Fatty acid profile and proliferation of bovine blood mononuclear cells after conjugated linoleic acid supplementation

BACKGROUND: Conjugated linoleic acids (CLA) are in focus of dairy cattle research because of its milk fat reducing effects. Little is known about the impact of CLA on immune function in dairy cows. Therefore, in the present study we investigated the effects of a long term supplementation of dairy cows with CLA on the fatty acid profile of peripheral blood mononuclear cells (PBMC) and their proliferation ex vivo. RESULTS: The supplementation of dairy cows with either 100 g/d of a control fat preparation (CON, n = 15), 50 g/d of the control fat preparation and 50 g/d CLA supplement – containing 12.0% cis-9, trans-11 and 11.9% trans-10, cis-12 CLA of total fatty acid methyl esters – (CLA-50, n = 15) or 100 g/d of the CLA supplement (CLA-100, n = 16) did not influence the major fatty acids (C18:0, C16:0, cis-9 C18:1, cis-9, cis-12 C18:2, cis-5, cis-8, cis-11, cis-14 C20:4) in the lipid fraction of PBMC. The proportion of trans-10, cis-12 CLA of total fatty acids was increased in both CLA supplemented groups, but there was no effect on the cis-9, trans-11 isomer. Furthermore, the proportion of trans-9 C18:1 and cis-12 C24:1 was reduced in the CLA-100 group. The mitogen stimulated cell proliferation was not influenced by CLA feeding. CONCLUSION: CLA supplementation influenced the FA profile of some minor FA in PBMC, but these changes did not lead to differences in the mitogen induced activation of the cells

Tuberculosis in Pediatric Antiretroviral Therapy Programs in Low- and Middle-Income Countries: Diagnosis and Screening Practices

Background The global burden of childhood tuberculosis (TB) is estimated to be 0.5 million new cases per year. Human immunodeficiency virus (HIV)-infected children are at high risk for TB. Diagnosis of TB in HIV-infected children remains a major challenge. Methods We describe TB diagnosis and screening practices of pediatric antiretroviral treatment (ART) programs in Africa, Asia, the Caribbean, and Central and South America. We used web-based questionnaires to collect data on ART programs and patients seen from March to July 2012. Forty-three ART programs treating children in 23 countries participated in the study. Results Sputum microscopy and chest Radiograph were available at all programs, mycobacterial culture in 40 (93%) sites, gastric aspiration in 27 (63%), induced sputum in 23 (54%), and Xpert MTB/RIF in 16 (37%) sites. Screening practices to exclude active TB before starting ART included contact history in 41 sites (84%), symptom screening in 38 (88%), and chest Radiograph in 34 sites (79%). The use of diagnostic tools was examined among 146 children diagnosed with TB during the study period. Chest Radiograph was used in 125 (86%) children, sputum microscopy in 76 (52%), induced sputum microscopy in 38 (26%), gastric aspirate microscopy in 35 (24%), culture in 25 (17%), and Xpert MTB/RIF in 11 (8%) children. Conclusions Induced sputum and Xpert MTB/RIF were infrequently available to diagnose childhood TB, and screening was largely based on symptom identification. There is an urgent need to improve the capacity of ART programs in low- and middle-income countries to exclude and diagnose TB in HIV-infected childre

Spatial-Explicit Climate Change Vulnerability Assessments Based on Impact Chains. Findings from a Case Study in Burundi

Climate change vulnerability assessments are an essential instrument to identify regions most vulnerable to adverse impacts of climate change and to determine appropriate adaptation measures. Vulnerability assessments directly support countries in developing adaptation plans and in identifying possible measures to reduce adverse consequences of changing climate conditions. Against this background, this paper describes a vulnerability assessment using an integrated and participatory approach that builds on standardized working steps of previously developed ‘Vulnerability Sourcebook’ guidelines. The backbone of this approach is impact chains as a conceptual model of cause–effect relationships as well as a structured selection of indicators according to the three main components of vulnerability, namely exposure, sensitivity and adaptive capacity. We illustrate our approach by reporting the results of a vulnerability assessment conducted in Burundi focusing on climate change impacts on water and soil resources. Our work covers two analysis scales: a national assessment with the aim to identify climate change ‘hotspot regions’ through vulnerability mapping; and a local assessment aiming at identifying local-specific drivers of vulnerability and appropriate adaptation measures. Referring to this vulnerability assessment in Burundi, we discuss the potentials and constraints of the approach. We stress the need to involve stakeholders in every step of the assessment and to communicate limitations and uncertainties of the applied methods, indicators and maps in order to increase the comprehension of the approach and the acceptance of the results by different stakeholders. The study proved the practical usability of the approach at the national level by the selection of three particularly vulnerable areas. The results at a local scale supported the identification of adaption measures through intensive engagement of local rural populations

Physiological Concentration of Exogenous Lactate Reduces Antimycin A Triggered Oxidative Stress in Intestinal Epithelial Cell Line IPEC-1 and IPEC-J2 In Vitro

Weaning triggers an adaptation of the gut function including luminal lactate generation by lactobacilli, depending on gastrointestinal site. We hypothesized that both lactobacilli and lactate influence porcine intestinal epithelial cells. In vivo experiments showed that concentration of lactate was significantly higher in gastric, duodenal and jejunal chyme of suckling piglets compared to their weaned counterparts. In an in vitro study we investigated the impact of physiological lactate concentration as derived from the in vivo study on the porcine intestinal epithelial cells IPEC-1 and IPEC-J2. We detected direct adherence of lactobacilli on the apical epithelial surface and a modulated F-actin structure. Application of lactobacilli culture supernatant alone or lactate (25 mM) at low pH (pH 4) changed the F-actin structure in a similar manner. Treatment of IPEC cultures with lactate at near neutral pH resulted in a significantly reduced superoxide-generation in Antimycin A-challenged cells. This protective effect was nearly completely reversed by inhibition of cellular lactate uptake via monocarboxylate transporter. Lactate treatment enhanced NADH autofluorescence ratio (F-cytosol/F-nucleus) in non-challenged cells, indicating an increased availability of reduced nucleotides, but did not change the overall ATP content of the cells. Lactobacilli-derived physiological lactate concentration in intestine is relevant for alleviation of redox stress in intestinal epithelial cells.Peer reviewe

Effects of conjugated linoleic acids on the function of bovine immune cells ex vivo and in vitro

Conjugated linoleic acids (CLA) are a group of C18:2 fatty acids (FA) that are characterized by the conjugated position of its 2 double bonds. Several health beneficial effects are proclaimed for CLA in animals and humans, such as anticancerogenic, antiatherosclerotic, antidiabetic and immunomodulating properties. Although CLA, especially the cis-9,trans-11 isomer, are intermediate products of biohydrogenation in the rumen and therefore naturally occur in dairy products and meat from ruminants, dairy cows feed is supplemented with CLA. This is done because CLA supplementation reduces the milk fat content. There is no information on the effects of CLA supplementation on the bovine immune system. Thus the following investigations were performed in order to clarify this open question. Two feeding trials were conducted to evaluate the effects of CLA on immune functions in dairy cows. First, a long term feeding study with 46 cows was performed. After parturition, the cows received a diet either supplemented with 100 g/d of a control fat preparation (Con), 50 g/d of the control fat preparation and 50 g/d of the CLA supplement, respectively (CLA-50) or 100 g/d of the CLA supplement (CLA-100). The supplement contained 12% of the cis-9,trans-11 and 12% of the trans-10,cis-12 isomer. Thus the daily CLA consumption was 4 and 8 g of each isomer in the CLA supplemented groups, respectively. The supplements were fed from calving onward until day 182 post partum (pp). Blood was taken at day 7, 21, 35, 49, 70, 105, 140 and 182 pp. From the whole blood peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. Samples from day 70 and 140 were pooled for each animal and the FA profile of the lipid fraction of PBMC was analyzed. The samples from the remaining 6 sampling days were used to evaluate the concanavalin A (ConA) stimulated proliferation ex vivo by Alamar blue (AB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The most abundant FA in the lipid fraction of PBMC were not altered by CLA supplementation (stearic acid, palmitic acid, oleic acid, linoleic acid and arachidonic acid), but effects on the proportions of some minor FA were detected. The percentage of the trans-10,cis-12 isomer was significantly increased in both CLA supplemented groups, but there was no effect on the cis-9,trans-11 isomer. However, the proportion of CLA isomers other than cis-9,trans-11 and trans-10,cis-12 was decreased in the CLA-100 group compared to Con, as well as the percentage of nervonic acid and elaidic acid. Although the FA profile was slightly altered, the mitogen stimulated proliferation of PBMC was not affected by CLA supplementation. The time in relation to calving had an effect on the stimulation index (SI, defined as optical density (OD) or fluorescence of ConA stimulated cells divided by OD or fluorescence of non-stimulated cells) of PBMC with a minimum at day 49 pp. The second feeding study was conducted as a slaughter trial. In that study 25 primiparous cows were investigated. The animals received either the 100 g/d of the control fat preparation or 100 g/d of the CLA supplement beginning at day 1 pp. An initial group (IG), which was not supplemented with CLA or the control fat, was slaughtered 1 day pp. Five animals of each feeding group were slaughtered 42 days pp (42/CON and 42/CLA) and 105 days pp (105/CON and 105/CLA), respectively. Fourteen days ante partum and immediately before slaughter, blood samples were taken to isolate PBMC. The spleen was removed during dissection to obtain splenocytes. Mitogen stimulated cell proliferation of splenocytes was analyzed by MTT assay and that of PBMC by MTT and AB assay. As an additional characterization of immune functions the basal expression of interleukin (IL) 4, IL-10, IL-12, interferon . (IFN-.), and the tumor necrosis factor a (TNF-a) from splenocytes and PBMC was analyzed by quantitative real time polymerase chain reaction (PCR). As in the previous study, there was no effect of CLA feeding on the SI of PBMC. The SI increased from day 1 pp to day 105 pp in the MTT assay, but only from day 42 pp until day 105 pp in the control fat fed groups in the AB assay. In contrast, there was no time effect on the SI of splenocytes, whereas CLA feeding significantly decreased the SI 105 days pp. Comparing the expression of the investigated cytokines at the same time point pp, there were no CLA effects. Increased expressions from day 1 pp (IG) up to day 105 pp were found within the CLA fed groups for IL-4 in splenocytes and for IFN-. and IL-12 in both cell types. The expression of IL-4 in PBMC and of IL-10 and TNF-a was not effected by CLA or the time in relation to calving. TNF-a was not expressed in splenocytes. In addition to the ex vivo analyses, the effects of CLA on the function of bovine PBMC was investigated in vitro. PBMC were obtained from 3 cows. The effect of linoleic acid (LA), cis-9,trans-11, and trans-10,cis-12 CLA, phytanic acid (PA), and a fatty acid mixture (mimicking the FA composition of the subcutaneous adipose tissue and containing 29.8% palmitic acid, 6.7% palmitoleic acid, 17.4% stearic acid and 46.1% oleic acid) on the ConA-stimulated proliferation of PBMC was tested by AB and 5-bromo-2'-deoxyuridine (BrdU) assay in a concentration range from 0 to 500 µM. Based on these results, a combination of the FA mixture (60%) with LA, cis-9,trans-11, trans-10,cis-12 (40%), and a combination of these 3 FA (each FA 13.3%) was tested in the AB assay (33, 66, 99 and 500 µM). The FA mixture (60%) was also combined with PA (40%), PA (20%), and cis-9,trans-11 (20%) as well as with PA (20%) and trans-10,cis-12 CLA (20%). Furthermore, the expression of PPAR-. and the cytokines IL-4, IL-10, IL-12, IFN-. and TNF-a from unstimulated and ConA-stimulated PBMC was analyzed by quantitative real-time PCR in response to cis-9,trans-11 and LA (concentrations: calculated IC50 values of the respective FA and 77 µM). The IC50 values, calculated from the SI obtained from the dose response studies using AB assay, were 100.67 µM for LA, 53.83 µM for cis-9,trans-11, 70.12 µM for trans-10,cis-12, 94.67 µM PA and 80.83 µM for the FA mixture. The IC50 values did not differ significantly between the investigated FA, but the ConA stimulated proliferation (% of control) was different between the various FA at certain concentrations in the AB (20 to 148 µM) and BrdU assay (44 to 99 µM). These differences were not observed in the SI when LA, cis-9,trans-11, trans-10,cis-12, and PA were combined with the FA mixture. As in the investigations using single FA the SI decreased with increasing FA concentrations. No effects of FA treatment were observed in the expression of IL-4, IL-10 and TNF-a. The expression of IFN-. was significantly reduced when PBMC were stimulated with ConA and incubated with DMSO or 77 µM cis-9,trans-11 compared to the ConA stimulated PBMC incubated without FA or DMSO (medium control). The expression significantly increased in response to ConA for TNF-a and IFN-. (medium control and IC50 value of LA). ConA decreased the expression of IL-10 in the medium control. The expression of PPAR-. was significantly lower in ConA treated cells incubated with 77 µM LA than in those treated with same concentration of cis-9,trans-11. In conclusion, the effects of CLA on the investigated immune functions in dairy cows are marginal and only inhibiting effects on the SI of splenocytes were observed. Although the FA composition of PBMC was slightly altered by CLA supplementation, these changes did not impair the mitogen stimulated proliferation of the cells. In vitro FA dose-dependently inhibit the proliferation of bovine PBMC. Differences in ConA-stimulated proliferation occur at certain concentrations when single FA are investigated, but not within the tested FA combinations. The effects of CLA on the expression of cytokines were marginal in ex vivo and in vitro investigations.Konjugierte Linolsäuren (CLA) werden in der Milchviehfütterung eingesetzt, weil sie den Milchfettgehalt senken. Bisher ist wenig darüber bekannt, ob dies das Immunsystem der Milchkühe beeinflusst. Deshalb hat die vorliegende Arbeit das Ziel, den Einfluss einer Langzeitsupplementierung mit CLA auf bovine Immunzellen im Zeitraum nach der Abkalbung zu untersuchen. Dazu wurden zwei Fütterungsversuche durchgeführt, um den Einfluss von CLA auf die Proliferation und Zytokinexpression von Splenozyten und peripheren mononukleären Blutzellen (PBMC) sowie deren Fettsäuremuster zu analysieren. Der Effekt einzelner CLA-Isomere auf die Proliferation und Zytokinexpression boviner PBMC wurde allein und in Kombination mit anderen Fettsäuren in vitro untersucht. Insgesamt wurden kleinere Effekte von CLA auf bovine Immunzellen gefunden, die sich hauptsächlich in einem erniedrigten Stimulationsindex der Splenozyten und kleineren Veränderungen im Fettsäuremuster der PBMC gezeigt haben

Effects of conjugated linoleic acids on the function of bovine immune cells ex vivo and in vitro - [kumulative Dissertation]

Konjugierte Linolsäuren (CLA) werden in der Milchviehfütterung eingesetzt, weil sie den Milchfettgehalt senken. Bisher ist wenig darüber bekannt, ob dies das Immunsystem der Milchkühe beeinflusst. Deshalb hat die vorliegende Arbeit das Ziel, den Einfluss einer Langzeitsupplementierung mit CLA auf bovine Immunzellen im Zeitraum nach der Abkalbung zu untersuchen. Dazu wurden zwei Fütterungsversuche durchgeführt, um den Einfluss von CLA auf die Proliferation und Zytokinexpression von Splenozyten und peripheren mononukleären Blutzellen (PBMC) sowie deren Fettsäuremuster zu analysieren. Der Effekt einzelner CLA-Isomere auf die Proliferation und Zytokinexpression boviner PBMC wurde allein und in Kombination mit anderen Fettsäuren in vitro untersucht. Insgesamt wurden kleinere Effekte von CLA auf bovine Immunzellen gefunden, die sich hauptsächlich in einem erniedrigten Stimulationsindex der Splenozyten und kleineren Veränderungen im Fettsäuremuster der PBMC gezeigt haben.Conjugated linoleic acids (CLA) are used in dairy cows feed because of its milk fat reducing abilities. Little is known about the effects of CLA feeding on the immune system of dairy cows. Therefore, the aim of the present study was to investigate the effects of long term CLA supplementation on the function of bovine immune cells after parturition. Two feeding trials with dairy cows were conducted to study the effects of CLA supplementation on the proliferation and cytokine expression of splenocytes and peripheral blood mononuclear cells (PBMC) as well as their fatty acid profile. The effect of different CLA isomers alone and in combination with other fatty acids on the proliferation and cytokine expression was tested in vitro. Altogether, the effects of CLA on dairy cows immune cells are marginal - seen as a decreased stimulation index of splenocytes after CLA supplementation and minor changes in the fatty acids profile of PBMC.von Lydia Renne

Genome evolution following an ecological shift in nectar-dwelling Acinetobacter

ABSTRACT The bacterial genus Acinetobacter includes species found in environmental habitats like soil and water, as well as taxa adapted to be host-associated or pathogenic. High genetic diversity may allow for this habitat flexibility, but the specific genes underlying switches between habitats are poorly understood. One lineage of Acinetobacter has undergone a substantial habitat change by evolving from a presumed soil-dwelling ancestral state to thrive in floral nectar. Here, we compared the genomes of floral-dwelling and pollinator-associated Acinetobacter, including newly described species, with genomes from relatives found in other environments to determine the genomic changes associated with this ecological shift. Following one evolutionary origin of floral nectar adaptation, nectar-dwelling Acinetobacter taxa have undergone reduction in genome size compared with relatives and have experienced dynamic gene gains and losses as they diversified. Gene content changes suggest a shift to metabolism of monosaccharides rather than diverse carbohydrates, and scavenging of nitrogen sources, which we predict to be beneficial in nectar environments. Gene gains appear to result from duplication events, evolutionary divergence, and horizontal gene transfer. Most notably, nectar-dwelling Acinetobacter acquired the ability to degrade pectin from plant pathogens, and the genes underlying this ability have duplicated and are under selection within the clade. We hypothesize that this ability was a key trait for adaptation to floral nectar, as it could improve access to nutrients in the nutritionally unbalanced habitat of nectar. These results identify the genomic changes and traits coinciding with a dramatic habitat switch from soil to floral nectar.IMPORTANCEMany bacteria, including the genus Acinetobacter, commonly evolve to exploit new habitats. However, the genetic changes that underlie habitat switches are often unknown. Floral nectar is home to specialized microbes that can grow in this nutritionally unbalanced habitat. Several specialized Acinetobacter species evolved from soil-dwelling relatives to become common and abundant in floral nectar. Here, we investigate the genomic adaptations required to successfully colonize a novel habitat like floral nectar. We performed comparative genomics analyses between nectar-dwelling Acinetobacter and Acinetobacter species from other environments, like soil and water. We find that although gene loss coincided with the switch to living in nectar, gains of specific genes from other bacteria may have been particularly important for this ecological change. Acinetobacter living in nectar gained genes for degrading pectin, a plant polysaccharide, which may improve access to nutrients in their environment. These findings shed light on how evolutionary novelty evolves in bacteria