Distinct neutrophil C5a receptor inflammatory events in cows initiated by chemoattractant C5a and lipopolysaccharide around parturition and in mid lactation.

In neutrophils, toll-like receptor and complement component 5a (C5a) signaling are critical pathways regulating innate immunity. In cows, not much is known about the second C5a receptor, complement component 5a receptor 2 (C5AR2). It is an interesting player in sepsis treatment because it is considered to have an anti-inflammatory effect during normal inflammation. Periparturient cows are prone to severe infections, and the objectives of this study were to investigate the expression and functionality of C5AR2 during peripartum. We investigated the effect of 2 major inflammatory stimuli, C5a and lipopolysaccharide (LPS), on the expression of a selected number of genes (C5AR1, C5AR2, TLR4, ITGAM, COX2, and CXCL8) and functions linked to these receptors. Overall, TLR4, ITGAM, and C5AR2, all of which are involved in early inflammation, showed a lower expression in periparturient cows. However, an overall lower expression seems not to be the only explanation for the increased risk of sepsis in periparturient cows. Normally, in response to inflammation and as seen in the mid-lactation group, the expression of these genes increases after stimulation with LPS. However, in periparturient cows, stimulation with LPS led to a decrease in expression of these receptors, indicating a different response of neutrophils in response to LPS during this period. A decrease in ITGAM (coding for CD11b) expression complicates correct neutrophil localization and phagocytosis. Its downregulation upon stimulation might be detrimental for adequate eradication of the pathogen and might increase the risk of an imbalanced inflammation; C5AR2 seems to play a central role in this altered response. In addition, myeloperoxidase (MPO) activity in periparturient cows is lower in response to C5a stimulation. It has been suggested that MPO plays an important role in neutrophil shutdown and, thereby, timely resolution of inflammation. A decreased MPO activity might thus prolong the inflammatory reaction of the neutrophils. This finding was supported by the increased viability of the neutrophils obtained from periparturient cows. Even after stimulation, we found a lower caspase-3 activity in this group, indicating that they might be activated for a longer time compared with the neutrophils from mid-lactation cows. Accordingly, these alterations might contribute to a temporal mismatch in inflammatory responses, as often seen in severe periparturient infections.

Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes.

The increasing demand for a sustainable larviculture has promoted research regarding environmental parameters, diseases and nutrition, intersecting at the mucosal surface of the gastrointestinal tract of fish larvae. The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling. This study aimed at optimizing an LCM protocol for intestinal tissue of sea bass larvae. Furthermore, a 3'/5' integrity assay was developed for LCM samples of fish tissue, comprising low RNA concentrations. Furthermore, reliable reference genes for performing qPCR in larval sea bass gene expression studies were identified, as data normalization is critical in gene expression experiments using RT-qPCR. We demonstrate that a careful optimization of the LCM procedure allows recovery of high quality mRNA from defined cell populations in complex intestinal tissues. According to the geNorm and Normfinder algorithms, ef1a, rpl13a, rps18 and faua were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass across a range of experimental settings. The methodology developed here, offers a rapid and valuable approach to characterize cells/tissues in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof.

The potential role of quorum-sensing peptides in oncology.

Cancer is a leading cause of death worldwide, with a limited cure rate and late diagnosis for certain types of cancer (e.g. pancreatic cancer). As this disease presents an enormous challenge for scientists, new paradigms in oncology are needed to defeat this serious disease. Currently, several peptide drugs are investigated for their preventive, diagnostic and therapeutic properties in oncology, with already 15 peptide drugs marketed for cancer therapy. However, we suggest that quorum-sensing peptide agonists and antagonists can be used in oncology as well, resulting in a larger potential peptide space. This hypothesis is based on (1) the recent evidence of prokaryote-eukaryote signalling by the use of quorum-sensing signalling molecules, (2) the apoptotic phenomenon seen in bacteria, (3) the clear similarities between the bacterial quorum-sensing mechanisms and the metastatic process tumor cells initiate, (4) the multiple receptor targeting and (5) the possibility of pharmacologic manipulation of peptides, resulting in increased receptor targeting. Up till now, however, the use of quorum-sensing signalling peptides in oncology has not yet been investigated, despite the urgent need for new insights in oncology and the promising perspectives.

Differential gene expression of the toll-like receptor-4 cascade and neutrophil function in early- and mid-lactating dairy cows.

During early lactation, neutrophils display several reduced immune functions. Particularly, a delayed recruitment of neutrophils into the infected udder seems to be one of the underlying events involved in the severity of postpartum Escherichia coli intramammary infections. The purpose of this study was to analyze the effect of in vitro chemotaxis and diapedesis on the expression of toll-like receptor-4 (TLR4)-related genes in bovine blood neutrophils isolated from 10 early-lactating (EL) and 10 mid-lactating (ML) cows. Functional characterization of the neutrophil population was performed by measuring phagocytosis and production of reactive oxygen species (chemiluminescence). Messenger RNA was extracted from neutrophils, and the expression of TLR4 and associated genes in EL and ML cows was analyzed by reverse-transcription quantitative PCR. To study the effect of chemotaxis and diapedesis on the expression of genes of the TLR4 cascade, neutrophils were stimulated to (trans)migrate in response to C5a using in vitro models. Our salient findings were that both neutrophil migration in vitro and lactation stage induced significant changes in the expression of several genes of the TLR4 signaling cascade. Before migration, expression of TRAF6, ATF3, RELA, IL8, and C5aR were lower in EL than in ML cows. Diapedesis and chemotaxis induced an increase in expression of TLR4, ATF3, and IL8 in both EL and ML cows. Diapedesis resulted in a downregulation of Syk, a TLR4-associated gene, in ML cows. This study shows that the perturbations in neutrophil functions during EL are accompanied by modulation of TLR4 pathway genes. These data can contribute to the understanding of the mechanisms explaining the relationship between stage of lactation and risk of severe E. coli mastitis.

Anaphylatoxin C5a-induced toll-like receptor 4 signaling in bovine neutrophils.

It is well known that signaling in neutrophils through both the complement component 5a (C5a) and C5a receptor (C5aR) and the toll-like receptor 4 (TLR4) pathways plays an essential role in innate defense. Neutrophil dysfunction, as seen during sepsis in severe mastitis during the periparturient period, is correlated with elevated concentrations of anaphylatoxin C5a. The aim of the current study was to elucidate the effect of C5a on TLR4 signaling in bovine neutrophils. Neutrophils were incubated with a high (but physiological) dose of purified C5a, and mRNA was extracted from neutrophils at different time points postincubation (PI). The incubation with C5a resulted in a biphasic C5aR expression profile, a phenomenon that might be explained by internalization (at 10 min PI) with subsequent reconstitution (starting at 40 min PI) of this receptor. The expression of TLR4, as well as its coreceptor, CD14, showed a similar biphasic change as observed with C5aR. In addition, changes in the mRNA expression levels of several genes belonging to the TLR4 pathway, such as TICAM-1, IKKα, and MAP3K7 were noted. The maximal expression of TLR4, CD14, and C5aR mRNA at 80 min PI was accompanied by a peak in IL8 mRNA, indicating that C5a is able to induce IL-8 production in neutrophils in vitro without the need of a costimulatory factor such as lipopolysaccharide. Moreover, a relatively constant expression of RELA was accompanied by increased expression of ATF3, an endogenous inhibitor of nuclear factor-κB mediated transcription, implying that C5a regulates TLR4 signaling and IL-8 synthesis independently. A significant time-dependent correlation was found between C5aR and TLR4, with the majority of the selected TLR4-dependent genes showing a significant correlation with C5aR at 80 min PI, when C5aR and TLR4 mRNA expression reached its maximum, suggesting crosstalk between both receptors. Taken together, this study showed that C5a is able to (1) alter the expression of genes belonging to the TLR4 pathway and (2) induce IL8 gene expression in bovine neutrophils. In addition, indications for cross-talk between complement activation and TLR4 signaling were found in the present study.

Detection of hypoxia inducible factors and angiogenic growth factors during foetal endochondral and intramembranous ossification.

During skeletogenesis, the development of a new vascular network, i.e. angiogenesis, is triggered by hypoxia through the activation of the hypoxia inducible factors (HIFs) HIF-1alpha and HIF-2alpha. HIFs regulate the expression of several genes, including those coding for angiogenic growth factors such as VEGFA, angiopoietin-1 (ANGPT1) and angiopoietin-2 (ANGPT2). The expression of HIFs and angiogenic growth factors is well documented in endochondral ossification, but few data exist on their expression during intramembranous ossification. In this study, the localization of these factors was examined using immunohistochemistry and RT-PCR in bones of porcine foetuses. Immunostaining for HIF-1alpha and HIF-2alpha was observed during endochondral ossification, whereas only HIF-2alpha was present at sites of intramembranous ossification. Furthermore, immunostaining for ANGPT2 was present at sites of endochondral and intramembranous ossification. In addition, gene transcripts for ANGPT1, ANGPT2 and VEGFA were detected with RT-PCR in laser capture microdissected isolates from both types of ossification. These results indicate that angiogenesis plays an important role during endochondral and intramembranous ossification. However, the different expression pattern of the HIF-alpha subunits suggests that alternative regulatory pathways trigger angiogenesis in these distinct types of ossification.

An integrated view on how the management of the dry period length of lactating cows could affect mammary biology and defence.

The dry period is necessary to facilitate cell turnover in the bovine mammary gland and to optimize milk production in the next lactation. An 8-week dry period has long been the golden standard of management for dairy cows. Genetic improvements and new management technologies have led to higher milk production and a need for re-evaluation of the dry period length. Over the last decade, dry period length has been proposed to be shortened or eliminated mainly from an economic point of view. However, the influence of modified dry period length on the immune defence of the bovine mammary gland and the occurrence of new intramammary infections has not yet been appreciated. The objective of this review is to discuss the bovine mammary gland biology, defence and systemic health when the dry period length is modified. Shortening or eliminating the dry period may minimize or remove the impact of milk accumulation at dry off, thereby lessening the immunodeficiency of the dam that is characteristic of this period. Composition of mammary secretions may change and the extent of tissue remodelling may be reduced when the dry period is reduced or eliminated. Additionally, impact of the dry period length on energy and nutritional status, and on hormonal and local regulatory factors, lead us to hypothesize that changing the dry period length might also affect the response to intramammary infection. It is concluded that there is a need to integrate mammary gland biology and defence mechanisms in studies dealing with modified dry period lengths.

LC-MS profiling of N-alkylamides in Spilanthes acmella extract and the transmucosal behaviour of its main bio-active spilanthol.

N-Alkylamides are a promising group of naturally occurring bio-actives, with evidence for immune stimulating properties, which find applications i.a. in buccal preparations. In Spilanthes extracts, these properties are mainly ascribed to the most abundant N-isobutylamide, spilanthol. Yet, other N-alkylamides present in these extracts may contribute to this effect, as well as to its potential toxicity and physiologic interactions. Therefore, N-alkylamide profiling of an ethanolic Spilanthes extract was performed using a gradient reversed phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry (HPLC/ESI-MS) method on an embedded polar column. MS(1) and MS(2) fragmentation data were used for identification purposes. Moreover, the transmucosal behaviour of spilanthol, formulated in this ethanolic extract and in two commercially available oral gels, was evaluated using porcine buccal mucosa in a Franz diffusion cell experimental set-up. A high-throughput HPLC-UV method was used for the quantification of spilanthol in the receptor phase. Fundamental permeation characteristics of spilanthol in a solvent-independent way (100% aqueous dose solution) were obtained using different propylene glycol/water ratios. Eight N-isobutylamides, two 2-methylbutylamides and one 2-phenylethylamide were detected, with spilanthol as most abundant N-alkylamide (88.8%). Up till now, two of these N-isobutylamides were not yet described in Spilanthes extracts. We demonstrated for the first time that spilanthol permeates the buccal mucosa. Depending on the formulation, a more pronounced local or systemic effect is achieved, which is important for the regulatory product classification. The purely aqueous permeation coefficient K(p,aq) (+/-SEM) was found to be 11.3 (+/-0.403)10(-3)cm/h.

Transdermal behaviour of the N-alkylamide spilanthol (affinin) from Spilanthes acmella (Compositae) extracts.

AIM OF THE STUDY: N-Alkylamides are a large group of bioactive molecules found in several plants from the genera Echinacea, Xanthoxylum and Spilanthes. Extracts and formulations derived from these plants are not only orally used, but also applied on the skin as well. However, there is currently no specific information available about the intrinsic local pharmacokinetics of N-alkylamides after topical application on human skin, questioning the role of this mode of administration. The present study investigates the transdermal behaviour of spilanthol, a prominent N-alkylamide. MATERIALS AND METHODS: Two pharmaceutically accepted dose solutions (ethanol and propylene glycol based aqueous donor vehicles), combined with three different receptor fluids (PBS, PBS+0.5% HPbetaCD, EtOH/H(2)O (30:70, v/v)), were applied on split-thickness human skin in a Franz diffusion cell (FDC) system. Fundamental permeation characteristics of spilanthol in a solvent-independent way (100% aqueous dose solution) were also obtained using an extrapolation approach with different organic solvent/H(2)O ratios. RESULTS AND CONCLUSIONS: We demonstrated for the first time that spilanthol permeates the skin. The following aqueous-extrapolated primary transdermal parameters were obtained (mean+/-SEM): K(p,aq)=3.31 (+/-0.29)x10(-3)cm/h, D(m,aq)=1.86 (+/-0.09)x10(-4)cm(2)/h and K(m,aq)=7.28 (+/-1.59)x10(-1). Partitioning (K(m)) was strongly dependent on the donor solution composition, while diffusion (D(m)) was mainly influenced by the receptor fluid composition.

Phagocytic and bactericidal activity of blood and milk-resident neutrophils against Staphylococcus aureus in primiparous and multiparous cows during early lactation.

To examine the effect of parity on polymorphonuclear neutrophils (PMN) function, phagocytic and bactericidal activity of the PMN isolated from blood and milk against Staphylococcus aureus was compared between groups of 6 primiparous and 6 multiparous healthy dairy cows during early lactation using bacteriological and PMN-pathogen interaction assays. Latex-stimulated luminol-amplified chemiluminescence (CL) and viability of these PMN were also investigated. The phagocytosis and killing of S. aureus by blood were remarkably higher than those of milk PMN. Similarly, the CL and viability in blood PMN were markedly higher than in milk PMN. Both in blood and in milk the phagocytosis of S. aureus by PMN in primiparous cows was substantially higher than in multiparous cows. The killing activity of blood PMN against S. aureus was 42.3+/-3.4% and 23.2+/-1.7% in primiparous and multiparous, respectively. Milk PMN killed only 20.7+/-2% S. aureus in primiparous and 10.2+/-1.3% in multiparous cows. Blood and milk PMN CL and milk PMN viability were significantly higher in primiparous cows. The pronounced reduction in phagocytic and bactericidal activity in blood and milk-resident PMN from multiparous cows, in part, resulted from the pronounced decrease of PMN viability and free radicals production capacity; this suggests that heifers' udders could be more protected against S. aureus, which remains to be tested in the field.

Analytical, biopharmaceutical and regulatory evaluation of topical testosterone preparations.

Testosterone-containing pharmaceutical products for topical use were obtained from the pharmacist or through the internet. The legal status of the different products obtained is discussed: some products through the internet were clearly a medicinal product according to the current definitions, while they are not registered as such. Assay and impurity profiles of each of the marketed samples were obtained using HPLC-UV and ESI-iontrap MS. The analytical results were evaluated relative to the reporting, identification and qualification thresholds as defined by the the International Conference on Harmonisation (ICH) and the European Pharmacopoeia (Ph. Eur.). Preparations with impurities above the qualification threshold were observed. Moreover, in vitro release profiles over an artificial membrane were obtained using a standardised cell in a paddle dissolution bath as well as in a static Franz diffusion cell, using phosphate buffered saline (PBS; pH 7.0) containing 5% bovine serum albumin (BSA) as dissolution or receptor fluid. This biopharmaceutical quality attribute differs significantly between the preparations tested. In conclusion, the equivalency of topical testosterone preparations is not assured, nor on their legal status, nor on their impurity profiling nor on their biopharmaceutical behaviour. This calls for an urgent trans-national product-class harmonisation approach.

Increase in Escherichia coli inoculum dose accelerates CD8+ T-cell trafficking in the primiparous bovine mammary gland.

Although migration of leukocytes into the mammary gland is pivotal for a cow's response against intramammary invading pathogens, the contribution of lymphocyte subsets to this response remains unclear. To investigate the dynamics of lymphocyte populations during Escherichia coli mastitis, T-lymphocyte subsets, CD4+/CD8+ ratio, CD21+ cells, and lymphoproliferation were studied in blood and milk of primiparous cows exposed to different quantities of bacteria. The cows were intramammarily inoculated with 10(4) cfu of E. coli (group A) and 10(6) cfu (group B). Compared with group A, a much greater number of lymphocytes migrated into the infected quarters at postinfection hour (PIH) 6 to 24 in group B, and the CD8+ cells were the first-recruited T cells in the milk. There was a significant decline in the CD4+/CD8+ ratios at PIH 6 to 24 in group B. The decrease of CD4+/CD8+ ratios at PIH 6 to 24 resulted mainly from greater CD8+ cell concentrations in milk. In contrast, at PIH 72, CD4+/CD8+ ratios increased about 2-fold in both groups. This increase was mainly due to the increase in CD4+ cell concentration. The increased concentration of CD4+ cells coincided with an increase in the CD21+ cell population in the milk. In blood, the increase of CD8+ cells appeared much faster in group B (PIH 6 and 12) than in group A. The results from lymphoproliferation also indicated a greater increase in the proliferative response in both blood and milk lymphocytes of group B. Our study demonstrates for the first time that an increase of E. coli inoculum dose accelerates the trafficking of CD8+ cells during initiation of E. coli mastitis, and these cells are the predominant T cells in milk during the early hours of bovine E. coli mastitis.

Effects of short dry periods on performance and metabolic status in Holstein dairy cows.

To evaluate effects of different dry period lengths on milk yield, milk composition, and energy balance of dairy cows, 122 multiparous and primiparous Holstein dairy cows were used in a completely randomized experimental design with 56-, 42-, and 35-d dry period lengths. Actual dry period lengths for respective treatments (TRT) were 56 +/- 5.1 d, 42 +/- 2.1 d, and 35 +/- 2.7 d. Overall, cows in the 42- and 56-d TRT gained more body condition than those in 35-d TRT during the dry period; however, postpartum body condition score did not change substantially among the TRT. Although from 3 to 210 DIM, differences were not detected in the milk yield of multiparous cows between the 35- and 56-d TRT, primiparous cows in the 35-d TRT produced less milk than those in 56-d TRT. In primiparous cows, the milk production at wk 9, 10, and 11 of lactation was lower in the 35-d compared with the 56-d TRT. Primiparous cows in the 35-d compared with the 56-d TRT produced less milk protein. In the 35-d TRT, serum triglyceride concentration was greater in primiparous cows than in multiparous cows during the peripartum period. Among primiparous cows, those in the 56-d TRT had greater concentrations of nonesterified fatty acids than those in the 35-d TRT during the peripartum period. No significant differences were observed in concentrations of serum glucose, insulin, and insulin-like growth factor-I during early lactation among TRT. There was also no difference among TRT for incidence of metabolic disorders. Thus, this study indicates that shortening the dry period to 35 d may be beneficial in multiparous and overconditioned cows, but not in primiparous cows.

Use of [123I]-2-iodo-L-phenylalanine as a tumor imaging agent in two dogs with synovial cell sarcoma.

[123I]-iodo-L-phenylalanine was successfully evaluated for gamma camera imaging in vivo in tumor-bearing athymic mice and in humans with brain tumors. Here, we report the use of this tracer in two dogs with synovial cell sarcoma of the tarsus. [123I]-iodo-L-phenylalanine was quantitatively prepared as a kit formulation using the Cu(1+) +-assisted nucleophilic exchange. Rapid [123I]-2-iodo-L-phenylalanine tumor accumulation was observed with good tumor to background contrast and rapid clearance in these two dogs. This radiopharmaceutical is a promising alternative tumor tracer to overcome the known limitations of 18F-fluorodeoxyglucose and, when labelled with radioiodine-131, has the potential to be used for therapeutic purposes.

Stability of the OECD model compound benzoic acid in receptor fluids of Franz diffusion cells.

The stability of benzoic acid, one of the model compounds recommended for skin absorption studies in the OECD guidelines, in Franz diffusion cell receptor fluids was studied. According to the results, addition of a preservative (sodium azide) to the solution is recommended for long-term skin permeation experiments.

Cumulative physiological events influence the inflammatory response of the bovine udder to Escherichia coli infections during the transition period.

A high proportion of intramammary coliform infections present at parturition develop disease characterized by severe inflammatory signs and sepsis during the first 60 to 70 d of lactation. In the lactating bovine mammary gland, the innate immune system plays a critical role in determining the outcome of these infections. Since the beginning of the 1990s, research has increased significantly on bovine mammary innate defense mechanisms in connection with the pathogenesis of coliform mastitis. Neutrophils are key effector cells of the innate immune response to intramammary infection, and their function is influenced by many physiological events that occur during the transition period. Opportunistic infections occur when the integrity of the host immune system is compromised by physical and physiological conditions that make the host more susceptible. The innate immune system of many periparturient cows is immunocompromised. It is unlikely that periparturient immunosuppression is the result of a single physiological factor; more likely, several entities act in concert, with profound effects on the function of many organ systems of the periparturient dairy cow. Their defense system is unable to modulate the complex network of innate immune responses, leading to incomplete resolution of the pathogen and the inflammatory reaction. During the last 30 yr, most efforts have been focused on neutrophil diapedesis, phagocytosis, and bacterial killing. How these functions modulate the clinical outcome of coliform mastitis, and how they can be influenced by hormones and metabolism has been the subject of intensive research and is the focus of this review. The afferent (sensing) arm of innate immunity, which enables host recognition of a diverse array of pathogens, is the subject of intense research interest and may contribute to the variable inflammatory response to intramammary infections during different stages of lactation. The development of novel interventions that modulate the inflammatory response or contribute to the elimination of the pathogen or both may offer therapeutic promise in the treatment of mastitis in periparturient cows.

Assessment of the solid-state composition of an active salicylanilide compound by FT-Raman spectroscopy.

A biologically active salicylanilide compound currently appears in three known solid-state forms: polymorph I (Pol I), polymorph II (Pol II) and the amorphous form (Amorph). The obtained FT-Raman spectra revealed several regions of interest (ROIs) qualitatively distinguishing the different forms, allowing samples with an unknown polymorphic composition to be quantitatively analysed by FT-Raman spectroscopy. The Markov-transformed peak areas of the Raman-bands in the ROIs from the samples were determined and compared with the transformed peak areas obtained for the reference solid-state forms. A constrainted linear regression model estimated the contribution of each reference to the different samples. The applicability of this approach was demonstrated by analysing commercially available batches.

Analysis of iodinated peptides by LC-DAD/ESI ion trap mass spectrometry.

The analysis of iodinated peptides resulting from chloramine-T (CAT), Iodo-Beads, Iodo-Gen and lactoperoxidase iodination reactions in the preparation of nanomole quantities 125I and 123I labelled tracers is described. Seven different model peptides were evaluated, varying in molecular weight from 294 (LY-dipeptide) to 2518 (obestatin containing 23 amino acid residues). Two different RP-C18 columns were used, each with a different gradient system based on aqueous formic acid and acetonitrile. Electrospray ionization (ESI) ion trap mass spectrometry was used for identification of the chromatographic eluting components of the reaction mixtures, while UV (DAD) served quantitative purposes. Non-, mono-, di-, tri- and tetra-iodinated peptides (respectively NIP, MIP, DIP, 3IP and 4IP) eluted in that order and were well separated from each other. An empirical model was derived. The applicability of this approach was demonstrated by the analysis of different reaction mixtures.

Technical note: validation of internal control genes for gene expression analysis in bovine polymorphonuclear leukocytes.

Analysis of gene expression is becoming more important in all areas of biological research to evaluate gene expression during physiological and pathological conditions (e.g., mastitis), not the least in the field of animal research. Presently, real-time gene expression analysis is considered to be the method of choice for accurate and sensitive quantification of mRNA transcripts. Because comparison of gene expression levels is frequently the aim of these experiments, there is a critical need to validate internal control genes. When studying gene expression in bovine polymorphonuclear leukocytes, special attention should be paid to this validation, because polymorphonuclear leukocytes are subjected to numerous physiological influences, depending on the stage of lactation. In this study, 8 commonly used reference genes (ACT, GAPD, H2A, TBP, HPRT1, SDHA, YWHAZ, and 18S rRNA) were evaluated in bovine polymorphonuclear leukocytes. The transcription levels of 6 reference genes were determined using real-time PCR. By geometrically averaging the expression levels of these genes, SDHA, YWHAZ, and 18S rRNA were selected as being the most stable genes for accurate normalization of real-time results of bovine polymorphonuclear leukocytes.

Mathematical model of the acute inflammatory response to Escherichia coli in intramammary challenge.

We constructed a mathematical model of the early response to Escherichia coli infection of the mammary gland and explored the roles and interactions between inflammatory cells and bacteria. The model incorporates 3 equations that describe the interactions among bacteria, milk somatic cells, and blood leukocyte densities. These 3 equations were fitted to cell densities observed during acute inflammatory responses in unvaccinated and vaccinated heifers inoculated with 10(4) or 10(6) cfu of E. coli. The rates computed for the cellular transit from the storage sites to the blood and from the blood to the milk were lower in cows receiving 10(4) cfu but increased at approximately 6 x 10(-6) and 30 x 10(-6) microL/cfu per h in nonvaccinated or vaccinated cows inoculated with 10(6) cfu, respectively. The cellular rates of bacterial killing were highest in unvaccinated cows ( approximately 400 x 10(-6) microL/cell per h) when compared with vaccinated cows (200 to 300 x 10(-6) microL/cell per h). A critical density of milk somatic cells at which bacteria density is constant was computed from the model at 2 x 10(6) cells/mL, and a one-way sensitivity analysis revealed that the changes in milk cellular densities were mostly sensitive to variations in the rate of bacterial killing and in the rate of production of inflammatory cells.