Biomedical subjects

L M Sordillo

Publications and source records attributed to L M Sordillo.

At least 19 recordsLinked to original sources

PubMed2005

Molecular characterization of a saposin-like protein family member isolated from bovine lymphocytes.

Human and porcine T lymphocytes and natural killer (NK) cells produce antibacterial proteins that belong to the saposin-like family of proteins (SAPLIP). The objective of this study was to determine if a bovine homolog of SAPLIP exists in lymphocytes that exhibit antibacterial activity. Following stimulation with IL-2, bactericidal activity against Staphylococcus aureus was detected to some extent in most major subpopulations of T lymphocytes including CD4+, CD8+, CD3+, and WC1+ gammadelta T lymphocytes. However, the majority of antibacterial activity was observed in the CD2+CD3- lymphocytes, which are similar phenotypically to NK cells. A partial sequence of a bovine SAPLIP was generated using low specificity primers designed from regions of homology between other SAPLIP including porcine NK-lysin and human granulysin. Enhanced expression of the bovine lysin gene was detected in mRNA isolated from IL-2-stimulated CD2+CD3- lymphocytes. The partial cDNA sequence was then used to make gene specific primers for a rapid amplification of cDNA ends (RACE) procedure that provided repeatable 5' and 3' cDNA ends. By examining overlapping regions from the RACE procedure, full-length sequence information was obtained for the bovine lysin homologue. Conceptual translation of the cDNA demonstrated conserved similarities to known SAPLIP members. Further characterization of the bovine lysin may facilitate its use in protecting dairy cattle against bacterial infections.

Amino Acid Sequence↗

PubMed2005

A survey on antibiotic usage in dairy herds in Pennsylvania.

A survey was conducted (July 2001 to June 2002) on antibiotic usage of 113 dairy herds from 13 counties in Pennsylvania. Fifty percent of dairy farms surveyed maintained antibiotic treatment records. Only 21% of dairy producers had written plans for treating sick animals. Thirty-two percent of dairy producers sought veterinarian advice before administering antibiotics and on most farms (93%), antibiotics were administered by the owner/manager or designated herdsman. Twenty-four percent of the dairy producers said they always completed the course of antibiotic treatment. Any extra-label use of antibiotics was administered only on the guidelines of a veterinarian on majority of the farms. Comprehensive records from 33 dairy farms indicated that antibiotic usage was largest for calves with enteritis (36%) followed by pneumonia in calves (25%) and foot rot in cattle (16%). Twenty-four antibiotics including beta-lactams, spectinomycin, florfenicol, and tetracyclines were used on these farms. Beta-lactam antibiotics were used mostly for dry cow therapy, clinical mastitis, and on some farms for pneumonia and metritis. On 18% of the dairy herds surveyed, ceftiofur was used in an extra-label manner to treat mastitis in lactating cattle. On 70% of farms, calves were fed medicated milk replacers containing oxytetracycline and neomycin. The results of this study suggest that antibiotics are used extensively on dairy herds for both therapeutic and prophylactic purposes. Beta-lactams and tetracyclines were the most widely used antibiotics. There is considerable variation in the management practices associated with antibiotic use on dairy farms. It is anticipated that the findings of this survey will permit developing new strategies for prudent use of antibiotics on dairy herds.

Animals↗

PubMed2003

Oxidant stress enhances Lyso-PAF-AcT activity by modifying phospholipase D and phosphatidic acid in aortic endothelial cells.

Oxidant stress, as a consequence of selenium (Se) deficiency, alters production of vasoactive compounds including platelet-activating factor (PAF). Recent studies report that enhanced PAF production during Se deficiency is a consequence of increased lyso-PAF:acetyl-coenzyme A acetyltransferase (Lyso-PAF-AcT) activity. To elucidate the mechanism behind increased Lyso-PAF-AcT activity during oxidant stress, phospholipase D (PLD) activity and phosphatidic acid (PA) production were examined. Increased PLD activity and PA production were exhibited in bovine aortic endothelial cells using a Se-deficient model of oxidant stress. The direct effects of PLD and PA on Lyso-PAF-AcT activity were assessed using selective inhibitors and repletion experiments. Following the inhibition of PLD and addition of exogenous PA, Lyso-PAF-AcT activity significantly decreased and increased, respectively. Therefore, Se deficiency enhances Lyso-PAF-AcT activity in part by modifying PLD and PA. This suggests a novel link between Se status and PAF production, providing potential upstream therapeutic targets for PAF regulation under conditions of oxidant stress.

Acetyltransferases↗

PubMed2003

Differential expression of the lactose transporter gene affects growth of Staphylococcus aureus in milk.

In this study, differential display polymerase chain reaction (PCR) was used to search for unique or enhanced expression of genes in prevalent bovine mastitis-causing Staphylococcus aureus strains. Comparison of a pair of prevalent and rare strains revealed the differential expression of several genes. The lactose-specific permease, enzyme II (EII), was highly expressed in the prevalent strain. This gene was selected for further study due to its potential influence on bacterial growth, because lactose is the primary carbohydrate in milk. Growth analysis illustrated that prevalent strains reach significantly higher growth densities sooner than rare strains. Quantitative competitive reverse transcription PCR (QC RT-PCR) revealed increased EII mRNA expression in prevalent strains as compared to rare strains. Mutation of the EII gene resulted in abrogated growth and decreased EII mRNA expression in media containing lactose. These data suggest that increased EII expression may facilitate the pathogenesis of S. aureus mastitis by enhancing growth. This study is the first to implicate EII as a potential virulence factor in mastitis, and therefore may be useful in the development of novel therapeutic strategies against S. aureus mastitis.

Animals↗

PubMed2001

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization.

A 25 kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na(2)CO(3), indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73-83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3 Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243-248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87-101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H(2)O(2). Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.

Amino Acid Sequence↗

PubMed2001

Increased 15-HPETE production decreases prostacyclin synthase activity during oxidant stress in aortic endothelial cells.

Selenium (Se) is an integral component of glutathione peroxidase and is able to detoxify peroxides that can affect arachidonic acid (AA) metabolism, thereby influencing eicosanoid biosynthesis. This study investigated the effects of oxidant stress, a consequence of Se deficiency, on eicosanoid formation and important key enzyme expression in bovine aortic endothelial cells (BAEC). Bovine aortic endothelial cells cultured in Se-deficient media and stimulated with tumor necrosis factor alpha or H2O2 produced significantly less prostacyclin (PGI(2)) and more 15-hydroxyeicosatetraenoic acid, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and thromboxane than Se-supplemented BAEC. Additionally, reverse transcription polymerase chain reaction and immunoblotting determined that the mRNA and protein levels of the eicosanoid forming enzymes cyclooxygenase-1 (COX1), cyclooxygenase-2 (COX2), and PGI synthase were not significantly changed. The addition of 15-HPETE to Se-supplemented BAEC inhibited the production of PGI(2) suggesting that the accumulation of lipid hydroperoxides during Se-deficiency may be the underlying factor in the altered eicosanoid production during Se deficiency. Furthermore, inhibition of COX and addition of PGH(2) to Se-deficient or Se-supplemented BAEC still resulted in lower PGI(2) formation by Se-deficient cells. Together, these results suggest that Se deficiency modifies eicosanoid production by affecting the activity of key enzymes, particularly PGI synthase, rather than their transcription or translation.

Animals↗

PubMed2001

Selenium modulates 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) biosynthesis in bovine aortic endothelial cells.

Selenium (Se) deficiency has been reported to increase platelet-activating factor (PAF) production in human endothelial cells; however, the mechanism is unclear. This study demonstrated that tumor necrosis factor-alpha (TNF-alpha) stimulated Se-deficient bovine aortic endothelial cells (BAEC) produced significantly more PAF than Se-supplemented cells. Moreover, the increase in the level of PAF was associated with enhanced activity of two anabolic enzymes in the remodeling pathway: phospholipase A2 and Lyso-PAF:acetyl-coenzyme A acetyltransferase (Lyso-PAF-AcT). In contrast, the activity of the PAF catabolic enzyme, PAF-acetylhydrolase, was not affected by Se status. Interestingly, prostacyclin, a potent vasodilator and inhibitor of platelet aggregation, inhibited the activity of Lyso-PAF-AcT and reduced the PAF production in TNF-alpha-stimulated BAEC. Therefore, we conclude that Se deficiency alters PAF production in TNF-alpha-stimulated BAEC by altering the activity of anabolic enzymes involved in the remodeling pathway partially through the inhibition of prostacyclin production.

Animals↗

PubMed2001

Staphylococcus aureus agr genotypes with enterotoxin production capabilities can resist neutrophil bactericidal activity.

Staphylococcus aureus pathogenicity is mainly due to the production of a number of secreted and cell surface-associated proteins under the regulation of the agr gene. A region of the agr gene was used to subgroup S. aureus strains according to restriction fragment length polymorphisms. Additionally, strains were subtyped according to the coagulase gene in order to strengthen discriminatory power. Virulence capabilities of agr genotype subgroups were evaluated using an in vitro neutrophil bactericidal assay, which showed that prevalent genotypes were significantly better at evading this primary host defense. Multiplex PCR was then used to detect enterotoxin genes among the genotype subgroups in order to determine possible virulence candidates that enable strains to combat neutrophil killing. The prevalent genotype strains were found to possess higher production capabilities for enterotoxin A than did low-prevalence strains. The significance of enterotoxin A production capabilities in affecting pathogenicity of S. aureus strains was evaluated and found to have a profound effect on neutrophil killing abilities. The use of a large epidemiological database as a tool for subgrouping strains with varying degrees of pathogenicity has allowed the identification of relevant and previously undefined virulence factors that affect a pathogen's capability to overcome host immune defenses.

Animals↗

PubMed2001

Application of differential inflammatory cell count as a tool to monitor udder health.

A flow cytometric technique called differential inflammatory cell count was standardized by staining bovine peripheral blood leukocytes with a combination of DNA binding dyes SYBR green 1 and propidium iodide in water. Leukocytes were also stained with propidium iodide in detergent to determine total cell count. Differential inflammatory cell count assay was evaluated with individual quarter milk samples from 13 cows. Cows were sampled at weekly intervals for 3 wk and assayed for total cell count, mononuclear leukocyte count, and polymorphonuclear leukocyte count. Simultaneously, milk samples were evaluated by the conventional electronic somatic cell count (SCC) technique. Somatic cell count positively correlated with total cell count (r = 0.9), mononuclear leukocyte count (r = 0.8), and polymorphonuclear leukocyte count (r = 0.89). Quarters with SCC > log10 5.4 had a higher total cell count, mononuclear leukocyte count, and polymorphonuclear leukocyte count and were more often culture positive compared with quarters with SCC < log10 5.4. Quarters that were culture positive on all three test occasions had a higher proportion of polymorphonuclear leukocytes (33 to 49%) compared with quarters that were culture negative on all three test occasions (17 to 25%). The findings of this study suggest that differential inflammatory cell count assay has the potential to evolve as a new technique for evaluation of udder health status.

Animals↗

PubMed2000

Altered eicosanoid biosynthesis in selenium-deficient endothelial cells.

Selenium (Se) is an integral part of the Se-dependent glutathione peroxidase (Se-GSH-Px) catalytic domain. By modulating the cellular levels of fatty acid hydroperoxides, Se-GSH-Px can influence key enzymes of arachidonic acid cascade, in this case cyclooxygenase (COX) and lipoxygenase (LOX). To investigate this phenomenon, the effects of cellular Se status on the enzymatic oxidation of arachidonic acid were investigated in bovine mammary endothelial cells (BMEC), which were cultured in either Se-deficient (-Se) or Se-adequate (+Se) media. When stimulated with calcium ionophore A23187, BMEC produced eicosanoids of both COX and LOX pathways. Compared with the Se-adequate cells, the production of prostaglandin I(2) (PGI(2)), prostaglandin F(2) (PGF(2alpha)), and prostaglandin E(2) (PGE(2)) was significantly decreased in Se-deficient cells, whereas the production of thromboxane A(2) (TXA(2)) was markedly increased in the -Se BMEC cultures. Although the enzymatic oxidation of arachidonic acid by the LOX pathway was found to be relatively less than by the COX pathway, the BMEC cultured in -Se media produced significantly more 15-hydroperoxyeicosatetraenoic acid (15-HPETE) than the +Se cells produced. Based on these results, we postulate that cellular Se status plays an important regulatory role in the enzymatic oxidation of arachidonic acid by the COX and LOX pathways. The altered eicosanoid biosynthesis, especially the overproduction of 15-HPETE, in -Se BMEC may be one of the underlying biochemical phenomena responsible for vascular dysfunction during Se deficiency.

Animals↗

PubMed2000

Phylogenetic relationships of Staphylococcus aureus from bovine mastitis based on coagulase gene polymorphism.

Coagulase gene restriction fragment length polymorphism (RFLP) patterns were analyzed to determine the phylogenetic relationship among isolates of Staphylococcus aureus from the Czech Republic (n = 27), France (n = 48), Korea (n = 115) and the United States (n = 278). A total of 468 isolates of S. aureus were subtyped into 41 coagulase genotypes. Cluster analysis placed the 41 types into nine clusters. Eighteen API Staph profiles were determined for 102 S. aureus isolates representing 1 to 4 isolates of each coagulase type. The results of the study suggest that based on coagulase gene RFLP analysis, several genetic variants of S. aureus are prevalent. Comparison of coagulase and API Staph profiles indicated that the two identification system were independent of each other.

Animals↗

PubMed1999

Increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells during selenium deficiency.

Leukocyte aggregation and activation on endothelial cells (EC) are important preliminary events in leukocyte migration into tissue and subsequent inflammation. Thus, an increase in leukocyte adherence has the potential to affect inflammatory disease outcome. Selenium (Se) is an integral part of the antioxidant enzyme glutathione peroxidase (GSH-Px) and plays an important role in the maintenance of the redox state of a cell. Se supplementation in the bovine has been shown to improve the outcome of acute mastitis caused by coliform bacteria, in part by enhancing the speed of neutrophil migration into the affected mammary gland. However, the mechanisms by which Se modulates neutrophil migration have not been elucidated. Therefore, an in vitro model of Se deficiency in primary bovine mammary artery EC was used to examine the impact of Se status on the adhesive properties of EC. The effect of Se on functional activities was examined by measuring neutrophil adherence to Se-deficient and Se-supplemented EC. Se-deficient EC showed significantly enhanced neutrophil adherence when stimulated with tumor necrosis factor alpha (TNF-alpha) for 4 or 24 h, interleukin-1 for 12 h, or H2O2 for 20 min (P < 0.05). To determine the mechanisms underlying these changes in neutrophil adherence, the expression of EC adhesion molecules, ICAM-1, E-selectin, and P-selectin were examined at the molecular level by a competitive reverse transcription-polymerase chain reaction. Results revealed higher mRNA expression for E-selectin and ICAM-1 in Se-deficient EC stimulated with TNF-alpha for 3 and 6 h, and greater expression of P-selectin mRNA in Se-supplemented EC with 3-h TNF-alpha stimulation. These studies provide new information to establish the role of Se nutrition in the initiation of leukocyte adherence to endothelium.

Animals↗

PubMed1999

Identification of in vitro cytochrome P450 modulators to detect induction by prototype inducers in the mallard duckling (Anas platyrhynchos).

Seven modulators of mammalian monooxygenase activity were screened for their ability to selectively stimulate or inhibit in vitro monooxygenase activities of hepatic microsomes from mallard ducklings treated with phenobarbital, beta-naphthoflavone, 3,3',4,4',5-pentachlorobiphenyl or vehicle. Microsomes were assayed fluorometrically for four monooxygenases: benzyloxy-, ethoxy-, methoxy-, and pentoxyresorufin-O-dealkylase, in combination with each of the seven modulators. Four combinations: alpha-naphthoflavone and 2-methylbenzimidazole with benzyloxyresorufin, and Proadifen with methoxy- and ethoxyresorufin, respectively, were evaluated further. beta-Naphthoflavone-treated groups were clearly distinguished from the corn oil vehicle control group by all of the assays and by the effects of the modulators in three of the four assay/modulator combinations. Enzyme activities of the phenobarbital and saline groups were statistically similar (P > or = 0.05) when assayed without modulator added, but each assay/modulator combination distinguished between these groups. The PCB-treated group was distinguished from the corn oil vehicle control group only for BROD activity, with or without the presence of modulator. Graphing of per cent modulation of BROD activity versus initial BROD activity provided the clearest distinction between all of the study groups. Identification of these selective in vitro modulators may improve detection and measurement of low level cytochrome P450 induction in avian species. Also, both the monooxygenase activities induced and the impacts of the modulators indicated differences between mammalian and avian cytochromes P450.

Animals↗

PubMed1999

Coagulase gene polymorphism of Staphylococcus aureus isolates from dairy cattle in different geographical areas.

The objectives of this study were to investigate the coagulase gene polymorphism of Staphylococcus aureus isolates obtained from bovine mastitic milk and to determine the resistance of predominant and rare coagulase genotypes to bovine blood neutrophil bactericidal activities. A total of 453 isolates were collected from four countries: the Czech Republic, France, Korea and the United States. The isolates were subtyped into 40 types by restriction fragment length polymorphism (RFLP) of the coagulase gene. Twenty-three strains from predominant and rare genotypes were evaluated for their ability to resist neutrophil bactericidal activities. There were significant (P < 0.01) differences in the average percent neutrophil killing of the predominant (16.7%) and rare (39.7%) genotypes when bacteria were opsonized with antiserum. The results indicate that the profiles of coagulase genotype differ among geographic locations, and only a few genotypes prevail in each location. In addition, the predominant genotypes were more resistant to neutrophil bactericidal activities than rare genotypes.

Animals↗

PubMed1999

Shifts in bovine CD4+ subpopulations increase T-helper-2 compared with T-helper-1 effector cells during the postpartum period.

This study determined the cytokine profile of CD4+ T-helper cells to elucidate the specific CD4+ T-helper phenotype during the postpartum period. Peripheral blood mononuclear cells were isolated from cows during periods of increased susceptibility (3 d postpartum, n = 7) and decreased susceptibility (mid- to late lactation, n = 6) to mastitis. Isolated mononuclear cells were magnetically separated into CD4(+)-enriched or CD4(+)-depleted populations using specific bovine monoclonal antibodies and were confirmed to be enriched or depleted by flow cytometric analysis. T-helper-1 and T-helper-2 subpopulations were distinguished by cytokine profiles, at both the molecular and protein level, by competitive quantitative reverse transcriptase-polymerase chain reaction and specific bioassays, respectively. The CD4(+)-enriched cultures isolated postpartum had enhanced interleukin-4 and interleukin-10 mRNA transcript expression; cultures isolated during the mid- to late lactating period had enhanced interleukin-2 mRNA transcripts. Depletion of CD4+ lymphocytes decreased, and enrichment of CD4+ lymphocytes increased interferon-gamma transcripts in cultures isolated from mid- to late lactation cows. Interferon-gamma and interleukin-2 bioassays revealed that cytokine secretion paralleled mRNA transcript levels. These data suggest that CD4+ lymphocytes act predominantly as T-helper-2 compared with T-helper-1 within 3 d after calving. Alterations in the T-helper-1 and T-helper-2 responses, and therefore the repertoire of cytokines produced, may be an underlying reason for diminished host immune response during the postpartum period.

Animals↗

PubMed1998

Selenium and vitamin E deficiency impair transferrin receptor internalization but not IL-2, IL-2 receptor, or transferrin receptor expression.

Vitamin E and Se deficiency increase the risk of disease by impairing the immune response. To aid in the understanding of how vitamin E and Se deficiency reduce immune competence, this study examined several mechanisms necessary for lymphocyte proliferation. Weanling rats were fed a vitamin E-deficient, selenium-deficient, or control diet for 8 weeks. At this time splenic mononuclear cells were isolated and stimulated with concanavalin A for 48 h. Although the percentage of lymphocytes and monocytes capable of proliferating were consistent among the dietary groups, lymphocyte proliferation was decreased significantly in vitamin E- and selenium-deficient rats. This decrease in proliferation was not associated with alterations in interleukin-2, interleukin-2 receptor, or transferrin receptor expression. However, stimulated cells from vitamin E- and Se-deficient rats internalized few if any transferrin receptors. Reduced transferrin receptor internalization may limit lymphocyte expansion by depleting the intracellular iron stores needed for cellular function and proliferation.

Animals↗

PubMed1998

A simple method to enrich mRNA from total prokaryotic RNA.

Isolation of prokaryotic mRNA by the poly(dT) method has been difficult, primarily due to the great instability of the poly(A) sequence in its mRNA. We developed a simple method to remove rRNA from total RNA of Staphylococcus aureus by cloning a PCR-amplified S. aureus rRNA gene fragment into a plasmid, and then synthesizing biotin-labeled antisense rRNA to subtract rRNA. By using this method, S. aureus rRNA is significantly reduced and mRNA is enriched. This method may be used to prepare prokaryotic mRNA for many molecular biology applications.

Cloning, Molecular↗

PubMed1997

Bovine CD8+ suppressor lymphocytes alter immune responsiveness during the postpartum period.

This study examined the immunoregulatory role of CD8+ lymphocytes during the postpartum period. Peripheral blood cells were isolated from postpartum and mid to late lactating animals. Flow cytometric analysis was performed to determine the frequencies of relevant cell populations. Depletion of CD8+ lymphocytes from whole cultures significantly decreased proliferation and cytotoxic ability of cells isolated from mid to late lactating animals. Enrichment of whole cultures with CD8+ lymphocytes further decreased their proliferative ability but pure CD8+ lymphocyte had increased cytotoxic activity. In contrast, neither depletion nor enrichment of whole cultures with CD8+ lymphocytes altered the already diminished proliferative responses of cells isolated from postpartum cows. No cytotoxic activity was observed by cells isolated from postpartum animals. Cultures from mid to late lactating cows mainly expressed IFN-gamma mRNA where as IL-4 mRNA was mainly expressed by cultures isolated from postpartum animals. Flow cytometric analysis revealed that CD8+ lymphocytes have a high level of activation and expression of the beta-chain during the postpartum compared with the mid to late lactating period. These data indicate that CD8+ lymphocytes are of the suppressor compared to the cytotoxic nature immediately following parturition.

Animals↗