P-Trifluoromethyl ligands derived from Josiphos in the Ir-catalysed hydrogenation of 3,4-dihydroisoquinoline hydrochlorides.

The synthesis of mono P-trifluoromethyl and therefore P-stereogenic Xyliphos-derived ligands 5 and their application in the Ir-catalyzed enantioselective hydrogenation of 1-substituted 3,4-dihydroisoquinolinium species (DHIQ) are reported. The ligands were prepared following previous procedures involving the reaction of a bistrifluoromethylphosphine with lithiated (R)-Ugi amine 1. Chloroiridium(i) cyclooctadiene precatalysts containing these new partially electron-poor ligands 9 were found to be poorly active in the hydrogenation of free 1-phenyl-3,4-dihydroisoquinoline 12a. However, the corresponding hydrochloride 12a·HCl was smoothly reduced at 55-60 °C and 100 bar hydrogen pressure. The (SP)-configured ligand (SP)-5 yielded significantly higher enantioselectivity in hydrogenation experiments than its P-stereoisomeric counterpart (RP)-5. These new ligands were subsequently applied in the hydrogenation of a series of different 1-substituted 3,4-DHIQ chlorides 12a-l·HCl. Good to excellent enantioselectivity was observed for substrates bearing relatively large substituents in position 1, reaching 96% ee for 1-Ph-DHIQ chloride 12a·HCl without the help of any additives. Furthermore, an interesting counter ion effect was found with chloride being the best and hexafluorophosphate being very detrimental to enantioselectivity.

Diet-dependent alterations of hepatic Scd1 expression are accompanied by differences in promoter methylation.

Obesity and alterations of lipid homeostasis are hallmarks of the metabolic syndrome and largely influenced by the dietary conditions of the individual. Although heritability is considered to be a major risk factor, the almost 40 candidate genes identified by genome-wide association studies (GWAS) so far account for only 5-10% of the observed variance in BMI in human subjects. Alternatively, diet-induced changes of epigenetic gene regulation might be involved in disturbed lipid homeostasis and weight development. The aim of this study was to investigate how a high-carbohydrate diet (HCD; 70 kcal% from carbohydrates, 10 kcal% from fat) or a high-fat diet (HFD; 20 kcal% from carbohydrates, 60 kcal% from fat) affects hepatic expression of genes involved in fatty acid metabolism and if these alterations are correlated to changes in promoter methylation. Expression of stearoyl-CoA desaturase 1 (Scd1) was lower in livers from HFD-fed C57BL/6 J mice compared to HCD-fed animals and correlated inversely with the degree of DNA methylation at 2 distinct, adjacent CpG sites in the Scd1 promoter. In contrast, expression of transcription factors peroxisome proliferator activated receptor alpha and gamma (Ppara, Pparg), and sterol regulatory element binding transcription factor 1 (Srebf1) was not affected. The degree of hepatic Scd1 promoter methylation at these CpG sites correlated positively to fat mass and serum leptin levels, whereas serum ghrelin levels were inversely correlated with methylation at both CpG sites. Taken together, hepatic expression of Scd1 is differentially affected by carbohydrate- and lipid content of the diet. These differences in Scd1 expression are associated with altered promoter methylation, indicating that diets affect lipid metabolism in the liver via epigenetic mechanisms.

[Comorbid depression mediates the association of childhood/adolescent maltreatment and fibromyalgia syndrome. A study with patients from different clinical settings]. / Komorbide depressive Störungen als Mediator der Assoziation von Misshandlungen in Kindheit/Jugend und Fibromyalgiesyndrom. Eine Fallserie von Patienten aus unterschiedlichen klinischen Kontexten.

BACKGROUND: Emotional and physical abuse and neglect in childhood and adolescence [childhood maltreatments (CMs)] are considered to play a role in the etiology of fibromyalgia syndrome (FMS). Whether the association between FMS and CMs can be explained by mediators has not been studied. METHODS: CMs of consecutive FMS patients from three different clinical settings were assessed by the German version of the Childhood Trauma Questionnaire. Clinical setting, gender, age, comorbid depression, and health care use were tested for mediators. RESULTS: Of 328 patients (86% women, mean age 50 years), 293 were analyzed; 16% of the patients reported severe emotional, 9% severe physical, and 11% severe sexual abuse and 25% severe emotional and 13% severe physical neglect during childhood. The frequency of CMs was not associated with setting, gender, age, and health care use. FMS patients with a comorbid depression more frequently reported physical abuse (p=0.01) and emotional abuse (p=0.001), as well as emotional neglect (p=0.0008) and physical neglect (p=0.001) compared with FMS patients without a comorbid depression. DISCUSSION: Comorbid depression is a mediator of the association between CMs and FMS. CONCLUSION: Prospective population-based studies controlling for depression are necessary to determine the role of CMs in the etiology of FMS.

Requirement for distinct vesicle-associated membrane proteins in insulin- and AMP-activated protein kinase (AMPK)-induced translocation of GLUT4 and CD36 in cultured cardiomyocytes.

AIMS/HYPOTHESIS: Upon stimulation of insulin signalling or contraction-induced AMP-activated protein kinase (AMPK) activation, the glucose transporter GLUT4 and the long-chain fatty acid (LCFA) transporter CD36 similarly translocate from intracellular compartments to the plasma membrane of cardiomyocytes to increase uptake of glucose and LCFA, respectively. This similarity in regulation of GLUT4 traffic and CD36 traffic suggests that the same families of trafficking proteins, including vesicle-associated membrane proteins (VAMPs), are involved in both processes. While several VAMPs have been implicated in GLUT4 traffic, nothing is known about the putative function of VAMPs in CD36 traffic. Therefore, we compared the involvement of the myocardially produced VAMP isoforms in insulin- or contraction-induced GLUT4 and CD36 translocation. METHODS: Five VAMP isoforms were silenced in HL-1 cardiomyocytes. The cells were treated with insulin or the contraction-like AMPK activator oligomycin or were electrically stimulated to contract. Subsequently, GLUT4 and CD36 translocation as well as substrate uptake were measured. RESULTS: Three VAMPs were demonstrated to be necessary for both GLUT4 and CD36 translocation, either specifically in insulin-treated cells (VAMP2, VAMP5) or in oligomycin/contraction-treated cells (VAMP3). In addition, there are VAMPs specifically involved in either GLUT4 traffic (VAMP7 mediates basal GLUT4 retention) or CD36 traffic (VAMP4 mediates insulin- and oligomycin/contraction-induced CD36 translocation). CONCLUSIONS/INTERPRETATION: The involvement of distinct VAMP isoforms in both GLUT4 and CD36 translocation indicates that CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process dependent on soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. The ability of other VAMPs to discriminate between GLUT4 and CD36 translocation allows the notion that myocardial substrate preference can be modulated by these VAMPs.

Serum adiponectin levels and enzyme markers of liver dysfunction in diabetic and non-diabetic Caribbean subjects.

Low adiponectin levels are associated with elevated plasma alanine aminotransferase, a marker of reduced hepatic insulin sensitivity and a risk factor for type 2 diabetes. This study aims to determine the relationship between serum adiponectin level and alanine aminotransferase in diabetic and non-diabetic subjects. Fifty-six type 2 diabetic patients and 33 non-diabetic subjects participate in the study. Baseline plasma concentrations of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose are measured on a chemistry analyser. Insulin and adiponectin are measured using enzyme-linked immunoassay techniques and insulin resistance is determined using the homeostatic model assessment method. Diabetic patients showed significantly lower levels of serum adiponectin than did the non-diabetic subjects, whereas levels of alanine aminotransferase and alkaline phosphatase were similar in both groups. While female non-diabetic subjects showed higher serum adiponectin levels than did female diabetic patients, alanine aminotransferase level did not differ (P>0.05). No significant relationship was seen between adiponectin and alanine aminotransferase in diabetic and non-diabetic subjects (P>0.05). Serum adiponectin levels were higher in non-diabetic subjects but there was no significant correlation between adiponectin and alanine aminotransferase in both groups of subjects. The data suggest that low serum adiponectin level may not be a suitable marker for impaired liver function in diabetic patients.

Serum adiponectin levels and enzyme markers of liver dysfunction in diabetic and non-diabetic Caribbean subjects

Low adiponectin levels are associated with elevated plasma alanine aminotransferase, a marker of reduced hepatic insulin sensitivity and a risk factor for type 2 diabetes. This study aims to determine the relationship between serum adiponectin level and alanine aminotransferase in diabetic and non-diabetic subjects. Fifty-six type 2 diabetic patients and 33 non-diabetic subjects participate in the study. Baseline plasma concentrations of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose are measured on a chemistry analyser. Insulin and adiponectin are measured using enzyme-linked immunoassay techniques and insulin resistance is determined using the homeostatic model assessment method. Diabetic patients showed significantly lower levels of serum adiponectin than did the non-diabetic subjects, whereas levels of alanine aminotransferase and alkaline phosphatase were similar in both groups. While female non-diabetic subjects showed higher serum adiponectin levels than did female diabetic patients, alanine aminotransferase level did not differ (P>0.05). No significant relationship was seen between adiponectin and alanine aminotransferase in diabetic and non-diabetic subjects (P>0.05). Serum adiponectin levels were higher in non-diabetic subjects but there was no significant correlation between adiponectin and alanine aminotransferase in both groups of subjects. The data suggest that low serum adiponectin level may not be a suitable marker for impaired liver function in diabetic patients.

The role of intrahepatic lymphocytes in mediating protective immunity induced by attenuated Plasmodium berghei sporozoites.

Exposure to irradiated Plasmodium sporozoites (gamma-spz) results in protection against malaria. Like infectious spz, gamma-spz colonize hepatocytes to undergo maturation. Disruption of liver stage development prevents the generation of protection, which appears, therefore, to depend on liver stage antigens. Although some mechanisms of protection have been identified, they do not include a role for intrahepatic mononuclear cells (IHMC). We demonstrated that P. berghei gamma-spz-immune murine IHMC adoptively transfer protection to naive recipients. Characterization of intrahepatic CD4+ T cells revealed an immediate, albeit transient, response to gamma-spz, while the response of CD8+ T cells is delayed until acquisition of protection. It is presumed that activated CD8+ T cells home to the liver to die; gamma-spz-induced CD8+CD45RB(lo)CD44(hi) T cells, however, persist in the liver, but not the spleen, during protracted protection. The association between CD8+CD45RB(lo)CD44(hi) T cells and protection has been verified using MHC class I and CD1 knockout mice and mice with disrupted liver stage parasites. Based on kinetic studies, we propose that interferon-gamma, presumably released by intrahepatic effector CD8+ T cells, mediates protection; the persistence of CD8+ T cells is, in turn, linked to Plasmodium antigen depots and cytokines released by CD4+ T cells and/or NK T cells.

Altered hepatic lymphocyte subpopulations in obesity-related murine fatty livers: potential mechanism for sensitization to liver damage.

Although obesity-related fatty livers are vulnerable to damage from endotoxin, the mechanisms involved remain obscure. The purpose of this study was to determine if immunologic priming might be involved by determining if fatty livers resemble normal livers that have been sensitized to endotoxin damage by Propionibacterium acnes infection. The latter induces interleukin (IL)-12 and -18, causing a selective reduction of CD4+NK T cells, diminished IL-4 production, deficient production of T-helper type 2 (Th-2) cytokines (e.g., IL-10), and excessive production of Th-1 cytokines (e.g., interferon gamma [IFN-gamma]). Liver and spleen lymphocyte populations and hepatic cytokine production were compared in genetically obese, ob/ob mice (a model for obesity-related fatty liver) and lean mice. Obese mice have a selective reduction of hepatic CD4+NK T cells. Serum IL-18 is also increased basally, and the hepatic mRNA levels of IL-18 and -12 are greater after endotoxin challenge. Thus, up-regulation of IL-18 and IL-12 in fatty livers may reduce hepatic CD4+NK T cells. In addition, mononuclear cells from fatty livers have decreased expression of the adhesion molecule, leukocyte factor antigen-1 (LFA-1), which is necessary for the hepatic accumulation of CD4+NK T cells. Consistent with reduced numbers of hepatic CD4+NK T cells, mononuclear cells from fatty livers produce less IL-4. Furthermore, after endotoxin treatment, hepatic induction of IL-10 is inhibited, while that of IFN-gamma is enhanced. Thus, fatty livers have inherent immunologic alterations that may predispose them to damage from endotoxin and other insults that induce a proinflammatory cytokine response.

Memory phenotype CD8(+) T cells persist in livers of mice protected against malaria by immunization with attenuated Plasmodium berghei sporozoites.

Natural exposure to Plasmodium parasites induces short-lived protective immunity. In contrast, exposure to radiation-attenuated sporozoites (gamma spz) promotes long-lasting protection that is in part mediated by CD8(+) T cells that target exoerythrocytic stage antigens. The mechanisms underlying the maintenance of long-lasting protection are currently unclear. The liver is a repository of Plasmodium antigens and may support the development and / or homing of memory T cells. While activated CD8(+) T cells are presumed to die in the liver, the fate of anti-Plasmodium CD8(+) T cells remains unknown. We propose that inflammatory conditions in the liver caused by Plasmodium parasites may allow some effector CD8(+) T cells to survive and develop into memory cells. To support this hypothesis, in this initial study we demonstrate that liver mononuclear cells from P. berghei gamma spz-immune mice transferred protection to naive recipients and moreover, that CD4(+) and CD8(+) T cells responded to Plasmodium antigens by up-regulating activation / memory markers. While CD4(+) T cells under went a transient activation following immunization with gamma spz, CD8(+) T cells expanded robustly after spz challenge and exhibited stable expression of CD44(hi) and CD45RB(lo) during protracted protection. These results establish a key role for intrahepatic T cells in long-lasting protection against malaria.

Pulmonary function after total laryngectomy.

OBJECTIVE: To present data on pulmonary function in 59 laryngectomees using a specially designed silicone adapter for connection of the stoma to the bodyplethysmograph. STUDY DESIGN: Prospective assessment of pulmonary function in 59 patients, and comparison of lung function before and after bronchodilator testing in selected cases. METHODS: The usefulness of the adapter was examined. Data of patients with airway obstruction were tabulated according to large airway obstruction (LAO), peripheral airway obstruction (PAO), and small airway disease (SAD) types. RESULTS: Findings show that pulmonary airway obstruction was present in 81% of patients and normal pulmonary function was present in only 11 of the 59 participants (19%). LAO was found in 25%, PAO in 17%, and SAD in 39% of cases. Emphysema was diagnosed in 14% of cases within the above-mentioned LAO and PAO groups. Improvement of pulmonary function was achieved in 12 of 16 laryngectomees with airway obstruction, when a bronchodilator aerosol was administered. In 60% of cases with LAO and PAO, the laryngectomees did not know of any marked obstruction of their airways, and only 10% of those knowing about their obstruction received appropriate medical treatment. CONCLUSIONS: These results suggest that 42% of the laryngectomees tested may have benefited from further medical treatment. After laryngectomy, pulmonary function assessment was performed elsewhere in 1 of 59 cases. In light of the high prevalence of airway obstruction in laryngectomees, more frequent postoperative assessments of pulmonary function should be offered to prevent or to reduce impairment of respiratory function in this postlaryngectomy vulnerable pulmonary status. Further studies are also needed to determine the effects of therapeutic intervention, e.g., assessment of therapy outcome and influence on quality of life.

Long-term efficacy and immune responses following immunization with the RTS,S malaria vaccine.

The malaria sporozoite vaccine candidate RTS,S, formulated with an oil-in-water emulsion plus the immunostimulants monophosphoryl lipid A and the saponin derivative QS21 (vaccine 3), recently showed superior efficacy over two other experimental formulations. Immunized volunteers were followed to determine the duration of protective immune responses. Antibody levels decreased to between one-third and one-half of peak values 6 months after the last dose of vaccine. T cell proliferation and interferon-gamma production in vitro were observed in response to RTS,S or hepatitis B surface antigen. Seven previously protected volunteers received sporozoite challenge, and 2 remained protected (1/1 for vaccine 1, 0/1 for vaccine 2, and 1/5 for vaccine 3). The prepatent period was 10.8 days for the control group and 13.2 days for the vaccinees (P < .01). Immune responses did not correlate with protection. Further optimization in vaccine composition and/or immunization schedule will be required to induce longer-lasting protective immunity.

[Parenteral nutrition associated cholestasis in the newborn]. / Cholestase bei parenteraler Ernährung Früh- und Neugeborener.

Parenteral nutrition associated cholestasis in preterm infants and newborn children is a frequent and serious disease with an incidence of 23% depended on duration of parenteral nutrition and birthweight. The incidence of liver cirrhosis is 40% when parenteral nutrition is given 74-242 days. The pathogenesis remains unclear. Several predisposing factors are discussed like immaturity, lack of hormonal stimulation by oral feeding, bacterial infection, liver toxicity of aminoacids and their products of photooxidation, lack of taurine, lack of antioxidation substances, hypermanganesaemia and pollution of infusion solutions. Furthermore sepsis during parenteral nutrition seems to multiply the risk of cholestasis. For prevention controlled studies recommend: 1. Early enteral nutrition. 2. The reduction of parenteral amino acids to less than 3 g/kg/d. 3. Light protection for parenteral solutions. 4. Cyclic infusion of parenteral nutrition. 5. The application of antibiotics (metronidazole, gentamicin) during parenteral nutrition. The most important therapeutic intervention is the beginning of oral feeding. Most of the time this leads to a decrease of icterus within two weeks. An icterus persisting longer than 3 weeks should be treated because of the risk of liver cirrhosis. Further therapeutic interventions are: 1. Cholecystokinin, good results in case studies which still has to be verified by a controlled study. 2. Ursodeoxycholic acid, its choleretic effectiveness is verified in several liver diseases by controlled studies, but it is not proven in parenteral nutrition associated cholestasis. 3. Laparoscopic biliary irrigation, successful in several case studies.

Immunochemical properties of some monoclonal IgE antibodies to 4-hydroxy-3-nitrophenylacetyl (NP).

Several hybridoma cell lines secreting NP-specific, murine IgE antibodies were generated by fusion of P3-X20 (gamma, kappa) tumour cells with spleen cells from (BALB/c X C57B1/6)F1 (CB6F1) mice previously immunized with NP-ovalbumin. Four subclones (designated NP-epsilon-3.57, NP-epsilon-15.88, NP-epsilon-91.58 and NP-epsilon-95.31) were propagated in vivo and milligram quantities of the corresponding IgE antibodies were purified from ascitic fluid by gel filtration, ion exchange chromatography and affinity chromatography. Immunological analyses and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that NP-epsilon-15.88, NP-epsilon-91.58 and NP-epsilon-95.31 all possessed lambda 1 (or possibly lambda 3) light chains; and that NP-epsilon-3.57 possessed lambda 2 light chains; NP-epsilon-95.31 also expressed the P3-X20 derived, MOPC-21 kappa light chain. Radioallergosorbent test (RAST) titration curves, generated from the interaction of the four monoclonal IgE antibodies with NP-BSA attached to paper discs (NP-BSA-P) were found to be non-overlapping. Measurements of the relative amounts of NP-epsilon-aminocaproic acid (NP-CAP) and 4-hydro-3-iodo-5-nitrophenylacetyl-epsilon-aminocaproic acid (NIP-CAP) that were required to inhibit by 50% the binding of the 4 IgE antibodies to NP-BSA-P indicated that these antibodies were all heteroclitic, since their affinity for NIP appeared to be higher than their affinity for NP. These results, in conjunction with other findings reported in the literature, suggested that the V regions of NP-specific IgE antibodies are similar to the V regions of NP-specific IgM and IgG antibodies, produced by the same mouse strains. Finally, in vitro histamine release measurements demonstrated that two of these monoclonal IgE antibodies could mediate antigen induced histamine release from passively sensitized rat peritoneal mast cells.

The suppression of insulin-specific immune responses by the administration of insulin-derivatized syngeneic cells and soluble lysates from these cells.

The results of this study demonstrated that the i.v. administration of insulin, in the form of a conjugate with either syngeneic spleen cells (SC) or peritoneal exudate cells (PEC), markedly reduced the capacity of recipient mice to develop insulin-specific immune responses, as manifested by diminished in vivo IgE antibody production and by depressed in vitro, lymph node cell proliferation responses, respectively. Furthermore, it was shown that i.v. injection of insulin-PEC conjugates induced the activation of suppressor cells that had the capacity to downregulate insulin-specific IgG plaque-forming cell (PFC) responses. Finally, it was also determined that freezing and thawing of the insulin-PEC conjugates resulted in the release of a soluble tolerogenic molecule and/or membrane preparation that could also markedly depress insulin-specific IgG antibody production.

Regulation of the murine IgE antibody response. III. Analysis of the IgE response to insulin species variants.

IgE responses to insulin species variants i.e. to bovine, ovine and porcine insulins (respectively BI, OI and PI), were investigated. H-2d mice developed an anti-BI IgE response when this antigen was administered i.p. with A1(OH)3 gel, and H-2b and H-2a,d,k mice generated anti-BI and anti-OI IgE responses, respectively, when these antigens were injected i.p. with Freund's complete adjuvant (FCA). H-2d mice, on the other hand, developed only weak to negligible anti-PI IgE responses, when this antigen was administered in A1(OH)3 or in FCA. Antibody cross-reactivity indicated that at least two populations of IgE antibodies were produced in response to BI and/or OI; one population specific for the A chain loop and a second population specific for a determinant common to several insulins. Finally, i.v. injections of conjugates of insulin with spleen lymphocytes markedly suppressed the capacity of mice to develop an anti-insulin IgE response.

The role of antigen-presenting cells in the IgE antibody response. I. The induction of high titer IgE antibody responses in IgE high-responder and low-responder mice by the administration of antigen-pulsed macrophages in the absence of adjuvants.

The administration of 2 i.p. injections of 2 x 10(7) ovalbumin- (OA) pulsed, syngeneic, adherent, peritoneal exudate cells (OA-pulsed macrophages [M phi]) to (C57BL/6 x DBA/2)F1 (B6D2F1) mice promoted a persistent and high-titer anti-OA IgE response in these mice. Similarly, 2 i.p. injections of OA-pulsed DBA/2 parental M phi also induced the generation of an anti-OA IgE response in B6D2F1 mice; an identical procedure using OA-pulsed C57BL/6 parental M phi, on the other hand, elicited in most cases a very weak to negligible anti-OA IgE response. The administration of 3 i.p. injections of OA-pulsed syngeneic M phi to SJL mice also induced these "IgE nonresponder" mice to develop an anti-OA IgE response that was both boosterable and of moderate to high titer. The significance of these results with regard to the induction and regulation of the IgE antibody response is discussed.

Regulation of the murine IgE antibody response. I. Characterization of suppressor cells regulating both persistent and transient responses and their sensitivity to low doses of X-irradiation.

Anti-ovalbumin (OA) IgE antibody responses were measured in B6D2F1 mice as a function of time and antigen dose. One hundred to 200 microgram of OA in Al(OH)3 elicited transient responses, whereas 1 to 10 microgram of OA in Al(OH)3 elicited persistent anti-OA IgE responses of high titer. T cells isolated from the spleens of mice mounting either a persistent or a transient response strongly suppressed primary anti-DNP IgE responses in unirradiated recipient mice that were immunized with DNP-OA in Al(OH)3; it was, therefore, concluded that suppressor T cells (Ts cells) were activated during both the persistent and transient IgE responses. Nevertheless, in the present study it was not possible to completely rule out the contention that IgG antibodies may also have been suppressing the IgE response. With a modified adoptive transfer system, it was shown that these Ts cells were sensitive to low doses (250 R) of x-irradiation. The suppressive activity of long-term OA primed cells was also shown to be markedly enhanced when cultured for 24 hr with soluble OA; this finding was interpreted to indicate the presence of memory suppressor cells.

Specific suppression of anti-hapten reaginic antibody titers with hapten-coated liposomes.

Reaginic antibodies to DNP and ovalbumin (OA) were induced in B6D2F1 mice by a single i.p. injection of 1 microgram of DNP3-OA suspended with 1 mg of A1(OH)3 in 0.5 ml of saline. The anti-DNP reaginic antibody titers were markedly depressed by treatment of mice with DNP-coated liposomes. This treatment, however, did not affect the level of antibody formation to OA.