The presence of an intrahepatic cytotoxic T lymphocyte response is associated with low viral load in patients with chronic hepatitis C virus infection

BACKGROUND/AIMS: The role of cytotoxic T lymphocytes (CTL) in limiting viral replication and producing hepatocellular injury in patients with chronic hepatitis C virus (HCV) infection is controversial. METHODS: Intrahepatic and peripheral blood HCV-specific CTL activity against the entire HCV polyprotein was assessed in 26 patients. CTL responses were assessed after effector lymphocytes were re-stimulated for 6 days in vitro using HCV-vaccinia virus-infected autologous cells expressing HCV antigens. Serum and hepatic viral loads were measured and immunohistochemistry for CD3 and CD8 was performed to localise and enumerate effector cells in liver. RESULTS: A positive CTL response was detected in 39/52 (75%) of assays conducted with intrahepatic mononuclear cells and 21/52 (40%) of peripheral blood assays (P<0.001). The presence of an intrahepatic CTL response was associated with low hepatic viral load (P=0.004). Hepatic lobular infiltration by CD8(+)T cells correlated weakly with serum alanine aminotransferase levels (r=0.42, P=0.04) and no relationship was demonstrated between CTL activity and histological evidence of liver damage. CONCLUSIONS: HCV-specific CTL activity is found more commonly in liver than in blood. An inverse relationship between CTL responses and viral load supports the hypothesis that HCV-specific CTL limit viral replication in patients with chronic HCV infection.     

Phospholamban gene ablation improves calcium transients but not cardiac function in a heart failure model

Decreased amplitude and slower kinetics of cardiomyocyte intracellular calcium (Ca(i)(2+)) transients may underlie the diminished cardiac function observed in heart failure. These alterations occur in humans and animals with heart failure, including the TNF1.6 mouse model, in which heart failure arises from cardiac-specific overexpression of tumor necrosis factor alpha (TNF alpha). OBJECTIVE: Since ablation of phospholamban expression (PLBKO) removes inhibition of the sarcoplasmic reticulum (SR) Ca(2+) pump, enhances SR Ca(2+) uptake and increases contractility, we assessed whether ablation of phospholamban expression could improve cardiac function, limit remodeling, and improve survival in the TNF1.6 model of heart failure. METHODS: We bred PLBKO with TNF1.6 mice and characterized the progeny for survival, cardiac function (echocardiography), cardiac remodeling (hypertrophy, dilation, fibrosis), and Ca(2+)(i) transients and contractile function of isolated cardiomyocytes. RESULTS: PLB ablation did not improve survival, cardiac function, or limit cardiac chamber dilation and hypertrophy in TNF1.6 mice (TKO mice). However, contractile function and Ca(2+)(i) transients (amplitude and kinetics) of isolated TKO cardiomyocytes were markedly enhanced. This discordance between unimproved cardiac function, and enhanced Ca(2+)(i) cycling and cardiomyocyte contractile parameters may arise from a continued overexpression of collagen and decreased expression of gap junction proteins (connexin 43) in response to chronic TNF alpha stimulation. CONCLUSIONS: Enhancement of intrinsic cardiomyocyte Ca(2+)(i) cycling and contractile function may not be sufficient to overcome several parallel pathophysiologic processes present in the failing heart.     

Familial leucine-sensitive hypoglycemia of infancy due to a dominant mutation of the beta-cell sulfonylurea receptor

Familial leucine-sensitive hypoglycemia of infancy was described in 1956 as a condition in which symptomatic hypoglycemia was provoked by protein meals or the amino acid, leucine. The purpose of this study was to determine the genetic basis for hypoglycemia in a family diagnosed with leucine-sensitive hypoglycemia in 1960. Recently diagnosed family members showed a dominantly transmitted pattern of diazoxide-responsive hyperinsulinism (HI). However, they did not fit the characteristics of HI caused by glutamate dehydrogenase gene mutations, previously felt to explain leucine-sensitive hypoglycemia. Islet function was examined using acute insulin response (AIR) tests to calcium, leucine, glucose, and tolbutamide as well as oral protein tolerance tests. Five of five affected family members showed an abnormal positive calcium AIR, and two of five showed a positive leucine AIR. Protein-induced hypoglycemia was demonstrated in five of six affected subjects. Mutation analysis of four known HI genes (sulfonylurea receptor 1, Kir6.2, glutamate dehydrogenase, and glucokinase) in family members identified an R1353H missense mutation in exon 33 of SUR1. (86)Rb(+) efflux and electrophysiological studies of R1353H SUR1 coexpressed with wild-type Kir6.2 in COSm6 cells demonstrated partially impaired ATP-dependent potassium channel function. Leucine-sensitive hypoglycemia in this family was found to result from a dominantly expressed SUR1 mutation.     

Evidence for fusion between cardiac and skeletal muscle cells

Cardiomyoplasty with skeletal myoblasts may benefit cardiac function after infarction. Recent reports indicate that adult stem cells can fuse with other cell types. Because myoblasts are "fusigenic" cells by nature, we hypothesized they might be particularly likely to fuse with cardiomyocytes. To test this, neonatal rat cardiomyocytes labeled with LacZ and green fluorescent protein (GFP) were cocultured with unlabeled C2C12 myoblasts. After 3 days, we observed a small population of skeletal myotubes that expressed LacZ and GFP, indicating cell fusion. To test whether such fusion occurred in vivo, LacZ-expressing C2C12 myoblasts were grafted into normal nude mouse hearts. At 2 weeks after grafting, cells at the graft-host interface expressed both LacZ and cardiac-specific myosin light chain 2v (MLC2v). To test more definitively whether fusion between skeletal and cardiac muscle could occur, we used a Cre/lox reporter system that activated LacZ only upon cell fusion. When neonatal cardiomyocytes from -myosin heavy chain promoter (-MHC)-Cre mice were cocultured with myoblasts from floxed-lacZ reporter mice, LacZ was activated in a subset of cells, indicating cell fusion occurred in vitro. Finally, we grafted the floxed-lacZ myoblasts into normal hearts of -MHC-Cre+ and -MHC-Cre- mice (n=5 each). Hearts analyzed at 4 days and 1 week after transplantation demonstrated activation of LacZ when the skeletal muscle cells were implanted into hearts of -MHC-Cre+ mice, but not after implantation into -MHC-Cre- mice. These data indicate that skeletal muscle cell grafting gives rise to a subpopulation of skeletal-cardiac hybrid cells with a currently unknown phenotype. The full text of this article is available online at http://circres.ahajournals.org.     

Proliferation of rat granulosa cells during the periovulatory interval

Granulosa cell proliferation during luteinization and terminal differentiation has historically been assumed to decline rapidly after an ovulatory stimulus. In contrast, terminal differentiation in other cell types has recently been associated with a transient increase in proliferation, suggesting that this may occur in the ovarian follicle. The goal of the current study was to test the hypothesis that an ovulatory stimulus to rats results in additional granulosa cell proliferation before cell cycle arrest. Immature rats were given a single injection of pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) to initiate periovulatory events. The proportion of granulosa cells in S phase did not change until 12 h after hCG, although the majority of the post-hCG proliferation was localized to cumulus granulosa cells for up to 10 h after hCG. The expression of cyclin D2 mRNA did not decline until 12 h after hCG, although both cyclin-dependent kinase (Cdk)4 and Cdk6 mRNA increased at 6 h. Protein levels of cyclin D2 and Cdk4 did not change as a result of hCG, whereas cyclin E increased 6 h after hCG. Kinase activity of Cdk2 dropped markedly by 4 h after hCG, but a slight increase in activity was evident 6-8 h after hCG. These data suggest that cumulus granulosa cells continue to proliferate for up to 10 h after an ovulatory stimulus, possibly via cyclin E/Cdk2. It is concluded that proliferation is maintained in granulosa cells in the proximity of the oocyte during luteinization of the rat follicle.     

Interleukin 2 production in bone marrow of normal individuals and patients associated with B-cell chronic lymphocytic leukaemia

T-cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL) have abnormal T4/T8 ratios and functions. Previously, we demonstrated that peripheral blood (PB) mononuclear cells from B-CLL patients secrete significant amounts of interleukin 2 (IL2) with an apparent dysregulation of accessory cells controlling this production. In this study, IL2 production was investigated in PB and in bone marrow (BM) from patients with previously untreated B-CLL, mostly in early stages of the disease, and compared to normal donors. A significant secretion was observed in both PB and BM from B-CLL patients after stimulation by phytohaemagglutinin (PHA), with lower amounts in patients with nodular involvement of BM, as compared to interstitial or diffuse involvements. To explore the role of accessory cells in controlling IL2 production, we added phorbol ester or indomethacin to the culture system or irradiated the cells before culture. Phorbol ester significantly increased the IL2 secretion in both the PB and the BM of B-CLL patients. The irradiation or the addition of indomethacin did not enhance the IL2 production in PB from B-CLL patients. However, IL2 secretion increased in the BM cells from B-CLL patients after addition of indomethacin or prior irradiation, in a similar way to that observed in PB and BM of normal controls, suggesting an apparent normal control of the IL2 production in BM from B-CLL patients. In normal controls, we demonstrated that IL2 secretion per T-cell from BM was 5.4-fold greater than that from normal PB, suggesting a very efficient role of accessory cells controlling IL2 production in normal BM.     

The role of non-esterified fatty acids in the deterioration of glucose tolerance in Caucasian subjects: results of the Paris Prospective Study

Summary Although an increased plasma non-esterified fatty acid (NEFA) concentration has been shown to increase insulin resistance (Randle cycle), decrease insulin secretion and increase hepatic gluconeogenesis, the effect of NEFA on the deterioration of glucose tolerance has not been studied prospectively in Caucasian subjects. Therefore, we investigated whether plasma NEFA may be regarded as predictors of deterioration of glucose tolerance in subjects with normal (NGT, n = 3671) or impaired (IGT, n = 418) glucose tolerance who were participants in the Paris Prospective study. The subjects were first examined between 1967 and 1972 and underwent two 75-g oral glucose tolerance tests 2 years apart with measurements of plasma glucose, insulin and NEFA concentrations. Glucose tolerance deteriorated from NGT to IGT or non-insulin-dependent diabetes (NIDDM) in 177 subjects and from IGT to NIDDM in 32 subjects. In multivariate analysis, high fasting plasma NEFA in NGT subjects and high 2-h plasma NEFA and low 2-h plasma insulin concentrations in IGT subjects were significant independent predictors of deterioration along with older age, high fasting and 2-h plasma glucose concentrations and high iliac to thigh ratio. When subjects were divided by tertiles of plasma NEFA concentration at baseline, there was an increase in 2-h glucose concentration with increasing NEFA in the subjects who did not deteriorate, but no effect of plasma NEFA in those who deteriorated. In subjects with IGT who deteriorated compared with those who did not 2-h plasma insulin concentration was lower but there was no evidence that this resulted from an effect of plasma NEFA. Our data suggest that a high plasma NEFA concentration is a risk marker for deterioration of glucose tolerance independent of the insulin resistance or the insulin secretion defect that characterize subjects at risk for NIDDM. [Diabetologia (1997) 40: 1101–1106] Received: 11 March 1997 and in revised form: 20 May 1997     

Pulmonary complications in bone marrow transplantation: a practical approach to diagnosis and treatment

Pulmonary complications occur in 40% to 60% of recipients of bone marrow trans-plants, account for more than 90% of mortality, and develop during identifiable phases. Phase 1 (Days 1-30) includes pulmonary edema; diffuse alveolar hemorrhage; and various bacterial, fungal, and viral infections; Phase 2 (Days 31-100) usually requires a distinction between cytomegalovirus pneumonitis and idiopathic pneumonia syndrome; and Phase 3 (Day 100+) includes complications that are due to chronic graft-versus-host disease and associated bronchiolitis obliterans. The spectrum of pulmonary complications has been influenced by changes in transplantation technique, prophylactic treatment for infections, and the use of new chemotherapeutic agents that contribute to lung injury. Nonetheless, infections remain a leading cause of morbidity and mortality. The most serious complications result in respiratory failure, for which the prognosis has not improved significantly over the last 2 decades. In this article, we describe our algorithmic approach to the diagnosis and management of these complications.     

Rapid and specific detection of clinically significant haemoglobinopathies using electrospray mass spectrometry-mass spectrometry

Increasing demand for population screening for the haemoglobinopathies gives rise to a requirement for high throughput systems, which allow for cost effective, rapid, sensitive and specific screening of clinically significant haemoglobins. We have developed a practical and efficient approach using tryptic digestion and electrospray triple quadrupole mass spectrometry-mass spectrometry (MSMS) in multiple reaction monitoring acquisition mode for the identification of the clinically important haemoglobin variants, S, C, DPunjab, OArab, and E. A total of 200 blood samples, comprising 52 haemoglobin AA, 57 AS (sickle cell trait), 44 AC (C trait), 16 SC (SC disease), 14 SS (sickle cell disease), 10 AE (E trait), 2 ADPunjab (DPunjab trait) and 1 each of AOArab (OArab trait), CC (C disease), DPunjabDPunjab (DPunjab disease), OArabOArab (OArab disease), and EE (E disease), have been analysed in parallel with existing phenotype and molecular methods. All haemoglobin variants were correctly identified by MSMS, with no false positives or false negatives. The system detects both heterozygotes and homozygotes and has potential applications in neonatal and antenatal screening.     

IL-1 regulates in vivo C-X-C chemokine induction and neutrophil sequestration following endotoxemia

The influx of neutrophils into tissues in response to inflammatory stimuli involves C-X-C chemokines. Interleukin-1 (IL-1) stimulates chemokine production in vitro, but its role in vivo on chemokine production is not as clearly understood. We hypothesized that IL-1 mediates in vivo tissue C-X-C chemokine production induced by systemic lipopolysaccharide (LPS). IL-1 activity was blocked by IL-1 receptor antagonist (IL-1Ra). Rats were injected with Salmonella typhi LPS (0.5 mg/kg) with and without prior administration of IL-1Ra. Cytokine-induced neutrophil chemoattractant-1 (CINC-1) and macrophage inflammatory protein-2 (MIP-2) protein and mRNA levels, tissue neutrophil accumulation, and indices of organ injury were measured. LPS administration resulted in increased plasma, lung, and liver IL-1beta that was decreased by Il-1Ra. LPS also induced an increase in plasma, lung, and liver CINC-1 and MIP-2 protein and mRNA. However, IL-1Ra had no effect on LPS-induced plasma or lung tissue CINC-1 levels. In contrast, IL-1Ra pretreatment did significantly decrease CINC-1 protein expression in the liver (45% decrease) and MIP-2 protein expression in plasma (100% decrease), lung (72% decrease) and liver (100% decrease) compared to LPS- treated controls. Steady-state mRNA levels by Northern blot analysis of both CINC-1 and MIP-2 in lung and liver were similar to the protein findings. Pretreatment with IL-1Ra also resulted in a 47% and 59% decrease in lung and liver neutrophil accumulation, respectively, following LPS. In addition, indices of both lung and liver injury were decreased in animals pretreated with IL-1Ra. In summary, LPS induces IL-1beta and MIP-2 expression in the lung and liver, both of which are IL-1 dependent. Although lung neutrophil accumulation in both lung and liver after LPS is also IL-1 mediated, lung CINC-1 levels were unaffected by IL-1Ra. These data suggest that IL-1 regulates tissue chemokine expression and neutrophil accumulation after LPS.