The Vampirome: Transcriptome and proteome analysis of the principal and accessory submaxillary glands of the vampire bat Desmodus rotundus, a vector of human rabies

Vampire bats are notorious for being the sole mammals that strictly feed on fresh blood for their survival. While their saliva has been historically associated with anticoagulants, only one antihemostatic (plasminogen activator) has been molecularly and functionally characterized. Here, RNAs from both principal and accessory submaxillary (submandibular) salivary glands of Desmodus rotundus were extracted, and ~. 200. million reads were sequenced by Illumina. The principal gland was enriched with plasminogen activators with fibrinolytic properties, members of lipocalin and secretoglobin families, which bind prohemostatic prostaglandins, and endonucleases, which cleave neutrophil-derived procoagulant NETs. Anticoagulant (tissue factor pathway inhibitor, TFPI), vasodilators (PACAP and C-natriuretic peptide), and metalloproteases (ADAMTS-1) were also abundantly expressed. Members of the TSG-6 (anti-inflammatory), antigen 5/CRISP, and CCL28-like (antimicrobial) protein families were also sequenced. Apyrases (which remove platelet agonist ADP), phosphatases (which degrade procoagulant polyphosphates), and sphingomyelinase were found at lower transcriptional levels. Accessory glands were enriched with antimicrobials (lysozyme, defensin, lactotransferrin) and protease inhibitors (TIL-domain, cystatin, Kazal). Mucins, heme-oxygenase, and IgG chains were present in both glands. Proteome analysis by nano LC-MS/MS confirmed that several transcripts are expressed in the glands. The database presented herein is accessible online at http://exon.niaid.nih.gov/transcriptome/D_rotundus/Supplemental-web.xlsx. These results reveal that bat saliva emerges as a novel source of modulators of vascular biology. Biological significance: Vampire bat saliva emerges as a novel source of antihemostatics which modulate several aspects of vascular biology. © 2013.

Recent mouse and rat methods for the study of experimental oral candidiasis

The Candida genus expresses virulence factors that, when combined with immunosuppression and other risk factors, can cause different manifestations of oral candidiasis. The treatment of mucosal infections caused by Candida and the elucidation of the disease process have proven challenging. Therefore, the study of experimentally induced oral candidiasis in rats and mice is useful to clarify the etiopathology of this condition, improve diagnosis, and search for new therapeutic options because the disease process in these animals is similar to that of human candidiasis lesions. Here, we describe and discuss new studies involving rat and mouse models of oral candidiasis with respect to methods for inducing experimental infection, methods for evaluating the development of experimental candidiasis, and new treatment strategies for oral candidiasis. © 2013 Landes Bioscience.

Proteinograma do soro lácteo de ovelhas da raça santa inês em diferentes fases de lactação

This study aimed to evaluate the influence of lactation phases on the proteinogram of whey protein in Santa Inês ewes. Ewes were accompanied in a semi-intensive system using the same sanitary and nutritional management evaluated at 15, 30, 60 and 90 days postpartum (end of weaning and lactation). Clinical examination of the mammary gland was carried out through and bacteriological culture. The screening of the material resulted in 44 milk samples of healthy glands concurrent negative by CMT and bacteriological culture exam. For obtaining the whey protein renin solution was used. The whey was fractionated into aliquots and kept in the -80C freezer to later separation of protein fractions. For determination of total protein of whey protein was employed the biuret, observing the linearity of the test. Separation of protein fractions was performed, using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). Eigth protein were observed including lactoferrin, serum albumin, IgA, IgG (heavy chain IgG (IgG CP), light chain IgG (IgG CL), ß-lactoglobulin, a-lactalbumin and proteins identified as PM 15000 and PM 29000. No significant difference was observed at different stages of lactation in the following protein: IgA (P>0.3895), lactoferrin (P>0.1611), PM 29000 (P>0.4879), α-lactalbumin (P>0.0799) and PM15000 (P>0.4494). In total protein (P<0.0022), albumin protein (P<0.0377) and IgG (P<0.0354) it was observed a significant variation in the first moments of observations, in the ß-lactoglobulin protein (P<0.0005) there was significant variation with reduction of 15 to 30 days postpartum with progressive elevation until the last stage of lactation (90 days postpartum). The SDS-PAGE technique allowed the quantification of eigth whey proteins in health ewes. The protein fractions identified reflect the profile of whey to ovine species, with influence of stages of lactation in albumin, IgG and ß-lactoglobulin.

Perfil bioquímico, condição microbiológica e celuridade do leite de búfalas

Pós-graduação em Medicina Veterinária - FCAV

Hemograma e perfil bioquímico de bezerros neonatos da raça Holandesa tratados com ferro suplementar

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Histologia e histomorfometria do papel da lactoferrina em importantes eventos de injúria intestinal e cutânea, em animais de experimentação

Pós-graduação em Alimentos e Nutrição - FCFAR

Avaliação colostral de cabras acometidas por mastite e sua influ~encia nos níveis imunes séricos de cabrito: Jefferson Filgueira Alcindo. -

Pós-graduação em Ciência Animal - FMVA

Padronização de técnica manual para obtenção de plasma rico em plaquetas de bovino

To evaluate the biochemical profile and protein concentration of whey from milk samples of healthy Murrah primiparous and pluriparous buffaloes, 30 female buffaloes were analyzed during a complete lactation. The animals were divided into three groups: G1 = 10 primiparous buffaloes, G2 = 10 pluriparous buffaloes with 2-3 lactations and G3 = 10 pluriparous buffaloes with > 3 lactations. The lactation period was divided into: early stage (I: 1-3 months of lactation), intermediate stage (T: 4-6 months of lactation) and final stage (F: 7-9 months of lactation). Before milk sampling, physical examination of the mammary gland, strip cup test and California Mastitis Test (CMT) were performed. After mammary quarters asepsis, 20mL of milk were collected monthly from each mammary quarter, during a complete lactation, in sterilized plastic bottles without preservative, in order to perform microbiological isolation, biochemical profile and protein electrophoresis in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 30mL of milk from each mammary quarter were collect, in sterilized plastic bottles containing preservative bronopol to perform the somatic cell count (SCC). A total of 1,042 milk samples were collected from the experimental groups during lactation, of which 923 samples showed negative reaction to CMT and negative microbiological isolation and were selected to biochemical profile analysis and protein electrophoresis in SDS-PAGE. There were influence of parity order and stage of lactation in biochemical profile and protein concentration of healthy Murrah buffaloes'whey. Primiparous buffaloes (G1) showed higher gamma-glutamyltransferase (GGT: 2,346 U/L), alkaline phosphatase (ALP: 181 U/L), phosphorus (P; 56.6mg/dL), potassium (K; 32.0mg/dL) and alpha-lactalbumin (458mg/dL). Buffaloes with 2-3 lactations (G2) showed higher SCC (70,700 cells/mL) and higher concentrations of total protein (1.55g/dL), albumin (100mg/dL), magnesium (Mg; 8.80mg/dL), chlorides (Cl; 176mg/dL), iron (Fe; 10.7 mu g/dL), sodium (Na; 178mMol/L) and lactoferrin (59.5mg/dL). Bufalloes with > 3 lactations (G3) showed higher concentrations of total calcium (Ca; 41.8mg/dL), ionized calcium (iCa; 2.92mMol/L), immunoglobulin A (IgA; 1.32mg/dL), serum albumin (99.1mg/dL), immunoglobulin G (IgG; 49.7mg/dL) and beta-lactoglobulin (1,068mg/dL). During lactation it was observed increase in SCC, GGT, ALP, total protein, albumin, P, Mg, Cl, Na, lactoferrin, serum albumin, IgG and alpha-lactalbumin, as well as decrease in concentrations of Ca, Fe, iCa, K, IgA and beta-lactoglobulin in buffaloes'whey. The results may be used as reference for buffaloes and to support diagnosis and prognosis of diseases common to lactation periods.

Physiologic cold shock of Moraxella catarrhalis affects the expression of genes involved in the iron acquisition, serum resistance and immune evasion

Background Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. It was previously shown that the prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis are greatest in winter. The aim of this study was to investigate how M. catarrhalis uses the physiologic exposure to cold air to upregulate pivotal survival systems in the pharynx that may contribute to M. catarrhalis virulence. Results A 26°C cold shock induces the expression of genes involved in transferrin and lactoferrin acquisition, and enhances binding of these proteins on the surface of M. catarrhalis. Exposure of M. catarrhalis to 26°C upregulates the expression of UspA2, a major outer membrane protein involved in serum resistance, leading to improved binding of vitronectin which neutralizes the lethal effect of human complement. In contrast, cold shock decreases the expression of Hemagglutinin, a major adhesin, which mediates B cell response, and reduces immunoglobulin D-binding on the surface of M. catarrhalis. Conclusion Cold shock of M. catarrhalis induces the expression of genes involved in iron acquisition, serum resistance and immune evasion. Thus, cold shock at a physiologically relevant temperature of 26°C induces in M. catarrhalis a complex of adaptive mechanisms that enables the bacterium to target their host cellular receptors or soluble effectors and may contribute to enhanced growth, colonization and virulence.

The airway epithelium: soldier in the fight against respiratory viruses

The airway epithelium acts as a frontline defense against respiratory viruses, not only as a physical barrier and through the mucociliary apparatus but also through its immunological functions. It initiates multiple innate and adaptive immune mechanisms which are crucial for efficient antiviral responses. The interaction between respiratory viruses and airway epithelial cells results in production of antiviral substances, including type I and III interferons, lactoferrin, β-defensins, and nitric oxide, and also in production of cytokines and chemokines, which recruit inflammatory cells and influence adaptive immunity. These defense mechanisms usually result in rapid virus clearance. However, respiratory viruses elaborate strategies to evade antiviral mechanisms and immune responses. They may disrupt epithelial integrity through cytotoxic effects, increasing paracellular permeability and damaging epithelial repair mechanisms. In addition, they can interfere with immune responses by blocking interferon pathways and by subverting protective inflammatory responses toward detrimental ones. Finally, by inducing overt mucus secretion and mucostasis and by paving the way for bacterial infections, they favor lung damage and further impair host antiviral mechanisms.

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 mug of LPS or 20 mug of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12h relative to the challenge to measure mRNA expression of tumor necrosis factor-alpha (TNFalpha), IL-1beta, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFalpha. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFalpha in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFalpha, IL-1beta, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.

Immune response of bovine milk somatic cells to endotoxin in healthy quarters with normal and very low cell counts

Low somatic cell count (SCC) is a reliable indicator of high-quality milk free of pathogenic microorganisms. Thus, an important goal in dairy practice is to produce milk with low SCC. Selection for cows with low SCC can sometimes lead to extremely low SCC in single quarters. The cells in milk are, however, predominantly immune cells with important immune functions. To investigate the mammary immune competence of quarters with very low SCC, healthy udder quarters of cows with normal SCC of (40-100) x 10(3) cells/ml and very low SCC of < 20 x 10(3) cells/ml were challenged with lipopolysaccharide (LPS) from Escherichia coli. In the first experiment, SCC and cell viability after a challenge with 50 ng of LPS/quarter was investigated. In the second experiment, tumour necrosis factor alpha (TNF-alpha) concentration and lactate dehydrogenase (LDH) activity in milk, and mRNA expression of various innate immune factors in milk cells were measured after a challenge with 100 mug LPS/quarter. LPS challenge induced an increase of SCC. SCC levels reached were higher in quarters with normal SCC and maximum SCC was reached 1 h earlier than in very low SCC quarters. The increase of TNF-alpha concentrations in milk in response to LPS challenge was lower in quarters with very low SCC than in quarters with normal SCC. The viability of cells and the LDH activity in milk increased in response to LPS challenge, however, without a difference between the groups. The mRNA expression of IL-1beta and IL-8 was increased in milk cells at 12 h after LPS challenge, whereas that of TNF-alpha and lactoferrin was not increased at the measured time points (12, 24 and 36 h after LPS challenge). No differences of mRNA expression of measured immune factors between normal and very low SCC samples were detected. The study showed that udder quarters with very low SCC responded with a less marked increase of SCC compared with quarters with normal SCC. This difference corresponded with simultaneously lower TNF-alpha concentrations in milk. However, the immune competence of the cells themselves based on mRNA expression of TNF-alpha, IL-8, IL-1beta, and lactoferrin, did not differ. The results may indicate that very low SCC can impair the immune competence of udder quarters, because the immune response in udder quarters with lower SCC is less efficient as fewer cells contribute to the production of immunoregulators.

Leukocyte populations and mRNA expression of inflammatory factors in quarter milk fractions at different somatic cell score levels in dairy cows

The effect of somatic cell count (SCC) and milk fraction on milk composition, distribution of cell populations, and mRNA expression of various inflammatory parameters was studied. Therefore, quarter milk samples were defined as cisternal (C), first 400 g of alveolar (A1), and remaining alveolar milk (A2) during the course of milking. Quarters were assigned to 4 groups according to their total SCC: 1) <12 x 10(3)/mL, 2) 12 to 100 x 10(3)/mL, 3) 100 to 350 x 10(3)/mL, and 4) >350 x 10(3)/mL. Milk constituents of interest were SCC, fat, protein, lactose sodium, and chloride ions as well as electrical conductivity. Cell populations were classified into lymphocytes, macrophages, and neutrophils (PMN). The mRNA expression of the inflammatory factors tumor necrosis factor-alpha, interleukin-1beta, cyclooxygenase-2, lactoferrin, and lysozyme was measured via real-time, quantitative reverse transcription PCR. Somatic cell count decreased from highest levels in C to lowest levels in A1 and increased thereafter to A2 in all groups. Fat content increased from C to A2 and with increasing SCC level. Lactose decreased with increasing SCC level but remained unchanged during milking. Concentrations of sodium and chloride, and electrical conductivity increased with increasing SCC but were higher in C than in A1 and A2. Protein was not affected by milk fraction or SCC level. The distribution of leukocytes was dramatically influenced by milk fraction and SCC. Lymphocytes were the dominating cell population in group 1, but the proportion of lymphocytes was low in groups 2, 3, and 4. Macrophage proportion was highest in group 2 and decreased in groups 3 and 4, whereas that of PMN increased from group 2 to 4. The content of macrophages decreased during milking in all SCC groups whereas that of PMN increased. The proportion of lymphocytes was not affected by milk fraction. The mRNA expression of all inflammatory factors showed an increase with increasing SCC but minor changes occurred during milking. In conclusion, milk fraction and SCC level have a crucial influence on the distribution of leukocyte populations and several milk constituents. The surprisingly high content of lymphocytes and concomitantly low mRNA expression of inflammatory factors in quarters with SCC <12 x 10(3)/mL indicates a different and possibly reduced readiness of the immune system to respond to invading pathogens.

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.

Differential expression of ABC transporters and their regulatory genes during lactation and dry period in bovine mammary tissue

ATP-binding cassette (ABC) transporters play a pivotal role in human physiology, and mutations in these genes often result in severe hereditary diseases. ABC transporters are expressed in the bovine mammary gland but their physiological role in this organ remains elusive. Based on findings in the context of human disorders we speculated that candidate ABC transporters are implicated in lipid and cholesterol transport in the mammary gland. Therefore we investigated the expression pattern of selected genes that are associated with sterol transport in lactating and nonlactating mammary glands of dairy cows. mRNA levels from mammary gland biopsies taken during lactation and in the first and second week of the dry period were analysed using quantitative PCR. Five ABC transporter genes, namely ABCA1, ABCA7, ABCG1, ABCG2 and ABCG5, their regulating genes LXRalpha, PPARgamma, SREBP1 and the milk proteins lactoferrin and alpha-lactalbumin were assessed. A significantly enhanced expression in the dry period was observed for ABCA1 while a significant decrease of expression in this period was detected for ABCA7, ABCG2, SREBP1 and alpha-lactalbumin. ABCG1, ABCG5, LXRalpha, PPARgamma and lactoferrin expression was not altered between lactation and dry period. These results indicate that candidate ABC transporters involved in lipid and cholesterol transport show differential mRNA expression between lactation and the dry period. This may be due to physiological changes in the mammary gland such as immigration of macrophages or the accumulation of fat due to the loss of liquid in the involuting mammary gland. The current mRNA expression analysis of transporters in the mammary gland is the prerequisite for elucidating novel molecular mechanisms underlying cholesterol and lipid transfer into milk.