Variable cartilage degradation in mice with diet-induced metabolic dysfunction: food for thought.

OBJECTIVE: Human cohort studies have demonstrated a role for systemic metabolic dysfunction in osteoarthritis (OA) pathogenesis in obese patients. To explore the mechanisms underlying this metabolic phenotype of OA, we examined cartilage degradation in the knees of mice from different genetic backgrounds in which a metabolic phenotype was established by various dietary approaches. DESIGN: Wild-type C57BL/6J mice and genetically modified mice (hCRP, LDLr-/-. Leiden and ApoE*3Leiden.CETP mice) based on C57BL/6J background were used to investigate the contribution of inflammation and altered lipoprotein handling on diet-induced cartilage degradation. High-caloric diets of different macronutrient composition (i.e., high-carbohydrate or high-fat) were given in regimens of varying duration to induce a metabolic phenotype with aggravated cartilage degradation relative to controls. RESULTS: Metabolic phenotypes were confirmed in all studies as mice developed obesity, hypercholesteremia, glucose intolerance and/or insulin resistance. Aggravated cartilage degradation was only observed in two out of the twelve experimental setups, specifically in long-term studies in male hCRP and female ApoE*3Leiden.CETP mice. C57BL/6J and LDLr-/-. Leiden mice did not develop HFD-induced OA under the conditions studied. Osteophyte formation and synovitis scores showed variable results between studies, but also between strains and gender. CONCLUSIONS: Long-term feeding of high-caloric diets consistently induced a metabolic phenotype in various C57BL/6J (-based) mouse strains. In contrast, the induction of articular cartilage degradation proved variable, which suggests that an additional trigger might be necessary to accelerate diet-induced OA progression. Gender and genetic modifications that result in a humanized pro-inflammatory state (human CRP) or lipoprotein metabolism (human-E3L.CETP) were identified as important contributing factors.

Butyrate restores HFD-induced adaptations in brain function and metabolism in mid-adult obese mice.

OBJECTIVE: Midlife obesity affects cognition and increases risk of developing dementia. Recent data suggest that intake of the short chain fatty acid butyrate could improve memory function, and may protect against diet-induced obesity by reducing body weight and adiposity. SUBJECTS: We examined the impact of a high-fat diet (HFD) followed by intervention with 5% (w/w) dietary butyrate, on metabolism, microbiota, brain function and structure in the low-density-lipoprotein receptor knockout (LDLr-/-) mouse model in mid and late life. RESULTS: In mid-adult mice, 15 weeks of HFD-induced adiposity, liver fibrosis and neuroinflammation, increased systolic blood pressure and decreased cerebral blood flow, functional connectivity assessed with neuroimaging. The subsequent 2 months butyrate intervention restored these detrimental effects to chow-fed control levels. Both HFD and butyrate intervention decreased variance in fecal microbiota composition. In late-adult mice, HFD showed similar detrimental effects and decreased cerebral white and gray matter integrity, whereas butyrate intervention attenuated only metabolic parameters. CONCLUSION: HFD induces detrimental effects in mid- and late-adult mice, which can be attenuated by butyrate intervention. These findings are consistent with reported associations between midlife obesity and cognitive impairment and dementia in humans. We suggest that butyrate may have potential in prevention and treatment of midlife obesity.

Surgical removal of inflamed epididymal white adipose tissue attenuates the development of non-alcoholic steatohepatitis in obesity.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is strongly associated with abdominal obesity. Growing evidence suggests that inflammation in specific depots of white adipose tissue (WAT) has a key role in NAFLD progression, but experimental evidence for a causal role of WAT is lacking. METHODS: A time-course study in C57BL/6J mice was performed to establish which WAT depot is most susceptible to develop inflammation during high-fat diet (HFD)-induced obesity. Crown-like structures (CLS) were quantified in epididymal (eWAT), mesenteric (mWAT) and inguinal/subcutaneous (iWAT) WAT. The contribution of inflamed WAT to NAFLD progression was investigated by surgical removal of a selected WAT depot and compared with sham surgery. Plasma markers were analyzed by enzyme-linked immunosorbent assay (cytokines/adipokines) and lipidomics (lipids). RESULTS: In eWAT, CLS were formed already after 12 weeks of HFD, which coincided with maximal adipocyte size and fat depot mass, and preceded establishment of non-alcoholic steatohepatitis (NASH). By contrast, the number of CLS were low in mWAT and iWAT. Removal of inflamed eWAT after 12 weeks (eWATx group), followed by another 12 weeks of HFD feeding, resulted in significantly reduced NASH in eWATx. Inflammatory cell aggregates (-40%; P<0.05) and inflammatory genes (e.g., TNFα, -37%; P<0.05) were attenuated in livers of eWATx mice, whereas steatosis was not affected. Concomitantly, plasma concentrations of circulating proinflammatory mediators, viz. leptin and specific saturated and monounsaturated fatty acids, were also reduced in the eWATx group. CONCLUSIONS: Intervention in NAFLD progression by removal of inflamed eWAT attenuates the development of NASH and reduces plasma levels of specific inflammatory mediators (cytokines and lipids). These data support the hypothesis that eWAT is causally involved in the pathogenesis of NASH.

Intervention with a caspase-1 inhibitor reduces obesity-associated hyperinsulinemia, non-alcoholic steatohepatitis and hepatic fibrosis in LDLR-/-.Leiden mice.

BACKGROUND/OBJECTIVES: Non-alcoholic steatohepatitis (NASH) is a serious liver condition, closely associated with obesity and insulin resistance. Recent studies have suggested an important role for inflammasome/caspase-1 in the development of NASH, but the potential therapeutic value of caspase-1 inhibition remains unclear. Therefore, we aimed to investigate the effects of caspase-1 inhibition in the ongoing disease process, to mimic the clinical setting. SUBJECTS/METHODS: To investigate effects of caspase-1 inhibition under therapeutic conditions, male LDLR-/-.Leiden mice were fed a high-fat diet (HFD) for 9 weeks to induce a pre-diabetic state before start of treatment. Mice were then continued on HFD for another 12 weeks, without (HFD) or with (HFD-YVAD) treatment with the caspase-1 inhibitor Ac-YVAD-cmk (40 mg kg(-1) per day). RESULTS: Nine weeks of HFD feeding resulted in an obese phenotype, with obesity-associated hypertriglyceridemia, hypercholesterolemia, hyperglycemia and hyperinsulinemia. Treatment with Ac-YVAD-cmk did not affect further body weight gain or dyslipidemia, but did attenuate further progression of insulin resistance. Histopathological analysis of livers clearly demonstrated prevention of NASH development in HFD-YVAD mice: livers were less steatotic and neutrophil infiltration was strongly reduced. In addition, caspase-1 inhibition had a profound effect on hepatic fibrosis, as assessed by histological quantification of collagen staining and gene expression analysis of fibrosis-associated genes Col1a1, Acta2 and Tnfa. CONCLUSIONS: Intervention with a caspase-1 inhibitor attenuated the development of NASH, liver fibrosis and insulin resistance. Our data support the importance of inflammasome/caspase-1 in the development of NASH and demonstrate that therapeutic intervention in the already ongoing disease process is feasible.

Combined analysis of pharmacokinetic and efficacy data of preclinical studies with statins markedly improves translation of drug efficacy to human trials.

Correct prediction of human pharmacokinetics (PK) and the safety and efficacy of novel compounds based on preclinical data, is essential but often fails. In the current study, we aimed to improve the predictive value of ApoE*3Leiden (E3L) transgenic mice regarding the cholesterol-lowering efficacy of various statins in humans by combining pharmacokinetic with efficacy data. The efficacy of five currently marketed statins (atorvastatin, simvastatin, lovastatin, pravastatin, and rosuvastatin) in hypercholesterolemic patients (low-density lipoprotein ≥ 160 mg/dl) was ranked based on meta-analysis of published human trials. Additionally, a preclinical combined PK efficacy data set for these five statins was established in E3L mice that were fed a high-cholesterol diet for 4 weeks, followed by 6 weeks of drug intervention in which statins were supplemented to the diet. Plasma and tissue levels of the statins were determined on administration of (radiolabeled) drugs (10 mg/kg p.o.). As expected, all statins reduced plasma cholesterol in the preclinical model, but a direct correlation between cholesterol lowering efficacy of the different statins in mice and in humans did not reach statistical significance (R(2) = 0.11, P < 0.57). It is noteworthy that, when murine data were corrected for effective liver uptake of the different statins, the correlation markedly increased (R(2) = 0.89, P < 0.05). Here we show for the first time that hepatic uptake of statins is related to their cholesterol-lowering efficacy and provide evidence that combined PK and efficacy studies can substantially improve the translational value of the E3L mouse model in the case of statin treatment. This strategy may also be applicable for other classes of drugs and other preclinical models.

Metabolic stress-induced inflammation plays a major role in the development of osteoarthritis in mice.

OBJECTIVE: Obesity is associated with systemic inflammation and is a risk factor for osteoarthritis (OA) development. We undertook this study to test the hypothesis that metabolic stress-induced inflammation, and not mechanical overload, is responsible for the development of high-fat diet-induced OA in mice. METHODS: Human C-reactive protein (CRP)-transgenic mice received a high-fat diet without or with 0.005% (weight/weight) rosuvastatin or 0.018% (w/w) rosiglitazone, 2 different drugs with antiinflammatory properties. Mice fed chow were included as controls. After 42 weeks, mice were killed and histologic OA grading of the knees was performed. To monitor the overall inflammation state, systemic human CRP levels were determined. RESULTS: Male mice on a high-fat diet had significantly higher OA grades than mice on chow and showed no correlation between OA severity and body weight. In male mice, high-fat diet-induced OA was significantly inhibited by rosuvastatin or rosiglitazone to OA grades observed in control mice. Both treatments resulted in reduced human CRP levels. Furthermore, a positive correlation was found between the relative individual induction of human CRP evoked by a high-fat diet on day 3 and OA grade at end point. CONCLUSION: High-fat diet-induced OA in mice is due to low-grade inflammation and not to mechanical overload, since no relationship between body weight and OA grade was observed. Moreover, the OA process was inhibited to a great extent by treatment with 2 drugs with antiinflammatory properties. The inflammatory response to a metabolic high-fat challenge may predict individual susceptibility to developing OA later in life. The use of statins or peroxisome proliferator-activated receptor γ agonists (e.g., rosiglitazone) could be a strategy for interfering with the progression of OA.

High fat diet-induced obesity prolongs critical stages of the spermatogenic cycle in a Ldlr-/-.Leiden mouse model.

Obesity can disturb spermatogenesis and subsequently affect male fertility and reproduction. In our study, we aim to elucidate at which cellular level of adult spermatogenesis the detrimental effects of obesity manifest. We induced high fat diet (HFD) obesity in low-density lipoprotein receptor knock-out Leiden (Ldlr-/-.Leiden) mice, and studied the morphological structure of the testes and histologically examined the proportion of Sertoli cells, spermatocytes and spermatids in the seminiferous tubules. We examined sperm DNA damage and chromatin condensation and measured plasma levels of leptin, testosterone, cholesterol and triglycerides. HFD-induced obesity caused high plasma leptin and abnormal testosterone levels and induced an aberrant intra-tubular organisation (ITO) which is associated with an altered spermatids/spermatocytes ratio (2:1 instead of 3:1). Mice fed a HFD had a higher level of tubules in stages VII + VIII in the spermatogenic cycle. The stages VII + VII indicate crucial processes in spermatogenic development like initiation of meiosis, initiation of spermatid elongation, and release of fully matured spermatids. In conclusion, HFD-induced obese Ldlr-/-.Leiden mice develop an aberrant ITO and alterations in the spermatogenic cycle in crucial stages (stages VII and VII). Thereby, our findings stress the importance of lifestyle guidelines in infertility treatments.

Lipid ratios representing SCD1, FADS1, and FADS2 activities as candidate biomarkers of early growth and adiposity.

BACKGROUND: Altered lipid metabolism in early life has been associated with subsequent weight gain and predicting this could aid in obesity prevention and risk management. Here, a lipidomic approach was used to identify circulating markers for future obesity risk in translational murine models and validate in a human infant cohort. METHODS: Lipidomics was performed on the plasma of APOE*3 Leiden, Ldlr-/-.Leiden, and the wild-type C57BL/6J mice to capture candidate biomarkers predicting subsequent obesity parameters after exposure to high-fat diet. The identified candidate biomarkers were mapped onto corresponding lipid metabolism pathways and were investigated in the Cambridge Baby Growth Study. Infants' growth and adiposity were measured at 0-24 months. Capillary dried blood spots were sampled at 3 months for lipid profiling analysis. FINDINGS: From the mouse models, cholesteryl esters were correlated with subsequent weight gain and other obesity parameters after HFD period (Spearman's r≥0.5, FDR p values <0.05) among APOE*3 Leiden and Ldlr-/-.Leiden mice, but not among the wild-type C57BL/6J. Pathway analysis showed that those identified cholesteryl esters were educts or products of desaturases activities: stearoyl-CoA desaturase-1 (SCD1) and fatty acid desaturase (FADS) 1 and 2. In the human cohort, lipid ratios affected by SCD1 at 3 months was inversely associated with 3-12 months weight gain (B±SE=-0.31±0.14, p=0.027), but positively with 12-24 months weight and adiposity gains (0.17±0.07, p=0.02 and 0.17±0.07, 0.53±0.26, p=0.04, respectively). Lipid ratios affected by SCD1 and FADS2 were inversely associated with adiposity gain but positively with height gain between 3-12 months. INTERPRETATION: From murine models to human setting, the ratios of circulating lipid species indicating key desaturase activities in lipid metabolism were associated with subsequent body size increase, providing a potential tool to predict early life weight gain.

Heat shock induces resistance in rat pancreatic islet cells against nitric oxide, oxygen radicals and streptozotocin toxicity in vitro.

When cultures of pancreatic islet cells are exposed to the nitric oxide donor sodium nitroprusside, to enzymatically generated reactive oxygen intermediates or to streptozotocin cell lysis occurs after 4-12 h. We report here that a heat shock at 43 degrees C for 90 min reduces cell lysis from nitric oxide (0.45 mM sodium nitroprusside) by 70%, from reactive oxygen intermediates (12 mU xanthine oxidase and 0.05 mM hypoxanthine) by 80% and from streptozotocin (1.5 mM) by 90%. Heat shock induced resistance was observed immediately after termination of the 90 min culture at 43 degrees C and correlated with enhanced expression of hsp70. The occurrence of DNA strand breaks, a major early consequence of nitric oxide, reactive oxygen intermediates, or streptozotocin action, was not suppressed by heat shock treatment. However, the depletion of NAD+, the major cause of radical induced islet cell death, was suppressed after heat shock (P < 0.01). We conclude that pancreatic islet cells can rapidly activate defence mechanisms against nitric oxide, reactive oxygen intermediates and streptozotocin by culture at 43 degrees C. Islet cell survival is due to the prevention of lethal NAD+ depletion during DNA repair, probably by slowing down poly(ADP-ribose)polymerase activation.

Fenofibrate reduces atherogenesis in ApoE*3Leiden mice: evidence for multiple antiatherogenic effects besides lowering plasma cholesterol.

OBJECTIVE: To demonstrate, quantify, and mechanistically dissect antiatherosclerotic effects of fenofibrate besides lowering plasma cholesterol per se. METHODS AND RESULTS: ApoE*3Leiden transgenic mice received either a high-cholesterol diet (HC) or HC containing fenofibrate (HC+FF) resulting in 52% plasma cholesterol-lowering. In a separate low-cholesterol diet (LC) control group, plasma cholesterol was adjusted to the level achieved in the HC+FF group. Low plasma cholesterol alone (assessed in LC) resulted in reduced atherosclerosis (lesion area, number and severity) and moderately decreased plasma serum amyloid-A (SAA) concentrations. Compared with LC, fenofibrate additively reduced lesion area, number and severity, and the total aortic plaque load. This additional effect in HC+FF was paralleled by an extra reduction of aortic inflammation (macrophage content; monocyte adhesion; intercellular adhesion molecule-1 [ICAM-1], soluble vascular cell adhesion molecule-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), MCP-1, and NF-kappaB expression), systemic inflammation (plasma SAA and fibrinogen levels), and by an upregulation of plasma apoE levels. Also, enhanced expression of ABC-A1 and SR-B1 in aortic macrophages may contribute to the antiatherosclerotic effect of fenofibrate by promoting cholesterol efflux. CONCLUSIONS: Fenofibrate reduces atherosclerosis more than can be explained by lowering total plasma cholesterol per se. Impaired recruitment of monocytes/macrophages, reduced vascular and systemic inflammation, and stimulation of cholesterol efflux may all contribute to these beneficial effect of fenofibrate.

Potential mechanisms by which certain foods promote or inhibit the development of spontaneous diabetes in BB rats: dose, timing, early effect on islet area, and switch in infiltrate from Th1 to Th2 cells.

Certain diets can have major effects on the development of IDDM in DP-BB rats, but data are scant on the timing, dose, and mechanisms involved. We therefore determined the dose response, timing, and duration of exposure required to induce diabetes, and characterized the effects of nutritionally adequate diets with widely different diabetogenicity on the pancreatic islet area and cytokines. DP-BB rats were fed a diabetogenic, cereal-based, NIH-07 (NIH) diet or a protective, casein or hydrolyzed casein (HC)-based, semipurified diet. Rats were fed from weaning to 50 or 100 days with the HC diet and then switched to the NIH diet, or fed the NIH diet from weaning to 50 days and switched to the HC diet. Pancreas histology and diabetes outcome were determined. Semiquantitative morphometric analyses of hematoxylin and eosin-stained sections of pancreas from 41-day-old rats were also carried out. Diet-induced effects on pancreatic cytokine levels were measured at 70 days using reverse transcriptase-polymerase chain reaction analysis of gamma-interferon (IFN-gamma), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-beta). Long-term daily exposure, particularly around the beginning of puberty to late adolescence (50-100 days), was important for development of diabetes. DP-BB rats could be rescued from diabetes development by feeding them a low-diabetogen HC diet as late as 50 days. Diabetes frequency was highest in rats fed 70% and 100% NIH diets. By age 41 days, before classic insulitis, the islet area in HC-fed DP-BB rats was 65% greater than in NIH-fed rats. By 70 days, when mononuclear cells were visible in the islets of most NIH-fed, but not HC-fed rats, the more pronounced inflammatory process in NIH-fed rats was associated with a Th1 cytokine pattern (high IFN-gamma and low IL-10 and TGF-beta), whereas the pancreases of HC-fed rats showed fewer infiltrating cells, low levels of IFN-gamma, and high levels of TGF-beta, typical of a Th2 cytokine pattern. Thus dietary modification can occur as late as puberty. Further, long-term exposure to sufficient amounts of food diabetogens between 50 and 100 days was required for maximum diabetes induction. The islet area was modified by diet before signs of classic insulitis. Pancreatic inflammation in NIH-fed animals is a Th1-dependent phenomenon. The HC diet inhibited insulitis and was associated with a Th2 cytokine pattern in the pancreas, protecting diabetes-prone rats from developing diabetes.

Disulfide analysis reveals a role for macrophage migration inhibitory factor (MIF) as thiol-protein oxidoreductase.

The molecular mechanism of action of macrophage migration inhibitory factor (MIF), a cytokine with a critical role in the immune and inflammatory response, has not yet been identified. Here we report that MIF can function as an enzyme exhibiting thiol-protein oxidoreductase activity. Using a decapeptide fragment of MIF (MF1) spanning the conserved cysteine sequence motif Cys57-Ala-Leu-Cys60 (CALC), Cys-->Ser mutants (C57S MIF, C60S MIF, and C57S/C60S MIF) of human MIF (wtMIF), and alkylated wtMIF, we show that this activity is mediated by the CALC region and is important for the macrophage-activating properties of MIF. Both wtMIF and MF1 were demonstrated to form an intramolecular disulfide bridge. Using two common oxidoreductase assays, MIF was shown to enzymatically catalyze the reduction of insulin and 2-hydroxyethyldisulfide (HED). Examination of wtMIF and the mutants by far-UV circular dichroism spectroscopy (CD) together with denaturation studies showed that substituting or reducing the cysteine residues of CALC led to a reduced conformational stability of MIF but did not significantly change its overall conformation. A functional role for the CALC region was revealed by subjecting the mutants and alkylated wtMIF to the enzymatic assays. Mutant C60S did not have any enzymatic activity while mutant C57S had a reduced activity. Thiol-modified wtMIF that was alkylated under oxidizing conditions was found to have full enzymatic activity, whereas alkylation of wtMIF under reducing conditions completely eliminated MIF-mediated redox activity. Importantly, further physiological relevance of the disulfide motif was obtained by examining the mutants and alkylated MIF in an immunological assay that involved the macrophage-activating properties of MIF. In this test, mutant C60S was essentially inactive and mutant C57S was partly active, indicating together that at least some of the cytokine-like biological activities of MIF are dependent on the presence of cysteine 57 and 60. Again, use of the alkylated MIF species confirmed the role of the cysteine motif for this MIF activity. In conclusion, our results argue (a) that MIF exhibits enzymatic oxidoreductase activity, (b) that this activity is dependent on the presence of the catalytic center that is formed by cysteine residues 57 and 60, and (c) that certain MIF-mediated immune processes are due to the cysteine-mediated redox mechanism.

HMG-CoA reductase inhibitors: effects on chronic subacute inflammation and onset of atherosclerosis induced by dietary cholesterol.

Besides classical risk factors such as hypercholesterolemia and hypertension, chronic subacute inflammation has recently been recognized as an important force driving the development of atherosclerosis, the most common underlying cause of myocardial infarction and stroke. There is compelling evidence that a disturbance of cholesterol homeostasis contributes to the development of a chronic inflammatory state and that inhibitors of HMG-CoA reductase (statins) may dampen inappropriate inflammatory responses. We review the evidence and suggest mechanisms by which dietary cholesterol can induce an atherogenic inflammatory response in liver and vessel wall, with particular emphasis on the time course of this inflammatory response during atherogenesis and the interplay between these tissues. We discuss how statins interfere in this process, and whether they may reduce chronic subacute inflammation via a) their cholesterol-lowering effect, and/or b) their cholesterol-independent (pleiotropic) vasculoprotective activities. Recent studies performed in (humanized) animal models allow us to distinguish the lipid-lowering-dependent from the lipid-lowering-independent functions of statins. Using these data, we discuss the degree to which the lipid-lowering-dependent and lipid-lowering-independent effects of statins contribute to a reduction of inflammation, allowing estimation of the relevance of pleiotropic statin effects for the human situation.

Cross-linking and mutational analysis of the oligomerization state of the cytokine macrophage migration inhibitory factor (MIF).

The structure of the cytokine MIF has been investigated by X-ray crystallography, NMR, and biochemical methods with conflicting results regarding the structural and functional oligomerization state of this protein. Determination of the oligomeric state(s) is important for understanding more precisely the molecular mechanism of MIF action. To address this issue, we performed cross-linking of human and mouse MIF and selected mutants by various methods and analyzed the oligomerization by SDS-PAGE and gel filtration. MIF was found to form a mixture of monomeric, dimeric, and trimeric states at physiological concentrations, with the monomer and dimer representing the major species. Similar results were obtained when the carboxy-truncated mutants MIF(1-104) and MIF(1-109) were examined, indicating that the C-terminus of MIF is not critical for trimer stabilization. Cross-linking analysis of the isosteric Cys --> Ser mutants C56S and C80S of human MIF resulted in a similar oligomer distribution, whereas substitution of Cys59 led to a significant reduction in the dimeric and trimeric forms, indicating that the hydrophobic region around Cys59 is important for the oligomerization of MIF. Together, our data argue that physiological MIF solutions contain a mixture of monomers, dimers, and trimers.

Specific reduction of insulin disulfides by macrophage migration inhibitory factor (MIF) with glutathione and dihydrolipoamide: potential role in cellular redox processes.

The molecular mechanism of action of MIF, a cytokine that plays a critical role in the host immune and inflammatory response, has not yet been identified. We recently demonstrated that MIF is an enzyme that exhibits oxidoreductase activity by a cysteine thiol-mediated mechanism. Here we further investigated this function by examining the reduction of insulin disulfides by wild-type human MIF (wtMIF) using various substrates, namely glutathione (GSH), dihydrolipoamide, L-cysteine, beta-mercaptoethanol and dithiothreitol. The activity of wtMIF was compared to that of the relevant cysteine mutants of MIF and to two carboxy-truncated mutants. Only GSH and dihydrolipoamide were found to serve as reductants, whereas the other substrates were not utilized by MIF. Reduction of insulin disulfides by MIF was closely dependent on the presence of the Cys57-Ala-Leu-Cys60 (CALC) motif-forming cysteines C57 and C60, whereas C81 was not involved (activities: 51+/-13%, 14+/-5%, and 70+/-12% of wtMIF, respectively, and 20+/-3% for the double mutant C57S/C60S). Confirming the notion that the activity of MIF was dependent on the CALC motif in the central region of the MIF sequence, the C-terminal deletion mutants MIF(1-105) and MIF(1-110) were found to be fully active. The favored use of GSH and dihydrolipoamide indicated that MIF may be involved in the regulation of cellular redox processes and was supported further by the finding that MIF expression by the cell lines COS-1 and RAW 264.7 was significantly induced upon treatment with the oxidant hydrogen peroxide.

Transcription and translation of inducible nitric oxide synthase in the pancreas of prediabetic BB rats.

The inducible NO synthase (iNOS) was found to be expressed in pancreatic lesions of adult diabetes-prone BB rats. Pancreatic iNOS mRNA was detected by reverse transcriptase PCR in pancreatic RNA of adult diabetes-prone BB rats but not in normal Wistar rats, young diabetes-prone BB rats without insulitis or in diabetes-resistant BB rats. Immunohistochemistry of pancreatic sections using an iNOS-specific antiserum labeled the pancreas of adult diabetes-prone BB rats but not Wistar rats. Parallel staining for ED1-positive macrophages showed restriction of iNOS expression to areas of islet infiltration by macrophages. In conclusion, the data provide direct evidence for enhanced expression of inducible NO synthase in tissue lesions during the development of autoimmune diabetes.

Structure activity studies of the cytokine macrophage migration inhibitory factor (MIF) reveal a critical role for its carboxy terminus.

Carboxy-truncated mutants of human MIF (MIF(1-104) and MIF(1-109)) were used in structure activity studies. CD spectroscopy revealed an overall structural similarity between the mutants and MIF. Denaturant-induced unfolding demonstrated that the C-terminus contributed significantly to the conformational stability of MIF. This appears to be due to the formation of two C-terminal beta-strands. The mutants were enzymatically active, exhibiting half of the enzymatic redox activity of MIF. However, immunological analysis showed that deletion of both 5 and 10 C-terminal residues resulted in loss of the macrophage activating properties of MIF, providing functional evidence that the C-terminus is important for immunological activity and trimer formation. A more detailed study of the C-terminus may assist in identifying the molecular basis for the immunological and enzymatic activities of MIF.

Insulin therapy of prediabetes suppresses TH1 associated gene expression in BB rat pancreas.

Subcutaneous insulin treatment of young diabetes prone BB rats has been shown previously to suppress the development of autoimmune diabetes. In this study the hypothesis was tested that exogenous insulin may deviate the autoimmune process by acting on the Th1/Th2 cytokine balance in the pancreas. BB rats were implanted with pellets which continuously released insulin, at 50 d of age. Three weeks later cytokine mRNA expression in the pancreas and insulitis score were determined. While in control BB rats high levels of IFNgamma mRNA were detectable by RT-PCR, insulin treatment almost completely suppressed IFNgamma mRNA levels without concomitant upregulation of counterregulatory IL-10 and TGFbeta gene expression. Insulin also suppressed gene expression of inducible nitric oxide synthase. Mean insulitis scores were decreased after insulin treatment. We conclude that the protective effects of insulin treatment may not be due to the induction of protective Th2 immune reactivity but to general downregulation of immune activation in the pancreas, and hence also of Th1 autoimmunity.

Formation of isomorphic desolvates: creating a molecular vacuum.

The objective of this work was to investigate a common but poorly understood category of crystalline organic substances: isomorphic desolvates. When solvent is lost from a crystal lattice but the lattice retains its three-dimensional order, a lattice is created which is in a high-energy state relative to the original solvate structure. The desolvated lattice can reduce its internal energy by either resorbing solvent or by relaxation processes which increase the packing efficiency of the solid by reducing the unit cell volume. In the following paper, solid-state properties of isomorphic desolvates of cephalexin, cefaclor, erythromycin A, and spirapril hydrochloride hydrates are investigated. The hygroscopicity of the compounds are evaluated using a vacuum moisture balance, and structural relaxation is measured using a combination of X-ray powder diffraction and isothermal microcalorimetry. The study results are explained in terms of Kitaigorodski's close packing principle.

Sephadex induced bronchial hyperreactivity in the rat: hematology, histology, histochemistry and immunohistology of the lung.

24 hours after an i.v. injection of 2 mg Sephadex G 200 particles ovalbumin sensitized Sprague Dawley rats show an antigen specific bronchial hyperreactivity and an unspecific hyperreactivity against serotonin. The aim of this study was to investigate the effects of Sephadex on blood parameters and lung pathology to find the morphological substrate of bronchial hyperreactivity in this animal model. In the blood neutrophilia (p < 0.01) but no eosinophilia was present. We conclude that a blood eosinophilia needs not to be necessarily correlated with hyperreactivity of the airways like claimed by other investigators for this animal model. Histologically we found that Sephadex particles are trapped in smaller-diameter arteries of the lung and lead to a granulomatous arteritis consisting mainly of ED1 positive and widely ED2 negative macrophages interspersed with eosinophils and neutrophils. Larger vessels not occluded by particles showed perivascular oedema with infiltration of eosinophils. We report here for the first time a significant hypertrophy of PAS positive goblet cells (p < 0.01) accompanied by a peribronchial infiltration with eosinophils (p < 0.01) and macrophages positive for ED1, ED2 and Ox-6 (p < 0.01) but not Ox-19 positive T-lymphocytes. The authors suggest that the peribronchial inflammation contributes importantly to the onset of bronchial hyperreactivity in this animal model and that the hypertrophy of goblet cells indicates the pathophysiological importance of peribronchial leukocytes.