (1000 nM) partially reversed the stimulation of glycolysis. These effects occur through activation of the key glycolytic enzyme 6-phosphofructo-1-kinase, which exhibits the same pattern of modulation by insulin as seen for glycolytic flux. This modulation also occurs physiologically since ex vivo experiments revealed 50% stimulation of 6-phosphofructo-1-kinase (PFK) activity following a high carbohydrate meal. Insulin increases phosphorylation of PFK and redistributes the enzyme in red blood cells, causing it to detach from the erythrocyte membrane: upon insulin stimulation, the amount of enzyme associated with the plasma decreases by 86%. Detachment is a common mechanism of enzyme activation. As a consequence, insulin prevents up to 68% of red cells hemolysis. These results show that insulin regulates erythrocyte glycolysis and viability and suggest that this regulation is associated to other erythrocyte functions such as oxygen transport. Finally, we suggest that this regulatory mechanism might be compromised in patients with diabetes mellitus.     

Cryptogenic chronic liver disease and hepatitis C virus infection in children.

The clinical features of 'cryptogenic' chronic liver disease and the prevalence of antibody to hepatitis C virus (HCV) in serum have been investigated in 33 Italian children (mean age 5 years). The diagnosis was based on the persistence of increased alanineaminotransferase values for longer than 6 months after the exclusion of biliary diseases, of extra-hepatic causes of hypertransaminasemia, of infection with known hepatotropic viruses and of autoimmune or metabolic disorders. Five patients had been transfused early in life, three had undergone surgery and one girl's mother had had acute non-A, non-B hepatitis during pregnancy. The remaining patients had no history of overt parenteral exposure. At presentation only 11 patients were symptomatic, the others had been referred after a check-up for intercurrent diseases. Liver histology performed in 21 cases showed persistent or mild active hepatitis in 18 cases and severe hepatitis or cirrhosis in three cases. Anti-HCV antibodies were found in 48% of the cases, including 88% with obvious exposure and 33% of the remaining cases. During a mean follow-up period of 5 years (range 1-14 years) only 11% of the cases achieved sustained biochemical remission, although none developed signs of liver failure. There was no significant difference in the clinical features and outcome of the disease between anti-HCV-positive and -negative patients. The results of this study suggest that HCV is implicated in most cases of 'cryptogenic' chronic liver disease observed in Italian children with a history of parenteral exposure and in at least one-third of the cases without overt exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary sclerosing cholangitis (PSC): clinical,laboratory and survival analysis in children and adults

Abstract: Background: Primary sclerosing cholangitis (PSC) is an uncommon disorder, rarely diagnosed in children, moreover, data on its natural history and survival are still lacking. Aim: The study was undertaken to compare clinical, laboratory and survival rates in two series of PSC: one in a pediatric group (group A) and the other in an adult population (group B). Methods: Group A included 9 patients (5 males, 4 females, mean age 10 yrs, range 7–15); group B included 28 patients (19 males, 9 females, mean age 32 years, range 19–60). The mean follow-up was 5.2 years in group A and 6.9 years in group B (range 1–14 years). ERCP and colonos-copy were performed in each case. Survival was analyzed using the Kaplan-Meier method. Results: At presentation children showed significantly higher levels of IgG and AST compared to adults (p<0.05); moreover, interface hepatitis occurred in 50% of children and in 14.2% in adults (p= ns). During follow-up the following major events occurred: oesophageal bleeding (n=2) in group A; progressive liver failure (n=6), cholangiocarcinoma (n=3), colonic cancer (n=1) in group B. Liver transplantation (OLTx) was performed in 4 adults (one died after a retransplantation). No deaths were observed in children. The Kaplan-Meier curve in adults shows a 65% rate of survival at 10 years. Conclusions: The present findings on PSC suggest a more severe activity of the disease in children than in adults at presentation; nonetheless, the prognosis seems to be better in children than in adults. The Mayo score prognostic index does not predict the development of liver/colonic cancer. A poor outcome (defined as death or being listed for OLTx) only occurred in adults.     

Transmission of hepatitis C virus from infected mother to offspring during subsequent pregnancies

BACKGROUND: Several studies have demonstrated that hepatitis C virus (HCV) may be transmitted from mother to offspring. To date, however, little is known about the risk of vertical transmission during subsequent pregnancies. The purpose of this study was to evaluate the risk of vertical HCV transmission in offspring in subsequent pregnancies of HCV infected women. METHODS: In a multicenter study, two groups of index cases were selected. Group 1 included 75 children investigated for HCV infection during prospective studies of vertical transmission. Group 2 included children born to HCV-infected mothers and found to be HCV infected, independent of studies on vertical transmission. All children in the index cases had one or more siblings. Anti-HCV, HCV-RNA (determined by polymerase chain reaction), and HCV genotype were evaluated in all the infected children, their mothers, and siblings. RESULTS: The results indicate that a mother who has already delivered an HCV-infected baby is not at greater risk of infecting her second child. Duration of maternal infection does not seem to be a risk factor in offspring infection, because HCV infection is equally distributed among first-born infants and infants of subsequent births. Because clustering of HCV infection among siblings appeared to be rare in this study, data also indirectly confirm that the risk of horizontal transmission of HCV among siblings is low. CONCLUSION: For practical purposes, the current observations indicate that mothers who have already delivered an HCV-infected child can be advised that this event does not increase the probability of infecting the second child.     

Isolated hepatocyte transplantation for Crigler-Najjar syndrome type 1

Crigler-Najjar syndrome type 1 (CN1) is an inherited disorder characterized by the absence of hepatic uridine diphosphoglucuronate glucuronosyltransferase (UDPGT), the enzyme responsible for the conjugation and excretion of bilirubin. We performed allogenic hepatocyte transplantation (AHT) in a child with CN1, aiming to improve bilirubin glucuronidation in this condition. A 9-year-old boy with CN1 was prepared with plasmapheresis and immunosuppression with prednisolone and tacrolimus. When a graft was made available, 7.5 x 10(9) hepatocytes were isolated and infused into the portal vein percutaneously. After 2 weeks phenobarbitone was added to promote the enzymatic activity of UDPGT of the transplanted hepatocytes. Nocturnal phototherapy was continued throughout the studied period. Total bilirubin was considered a reliable marker of allogenic cell function. There was no significant variation of vital signs nor complications during the infusion. Mean +/- SD bilirubin level was 530 +/- 38 micromol/L before and 359 +/- 46 micromol/L after AHT (t-test, p < 0.001). However, the introduction of phenobarbitone was followed by a drop of tacrolimus level with increase of alanine aminotransferase (ALT) and increase of bilirubin. After standard treatment of cellular rejection bilirubin fell again but from then on it was maintained at a greater level. After discharge the patient experienced a further increase of bilirubin that returned to predischarge levels after readmission to the hospital. This was interpreted as poor compliance with phototherapy. Only partial correction of clinical jaundice and the poor tolerability to nocturnal phototherapy led the parents to refuse further hepatocyte infusions and request an orthotopic liver transplant. After 24 months the child is well, with good liver function on tacrolimus and prednisolone-based immunosuppression. Isolated AHT, though effective and safe, is not sufficient to correct CN1. Maintenance of adequate immunosuppression and family compliance are the main factors hampering the success of this procedure.     

Hepatitis C virus infection associated with liver-kidney microsomal antibody type 1 (LKM1) autoantibodies in children

OBJECTIVE: To evaluate the clinical pattern and evolution of chronic hepatitis C in children with liver/kidney microsomal antibody type 1 autoantibodies (LKM1). STUDY DESIGN: A multicenter, retrospective study, including the following groups of children with hepatitis C virus infection: (1). 21 consecutive LKM1-positive patients, (2). 42 age- and sex- matched LKM1-negative patients, and (3). 4 interferon-induced LKM1-positive cases. LKM1 reactivity to human microsomes and recombinant cytochrome P450IID6 (CYP2D6) was assayed by immunoblotting. RESULTS: Clinical and biochemical features overlapped in LKM1-positive and LKM1-negative children, but a fibrosis score >3 (range 0-6) was significantly more frequent (P =.04) in the former. Reactivity to microsomal protein and CYP2D6 was significantly (P =.02) associated with LKM1 titers >or=1:320 and was found in 39% of patients, including severe cases and both children (of 4 treated) who achieved a sustained alanine aminotransferase (ALT) normalization after steroid treatment. Five of 7 LKM1-positive children treated with interferon had an ALT exacerbation. CONCLUSIONS: LKM1-positive hepatitis C in children is characterized by a wide spectrum of biochemical, serologic, and histologic features. Whether autoimmunity may contribute to liver damage in a subgroup of patients with more severe liver disease, high LKM1 titers, and reactivity to CYP2D6 is a question deserving further investigation.     

Clinical features and progression of perinatally acquired hepatitis C virus infection

The purpose of this prospective-retrospective study was to provide information about the clinical features and progression of hepatitis C virus (HCV) infection transmitted perinatally. Seventy children born to HCV infected woman were enrolled consecutively in five European centers between 1990 and 1999, provided they had HCV RNA in the serum during the first year of life and/or were still anti-HCV positive at 18 months. Sixty-two infants were followed up to 24 months of age or more (range, 24 months-11 years; average, 4.8 +/- 2.3 years). A wide range of ALT elevation was observed in 93% of the infants in the first year of life. During the follow-up, a sustained ALT normalization with loss of HCV RNA was seen in 12/62 (19%) of the children within 30 months of life; 66% of the infants had developed an ALT peak greater than 5x normal at onset (vs. 28% of children with persistent viremia; P < 0.05), and 50% had HCV genotype 3 (vs. 17% of viremic children). Conversely the cumulative probability of chronic progression was 81%. Chronic infection was asymptomatic and liver disease was mild in all 11 children who underwent a biopsy. In conclusion the early stage of acquired perinatally HCV infection is characterized by a wide range of ALT abnormalities, suggesting the interaction of multiple host and virus factors. The chronic progression rate of infection is high, but the associated liver disease is usually mild. High ALT levels at onset seem to offer greater opportunity of biochemical remission and loss of viremia during follow-up.     

Unbalanced effects of dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding

We compared the anticoagulant, antithrombotic and bleeding effects of highly sulfated dermatan sulfates from invertebrates and their mammalian counterpart. An invertebrate dermatan sulfate containing 2-O-sulfated alpha-L-iduronic acid and 4-O-sulfated N-acetyl-beta-D-galactosamine residues is a potent anticoagulant due to a high heparin cofactor II activity. It inhibits thrombin due to the formation of a covalent complex with heparin cofactor II, as in the case of mammalian dermatan sulfate, but the effect occurs at lower concentrations for the invertebrate polysaccharide. Surprisingly, the invertebrate dermatan sulfate has a lower potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the mammalian dermatan sulfate. In contrast, another invertebrate dermatan sulfate, also enriched in 2-O-sulfated alpha-L-iduronic acid, but in this case sulfated at O-6 position of the N-acetyl-beta-D-galactosamine units, has no in vitro or in vivo anticoagulant activity, does not prevent thrombus formation but shows a bleeding effect similar to the mammalian glycosaminoglycan. Overall, these results demonstrate unbalanced effects of dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding, and raise interesting questions concerning the relationship among these three biological actions of sulfated polysaccharides.     

Androgen deprivation, estrogen treatment and vascular function in male rat aorta

The beneficial effects of estrogen on arterial function in women are well established, whereas studies concerning the vascular role of androgens have produced conflicting results. In the present study, we examined the effects of androgen deprivation and of estrogen treatment on vascular responses in male rats. Vascular reactivity was studied in aortic rings excised from intact and castrated rats, which had been implanted with capsules containing either 17beta-estradiol (E2) or its vehicle for 5 days. Contractile responses to noradrenaline were potentiated by castration and by E2 treatment. Concentration-response curves for N-methyl-L-arginine and superoxide dismutase indicated that the tone-related release of NO increased in tissues from castrated, compared with intact rats, but was not affected by E2 treatment. Endothelium-dependent relaxation elicited by carbachol and histamine were not altered by castration and were attenuated by E2 in preparations from intact, but not from castrated rats. Moreover, aortic prostacyclin release dropped by about 40% after E2 treatment in tissues from both intact and castrated animals. Similarly, smooth muscle sensitivity to NO significantly decreased following castration and E2 treatment, as assessed by responses to sodium nitroprusside. Finally, no differences among groups were detected in platelet thromboxane A2 production. Thus, vascular responses in male rats were not improved by androgen deprivation alone or by E2 treatment, whose effects differed in the presence or absence of androgens. These findings provide evidence for the gender specificity of the vascular effects of estrogen and may be consistent with a beneficial role of physiologic levels of male sex hormones in arterial function.