Increasing the understanding of nutrient-transport capacity of the ovine placentome

Placental nutrient transport capacity influences fetal growth and development; however, it is affected by environmental factors, which are poorly understood. The objective of this study was to understand the impact of the ovine placentome morphological subtype, tissue type, and maternal parenteral supplementation of arginine mono-hydrochloride (Arg) on nutrient transport capacity using a gene expression approach. Placentomal tissues of types A, B, and C morphologic placentome subtypes were derived from 20 twin-bearing ewes, which were infused thrice daily with Arg (n = 9) or saline (Ctrl, n = 11) from 100 to 140 days of gestation. Samples were collected at day 140 of gestation. Expression of 31 genes involved in placental nutrient transport and function was investigated. Differential expression of specific amino acid transporter genes was found in the subtypes, suggesting a potential adaptive response to increase the transport capacity. Placentomal tissues differed in gene expression, highlighting differential transport capacity. Supplementation with Arg was associated with differential expressions of genes involved in amino acid transport and angiogenesis, suggesting a greater nutrient transport capacity. Collectively, these results indicate that the morphological subtype, tissue type, and maternal Arg supplementation can influence placental gene expression, which may be an adaptive response to alter the transport capacity to support fetal growth in sheep.</p

Feeding synthetic zeolite to transition dairy cows alters neutrophil gene expression.

Synthetic zeolites are used to control the availability of dietary minerals (e.g., Ca, Mg, and P) in dairy cows. Due to calcium demand increasing with lactation onset, most cows become hypocalcemic immediately postpartum, which likely contributes to poorer immune function because calcium is important for immune cell signaling. To overcome postpartum hypocalcemia, we fed transition cows synthetic zeolite A (sodium aluminosilicate) precalving and hypothesized that it would alter calcium and thus neutrophil function during the transition period. Multiparous Holstein-Friesian cows in late gestation were randomly allocated to an untreated control group (n = 10) or a treatment group in which each cow received 500 g of zeolite A daily (n = 10) for 14 d prior to the expected calving date (actual duration = 17 ± 3 d prepartum). The cows grazed pasture, and each was supplemented with 2 kg/d of maize silage (dry matter basis), with or without zeolite, until calving. Blood samples for neutrophil isolation and analysis of plasma indicators of mineral status, energy status, liver function, and inflammation were collected pretreatment (covariate; d -19); on d -14 and -7 precalving; on the day of calving (d 0); and on d 1, 4, 7, and 28 postcalving. Neutrophils were isolated and gene expression was analyzed using microfluidic gene expression arrays. Neutrophil respiratory burst was assessed using stimulation with phorbol 12-myristate 13-acetate and flow cytometry. Plasma calcium and phosphorus revealed a treatment by time interaction; cows offered zeolite had greater plasma calcium concentrations at d 0, 1, and 4 postcalving and plasma phosphorus concentrations were lower in zeolite-treated cows during the precalving period until d 1 postcalving compared with control animals. Zeolite treatment downregulated neutrophil gene expression of CXCR4 and S100A8 and tended to lower gene expression for other immune mediators (CXCR1, IFNG, S100A12, and S100A9) compared with the control. Zeolite treatment did not affect neutrophil respiratory burst or expression of the other genes investigated. Plasma concentrations of cytokine IL-6 were reduced with zeolite treatment, which was most evident immediately postcalving (d 0, 1, and 7). Overall, feeding zeolite precalving had few effects on neutrophil gene expression and function; however, the lower gene expression of neutrophil inflammatory mediators may be due to altered availability of dietary minerals prepartum and indicates that zeolite A may control inflammation during the transition period

Mycobacterium avium subsp. paratuberculosis antigens induce cellular immune responses in cattle without causing reactivity to tuberculin in the tuberculosis skin test

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhea, weight-loss, and eventual death in ruminants. Commercially available vaccine provides only partial protection against MAP infection and can interfere with the use of current diagnostic tests for bovine tuberculosis in cattle. Here, we characterized immune responses in calves to vaccines containing four truncated MAP antigens as a fusion (Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+669-786), either displayed on protein particles, or expressed as a soluble recombinant MAP (rMAP) fusion protein as well as to commercially available Silirum® vaccine. The rMAP fusion protein elicited the strongest antigen-specific antibody responses to both PPDA and recombinant antigen and strong and long-lasting T-cell immune responses to these antigens, as indicated by increased production of IFN-γ and IL-17A in antigen-stimulated whole blood cultures. The MAP fusion protein particle vaccine induced minimal antibody responses and weak IFN-γ responses but stimulated IL-17A responses to recombinant antigen. The immune response profile of Silirum® vaccine was characterized by weak antibodies and strong IFN-γ and IL-17A responses to PPDA. Transcription analysis on antigen-stimulated leukocytes from cattle vaccinated with rMAP fusion protein showed differential expression of several immune response genes and genes involved in costimulatory signaling, TLR4, TLR2, PTX3, PTGS2, PD-L1, IL1B, IL2, IL6, IL12B, IL17A, IL22, IFNG, CD40, and CD86. Moreover, the expression of several genes of immune pathways correlated with cellular immune responses in the rMAP fusion protein vaccinated group. These genes have key roles in pathways of mycobacterial immunity, including autophagy, manipulation of macrophage-mediated killing, Th17- and regulatory T cells- (Treg) mediated responses. Calves vaccinated with either the rMAP fusion protein or MAP fusion protein particle vaccine did not induce reactivity to PPDA and PPDB in a comparative cervical skin test, whereas Silirum® induced reactivity to these tuberculins in most of the vaccinated animals. Overall, our results suggest that a combination of recombinant MAP antigens in the form of a soluble fusion protein vaccine are capable of inducing strong antigen-specific humoral and a balanced Th1/Th17-cell immune response. These findings, together with the absence of reactivity to tuberculin, suggest this subunit vaccine could provide protective immunity against intracellular MAP infection in cattle without compromising the use of current bovine tuberculosis surveillance test

Far-off and close-up dry matter intake modulate indicators of immunometabolic adaptations to lactation in subcutaneous adipose tissue of pasture-based transition dairy cows

The common practice of increasing dietary energy density during the close-up dry period (last ∼3 wk prepartum) has been recently associated with a higher incidence of metabolic disorders after calving. Despite these reports, over-feeding of metabolizable energy (ME) during the far-off, nonlactating period is a common management policy aimed at achieving optimum calving body condition score (BCS) in pasture-based systems, as cows are generally thinner than total mixed ration cows at the end of lactation. Our hypothesis was that both far-off and close-up overfeeding influence the peripartum adipose tissue changes associated with energy balance and inflammatory state. Sixty mid-lactation, grazing dairy cows of mixed age and breed were randomly allocated to 1 of 2 groups that were managed through late lactation to achieve a low and high BCS (approximately 4.25 and 5.0 on a 10-point scale) at dry-off. The low BCS cows were then overfed ME to ensure that they achieved the same BCS as the higher BCS group by calving. Within each rate of BCS gain treatment, cows were offered 65, 90, or 120% of their pre-calving ME requirements for 3 wk pre-calving in a 2 × 3 factorial arrangement of treatments (i.e., 10 cows/treatment). Subcutaneous adipose tissue was collected via biopsy at −1, 1, and 4 wk relative to parturition. Quantitative PCR was used to measure mRNA and microRNA expression of targets related to adipogenesis and inflammation. Cows overfed in the far-off period had increased expression of miR-143 and miR-378 prepartum (−1 wk) indicating greater adipogenesis, consistent with their rapid gain in BCS following dry-off. Furthermore, the lower postpartum expression of IL6, TNF, TLR4, TLR9, and miR-145, and a higher abundance of miR-99a indicated lower body fat mobilization in early lactation in the same group. In the close-up period, feeding either 65 or 120% of ME requirements caused changes in FASN, IL1B, IL6R, TLR9, and the microRNA miR-143, miR-155, and miR-378. Their respective expression patterns indicate a tentative negative-feedback mechanism in metabolically compromised, feed-restricted cows, and a possible immune-related stimulation of lipolysis in apparently static adipocytes in overfed cows. Data from cows fed 90% of ME requirements indicate the existence of a balance between lipolytic (inflammatory-related) and anti-lipolytic signals, to prime the mobilization machinery in light of imminent lactation. Overall, results indicate that far-off dry cow nutrition influences peripartum adipose tissue metabolism, with neither strategy negatively affecting the physiological adaptation to lactation. Furthermore, to ensure a favorable transition, cows should be subjected to a small feed restriction in the close-up period, irrespective of far-off nutritional management

A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum

Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M’s impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein–isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity

Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors

Autologous dendritic cells (DCs) transfected with mRNA encoding prostate-specific antigen (PSA) are able to stimulate potent, T cell–mediated antitumor immune responses in vitro. A phase I trial was performed to evaluate this strategy for safety, feasibility, and efficacy to induce T cell responses against the self-protein PSA in patients with metastatic prostate cancer. In 13 study subjects, escalating doses of PSA mRNA–transfected DCs were administered with no evidence of dose-limiting toxicity or adverse effects, including autoimmunity. Induction of PSA-specific T cell responses was consistently detected in all patients, suggesting in vivo bioactivity of the vaccine. Vaccination was further associated with a significant decrease in the log slope PSA in six of seven subjects; three patients that could be analyzed exhibited a transient molecular clearance of circulating tumor cells. The demonstration of vaccine safety, successful in vivo induction of PSA-specific immunity, and impact on surrogate clinical endpoints provides a scientific rationale for further clinical investigation of RNA-transfected DCs in the treatment of human cancer

Antisense repression of the mitochondrial nadh-binding subunit of complex I in transgenic potato plants affects male fertility

Mitochondrial respiratory chain complex I is a large multi-subunit enzyme composed of both organellar and nuclear encoded proteins. To investigate the role of the nuclear encoded components, expression of the gene for the 55 kDa NADH-binding subunit of complex I was disturbed by antisense repression in transgenic potato plants. The antisense construct driven by the CaMV 35S promoter decreases the steady-state mRNA levels of transcripts for the 55 kDa subunit to 33% of the wild type levels. Quantities of the 55 kDa protein in mitochondrial protein extracts are lowered to about 50% in these plants. Transgenic plants show normal vegetative growth and tuber formation, but pollen maturation is found to be disturbed. The reduced male fertility of the transgenic 55 kDa antisense plants may be caused by an insufficient mitochondrial respiratory chain, impaired by the decreased expression of the NADH-binding component of mitochondrial complex I

Physiological, biochemical and molecular aspects of mitochondrial complex I in plants

Respiratory complex I of plant mitochondria has to date been investigated with respect to physiological function, biochemical properties and molecular structure. In the respiratory chain complex I is the major entry gate for low potential electrons from matrix NADH, reducing ubiquinone and utilizing the released energy to pump protons across the inner membrane. Plant complex I is active against a background of several other NAD(P)H dehydrogenases, which do not contribute in proton pumping, but permit and establish several different routes of shuttling electrons from NAD(P)H to ubiquinone. Identification of the corresponding molecular structures, that is the proteins and genes of the different NADH dehydrogenases, will allow more detailed studies of this interactive regulatory network in plant mitochondria. Present knowledge of the structure of complex I and the respective mitochondrial and nuclear genes encoding various subunits of this complex in plants is summarized here. Copyright (C) 1998 Elsevier Science B.V

Molecular characterisation of the 76 kDa iron-sulphur protein subunit of potato mitochondrial complex I

Genes encoding subunits of complex I (EC 1.6.5.3) of the mitochondrial respiratory chain vary in their locations between the mitochondrial and nuclear genomes in different organisms, whereas genes for a homologous multisubunit complex in chloroplasts have to date only been found on the plastid genome. In potato (Solanum tuberosum L.), the gene coding for the mitochondrial 76 kDa iron-sulphur protein is identified in the nuclear genome. The gene is transcribed into polyadenylated mRNA which is most abundant in flowers, and more frequent in tubers than in leaves. The amino acid sequence is well conserved relative to the nuclear-encoded 75 kDa and 78 kDa subunits of Bos taurus and Neurospora crassa, respectively, and to the Paracoccus denitrificans homologue, most prominently in the region presumed to carry the iron-sulphur clusters. Polyclonal antibodies directed against the 78 kDa complex I subunit of N. crassa recognise the 76 kDa polypeptide in potato mitochondrial complex I, and additionally a polypeptide of 75 kDa in solubilised stroma thylakoids from spinach chloroplasts. The 32 amino acid residues long presequence of the potato mitochondrial 76 kDa complex I subunit targets the precursor polypeptide into isolated potato mitochondria but not into isolated chloroplasts. These results suggest that chloroplast stroma thylakoids contain a protein similar in size and antigenicity to, but genetically distinct from, the mitochondrial subunit