Granule exocytosis of granulysin and granzyme B as a potential key mechanism in vaccine-induced immunity in cattle against the nematode Ostertagia ostertagi.

Ostertagia ostertagi is considered one of the most economically important bovine parasites. As an alternative to anthelmintic treatment, an experimental host-protective vaccine was previously developed on the basis of ASP proteins derived from adult worms. Intramuscular injection of this vaccine, combined with QuilA as an adjuvant, significantly reduced fecal egg counts by 59%. However, the immunological mechanisms triggered by the vaccine are still unclear. Therefore, in this study, the differences in immune responses at the site of infection, i.e., the abomasal mucosa, between ASP-QuilA-vaccinated animals and QuilA-vaccinated control animals were investigated on a transcriptomic level by using a whole-genome bovine microarray combined with histological analysis. Sixty-nine genes were significantly impacted in animals protected by the vaccine, 48 of which were upregulated. A correlation study between the parasitological parameters and gene transcription levels showed that the transcription levels of two of the upregulated genes, those for granulysin (GNLY) and granzyme B (GZMB), were negatively correlated with cumulative fecal egg counts and total worm counts, respectively. Both genes were also positively correlated with each other and with another upregulated gene, that for the IgE receptor subunit (FCER1A). Surprisingly, these three genes were also correlated significantly with CMA1, which encodes a mast cell marker, and with counts of mast cells and cells previously described as globule leukocytes. Furthermore, immunohistochemical data showed that GNLY was present in the granules of globule leukocytes and that it was secreted in mucus. Overall, the results suggest a potential role for granule exocytosis by globule leukocytes, potentially IgE mediated, in vaccine-induced protection against O. ostertagi.

Analysis of cell hyperplasia and parietal cell dysfunction induced by Ostertagia ostertagi infection.

Infections in cattle with the gastric nematode Ostertagia ostertagi are associated with decreased acid secretion and profound physio-morphological changes of the gastric mucosa. The purpose of the current study was to investigate the mechanisms triggering these pathophysiological changes. O. ostertagi infection resulted in a marked cellular hyperplasia, which can be explained by increased transcriptional levels of signaling molecules related to the homeostasis of gastric epithelial cells such as HES1, WNT5A, FGF10, HB-EGF, AREG, ADAM10 and ADAM17. Intriguingly, histological analysis indicated that the rapid rise in the gastric pH, observed following the emergence of adult worms, cannot be explained by a loss of parietal cells, as a decrease in the number of parietal cells was only observed following a long term infection of several weeks, but is likely to be caused by an inhibition of parietal cell activity. To investigate whether this inhibition is caused by a direct effect of the parasites, parietal cells were co-cultured with parasite Excretory/Secretory products (ESP) and subsequently analyzed for acid production. The results indicate that adult ESP inhibited acid secretion, whereas ESP from the L4 larval stages did not alter parietal cell function. In addition, our data show that the inhibition of parietal cell activity could be mediated by a marked upregulation of inflammatory factors, which are partly induced by adult ESP in abomasal epithelial cells. In conclusion, this study shows that the emergence of adult O. ostertagi worms is associated with marked cellular changes that can be partly triggered by the worm's Excretory/secretory antigens.

Antielastin B-cell and T-cell immunity in patients with chronic obstructive pulmonary disease.

RATIONALE: Antielastin autoimmunity has been hypothesised to drive disease progression in chronic obstructive pulmonary disease (COPD). The proposed mechanism is currently disputed by conflicting data. The authors aimed to explore antibody responses against elastin in a large and extensively characterised COPD population and to assess elastin-specific peripheral T-cell reactivity in a representative subgroup. METHODS: Antielastin antibodies were analysed with indirect ELISA on the plasma of 320 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease 1-4) and 143 smoking controls. In a second group of 40 patients with COPD and smoking controls, T-cell responses against extracellular matrix (elastin, collagen I and collagen V) were determined with enzyme-linked immunosorbent spot (EliSpot) (interferon γ (IFNγ) and interleukin-2) on peripheral blood mononuclear cells and compared with the responses of 11 never-smoking controls. RESULTS: Antielastin antibody titres were not elevated in patients with COPD compared with smoking controls and even decreased significantly with increasing severity of COPD (p<0.001). Lower antielastin antibody titres were also found in a subgroup of patients with CT-proven emphysema. Elastin-specific INFγ-mediated T helper 1 responses could not be revealed in smoking subjects with and without COPD. Collagen I-mediated T-cell responses were also absent, which contrasted with a significant increased anticollagen V response in the smoking controls and patients with COPD compared with the never smokers (p=0.008). Collagen V-mediated T-cell responses could not discriminate between patients with COPD and smoking controls. CONCLUSION: A systemic immune response against elastin could not be identified in patients with COPD. By contrast, collagen V-mediated autoimmunity was increased in the subgroup of smokers and may potentially contribute to the pathogenesis of COPD.

Genome wide analysis of the bovine mucin genes and their gastrointestinal transcription profile.

BACKGROUND: Mucins are large glycoproteins implicated in protection of all mucosal surfaces. In humans and rodents, the mucin gene family has been well described and previous studies have investigated the distribution and function of mucins in the gastrointestinal (GI) tract. In contrast, little data is available on the mucin gene family in polygastric species, such as cattle. The aim of the current study was to identify all members of the bovine mucin family by genome mining and subsequently investigate the transcription pattern of these mucins in the GI tract. RESULTS: Nine bovine membrane-associated mucins (MUC1, MUC3A, MUC4, MUC12, MUC13, MUC15, MUC16, MUC20 and MUC21) and six secreted mucins (MUC2, MUC5AC, MUC5B, MUC6, MUC7 and MUC19) were identified in the bovine genome. No homologues could be identified for MUC3B, MUC8 and MUC17. In general, domain architecture of the membrane-associated mucins was found to be similar between humans and cattle, while the protein architecture of the gel-forming mucins appeared to be less conserved. Further analysis of the genomic organization indicated that the previously reported bovine submaxillary mucin (BSM) may be part of a larger gene encoding for MUC19. Analysis of the transcription profile showed that the secreted mucins were transcribed from the abomasum onwards, whereas the membrane associated mucins MUC1 and MUC20 were transcribed throughout the whole GI tract. In contrast to humans, MUC5B transcript was found in both the small and large intestine, but was absent in oesophageal tissue. CONCLUSIONS: This study provides the first characterization of the mucin gene family in cattle and their transcriptional regulation in the GI tract. The data presented in this paper will allow further studies of these proteins in the physiology of the GI tract in ruminants and their interactions with pathogens.

Characterization of the abomasal transcriptome for mechanisms of resistance to gastrointestinal nematodes in cattle.

The response of the abomasal transcriptome to gastrointestinal parasites was evaluated in parasite-susceptible and parasite-resistant Angus cattle using RNA-seq at a depth of 23.7 million sequences per sample. These cattle displayed distinctly separate resistance phenotypes as assessed by fecal egg counts. Approximately 65.3% of the 23,632 bovine genes were expressed in the fundic abomasum. Of these, 13,758 genes were expressed in all samples tested and likely represent core components of the bovine abomasal transcriptome. The gene (BT14427) with the most abundant transcript, accounting for 10.4% of sequences in the transcriptome, is located on chromosome 29 and has unknown functions. Additionally, PIGR (1.6%), Complement C3 (0.7%), and Immunoglobulin J chain (0.5%) were among the most abundant transcripts in the transcriptome. Among the 203 genes impacted, 64 were significantly over-expressed in resistant animals at a stringent cutoff (FDR < 5%). Among the 94 224 splice junctions identified, 133 were uniquely present: 90 were observed only in resistant animals, and 43 were present only in susceptible animals. Gene Ontology (GO) enrichment of the genes under study uncovered an association with lipid metabolism, which was confirmed by an independent pathway analysis. Several pathways, such as FXR/RXR activation, LXR/RXR activation, LPS/IL-1 mediated inhibition of RXR function, and arachidonic acid metabolism, were impacted in resistant animals, which are potentially involved in the development of parasite resistance in cattle. Our results provide insights into the development of host immunity to gastrointestinal nematode infection and will facilitate understanding of mechanism underlying host resistance.

Infection with the gastrointestinal nematode Ostertagia ostertagi in cattle affects mucus biosynthesis in the abomasum.

The mucus layer in the gastrointestinal (GI) tract is considered to be the first line of defense to the external environment. Alteration in mucus components has been reported to occur during intestinal nematode infection in ruminants, but the role of mucus in response to abomasal parasites remains largely unclear. The aim of the current study was to analyze the effects of an Ostertagia ostertagi infection on the abomasal mucus biosynthesis in cattle. Increased gene expression of MUC1, MUC6 and MUC20 was observed, while MUC5AC did not change during infection. Qualitative changes of mucins, related to sugar composition, were also observed. AB-PAS and HID-AB stainings highlighted a decrease in neutral and an increase in acidic mucins, throughout the infection. Several genes involved in mucin core structure synthesis, branching and oligomerization, such as GCNT3, GCNT4, A4GNT and protein disulphide isomerases were found to be upregulated. Increase in mucin fucosylation was observed using the lectin UEA-I and through the evaluation of fucosyltransferases gene expression levels. Finally, transcription levels of 2 trefoil factors, TFF1 and TFF3, which are co-expressed with mucins in the GI tract, were also found to be significantly upregulated in infected animals. Although the alterations in mucus biosynthesis started early during infection, the biggest effects were found when adult worms were present on the surface of the abomasal mucosa and are likely caused by the alterations in mucosal cell populations, characterized by hyperplasia of mucus secreting cells.

Noninvasive and invasive pulmonary function in mouse models of obstructive and restrictive respiratory diseases.

Pulmonary function analysis is an important tool in the evaluation of mouse respiratory disease models, but much controversy still exists on the validity of some tests. Most commonly used pulmonary function variables of humans are not routinely applied in mice, and the question of which pulmonary function is optimal for the monitoring of a particular disease model remains largely unanswered. Our study aimed to delineate the potential and restrictions of existing pulmonary function techniques in different respiratory disease models, and to determine some common variables between humans and mice. A noninvasive (unrestrained plethysmography) and two invasive pulmonary function devices (forced maneuvers system from Buxco Research Systems [Wilmington, NC] and forced oscillation technique from SCIREQ [Montreal, PQ, Canada]) were evaluated in well-established models of asthma (protein and chemical induced): a model of elastase-induced pulmonary emphysema, and a model of bleomycin-induced pulmonary fibrosis. In contrast to noninvasive tests, both invasive techniques were efficacious for the quantification of parenchymal disease via changes in functional residual capacity, total lung capacity, vital capacity, and compliance of the respiratory system. Airflow obstruction and airflow limitation at baseline were only present in emphysema, but could be significantly induced after methacholine challenge in mice with asthma, which correlated best with an increase of respiratory resistance. Invasive pulmonary functions allow distinction between respiratory diseases in mice by clinically relevant variables, and should become standard in the functional evaluation of pathological disease models.

A sentinel function for teat tissues in dairy cows: dominant innate immune response elements define early response to E. coli mastitis.

Escherichia coli intramammary infection elicits localized and systemic responses, some of which have been characterized in mammary secretory tissue. Our objective was to characterize gene expression patterns that become activated in different regions of the mammary gland during the acute phase of experimentally induced E. coli mastitis. Tissues evaluated were from Fürstenburg's rosette, teat cistern (TC), gland cistern (GC), and lobulo-alveolar (LA) regions of control and infected mammary glands, 12 and 24 h after bacterial (or control) infusions. The main networks activated by E. coli infection pertained to immune and inflammatory response, with marked induction of genes encoding proteins that function in chemotaxis and leukocyte activation and signaling. Genomic response at 12 h post-infection was greatest in tissues of the TC and GC. Only at 24 h post-infection did tissue from the LA region respond, at which time the response was the greatest of all regions. Similar genetic networks were impacted in all regions during early phases of intramammary infection, although regional differences throughout the gland were noted. Data support an important sentinel function for the teat, as these tissues responded rapidly and intensely, with production of cytokines and antimicrobial peptides.

Gait analysis in children treated by surgery followed by adjuvant therapy for posterior fossa tumors.

Children affected by posterior fossa tumors show signs and symptoms of neurological dysfunction, associated both to cancer itself and to cancer therapies. Abnormal gait and coordination difficulties are frequent presenting features. Radiation therapy represents the main adjuvant treatment for these patients. However it can produce significant neurologic injury, also manifested as gait disturbance months after treatment. We have analyzed temporo-spatial parameters of gait in sixteen children treated for posterior fossa tumors, mainly medulloblastomas. In all children we found a typical gait pattern of cerebellar ataxia. There were no significant differences between data obtained in children affected by severe neurological impairment and those having slight or even hardly recognizable physical signs. Although the number of patients studied is not large enough to allow definitive conclusions and gait analyses were performed after treatment, our results suggest that most children treated for posterior fossa tumors have gait disturbances and in some of them these are subclinical. Standard gait analysis laboratory methods can thus be a valuable tool for the careful assessment and follow-up of these patients.

Effects of intramammary infusion of cis-urocanic acid on mastitis-associated inflammation and tissue injury in dairy cows.

OBJECTIVE: To evaluate the effects of cis-urocanic acid (cis-UCA) on mammary gland (MG) inflammation and injury associated with Escherichia coli-induced mastitis in dairy cows. ANIMALS: 12 lactating dairy cows (36 MGs). PROCEDURES: At 7-week intervals, a different MG in each cow was experimentally inoculated with E coli. At 6-hour intervals from 6 to 36 hours after inoculation, the inoculated MG in each cow was infused with 40 mL of saline (0.9% NaCl) solution, 12.5mM cis-UCA, or 25mM cis-UCA (4 cows/group); ultimately, each cow received each treatment. Immediately prior to and at various time points after inoculation and treatment, milk samples were collected. Bacterial CFUs, somatic cell counts (SCCs), N-acetyl-beta-D-glucosaminidase (NAGase) and lactate dehydrogenase (LDH) activities, and concentrations of bovine serum albumin, tumor necrosis factor-alpha, and cis-UCA were quantified in each milk sample. Results-Compared with findings in saline solution-treated MGs, NAGase and LDH activities in milk samples from cis-UCA-treated MGs were lower. Cis-UCA had no effect on milk SCCs and milk concentrations of bovine serum albumin and tumor necrosis factor-alpha. Furthermore, cis-UCA had no adverse effect on bacterial clearance; CFUs of E coli in MGs treated with saline solution or cis-UCA were equivalent. CONCLUSIONS AND CLINICAL RELEVANCE: In cows, milk NAGase and LDH activities were both lower in E coli-infected MGs infused with cis-UCA than in those infused with saline solution, which suggests that cis-UCA reduced mastitis-associated tissue damage. Furthermore, these data indicated that therapeutic concentrations of cis-UCA in milk can be achieved via intramammary infusion.

The epidemiology of amyotrophic lateral sclerosis in Reggio Emilia, Italy.

Incidence and mortality rates of amyotrophic lateral sclerosis (ALS) vary between countries, and in some studies appear to increase over time. We performed a study to assess ALS incidence in a northern Italy area over a 10-year period. We identified the new cases of probable or definite ALS diagnosed among residents in Reggio Emilia province between 1996 and 2005 using several sources of data, such as death certificates, clinical records, hospital discharge registers and drug prescriptions. A total of 94 newly-diagnosed patients were identified. The average standardized incidence in the period was 2.0 and 1.0 cases/100,000/year, using the Italian and the world population, respectively, as reference. There was no variation in rates over time. Incidence was 1.3 in males and 0.8 in females. No cases were observed in patients under 35 years of age. Incidence increased after the age of 55 years, reaching a peak in the group aged 70-74 years and declining thereafter. We concluded that ALS incidence in this population was similar to that observed in other Italian regions and European countries, and no variation was identified during the study period.

Differential effects of alpha1-acid glycoprotein on bovine neutrophil respiratory burst activity and IL-8 production.

During bacterial-mediated diseases, neutrophils (PMNs) play a critical role in defending the host against invading pathogens. PMN production of reactive oxygen species (ROS) contributes to the bactericidal capabilities of these cells. ROS are produced intracellularly and can be released extracellularly. The aberrant extracellular release of ROS, however, has been reported to induce injury to host tissues during mastitis and other inflammatory-mediated diseases of cattle. The acute phase response, which occurs shortly after infection or tissue injury, is characterized by the induction of a large number of plasma proteins referred to as acute phase proteins (APP). alpha1-Acid glycoprotein (AGP) is an APP that increases in response to infection or injury in cattle and humans. The precise function of AGP is unknown, but it has been reported to possess anti-inflammatory properties. The objective of this study was to evaluate the effects of bovine AGP on PMN pro-inflammatory responses, including respiratory burst activity and cytokine production. Bovine AGP dose-dependently inhibited zymosan-induced PMN extracellular release of superoxide anion and hydrogen peroxide without affecting the capacity of PMN to engulf and kill Staphylococcus aureus. Moreover, AGP exerted its effect on ROS production regardless of whether PMNs were exposed to AGP prior to or after activation. In contrast to respiratory burst activity, AGP enhanced PMN production of IL-8. The precise mechanism by which AGP regulates PMN functions remains unknown, but data presented in this study suggest that AGP may have a complex role by differentially regulating PMN pro-inflammatory activities.

Differential alterations in the ability of bovine neutrophils to generate extracellular and intracellular reactive oxygen species during the periparturient period.

The periparturient period of a dairy cow is associated with increased incidence and/or severity of certain infectious diseases, including mastitis. It is believed that the heightened physiological demands of calving and initiation of milk production contribute to a state of immunosuppression during this period. Previous studies have indicated that neutrophil production of reactive oxygen species (ROS), which is a critical element of the host innate immune response to bacterial infection, is impaired in the 1-2week period following calving. However, whether there is comprehensive inhibition of ROS production or selective inhibition of particular ROS remains unknown. The present study provides evidence that neutrophils isolated from cows (n=20) after calving have an increased capacity to generate intracellular ROS and an impaired ability to release extracellular superoxide anion and hydrogen peroxide.

A peptide derived from human bactericidal/permeability-increasing protein (BPI) exerts bactericidal activity against Gram-negative bacterial isolates obtained from clinical cases of bovine mastitis.

Gram-negative bacteria are responsible for approximately one-third of the clinical cases of bovine mastitis and can elicit a life-threatening, systemic inflammatory response. Lipopolysaccharide (LPS) is a membrane component of Gram-negative bacteria and is largely responsible for evoking the inflammatory response. Antibiotic and anti-inflammatory therapy for treating Gram-negative infections remains suboptimal. Bactericidal/permeability-increasing protein (BPI) is a neutrophil-derived protein with antimicrobial and LPS-neutralizing properties. Select peptide derivatives of BPI are reported to retain these properties. The objective of this study was to evaluate the antimicrobial activity of a human BPI-derived synthetic peptide against clinical bovine mastitis isolates of Gram-negative bacteria. A hybrid peptide was synthesized corresponding to two regions of human BPI (amino acids 90-99 and 148-161), the former of which has bactericidal activity and the latter of which has LPS-neutralizing activity. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of this peptide against various genera of bacteria were determined using a broth microdilution assay. The MIC's were determined to be: 16-64 microg/ml against Escherichia coli; 32-128 microg/ml against Klebsiella pneumoniae and Enterobacter spp.; and 64-256 microg/ml against Pseudomonas aeruginosa. The MBC's were equivalent to or 1-fold greater than corresponding MIC's. The peptide had no growth inhibitory effect on Serratia marcescens. The antimicrobial activity of the peptide was retained in the presence of serum, but severely impaired in milk. Further functional evaluation of the peptide demonstrated its ability to completely neutralize LPS. Together, these data support additional investigations into the therapeutic application of BPI to the treatment of Gram-negative infections in cattle.

Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species.

During mastitis and other bacterial-mediated diseases of cattle, neutrophils play a critical role in the host innate immune response to infection. Neutrophils are among the earliest leukocytes recruited to the site of infection and contribute to host innate immune defenses through their ability to phagocytose and kill bacteria. The bactericidal activity of neutrophils is mediated, in part, through the generation of reactive oxygen species (ROS). Extracellular release of ROS can induce injury to host tissue as well, and aberrant release of ROS has been implicated in the pathogenesis of certain inflammatory-mediated diseases. Due to their essential role in bacterial clearance and implicated involvement in the pathogenesis of other diseases, there is much interest in the study of neutrophil-generated ROS. Several assays have been developed to measure ROS production, however, many of these have not been evaluated with bovine neutrophils. The objectives of the current study were to evaluate different assays capable of measuring bovine neutrophil ROS, and to compare the results of assays never previously tested with bovine neutrophils to those obtained from more well-established assays frequently used with these cells. Eight different assays were evaluated, including: luminol, isoluminol, and methyl cypridina luciferin analog (MCLA) chemiluminescence assays; Amplex Red, dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), and dihydrorhodamine 123 fluorescence assays; and the cytochrome c absorbance assay. The assays were evaluated in the context of their abilities to detect ROS produced in response to two agonists commonly used to induce neutrophil activation, phorbol 12-myristate, 13-acetate (PMA) and opsonized zymosan. Diphenyleneiodonium chloride, a NADPH oxidase inhibitor, was used to assess the specificity of the assays to detect ROS. The ability of these assays to discriminate between intra- and extracellular ROS and to specifically detect distinct ROS was evaluated using superoxide dismutase and catalase, which scavenge extracellular superoxide and hydrogen peroxide, respectively. With the exception of the DHE assay, all assays detected bovine neutrophil ROS generation elicited by PMA and zymosan. PMA, but not zymosan, was able to stimulate neutrophil generation of ROS at levels that were detectable with DHE. The MCLA chemiluminescence assay was the only assay that detected ROS produced in response to each of the lowest concentrations of PMA and zymosan tested. To our knowledge, this is the first study to evaluate DHE-, MCLA-, Amplex Red-, and isoluminol-based assays for the measurement of bovine neutrophil ROS, and the most comprehensive comparative study of ROS assays under similar experimental conditions.

Long-term nose-only cigarette smoke exposure induces emphysema and mild skeletal muscle dysfunction in mice.

Mouse models of chronic obstructive pulmonary disease (COPD) focus on airway inflammation and lung histology, but their use has been hampered by the lack of pulmonary function data in their assessment. Systemic effects such as muscle dysfunction are also poorly modeled in emphysematous mice. We aimed to develop a cigarette-smoke-induced emphysema mouse model in which serial lung function and muscular dysfunction could be assessed, allowing the disease to be monitored more appropriately. C57Bl6 mice were nose-only exposed to cigarette smoke or filtered air for 3-6 months. Lung function tests were repeated in the same mice after 3 and 6 months of cigarette smoke or air exposure and compared with lung histological changes. Contractile properties of skeletal muscles and muscle histology were also determined at similar time points in separate groups of mice. Serial lung function measurements documented hyperinflation after 3 and 6 months of cigarette smoke exposure, with a significant 31-37% increase in total lung capacity (TLC) and a significant 26-35% increase in compliance (Cchord) when compared with animals exposed to filtered air only (P<0.001 after 3 and after 6 months). These functional changes preceded the changes in mean linear intercept, which became only significant after 6 months of cigarette smoke exposure and which correlated very well with TLC (r=0.74, P=0.004) and Cchord (r=0.79, P=0.001). After 6 months of cigarette smoke exposure, a significant fiber-type shift from IIa to IIx/b was also observed in the soleus muscle (P<0.05), whereas a 20% reduction of force was present at high stimulation frequencies (80 Hz; P=0.09). The extensor digitorum longus (EDL) muscle was not affected by cigarette smoke exposure. These serial pulmonary function variables are sensitive outcomes to detect emphysema progression in a nose-only cigarette-smoke-exposed animal model of COPD. In this model, muscular changes became apparent only after 6 months, particularly in muscles with a mixed fiber-type composition.

Microarray analysis of the intestinal host response in Giardia duodenalis assemblage E infected calves.

Despite Giardia duodenalis being one of the most commonly found intestinal pathogens in humans and animals, little is known about the host-parasite interactions in its natural hosts. Therefore, the objective of this study was to investigate the intestinal response in calves following a G. duodenalis infection, using a bovine high-density oligo microarray to analyze global gene expression in the small intestine. The resulting microarray data suggested a decrease in inflammation, immune response, and immune cell migration in infected animals. These findings were examined in more detail by histological analyses combined with quantitative real-time PCR on a panel of cytokines. The transcription levels of IL-6, IL-8, IL-13, IL-17, and IFN-γ showed a trend of being downregulated in the jejunum of infected animals compared to the negative controls. No immune cell recruitment could be seen after infection, and no intestinal pathologies, such as villus shortening or increased levels of apoptosis. Possible regulators of this intestinal response are the nuclear peroxisome proliferator-activated receptors alpha (PPARα), and gamma (PPARγ) and the enzyme adenosine deaminase (ADA), all for which an upregulated expression was found in the microarray and qRT-PCR analyses.

Chronic rejection pathology after orthotopic lung transplantation in mice: the development of a murine BOS model and its drawbacks.

Almost all animal models for chronic rejection (CR) after lung transplantation (LTx) fail to resemble the human situation. It was our attempt to develop a representative model of CR in mice. Orthotopic LTx was performed in allografts receiving daily immunosuppression with steroids and cyclosporine. Controls included isografts and mice only undergoing thoracotomy (SHAM). Allografts were sacrificed 2, 4, 6, 8, 10 or 12 weeks after LTx. Pulmonary function was measured repeatedly in the 12w allografts, isografts and SHAM mice. Histologically, all allografts demonstrated acute rejection (AR) around the blood vessels and airways two weeks after LTx. This decreased to 50-75% up to 10 weeks and was absent after 12 weeks. Obliterative bronchiolitis (OB) lesions were observed in 25-50% of the mice from 4-12 weeks. Isografts and lungs of SHAM mice were normal after 12 weeks. Pulmonary function measurements showed a decline in FEV(0.1), TLC and compliance in the allografts postoperatively (2 weeks) with a slow recovery over time. After this initial decline, lung function of allografts increased more than in isografts and SHAM mice indicating that pulmonary function measurement is not a good tool to diagnose CR in a mouse. We conclude that a true model for CR, with clear OB lesions in about one third of the animals, but without a decline in lung function, is possible. This model is an important step forward in the development of an ideal model for CR which will open new perspectives in unraveling CR pathogenesis and exploring new treatment options.

Effect of an Ostertagia ostertagi infection on the transcriptional stability of housekeeping genes in the bovine abomasum.

Quantitative Real-Time PCR (qRT-PCR) is a widely used tool to study host responses against parasites. A crucial step in the gene quantification process is the normalization of the expression data against stable housekeeping genes (HKGs). However, in recent years, several reports have showed that the transcriptional levels of such HKGs can change dramatically, especially when cellular changes appear in the tissues investigated. The aim of the current study was to assess the variability of 11 putative HKGs in bovine abomasal tissue during an infection with the parasitic nematode Ostertagia ostertagi. Gene transcription levels of selected potential HKGs were measured by qRT-PCR and the expression stabilities evaluated using geNorm, NormFinder, and The Mann-Whitney-U test. The analysis showed that all the putative HKGs considered in this study, including the ones selected by geNorm and NormFinder, were found to be significantly upregulated in infected animals compared to the controls, clearly suggesting that none of these genes can actually be used as a HKG. The greatest alterations in gene transcription levels appeared at 24 dpi, which might be due to the dramatic changes in cell populations occurring in the abomasal tissue at this infection time point. To demonstrate the effect of normalizing target gene transcription levels with unstable HKGs, IL4 transcription levels were assessed using different normalization procedures. Our findings clearly showed that gene expression levels determined using HKGs differed significantly from those determined without normalization. The potential for HKG selection to impact candidate transcript levels is therefore an important consideration for studies of parasite infected tissue.

Repeated invasive lung function measurements in intubated mice: an approach for longitudinal lung research.

Invasive lung function measurements are useful tools to describe respiratory disease models in mice but only result in one time-point measurements because of tracheostomy. We explored if intubation may overcome the need for tracheostomy thereby allowing invasive lung function monitoring of individual mice over time. Repeated invasive lung function measurements with Scireq(©) - FlexiVent or Buxco(©) - Forced Pulmonary Maneuvers(®) were performed three times in BALB/c mice with intervals of 10 days. Each lung function assessment following intubation was compared with a similar measurement in age-matched tracheostomized mice, the golden standard in lung function measurements. Tracheostomy and intubation gave similar results for resistance, elastance and compliance of the whole respiratory system as assessed by Flexivent. Likewise, Forced Pulmonary Maneuvers used to measure lung volumes such as total lung capacity, functional residual capacity, forced expiratory volume in 0.1 s and forced vital capacity, resulted in identical outcomes for both airway approaches. No interaction was found between the procedures for any of the pulmonary function variables. The observed changes over time were rather related to animal growth than to repetitive intubation. Eighty percent of the animals survived three consecutive intubations, which were hampered by transient breathing difficulties, weight loss and neutrophilic bronchoalveolar lavage immediately postextubation. Repetitive invasive lung function measurements by intubation are feasible and reproducible in healthy mice and results are comparable to the standard method. This may open new perspectives for longitudinal research in animal models of respiratory diseases.