Anti-Inflammatory benefits of antibiotic-induced neutrophil apoptosis: tulathromycin induces caspase-3-dependent neutrophil programmed cell death and inhibits NF-kappaB signaling and CXCL8 transcription.

Clearance of apoptotic neutrophils is a central feature of the resolution of inflammation. Findings indicate that immuno-modulation and induction of neutrophil apoptosis by macrolide antibiotics generate anti-inflammatory benefits via mechanisms that remain obscure. Tulathromycin (TUL), a new antimicrobial agent for bovine respiratory disease, offers superior clinical efficacy for reasons not fully understood. The aim of this study was to identify the immuno-modulating effects of tulathromycin and, in this process, to establish tulathromycin as a new model for characterizing the novel anti-inflammatory properties of antibiotics. Bronchoalveolar lavage specimens were collected from Holstein calves 3 and 24 h postinfection, challenged intratracheally with live Mannheimia haemolytica (2 × 10(7) CFU), and treated with vehicle or tulathromycin (2.5 mg/kg body weight). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and enzyme-linked immunosorbent assay (ELISA) revealed that tulathromycin treatment significantly increased leukocyte apoptosis and reduced levels of proinflammatory leukotriene B(4) in M. haemolytica-challenged calves. In vitro, tulathromycin concentration dependently induced apoptosis in freshly isolated bovine neutrophils from healthy steers in a capase-3-dependent manner but failed to induce apoptosis in bovine fibroblasts, epithelial cells, and endothelial cells, as well as freshly isolated bovine blood monocytes and monocyte-derived macrophages. The proapoptotic effects of TUL were also, in part, drug specific; equimolar concentrations of penicillin G, oxytetracycline, and ceftiofur failed to cause apoptosis in bovine neutrophils. In addition, tulathromycin significantly reduced levels of phosphorylated IκBα, nuclear translocation of NF-κB p65, and mRNA levels of proinflammatory interleukin-8 in lipopolysaccharide (LPS)-stimulated bovine neutrophils. The findings illustrate novel mechanisms through which tulathromycin confers anti-inflammatory benefits.

Apoptosis induced by inhibition of intercellular contact.

The LIM 1863 colon carcinoma cell line grows as structural organoids of goblet and columnar cells around a central lumen and provides a model for the development of stem cells in the normal colon. The organoid structure can be disrupted by removal of calcium from the medium, resulting in a suspension of single cells. Upon readdition of calcium, the cells reform the organoid structure over a period of 24 h, and ultrastructural examination of the reforming cells reveals that this involves a complex process that we have termed clutching. To determine the adhesion molecules involved in organoid formation we attempted to block this process by single cell suspensions of LIM 1863 reseeded in the presence of monoclonal antibodies. An anti-integrin antibody directed against a conformational epitope on the alpha v subunit totally inhibited organoid reformation. As a consequence of this inhibition of cell contact the colon carcinoma cells rapidly underwent apoptosis. Investigations of the apoptotic pathway involved suggested an induction mechanism since the onset of apoptosis in the contact-inhibited cells showed specific increased synthesis of 68- and 72-kD proteins. In addition, immunoblotting of cytosolic and nuclear extracts of the cells revealed the rapid translocation of the tumor suppressor gene product, p53 to the cell nucleus upon induction of apoptosis. These results suggest that cell-cell adhesion may be a vital regulator of colon development overcome in tumor cells by loss of adhesion molecules or of functional p53 protein.

Immuno-modulating properties of Tulathromycin in porcine monocyte-derived macrophages infected with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus that grows in macrophages and causes acute pneumonia in pigs. PRRSV causes devastating losses to the porcine industry. However, due to its high antigenic variability and poorly understood immunopathogenesis, there is currently no effective vaccine or treatment to control PRRSV infection. The common occurrence of PRRSV infection with bacterial infections as well as its inflammatory-driven pathobiology raises the question of the value of antibiotics with immunomodulating properties for the treatment of the disease it causes. The macrolide antibiotic Tulathromycin (TUL) has been found to exhibit potent anti-inflammatory and immunomodulating properties in cattle and pigs. The aim of this study was to characterize the anti-viral and immunomodulating properties of TUL in PRRSV-infected porcine macrophages. Our findings indicate that blood monocyte-derived macrophages are readily infected by PRRSV and can be used as an effective cellular model to study PRRSV pathogenesis. TUL did not change intracellular or extracellular viral titers, not did it alter viral receptors (CD163 and CD169) expression on porcine macrophages. In contrast, TUL exhibited potent immunomodulating properties, which therefore occurred in the absence of any direct antiviral effects against PRRSV. TUL had an additive effect with PRRSV on the induction of macrophage apoptosis, and inhibited virus-induced necrosis. TUL significantly attenuated PRRSV-induced macrophage pro-inflammatory signaling (CXCL-8 and mitochondrial ROS production) and prevented PRRSV inhibition of non-opsonized and opsonized phagocytic function. Together, these data demonstrate that TUL inhibits PRRSV-induced inflammatory responses in porcine macrophages and protects against the phagocytic impairment caused by the virus. Research in live pigs is warranted to assess the potential clinical benefits of this antibiotic in the context of virally induced inflammation and tissue injury.

Pathophysiology of enteric infections with Giardia duodenalius.

Giardia is the most prevalent human intestinal parasitic protist in the world, and one of the most common parasite of companion animals and young livestock. Giardia is a major cause of diarrhea in children and in travelers. The host-microbial interactions that govern the outcome of infection remain incompletely understood. Findings available to date indicate that the infection causes diarrhea via a combination of intestinal malabsorption and hypersecretion. Malabsorption and maldigestion mainly result from a diffuse shortening of epithelial microvilli. This enterocytic injury is mediated by activated host T lymphocytes. Pathophysiological activation of lymphocytes is secondary to Giardia-induced disruption of epithelial tight junctions, which in turn increases intestinal permeability. Loss of epithelial barrier function is a result of Giardia-induced enterocyte apoptosis. Recent findings suggest that these effects may facilitate the development of chronic enteric disorders, including inflammatory bowel disease, irritable bowel syndrome, and allergies, via mechanisms that remain poorly understood. A newly discovered SGLT-1 glucose uptake-mediated host cytoprotective mechanism may represent an effective modulator of the epithelial apoptosis induced by this parasite, and, possibly, by other enteropathogens. A better understanding of the pathogenesis of giardiasis will shed light on new potential therapeutic targets.

Exacerbation of inflammation-associated colonic injury in rat through inhibition of cyclooxygenase-2.

Cyclooxygenase type 1 is constitutively expressed and accounts for synthesis of prostaglandins in the normal gastrointestinal tract. Cyclooxygenase-2 is expressed at sites of inflammation. Selective inhibitors of cyclooxygenase-2 have been suggested to spare gastrointestinal prostaglandin synthesis, and therefore lack the ulcerogenic effects associated with standard nonsteroidal antiinflammatory drugs. However, the effects of cyclooxygenase-2 inhibitors on inflamed gastrointestinal mucosa have not been examined. We examined cyclooxygenase-2 mRNA and protein expression before and after induction of colitis in the rat, the contribution of cyclooxygenase-2 to colonic prostaglandin synthesis during colitis and the effects of selective inhibitors of cyclooxygenase-2 on colonic injury in this model. Cyclooxygenase-2 mRNA expression increased three to sixfold during the period 24 h to 1 wk after induction of colitis, with marked increases in cyclooxygenase-2 protein expression in the lamina propria and muscularis of the colon during colitis. Cyclooxygenase-1 expression (mRNA and protein) was not affected by the induction of colitis. The prostaglandins produced during colitis were largely derived from cyclooxygenase-2. Treatment with selective cyclooxygenase-2 inhibitors resulted in exacerbation of colitis, with perforation occurring when the compounds were administered for a week. These studies demonstrate that suppression of cyclooxygenase-2 can result in exacerbation of inflammation-associated colonic injury.

The role of the epidermal growth factor receptor in microbial infections of the gastrointestinal tract.

The epidermal growth factor receptor (EGFr) is a transmembrane glycoprotein with an intrinsic tyrosine kinase. Ligand-binding to the EGFr activates cell signaling, phosphorylates protein kinases, and rearranges cytoskeletal proteins - responses that resemble those induced by microbial attachment to cell surfaces, a process known to be mediated by host cell receptors in a number of cases. This article critically reviews the possible role played by the EGFr in microbial colonization, and discusses how modulation of the EGF-EGFr axis may affect infection of the gastrointestinal tract.

Giardiasis in dairy calves: effects of fenbendazole treatment on intestinal structure and function.

Twelve Giardia duodenalis-infected Holstein dairy calves were allocated into a treatment (n=6) and placebo group (n=6) according to pre-study faecal cyst counts. Calves in the treatment group received an oral dose of 5 mg/kg fenbendazole once daily for 3 days, while placebo calves received a sterile saline solution. Calves were euthanised 7 days following the initiation of treatment and intestinal were collected and prepared for trophozoite quantitation, histology, electron microscopy, and disaccharidase assays. In all calves treated with fenbendazole, intestinal trophozoites were below detection limits, while in saline-treated calves, trophozoites were observed in all intestinal segments. Histologically, no significant difference was observed between treatment groups with respect to intestinal villus height or crypt depth. However, a significant decline in the number of intraepithelial lymphocytes (IEL) was observed in fenbendazole-treated calves when compared with placebo-treated calves in the duodenum (13.9+/-1.2 vs. 17.0+/-1.1 IEL/100 enterocytes) and jejunum (21.6+/-0.8 vs. 30.7+/-1.0 IEL/100 enterocytes). In addition, measurements from TEM micrographs demonstrated a significant increase in microvillus surface area in the jejunum of fenbendazole-treated calves compared with saline-treated calves (31.2+/-10.2 vs. 22.8+/-7.6 microm(2)). This increase in microvillus surface area was also associated with an increase in jejunal maltase activity in fenbendazole-treated calves compared with calves treated with saline. These results demonstrate that fenbendazole is an effective treatment for giardiasis in calves. fenbendazole treatment eliminated Giardia trophozoites from the small intestine of calves resulting in increased microvillus surface area and greater intestinal enzyme activity. This study also demonstrates that the pathogenesis of giardiasis in calves is similar to that observed in humans and laboratory animals, and provides further evidence that Giardia is a pathogen of cattle with potential economic importance.

Giardia duodenalis trophozoites isolated from a parrot (Cacatua galerita) colonize the small intestinal tracts of domestic kittens and lambs.

This study examines the ability of Giardia duodenalis trophozoites, isolated from a wild bird, to colonize the intestinal tracts of companion animals (kittens) and domestic ruminants (lambs). Trophozoites colonized the intestinal tracts of intraduodenally inoculated animals as demonstrated by increasing parasite burdens within the duodenum and jejunum and by fecal passage of cysts within 4 days post-inoculation. The pathogenesis of the trophozoites was further investigated in kittens. In these animals, infection significantly reduced jejunal brush border microvillous length and density, which resulted in a loss of overall epithelial brush border surface area. This injury was associated with the production of diarrhea in four of five infected kittens. These findings indicate that some bird species may carry G. duodenalis that represent a possible health threat to companion animals and livestock. Our results describe the first successful colonization of avian-derived G. duodenalis trophozoites in the small intestines of domestic kittens and lambs.

Effects of murine giardiasis on growth, intestinal morphology , and disaccharidase activity.

The aim of this study was to assess the effects of Giardia muris on host growth and food intake, small intestinal morphometrics, mucosal enzyme activities, and brush border ultrastructure. Weanling mice infected with 1,000 G. muris cysts were compared to control and pair-fed sham-treated animals. Infection with G. muris resulted in decreased food intake and retarded growth. In infected animals, villus atrophy was observed in the duodenum throughout the study period and in the jejunum on days 8 and 50. On day 30, whereas jejunal architecture returned to normal in infected animals, malnourished pair-fed animals exhibited a compensatory increase in villus height. Sucrase and maltase were depressed in infected animals on days 2-24. On day 8 jejunal disaccharidases in pair-fed animals were also decreased but to a lesser extent than in infected animals. On day 24, disaccharidase values for control and infected mice were similar, whereas values in pair-fed animals were increased. On day 8, jejunal microvilli were shorter in infected animals than in control and pair-fed animals. This brush border injury was present throughout the jejunum and was also observed in pair-fed animals, but to a lesser extent. These findings suggest that G. muris retards growth in weanling mice, results in small intestinal injury, and interferes with the compensatory response to malnutrition of the infected host. Villus atrophy and brush border enzyme deficiencies associated with the disease mainly occur in the duodenum and jejunum, where trophozoites are most numerous. In infected and in pair-fed animals, the decrease in jejunal disaccharidase activities correlated with a diffuse shortening of brush border microvilli.

Axenic isolation of viable Giardia muris trophozoites.

Large numbers of viable Giardia muris trophozoites were isolated from the duodenum of experimentally infected mice 6 days after inoculation with 1,000 G. muris cysts. A series of shaking, incubation, and washing steps in the presence of the broad-spectrum antibiotic piperacillin readily provided 4.9 +/- 1.5 x 10(5) G. muris trophozoites per mouse, free of detectable contaminant organisms. Anaerobic and microaerophilic culturing and scanning electron microscopy demonstrated axenic status and high purity of the isolates. The viability of trophozoites was 98 +/- 2%. Application of this technique should permit novel immunological and epidemiological analyses of G. muris infection and biochemical investigations of this protozoan parasite.

Giardia lamblia rearranges F-actin and alpha-actinin in human colonic and duodenal monolayers and reduces transepithelial electrical resistance.

The mechanisms of epithelial injury in giardiasis remain unknown. The effects of live Giardia lamblia on cellular G-actin, F-actin, alpha-actinin, and electrical resistance of human intestinal epithelial monolayers were investigated using SCBN and Caco2 cell lines grown on chamber slides or Transwell filter membranes. In separate experiments, some monolayers were also exposed to sonicated trophozoites, some to supernatant from live G. lamblia cultures, and some with or without the Ca2+ channel blocker verapamil. After 2, 24, or 48 hr of coincubation with G. lamblia, monolayers were assessed for cytoskeletal arrangement under fluorescence and confocal laser microscopy, and transepithelial electrical resistance was measured. Exposure to live G. lamblia trophozoites induced localized condensation of F-actin and loss of perijunctional alpha-actinin while G-actin remained unchanged. Confocal laser microscopy indicated that F-actin rearrangement was not affected by verapamil and was localized within the terminal web area. Coincubation of monolayers with G. lamblia lysates or with spent medium alone similarly rearranged F-actin. Verapamil alone did not alter F-actin. Electrical resistance of SCBN and Caco2 monolayers exposed to G. lamblia was significantly decreased versus controls regardless of whether live or lysed trophozoite samples were used. The results indicate that G. lamblia-induced epithelial injury is associated with F-actin and alpha-actinin rearrangements in the terminal web area via mechanisms independent of extracellular Ca2+. These alterations are associated with reduced transepithelial electrical resistance and are due at least in part to trophozoite products.

Mast cell hyperplasia and increased macromolecular uptake in an animal model of giardiasis.

Giardiasis has been associated with an increase in allergic disease following infection suggesting an alteration in mucosal immune function. Jejunal in vivo and in vitro macromolecular transport, epithelial permeability, and mucosal and connective tissue mast cell counts were examined in Mongolian gerbils (35-45 g) orogastrically inoculated (I) with a pathogenic strain of Giardia lamblia and compared to age- and weight-matched, sham-treated controls (C) 6 and 21 days postinoculation. Macromolecular uptake was significantly increased in infected tissue at 6 days both in vivo (I 134 +/- 19 vs. C 74 +/- 17 ng/hr; n = 8; P < 0.05) and in vitro (I 125 +/- 17 vs. C 67 +/- 8 ng/hr/cm; n = 12; P < 0.05). Macromolecular uptake did not differ between groups at 21 days. Infection had no effect on mucosal permeability of [51Cr]EDTA. Mucosal mast cell counts did not differ at 6 days but were significantly elevated in infected tissue at 21 days (I 33.3 +/- 6.8 vs. C 2.7 +/- 0.4 per high magnification field; n = 5; P < 0.01) as were connective tissue mast cell counts (I 1.7 +/- 0.2 vs. C 1.0 +/- 0.1 per high magnification field; n = 13; P < 0.005). The findings indicate that during the peak phase of giardiasis, jejunal active antigen uptake is increased leading to a delayed recruitment of mucosal and connective tissue mast cells. These changes may play a role in the increased incidence of hypersensitivity reactions associated with Giardia infection.

Tilmicosin does not inhibit interleukin-8 gene expression in the bovine lung experimentally infected with Mannheimia (Pasteurella) haemolytica.

The expression of the interleukin-8 (IL-8) gene was examined by in situ hybridization in lung tissues from calves experimentally infected with Mannheimia (Pasteurella) haemolytica and treated with tilmicosin. Interleukin-8 mRNA expression was detected in alveolar areas, particularly along interlobular septa, in the lumen, and in the epithelial cells of some bronchioles. In lesional lung tissues from animals that had received tilmicosin, we found large areas with limited inflammation. There was no staining for IL-8 mRNA in these areas. In contrast, in strongly inflamed areas, the same patterns and intensities of staining for IL-8 mRNA were detected in tilmicosin- and sham-treated animals. We conclude that tilmicosin does not affect the expression of IL-8 mRNA in tissue showing microscopic signs of inflammation. Together with previous reports, this supports the view that the pro-apoptotic properties of tilmicosin on neutrophils do not compromise the host defense mechanisms required to control the infection.

Anti-inflammatory benefits of tilmicosin in calves with Pasteurella haemolytica-infected lungs.

OBJECTIVES: To determine whether tilmicosin alters neutrophil infiltration or function, induces neutrophil apoptosis, and affects accumulation of leukotriene B4 (LTB4) or tumor necrosis factor-alpha (TNF-alpha) in lungs of calves experimentally infected with Pasteurella haemolytica. ANIMALS: 12 weight-ranked Holstein calves. PROCEDURE: Calves were given 25% propylene glycol vehicle (n = 5) or tilmicosin (10 mg/kg of body weight; n = 6) subcutaneously, 18 hours and 15 minutes before intratracheal infection with 2 x 10(8) P haemolytica organisms. Two unmanipulated calves served as controls in some experiments. Rectal temperatures were recorded 15 minutes before, and at 3-hour intervals after infection for 24 hours. Samples obtained from bronchoalveolar lavage performed 3 and 24 hours after infection were used to assess colonization by P haemolytica, and neutrophil infiltration. Neutrophil phagocytosis of P haemolytica, membrane leakage as determined by trypan blue exclusion, oxidative function as determined by nitro blue tetrazolium reduction, and apoptosis, using electron microscopy and DNA fragmentation ELISA, were determined. SOluble TNF-alpha and LTB4 were measured from supernatants from bronchoalveolar lavage samples, using ELISA. RESULTS: Treatment with tilmicosin resulted in significant (P < 0.05) clearance of P haemolytica and neutrophil apoptosis at 3 hours, and decreased concentration of LTB4 at 24 hours. Rectal temperatures, neutrophil infiltration, phagocytosis, oxidative functions, membrane leakage, and soluble TNF-alpha concentrations were not significantly affected by tilmicosin. CONCLUSION: Tilmicosin effectively controlled P haemolytica infection, induced neutrophil apoptosis, reduced pulmonary inflammation, and did not affect neutrophil infiltration or function. CLINICAL RELEVANCE: By inducing neutrophil apoptosis, tilmicosin prevents further amplification of inflammatory injury in P haemolytica-infected lungs.

Effects of giardiasis on production in a domestic ruminant (lamb) model.

OBJECTIVE: To examine the effects of giardiasis on production and carcass quality, using growing lambs as a domestic ruminant model. DESIGN: Randomized block. ANIMALS: Giardia-free lambs: 23 in infected group, 24 in control group. PROCEDURE: Six-week-old, specific-pathogen-free lambs were infected with Giardia trophozoites; control lambs received saline solution. Clinical signs of infection, body weight, and feed intake were determined for 10 weeks. Carcass weight and quality were determined at slaughter weight of 45 kg. RESULTS: Giardia infection persisted from weeks 7 to 16. For 5 weeks after challenge exposure, abnormal feces were more frequently observed in infected lambs. Giardia infection was associated with a decrease in rate of weight gain and impairment in feed efficiency. Time to reach slaughter weight was extended in infected lambs, and the carcass weight of Giardia-infected lambs was lower than that of control lambs. CONCLUSION: Giardiasis has a negative effect on domestic ruminant production. CLINICAL RELEVANCE: Giardiasis in domestic ruminants is an economically important disease, thus necessitating control or elimination of the infection.

In vitro anaerobic biofilms of human colonic microbiota.

The human gastrointestinal tract hosts a complex community of microorganisms that grow as biofilms on the intestinal mucosa. These bacterial communities are not well characterized, although they are known to play an important role in human health. This study aimed to develop a model for culturing biofilms (surface-adherent communities) of intestinal microbiota. The model utilizes adherent mucosal bacteria recovered from colonic biopsies to create multi-species biofilms. Culture on selective media and confocal microscopy indicated the biofilms were composed of a diverse community of bacteria. Molecular analyses confirmed that several phyla were represented in the model, and demonstrated stability of the community over 96 h when cultured in the device. This model is novel in its use of a multi-species community of mucosal bacteria grown in a biofilm mode of growth.

Immunopathology of giardiasis: the role of lymphocytes in intestinal epithelial injury and malfunction.

T lymphocyte-mediated pathogenesis is common to a variety of enteropathies, including giardiasis, cryptosporidiosis, bacterial enteritis, celiac's disease, food anaphylaxis, and Crohn's disease. In giardiasis as well as in these other disorders, a diffuse loss of microvillous brush border, combined or not with villus atrophy, is responsible for disaccharidase insufficiencies and malabsorption of electrolytes, nutrients, and water, which ultimately cause diarrheal symptoms. Other mucosal changes may include crypt hyperplasia and increased infiltration of intra-epithelial lymphocytes. Recent studies using models of giardiasis have shed new light on the immune regulation of these abnormalities. Indeed, experiments using an athymic mouse model of infection have found that these epithelial injuries were T cell-dependent. Findings from further research indicate that that the loss of brush border surface area, reduced disaccharidase activities, and increase crypt-villus ratios are mediated by CD8+ T cells, whereas both CD8+ and CD4+ small mesenteric lymph node T cells regulate the influx of intra-epithelial lymphocytes. Future investigations need to characterize the CD8+ T cell signaling cascades that ultimately lead to epithelial injury and malfunction in giardiasis and other malabsorptive disorders of the intestine.