Microarray analysis reveals influence of the sesquiterpene lactone parthenolide on gene transcription profiles in human epithelial cells.

Sesquiterpene lactones are known for their anti-inflammatory activity which has been proven in various assays on DNA, mRNA and protein level. Here we report on the change in the gene expression profile in TNF-alpha stimulated human 293 cells after treatment with parthenolide using a cDNA microarray analysis. Twenty-one of 7028 genes were found to be up- and 18 down-regulated. They encode for chemoattractants, immune system proteins, glycoproteins, metabolism, serine proteinases, and transcription factors. Confirmatory analyses were carried out using quantitative real-time RT-PCR (TaqMan). Additional studies with selected genes revealed the concentration-dependent influence of parthenolide on the expression of these genes.

Biochemical studies of Xenopus laevis lymphocytes surface immunoglobulins.

Cell surface immunoglobulins of adult Xenopus laevis splenic small lymphocytes were analysed utilizing direct immunofluorescent staining and lactoperoxidase-catalysed radioiodination followed by immunoprecipitation of Ig molecules and characterization on SDS-PAGE. Nearly 30% of splenic lymphocytes are surface Ig positive. The HMW and LMW Ig classes are present on the surface of 23% and less than 5% of the cells, respectively. The mu chains of membrane HMW Ig have an apparent m.w. of 84,000 versus 73,000 for the mu chains of serum HMW Ig. Using immunofluorescent technique, we previously reported the absence of Ig molecules on the surface of larval Xenopus thymocytes. When the lactoperoxidase radioiodination technique was used, no cell surface Ig molecules could be isolated from Xenopus thymocytes.

Relation between the heavy chain complementarity region 3 characteristics and rheumatoid factor binding properties.

Among the rheumatoid factors (RFs), monospecific and polyspecific types can be distinguished. However the molecular basis responsible for their different specificity is not well understood. In a previous report, we have shown that the binding of the majority of the polyspecific antibodies is salt-sensitive. No binding to IgG was observed under high ionic strength (0.3-0.5 M NaCl). This salt-sensitivity was only observed for 18% of the monospecific RFs. Here, we have analyzed 14 RFs representing the 3 different groups (6 salt-insensitive monospecific, 4 salt-sensitive monospecific and 4 salt-sensitive polyspecific RFs). By analysis of the amino acid composition and the distribution of polar and non-polar residues of their heavy chain complementarity-determining region 3 (H-CDR3) in relation to mono/polyspecificity, salt-sensitivity and reactivity against human IgG subclasses, we have identified common structural features responsible for their different binding properties. Salt-sensitive RFs (mono as well as polyspecific antibodies) were characterized by long H-CDR3's (15.3+/-2.7) that contained large numbers of hydrophilic residues such as arginine and serine, while salt-insensitive RFs had more hydrophobic H-CDR3's of smaller length (11.3+/-2.4). In addition, for the monospecific RFs, remarkably similar hydrophilicity H-CDR3 profiles were found that were correlated with their specificity for IgG subclasses. These observations confirm the importance of the H-CDR3 for the binding of RFs to IgG. Furthermore, on the basis of their shorter H-CDR3's and their rather unique H-CDR3 hydrophilicity profiles, it is likely that the majority of the monospecific RFs should be considered as a group of RFs that is independent of the polyspecific RF repertoire.

[Is HbA1c appropriate for the screening of diabetes in general pratice?]. / L'HbA1c peut-elle être utilisée par le praticien pour le dépistage du diabète?

The measurement of glycated haemoglobin (HbA(1c)) is a practical and more sensitive tool than fasting plasma glucose (FPG) in screening type 2 diabetes in current practice. Its use has been limited so far by the variability of the analytical methods. The standardization process is going on, and many laboratories are currently using valid methods. Our study is consistent with the results of other groups who recommended this measurement to identify undiagnosed diabetic patients, that are about 25% to 30% in the French population. The demonstration was provided through a survey including a screening step by both HbA(1c) and G0, and a second exam with a 2 hr OGTT in a sample of positive screenees according to at least one criterion (HbA(1c) >=6% or G0 >=1.26 g/L), as well as in a sample of negative screenees. We showed that nine confirmed diabetic subjects out of ten had HbA(1c) >=6% at the screening step, while only a half had G0 >=1.26 g/L. Conversely, 22% of the positive screenees according to HbA(1c) were not confirmed as diabetic by the OGTT, including however more than half with abnormal glucose values. A chart for practical use is proposed to define patients at risk, the process of screening, and the patient follow-up according to the results of the tests.

Maturation of the mitogen responsiveness, and IL2 and IL6 production by neonatal swine leukocytes.

The maturation of the immune system of neonatal piglets was studied by following changes in the phenotypic composition and function of blood-borne leukocytes. The proportion of mature T and B lymphocytes decreased in the first week of birth and the circulating cells had poorly developed capacities to respond to mitogens and to secrete interleukins. From the end of the first week, however, there was a steady increase in the proportion of mature T cells (CD4+ and CD8+) and B cells in blood until 6-7 weeks after birth, when the study was ended. By 3-4 weeks, the relative proportions of different lymphocyte subsets resembled an adult-type composition. As they increased in prevalence, lymphocytes also developed capacities to proliferate and secrete interleukins. Proliferative responses to T-cell and B-cell mitogens reached adult levels within 2 weeks and 4-5 weeks, respectively. Blood leukocytes produced large quantities of IL6 by 1-2 weeks after birth and IL2 by 2-3 weeks. In contrast to lymphocyte patterns, the myeloid and granulocyte lineages were dominant at birth but then declined steadily. Unlike lymphocytes, the monocytes, macrophages and granulocytes appeared to be fully functional from the time of birth and exhibited a strong oxidative burst after appropriate stimulations. The magnitude of this response remained constant over the first 6-7 weeks. These results indicate that the first 3-4 weeks of post-natal life are a particularly susceptible interval for newborn piglets because constitutive and functional components necessary for specific cellular immune responses remain immature. This deficit may be offset by non-specific cellular mechanisms and maternally derived antibodies.

Modulation of interleukin production by ascorbic acid.

We studied the influence of ascorbate (vitamin C) on peripheral blood mononuclear cells (PBMC) of pigs with hereditary deficiency in ascorbate synthesis. Groups of animals were depleted of, or supplemented with dietary ascorbate for up to 5 weeks. B lymphocytes and T lymphocyte subsets differed in the two experimental groups only marginally and transiently as determined by analysis of cell surface markers. The proliferative response of PBMC to B and T lymphocyte mitogens was lower in depleted as compared to supplemented animals. Interleukin (IL)-2 and IL-6 were determined by bioassays and were secreted within few hours after mitogenic activation of PBMC which contained normal physiological concentrations of ascorbate. IL-2 production peaked at about 24 h of in vitro culture after Con A activation, but it lasted for 2-3 days after PWM activation. The production of IL-2 and IL-6 were compared during systemic depletion and supplementation with ascorbate. Depleted PBMC produced IL-2 which accumulated in cultures instead of being rapidly consumed by IL-2 dependent cell growth. This suggests that cellular ascorbate influences the production of IL-2. Secretion of IL-6 by mitogen activated PBMC was also affected by prolonged dietary ascorbate depletion. The results suggest that ascorbate levels exert an early effect on immune homeostasis via reactive oxygen intermediates (ROI)-dependent expression of interleukin genes, since the transcription factor NF-kappa B is sensitive to ROI and regulates the expression of interleukin genes.

Dependence of growth, bone metabolism and functions of polymorphonuclear leukocytes on ascorbic acid in pigs.

Pigs with hereditary ascorbate deficiency (OD pigs) were depleted of, or supplemented with, ascorbic acid by respective diets. Depletion of young (i.e. 5-7 weeks old) animals for at least three weeks had a negative effect on growth, body temperature and levels of bone alkaline phosphatase and induced symptoms of scurvy. Doses of 5 mg ascorbic acid kg-1 body weight day-1 were sufficient to reverse these effects. The level of ascorbic acid sharply decreased in plasma within one week of depletion, whereas in leukocytes it declined more slowly and to a lower extent. Bone alkaline phosphatase levels substantially declined in ascorbic acid depleted animals. Supplementation with > 100 mg ascorbic acid kg-1 body weight day-1 did not improve growth. Dietary ascorbic acid was absorbed from the intestinal lumen into the blood within less than 1 hour and reached a peak 5-6 hours after the meal. The extent of this absorption depended on the systemic ascorbic acid level. Ascorbic acid influenced leukocyte function, since the production of reactive oxygen intermediates by polymorphonuclear leukocytes decreased in supplemented animals. Thus, this animal model permits to establish the level of dietary ascorbic acid that is critical for growth of pigs as well as to study its absorption into the blood and the associated alterations in polymorphonuclear leukocytes and bone metabolism.

Influence of ascorbic acid on the response to mitogens and interleukin production of porcine lymphocytes.

The influence of ascorbic acid (AA) on lymphocyte functions was examined in vitro and ex vivo in peripheral blood mononuclear cells (PBMC) of vitamin C-deficient pigs, which are unable to synthesise ascorbic acid. AA is accumulated to physiological levels in PBMC in vitro. The cell proliferation induced by T lymphocyte mitogens was unaltered at all AA concentrations tested (0-400 micrograms/ml, i.e., 0-2.3 mM). Conversely, the response to pokeweed mitogen (PWM) which activates T and B lymphocytes was significantly reduced with increasing intracellular and extracellular AA concentrations. The response to lipopolysaccharide (LPS) showed a tendency to increase at low (9 microM) and was significantly reduced at high AA concentrations (> 36 microM). The IL2 production induced by PWM (but not by concanavalin A (Con A) or phytohemagglutinin (PHA)) decreased at high AA (> 142 microM). In contrast, IL6 production induced by mitogens was not dependent on AA concentrations. In concordance with these results, AA-depleted PBMC which were obtained from pigs that were fed an AA-free diet, displayed an increasing response to LPS and PWM. Collectively, the data indicate that ascorbic acid selectively influences the proliferation of B lymphocytes and negatively acts on IL2 production by T lymphocytes when a threshold of saturation is exceeded.

[Clinical, biological and developmental aspects of alcoholic ketoacidosis]. / Aspects cliniques, biologiques et évolutifs de l'acidocétose alcoolique.

OBJECTIVES: To review cases of alcoholic ketoacidosis in order to better ascertain therapeutic management. METHODS: The medical files of 32 alcoholic patients with ketoacidosis hospitalized in the Saint-Pierre general hospital of the Reunion island from January 1, 1991 through 31 August 1994 were analyzed. RESULTS: There were 18 women and 14 men, mean age 47 years. The first clinical signs were predominated by digestive (n = 22) or neurological disorders (n = 10). Acidosis was severe (mean pH = 7.12) and always associated with a wide anion gap (mean anion gap = 35). There were 3 types of glycemic status: hypoglycemia 10 cases, normal or subnormal glycemia in 19 cases (mean glycemia = 9.3 mmol/l) and hyperglycemia above 20 mmol/l in 3 cases. Hypophosphatemia, elevated serum lactate levels and cytolytic hepatitis were the main abnormalities associated. CONCLUSION: Short-term outcome was favorable in all cases after rehydration. The use of insulin may be dangerous and needs to be avoided.

[Congenital diverticuli of the common bile duct. Review of the literature]. / Diverticules congénitaux de la voie biliaire principale. Revue de la littérature

Among congenital dilatations of the common bile duct, a congenital diverticulum of the common bile duct may remain clinically latent for a long period. One case is reported in a 77 year old subject. Precise diagnosis of the disease is often made at operation and on pathological examination. The diverticulum is cured in most cases after surgery which consists of simple removal, but which may sometimes require reconstruction of the common bile duct.

Quarterly individual outpatients lifestyle counseling after initial inpatients education on type 2 diabetes: the REDIA Prev-2 randomized controlled trial in Reunion Island.

AIMS: This study aimed to describe the 1-year evolution of type 2 diabetes (T2D) patients who attended inpatients education, and to assess whether quarterly outpatients counseling visits by nurses and dietitians can improve metabolic control and health-related behaviours. METHODS: Following in-hospital educational sessions, 398 adult T2D patients were randomized to either attend quarterly individual lifestyle counseling visits by a nurse and a dietitian (intervention group), or receive the usual care (control group). Primary (HbA(1c)) and secondary endpoints (fasting blood glucose, lipids, body mass index, waist circumference, fat mass, blood pressure, diet, physical activity) were assessed at baseline and at 12 months. RESULTS: HbA(1c) changes from baseline to 12 months were -1.74±2.64% (P<0.0001) for the intervention group and -2.02±2.57% (P<0.0001) for the control group. There was no statistically significant difference between the intervention group (n=153) and the controls (n=166) for any of the clinical and biological outcomes. In both groups, total energy and fat intakes decreased significantly from baseline levels. Also, no difference was found between the groups for any dietary outcome. A slight enhancement in sports activity was observed in the intervention group, but the difference between the two groups did not reach statistical significance, and no difference was found concerning any other physical activity scores. CONCLUSION: In this study of adults with T2D, patients significantly improved their metabolic control, and dietary and exercise habits, 1 year after receiving intensive inpatients education, whereas subsequent quarterly outpatients counseling visits with nurses and dietitians have not demonstrated any superiority compared with the usual care.

A theoretical models of oncolysis by Clostridium oncolyticum M 55 ATCC 13,732.

Based on experimental data obtained by own experiments and investigations of other authors the first time it is tried to develop a new theoretical model of oncolysis by Clostridium oncolyticum M 55. The correlations between tumour, clostridial cells, the tumour kinin system and the immune system of the host are represented in their influence on oncolysis. The consequences for a therapeutic application of the tumour clostridia phenomenon are discussed.

[Enzymatic mechanisms of the oncolysis by Clostridium oncolyticum M 55 ATCC 13.732 (author's transl)]. / Enzymatische Mechanismen der Onkolyse durch Clostridium oncolyticum M 55 ATCC 13.732.

After a short review of older attempts at explanation of the oncolysis by Cl. oncolyticum M 55 a new theory will be presented. In connection with new results of enzymological investigations the metabolic correlations between the tumor cell and the vegetative clostridial cell and the consequences of the results for an oncolytic therapy will be demonstrated.

[The use of clostridial antigens for a serological and autoradiographical early diagnosis of tumours (author's transl)]. / Die Verwendung von Clostridien-Antigenen für eine serologische und autoradiographische Frühdiagnose von Tumoren.

In continuation of the examinations on the usefulness of Cl. oncolyticum M 55 spores reported by Möse new experiments with an auto-radiographical method are plannes. After the isolation, chemical characterization of the cell wall antigens and the exo-antigens and the formation of antibodies, these antibodies should be at first labelled with 125 J and henceforth with 131 J. The iodine-labelled antibodies should allow the diagnosis and localization of the tumours.

Anti-immunoglobulin M induces both B-lymphocyte proliferation and differentiation in Xenopus laevis.

Anti-IgM induced the proliferation of spleen lymphocytes of the amphibian Xenopus laevis as determined by 3H-thymidine uptake. The responding cells were B lymphocytes, since lymphocyte populations enriched in surface-Ig-positive cells exhibited an increased proliferative response, and spleen cells from larvally thymectomized animals still responded to anti-IgM. Immunofluorescence analysis and gel electrophoresis of biosynthetically labeled Ig polypeptides revealed that lymphoblasts induced by anti-IgM differentiated into plasmablasts that synthesized and secreted mainly IgM and small amounts of IgY. The in vitro differentiation of B lymphocytes also occurred in spleen cells obtained from thymectomized animals. These findings are in contrast with those obtained in mammals and suggest that the differentiation of B lymphocytes in X. laevis is subject to different regulatory mechanisms.

Mitogen-induced B-cell differentiation in Xenopus laevis.

Spleen cells from the South African clawed toad Xenopus laevis proliferate vigorously upon pokeweed mitogen (PWM) stimulation, as measured by 3H-thymidine uptake. The peak response is observed after 6-8 days of culture, and both light- and electron-microscopy examinations of stimulated cells reveal the presence of a large number of lymphoblasts. Beside proliferation, PWM induces the in vitro differentiation of a large proportion of lymphoblasts into immunoglobulin (Ig)-producing plasmablasts, as shown by direct immunofluorescence. On average, 25% of the lymphoblasts contain cytoplasmic high-molecular-weight Ig (IgM) at days 8-10, whereas less than 1% of the lymphoblasts have cytoplasmic low-molecular-weight Ig (IgY). Ig's of mitogen-stimulated cells were labeled biosynthetically and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE). The results confirm that Xenopus lymphoblasts induced in vitro synthesize Ig polypeptide chains. These are assembled into monomers of H2L2 units or polymerized hexameric forms. Both the monomer and hexamer forms are secreted. Two Ig heavy chains with apparent molecular weights (MW) of 74 and 81 daltons (kd) are detected in the cell lysates. The secreted chains have respectively an MW which is 3 kd and 5 kd greater than the intracellular forms. The two-dimensional gel electrophoretic pattern of PWM-induced Ig's is as heterogeneous as that of serum IgM. We conclude that PWM induces the in vitro proliferation and polyclonal differentiation of a large proportion of splenic B lymphocytes into plasmablasts.

The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus.

Since the larval and adult antibody responses are distinct and restricted in the clawed toad Xenopus, it offers a near ideal model for studying the ontogeny of antibody repertoires and the mechanisms involved. Immunoglobulin heavy chain (IgH) cDNA clones and B cell IgH DNA clones from various larval and adult libraries have been analysed in isogenic Xenopus. Some features are similar in adults and tadpoles, while others differ and explain the particularities observed previously at the protein level. Among the similarities we found are: (i) the mode of rearrangements (there are approximately 50% abortive events in B cells from both stages), (ii) VH family usage (10 of 11 known VH families are expressed proportionally to the number of VH elements per family), and (iii) JH usage (of the eight to nine Xenopus JH elements, two are used in approximately 70% of the VH regions in both stages of development). We found that there is relatively higher membrane exon expression in tadpoles compared with adults; and that most of the differences come from the diversification of CDR3 through DH usage and N diversification. Unlike in mammals, Xenopus DH elements are used with a remarkable flexibility with inversion, fusions and usage in different reading frames, but tadpoles show a strong bias for the usage of only a few DH elements and of a preferred reading frame. There is N diversification, which further increases CDR3 heterogeneity, in adult Xenopus but virtually none in tadpoles. These observations can account for the fact that larval antibody responses are less heterogeneous than those of adults.

Evolution of immunoglobulin light chain genes: analysis of Xenopus IgL isotypes and their contribution to antibody diversity.

The amphibian Xenopus laevis expresses several types of immunoglobulin light chain (IgL). cDNA clones for two IgL isotypes, C sigma 1 and C sigma 2, were analysed. C sigma is expressed in spleen and mitogen-stimulated B cells, like another Xenopus IgL type, termed C rho. C sigma shares less than 33% residues with C rho or with CL regions of shark, chicken and mammals. This suggests that C sigma diverged from a common ancestor of CL regions before or at the emergence of amphibians. Two families of VL elements, V sigma 1 and V sigma 2 are associated with C sigma (but not with C rho). They rearrange to their own set of JL elements, J sigma 1 and J sigma 2, which are poorly related to other J elements of the Ig gene family. The Xenopus genome contains a few V sigma 2 and multiple V sigma 1 elements (comparable with mammalian V kappa), but only two C sigma genes. Thus, the organization and expression of Xenopus IgL loci are apparently similar to mammalian IgL loci but different from shark and chicken IgL loci. Only a few VL elements are expressed, since cDNA clones show extensive sharing of CDR1 and CDR2 sequences; some clones differ only in CDR3. Rearranging VL and JL elements increases CDR3 diversity in both V sigma families, but abortive rearrangements are frequent in V sigma 1 regions. The very poor heterogeneity of expressed VL elements therefore appears to limit antibody diversity in Xenopus.