Construction, expression and characterization of human interferon α2b-（G4S）n-thymosin α1 fusion proteins in Pichia pastoris

AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNc(2b and To(1 linked by different lengths of (G4S)n (n=1-3) were constructed and expressed in Pichia pastoris.METHODS: Using PCR and molecular clone techniques,the fusion genes of IFNα2b-(G4S)n-Tα1(n=1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1(n=1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex^TM 75 gel filtration and analyzed by SDSPAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins.RESULTS: DNA sequencing confirmed that the fusion genes of IFNα2b-(G4S)n-Tα1(n=1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1(n=1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex^TM 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6ku, respectively, and reacted to the IFNα2b monoclonal antibody and Tα1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay.CONCLUSION: The recombinant IFNα2b-(G4S)n-Tα1(n=1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins‘ function in vivo.     

Molecular genetics of diabetes mellitus in Chinese subjects: identification of mutations in glucokinase and hepatocyte nuclear factor-1alpha genes in patients with early-onset type 2 diabetes mellitus/MODY.

AIMS: To examine the prevalence of identified MODY-related genes in Chinese subjects with early onset Type 2 diabetes mellitus and a positive family history of diabetes and to look for possible associations between the gene mutations and the development of diabetes. METHODS: Ninety-two unrelated Chinese subjects with diabetes diagnosed before the age of 40 years who had a positive family history of diabetes were screened for mutations in hepatocyte nuclear factors (HNF-1alpha and HNF-4alpha) and glucokinase genes by direct sequencing. The family members of patients with mutations and 100 healthy controls were also examined. RESULTS: Mutations in the HNF-1alpha and the glucokinase genes were found in 5% and 3% of the diabetic subjects, respectively but no mutations were found in the coding region of the HNF-4alpha gene. Three mutations found in the glucokinase gene were novel missense mutations (I110T, A119D and G385V). The mutations in the HNF-1alpha gene were also new and included four missense mutations (G20R, R203H, S432C, I618M) and one splice acceptor site mutation (IVS2nt-1G-->A). Patients with mutations in these genes were clinically heterogeneous with respect to phenotype and basal pancreatic beta cell function. CONCLUSIONS: Genetic factors such as mutations in the HNF-1alpha and glucokinase genes may be important in the development of diabetes in Chinese people, especially when the disease is of early onset.     

吲哒帕胺对原发性高血压患者TXA2、PGI2含量的影响

目的研究轻中度原发性高血压患者血栓索A2(TXA2)和前列环素(PGI2)含量及二者比值的变化,评价吲哒帕胺对轻、中度原发性高血压的降压疗效以及对TXA2、PGI2和TXA2/PGI2的影响,探讨吲哒帕胺对血管内皮功能和血小板活性影响的机制.方法对30例轻、中度高血压患者和20例正常对照者的血浆采用放射免疫法测定TXA2、PGI2的水平,对高血压患者给予吲哒帕胺治疗2周,并进行治疗前、后对照比较.结果高血压组TXA2水平明显高于对照组,而PGI2水平明显低于对照组,TXA2/PGI2明显高于对照组(P均＜0.01).吲哒帕胺治疗组,降压有效率为60%,治疗后TXA2水平明显降低,PGI2水平明显升高,TXA2/PGI2明显下降(P均＜0.01).结论原发性高血压患者TXA2及TXA2/PGI2升高,PGI2水平下降.吲哒帕胺对轻、中度原发性高血压患者疗效显著,可引起TXA2水平显著下降,PGI2水平显著升高,对高血压所致的血管内皮损害具有保护作用.     

Gene mapping of Malaysian alpha thalassemias with alpha and zeta globin gene probes

Restriction enzyme analysis of the alpha and zeta globin genes was carried out in four cases of Hb Bart's hydrops fetalis, in three patients with Hb H disease without Hb CoSp, in three patients with Hb H disease with Hb CoSp, in 47 individuals with alpha thalassemia trait, and in 47 normal individuals. All four cases of Hb Bart's hydrops fetalis resulted from deletions of alpha 1 and alpha 2 globin genes which did not extend to the psi zeta 1 and zeta 2 globin genes. The same type of deletion was observed in alpha thal1 carriers, but two newborns (one Malay and one of Chinese extraction) had a nondeletion type of alpha thal1 which was confirmed by quantitative alpha globin gene analysis. In addition, two other newborns diagnosed as alpha thal1 trait carriers (one Malay, one Chinese) were shown to have a deletion of both alpha globin genes by quantitative alpha globin gene analysis, but further testing with zeta globin gene probe failed to reveal an abnormal fragment length characteristic of an alpha globin gene deletion. We believe that this last condition is due to a large deletion which includes all alpha globin genes and all zeta globin genes on the same chromosome. On another front, Bgl II restriction analysis of all four Hb Bart's hydrops fetalis cases and the alpha thal1 trait carriers showed a 10.5-kb Bgl II restriction fragment, in the hydrops fetalis as a single band, while in the carriers this 10.5-kb fragment was accompanied by the usual normal 12.5-kb and 11.3-kb fragments. We report that this 10.5-kb fragment, previously thought to be specific for the Southeast Asian alpha thal1 gene deletion, is also common in normal individuals. Nevertheless, digestion with other enzymes can clearly differentiate the alpha thal1 and normal genotypes. We distinguish the findings in the alpha thalassemias from the extensive DNA polymorphism in the region of the alpha and zeta globin genes.     

Pulmonary side effects of interferon-alpha therapy in patients with hematological malignancies

Several side effects of interferon-alpha-2b (IFN-alpha) therapy have been described. Pulmonary side effects have seldom been reported. The four patients we describe all developed respiratory disorders while being treated with IFN-alpha for hematological malignancies. We point out the similarities and differences noticed concerning the clinical course, pathological findings, and prognosis in the four different cases. Also, in addition to our review of the literature, we discuss the possible mechanisms involved in development of lung symptoms. In three patients the pulmonary disorder seems to have been caused by a cell-mediated immunological side effect in the form of interstitial pneumonitis. In one patient the symptoms were most likely caused by an autoimmunologic reaction, primarily engaging the vascular system, initially in the lungs. The single pathological finding existing in all four cases was the marked decrease in carbon monoxide diffusion capacity when performing spirometry. This was not necessarily associated with the existence of radiological findings. The decrease in diffusion capacity and the clinical symptoms were completely reversible in three of the patients, either spontaneously after the withdrawal of IFN-alpha or after treatment with corticosteroids. Our conclusion is that IFN-alpha, on rare occasions, can cause serious pulmonary side effects when used to treat both lympho- and myeloproliferative hematological malignancies. When pulmonary symptoms are evaluated during IFN-alpha therapy, spirometry, including estimation of carbon monoxide diffusion capacity, high-resolution computerized tomography, and ultracardiography should be used.     

Tumour necrosis factor-alpha plasma levels in elderly patients with Type 2 diabetes mellitus-observations over 2 years.

AIMS: The cytokine tumour necrosis factor-alpha (TNF-alpha) is involved in the development of obesity-linked insulin resistance. TNF-alpha plasma levels rise with increasing age and might thus also be related to metabolic control in Type 2 diabetes mellitus. We have studied the relationship of TNF-alpha plasma levels to glycaemic control in elderly patients with Type 2 diabetes over 2 years. METHODS: Clinical and laboratory data of 53 patients (26 women, 27 men) with Type 2 diabetes (mean age 71.6 +/- 5.6 years) were regularly evaluated over 2 years, and the relationship to anti-diabetic treatment regimens analysed. TNF-alpha plasma level was measured by a solid-phase enzyme amplified sensitivity immunoassay. RESULTS: TNF-alpha plasma levels increased significantly from 16.2 +/- 9.6 pg/ml at baseline to 28.0 +/- 13.8 pg/ml after 2 years (P = 0.028). HbA1c values also increased from 6.4 +/- 1.2% to 7.7 +/- 1.6% (P = 0.046). Mean body mass index of the patients remained almost constant, while a moderate increase in the percentage of body fat (34.5 +/- 7.0% to 35.3 +/- 6.9%; P= 0.061) and in waist-hip ratio was observed (0.86 +/- 0.04 to 0.88 +/- 0.04; P= 0.052). After adjustment for covariates multivariate analysis demonstrated that TNF-alpha plasma levels are positively related to the HbA1c values of the whole study population at the baseline control and after 2 years. TNF-alpha also revealed a positive correlation to the percentage of body fat. CONCLUSIONS: In elderly patients with Type 2 diabetes TNF-alpha plasma levels revealed a continuous increase during an observation period of 2 years. This increase in TNF-alpha plasma levels might add another aspect to the worsening of glycaemic control in the progression of Type 2 diabetes.     

Occurrence of clinical depression during combination therapy with pegylated interferon alpha or natural human interferon beta plus ribavirin

Aim: The onset of depression symptoms during pegylated interferon α plus ribavirin (PEG‐IFN/RBV) combination therapy has led to treatment discontinuation in some cases. In the present study, we conducted a questionnaire survey during treatment to determine whether natural human interferon β plus ribavirin (IFNβ/RBV) therapy is associated with a lower incidence of depression symptom onset compared with PEG‐IFN/RBV therapy. Methods: Seventy‐seven patients with chronic hepatitis C received PEG‐IFN/RBV (PR) or IFNβ/RBV (FR) therapy. A questionnaire survey was administered at the start of treatment, and at 4 and 12 weeks, using the Beck Depression Inventory II (BDI‐II) and the Pittsburgh Sleep Quality Index (PSQI). Results: BDI‐II scores in the PR group increased at 4 and 12 weeks, but remained unchanged in the FR group. At 12 weeks, the mean BDI‐II score and incidence of abnormalities with a BDI‐II score of ≥14 were significantly lower in the FR group than in the PR group. BDI‐II scores during IFNβ/RBV therapy in 11 patients currently using antidepressants remained unchanged up to 12 weeks. None of these 11 patients required addition or dose increases of antidepressants, and there was no evidence of worsened depression symptoms. Nine PR patients had BDI‐II scores of ≥14 and PSQI scores of ≥11 at 12 weeks. Conclusions: IFNβ/RBV therapy was associated with a lower incidence of depression symptom onset during treatment. In patients already diagnosed with depression, there was no evidence that IFNβ/RBV therapy caused any worsening of symptoms, indicating that IFNβ/RBV therapy is safe for patients with depression.     

Role of beta1- and alpha2c-adrenergic receptor polymorphisms and their combination in heart failure: a case-control study

BACKGROUND: Adrenergic activation has a central role in the development of HF. The function of the beta1- and the alpha2C-adrenergic receptors is influenced by gene polymorphisms: the beta1Arg389 variant is associated with increased beta1-receptor sensitivity and the alpha2C-receptor Del322-325 variant is associated with decreased alpha2C receptor function and increased norepinephrine release. We hypothesised that these polymorphisms could influence the prevalence of heart failure. METHODS: The role of the beta1- and alpha2C-adrenergic receptor gene polymorphisms as risk factors for heart failure (HF) was assessed in an Italian white Caucasian population using a case-control study design. Genomic DNA was analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RLFP). RESULTS: We compared 260 Caucasian patients with HF and 230 normal subjects. The beta1Arg389 allele was frequent both in the patients with HF (69%) and in the normal subjects (73%). The alpha2CDel322-325 variant was rare in both groups (9% and 8%, respectively). Patients homozygotes for either the beta1Arg389 or the alpha(2C)Del322-325 alleles had no increased risk of HF (odds ratio [OR], 0.8; 95%CI: 0.5-1.2 and OR, 0.8; 95% CI: 0.4-1.8, respectively). Patients homozygotes for both the beta1Arg389 and the alpha(2C)Del322-325 alleles had no increased risk of HF as well (OR: 0.6; 95% CI: 0.2-2.1). CONCLUSIONS: Beta1-ARs and alpha2C-ARs polymorphisms are not associated with an increased risk of HF in an Italian white Caucasian population.     

Similar effects of recombinant interferon-alpha-2b and natural interferon-alpha when combined with intra-arterial 5-fluorouracil for the treatment of advanced hepatocellular carcinoma.

AIM: Intra-arterial 5-fluorouracil (5-FU) plus interferon (IFN) combination therapy is effective against advanced hepatocellular carcinoma (HCC) with portal vein tumour thrombosis. In this study, we compared the efficiency and safety of recombinant IFN-alpha-2b with natural IFN-alpha as components of the combination therapy. METHODS: Consecutive HCC patients (n=31) with portal vein tumour thrombosis were enrolled in this prospective study. They received combination therapy of 5-FU and either recombinant IFN-alpha-2b (R group, n=15) or natural IFN-alpha (N group, n=16). We compared the two groups for the early response rate, adverse reactions, time to progression (TTP) and survival rates. In addition, we assessed the cost-effectiveness of each protocol. RESULTS: The early response rate (R: 26.7%, N: 31.2%), median TTP (R: 5.8 months, N: 5.6 months) and median survival time (R: 7.5 months, N: 6.5 months) were not significantly different between the R and N groups. There were no differences in adverse reactions between the two groups. The estimated cost-effectiveness ratio of recombinant IFN-alpha-2b was better than natural IFN-alpha. CONCLUSIONS: In our protocol of combination therapy, there were no significant differences between recombinant IFN-alpha-2b and natural IFN-alpha with regard to early response to therapy, adverse effects, TTP and survival rates. 5-FU could be combined with either recombinant IFN-alpha-2b or natural IFN-alpha, although the cost-effectiveness of the former warrants its use clinically.     

In vitro effect of thymosin-alpha1 and interferon-alpha on Th1 and Th2 cytokine synthesis in patients with chronic hepatitis C

Current evidence suggests that increased expression of Th1-associated cytokines is important for immune-mediated eradication of hepatitis C infection, while an increase in Th2-associated cytokines is associated with persistence of infection. In this study we evaluated the effects of thymosin-alpha1 (TA1), a naturally occurring thymic peptide, and interferon-alpha (IFN-alpha) on cytokine production in peripheral blood mononuclear cells from untreated patients with chronic hepatitis C. We examined the effect of incubation with TA1, IFN-alpha, or both, on production of Th1-associated cytokines (IL-2, IFN-gamma), Th2-associated cytokines (IL-4, IL-10), and synthesis of the antiviral protein 2',5'-oligoadenylate synthetase. TA1 treatment induced a significant increase in production of IL-2 and 2',5'-oligoadenylate synthetase. Smaller increases were also seen after treatment with IFN-alpha, while incubation with TA1 and IFN-alpha together led to an additive or synergistic effect. Incubation with TA1 resulted in a decrease in IL-4 and IL-10, whereas IFN-alpha increased these cytokines. The addition of TA1 to IFN-alpha significantly reversed this IFN-alpha-induced increase. Hence, TA1 treatment could benefit patients with hepatitis C infection by increasing the Th1-type response, fundamental for sustained clearance of hepatitis C; and by decreasing the Th2-type response, associated with persistence of viraemia.     

Peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1alpha) gene polymorphisms and their relationship to Type 2 diabetes in Asian Indians.

AIMS: The objective of the present investigation was to examine the relationship of three polymorphisms, Thr394Thr, Gly482Ser and +A2962G, of the peroxisome proliferator activated receptor-gamma co-activator-1 alpha (PGC-1alpha) gene with Type 2 diabetes in Asian Indians. METHODS: The study group comprised 515 Type 2 diabetic and 882 normal glucose tolerant subjects chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The three polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Haplotype frequencies were estimated using an expectation-maximization (EM) algorithm. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. RESULTS: The three polymorphisms studied were not in linkage disequilibrium. With respect to the Thr394Thr polymorphism, 20% of the Type 2 diabetic patients (103/515) had the GA genotype compared with 12% of the normal glucose tolerance (NGT) subjects (108/882) (P = 0.0004). The frequency of the A allele was also higher in Type 2 diabetic subjects (0.11) compared with NGT subjects (0.07) (P = 0.002). Regression analysis revealed the odds ratio for Type 2 diabetes for the susceptible genotype (XA) to be 1.683 (95% confidence intervals: 1.264-2.241, P = 0.0004). Age adjusted glycated haemoglobin (P = 0.003), serum cholesterol (P = 0.001) and low-density lipoprotein (LDL) cholesterol (P = 0.001) levels and systolic blood pressure (P = 0.001) were higher in the NGT subjects with the XA genotype compared with GG genotype. There were no differences in genotype or allelic distribution between the Type 2 diabetic and NGT subjects with respect to the Gly482Ser and +A2962G polymorphisms. CONCLUSIONS: The A allele of Thr394Thr (G --> A) polymorphism of the PGC-1 gene is associated with Type 2 diabetes in Asian Indian subjects and the XA genotype confers 1.6 times higher risk for Type 2 diabetes compared with the GG genotype in this population.     

In vitro effect of granulocyte-colony stimulating factor and all-trans retinoic acid on the expression of inflammatory cytokines and adhesion molecules in acute promyelocytic leukemic cells.

Differentiation therapy with all-trans retinoic acid (ATRA) represents a landmark approach in the treatment of acute promyelocytic leukemia (APL). However, a potentially fatal complication of retinoic acid (RA) syndrome occurs in about a quarter of patients and its pathophysiology is still unclear. In order to investigate whether or not the treatment with ATRA leads to increased elaboration of inflammatory cytokines and adhesion molecules by the APL cells, the expression of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-8, L-selectin and intercellular adhesion molecule-1 (ICAM-1) was examined in the APL cells after induction of differentiation with ATRA in the presence or absence of granulocyte-colony stimulating factor (G-CSF) or IL-3 in the present study. Cytokine elaboration by the treated cells was detected using both Northern blotting and enzyme-linked immunosorbent assay. Our results have shown that ATRA induces an increased expression of IL-8, IL-1beta, TNF-alpha and ICAM-1 in APL cells, which can be amplified by the addition of G-CSF. These data imply that the induction of inflammatory cytokines in APL cells may play an important role in the pathogenesis of RA syndrome. Furthermore, G-CSF, through its potent differentiating activity, may increase the risk of such complications during ATRA treatment.     

The level of Hb F-Sardinia (alpha 2A gamma 2(75)Ile----Thr) in the fetal hemoglobin of Sardinian beta-thalassemic homozygotes determined by isoelectric focusing

A simple thin-layer isoelectric focusing technique was used to separate Hb F-Sardinia, containing the A gamma T-globin chain, from the Hb F containing the G gamma- and the A gamma I-globin chains. The identity of the slow-moving Hb F fraction as Hb F-Sardinia was verified by PAGE. A negative correlation (R2 = 0.747, p less than 0.001) was found between the percent Hb F-Sardinia and percent G gamma-chain in homozygotes for beta-thalassemia. Of 31 Sardinian beta-thalassemic patients studied, 21 were homozygous and eight heterozygous for the A gamma T polymorphism with a gene frequency of 0.823. The mean values of Hb F-Sardinia were 39.1 +/- 5.9% for the homozygotes and 17.1 +/- 3.6% for the heterozygotes. The percentage of Hb F-Sardinia found in beta o-thalassemic newborns was similar to that of corresponding normal newborns who also had the A gamma T polymorphism. No measurable differences in the percent Hb F-Sardinia level were observed among beta o-thal patients who were polytransfused, beta o-thal patients studied before transfusion, and beta o-thal patients exhibiting the intermediate form of the disease who had never been transfused.     

Pioglitazone increases circulating adiponectin levels and subsequently reduces TNF-alpha levels in Type 2 diabetic patients: a randomized study.

BACKGROUND: Adipocytokines are involved in the development of insulin resistance and endothelial dysfunction in diabetic patients. However, the relationship between these factors remains unclear. We observed a chronological change in circulating adipocytokines and blood pressure levels with administration of oral hypoglycaemic agents in Type 2 diabetic (T2DM) subjects. METHODS: Thirty poorly controlled T2DM subjects (aged 60.1 +/- 1.5 years, 11 males and 19 females) were randomized into two groups: voglibose (initial dose 0.6 mg/day, increased to 0.9 mg/day) and pioglitazone (initial dose 15 mg/day, increased to 30 mg/day). RESULTS: Both treatment groups showed a similar improvement in glycaemic control. In pioglitazone-treated patients, circulating adiponectin levels were significantly increased from 4 weeks after the start of treatment, and until the end of the study at 12 weeks. Plasma tumour necrosis factor-alpha (TNF-alpha) levels were significantly decreased only at 12 weeks. In contrast, no significant changes in plasma adiponectin or TNF-alpha levels were observed in voglibose-treated patients. Plasma PAI-1 and leptin levels were not significantly changed at 12 weeks in either treatment group. Pioglitazone significantly decreased systolic and diastolic blood pressure levels at 12 weeks, but voglibose had no effect. CONCLUSION: In summary, pioglitazone caused an immediate increase in circulating adiponectin levels, followed by a reduction of TNF-alpha. The observed increase in circulating adiponectin could be related to decreases in both systolic and diastolic blood pressure.     

A novel mutation in the hepatocyte nuclear factor-1alpha/MODY3 gene in Chinese subjects with early-onset Type 2 diabetes mellitus in Taiwan.

AIMS: The goal of this study was to determine the frequency of mutation in hepatic nuclear factor (HNF)-1alpha, a gene recently implicated as causing maturity-onset diabetes of the young (MODY) and to analyse the respective clinical presentations in an ethnically Chinese population. METHODS: Fifteen unrelated subjects (nine females and six males) aged less than 35 years who had early-onset diabetes were analysed to test the possibility that mutation of the HNF-1alpha gene was responsible for this disorder. Genomic DNA extraction, polymerase chain reaction and DNA sequence analysis were performed accordingly. RESULTS: One patient with MODY had a novel missense mutation in exon 3 of the HNF-1alpha gene (Y218C) in a region of the protein that corresponds to a predicted DNA binding domain. CONCLUSIONS: A Y218C mutation in HNF-1alpha gene was identified in one family in Taiwan.     

Identification of new mutations in the hepatocyte nuclear factor 4alpha gene among families with early onset Type 2 diabetes mellitus.

AIMS: Mutations in hepatocyte nuclear factor (HNF)-4alpha gene located on chromosome 20q have been found to be responsible for the development of early onset Type 2 diabetes mellitus (DM). Through a national campaign, 53 families with autosomal dominant, early onset Type 2 DM (n=654) were assembled to determine the frequency of mutations in the HNF-4alpha gene and their contribution to the development of diabetes. METHODS: Twelve exons and the promoter region of the HNF-4alpha gene were screened in probands of the families by a double gradient, denaturing gradient gel electrophoresis (DG-DGGE) protocol combined with automated bi-directional sequencing of the PCR products of all heterozygous individuals. RESULTS: We detected two new mutations in the HNF-4alpha gene that changed the amino-acid sequence. The first mutation was a Gly-->Ser substitution in codon 115 within a highly conserved DNA binding domain, and all six carriers of this mutation had diabetes and low insulin secretion. The second mutation was an Ile-->Val substitution in codon 454 within the transactivation domain. It was carried by four family members, two of whom also carried a mutation in the HNF-1alpha gene. Of those having only the mutation in HNF-4alpha one had diabetes and the other had normal glucose tolerance and both were obese and hyperinsulinaemic. Thus, it is uncertain that this mutation is responsible for any of the diabetes in this family. CONCLUSION: We have found that mutations in the HNF-4alpha gene account for a small proportion, about 2-4%, of families with early onset, autosomal dominant, Type 2 DM in US Caucasians.     

Interferon alpha2b directly induces fibroblast proliferation and transforming growth factor beta secretion of macrophages

To elucidate the effects of interferon alpha2b (IFN-alpha) on normal human bone marrow, fibroblasts from patients without haematopoietic pathology were cultivated and used in stimulation experiments. Further, co-cultures with highly enriched fractions of megakaryocytes and bone marrow macrophages were analysed. In this context, the influence of cell-to-cell interactions and humoral factors was determined in transwell and neutralization studies. Finally, secretion of platelet-derived growth factor (PDGF) and transforming growth factor beta1 (TGF-beta1) by single megakaryocytes and macrophages was examined by using the reverse haemolytic plaque assay (RHPA). Following these experimental designs, a direct proliferative activity of IFN-alpha on bone marrow fibroblasts could be demonstrated. In the unstimulated co-cultures, the megakaryocyte- but not the macrophage-enriched fraction induced fibroblast growth and [3H]-thymidine uptake. This effect was dependent on cell-to-cell contact and also on the influence of TGF-beta and PDGF. In the megakaryocyte-enriched co-cultures, the fibroblast proliferation was not altered by IFN-alpha, but in the macrophage fibroblast cultures addition of IFN-alpha enhanced fibroblast growth and [3H]-thymidine uptake was distinctively higher than in the monocultures. This effect was not obvious in the transwell or neutralization experiments. Finally, IFN-alpha treatment exerted a significantly elevated TGF-beta1 secretion in single macrophages. Our findings are in keeping with the assumption that several pathomechanisms participate in IFN-alpha-induced myelofibrosis, including direct and indirect effects.     

Differential effects of interferon alpha-2b and beta on the signaling pathways in human liver cancer cells

Background Interferon (IFN) has been reported to reduce the incidence of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C and the recurrence of HCC after effective treatment. We examined the effect of IFNs on the proliferation and the signaling pathways of human HCC cells.Methods Cellular proliferation was examined by a modified 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Activities of signaling molecules were evaluated by Western blot analysis.Results Cellular growth was not significantly modulated by IFNα-2b or by IFN-β, even though the HCC cells expressed the IFN receptors. However, extracellular signal-regulated kinase (ERK)1/2 was activated by treatment with IFNα-2b, and both ERK1/2 and AKT were activated by treatment with IFN-β, implying a possible role in resistance to IFNs. Contrary to our expectations, inhibition of mitogen-activated ERK-regulating kinase (MEK) or phosphatidylinositol-3-OH kinase (PI3K) did not modulate the proliferation of HCC cells. Also, abrogation of the ERK1/2 and AKT signaling pathways did not affect cell-cycle arrest at the G1/S phase caused by IFNα-2b.Conclusions IFNα-2b and IFN-β activated ERK1/2 and/or AKT independently of modulating the proliferation of HCC cells and the cell-cycle machinery. A signal transduction-based approach for HCC treatment needs to focus on other possible signaling molecules besides ERK1/2 and AKT when challenged with IFNs.     

Enhanced oxidative stress in coronary heart disease and chronic heart failure as indicated by an increased 8-epi-PGF(2alpha)

The role of oxidation injury as an important factor in the pathophysiology of cardiomyopathy (CMP) has recently gained increasing interest. Semiquantitative analysis for isoprostane, 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)), and oxidised low-density lipoprotein (ox-LDL) of coronary vascular tissue samples derived from CMP patients revealed an increased extent and intensity of uptake as compared to the respective controls. To evaluate oxidative stress in vivo, we examined plasma, serum, salivary, and urinary 8-epi-PGF(2alpha) in patients with dilated CMP (n=20) and ischemic CMP (n=20) with decreased left ventricular ejection fraction (LVEF). Patients with coronary heart disease (CHD) (n=20) and 20 healthy, age-matched, and sex-matched controls were investigated in parallel. 8-Epi-PGF(2alpha) levels were correlated with the functional severity of heart failure [New York Heart Association (NYHA) classification] and LVEF. 8-Epi-PGF(2alpha) levels were matched according to risk factors (smoking and hypercholesterolemia) and were significantly higher in patients with CMP as compared to healthy controls and patients with CHD in all investigated compartments. A positive correlation between NYHA stages and 8-epi-PGF(2alpha), as well as a negative correlation to LVEF, could be demonstrated in a subgroup analysis. These findings reflect the enhanced oxidation injury in patients with CMP and, to a lesser extent, in CHD as compared to healthy controls, thus highly indicating the relevance of oxidative stress for the pathogenesis and progression of cardiovascular disease.     

Alpha-adrenergic blockers: mechanism of action, blood pressure control, and effects of lipoprotein metabolism

The sympathetic nervous system plays a major role in the pathogenesis of essential hypertension and is mediated by the alpha and beta receptors. The alpha receptor is divided into two types, alpha 1 and alpha 2, based on response to epinephrine and norepinephrine. alpha 1-Adrenergic receptors have a high affinity for drugs such as prazosin, doxazosin, and terazosin, which act to reduce blood pressure by selective blockade of the receptor. These agents provide a rational approach to the treatment of hypertension by correcting elevated total peripheral resistance, the fundamental hemodynamic abnormality in essential hypertension. In contrast, early alpha-adrenergic receptor blockers nonselectively blocked both alpha 1 and alpha 2 receptors and were unsuitable as antihypertensive agents because they induced tachycardia and patients developed a tolerance to them rapidly. alpha 1-Adrenergic blockers also have beneficial effects on plasma lipoproteins, tending to decrease levels of triglycerides and cholesterol and increase levels of high-density lipoprotein (HDL) cholesterol and the HDL cholesterol/total cholesterol ratio. beta-Adrenergic blockers, such as propranolol and atenolol, have been shown to have an adverse effect on the lipid profile by tending to increase levels of triglycerides and decrease HDL cholesterol. A number of mechanisms contribute to these effects, in particular, adrenergic modulation of lipoprotein lipase and the triglyceride secretion rate. Doxazosin has been shown to increase the activity of LDL receptors, which may be partly responsible for its beneficial effect on plasma lipids and lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)