A novel binding site for the native hepatic acute-phase protein alpha1-antitrypsin expressed on the human hepatoma cell line HepG 2 and intestinal cell line Caco 2

AIMS/BACKGROUND: alpha1-antitrypsin (alpha1-AT) is a hepatic acute phase protein which predominantly inhibits neutrophil elastase. Besides this major function, we have also previously shown that alpha1-AT markedly increased H-ferritin mRNA expression and ferritin synthesis in the human hepatoma cell line HepG 2. These actions suggest that alpha1-AT might interact with HepG 2 cells via a specific cell surface binding site. METHODS AND RESULTS: Using radio-labelled native alpha1-AT, we observed saturable binding to HepG 2 cells with a dissociation constant (Kd) of 63.3+/-6.9 nM and a maximal density of binding sites (Bmax) of 0.34+/-0.05 pmol/10(6) cells equivalent to 195,800+/-29,200 sites/cell. The binding of [125I]alpha1-AT was time dependent with a calculated association rate constant of 9.22+/-1.84x10(4)xM(-1)xmin(-1). Binding was highly specific since other acute phase proteins or protease inhibitors failed to block binding. Although alpha1-AT-trypsin, alpha1-AT-elastase and the pentapeptide FVYLI, the minimal binding sequence for the SEC receptor, increased [125I]alpha1-AT binding, in long term experiments these complexes failed to influence the number of alpha1-AT binding sites. Specific, saturable binding of [125I]alpha1-AT was also found on the human intestinal epithelial Caco 2 cells, but not on fibroblast or leukaemic cell lines. CONCLUSION: These experiments demonstrate a specific, high affinity binding site for native alpha1-AT on HepG 2 and Caco 2 cells, cell lines derived from tissues involved in the acute phase response.     

Effects of non‐cytotoxic drugs on the growth of multidrug‐resistance human gastric carcinoma cell line

OBJECTIVE: To investigate the effects of the non‐cytotoxic drug (cyclooxygenase‐2 (COX‐2) inhibitor and octreotide) on growth of the multidrug‐resistant human gastric carcinoma cell line SGC‐7901/ADR. METHODS: The effects of non‐cytotoxic drug on the growth of SGC‐7901 and SGC‐7901/ADR cells were evaluated by ³H‐thymidine incorporation assay. The apoptosis of cells was measured by the TdT‐mediated dUTP nick end‐labeling assay (TUNEL) and flow cytometric assay. Western blotting was used to analysis the expression of cyclooxygenase (COX‐2) protein in SGC‐7901 cells and SGC‐7901/ADR cells and P‐glycoprotein (P‐gp) from SGC‐7901/ADR cells with variable treatments. Activator protein‐1 binding activity was examined by electrophoretic mobility shift assay. RESULTS: ³H‐thymidine incorporation into SGC‐7901/ADR cells treated with celecoxib was significantly lower than that of control group (471.3 ± 79.7 cpm vs 917.5 ± 130.8 cpm, P < 0.05). When combined with octreotide, celecoxib presented lower ³H‐thymidine incorporations than its used alone and decreased to 53.3% of that amount original. Either celecoxib or the combination group markedly induced apoptosis in SGC‐7901/ADR cells. COX‐2 protein in the SGC‐7901/ADR cells was higher than in that of the SGC‐7901 cells (1.543 ± 0.052 vs 0.564 ± 0.021, P < 0.05). The inhibition of P‐gp could be achieved with celecoxib alone and combination with octreotide (0.486 ± 0.012, 0.252 ± 0.014 vs 0.941 ± 0.033, P < 0.05). Moreover, AP‐1 binding activity could be suppressed by non‐cytotoxic drug and showed a synergistic effect. CONCLUSION: The combination of non‐cytotoxic drug significantly improved the inhibitive effects on the growth of multidrug‐resistant human gastric cancer cells.     

Effect of indomethacin on Bfl‐1, WISP‐1 and proliferating cell nuclear antigen in colon cancer cell line HCT116 cells

BACKGROUND: Non‐steroidal anti‐inflammatory drugs such as indomethacin can inhibit the growth of tumors through both the cyclooxygenase‐2 (COX‐2) dependant and COX‐2 independent pathways, but the exact mechanism has not yet been shown. In our previous study, COX‐2 independent proteins (Bfl‐1, WISP‐1 and proliferating cell nuclear antigens [PCNA]) in indomethacin‐treated colorectal cancer cells with the use of proteomics technology had been identified. OBJECTIVES: To study and confirm the effect of indomethacin on the expression of Bfl‐1, WISP‐1 and PCNA in human colon cancer line HCT116 cells and the COX‐2 independent tumor inhibiting pathway. METHODS: Human colon cancer cell line HCT116 cells were divided into a treatment with indomethacin (IC 50) group, and a treatment with dimethyl sulfoxide (DMSO) as a control group for 48 h. The expression of Bfl‐1, WISP‐1 and PCNA, mRNA and protein were determined by a real‐time quantitative PCR and Western blot, respectively. RESULTS: Indomethacin down‐regulated the expression of Bfl‐1, WISP‐1 and PCNA mRNA in vitro (9.53 ± 0.15 vs 27.87 ± 0.12, 7.37 ± 0.58 vs 20.17 ± 0.58, 5.17 ± 0.06 vs 0.87 ±  0.06). Indomethacin also down‐regulated the expression of Bfl‐1, WISP‐1 and PCNA protein (40.01 ± 1.61 vs 43.76 ± 1.63, 22.50 ± 1.17 vs 30.30 ± 1.55, 17.69 ± 1.18 vs 20.80 ± 1.08). CONCLUSIONS: Inducing apoptosis and inhibiting proliferation contribute to the anticancer activity of indomethacin via COX‐2 independent pathway of Bfl‐1, WISP‐1 and PCNA. This further confirms the results of our previous study.     

Expansion of circulating CD56bright natural killer cells in patients with JAK2‐positive chronic myeloproliferative neoplasms during treatment with interferon‐α

In recent years, major molecular remissions have been observed in patients with JAK2‐positive chronic myeloproliferative neoplasms (MPNs) after therapy with IFN‐α. IFN‐α is known to have altering effects on immune cells involved in immune surveillance and might consequently enhance anti‐tumor immune response against the JAK2‐mutated clone. The objective of this study was to investigate circulating levels and phenotype of natural killer cells in 29 JAK2‐positive MPN patients during IFN‐α treatment. Furthermore, functional studies of NK cells upon target‐cell recognition and cytokine stimulation were performed. The CD56^bright and CD56^dim NK cell subtypes display different properties in terms of cytokine production and cytotoxicity, respectively. Our results show a significant increase in the proportion of CD56^bright NK cells and a decreasing CD56^dim population during treatment with IFN‐α compared to patients that are untreated, treated with hydroxyurea and healthy controls, P < 0.0001. Furthermore, an overall increase in cytokine‐dependent (IL‐12 and IL‐15) IFN‐γ expression by CD56^dim NK cells during IFN‐α treatment was observed. In contrast, our data indicate a compromised NK cell response to target‐cell recognition during treatment with IFN‐α in four patients. We also report low levels of circulating NK cells in untreated patients compared to healthy donors, patients treated with hydroxyurea and IFN‐α, P = 0.02. Based on our findings, one might speculate whether treatment with IFN‐α skews the human NK population toward a helper type that may assist in CD8⁺ T cell priming in lymphoid tissues at the expense of their immediate cytotoxic functions in peripheral blood and tissues.     

Phytomelatonin receptor PMTR1‐mediated signaling regulates stomatal closure in Arabidopsis thaliana

Melatonin has been detected in plants in 1995; however, the function and signaling pathway of this putative phytohormone are largely undetermined due to a lack of knowledge about its receptor. Here, we discovered the first phytomelatonin receptor (CAND2/PMTR1) in Arabidopsis thaliana and found that melatonin governs the receptor‐dependent stomatal closure. The application of melatonin induced stomatal closure through the heterotrimeric G protein α subunit‐regulated H₂O₂ and Ca²⁺ signals. The Arabidopsis mutant lines lacking AtCand2 that encodes a candidate G protein‐coupled receptor were insensitive to melatonin‐induced stomatal closure. Accordingly, the melatonin‐induced H₂O₂ production and Ca²⁺ influx were completely abolished in cand2. CAND2 is a membrane protein that interacts with GPA1 and the expression of AtCand2 was tightly regulated by melatonin in various organs and guard cells. CAND2 showed saturable and specific ¹²⁵I‐melatonin binding, with apparent K_d (dissociation constant) of 0.73 ± 0.10 nmol/L (r² = .99), demonstrating this protein is a phytomelatonin receptor (PMTR1). Our results suggest that the phytomelatonin regulation of stomatal closure is dependent on its receptor CAND2/PMTR1‐mediated H₂O₂ and Ca²⁺signaling transduction cascade.     

Effect of short‐chain fatty acids on the proliferation and differentiation of the human colonic adenocarcinoma cell line Caco‐2

OBJECTIVE: Short‐chain fatty acids (SCFA) in the colon may maintain colonocyte differentiation and oppose carcinogenesis. The purpose of this study was to investigate the effects of three SCFA, butyrate, propionate and acetate, on the differentiation, proliferation, and matrix interactions of the Caco‐2 human colonic adenocarcinoma cell line. METHODS: Differentiation was assessed by brush border enzyme expression and the doubling time (proliferation) was calculated directly from serial cell counts and by the logarithmic transformation method. Cell motility (migration) was quantitated by the expansion of a confluent Caco‐2 monolayer (after release from a constraining fence) across bacteriologic plastic dishes precoated with saturating concentrations of type I collagen. Results were expressed as mean ± SE and were analyzed by anova and Bonferroni's modified t‐test. RESULTS: All three SCFA studied altered the Caco‐2 phenotype. Treatment with 10 mmol SCFA significantly prolonged the cell doubling time, promoted brush border enzyme expression (cathepsin C), and inhibited the motility of the Caco‐2 cells. CONCLUSIONS: Butyrate, propionate and acetate inhibited the proliferation and motility of a well‐differentiated human colonic cancer cell line while promoting the expression of the differentiation marker, cathepsin C. Thus the SCFA produced by bacterial fermentation of dietary fiber may exert a protective effect against the development of colon cancer.     

Eukaryotic expression of extracellular ligand binding domains of murine Tie‐2 and its anti‐angiogenesis effect in SGC‐7901 cell lines

Background and Aims: Researches about blocking angiogenesis to treat tumor have become one of the most promising and active fields in anticancer research. This study aimed to investigate the eukaryotic expression of extracellular ligand binding domains of murine Tie‐2 and its anti‐angiogenesis effect. Methods: A eukaryotic expression vector pcDNA3.1⁺ integrating with a DNA fragment which encode extracellular ligand binding domains of murine Tie‐2 was transfected into SGC‐7901 gastric cancer cell line. The protein expression was detected by western blot analysis and immunocytochemistry staining. Following the construction of nude mouse tumor xenograft model with and without transfected cells, tumor microvessel density was determined by counting per high power field in the sections stained with an antibody to CD31 to test its inhibition of angiogenesis. Results: The extracellular ligand binding domains of murine Tie‐2 receptor was highly expressed in SGC‐7901 gastric cancer cells with plasmid transfection. The mean tumor sizes of groups with and without transfection were 1.27 ± 0.35 and 1.75 ± 0.17 cm³, respectively (P = 0.025). The mean inhibitory rate of tumor was 27.18 ± 19.93%. The comparison between highest microvessel density of group with transfection (14.00 ± 3.80) and that of group without transfection (22.30 ± 5.91) was statistically significant at P = 0.030. Conclusion: The protein of extracellular ligand binding domains of murine Tie‐2 can be expressed at high level in the eukaryotic expression system, and the expressed protein may have the anti‐angiogenesis effect.     

Metabolic Syndrome Enhances Prostate Contractility and In Vitro Phenylephrine‐induced α1‐Adrenoceptor Protein Expression in the Fructose‐fed Rat

Objectives: To study the effects of metabolic syndrome on prostate α‐adrenergic contractile function using fructose‐fed rats (FR). Methods: Age‐matched male Wistar rats were divided into two groups: group I, normal control rats; and group II, 9‐week FR. Animal body weight, blood pressure and serum metabolic parameters were monitored. The prostate was removed 9 weeks after induction of metabolic syndrome in the FR. The contractile responses of prostatic strips to phenylephrine (10^?7 to 10^?6 M) and KCl (50 mM) were tested. Prostate α₁‐adrenoceptor (α₁‐AR) protein expression was studied by Western blotting analysis with a polyclonal antiserum. Results: At week 9, the FR showed significant increases in body weight, blood pressure, plasma glucose, insulin and triglyceride levels. The FR prostate weight was significantly higher than that of the controls (610.5 ± 13.2 vs 422.3 ± 7.7 mg, P < 0.05 for n = 8). FR prostate contractile responses to phenylephrine and KCl were both significantly increased. Interestingly, prostate α₁‐AR protein expression level was lower in the FR. However, after in vitro 10^?6 M phenylephrine stimulation, FR prostate α₁‐AR protein expression was significantly increased. Conclusion: Metabolic syndrome in FR significantly increases prostate contractile responses to KCl and α‐adrenergic stimulation. Paradoxically, FR prostate α₁‐AR protein expression is decreased, but significantly enhanced after in vitro phenylephrine stimulation.     

Deleted in liver cancer 2 suppresses cell growth via the regulation of the Raf‐1–ERK1/2–p70S6K signalling pathway

Background: Deleted in liver cancer 2 (DLC2) gene, a putative tumour suppressor gene, encodes a Rho GTPase‐activating protein (RhoGAP) with GAP activity specific for RhoA. It exhibits tumour suppressor functions and inhibits tumour cell proliferation, migration as well as transformation. Aims: In this study, we aimed to investigate the underlying mechanisms of the DLC2 gene in suppressing cell migration and cell growth. HepG2 hepatoma cells were stably transfected with the DLC2γ isoform, which contains the RhoGAP domain. Methods and results: On performing immunofluorescence staining and Western blot analysis, the expression of the focal adhesion protein paxillin was found to be much reduced in DLC2γ‐stable clones. Upon flow cytometric analysis of the cell cycle profiles, the DLC2γ‐stable clones were shown to have a higher population of cells arrested at the G1 phase than the EGFP vector‐stable clone, suggesting that downregulation of RhoA activity in DLC2γ‐stable clones inhibited cell cycle progression. In the DLC2γ‐stable clone, the levels of Raf‐1 and extracellular signal‐regulated kinase (ERK) 1/2 were decreased as compared with those of the parental HepG2, EGFP vector and DLC2γ–GAP defective mutant‐stable clones. Furthermore, the ribosomal kinase p70S6K, a downstream target of ERK1/2, was suppressed in the DLC2‐stable clones. On the contrary, when DLC2 was knocked down by siRNA in HepG2 cells, the expression levels of phospho‐p70S6K and phospho‐ERK1/2 were upregulated. Conclusion: Our data show that DLC2 inhibits the activity of Raf‐1–ERK1/2–p70S6K via its RhoGAP function, resulting in the suppression of cell growth. Further studies on the molecular signalling between DLC2 and p70S6K may provide an insight into its growth suppressor function.     

Four weeks of near‐normalization of blood glucose has no effect on postprandial GLP‐1 and GIP secretion,but augments pancreatic B‐cell responsiveness to a meal in patients with Type 2 diabetes

Objective The aim of the present study was to investigate whether 4 weeks of near‐normalization of blood glucose (BG) improves incretin hormone secretion and pancreatic B‐cell function during a mixed meal. Research design and methods Nine patients with Type 2 diabetes in poor glycaemic control [glycated haemoglobin (HbA_1c) 8.0 ± 0.4%] were investigated before and after 4 weeks of near‐normalization of BG (mean BG 6.4 ± 0.3 mmol/l) using insulin treatment. HbA_1c after insulin treatment was 6.6 ± 0.3%. For comparison, nine healthy control subjects were also studied. Postprandial glucagon‐like peptide 1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP) incremental responses were assessed during a mixed meal test. Fasting and postprandial pancreatic B‐cell function was determined from calculations of insulin secretion rates in relation to plasma glucose. Results There was no difference in IAUC_totalGLP‐1 or in IAUC_totalGIP between the two experimental days. B‐cell sensitivity to glucose (insulinogenic index) did not differ before and after insulin treatment in the fasting state (0.21 ± 0.17 vs. 0.25 ± 0.10 pmol kg^?1 min^?1/mmol l^?1), but improved significantly during the first 30 min after start of the meal (0.28 ± 0.07 vs. 0.46 ± 0.06 pmol kg^?1 min^?1/mmol l^?1) and during the following 4 h (0.34 ± 0.09 vs. 0.56 ± 0.07 pmol kg^?1 min^?1/ mmol l^?1). The B‐cell responsiveness to changes in plasma glucose, expressed as the slope of the linear relationship between the insulin secretion rate and the concomitant plasma glucose increased from 0.59 ± 0.16 to 0.94 ± 0.13 pmol kg^?1 min^?1/ mmol l^?1 (P < 0.07). Conclusions Four weeks of near‐normalization of BG had no effect on postprandial secretion of incretin hormones. Nevertheless, several parameters of meal‐induced insulin secretion improved after insulin treatment.     

Relationship between α1-antitrypsin inactivation and tumor necrosis factor α concentration in the synovial fluid of patients with rheumatoid arthritis

Objective. To examine the relationship between α₁-antitrypsin (α₁AT) specific activity and tumor necrosis factor α (TNFα) concentration in synovial fluid from 48 patients with rheumatoid arthritis. Methods. The specific activity of α₁AT was calculated from the measurement of α₁AT concentration (by rocket immunoelectrophoresis) and elastase inhibitory capacity. TNFα was detected by enzyme-linked immuno-sorbent assay. Results. TNFα concentrations correlated with the extent of α₁AT inactivation. Conclusion. Our findings are consistent with a role of elastase in TNFα release within the inflamed joint.     

The role of IL‐1β in reduced IL‐7 production by stromal and epithelial cells: a model for impaired T‐cell numbers in the gut during HIV‐1 infection

Abstract. Thang PH, Ruffin N, Brodin D, Rethi B, Cam PD, Hien NT, Lopalco L, Vivar N, Chiodi F (Karolinska Institutet, Stockholm, Sweden; National Institute of Hygiene and Epidemiology, Hanoi, Vietnam; San Raffaele Scientific Institute, Milan, Italy). The role of IL‐1β in reduced IL‐7 production by stromal and epithelial cells: a model for impaired T‐cell numbers in the gut during HIV‐1 infection. J Intern Med 2010; 268 : 181–193. Objectives. Interleukin (IL)‐7 is a key cytokine in T‐cell homeostasis. Stromal cells, intestinal epithelial cells and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL‐7 production are still unclear. We assessed whether IL‐1β and interferon (IFN)‐γ, cytokines produced during inflammatory conditions, may impact on IL‐7 production. Design. We used human intestinal epithelial cells (DLD‐1 cell line) and bone marrow stromal cells (HS27 cell line), known to produce IL‐7; IL‐7 production was evaluated at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL‐1β and/or IFN‐γ leads to changes in the gene expression of cytokines, Toll‐like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole‐genome microarray Human Gene 1.0 ST. Results. We found that IFN‐γ enhanced the expression of IL‐7 mRNA (P < 0.001) in both cell lines. IL‐1β treatment led to a significant down‐regulation (P < 0.001) of IL‐7 mRNA expression in both cell lines. The IL‐7 concentration in supernatants collected from treated DLD‐1 and HS27 cell cultures reflected the trend of IL‐7 mRNA levels. The gene profiles revealed dramatic changes in expression of cytokines and their receptors (IL‐7/IL‐7Rα; IL‐1α,IL‐1β/IL‐1R1; IFN‐γ/IFN‐γR1), of IFN regulatory factors (IRF‐1 and 2), of TLRs and of important chemo‐attractants for T cells. The microarray results were verified by additional methods. Conclusions. Our results are discussed in the setting of inflammation and T‐cell survival in the gut compartment during HIV‐1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL‐7 homeostasis and homing of T cells.     

Melatonin reverses H2O2‐induced premature senescence in mesenchymal stem cells via the SIRT1‐dependent pathway

Mesenchymal stem cells (MSCs) represent an attractive source for stem cell‐based regenerative therapy, but they are vulnerable to oxidative stress‐induced premature senescence in pathological conditions. We previously reported antioxidant and antiarthritic effects of melatonin on MSCs against proinflammatory cytokines. In this study, we hypothesized that melatonin could protect MSCs from premature senescence induced by hydrogen peroxide (H₂O₂) via the silent information regulator type 1 (SIRT1)‐dependent pathway. In response to H₂O₂ at a sublethal concentration of 200 μm , human bone marrow‐derived MSCs (BM‐MSCs) underwent growth arrest and cellular senescence. Treatment with melatonin before H₂O₂ exposure cannot significantly prevent premature senescence; however, treatment with melatonin subsequent to H₂O₂ exposure successfully reversed the senescent phenotypes of BM‐MSCs in a dose‐dependent manner. This result was made evident by improved cell proliferation, decreased senescence‐associated β‐galactosidase activity, and the improved entry of proliferating cells into the S phase. In addition, treatment with 100 μm melatonin restored the osteogenic differentiation potential of BM‐MSCs that was inhibited by H₂O₂‐induced premature senescence. We also found that melatonin attenuated the H₂O₂‐stimulated phosphorylation of p38 mitogen‐activated protein kinase, decreased expression of the senescence‐associated protein p16^INK4α, and increased SIRT1. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked melatonin‐mediated antisenescence effects. Inhibition of SIRT1 by sirtinol counteracted the protective effects of melatonin, suggesting that melatonin reversed the senescence in cells through the SIRT1‐dependent pathway. Together, these findings lay new ground for understanding oxidative stress‐induced premature senescence and open perspectives for therapeutic applications of melatonin in stem cell‐based regenerative medicine.     

Proteolytic inactivation of α1-antitrypsin and α1-antichymotrypsin by neutrophils in arthritic joints

Objective. In vitro, activated neutrophils create a microenvironment in which proteinase inhibitors are inactivated through the coordinate action of reactive oxygen species and released elastase. We investigated whether such a mechanism may contribute to the destruction of the joint tissues in arthritis. Methods. We analyzed the state of α₁-antitrypsin (α₁AT) and α₁-antichymotrypsin (α₁ACT), the two major inhibitors of the neutrophilic serine proteinases, in synovial fluid (SF) from patients with inflammatory arthropathies (n = 71) and osteoarthritis (OA) (n = 11), and related the results to neutrophil activation in SF. Results. The ratio of functional to antigenic levels of α₁AT in SF of patients with inflammatory joint diseases was similar to that of α₁AT in normal plasma, whereas that of α₁ACT was significantly decreased. Patients with inflammatory arthropathies had significantly higher levels of inactivated α₁AT (iα₁AT) and inactivated α₁ACT (iα₁ACT) in SF (as determined with monoclonal antibodies specific for the inactivated [i.e., proteolytically inactivated and/or complexed] forms of these inhibitors) than patients with OA (P < 0.005). Inactivated α₁AT and iα₁ACT levels corresponded to 0.3–11% and 3–99%, respectively, of the total amount of these inhibitors in SF. Most of the iα₁AT in SF had a lower M_r than that of native α₁AT. Inactivated α₁ACT in SF had an M_r identical to that of nonfunctional α₁ACT in plasma treated with chymotrypsin. Levels of both iα₁AT and iα₁ACT correlated significantly with lactoferrin and elastase levels. Conclusion. These results suggest that α₁AT and α₁ACT in arthritic joints are inactivated in part by activated neutrophils, suggesting a role for these cells in impairment of the local balance between proteinases and their inhibitors in arthritis.     

Investigation of the effect of oral metformin on dipeptidylpeptidase‐4 (DPP‐4) activity in Type 2 diabetes1

Aims Glucagon‐like peptide‐1 (GLP‐1) is an insulinotropic hormone and major component of the enteroinsular axis. Its therapeutic potential in human diabetes is limited by rapid degradation and inactivation by the enzyme dipeptidylpeptidase‐4 (DPP‐4). We investigated the acute effects of metformin with and without food on DPP‐4 activity in Type 2 diabetes. Methods Ten subjects with Type 2 diabetes (6 male/4 female, age 65.8 ± 2.6 years, body mass index 30.0 ± 1.2 kg/m², glycated haemoglobin (HbA_1c) 6.3 ± 0.2%, mean ± sem ) received metformin 1 g orally or placebo together with a standard mixed meal (SMM) in a random crossover design. Six subjects re‐attended fasting and received metformin 1 g without a SMM. Results Following SMM (n = 10), DPP‐4 activity was not suppressed by metformin compared with placebo [area under curve (AUC)_{0–4 h} 1574 ± 4 vs. 1581 ± 8 μmol/ml/min, respectively]. Plasma glucose, insulin and active GLP‐1 were not different. However, DPP‐4 activity was suppressed with metformin following fasting compared with a SMM (n = 6) (AUC_{0–4 h} 1578 ± 4 vs. 1494 ± 9 μmol/min, P < 0.02). Metformin serum levels were significantly lower (P < 0.001) after SMM than fasting (AUC_{0–4 h} 350 ± 66 vs. 457 ± 55 mg/ml/min). Conclusion Metformin inhibits DPP‐4 activity in Type 2 diabetic patients in the fasting state but not when taken with a standard mixed meal. Metformin serum concentrations are lower if the drug is taken with food. These findings should be taken into account in establishing how to maximize efficacy of the drug.     

Regression equations to estimate the 2‐min walk distance in an adult Asian population aged 40–75 years

Background and objective

There is increased use of the 2‐min walk test (2MWT) to assess functional exercise capacity. However, the distance achieved during this test may be difficult to interpret in the absence of reference values from a local population. Regression equations to estimate the 2‐min walk distance (2MWD) only exist for American and Brazilian populations. The objective of this study was to develop regression equations to estimate the 2MWD in Malaysian adults who were free from major health problems.

Methods

Eighty‐seven adults (43 males; mean ± SD age: 57.1 ± 9.6 years) performed two 2MWT using a standardized protocol. Heart rate (HR) was recorded every 30 s during the test. Stepwise multiple regression analysis was performed using age, gender, height, weight and change in HR (ΔHR) as independent variables, and better of the two 2MWD as the dependent variable. A second regression equation, without ΔHR, was planned if ΔHR was retained as one of the predictors of the 2MWD in the first equation.

Results

The better of the two 2MWD was 200 ± 34 m. Males walked 33 ± 6 m further than females (P < 0.001). The two regression equations were 196 − 1.1 × age, years + 1.0 × ΔHR, bpm + 31.2 × gender (R² = 0.73) and 279 − 1.7 × age, years + 35.9 × gender (R² = 0.47) with females = 0 and males = 1.

Conclusion

The equations derived in this study may facilitate the interpretation of the 2MWD in clinical populations in Malaysia, as well as in countries with similar cultural backgrounds to Malaysia.

     

Evidence of differential selection for the −α3.7 and −α4.2 single‐α‐globin gene deletions within the same population

Since the 1950s, a strong correlation between high carrier rates for β‐thalassemia mutations and selective survival advantage in tropical and subtropical ‘malarial belt’ regions has been established. Due to the relatively more complex genetics of α‐thalassemia, a similar relationship was demonstrated for α‐globin gene mutations only from the 1980s, with both single‐ and double‐α‐globin gene deletions prevalent in the malarial belt. Mechanistically, the single‐α‐globin gene deletions arise from non‐allelic recombination between the homologous α1 (HBA1) and α2 (HBA2) globin genes. Compared to the ?α^3.7 and ααα^anti3.7 rightward crossover alleles, much less is known about the ?α^4.2 and ααα^anti4.2 leftward crossover alleles. We performed a survey of 1,285 unselected cord blood samples from the 3 major ethnic groups in Singapore. Overall, the frequency of the ?α^3.7 deletion was significantly higher than its reciprocal ααα^anti3.7 triplication, consistent with positive selection for the ?α^3.7 single‐gene deletion. In marked contrast, there was no significant difference in frequency between the ?α^4.2 and reciprocal ααα^anti4.2 alleles, suggesting the absence of positive selection for the ?α^4.2 single‐gene deletion. The similar ααα^anti3.7 and ααα^anti4.2 allele frequencies also suggested that the crossover rates at X and Z homology boxes are similar. Taken together, these observations suggest a differential positive selection for the ?α^3.7 and ?α^4.2 alleles within the same population. Further population and biological studies may be required to explain these current observations.     

Comprehensive longitudinal analysis of hepatitis C virus (HCV)‐specific T cell responses during acute HCV infection in the presence of existing HIV‐1 infection

Summary. The aim of this study was to study the development of HCV‐specific T cell immunity during acute HCV infection in the presence of an existing HIV‐1 infection in four HIV‐1 infected men having sex with men. A comprehensive analysis of HCV‐specific T cell responses was performed at two time points during acute HCV infection using a T cell expansion assay with overlapping peptide pools spanning the entire HCV genome Three patients with (near) normal CD4+ T cell counts (range 400–970 × 10⁶/L) either resolved (n = 1) or temporary suppressed HCV RNA. In contrast, one patient with low CD4+ T cell counts (330 × 10⁶/L), had sustained high HCV RNA levels. All four patients had low HCV‐specific CD8+ T cell responses, and similar magnitudes of CD4+ T cell responses. Interestingly, individuals with resolved infection or temporary suppression of HCV‐RNA had HCV‐specific CD4+ T cell responses predominantly against nonstructural (NS) proteins. While the individual with high HCV RNA plasma concentrations had CD4+ T cell responses predominantly directed against Core. Our data show that an acute HCV infection in an HIV‐1 infected person can be suppressed in the presence of HCV‐specific CD4+ T cell response targeting non‐structural proteins. However further research is needed in a larger group of patients to evaluate the role of HIV‐1 on HCV‐specific T cell responses in relation to outcome of acute HCV infection.     

Induction of lipocalin‐2 expression in acute and chronic experimental liver injury moderated by pro‐inflammatory cytokines interleukin‐1β through nuclear factor‐κB activation

Background: Lipocalin‐2 (LCN2) belongs to the lipocalin superfamily, sharing a barrel‐shaped tertiary structure with a hydrophobic pocket and an ability to bind lipophilic molecules. LCN2 has recently emerged as an important modulator of cellular homeostasis in several organs, i.e. heart, lung and kidney, but little is known about the expression of LCN2 in acute and chronic liver injury. Aims: In this study, we wanted to analyse the expression and regulation of LCN2 in models of acute and chronic experimental liver injury. Materials and methods: We analysed LCN2 expression in livers of rats subjected to bile duct ligation or repeated doses of carbon tetrachloride and tested the impact of various pro‐inflammatory cytokines in cultured primary liver cells. Results: By using primary cultures of hepatic stellate cells and hepatocytes isolated from normal and injured rat livers, we found a significant LCN2 expression in early hepatic stellate cell cultures, a lower expression in fully transdifferentiated myofibroblasts and no expression in freshly isolated hepatocytes. However, LCN2 expression and secretion in hepatocytes increased dramatically during culturing. In addition, chronic in vivo liver injury resulting from both bile duct ligation and repeated application of carbon tetrachloride resulted in rapid and well‐sustained induction of LCN2 expression. Immunohistochemistry and primary liver cell isolation identified injured hepatocytes as the main source of LCN2 production. LCN2 is strongly induced in both primary hepatocytes and immortalized hepatocellular carcinoma cell line HepG2 by the pro‐inflammatory cytokine interleukin‐1β via nuclear factor‐κB activation, but not by the profibrotic cytokines platelet‐derived growth factor and transforming growth factor‐β. Conclusion: LCN2 expression shows clear correlation to liver damage and resulting inflammatory responses, rather than to the degree of liver fibrosis, which in fact may imply a distinct diagnostic value as an early biomarker of liver inflammation.