Lactation Affects Isolated Mitochondria and Its Fatty Acid Composition but Has No Effect on Tissue Protein Oxidation, Lipid Peroxidation or DNA-Damage in Laboratory Mice.

Linking peak energy metabolism to lifespan and aging remains a major question especially when focusing on lactation in females. We studied, if and how lactation affects in vitro mitochondrial oxygen consumption and mitochondrial fatty acid composition. In addition, we assessed DNA damage, lipid peroxidation and protein carbonyls to extrapolate on oxidative stress in mothers. As model system we used C57BL/6NCrl mice and exposed lactating females to two ambient temperatures (15 °C and 22 °C) while they nursed their offspring until weaning. We found that state II and state IV respiration rates of liver mitochondria were significantly higher in the lactating animals than in non-lactating mice. Fatty acid composition of isolated liver and heart mitochondria differed between lactating and non-lactating mice with higher n-6, and lower n-3 polyunsaturated fatty acids in the lactating females. Surprisingly, lactation did not affect protein carbonyls, lipid peroxidation and DNA damage, nor did moderate cold exposure of 15 °C. We conclude that lactation increases rates of mitochondrial uncoupling and alters mitochondrial fatty acid composition thus supporting the "uncoupling to survive" hypothesis. Regarding oxidative stress, we found no impact of lactation and lower ambient temperature and contribute to growing evidence that there is no linear relationship between oxidative damage and lactation.

Investigations into cytotoxic effects of the herbal preparation Abnormal Savda Munziq.

OBJECTIVE: To evaluate the effects of Abnormal Savda Munziq (ASMq), a traditional herbal medicine, for the prevention and treatment of human diseases, e.g. bowel cancer. METHODS: The parameters total polyphenol content, cell proliferation and DNA-damage as well as RNA and protein-oxidation were analysed in vitro. Besides, the expressions of miRNA and tumor suppressor genes as well as cellular senescence were evaluated. RESULTS: ASMq had a high polyphenol content and induced damage to proteins, RNA as well as to DNA, which is correlated with its cytotoxicity. Furthermore ASMq up-regulated the tumor suppressor genes p21, p53 and p16 and down-regulated the micro-RNAs hsa-mir-17 and hsa-mir-106b. In addition cellular growth arrest and SA-ß-gal-staining were induced. CONCLUSION: ASMq has the ability to induce DNA damage and cellular senescence, which are double-edged mechanisms in fighting cancer, as they might also have harmful side effects.

Effects of feed contaminant deoxynivalenol on plasma cytokines and mRNA expression of immune genes in the intestine of broiler chickens.

An experiment was conducted to investigate the individual and combined effects of dietary deoxynivalenol (DON) and a microbial feed additive on plasma cytokine level and on the expression of immune relevant genes in jejunal tissues of broilers. A total of 40 broiler chicks were obtained from a commercial hatchery and divided randomly into four groups (10 birds per group). Birds were reared in battery cages from one day old for 5 weeks. The dietary groups were 1) control birds fed basal diet; 2) DON group fed basal diet contaminated with 10 mg DON/ kg feed; 3) DON + Mycofix group fed basal diet contaminated with 10 mg DON/ kg feed and supplemented with a commercial feed additive, Mycofix® Select (MS) (2.5 kg/ton of feed); 4) Mycofix group fed basal diet supplemented with MS (2.5 kg/ton of feed). At 35 days, the plasma levels of tumor necrosis factor alpha (TNF-α) and interleukin 8 (IL-8) were quantified by ELISA test kits. Furthermore, the mRNA expression of TNF-α, IL-8, IL-1ß, interferon gamma (IFNγ), transforming growth factor beta receptor I (TGFBR1) and nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κß1) in jejunum were quantified by qRT-PCR. The results showed that the plasma TNF-α decreased in response to DON, while in combination with MS, the effect of DON was reduced. DON down-regulated the relative gene expression of IL-1ß, TGFBR1 and IFN-γ, and addition of MS to the DON contaminated diet compensates these effects on IL-1ß, TGFBR1 but not for IFN-γ. Furthermore, supplementation of MS to either DON contaminated or control diet up-regulated the mRNA expression of NF-κß1. In conclusion, DON has the potential to provoke and modulate immunological reactions of broilers and subsequently could increase their susceptibility to disease. The additive seemed to have almost as much of an effect as DON, albeit on different genes.

Protective effects of antioxidants on deoxynivalenol-induced damage in murine lymphoma cells.

As contradictory results have been reported on the immunotoxic properties of deoxynivalenol (DON) in animal studies, we introduced a lymphoblast cell culture model in order to examine the effects of DON on lymphoblastic cell growth and metabolism as well as the preventive properties of free radical scavenger molecules against the DON-induced cell damage. Murine YAC-1 lymphoma cells were used because lymphoblasts have been shown to be sensitive to DON-induced immunotoxicity. Cells were quantified and their proliferative activity was measured by a proliferation test. Lipid peroxidation and protein oxidation were determined using assays quantifying thiobarbituric acid reactive substances (TBARS) and carbonylated proteins. Severely reduced cell counts were detected in DON-treated samples, confirmed by a 5-10 times lower proliferative activity. Significant increases in lipid peroxidation and protein oxidation were found in parallel incubated samples. The pre-incubation with free radical scavengers significantly reduced DON-induced changes to proteins and lipids as well as the tarnished cell viability and cell proliferation. These results suggest that YAC-1 lymphoma cells are a suitable model to investigate and elucidate the basic molecular and cellular mechanisms for possible immunotoxic effects of DON. With regard to the impact of free radical scavengers, the applied in-vitro model might enable the investigation of potential prophylactic and therapeutic effects before or even without harmful animal experiments and cost- and time-intensive expenses.

Abnormal Savda syndrome: long-term consequences of emotional and physical stress on endocrine and immune activities in an animal model.

OBJECTIVE: To investigate the relationship between emotional status, cold-dry environment and long-term immune responses to the stressors, and the potential pathological mechanisms between causative factors of abnormal Savda syndrome (ASS) and the susceptibility to disease; thus to clarify the ASS, and secondly to identify the optimal ASS animal model for further studies on traditional Uighur therapeutical formulations. METHODS: Sixty mice were randomly and equally divided into 4 groups: control and 3 stress groups. The cold-dry environment was applied by keeping the mice in a climatic chamber. The emotional stress was induced by the application of the repeated electric foot-shocks in the electric foot-shock apparatus. The mice of the combined stress group underwent the repeated electric foot-shock treatment before being housed in the climatic chamber. The experimental routine was repeated for 21 days. In order to look into endocrine and immune stress responses, ELISA was used to determine the serum levels of the hormones corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), Beta-endorphin (ß-END) and corticosterone (CORT), of the cytokines interleukin 2 (IL-2), interleukin 6 (IL-6), interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α), and of the immunoglobulins immunoglobulin A (IgA), immunoglobulin M (IgM) and immunoglobulin G (IgG). Lymphocyte subsets were analyzed in duplicate in order to determine differences in the T cell ratio. RESULTS: In the cold-dry environment group, the serum levels of CRH, ACTH and CORT were significantly higher than those of the control group, whereas serum ß-END was not found significantly different. In both the repeated electric foot-shock group as well as in the combined stress group the serum levels of CRH, ACTH, ß-END and CORT were significantly higher. Compared to the control animals, the serum concentration of INF-γ was significantly lower in all three different stress groups. The serum level of IL-2 was decreased in the combined stress group whereas the serum TNF-α level was significantly higher. The serum IgG level was significantly higher in all three stress groups, whereas the IgA level was lower in both chronic electric foot-shock group and combined stress group. The IgM level was found significantly higher in the combined stress group only. The percentage of CD4(+) cells in peripheral blood was dramatically decreased in mice exposed to colddry environment, chronic electric foot-shock and combined stress, whereas the percentage of the CD8(+) subset was not significantly different. The CD4(+)/CD8(+) ratios were markedly lower in both cold-dry environment group and combined stress group. CONCLUSIONS: Combined stress can cause hyperactivity of the HPA axis, and an imbalance in the Th1/Th2 cell subset may contribute to illustrate the partial pathological mechanisms of ASS. This study identified this animal model of a combination of physical and emotional stress as an optimal model for further studies on ASS and relative therapies.

Highly deoxynivalenol contaminated oats and immune function in horses.

The aim of the present study was to investigate the impact of deoxynivalenol (DON) on cellular and humoral immune parameters in horses. A feeding trial using naturally contaminated oats with high (20.2 mg/kg) and low (0.49 mg/kg) levels of DON was conducted. Two groups of five mares were fed 2 kg oats daily with high or low DON levels for two weeks, using a crossover design with a three-week wash-out period. No adverse effects on general health were observed. Only minor diet-related changes in differential blood counts and serum biochemistry were noted. Serum haptoglobin concentration was significantly elevated after feeding DON (p = 0.04). Lymphocyte subsets (CD4+ CD8+, CD2+, CD21+, MHCII+) and lymphocyte proliferation data (concanavalin A, phytohemagglutinin, pokeweed mitogen) were not different between feeding-groups. It can be concluded that daily DON intakes as high as 6.9 to 9.5 mg/100 kg BW appear to have no major impact on the measured immune response of horses, indicating that this species has a high tolerance for DON.

Reversal of postischemic hypoperfusion by tempol: endothelial signal transduction mechanism.

This report entails in vivo and in vitro studies concerned with free radical species involved in brain ischemia. The participation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the early manifestation of cerebral ischemia/reperfusion was investigated in gerbils exposed to transient global ischemia using 4-OH-2,2,6,6-tetramethylpiperidine-1-oxyl (TPL), a well-known antioxidant. TPL treatment reversed cerebral postischemic hypoperfusion and tissue edema in these animals. The findings are consistent with ROS/RNS participation in tissue injury and the reduction of cerebromicrovascular blood flow (CBF) during postischemic recirculation. The activation/deactivation of signal transduction pathway by oxidation/antioxidation [i.e., using hydrogen peroxide (H2O2)/TPL] was evaluated in cultured human brain endothelial cells (HBEC) to assess the involvement of endothelial-dependent mechanisms. The data showed that H2O2 activates various "stress" kinases and vasodilalator-stimulated phosphoprotein (VASP); activation of this pathway was reduced by inhibitors of Rho- or IP-3 kinases, as well as TPL. H2O2 also induced cytoskeleton (actin) rearrangements in HBEC; this effect was prevented by inhibitors of Rho/IP3 kinase or TPL. The observed activation/deactivation of H2O2-induced "stress" kinase is in agreement with the reported capacity of ROS/RNS to stimulate the oxidative signal transduction pathway. The noted TPL reduction of H2O2-induced phosphorylation of kinase strongly suggests that the beneficial effect of TPL implicates the stress signal transduction pathway. This may represent a mechanism for the cerebral postischemic manifestations observed by in vivo experiments.

miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging.

Aging is a multifactorial process where deterioration of body functions is driven by stochastic damage while counteracted by distinct genetically encoded repair systems. To better understand the genetic component of aging, many studies have addressed the gene and protein expression profiles of various aging model systems engaging different organisms from yeast to human. The recently identified small non-coding miRNAs are potent post-transcriptional regulators that can modify the expression of up to several hundred target genes per single miRNA, similar to transcription factors. Increasing evidence shows that miRNAs contribute to the regulation of most if not all important physiological processes, including aging. However, so far the contribution of miRNAs to age-related and senescence-related changes in gene expression remains elusive. To address this question, we have selected four replicative cell aging models including endothelial cells, replicated CD8(+) T cells, renal proximal tubular epithelial cells, and skin fibroblasts. Further included were three organismal aging models including foreskin, mesenchymal stem cells, and CD8(+) T cell populations from old and young donors. Using locked nucleic acid-based miRNA microarrays, we identified four commonly regulated miRNAs, miR-17 down-regulated in all seven; miR-19b and miR-20a, down-regulated in six models; and miR-106a down-regulated in five models. Decrease in these miRNAs correlated with increased transcript levels of some established target genes, especially the cdk inhibitor p21/CDKN1A. These results establish miRNAs as novel markers of cell aging in humans.

Effects of bovine lactoferrin on the immune system and the intestinal microflora of adult dogs.

Eighteen Beagle dogs were used to evaluate the effects of bovine lactoferrin (bLF) on immune function and faecal microbial populations. The study comprised three feeding periods, each lasting four weeks. After an initial control Period 1, six dogs per group were supplemented with 0, 120 and 1800 mg bLF/kg dry diet, respectively (Period 2). In Period 3 dogs received again control diets. Peripheral blood mononuclear cell subsets, lymphocyte proliferative response to concanavalin A, phytohaemagglutinin and pokeweed mitogen and plasma IgA and IgG concentrations were analysed. The faecal concentrations of aerobic and anaerobic bacteria, Escherichia coli, Clostridium perfringens, Lactobacillus spp. and Bifidobacterium spp. were determined by cultural methods. Supplementation of bLF increased the number of monocytes, T cells and cytotoxic T cells in the blood and the proliferative response of peripheral blood mononuclear cells. The leukocyte counts were not affected, except monocytes that increased after the supplementation with bLF. Plasma immunoglobulin concentrations were unchanged by treatment. Dogs supplemented with bLF tended to have lower faecal concentrations of E. coli and Clostridium perfringens. In conclusion, bLF seems to alter indices of the cellular immune response and faecal microbial populations of healthy adult dogs.

L-Carnitine L-tartrate (LCLT) and dehydroepiandrosterone sulfate (DHEAS) affect red and white blood cells in aged Sprague-Dawley rats.

Supplementation with either L-carnitine or DHEAS was separately suggested to counteract age-related declines. However, little is known about any interactive effects of these substances, independently promoting mitochondrial energy metabolism, in older individuals. We thus studied the effects of 3 months of daily oral combined supplementation with LCLT and DHEAS on red (RBCs) and white blood cells (WBCs) in male Sprague-Dawley rats by determining RBC and WBC counts, lymphocyte proliferation and interleukin-2 (IL-2) synthesis in spleen lymphocytes after Concanavalin A (ConA) stimulation. Supplementation with LCLT in addition to DHEAS decreased RBCs and increased platelets in the blood of 25-month-old Sprague-Dawley rats, whereas supplementation with DHEAS alone shifted the balance from segmented neutrophile granulocytes to large lymphocytes in differential WBC counts. Based on these results, interactive effects of supplementation with L-carnitine and DHEAS on RBCs and platelets are suggested.

The impact of environment in comparison with moderate physical exercise and dietary restriction on BDNF in the cerebral parietotemporal cortex of aged Sprague-Dawley rats.

BACKGROUND AND AIMS: Moderate physical exercise, dietary restriction as well as enriched environment in separate studies have been reported to delay some of the adverse effects of aging on brain function, parallel to an increase in brain-derived neurotrophic factor (BDNF). In order to elucidate these influences in a comparative setting, we examined the tissue concentrations of BDNF in the cerebral parietotemporal cortex of old Sprague-Dawley rats. METHODS: Male Sprague-Dawley rats were divided randomly into six groups, living from 5 months (baseline group BL) up to 23 months of age as follows: voluntary running in wheels (RW), food restricted by feeding to pair weight with RW animals (PW), forced running on treadmills (TM), and sedentary controls with ad libitum access to food, either housed individually (S1) or in groups of 4 animals (S4). BDNF concentrations were determined by a commercially available ELISA. RESULTS: We found higher BNDF concentrations in the 5 months old animals than in the 23 months old animals of group S1. The old sedentary group S4 showed significantly higher BNDF concentrations in comparison with the old individually caged groups RW, TM, PW and S1. Their BNDF concentrations were even higher than those of the young baseline group. CONCLUSIONS: Our data suggest that housing and social interactions have more influence on BDNF concentrations in the cerebral parietotemporal cortex of aging Sprague-Dawley rats than physical exercise and food restriction.

Impact of moderate physical exercise--in comparison with dietary restrictions--on age-associated decline in cell-mediated immunity of Sprague-Dawley rats.

BACKGROUND AND AIMS: Moderate physical exercise and dietary restriction have both been demonstrated to delay some of the adverse effects of aging. In order to elucidate similarities or dissimilarities in their mode of action on the aging immune system in a comparative setting, we examined significant parameters of cell-mediated immunity in Sprague- Dawley rats. METHODS: Male Sprague-Dawley rats, housed individually, were divided into four groups, living from 5 months (baseline group BL) up to 15, 19 and 23 months of age as follows: voluntary running in wheels (RW), food restricted by feeding to pair weight with RW animals (PW), forced running on treadmills (TM), and sedentary controls with ad libitum access to food (S1). White blood cell counts, capacity for lymphocyte proliferation in response to Concanavalin A, and interleukin-2 (IL-2) plasma concentrations were determined. RESULTS: White blood cell counts and the cell numbers of lymphocytes, neutrophil and eosinophil granulocytes were significantly lower in the older RW and PW groups. We observed influences of forced exercise on lymphocyte proliferation: blastogenic reactivity was higher in TM animals compared with RW and PW animals at 23 months of age. Exclusively for RW animals, we found lower plasma concentrations of IL-2 at 23 months. CONCLUSIONS: Our data support the idea that moderate physical exercise modulates age-associated decline in the cell-mediated immunity of old Sprague-Dawley rats significantly more than corresponding dietary restrictions.

Immune modulation following immunization with polyvalent vaccines in dogs.

A decline in T-cell-mediated immunity and transient state of immunosuppression after immunization has been reported in dogs. Nevertheless, dogs are still routinely vaccinated with polyvalent live vaccines and severe disease does not generally occur. In order to investigate these effects on the canine immune system and to elucidate possible mechanisms we determined the following immune parameters in the blood of 33 clinically sound German shepherd dogs before and after standard vaccination with a polyvalent vaccine against distemper, parvovirus, viral hepatitis, leptospirosis, kennel cough and rabies: white and differential blood cell count, the serum concentrations and/or activities of IL-1, IL-2, IFN-gamma, TNF-alpha, neopterin and IgG, natural killer (NK) cell activity, bactericidal activity and complement hemolytic activity, lymphocyte proliferation test (LPT) and nitroblue tetrazolium test (NBT). Our major findings were that significant postvaccinal decreases in T-cell mitogenic response to PHA and in neutrophil function and neopterin serum concentration were accompanied by simultaneous increase in plasma IgG and hemolytic complement activity. This suggests a transient shift in the balance between cell-mediated and humoral (T(H)1/T(H)2) immunity rather than immunosuppression. These results do not imply that dogs should not receive live vaccines, as the response to vaccines just seems to create a state of altered homeostasis when immunization elicits protection by humoral and cell-mediated immunity. However, these recognized compromises of immune function should be considered and vaccines still be applied only in healthy animals and strictly according to the rules and regulations given by the manufacturer.