Elevated nitric oxide and 3′,5′ cyclic guanosine monophosphate levels in patients with alcoholic cirrhosis

AIM： To evaluate whether serum levels of nitric oxide （NO^.） and plasma levels of cyclic guanosine monophosphate （cGMP） and total glutathione （GSH） are altered in patients with alcoholic cirrhosis and to examine their correlation with the severity of liver disease.
METHODS： Twenty-six patients with alcoholic liver cirrhosis were studied. Serum levels of NO^. and plasma levels of cGMP and GSH were measured in 7 patients with compensated alcoholic cirrhosis （Child-Pugh A） and 19 patients with advanced cirrhosis （Child-Pugh B and C）. The model for end-stage liver disease （MELD） score was evaluated. Sixteen healthy volunteers served as controls. Liver enzymes and creatinine levels were also tested.
RESULTS： NO^. and cGMP levels were higher in patients with Child-Pugh B and C cirrhosis than in Child-Pugh A cirrhosis or controls （NO^.： 21.70 ± 8.07 vs 11.70 ± 2.74; 21.70± 8.07 vs 7.26 ± 2.47 μmol/L, respectively; P 〈 0.001） and （cGMP： 20.12 ± 6.62 vs 10.14 ± 2.78; 20.12 ± 6.62 vs 4.95 ± 1.21 pmol/L, respectively; P 〈 0.001）. Total glutathione levels were lower in patients with Child-Pugh B and C cirrhosis than in patients with Child-Pugh A cirrhosis or controls （16.04 ± 6.06 vs 23.01 ± 4.38 or 16.04 ± 6.06 vs 66.57 ±26.23 μmol/L, respectively; P 〈 0.001）. There was a significant correlation between NO^. and cGMP levels in all patients with alcoholic cirrhosis. A significant negative correlation between reduced glutathione/glutathione disulfide and the MELD score was found in all cirrhotic patients.
CONCLUSION： Our results suggest a role for oxidative stress in alcoholic liver cirrhosis, which is more significant in decompensated patients with higher levels of NO^. and cGMP and lower GSH levels than in compensated and control patients. Altered mediator levels in decompensated patients may influence the hemodynamic changes in and progression of liver disease.     

Human vascular smooth muscle cells express a constitutive nitric oxide synthase that insulin rapidly activates, thus increasing guanosine 3′ : 5′-cyclic monophosphate and adenosine 3′ : 5′-cyclic monophosphate concentrations

Aims/hypothesis. Insulin incubation of human vascular smooth muscle cells (hVSMC) for 120 min increases both guanosine 3′ : 5′-cyclic monophosphate (cGMP) and adenosine 3′ : 5′-cyclic monophosphate (cAMP) and these effects are blocked by inhibiting nitric oxide synthase (NOS). These data suggest that insulin activates a constitutive Ca²⁺-dependent NOS (cNOS), not described at yet in hVSMC. To test this hypothesis, we evaluated in hVSMC: i) the kinetics of the insulin-induced enhancement of the two cyclic nucleotides; ii) the ability of nitric oxide (NO) to increase both cyclic nucleotides; iii) NO involvement in the short-term influence of insulin on both cyclic nucleotides; iv) the ability of insulin to increase NO production in a few minutes; v) the presence of a cNOS activity; vi) the expression of mRNA for cNOS. Methods. In hVSMC incubated with insulin, NO donors and the Ca²⁺ ionophore ionomycin, we measured cAMP and cGMP (RIA); in hVSMC incubated with insulin and ionomycin we measured NO, evaluated as l-(³H)-citrulline production from l-(³H)-arginine; by northern blot hybridization, we measured the expression of cNOS mRNA. Results. i) By incubating hVSMC with 2 nmol/l insulin for 0–240 min, we observed an increase of both cGMP and cAMP (ANOVA: p = 0.0001). Cyclic GMP rose from 0.74 ± 0.01 to 2.62 ± 0.10 pmol/10⁶ cells at 30 min (p = 0.0001); cAMP rose from 0.9 ± 0.09 to 11.65 ± 0.74 pmol/10⁶ cells at 15 min (p = 0.0001). ii) Sodium nitroprusside (100 μmol/l) and glyceryltrinitrate (100 μmol/l) increased both cGMP and cAMP (p = 0.0001). iii) The effects of insulin on cyclic nucleotides were blocked by NOS inhibition. iv) An increase of NO was observed by incubating hVSMC for 5 min with 2 nmol/l insulin (p = 0.0001). v) Ionomycin (1 μmol/l) enhanced NO production (p = 0.0001) and increased both cyclic nucleotides (p = 0.0001). vi) hVSMC expressed mRNA of cNOS. Conclusion/interpretation. Human VSMC express cNOS, which is rapidly activated by insulin with a consequent increase of both cGMP and cAMP, suggesting that insulin-induced vasodilation in vivo is not entirely endothelium-mediated. [Diabetologia (1999) 42: 831–839] Received: 30 November 1998 and in revised form: 25 January 1999     

Involvement of α5 integrin in survivin-mediated osteosarcoma metastasis

Objective: To investigate the role of survivin in osteosarcoma metastasis. Methods: Small interfering RNA(si RNA) was used to knockdown the expression of survivin and α5 integrin in the human osteosarcoma cell line MG63. Western blotting and immunostaining methods was used to assessed the effect of survivin knockdown on the expression of α5 integrin through flow cytometry and fluorescence microscopy detection. Meanwhile, the invasion and migration of transfected cells in Transwell and wound healing assays were probed, and the growth situation of these cells transplanted into nude mice was monitored. Results: Knockdown of survivin expression could inhibit the invasion and migration of osteosarcoma MG64 cells in vitro and the expression of α5 integrin on osteosarcoma MG64 cell surface, suggesting that survivin can inhibit the invasion and migration of osteosarcoma cells through downregulation of α5 integrin. Anti-α5 integrin antibody could also markedly decrease the capability of invasion and migration of osteosarcoma MG64 cells. Additionally, knockdown of survivin expression could slow the growth of osteosarcoma MG63 cells transplanted into nude mice. Conclusions: Survivin-directed anti-tumor strategies might be an effective method in the treatment of osteosarcoma.     

Sensitivity of CFU-GM from normal human bone marrow and leukaemic clonogenic cells (CFU-L) from blood of patients with myelogenous leukaemia to 3′-deoxy-3′-fluorothymidine in comparison to 3′-azido-3′-deoxythymidine

Summary 3-Deoxy-3-fluorothymidine (FT), a thymidine analogue highly effective against HIV 1 in vitro was investigated as to its in vitro effect on normal human bone marrow CFU-GM (agar colony assay) and on human peripheral myeloid leukaemic clonogenic cells (CFU-L, colony assay in methylcellulose). For comparison, 3-azido-3-deoxythymidine (AZT), structurally related and used in AIDS treatment, was included in the study. Both compounds inhibit the formation of clusters and colonies from bone marrow stem cells with an [IC]₅₀ between 10^–6 and 10^–5M. In concentrations only 5–10 times lower than the [IC]₅₀, FT begins to stimulate cluster and colony formation. AZT and FT also inhibit the formation of clusters and colonies from CFU-L. Compared to CFU-GM, CFU-L were more sensitive to FT, and a stimulation was not seen. It is concluded that similar side effects on bone marrow could be expected for possible use of FT against AIDS as have been found for AZT and that both compounds are potential candidates for antileukaemic drugs.     

Flow-cytometric enumeration of reticulocytes with the new fluorochrome 1′,3′-diethyl-4,2′-quinolylthiacyanine

Summary Several flow-cytometric methods for reticulocyte enumeration in whole blood have been developed, with different degrees of practical use. Recently, a new fluorochrome, 1,3-diethyl-4,2-quinolylthyacianine (DEQTC) was proposed in a brief report, as an alternative to thiazole orange for reticulocyte counting. We have evaluated the usefulness of this fluorescent stain by assessing the optimal conditions for the flow-cytometric analysis, and by comparing in double-blind assays the quantitative results of this technique with those obtained by manual counting with brilliant cresyl blue. Our results show that flow cytometry with DEQTC is highly correlated to the manual method (r=0.95–0.99), supporting the interest of this particular stain and of flow cytometry for routine laboratory work in hematology.     

Effect of salt on cell proliferation andN-methyl-N′-nitro-N-nitrosoguanidine penetration to proliferative cells in the forestomach of rats

We have studied the effect of intragastric instillation of 4.5M NaCl on cell proliferation and carcinogen penetration into the forestomach of Wistar rats at different time intervals after salt exposure. Cells in the S-phase were labelled by incorporation of bromodeoxyuridine and located after immunohistochemistry.N-[³H]Methyl-N-nitro-N-nitrosoguanidine ([³H]MNNG) was used as the carcinogen and penetration of [³H]MNNG to proliferative cells was determined by autoradiography. The number of cells in S-phase per millimetre epithelium length 12 h and 24 h after salt exposure (32.2±11.9 and 20.6±7.4) was significantly higher than in the control animals (9.4±3.6). No increase in cell proliferation occurred during the first 2 h after salt exposure. Microscopy also revealed oedema in the lamina propia. The forestomach blood flow was not influenced by the application of hypertonic saline. [³H]MNNG at a concentration of 40 g/ml did not penetrate to the proliferative cells in the forestomach and no effect of salt pretreatment on carcinogen penetration was seen. The low penetration of [³H]MNNG to proliferative cells in the forestomach epithelium may explain why this concentration of MNNG given in drinking water over several weeks usually does not induce squamous cell carcinomas in the forestomach. The previously observed cocarcinogenic effect of salt on squamous cell cercinogenesis in the upper gastrointestinal tract could be due to the observed increase in cell proliferation after salt exposure.Abbreviations [³H]MNNG N-[³H]methyl-N-nitro-N-nitrosoguanidine - MNNG N-methyl-N-nitro-N-nitrosoguanidine This work was supported by grants from the Norwegian Cancer Society and the Helga Semb Fund. Dr. Sørbye is a Research Fellow of the Norwegian Cancer Society     

Pyrimidine 5′-nucleotidase activity in normal and deficient human lymphoblastoid cells

Summary Two distinct pyrimidine 5-nucleotidases (UMPH-1 and UMPH-2) have previously been detected in human erythrocytes; UMPH-1 is deficient in a haemolytic anaemia, while UMPH-2 is unaffected. Only the erythrocyte shows pathological effects in this disorder. Here we have studied lymphoblastoid cell lines from control and UMPH-1 deficient patients to determine whether UMPH-1 can be detected in lymphoblastoid cells and whether the deficiency of UMPH-1 results in any measurable metabolic effects. Both UMPH-1 and a UMPH-2-like activity were found to be present in control lymphoblastoid cells. UMPH-1 was undetectable in the patients' cells; minor but significant changes were found in the pyrimidine nucleotide and nucleoside pools in the cells.     

Thymosin β10 as a predictive biomarker of response to 5-fluorouracil chemotherapy in cholangiocarcinoma

Introduction and aim. 5-Fluorouracil (5-FU) is the most commonly used chemotherapeutic drug in the treatment of cholangiocarcinoma (CCA). Since development of drug resistance to 5-FU in CCA patients is the primary cause of treatment failure, a better understanding of the mechanism of drug resistance of this cancer is essential to improve the efficacy of 5-FU in CCA therapy.Material and methods. A 5-FU resistant CCA cell line (M214-5FUR) for a comparative chemo-resistance study was established. Real time RT-PCR was used to determine gene expression levels. Cell cytotoxicity was measured by the MTT assay. Protein expression levels were detected by the immunofluorescene method.Results. It was found that 5-FU resistance was associated with the overexpression of Tβ10 in CCA cell lines. 5-FU treatment at various concentrations induced the expressions of Tβ10 and ABC transporters (ABCB1, ABCG2 ABCA3) in two CCA cell lines, KKU-M055 and KKU-M214. M214-5FUR, a 5-FU-resistant cell line, exhibited a 5-FU resistant phenotype with a 16-fold extremely high expression of Tβ10 and ABC transporters, as compared to the parental cells, KKU-M214. siRNA targeted to Tβ10 significantly reduced expression of ABC transporters tested in the M214-5FUR cells (P < 0.05).Conclusions. The present novel findingsof Tβ10 connected with drug resistance as shown in this study provides a new insight for the therapeutic value of Tβ10 as a predictive biomarker of 5-FU chemoresistance. Inhibiting Tβ10 may be a valuable adjunct for suppression of ABC transporters and sensitizing chemotherapy treatment, especially 5-FU in CCA patients.     

Polymorphisms of Thymidylate Synthase in the 5′- and 3′-Untranslated Regions and Gastric Cancer

Studies investigating the association of polymorphisms in the 5′-untranslated regions (5′UTR) and 3′-untranslated regions (3′UTR) of thymidylate synthase with gastric cancer susceptibility and sensitivity to fluoropyrimidine-based chemotherapy report conflicting results. The objective of this study was to quantitatively summarize the evidence for such a relationship. This meta-analysis included ten studies, which included 1,730 gastric cancer cases and 1,843 controls. The combined results based on all studies showed that there was no significant difference in genotype distribution of 5′UTR or 3′UTR between gastric cancer and noncancer patients. When stratifying for race, we found that: (1) among Asians, patients with gastric cancer had significantly higher frequency of 2R/2R of 5′UTR than did noncancer patients, and (2) among Caucasians, patients with gastric cancer had significantly lower frequency of ins6/ins6 and higher frequency of ins6/del6 of 3′UTR than did noncancer patients. No significantly different response rate or survival of gastric cancer with fluoropyrimidine-based chemotherapy were observed with genotype distribution of 5′UTR or 3′UTR among Caucasians or Asians. This meta-analysis suggests that polymorphisms in the 5′UTR and 3′UTR of thymidylate synthase may be associated with gastric cancer susceptibility, but are not correlated with sensitivity of gastric cancer to fluoropyrimidine-based chemotherapy.     

Deficiency of pyrimidine 5′-nucleotidase in human leukocytes

Summary Both erythrocytes and leukocytes from a patient with erythrocyte pyrimidine 5-nucleotidase (P5N) deficiency were shown to contain increased amounts of pyrimidine nucleotides. These findings suggested that the leukocytes were also deficient for P5N. Measurement of the P5N activity in lysates from lymphocytes or granulocytes, in the presence of inhibitors for non-specific 5-nucleotidase or alkaline phosphatase, indeed showed a deficiency for P5N in lymphocytes and granulocytes of the patient with erythrocyte P5N deficiency. However, the P5N deficiency in the leukocytes did not cause clinical disturbances in addition to the weak haemolytic anaemia.     

Granulocytes: effector cells or immunomodulators in the immune response to helminth infection?

Granulocytes are effector cells in defence against helminth infections. We review the current evidence for the role of granulocytes in protective immunity against different helminth infections and note that for each parasite species the role of granulocytes as effector cells can vary. Emerging evidence also points to granulocytes as immunomodulatory cells able to produce many cytokines, chemokines and modulatory factors which can bias the immune response in a particular direction. Thus, the role of granulocytes in an immunomodulatory context is discussed including the most recent data that points to an important role for basophils under this guise.     

Characterization of the 5′ and 3′ Breakpoints of the Spanish (δβ)0-Thalassemia Deletion in Mexican Patients

We studied five unrelated Mexican carriers of the Spanish (δβ)⁰-thalassemia [(δβ)⁰-thal] mutation to characterize the size of the deletion, the 5′ and 3′ breakpoints and the 5′ β-globin haplotype. Sequence analysis revealed the presence of an 89,548 bp deletion. The δ- and β-globin genes, two olfactory receptor genes (OR51V1 and OR52A1) and two pseudogenes (OR52Z1P and OR51A1P) were deleted. The 5′ breakpoint was located at the same position as previously reported, and the 3′ breakpoint was situated 7.0 kb downstream of OR52A1 and 11.7 kb upstream of OR52A5. The Spanish (δβ)⁰-thal allele was associated with the 5′ haplotype 2 [– + + – +] in the studied patients. Because this mutation is relatively frequent in Spain, and the Mexican population contains a high level of Spanish genetic background, we propose that the mutation in both populations share a common ancestral origin.     

Analysis of linkage disequilibrium between the 5′ and 3′ haplotypes of the β-globin gene cluster in Mexican afromestizos

Analysis of the 5′ and 3′ haplotypes (Hps) of the β-globin gene cluster was performed in 110 β^A chromosomes from unrelated Mexican afromestizo individuals in order to determine Hardy-Weinberg equilibrium, allelic frequencies, linkage disequilibrium (LD) and association between the 5′ and 3′ haplotypes. All sites were found to be in Hardy–Weinberg equilibrium (p > 0.05). In the whole β-cluster, only 12.87% of the pairs of loci exhibited significant LD (22/171) (r² > 0.33). Within the 5′Hp, three pairs of loci were associated (?HincII/^GγHindIII, ?HincII/3′ψβHincII and ^GγHindIII/3′ψβHincII). In the 3′Hp, 19 pairs of loci showed significant LD and were distributed mostly among the ? 551, ? 340, Exon1nt6 and IVS2nt16 polymorphisms. The absence of pairs of loci significantly linked between both 5′ and 3′ Hps is noteworthy. The allelic combinations of the 40 studied polymorphisms (5 sites in the 5′ Hp and 35 sites in the 3′ Hp) displayed 69 distinct haplotypes, 22 of them belonging to group A, 27 to B, 18 to C and 2 to D, which denoted the great heterogeneity of our population. Further, 1a7A1, 1a7B1 and 1a1C1 were the most common sequences with 8, 9 and 9 chromosomes each. Association analysis between both 5′ and 3′ Hps revealed strong coherence with the proposed evolutionary histories for the β-globin gene polymorphisms. 5′Hp1 (+––––), which is considered to be an ancestral haplotype, was the most frequent haplotype found in our population and was linked to 24 different sequences in the 3′Hp, demonstrating great heterogeneity. A similar result was found in the 3′ Hps, where older alleles (a17A1 and a7B1) were linked to a higher number of 5′Hps.This is the first time that an analysis of association among the 5′ and 3′ haplotypes and the LD has been performed with 40 polymorphisms distributed in the β-globin gene cluster in the Mexican afromestizo population. The poor LD observed between and within the 5′ and 3′ Hps show that this region is very prone to recombination events.     

Homeostatic expansion and phenotypic conversion of naïve T cells in response to self peptide/MHC ligands

Recent data suggest that survival of resting, na?ve T cells requires an interaction with self MHC molecules. From analysis of the class I MHC-restricted T cell receptor transgenic strain OT-I, we report a different response. Rather than merely surviving, these T cells proliferated slowly after transfer into T-depleted syngeneic hosts. This expansion required both T cell "space" and expression of normal levels of self class I MHC molecules. Furthermore, we demonstrate that during homeostatic expansion in a suitable environment, na?ve phenotype (CD44(low)) OT-I T cells converted to memory phenotype (CD44(med/high)), despite the absence of foreign antigenic stimulation. On the other hand, cells undergoing homeostatic expansion did not acquire cytolytic effector function. The significance of these data for reactivity of T cells with self peptide/MHC ligands and the implications for normal and abnormal T cell homeostasis are discussed.