Radiation produces differential changes in cytokine profiles in radiation lung fibrosis sensitive and resistant mice

We studied serum proteomic profiling in patients with graft versus host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) by two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis. The expression of a group of proteins, haptoglobin (Hp), alpha-1-antitrypsin, apolipoprotein A-IV, serum paraoxonase and Zn-alpha-glycoprotein were increased and the proteins, clusterin precursor, alpha-2-macroglobulin, serum amyloid protein precursor, sex hormone-binding globulin, serotransferrin and complement C4 were decreased in patients with extensive chronic GVHD (cGVHD). Serum haptoglobin (Hp) levels in patients with cGVHD were demonstrated to be statistically higher than in patients without cGVHD and normal controls (p < 0.01). We used immunoblotting and PCR in combination with 2-DE gel image analysis to determine Hp polymorphisms in 25 allo-HCT patients and 16 normal donors. The results demonstrate that patients with cGVHD had a higher incidence of HP 2-2 phenotype (43.8%), in comparison to the patients without cGVHD (0%) and normal donors (18.7%), suggesting the possibility that specific Hp polymorphism may play a role in the development of cGVHD after allo-HCT. In this study, quantitative serum Hp levels were shown to be related to cGVHD development. Further, the data suggest the possibility that specific Hp polymorphisms may be associated with cGVHD development and warrant further investigation.     

The toxic oil syndrome: An example of an exogenously induced autoimmune reaction

The toxic oil syndrome (TOS) is a chemically induced autoimmune-like reaction in humans. The etiologic agent(s) have so far not clearly been identified. A short overview is given on this disease which is associated with a unique antibody specificity to cryptic epitopes of C-reactive protein in affected patients. In addition a murine model for TOS is described which suggests that genetic suscepbility might play a role in inducing a similar syndrome in mice. These differences are reflected in the immunological alterations and cytokine production caused by the toxicant.Abbreviations CRP C-reactive protein - OAA oleic acid anilide - PAP 3-phenylamino-1,2-propanediol - TOS toxic oil syndrome     

Glycosylation of alpha-1-proteinase inhibitor and haptoglobin in ovarian cancer: evidence for two different mechanisms

The change in glycosylation of the two acute-phase proteins, alpha-1-proteinase inhibitor (API) and haptoglobin (Hp), in progressive ovarian cancer is different. This has been shown by monosaccharide analysis and lectin-binding studies of proteins purified from serum. In the glycan chains of API, there is decreased branching (more biantennary chains), less branches ending in alpha 2-3 sialic acid, more branches ending in alpha 2-6 sialic acid and more fucose, probably linked alpha 1-6 to the core region. On the other hand, Hp shows increased branching (more triantennary chains), more branches ending in alpha 2-3 sialic acid, less branches ending in alpha 2-6 sialic acid, and more fucose, probably in the alpha 1-3 linkage at the end of the chains. This is surprising because API and Hp are thought to be glycosylated by a common pathway in the liver. We have also shown that the fucose-specific lectin,lotus tetragonolobus, extracts abnormal forms of both Hp and API in ovarian cancer, but the expression of this Hp is related to tumour burden and the expression of this API is related to lack of response to therapy. It is suggested that this difference in the behaviour of API and Hp in ovarian cancer may be associated with the different changes in their glycosylation. Of the many mechanisms that could explain these findings, a likely one is that a pathological process is removing API with triantennary chains from the circulation. In addition to their normal roles (API-enzyme inhibitor and Hp-transport protein) these proteins are reported to have many other effects in biological systems, such as immunosuppression. As correct glycosylation of API and Hp is required for their normal stability/activity, changes in glycosylation could affect their functions in ovarian cancer and these modifications could alter the course of the disease.     

Genetic serum protein markers (HP,TF, GC and PI) in eight Slovakian population samples

1194 individuals from eight different regions of Slovakia have been typed for haptoglobin (HP) types and for transferrin (TF), group specific component (GC) and alpha-1-antitrypsin (PI) subtypes. Whereas the HP allele frequencies do not show a remarkable regional variability within Slovakia, this could be demonstrated concerning the TF, GC and PI allele frequencies. The reason for these distribution heterogeneities seems to be due to the incomplete panmixia of the Slovakian population by which local variations in the distribution of genetic markers could be maintained.     

Induction of c-fos messenger RNA by 3-(N-phenylamino)-1,2-propanediol esters, compounds related to Toxic Oil Syndrome

The Toxic Oil Syndrome (TOS) was a toxic epidemic disease, related to the consumption of rapeseed oil denatured with aniline that affected more than 20,000 people in Spain and resulted in more than 330 deaths after its sudden appearance in 1981. It has been reported that the fatty acid esters of 3-(N-phenylamino)-1,2-propanediol (PAP) have shown a strong association with TOS. These PAP-esters could be absorbed and metabolized in a similar way than phospholipids. This is of interest because some products of phospholipid metabolism are important mediators in downstream pathways involved in the regulation of different nuclear factors. In particular, phospholipase D activity is involved in the activation of c-fos. Thus, we have investigated the effect of different PAP-esters in the induction of c-fos in lung fibroblasts. Results indicate that PAP-esters rapidly induced the expression of c-fos in a dose-dependent manner. In addition, both butanol and propranolol prevent this induction pointing to the involvement of phospholipase D in this activation. These results suggest that deregulation of some nuclear factors such as AP-1 could be involved in the pathogenesis of TOS.     

Relationship between Hp1S and Hp2 gene frequencies among human populations

In this work, we present new data on the Hp1alpha- and Hp2alpha-chains polymorphism in different populations. We confirm the singularity of the geographical distribution of the Hp2 alleles in our samples. The analysis of the results shows that a significant correlation exists in the population between the Hp1S and Hp2 gene frequencies. An additional Hp1alpha-chain variant is described in a Pyrenean sample.     

Distribution of haptoglobin subtypes in French Basques.

THE Hl1f, Hp1s and Hp2 gene frequencies were studied in two French population samples: one from the Toulouse area and one from a Basque district. The hp alpha1F and alpha1S polypeptide chains were determined by a simple technique. The observations were in accordance with previous findings in Caucasoid populations. The frequency of the Hp1S gene was slightly higher in the Basque sample than in the group from Toulouse.     

Purification of human plasma haptoglobin by hemoglobin-affinity column chromatography

Haptoglobin (Hp) is an acute-phase protein; its plasma levels increase consistently in response to infection and inflammation. The concentration of human plasma Hp is ranged between 1 and 1.5 mg/ml. Similar to blood type, individual human Hp is classified as Hp 1-1, 2-1, or 2-2. The structural and functional analysis of the Hp, however, has not been studied in detail due to its difficult isolation procedure. Previously, we reported a single step for the purification of porcine Hp. In this study, we established a purification method using a high capacity hemoglobin-affinity column. Briefly, DEAE-purified human hemoglobin was first coupled to Sepharose 4B to prepare an affinity column in a 15-ml bed volume. Following a flow through of human plasma and an extensive wash, the bound material was eluted with a solution of 0.15 M NaCl, pH 11 (adjusted by ammonium), to remove low-affinity bound proteins. The high-affinity bound Hp was then eluted with 0.15 M NaCl containing 5 M urea, pH 11, and collected in tubes containing 100 microl of 1 M Tris buffer, pH 7.0. The biological activity of dialyzed Hp was retained as it formed a complex with hemoglobin on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure, approximately 10 mg of Hp 1-1, with homogeneity greater than 96%, was obtained from 15 ml of human plasma. Affinity purified Hp 2-1 or 2-2, however, contained trace amounts of apoA-I with the similar approach. The Hp could be further purified by HPLC using a Superose 12 gel-permeation chromatography, if desired, to achieve 100% purity. All the phenotypes of purified Hp consisted of alpha and beta chains on SDS-PAGE in the presence of a reducing reagent, further confirmed by a Western blot analysis. We conclude that human hemoglobin-affinity column was most suitable for the isolation of Hp 1-1 in large quantities. Whereas, one additional step using a gel-permeation was necessary for that of Hp 2-1 and 2-2.     

Study of serum haptoglobin and its glycoforms in the diagnosis of hepatocellular carcinoma: a glycoproteomic approach

Increased serum haptoglobin concentration and changes in its glycosylation have been reported in certain cancer types. Information for hepatocellular carcinoma (HCC) has not yet been available. In this study, we aimed to carry out a systematic analysis of serum concentrations of haptoglobin (Hp) and its glycoforms in the patients with HCC and noncancer patients only with chronic liver diseases (CLD) and to examine their clinical values. This study was divided into two major parts, (1) measurement of serum Hp concentration, and investigation of its value in the diagnosis of HCC, and (2) quantitative analysis of Hp glycoforms with alpha-2,6-sialylation and/or alpha-1,6-fucosylation by using lectin affinity purification and 2D gel electrophoresis and investigation of their relationships with tumor stage. The concentrations of serum Hp in HCC patients were significantly higher than those in noncancer patients with CLD. With the use of serum concentrations of Hp and alpha-fetoprotein, a logistic regression (LR) model was developed from the training data set and used to classify the validation cases. At a specificity of 95%, the sensitivity for HCC detection was 79%. Comparing serum concentrations of alpha-2,6-sialylated Hp (S-Hp) and alpha-1,6-fucosylated Hp (F-Hp) between HCC and CLD patients suggests that purification of S-Hp and F-Hp could enrich the glycosylation variants associated with HCC. 2D gel analysis of S-Hp and F-Hp identified a total of 18 glycoforms. A unique pattern of Hp glycoforms comprising both hypersialylated fucosylated and hyposialylated fucosylated species was found in the HCC patients. Serum concentrations of these glycoproteins were significantly higher in the patients with advanced tumors, suggesting their tumor-specific nature. We have shown that serum Hp is a potential biomarker in the diagnosis of HCC. The combined use of Hp and AFP could greatly improve the diagnostic accuracy. A unique pattern of Hp glycoforms with altered sialylation and fucosylation is specific to HCC and associated tumor progression.     

Haptoglobin polymorphism--not only a genetic marker]

The biological activities of the haptoglobin polymorphism are controlled by continuous DNA sequences coding for the HP alpha and Hp beta polypeptide chains and forming with a linked Hp related gene the haptoglobin gene complex on chromosome 16. Probably, this DNA domain originates from the gene family of the serine proteases after having lost the informations for the proteolytic functions. Instead of this, the haptoglobins have acquired other qualities, among them the hemoglobin binding capacity, inserted into the Hp beta chain. The Hp polymorphism is constituted by the evolutionary progressive DNA sequences for the Hp alpha chains, which probably have activation functions. The haptoglobins display immunoregulative abilities, which can be immunosuppressive by inhibition of the lymphocyte reactivity or immunoinductive by influencing the IgM biosynthesis, adapted to the functional requirements. In this field, Hp 2-2 has a stronger effect than the two other Hp types. Moreover, the haptoglobins inhibit the prostaglandin synthesis and protect against harmful oxidation processes. These qualities are based on the hemoglobin binding ability and can be realized by Hp 1-1 with the comparatively highest efficacy. Further on, the haptoglobins are protease inhibitors. Finally, Hp 2-2 is associated with higher albumin and ceruloplasmin serum levels than Hp 2-1 and Hp 1-1. Evidently, the haptoglobins are inserted into a widely ramified network of biological functions. The selective advantages and disadvantages of the Hp polymorphism are noticeable under pathological conditions in case of malignant tumors, inflammations, autoimmune diseases, allergic illness, affective psychoses and affective lability favouring addiction.     

Comparison of the chemical, physical, and survival properties of normal and Z-variant alpha-1-antitrypsins.

A procedure has been developed for the purification of Z-type alpha-1-antitrypsin (alpha-1-AT) which is rapid, gentle, and results in good yields. From 4 units (750 ml) of fresh human plasma, obtained from two individuals possessing the Pizz phenotype, 53 mg of pure Z-type alpha-1-AT was obtained. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis, both in the presence and absence of sodium dodecyl sulfate, and by analytical ultracentrifugation. When compared to pure alpha-1-AT from plasma of individuals possessing the normal PiMM phenotype, the two proteins were indistinguishable with respect to amino acid composition, sedimentation coefficient (s20w of 3.33 for both M and Z), molecular weight (51,000 by sodium dodecyl sulfate gel electrophoresis and 47,000 by sedimentation equilibrium for both M and Z), and trypsin-combining ratio (0.91 for Z and 0.99 for M). The only difference which was observed between the variant forms of alpha-1-AT was in the carbohydrate composition. The Z-type alpha1-AT contains between 20 and 25% less carbohydrate than the M-type alpha-1-AT. Specifically, the Z-type alpha-1-AT is deficient in 1 glucosamine residue, 3 neutral sugar residues (1 mannose and 2 galactose), and 2 sialic acid residues. Although the Z-variant is deficient in sialic acid, its survival time in the serum of a rabbit was not significantly different from that of M-type alpha-1-AT.     

Influence of N-phenyllinoleamide from toxic oil samples on the lipoxygenase metabolism of exogenous arachidonic acid in mouse peritoneal macrophages.

N-phenyllinoleamide (NPLA) is a useful marker for adulterated oil samples associated with cases of toxic oil syndrome (TOS). To date, NPLA has not reproduced the human poisoning episode in experimental animal models and, thus, its pathological role in the syndrome remains controversial. The present report describes the effect of NPLA on the lipoxygenase metabolism of exogenous arachidonic acid (AA) in mouse peritoneal macrophages (MPM). Results show that MPM cells exposed to 1mM NPLA for 2 h, when subsequently incubated with exogenous 3H-AA, undergo a significant increase in the biosynthesis of 3H-12-hydroxyeicosatetraenoic acid (3H-12-HETE) whereas levels of 3H-15-HETE are relatively stable. These data indicate that NPLA selectively potentiates the lipoxygenase metabolism of exogenous AA, supporting the possible implication of lipid peroxidative processes in the ethiopathology of TOS, although the relatively high NPLA concentration required 'in vitro' makes it unlikely that this xenobiotic could be directly related to human toxicity.     

Isolation of tri-antennary enriched and tri-antennary depleted alpha-1-protease inhibitor

Alpha-1-protease inhibitor, (alpha-1-PI), the major inhibitor of serine proteases in human plasma, has three asparagine-linked carbohydrate chains located at positions 46, 83 and 247. The protein has a microheterogeneity which is seen on isoelectric focusing and which is a result of whether the various carbohydrate chains are in bi- or tri-antennary forms. Tri-antennary enriched forms of alpha-1-PI are associated with inflammation. By using a combination of three methods, reductive salting out, Sepharose-bound Concanavalin A affinity chromatography, and Sepharose-bound anhydrochymotrypsin, biologically active alpha-1-PI was obtained in tri-antennary enriched and tri-antennary depleted forms. These preparations should be useful for studies on the physiological role of the carbohydrate moiety in alpha-1-PI.     

The sugar chain structures of ABO blood group active glycoproteins obtained from human erythrocyte membrane

The glycoproteins of human erythrocyte membrane have two groups of sugar chains with blood type ABH determinants, which are quite distinct in their molecular sizes. A neutral sugar chain and an acidic sugar chain, which belong to the small size group, were isolated from the glycoproteins obtained from the erythrocyte of blood type O individuals, and their structures were elucidated as Fucalpha1 leads to 2Galbeta1 leads to 3N-acetylgalactosaminitol and Fucalpha1 leads to 2Galbeta1 leads to 3(AcNeualpha2 leads to 6)N-acetylgalactosaminitol, respectively. The molecular weight of the large sugar chains with ABH determinants were estimated to be more than 4000. Both large and small neutral sugar chains of membrane glycoproteins obtained from blood type O erythrocyte could serve as acceptors of alpha-N-acetylgalactosaminyltransferases purified from milk of blood type A1 and A2 individuals, producing the same radioactive sugar chain distribution patterns. However, the acidic sugar chain with the H determinant could not serve as an acceptor of these enzymes.     

Androgen metabolism in the juvenile oscine forebrain: a cross-species analysis at neural sites implicated in memory function.

Juvenile songbirds are useful models for studying the neural bases of memory. Memory-reliant behaviors demonstrated at this stage include song learning (most songbirds) and food caching (food-storing songbirds). Sex steroids are implicated in the modulation of memory processes in several vertebrates. The songbird forebrain expresses aromatase, 5alpha-reductase and 5beta-reductase, enzymes which convert testosterone to estradiol, 5alpha-, and 5beta-dihydrotestosterone, respectively. To explore the role of local androgen metabolism on memory processes, we documented the activities of these enzymes in the anterior neostriatum (NAN), caudomedial neostriatum (NCM), and hippocampus (HP) of four species of juvenile songbird, two of which are food storers. Areas were dissected, homogenized, and provided with radiolabeled substrate; and formed estrogens, and 5alpha- and 5beta-reduced androgens were measured. In the NAN, 5beta-reductase was the predominant enzyme, suggesting that local inactivation of testosterone may preserve the sensitive period of song acquisition. In the NCM, estrogens were formed in abundance despite high 5beta-reductase, suggesting that locally high estrogen synthesis may play a role in processes of song perception. In the HP, both estrogens and 5alpha reduced androgens were formed, suggesting that HP function may be modulated by both estrogens and androgens. Finally, a derived measure of steroid-differential reveals that food-storing songbirds differ from nonstorers in the steroidal milleiu within the HP, but not in the NAN or NCM. Thus, distinct loci within the juvenile songbird forebrain are exposed to different patterns of androgen metabolites. This local conversion may play a role in the neuroendocrine modulation of memory in these birds.     

Genetic markers in alcoholic liver cirrhosis.

11 genetic markers were typed in 157 individuals suffering from alcoholic cirrhosis, and compared with a random sample of healthy individuals. No significant differences were found for transferrin, specific group component, orosomucoid, esterase D, phosphogluconate dehydrogenase and adenylate kinase. Strong associations between alcoholic cirrhosis and alpha-1-antitrypsin PI*Z allele, haptoglobin HP*1 allele and acid phosphatase ACP AC phenotype were observed. The biological significance of these associations and their relationships with the development of alcoholic cirrhosis are also discussed.     

Effect of haptoglobin subtypes on serum lipid levels

Haptoglobin (Hp) subtypes were analysed by two-dimensional high-resolution gel electrophoresis in 81 Norwegian individuals with moderate hypercholesterolemia and in 316 Norwegian control subjects. The frequencies of the genes Hp2SS and Hp2SF were higher in individuals with hypercholesterolemia than in controls but the differences did not reach statistical significance (p = 0.087). Within the control population, no effect of the different Hp subtypes was found on total serum cholesterol, triglycerides or high-density lipoprotein (HDL) cholesterol. However, in the controls a significantly higher frequency of Hp2-2 types was found among those with HDL cholesterol values in the upper quartile as compared to those with HDL cholesterol in the lower quartile. A similar phenomenon was not uncovered in analyses of total serum cholesterol or triglycerides. Our results are in agreement with others which indicate that genes belonging to the Hp polymorphism play a role in predicting an individual's total serum cholesterol level. However, our data indicate that the cholesterol effect is on the HDL rather than on the total cholesterol level.     

Vacuolating cytotoxin A is associated with increased thrombin generation in gastric mucosa

BACKGROUND: Activation of the coagulation system is a critical response for both the repair of tissue injury and the host defense against microbial pathogens. Activation of the coagulation cascade culminates with the generation of thrombin. In vitro studies have shown that thrombin protects gastric epithelial cells from injury. The present study was undertaken to assess in vivo the relationship between gastric intramucosal generation of thrombin and Helicobacter pylori infection. MATERIALS AND METHODS: This study comprised 59 patients with gastroduodenal disorders. There were 27 patients with H. pylori infection (Hp+), 14 without it (Hp-), and 18 patients with cured H. pylori infection (Hp c). The gastric intramucosal concentrations of thrombin-antithrombin complex (TAT), epidermal growth factor (EGF), prostaglandin E2 (PGE2), and vacuolating cytotoxin A (VacA) were measured by specific immunoassays. RESULTS: The level of TAT was significantly increased in patients with Hp+ compared to Hp- and Hp c. The levels of TAT, EGF and PGE2 were higher in VacA (+) patients than in those with VacA (-). VacA induced significant expression of tissue factor in gastric epithelial cells in vitro. The gastric intramucosal level of VacA antigen was proportionally and significantly correlated with TAT, EGF and PGE2 in Hp+ patients. The level of TAT was proportionally and significantly correlated with EGF in Hp+ patients but not in Hp- and HP c patients. CONCLUSIONS: These results showed that VacA produced by H. pylori is associated with increased thrombin generation, and that thrombin may play a protective role in H. pylori-associated gastroduodenal disorders.     

Altered glycosylation and expression of plasma alpha-1-acid glycoprotein and haptoglobin in rheumatoid arthritis

Altered glycosylation patterns in plasma proteins are found to be associated with the pathogenesis of various malignancies and autoimmune disorders. Our previous studies demonstrated the occurrence of some differentially glycosylated plasma proteins in rheumatoid arthritis (RA) patients. The current study was conducted to evaluate the alterations in expression and glycosylation of major acute phase proteins from wheat germ agglutinin enriched RA patients' plasma. Immunoblotting studies revealed a significant enhancement in the plasma levels of alpha-1 acid glycoprotein (AGP) and haptoglobin (Hp) in RA patients with respect to healthy controls. Monosaccharide analysis by high performance anion exchange-chromatography with pulse amperometric detection showed significant variations in the relative percentage of galactose, glucosamine and mannose in AGP and of mannose in Hp in RA patients. Altered patterns of mannosylation in AGP and Hp were also established by enzyme linked immunosorbent assay and Western blotting using Concanavalin-A lectin. These results could give information for understanding the disease pathogenesis and may provide an insight into the development and progression of the disease.     

Gas chromatographic-mass spectrometric determination of brain levels of alpha-cholest-8-en-3beta-ol (lathosterol)

A gas chromatographic-mass spectrometric (GC-MS) method is proposed for the detection and quantification of lathosterol in rabbit brain. This compound is one of the most important precursors of the cholesterol synthesis. The interest in brain cholesterol metabolism is growing nowadays since it was described to play an important role in some neurodegenerative disorders such as Alzheimer's disease and Multiple Sclerosis. The analytical methodology proposed involves a liquid-liquid extraction procedure (LLE) followed by a silylation step previous to the GC-MS analysis. The chromatographic separation was performed by using a low bleed HP5-MS fused silica capillary column. A clean up is not necessary when using single-ion monitoring (SIM) mode. The molecular ion appears at 458 m/z; being as well the base peak. Alpha-naphtol was used as an internal standard. The detection limit obtained was 0.09 microg mL(-1). The method was applied to the determination of brain lathosterol levels in rabbits fed with different types of diets (control and atherogenic, supplemented or not with natural polyphenolic antioxidants). The quantification of the compound in samples showed a reduction, after 1 month, of this precursor of cholesterol synthesis in groups fed with antioxidant supplemented diets.