Polymorphonuclear Leukocyte Phagocytosis Research Articles

Bacteria and Viruses in Periodontics- A review

Periodontal diseases are infectious diseases, but the specific mechanism by which the tooth-supportive tissue is destroyed is not clearly understood. Periodontitis is a multifactorial, chronic disease followed by destruction of encompassing structures of teeth and when left untreated leads to loss of alveolar bone and exfoliation of the involved teeth. The main etiological factor for development of periodontitis is oral biofilm containing anaerobic microorganisms. Microbiological culture studies have identified more than 1200 bacterial species in the oral cavity. Although the role of bacterial plaque in general seems to be evident, on the contrary the role of virus has been largely unexplored. Viral infection impairs periodontal defenses, thereby permitting subgingival overgrowth of periodontopathic bacteria. The role of viruses is significant, as they may induce abnormalities in the adhesion, chemotaxis, phagocytosis, and bactericidal activities of polymorphonuclear leukocytes. When associated with one another, viruses and bacteria have stronger periodonto-pathogenic potential than individually. Therefore, it is significant to know all etiologic factors and such an insight would lead to the better treatment of the disease.

Effect of injectable trace mineral supplementation on peripheral polymorphonuclear leukocyte function, antioxidant enzymes, health, and performance in dairy cows in semi-arid conditions

The objective of this study was to evaluate the effect of subcutaneous injections of 15 mg/mL Cu, 5 mg/mL Se, 60 mg/mL Zn, and 10 mg/mL Mn on health, performance, polymorphonuclear leukocyte (PMNL) function, circulating glutathione peroxidase (GPx) and superoxide dismutase (SOD) concentrations, and inflammation of dairy cows undergoing the transition period in high temperature-humidity index. A total of 923 multiparous cows from 2 commercial dairy farms were randomly allocated into 1 of 2 treatment groups as follows: control and injectable trace mineral supplementation (ITMS). Cows in the ITMS group received 7 mL of subcutaneous injections at dry-off (208 ± 3 d of gestation), 260 ± 3 d of gestation, and at 35 ± 3 d in milk (DIM). Data regarding health traits, reproductive performance, milk yield, and survivability were extracted from farm database software, and animals were followed-up until 300 DIM. For a subset of 142 cows from one herd, blood samples were collected at enrollment, and at 3 ± 1, 7 ± 1, 10 ± 1, and 35 ± 3 DIM to evaluate hematology, PMNL function, GPx and SOD concentrations, and circulating haptoglobin. Logistic regression was used to assess health and pregnancy per artificial insemination at first service. Cox proportional hazards models were used to evaluate hazard of pregnancy and culling. Mixed linear regression models accounting for repeated measures were used to assess all continuous variables collected over time. Parity, twinning, and previous gestation length were considered as potential confounders. Farm was included as a random effect. The ITMS cows tended to have lower incidence of metritis and stillbirth compared with control group. However, ITMS treatment did not influence the incidence of other diseases (e.g., mastitis, retained placenta), milk yield, reproductive performance, culling, and leukocyte count. Neutrophil-to-lymphocyte ratio, PMNL phagocytosis, and oxidative burst as well as intensity of the oxidative burst were greater for ITMS-treated cows in comparison to control cows. The ITMS cows had decreased expression of the adhesion molecule L-selectin on PMNL surface. The serum concentration of GPx and SOD were not affected by ITMS treatment. In conclusion, ITMS tended to reduce the incidence of metritis and stillbirth parturition, improved PMNL function, and improved the inflammatory status of dairy cows undergoing the transition period in high temperature-humidity index conditions. However, these findings did not translate into improved milk yield, reproductive performance, and survivability.

Predicting the risk of retained fetal membranes and metritis in dairy cows according to prepartum hemogram and immune and metabolic status

Uterine diseases affect a significant proportion of dairy cows, causing significant economic losses. Immune and metabolic statuses are associated with the risk of retained fetal membranes (RFM) and metritis. The hypothesis of this study was that it is possible to use such responses to predict the risk of RFM and metritis. Data from cows (Jersey = 143, Holstein = 116) previously used in four experiments were used. Cow factors [parity, BCS change from −28 to 0 d relative to calving, calf sex, calving problems (twins, stillbirth, dystocia)] were evaluated for their association with the risk of RFM and metritis. Blood samples collected in the last week of gestation were used to measure polymorphonuclear leukocyte phagocytosis, oxidative burst, and expression of CD18 and CD62L, total blood count, haptoglobin optical density, and concentrations of glucose, non-esterified fatty acids, and β-hydroxybutyrate. Cows were treated with egg ovalbumin at −21, −7, and 7 d relative to calving and blood samples were used to determine the anti-ovalbumin IgG optical density. Univariable analyses were carried out to identify variables associated with the risk of RFM and metritis. The significant (P ≤ 0.15) variables were included in multivariable models from which variables with P > 0.15 were removed in a backward stepwise fashion. Risk of RFM was reduced in the absence of calving problems [adjusted odds ratio (AOR) 95 % confidence interval (CI) = 0.13 (0.02, 0.86)], when intensity of phagocytosis [AOR (95 % CI) = 0.50 (0.25, 1.04)] and expression of CD62L [AOR (95 % CI) = 0.71 (0.46, 1.09)] by polymorphonuclear leukocyte were greater, and when prepartum non-esterified fatty acid concentration was lower [AOR (95 % CI) = 8.71 (0.49, 153.84)]. Calf sex [AOR (95 % CI) = 0.35 (0.10, 1.25)], calving problem [AOR (95 % CI) = 0.14 (0.02, 1.08)], PMNL phagocytosis intensity [AOR (95 % CI) = 0.72 (0.47, 1.11)], polymorphonuclear leukocyte intensity of expression CD18 [AOR (95 % CI) = 0.78 (0.60, 1.00)] and CD62L [AOR (95 % CI) = 0.77 (0.60, 0.98)], and haptoglobin optical density [AOR (95 % CI) = 1.16 (0.97, 1.39)] were associated with metritis. Indexes generated from the estimates of the multivariable analyses to predict the risk of RFM [area under the curve = 0.77 (95 % CI = 0.70 to 0.84)] and metritis [area under the curve = 0.76 (95 % CI = 0.70, 0.81)] demonstrated that polymorphonuclear leukocyte function, non-esterified fatty acid concentration, and haptoglobin optical density prepartum may be used as predictors of uterine diseases.

New Advances in Targeting the Resolution of Inflammation: Implications for Specialized Pro-Resolving Mediator GPCR Drug Discovery.

Chronic inflammation is a component of numerous diseases including autoimmune, metabolic, neurodegenerative, and cancer. The discovery and characterization of specialized pro-resolving mediators (SPMs) critical to the resolution of inflammation, and their cognate G protein-coupled receptors (GPCRs) has led to a significant increase in the understanding of this physiological process. Approximately 20 ligands, including lipoxins, resolvins, maresins, and protectins, and 6 receptors (FPR2/ALX, GPR32, GPR18, chemerin1, BLT1, and GPR37) have been identified highlighting the complex and multilayered nature of resolution. Therapeutic efforts in targeting these receptors have proved challenging, with very few ligands apparently progressing through to preclinical or clinical development. To date, some knowledge gaps remain in the understanding of how the activation of these receptors, and their downstream signaling, results in efficient resolution via apoptosis, phagocytosis, and efferocytosis of polymorphonuclear leukocytes (mainly neutrophils) and macrophages. SPMs bind and activate multiple receptors (ligand poly-pharmacology), while most receptors are activated by multiple ligands (receptor pleiotropy). In addition, allosteric binding sites have been identified signifying the capacity of more than one ligand to bind simultaneously. These fundamental characteristics of SPM receptors enable alternative targeting strategies to be considered, including biased signaling and allosteric modulation. This review describes those ligands and receptors involved in the resolution of inflammation, and highlights the most recent clinical trial results. Furthermore, we describe alternative mechanisms by which these SPM receptors could be targeted, paving the way for the identification of new therapeutics, perhaps with greater efficacy and fidelity.

Tamm-Horsfall Protein is a Potent Immunomodulatory Molecule and a Disease Biomarker in the Urinary System.

Tamm–Horsfall protein (THP), or uromodulin (UMOD), is an 80–90-kDa phosphatidylinositol-anchored glycoprotein produced exclusively by the renal tubular cells in the thick ascending limb of the loop of Henle. Physiologically, THP is implicated in renal countercurrent gradient formation, sodium homeostasis, blood pressure regulation, and a defense molecule against infections in the urinary system. Investigations have also revealed that THP is an effective binding ligand for serum albumin, immunoglobulin G light chains, complement components C1 and C1q, interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, and interferon-γ through its carbohydrate side chains for maintaining circulatory and renal immune homeostasis. Thus, THP can be regarded as part of the innate immune system. UMOD mutations play crucial roles in congenital urolithiasis, hereditary hyperuricemia/gout, and medullary cystic kidney diseases. Recent investigations have focused on the immunomodulatory effects of THP on immune cells and on THP as a disease biomarker of acute and chronic kidney diseases. Our studies have suggested that normal urinary THP, through its epidermal growth factor (EGF)-like domains, binds to the surface-expressed EGF-like receptors, cathepsin G, or lactoferrin to enhance polymorphonuclear leukocyte phagocytosis, proinflammatory cytokine production by monocytes/macrophages, and lymphocyte proliferation by activating the Rho family and mitogen-activated protein kinase signaling pathways. Furthermore, our data support both an intact protein core structure and carbohydrate side chains are important for the different protein-binding capacities of THP. Prospectively, parts of the whole THP molecule may be used for anti-TNF-α therapy in inflammatory diseases, autoantibody-depleting therapy in autoimmune disorders, and immune intensification in immunocompromised hosts.

Effect of recombinant bovine somatotropin on leukocyte mRNA expression for genes related to cell energy metabolism, cytokine production, phagocytosis, oxidative burst, and adaptive immunity

Objectives of the current experiment were to evaluate the effects of treatment of periparturient dairy cows with recombinant bovine somatotropin (rbST) on mRNA expression in peripheral leukocytes for genes related to the somatotropic axis, cell energy metabolism, and innate and adaptive immune responses. Cows were enrolled in the experiment at 253 ± 3 d of gestation and assigned to an untreated control group (n = 98) or to receive 125 mg of rbST weekly from -21 to 21 d relative to calving (rbST125; n = 98). Data from a subsample of cows (control = 16, rbST125 = 16) are reported herein. Hemogram and polymorphonuclear leukocyte (PMNL) phagocytosis, oxidative burst, and expression of adhesion molecules were determined weekly, from -28 ± 3 to 24 ± 3 d relative to calving. Cows were vaccinated with ovalbumin at -21 ± 3, -7 ± 3, and 7 ± 3 d relative to calving. Serum IgG anti-ovalbumin and haptoglobin optical densities were determined weekly, from -28 ± 3 to 24 ± 3 d relative to calving. Leukocytes were isolated from whole blood on d -21 ± 3, -7 ± 3, and 7 ± 3 relative to calving to determine leukocyte mRNA expression for 66 genes. Cows in the rbST125 treatment had greater concentration of granulocytes 1 wk prepartum (control = 3.7 ± 0.4 vs. rbST125 = 4.6 ± 0.4 × 109 cells/L). Expression of CD18 by PMNL during the prepartum (control = 3,262 ± 280 vs. rbST125 = 3,926 ± 260 geometric mean fluorescence intensity) and percentage of PMNL positive for phagocytosis and oxidative burst 1 wk postpartum (control = 59.2 ± 2.8 vs. rbST125 = 67.6 ± 3.1%) were increased in rbST125 cows. Postpartum IgG anti-ovalbumin optical density was higher in rbST125 cows than control cows (control = 1.5 ± 0.1 vs. rbST125 = 1.9 ± 0.1 optical density). On d -7 relative to calving, leukocyte mRNA expression of IGF1R and JAK1 tended to be downregulated and expression of DEFB3 tended to be upregulated by rbST treatment. Expression of mRNA for STAT1, RELA, and NOD2 tended to be upregulated, expression of RAC2 was downregulated, and expression of JAK3, BLK, and TNFα tended to be downregulated on d 7 relative to calving by rbST treatment. Effects of treatment of periparturient cows with 125 mg of rbST on leukocyte gene expression were minute; thus, additional experiments are necessary to elucidate how rbST-induced increases in growth hormone and insulin-like growth factor-1 concentrations may modulate the immune response of periparturient cows.

Role of periodontopathogenic virus in periodontal disease: a review

Periodontal diseases are infectious diseases, but the specific mechanism by which the tooth-supportive tissue is destroyed is not clearly understood. Viral infection impairs periodontal defenses, thereby permitting subgingival overgrowth of periodontopathic bacteria. The role of viruses is significant, as they may induce abnormalities in the adhesion, chemotaxis, phagocytosis, and bactericidal activities of polymorphonuclear leukocytes. When associated with one another, viruses and bacteria have stronger periodontopathogenic potential than individually. Therefore, it is significant to know all etiologic factors and such an insight would lead to the better treatment of the disease.

Rumen-protected methionine compared with rumen-protected choline improves immunometabolic status in dairy cows during the peripartal period.

The immunometabolic status of peripartal cows is altered due to changes in liver function, inflammation, and oxidative stress. Nutritional management during this physiological state can affect the biological components of immunometabolism. The objectives of this study were to measure concentrations of biomarkers in plasma, liver tissue, and milk, and also polymorphonuclear leukocyte function to assess the immunometabolic status of cows supplemented with rumen-protected methionine (Met) or choline (CHOL). Forty-eight multiparous Holstein cows were used in a randomized complete block design with 2×2 factorial arrangement of Met (Smartamine M, Adisseo NA, Alpharetta, GA) and CHOL (ReaShure, Balchem Inc., New Hampton, NY) level (with or without). Treatments (12 cows each) were control (CON), no Met or CHOL; CON and Met (SMA); CON and CHOL (REA); and CON and Met and CHOL (MIX). From -50 to -21d before expected calving, all cows received the same diet [1.40Mcal of net energy for lactation (NEL)/kg of DM] with no Met or CHOL. From -21d to calving, cows received the same close-up diet (1.52Mcal of NEL/kg of DM) and were assigned randomly to each treatment. From calving to 30d, cows were on the same postpartal diet (1.71Mcal of NEL/kg of DM) and continued to receive the same treatments until 30d. The Met supplementation was adjusted daily at 0.08% DM of diet, and CHOL was supplemented at 60g/cow per day. Liver (-10, 7, 21, and 30d) and blood (-10, 4, 8, 20, and 30d) samples were harvested for biomarker analyses. Neutrophil and monocyte phagocytosis and oxidative burst were assessed at d 1, 4, 14, and 28d. The Met-supplemented cows tended to have greater plasma paraoxonase. Greater plasma albumin and IL-6 as well as a tendency for lower haptoglobin were detected in Met- but not CHOL-supplemented cows. Similarly, cows fed Met compared with CHOL had greater concentrations of total and reduced glutathione (a potent intracellular antioxidant) in liver tissue. Upon a pathogen challenge in vitro, blood polymorphonuclear leukocyte phagocytosis capacity and oxidative burst activity were greater in Met-supplemented cows. Overall, liver and blood biomarker analyses revealed favorable changes in liver function, inflammation status, and immune response in Met-supplemented cows.

Effect of probiotic bacteria on phagocytosis and respiratory burst activity of blood polymorphonuclear leukocytes (PMNL) in mice infected with Trichinella spiralis

This study focusses on the effect of probiotic (bacteriocinogenic) strains on parasite infection and innate immunity – phagocytosis and oxidative burst of blood monocytes and polymorphonuclear leukocytes (PMNL) in mice infected with Trichinella spiralis. Bacteriocinogenic and probiotic strains of different origin (Enterococcus faecium AL41=CCM8558, Enterococcus durans ED26E/7, Lactobacillus fermentum AD1=CCM7421, Lactobacillus plantarum 17L/1) were administered daily in dose of 109CFU/ml in 100μl and mice were infected with 400 larvae of T. spiralis on 7th day of treatment. Phagocytic activity of blood leukocytes was inhibited at week 3 and 4 post infection (p.i.), i.e. in the time of massive muscle invasion with larvae T. spiralis. Administration of bacterial strains to mice prior to T. spiralis infection elevated and prolonged phagocytic activity of blood leukocytes and their ingestion capability from week 1 to 3 of the infection and the phagocytosis was inhibited only at week 4 p.i. The highest stimulative effect on phagocytosis was induced by strains E. durans ED26E/7, L. fermentum AD1=CCM7421, and L. plantarum 17L/1. The percentage of cells with respiratory burst and their enzymatic activity was increased after T. spiralis infection with the exception of week 3 p.i. In contrast, in all mice treated with bacterial strains the enzymatic stimulation was observed after the infection, with the highest intensity caused by strains E. durans ED26E/7, L. fermentum AD1=CCM7421 and L. plantarum 17L/1. The administration of probiotic strains stimulated phagocytosis and respiratory burst of blood PMNL that could contribute to a decreased larval migration and a destruction of muscle larvae and then reduced parasite burden in the host. The protective effect against T. spiralis infection was induced by all strains, but the highest reduction was recorded by E. faecium AL41=CCM8558.

Effects of prepartum stocking density on innate and adaptive leukocyte responses and serum and hair cortisol concentrations

Objectives were to evaluate the effects of prepartum stocking density on innate and adaptive leukocyte responses, serum cortisol and haptoglobin concentrations and hair cortisol concentration of Jersey cows. The cows (254±3d of gestation) were balanced for parity (nulliparous vs. parous) and previous lactation projected 305-d mature equivalent milk yield and assigned to one of two treatments: 80SD=80% stocking density (38 animals/48 headlocks) and 100SD=100% stocking density (48 animals/48 headlocks). Pens (n=4) were identical in size and design and each pen received each treatment a total of 2 times (4 replicates; 80SD: n=338; 100SD: n=418). A sub-group of cows (n=48/treatment per parity) was randomly selected on week 1 of each replicate from which blood was sampled weekly from d −14 to 14 (d 0=calving) to determine polymorphonuclear leukocyte (PMNL) phagocytosis, oxidative burst, and expression of CD18 and L-selectin, and hemogram. The same sub-group of cows was treated with chicken egg ovalbumin on d −21, −7, and 7 and had blood sampled weekly from d −21 to 21 for determination of serum IgG anti-ovalbumin concentration. Blood was sampled weekly from d −21 to 21 to determine glucose, cortisol, and haptoglobin concentrations in serum. Hair samples collected at enrollment and within 24h of calving were analyzed for cortisol concentration. The percentage of leukocytes classified as granulocyte and the granulocyte to the lymphocyte ratio were not affected by treatment. Treatment did not affect the percentage of PMNL positive for phagocytosis and oxidative burst or the intensity of phagocytosis and oxidative burst. Similarly, treatment did not affect the percentage of PMNL expressing CD18 and L-selectin or the intensity of expression of CD18 and L-selectin. Concentration of IgG anti-ovalbumin was not affected by treatment. Serum concentrations of haptoglobin and cortisol were not affected by treatment. Similarly, hair cortisol concentration at calving was not affected by treatment. According to the current experiment, a target stocking density of 80% did not improve leukocyte responses compared with 100% target stocking density.

Glucose supplementation has minimal effects on blood neutrophil function and gene expression in vitro

During early lactation, glucose availability is low and the effect of glucose supply on bovine polymorphonuclear leukocyte (PMNL) function is poorly understood. The objective of this study was to determine the effect of glucose supplementation on the function and transcriptomic inflammatory response of PMNL from cows in early and mid-lactation in vitro. Twenty Holstein cows in early (n=10; days in milk=17±3.1) and mid-lactation (n=10; days in milk=168±14.8) were used for this study. Jugular blood was analyzed for serum concentrations of nonesterified fatty acids, β-hydroxybutyrate, and glucose. Polymorphonuclear leukocytes were isolated and diluted using RPMI (basal glucose concentration was 7.2 mM) to different concentrations of PMNL/mL for phagocytosis, chemotaxis, gene expression, and medium analyses. Working solutions of glucose (0 or 4 mM of d-glucose) and lipopolysaccharide (0 or 50μg/mL) were added and tubes were incubated for 120 min at 37°C. Media were analyzed for concentrations of glucose and tumor necrosis factor-α (TNF-α). Data were analyzed in a randomized block (stage of lactation) design. Challenge with lipopolysaccharide increased the expression of the genes encoding for nuclear factor kappa B (NFKB1), IL-10 (IL10), IL1B, IL6, IL8, TNF-α (TNFA), glucose transporter 3 (SLC2A3), and the concentration of TNF-α in medium (147.3 vs. 72.5 pg/mL for lipopolysaccharide and control, respectively). Main effect of stage of lactation was minimal where the expression of IL10 increased for cows in early compared with cows in mid-lactation. After lipopolysaccharide challenge, cows in early lactation experienced more marked increases in the expression of IL6, TNFA, and IL8 when compared with cows in mid-lactation. Glucose supplementation had minimal effects on gene expression where glucose supplementation increased the expression of lysozyme (LYZ). Glucose supplementation increased PMNL phagocytosis but did not alter chemotaxis, morphology, or concentration of TNF-α in the medium. Under the conditions of the experiment, stage of lactation had minimal effects on PMNL response to glucose supply where only the expression of NFKB1 and the production of TNF-α were greater for cows in mid-lactation when compared with early lactation. Metabolic profiles for cows in early lactation did not parallel those for cows during the early postpartum period and may partly explain results for this study. Future studies investigating the effect of glucose supply on bovine PMNL function in vivo and how this may be altered by stage of lactation are warranted.

Effects of tramadol and o-desmethyltramadol on canine innate immune system function

ObjectiveTramadol is a commonly used opioid analgesic in dogs, particularly in dogs with a compromised immune system. An opioid may be selected for its immunomodulatory effects. Consequently, the objective of this study was to investigate the effects of tramadol on immune system function by evaluating the effect of tramadol and o-desmethyltramadol (M1) on the function of canine leukocytes in vitro. The hypothesis was that tramadol and M1 would not alter polymorphonuclear leukocyte (PMN) phagocytosis, PMN oxidative burst, or stimulated leukocyte cytokine production capacity of tumor necrosis factor (TNF)-a, interleukin (IL)-6, and IL-10. Study designIn vitro pharmacodynamic study. AnimalsSix healthy dogs. MethodsBlood from six dogs was obtained and incubated with various concentrations of tramadol and M1. Phagocytosis and oxidative burst were assessed using flow cytometry, and lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PG)-stimulated leukocyte production of TNF, IL-6, and IL-10 were measured using a canine specific multiplex assay. ResultsNo differences were detected in phagocytosis or oxidative burst with any drug concentration. Tramadol did not alter leukocyte cytokine production, however, M1 significantly blunted IL-10 production. ConclusionsTramadol and its metabolite M1 were sparing to PMN phagocytosis and oxidative burst in dogs in vitro. Tramadol did not alter leukocyte cytokine production, however, M1 blunted IL-10 production at clinically achievable concentrations suggesting that M1 may promote a proinflammatory shift. Clinical relevanceThese data suggest that tramadol has minimal effect on phagocytosis and oxidative burst, and may promote a proinflammatory shift. Therefore, tramadol may be an ideal opioid analgesic in dogs at high risk of infection. Further investigation in vivo is warranted.

The effect of citrus-derived oil on bovine blood neutrophil function and gene expression in vitro

Research on the use of natural products to treat or prevent microbial invasion as alternatives to antibiotic use is growing. Polymorphonuclear leukocytes (PMNL) play a vital role with regard to the innate immune response that affects severity or duration of mastitis. To our knowledge, effect of cold-pressed terpeneless Valencia orange oil (TCO) on bovine PMNL function has not been elucidated. Therefore, the objective of this study was to investigate the effect of TCO on bovine blood PMNL chemotaxis and phagocytosis capabilities and the expression of genes involved in inflammatory response in vitro. Polymorphonuclear leukocytes were isolated from jugular blood of 12 Holstein cows in mid-lactation and were incubated with 0.0 or 0.01% TCO for 120min at 37°C and 5% CO2, and phagocytosis (2×106 PMNL) and chemotaxis (6×106 PMNL) assays were then performed in vitro. For gene expression, RNA was extracted from incubated PMNL (6×106 PMNL), and gene expression was analyzed using quantitative PCR. The supernatant was stored at −80°C for analysis of tumor necrosis factor-α. Data were analyzed using a general linear mixed model with cow and treatment (i.e., control or TCO) in the model statement. In vitro supplementation of 0.01% of TCO increased the chemotactic ability to IL-8 by 47%; however, migration of PMNL to complement 5a was not altered. Treatment did not affect the production of tumor necrosis factor-α by PMNL. Expression of proinflammatory genes (i.e., SELL, TLR4, IRAK1, TRAF6, and LYZ) coding for proteins was not altered by incubation of PMNL with TCO. However, downregulation of TLR2 [fold change (FC=treatment/control)=−2.14], NFKBIA (FC=1.82), IL1B (FC=−2.16), TNFA (FC=−9.43), and SOD2 (FC=−1.57) was observed for PMNL incubated with TCO when compared with controls. Interestingly, expression of IL10, a well-known antiinflammatory cytokine, was also downregulated (FC=−3.78), whereas expression of IL8 (FC=1.93), a gene coding for the cytokine IL-8 known for its chemotactic function, tended to be upregulated in PMNL incubated with TCO. Incubation of PMNL with TCO enhanced PMNL chemotaxis in vitro. The expression of genes involved in the inflammatory response was primarily downregulated. Results showed that 0.01% TCO did not impair the function of PMNL in vitro. Future studies investigating the use of TCO as an alternative therapy for treatment of mastitis, including dose and duration, for cows during lactation are warranted.

Effects of weekly regrouping of prepartum dairy cows on innate immune response and antibody concentration

Objectives were to evaluate the effects of a stable prepartum grouping strategy on innate immune parameters, antibody concentration, and cortisol and haptoglobin concentrations of Jersey cows. Cows (253±3d of gestation) were paired by gestation length and assigned randomly to the stable (all-in-all-out; AIAO) or traditional (TRD) treatment. In the AIAO treatment, groups of 44 cows were moved into a pen where they remained for 5wk, whereas in the TRD treatment, approximately 10 cows were moved into a pen weekly to maintain stocking density (44 cows for 48 headlocks). Pens were identical in size and design and each pen received each treatment a total of 3 times (6 replicates; AIAO, n=259; TRD, n=308). A subgroup of cows (n=34/treatment) was selected on wk 1 of each replicate from which blood was sampled weekly from d −14 to 14 (d 0=calving) to determine polymorphonuclear leukocyte (PMNL) phagocytosis, oxidative burst, and expression of CD18 and L-selectin, hemogram, cortisol and glucose concentrations, and haptoglobin concentration. Another subgroup of cows (n=40/treatment) selected on wk 1 of each replicate was treated with chicken egg ovalbumin on d −21, −7, and 7 and had blood sampled weekly from d −21 to 21 for determination of immunoglobulin G anti-ovalbumin. All cows (n=149) had blood sampled weekly for nonesterified fatty acid (NEFA) and β-hydroxybutyrate (BHBA) concentrations from d −21 to 21. Treatment did not affect percentage of PMNL positive for phagocytosis and oxidative burst (AIAO=64.3±2.9 vs. TRD=64.3±2.9%) and intensity of phagocytosis [AIAO=2,910.82±405.99 vs. TRD=2,981.52±406.87 geometric mean fluorescence intensity (GMFI)] and oxidative burst (AIAO=7,667.99±678.29 vs. TRD=7,742.70±682.91GMFI). Similarly, treatment did not affect the percentage of PMNL expressing CD18 (AIAO=96.3±0.7 vs. TRD=97.8±0.7%) and L-selectin (AIAO=44.1±2.8 vs. TRD=45.1±2.8%) or the intensity of expression of CD18 (AIAO=3,496.2±396.5 vs. TRD=3,598.5±396.9GMFI) and L-selectin (AIAO=949.8±22.0 vs. TRD=940.4±22.3GMFI). Concentration of immunoglobulin G anti-ovalbumin was not affected by treatment (AIAO=0.98±0.05 vs. TRD=0.98±0.05 OD). The percentage of leukocytes classified as granulocyte (AIAO=38.9±1.5 vs. TRD 38.2±1.5%) and the granulocyte:lymphocyte ratio (AIAO=0.75±0.04 vs. TRD=0.75±0.04) were not affected by treatment. Concentrations of cortisol (AIAO=14.95±1.73 vs. TRD=18.07±1.73ng/mL), glucose (AIAO=57.6±1.5 vs. TRD=60.0±1.5ng/mL), and haptoglobin (AIAO=3.09±0.48 vs. TRD=3.51±0.49 OD) were not affected by treatment. According to the current experiment, a stable prepartum grouping strategy does not improve innate immune parameters or antibody concentration compared with weekly prepartum regrouping.

Virulence Mechanism of Bacteria in Mixed Infection: Attenuation of Cytokine Levels and Evasion of Polymorphonuclear Leukocyte Phagocytosis

The objective of the present study is to evaluate the effect of bacterial viability on the virulence of mixed infection. Expression of pro- and anti-inflammatory cytokines (interleukin [IL]-1β and IL-10, respectively) was tested in vivo, following live versus heat-killed infection (mono or mixed), using the mouse chamber model of infection. Ex vivo, phagocytosis of fluorescently labeled bacteria was tested in primary mouse polymorphonuclear leukocytes by flow cytometry. In monoinfection, heat-killed Porphyromonas gingivalis led to augmented levels of IL-1β 2 hours postinfection, whereas IL-10 levels remained unaffected. Phagocytosis of heat-killed P. gingivalis was reduced compared with that of the live P. gingivalis, whereas phagocytosis of heat-killed Fusobacterium nucleatum was augmented compared with that of live F. nucleatum. In mixed infection, both IL-1β and IL-10 levels were augmented 24 hours postinfection when the bacteria were heat-killed. Although the phagocytosis pattern of F. nucleatum in the mixed infection remained similar to that upon monoinfection, phagocytosis of P. gingivalis was reduced following mixed infection. The inflammatory response to live mixed infection is attenuated with reduced phagocytosis, compared with that of heat-killed mixed infection. The lower response to live mixed infection could stem from a mechanism enabling the bacteria to evade the host response, thereby increasing bacterial survival.

Cinacalcet effect on polymorphonuclear leucocytes of kidney transplant patients

Polymorphonuclear leucocytes (PMNLs) play a key role in the nonspecific immune defence. Cinacalcet reduces serum calcium levels in kidney transplant recipients with mineral bone disorder associated with chronic kidney disease. We investigated essential functions of PMNLs of kidney transplant recipients with and without hypercalcaemia and with and without cinacalcet therapy. Oxidative burst, phagocytosis, apoptosis and intracellular calcium concentrations of PMNLs from normocalcaemic kidney transplant patients without (KT-NC) or with cinacalcet intake (KT-NC/CI), hypercalcaemic kidney transplant patients (KT-HC) and healthy subjects (HS) were investigated. Stimulation of oxidative burst of PMNLs from KT-HC patients by phorbol-12-myristate-13-acetate or Escherichia coli was significantly attenuated compared with PMNLs from KT-NC, KT-NC/CI and HS. Apoptosis of PMNLs from KT-HC patients was significantly decreased compared with cells from KT-NC, KT-NC/CI and HS. Apoptosis correlated significantly with serum calcium concentrations. Intracellular calcium concentrations and phagocytosis of PMNLs did not differ between groups. Our data indicate that stimulation of PMNL oxidative burst and apoptosis is significantly diminished in kidney transplant patients with hypercalcaemia, while kidney transplant patients with serum calcium levels normalized by cinacalcet have normal PMNL functions despite immunosuppressive therapy.

The effect of incomplete milking or nursing on milk production, blood metabolites, and immune functions of dairy cows

During the transition from pregnancy to lactation, the sudden increase in nutrient demand for milk production causes metabolic perturbations and is associated with immunosuppression and a high incidence of metabolic and infectious diseases in high-yielding cows. In this study, we examined whether limiting milk harvest postpartum while maintaining milking stimulus could improve the metabolic status of cows without reducing overall milk production. Forty-seven Holstein cows were milked completely twice a day from calving (control); milked incompletely (about one-third of expected milk production was collected) twice a day until d 5 after calving (incomplete); or left to nurse their calf until d 5 and milked once a day from d 3 to d 5 (nursing). All cows were milked twice a day from d 6 to the end of the experiment (d 61). During the treatment period (d 1 to 5), milk production averaged 27.3 and 9.7kg/d for the control and incomplete treatments, respectively. We observed no residual effect of treatment on milk production, which averaged 47.8, 45.7, and 46.4 kg/d for the control, incomplete, and nursing treatments, respectively, between wk 2 and 9. The dry matter intake of the cows was similar during and after treatment. From wk 2 to 9, milk protein and lactose percentage were not affected by treatments, but milk fat tended to be higher in control cows than in cows milked partially (incomplete + nursing). Blood concentrations of glucose and phosphorus were lower and concentrations of nonesterified fatty acids and β-hydroxybutyrate were higher in control cows than in partially milked cows during the treatment period. The positive effects on glucose and β-hydroxybutyrate remained significant up to d 28. Peripheral blood mononuclear cell (PBMC) proliferation and secretion of IL-4 were depressed during the postpartum period, and proliferation tended to be greater for cells incubated in serum from cows in the incomplete treatment on d 5 but lower on d 61. We observed no effect of treatments on polymorphonuclear neutrophilic leukocyte chemotaxis or phagocytosis. Proliferation and IL-4 secretion of PBMC were negatively correlated with concentration of serum nonesterified fatty acids. Reducing milk harvest postpartum while maintaining milking stimuli reduced metabolic stress without compromising productivity of high-yielding dairy cows.

Decoration of Histophilus somni lipooligosaccharide with N-acetyl-5-neuraminic acid enhances bacterial binding of complement factor H and resistance to killing by serum and polymorphonuclear leukocytes

The incorporation of N-acetyl-5-neuraminic acid (Neu5Ac), or sialic acid, onto surface components of some bacterial species may enhance their virulence. We have previously shown that Neu5Ac can be incorporated onto the lipooligosaccharide (LOS) of the bovine pathogen Histophilus somni, resulting in diminished antibody binding and enhanced serum resistance (Inzana et al., 2002. Infect. Immun. 70, 4870). In the present study, we assessed the effect of sialylation of H. somni LOS on the interaction with bovine innate host defenses. Incubation of non-sialylated H. somni with pre-colostral calf serum (PCS) resulted in dose-dependent, complement-mediated killing of the bacteria by the alternative pathway. However, sialylated H. somni was significantly more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated and consumed less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized red blood cells, and by Western blotting of C3 activation products. Sialylated H. somni bound more factor H and iC3b and less C3 than non-sialylated bacteria, as determined by enzyme-linked immunosorbent assay, supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni LOS inhibited both polymorphonuclear leukocyte phagocytosis of 3H-thymidine-labeled bacteria and intracellular killing of the bacteria, compared to non-sialylated bacteria. Furthermore, sialylated H. somni bound less non-specific antibodies in normal bovine sera than non-sialylated bacteria. Therefore, sialylation of H. somni LOS had profound effects on resistance of the bacteria to innate bovine host defenses, which should be taken into consideration during in vitro studies of H. somni.

Effect of postcalving serum nonesterified fatty acids concentration on the functionality of bovine immune cells

The periparturient period is marked by metabolic, hormonal, and immunological changes, which have an effect on the incidence of infectious and metabolic diseases. In a previous study, a slower increase in milk production was induced by milking cows once daily during the first week of lactation, leading to an improvement in levels of several metabolites, including nonesterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA). The aim was to determine the influence of serum collected on d 2, 5, and 61 postpartum from cows milked once or twice daily on immune cell functions and to determine which of the constituents were responsible for these effects. Peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes were collected from healthy midlactation cows and their immune functions (i.e., proliferation and interferon-γ production and chemotaxis, phagocytosis, and oxidative burst, respectively), were evaluated in presence of serum, NEFA, and BHBA. Proliferation of PBMC was greater with d-61 (65.1±1.6%) serum than with d-2 (37.3±2.4%) or d-5 (48.4±1.6%) serum and greater with d-2 and -5 serum from cows milked once (42.2±3.7 and 54.0±2.5) compared with cows milked twice daily (32.4±3.0 and 42.9±2.1). Proliferation was inversely correlated with the concentration of NEFA and BHBA in the serum (r=−0.86). Adding NEFA to d-61 serum to reach the level present in d-5 serum decreased proliferation to the level observed with d-5 serum. No effect of BHBA addition was observed. The release of interferon-γ by PBMC was lower in d-5 serum (766±63 pg/mL) than in d-61 serum (1,187±90 pg/mL) and by NEFA. Milking frequency did not affect chemotaxis, phagocytosis, or oxidative burst of polymorphonuclear leukocytes. Phagocytosis decreased over time in serum from d 2 to 61. Similarly, oxidative burst was greater with d-5 serum (12.7×108 ± 1.6×108 relative light units) than with d-61 serum (9.0×108 ± 1.6×108 relative light units). The NEFA had a negative effect on oxidative burst, but BHBA did not. In conclusion, several immune cell functions appear affected by the NEFA concentration. Therefore, strategies that prevent increases in blood NEFA during the transition period may limit postpartum immunosuppression.

Human viruses and periodontal disease

Periodontitis is a multifactorial, chronic disease followed by destruction of supporting structures of teeth. The main etiological factor for development of periodontitis is oral biofilm containing anaerobic microorganisms. Besides bacteria, viruses can also be present within periodontal pockets. The role of viruses is significant, as they may induce abnormalities in the adhesion, chemotaxis, phagocytosis, and bactericidal activities of polymorphonuclear leukocytes. Associated with one another, viruses and bacteria have stronger periodontopathogenic potential than individually. The synergism between periodontopathogenic bacteria, Epstein-Barr virus, and Cytomegalovirus stands out. For full understanding of the pathogenesis of periodontitis, it is significant to know all etiologic factors and host immune response; such an understanding should also guide the adequate treatment of this disease.