Metformin Alters Human Host Responses to Mycobacterium tuberculosis in Healthy Subjects.

BACKGROUND: Metformin, the most widely administered diabetes drug, has been proposed as a candidate adjunctive host-directed therapy for tuberculosis, but little is known about its effects on human host responses to Mycobacterium tuberculosis. METHODS: We investigated in vitro and in vivo effects of metformin in humans. RESULTS: Metformin added to peripheral blood mononuclear cells from healthy volunteers enhanced in vitro cellular metabolism while inhibiting the mammalian target of rapamycin targets p70S6K and 4EBP1, with decreased cytokine production and cellular proliferation and increased phagocytosis activity. Metformin administered to healthy human volunteers led to significant downregulation of genes involved in oxidative phosphorylation, mammalian target of rapamycin signaling, and type I interferon response pathways, particularly following stimulation with M. tuberculosis, and upregulation of genes involved in phagocytosis and reactive oxygen species production was increased. These in vivo effects were accompanied by a metformin-induced shift in myeloid cells from classical to nonclassical monocytes. At a functional level, metformin lowered ex vivo production of tumor necrosis factor α, interferon γ, and interleukin 1ß but increased phagocytosis activity and reactive oxygen species production. CONCLUSION: Metformin has a range of potentially beneficial effects on cellular metabolism, immune function, and gene transcription involved in innate host responses to M. tuberculosis.

Epstein-Barr virus-induced gene 3 suppresses T helper type 1, type 17 and type 2 immune responses after Trypanosoma cruzi infection and inhibits parasite replication by interfering with alternative macrophage activation.

The Epstein-Barr virus-induced gene 3 (EBI3) is a member of the interleukin-12 (IL)-12) family structurally related to the subunit p40 of IL-12 and forms a heterodimer either with the p28 subunit to build IL-27 or with p35 to form IL-35. Interleukin-27 is secreted by antigen-presenting cells whereas IL-35 appears to be produced mainly by regulatory T cells and regulatory B cells but both cytokines negatively regulate inflammatory immune responses. We here analysed the function of EBI3 during infection with the intracellular parasite Trypanosoma cruzi. Compared with C57BL/6 wild-type mice, EBI3-deficient (EBI3(-/-) ) mice showed a higher parasitaemia associated with an increased mortality rate. The EBI3(-/-) mice displayed an elevated inflammatory immune response with an increased production of T helper type 1 (Th1-), Th2- and Th17-derived cytokines. The increased Th2 immune response appears to have over-ridden the otherwise protective Th1 and Th17 immune responses by the induction of arginase-1-expressing alternatively activated macrophages in these mice. Hence, neutralization of IL-4 and arginase-1 activity partially restored protective immune responses in EBI3(-/-) mice. So far, our results demonstrate that EBI3 is an essential general regulator of inflammatory immune responses in experimental Chagas disease and is required for control of T. cruzi infection by inhibiting Th2-dependent alternative macrophage activation. Further studies are needed to dissect the underlying mechanisms and clarify whether EBI3 association with IL-27 or/and IL-35 accounts for its anti-inflammatory character in parasitic disease.

Sulfate determines the glucosinolate concentration of horseradish in vitro plants (Armoracia rusticana Gaertn., Mey. & Scherb.).

BACKGROUND: Horseradish plants (Armoracia rusticana) contain high concentrations of glucosinolates. Former studies have revealed that Armoracia plants cultivated in vitro have markedly lower glucosinolate concentrations than those grown in soils. Yet, these studies neglected that the sulfate concentration in the growth medium may have had a strong impact on glucosinolate metabolism. Accordingly, in this study horseradish in vitro plants were cultivated with differing sulfate concentrations and the glucosinolate concentrations were quantified by ion pair HPLC. RESULTS: Cultivation in 1.7 mmol L(-1) sulfate (as used in the prior studies) resulted in the accumulation of 16.2 µmol g(-1) DW glucosinolates, while the glucosinolate concentration increased to more than 23 µmol g(-1) DW when 23.5 mmol L(-1) sulfate was used in the medium. Correspondingly, the glucosinolate concentration decreased to 1.6 µmol g(-1) DW when sulfate concentration was lowered to 0.2 mmol L(-1). CONCLUSION: Since the glucosinolate accumulation in relation to the sulfate concentration follows a typical saturation curve, we deduce that the availability of sulfate determines the glucosinolate concentration in horseradish in vitro plants.

A complicated, metastatic, humeral air sac cystadenocarcinoma in a timneh African grey parrot (Psittacus erithacus timneh).

A 9-year-old male timneh African grey parrot (Psittacus erithacus timneh) was presented because of inability to fly and suspected trauma. The owner also had observed dyspnea, with tail bobbing and open-beak breathing. On clinical examination, a hard, painful mass was palpable in the left proximal humerus and axillary area. Radiographs revealed a radiodense soft tissue mass of the left humerus with no bony involvement, multifocal opacities in lung and air sacs, and an enlarged spleen. An asymmetric, vascularized cyst was detected in the mass by ultrasound examination. Results of biopsy of the mass revealed multifocal cysts composed of unilayer isoprismatic cells laying in vascularized connective tissue. Because of the severity of clinical signs and the poor clinical condition, the bird was euthanatized. On postmortem examination, the findings were air sac cystadenocarcinoma involving the humeral air sac with metastases in the spleen and kidneys, atherosclerosis, pneumoconiosis, and mycotic granulomatous pneumonia and airsacculitis with isolation of Aspergillus niger.

31P and 13C solid-state NMR spectroscopy to study collagen synthesis and biomineralization in polymer-based bone implants.

A combination of solid-state NMR spectroscopy and MRI was used to evaluate the formation of extracellular matrix in poly(D,L-lactide-co-glycolide) (PLGA) bone implants. Porous PLGA scaffolds were implanted into rat tibiae and analysed after 2, 4 or 8 weeks. MRI clearly delineated the implants within the cancellous bone. Differences in the trabecular structure of the implanted material and native bone were demonstrated. In addition, implants were analyzed by solid-state NMR spectroscopy under magic angle spinning. (13)C NMR spectra showed the unambiguous signature of collagen formed in the scaffolds, but also the characteristic signals of the PLGA matrix, indicating that resorption was not complete after 8 weeks. Furthermore, (31)P NMR spectroscopy detected the inorganic component of the matrix, which is composed of bioapatite. (31)P NMR spectra were quantified and this analysis revealed that the amount of inorganic extracellular matrix formed de novo was significantly lower than in native bone. This demonstrates that solid-state NMR spectroscopy, in particular in combination with MRI, can provide useful information on the composition and structure of the extracellular matrix, and serve as a tool to evaluate the quality of tissue engineering strategies.

De novo biosynthesis of glycosaminoglycans in the extracellular matrix of skin studied by matrix-assisted laser desorption/ionization mass spectrometry.

The self-healing capacity of skin is limited, and medical intervention is often unavoidable. Skin may be generated ex vivo from cultured fibroblasts. Because the molecular composition of de novo formed skin (mostly collagen and glycosaminoglycans [GAGs]) is crucial, analytical methods are required for the quality control of tissue-engineered products. Here, we show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of fibroblast cultures subsequent to digestion with chondroitinase ABC is a reliable and fast method to monitor the GAG content of native and bioengineered skin. Furthermore, the supplementation of the fibroblast medium with ¹³C-labeled glucose provides insights into the biosynthesis of GAGs.

Gut-Evolved Candida albicans Induces Metabolic Changes in Neutrophils.

Serial passaging of the human fungal pathogen Candida albicans in the gastrointestinal tract of antibiotics-treated mice selects for virulence-attenuated strains. These gut-evolved strains protect the host from infection by a wide range of pathogens via trained immunity. Here, we further investigated the molecular and cellular mechanisms underlying this innate immune memory. Both Dectin-1 (the main receptor for ß-glucan; a well-described immune training molecule in the fungal cell wall) and Nod2 (a receptor described to mediate BCG-induced trained immunity), were redundant for the protection induced by gut-evolved C. albicans against a virulent C. albicans strain, suggesting that gut-evolved C. albicans strains induce trained immunity via other pathways. Cytometry by time of flight (CyTOF) analysis of mouse splenocytes revealed that immunization with gut-evolved C. albicans resulted in an expansion of neutrophils and a reduction in natural killer (NK) cells, but no significant numeric changes in monocytes, macrophages or dendritic cell populations. Systemic depletion of phagocytes or neutrophils, but not of macrophages or NK cells, reduced protection mediated by gut-evolved C. albicans. Splenocytes and bone marrow cells of mice immunized with gut-evolved C. albicans demonstrated metabolic changes. In particular, splenic neutrophils displayed significantly elevated glycolytic and respiratory activity in comparison to those from mock-immunized mice. Although further investigation is required for fully deciphering the trained immunity mechanism induced by gut-evolved C. albicans strains, this data is consistent with the existence of several mechanisms of trained immunity, triggered by different training stimuli and involving different immune molecules and cell types.

CD27hiCD38hi plasmablasts are activated B cells of mixed origin with distinct function.

Clinically important broadly reactive B cells evolve during multiple infections, with B cells re-activated after secondary infection differing from B cells activated after a primary infection. Here we studied CD27highCD38high plasmablasts from patients with a primary or secondary dengue virus infection. Three transcriptionally and functionally distinct clusters were identified. The largest cluster 0/1 was plasma cell-related, with cells coding for serotype cross-reactive antibodies of the IgG1 isotype, consistent with memory B cell activation during an extrafollicular response. Cells in clusters 2 and 3 expressed low levels of antibody genes and high levels of genes associated with oxidative phosphorylation, EIF2 pathway, and mitochondrial dysfunction. Clusters 2 and 3 showed a transcriptional footprint of T cell help, in line with activation from naive B cells or memory B cells. Our results contribute to the understanding of the parallel B cell activation events that occur in humans after natural primary and secondary infection.

Metformin enhances anti-mycobacterial responses by educating CD8+ T-cell immunometabolic circuits.

Patients with type 2 diabetes (T2D) have a lower risk of Mycobacterium tuberculosis infection, progression from infection to tuberculosis (TB) disease, TB morality and TB recurrence, when being treated with metformin. However, a detailed mechanistic understanding of these protective effects is lacking. Here, we use mass cytometry to show that metformin treatment expands a population of memory-like antigen-inexperienced CD8+CXCR3+ T cells in naive mice, and in healthy individuals and patients with T2D. Metformin-educated CD8+ T cells have increased (i) mitochondrial mass, oxidative phosphorylation, and fatty acid oxidation; (ii) survival capacity; and (iii) anti-mycobacterial properties. CD8+ T cells from Cxcr3-/- mice do not exhibit this metformin-mediated metabolic programming. In BCG-vaccinated mice and guinea pigs, metformin enhances immunogenicity and protective efficacy against M. tuberculosis challenge. Collectively, these results demonstrate an important function of CD8+ T cells in metformin-derived host metabolic-fitness towards M. tuberculosis infection.

Needle-based storage-phosphor detector radiography is superior to a conventional powder-based storage phosphor detector and a high-resolution screen-film system in small patients (budgerigars and mice).

This method comparison study used radiographs of 20 mice and 20 budgerigars to investigate comparability between computed radiography (CR) and high-resolution screen-film systems and study the effects of reduced radiation doses on image quality of digital radiographs of small patients. Exposure settings used with the mammography screen-film system (SF) were taken as baseline settings. A powder-based storage-phosphor system (CRP) and a needle-based storage-phosphor system (CRN) were used with the same settings (D/100%) and half the detector dose (D/50%). Using a scoring system four reviewers assessed five criteria per species covering soft tissue and bone structures. Results were evaluated for differences between reviewers (interobserver variability), systems and settings (intersystem variability, using visual grading characteristic analysis). Correlations were significant (p ≤ 0.05) for interobserver variability in 86.7% of the cases. Correlation coefficients ranged from 0.206 to 0.772. For mice and budgerigars, the CRN system was rated as superior to the SF and CRP system for most criteria, being significant in two cases each. Comparing the SF and CRP system, the conventional method scored higher for all criteria, in one case significantly. For both species and both digital systems, dose reduction to 50% resulted in significantly worse scores for most criteria. In summary, the needle-based storage-phosphor technique proved to be superior compared to the conventional storage-phosphor and mammography screen-film system. Needle-based detector systems are suitable substitutes for high-resolution screen-film systems when performing diagnostic imaging of small patients. Dose reduction to 50% of the corresponding dose needed in high-resolution film-screen systems cannot be recommended.

Metabolic energy sensors as targets for designing host-directed therapies for tuberculosis.

A wealth of scientific and clinical evidence during the past few years has lent credence to the idea that key components of the host immune effector mechanisms can be targeted to boost current tuberculosis (TB) treatment and control patient relapse. These host-directed strategies not only accelerate the clearance of pathogens but also have the ability to limit overt inflammation and pathology, which are associated with the tissue damage. Studies have indicated that inflammatory responses are intrinsically linked to cellular metabolism and together drive the fate of many host responses, coupling host survival with the capacity to respond to infectious insult. Metabolic sensors such as mammalian target of rapamycin, AMP-activated protein kinase, and sirtuin 1 are central regulators of host metabolic alterations and play important roles in immune responses against infections. The present review discusses the functions of AMP-activated protein kinase and sirtuin 1, with a focus on their role in immune homeostasis and how manipulating the AMP-activated protein kinase-sirtuin 1 axis with drugs can modulate immunity to tuberculosis.

Breed-related differences in age-dependent down-regulation of the ß1-adrenoceptor and adenylate cyclase activity in atrial and ventricular myocardium of Cröllwitzer ("wild-type") turkeys.

In conventional meat-type (British United Turkey (B.U.T.) Big 6) turkey hearts, it has been shown that all cardiac chambers exhibit down-regulation of the ß1-adrenoceptors (ß1-AR) and concomitantly cAMP accumulation with increasing age regardless of sex. In this study we proved the hypothesis that breed differences exist in age-dependent alterations in the ß1-AR system. Right (RA) and left (LA) atrial as well as right (RV) and left (LV) ventricular tissues were collected from male and female Cröllwitzer "wild-type" turkey poults of increasing age (6 wk, 12 wk, 16 wk, 21 wk). The ß1-AR density and function were quantified by (-)-[125I]-iodocyanopindolol (ICYP) radioligand binding analysis in cell membranes from 4 cardiac chambers. Basal and stimulated cAMP production was determined as indicator of the receptor function. Wild-type turkeys showed significantly higher heart to body weight ratio than the meat-type B.U.T. Big 6 turkeys. In both sexes of Cröllwitzer turkey hearts, the ß1-AR density decreased with age but significance was reached in male cardiac chambers. The receptor affinity (KD) and subtype distribution were not altered. Sex had no effect on age-related decrease in receptor density but had an effect on adenylate cyclase (AC) activity and subsequently cAMP production. In male Cröllwitzer turkey hearts of all ages, cAMP remained at same level, whereas this was even increased in female cardiac chambers. Thus, breed affected age-related receptor-, G-protein and AC-stimulated cAMP formation in normal ventricles and atria, with females exhibiting pronounced increase with age. This suggests that the receptor signaling in wild-type turkey hearts is not as blunted as in hearts of meat-type turkey poults in which stressful farming conditions and fast growing lead to receptor down-regulation.

NLRP10 Enhances CD4+ T-Cell-Mediated IFNγ Response via Regulation of Dendritic Cell-Derived IL-12 Release.

NLRP10 is a nucleotide-binding oligomerization domain-like receptor that functions as an intracellular pattern recognition receptor for microbial products. Here, we generated a Nlrp10-/- mouse to delineate the role of NLRP10 in the host immune response and found that Nlrp10-/- dendritic cells (DCs) elicited sub-optimal IFNγ production by antigen-specific CD4+ T cells compared to wild-type (WT) DCs. In response to T-cell encounter, CD40 ligation or Toll-like receptor 9 stimulation, Nlrp10-/- DCs produced low levels of IL-12, due to a substantial decrease in NF-κB activation. Defective IL-12 production was also evident in vivo and affected IFNγ production by CD4+ T cells. Upon Mycobacterium tuberculosis (Mtb) infection, Nlrp10-/- mice displayed diminished T helper 1-cell responses and increased bacterial growth compared to WT mice. These data indicate that NLRP10-mediated IL-12 production by DCs is critical for IFNγ induction in T cells and contributes to promote the host defense against Mtb.

Differential regulation of the ß-adrenoceptor density and cyclic AMP level with age and sex in turkey cardiac chambers.

Decreased responses of the heart to ß-adrenoceptor stimulation with aging have been shown to occur merely in selected heart chambers in relation to increased catecholamine levels. However, there are no systematic studies that investigate all cardiac chambers with regard to receptor density and cAMP (adenosine 3', 5'-cyclic monophosphate) responses. We used meat-type turkey poults (British United Turkey (B.U.T.) Big 6) with increasing age because their heart seems to decrease in weight in relation to body weight and they are often used as an animal model for heart failure. The receptor density and distribution were quantified by radioligand binding analysis using (-)-[(125)I]-iodocyanopindolol and ß-adrenoceptor subtype-specific antagonists (ICI 118.551 and CGP 20712 A) in membranes of four cardiac chambers (right and left atria and ventricles) of 6-week-, 12-week-, 16/21-week-, and 57-week-old B.U.T. BIG 6 turkeys. Receptor function was determined by measuring basal and stimulated cAMP production. In both sexes, the ß-adrenoceptor density decreased significantly in all chambers with age without altered ß-adrenoceptor subtype distribution. The receptor affinity (KD) to the radioligand was similar in hearts of all age groups. ß-adrenoceptor-(isoproterenol and guanosine 5'-triphosphate), G-protein-(NaF) and catalytic unit of adenylate cyclase (forskolin, Mn(2+)) mediated cAMP responses were not chamber-dependent. Indeed, the cAMP level was significantly lower in 57-week-old hearts than in 6-week-, 12-week-, 16/21-week-old hearts. These data suggest that with increasing age and body weight, the ß-adrenoceptor signal transduction pathway was highly blunted in all cardiac chambers, occurring by decreased receptor density and cAMP responses.

IL-17A promotes macrophage effector mechanisms against Trypanosoma cruzi by trapping parasites in the endolysosomal compartment.

The contribution of the IL-23-IL-17A pathway to resistance against extracellular bacterial infections is well established, whereas its role in immunity to intracellular pathogens is much less clear. To analyze the contribution of the IL-23-IL-17A-axis to resistance against Trypanosoma cruzi infection, we infected IL-23p19(-/-) mice and IL-17A(-/-) mice with T. cruzi. Both mouse strains were susceptible to T. cruzi infection despite strong Th1 immune responses. In vitro experiments revealed that IL-17A, but not IL-23, directly stimulates macrophages to internalize T. cruzi parasites by phagocytosis, which is in contrast to the active invasion process normally used by T. cruzi. In contrast to the active entry of parasites into macrophages, the IL-17A-driven phagocytosis prolonged residency of parasites in the endosomal/lysosomal compartment of the macrophage, which subsequently led to eradication of parasites. This IL-17A-dependent mechanism represents a novel function of IL-17A trapping pathogens in endosomal/lysosomal compartments and enhancing exposure time to antimicrobial effectors of the macrophage.

[Examination of rearing standards and health status in turkeys in Germany]. / Erhebungen zur Haltung und Gesundheit bei Mastputen in Deutschland.

Within the framework of a cooperative research project, sponsored by the Federal Ministry of Food, Agriculture and Consumer Protection in Germany (BMELV), turkey health as well as numerous aspects of animal welfare in various intensive commercial farms with varying rearing forms were examined. For this purpose extensive documentation of flock management and health status information was conducted over the past two years at the farms. Additional information was ascertained from the carcasses at the slaughterhouse. The first results of this study, which look at flock management, will be presented here. Upon reviewing the questionnaires, it is clear that on one hand almost all farmers orientate there stocking rate upon the allowed limits, on the other hand, however, when considering national general requirements, which are very important parameters thatargely influence animal health (i. e. litter quality and care of sick/injured animals) farmer compliance lacks. Especially the litter quality, which, among other parameters, plays a very important role in food pad health, proved to be unsatisfactory. Unfortunately, no instruments are available that measure the litter quality, yet. The status of footpad health was a major problem in all the rearing systems. However, the prevalence of epithelial necrosis and deep skin lesions varied immensely among different flocks of the same age.