The effect of levofloxacin on the lung microbiota of laboratory rats.

Aim: The aim of this study was to investigate the short-term effect of levofloxacin on the microbiota of healthy lungs. Material and methods: Male F344 rats received either no levofloxacin (n = 9), intravenous levofloxacin (n = 12), oral levofloxacin (n = 12), or subcutaneous levofloxacin (n = 14). Rats received a clinically applicable dose (5.56 mg/kg) of levofloxacin via the assigned delivery route once daily for three days. On day four, lung tissue was collected and the lung microbiota composition was investigated using 16S ribosomal RNA gene sequencing. Results: Untreated lungs showed a microbiota dominated by bacteria of the genera Serratia. After treatment with levofloxacin, bacteria of the genus Pantoea dominated the lung microbiota. This was observed for all routes of antibiotic administration, with a significant difference compared to no-antibiotic control group (PERMANOVA: P < 0.001; homogeneity of dispersions: P = 0.656). Conclusion: Our study is the first to demonstrate the effects of levofloxacin therapy on lung microbiota in laboratory rats. Levofloxacin treatment by any route of administration leads to profound changes in the rat lung microbiota, resulting in the predominance of bacteria belonging to the genus Pantoea. Further studies regarding the role of long-term application of broad spectrum antibiotics on induction of lung, allergic and autoimmune diseases are indicated.

Effect of gastric fluid aspiration on the lung microbiota of laboratory rats.

AIM OF THE STUDY: The pulmonary microbiota is important for both normal homeostasis and the progression of disease, and may be affected by aspiration of gastric fluid. The aim of this study was to investigate changes in the lung microbiota induced by aspiration of gastric fluid in a laboratory rat model. MATERIAL AND METHODS: Using the intratracheal application method, male rats received aspiration with 0.9% normal saline (n = 11); gastric fluid (n = 24) or sterilized (gamma-irradiated) gastric fluid (n = 12) once-weekly for four weeks. On the fifth week, the animals were sacrificed, and the microbiota of the lung was assessed by 16S ribosomal RNA gene sequencing. RESULTS: Lungs without aspiration and lungs after aspiration with normal saline had similar microbial compositions, dominated by bacteria of the genera Serratia, Ralstonia and Brucella. Evaluation of the microbiota following aspiration of gastric fluid revealed a much different profile that was dominated by bacteria from the genera Romboutsia and Turicibacter and largely independent of sterilization of the gastric fluid. CONCLUSION: In a laboratory rat model, aspiration with gastric fluid caused a substantial shift of the lung microbiota that could be characterized as a shift from Proteobacteria towards Firmicutes, possibly of enteric origin. Bacteria contained in the gastric fluid are not apparently responsible for this change.

Murine model of oropharyngeal gastric fluid aspiration-A new assessment method for intrapulmonary liquid distribution using digital pixel calculation.

AIM OF THE STUDY: The aim of this study was to investigate a new method for visualization and quantification of intrapulmonary liquid distribution after oropharyngeal gastric fluid aspiration in mice. MATERIAL AND METHODS: Eleven mice received oropharyngeal aspiration with a gastric fluid, India ink, and saline solution. Digital imaging and pixel calculation were used to analyze intrapulmonary fluid distribution selectively. RESULTS: Digital pixel analysis and orophanryngeal aspiration are both safe techniques in mice and deliver reproducible/valid results. Analysis revealed an average aspirate distribution of 86.8% of the total lung area. The proportional amount of the left lung was significantly greater than that of the right lung (P = 0.023). The lobe with the lowest mean distribution was the right lower lobe (79.2% ± 4.4%). CONCLUSION: Digital pixel calculation is a reliable method for quantitative, macroscopic evaluation of fluid distribution in the lung. This method is a useful tool for training purposes and it can be used to ensure interinvestigator reproducibility.

Got worms? Perinatal exposure to helminths prevents persistent immune sensitization and cognitive dysfunction induced by early-life infection.

The incidence of autoimmune and inflammatory diseases has risen dramatically in post-industrial societies. "Biome depletion" - loss of commensal microbial and multicellular organisms such as helminths (intestinal worms) that profoundly modulate the immune system - may contribute to these increases. Hyperimmune-associated disorders also affect the brain, especially neurodevelopment, and increasing evidence links early-life infection to cognitive and neurodevelopmental disorders. We have demonstrated previously that rats infected with bacteria as newborns display life-long vulnerabilities to cognitive dysfunction, a vulnerability that is specifically linked to long-term hypersensitivity of microglial cell function, the resident immune cells of the brain. Here, we demonstrate that helminth colonization of pregnant dams attenuated the exaggerated brain cytokine response of their offspring to bacterial infection, and that combined with post-weaning colonization of offspring with helminths (consistent with their mothers treatment) completely prevented enduring microglial sensitization and cognitive dysfunction in adulthood. Importantly, helminths had no overt impact on adaptive immune cell subsets, whereas exaggerated innate inflammatory responses in splenic macrophages were prevented. Finally, helminths altered the effect of neonatal infection on the gut microbiome; neonatal infection with Escherichia coli caused a shift from genera within the Actinobacteria and Tenericutes phyla to genera in the Bacteroidetes phylum in rats not colonized with helminths, but helminths attenuated this effect. In sum, these data point toward an inter-relatedness of various components of the biome, and suggest potential mechanisms by which this helminth might exert therapeutic benefits in the treatment of neuroinflammatory and cognitive disorders.

The role of soluble and insoluble gastric fluid components in the pathogenesis of obliterative bronchiolitis in rat lung allografts.

Repetitive gastric fluid aspirations have been shown to lead to obliterans bronchiolitis (OB), but the component or components of gastric fluid that are responsible are unknown. This study investigates the role of particulates and, separately, soluble material in gastric fluid during the development of OB. Whole gastric fluid (WGF) was collected from male Fischer 344 (F344) rats and separated by centrifugation into particle reduced gastric fluid (PRGF) and particulate components resuspended in normal saline (PNS). Orthotopic left lung transplants from male Wistar-Kyoto rats into F344 rats were performed using a modification of the nonsuture external cuff technique with prolonged cold ischemia. Rats were subjected to weekly aspiration of 0.5 ml/kg of WGF (n = 9), PRGF (n = 10), PNS (n = 9), or normal saline (control, NS; n = 9) for 8 weeks following transplantation. Lung allografts treated with WGF, PRGF, or PNS developed a significantly greater percentage of OB-like lesions compared with the control. No statistical difference was observed when comparing the fibrosis grades or the percentage of OB lesions of WGF, PRGF, and PNS groups, suggesting that both soluble and insoluble components of gastric fluid can promote the development of aspiration-induced OB and fibrosis in lung allografts.

Clearance of bile and trypsin in rat lungs following aspiration of human gastric fluid.

PURPOSE: In the clinical setting, there is no reliable tool for diagnosing gastric aspiration. A potential way of diagnosing gastric fluid aspiration entails bronchoalveolar lavage (BAL) with subsequent examination of the BAL fluid for gastric fluid components that are exogenous to the lungs. The objective of this study was to determine the longevity of the gastric fluid components bile and trypsin in the lung, in order to provide an estimate of the time frame in which assessment of these components in the BAL might effectively be used as a measure of aspiration. MATERIALS AND METHODS: Human gastric fluid (0.5 mg/kg) was infused in the right lung of intubated male Fischer 344 rats (n = 30). Animals were sacrificed at specified times following the experimentally induced aspiration, and bronchoalveolar lavage fluid (BALF) was collected. Bile concentrations were analyzed by an enzyme-linked chromatogenic method, and the concentration of trypsin was quantified using an ELISA. Data were analyzed using non-linear regression and a one-phase decay equation. RESULTS: In this experimental model, the half-life of bile was 9.3 hours (r(2) = 0.81), and the half-life of trypsin was 9.0 hours (r(2) = 0.68). CONCLUSIONS: The half-lives of bile and trypsin in the rodent aspiration model suggest that the ability to detect aspiration may be limited to a few days post-aspiration. If studies using rats are any indication, it may be most effective to collect BAL samples within the first 24 hours of suspected aspiration events in order to detect aspiration.

Association of age-dependent liver injury and fibrosis with immune cell populations.

BACKGROUND & AIMS: The liver's response to injury is fibrosis, and when chronic, cirrhosis. Age is a critical factor impacting many immune-mediated processes, potentially including the liver's wounding response to injury. METHODS: The effects of age on acute and chronic liver injury were evaluated using a carbon tetrachloride model in mice. Lymphocyte and macrophage populations were assessed by flow cytometry and immunohistochemical analysis. RESULTS: Acute liver injury was greater in 18-month-old (old) mice than in 9-month-old (middle-aged) mice as judged by changes in aminotransferases. Similarly, livers of 18-month-old mice had a significantly greater fibrogenic response to injury than did livers of 9-month-old mice after chronic injury (assessed by col1α1 mRNA expression, morphometric analysis and hydroxyproline measurement). Interestingly, livers from young mice (6 weeks old) also exhibited an increase in fibrogenesis compared to 9-month-old mice, albeit not to the same degree as in old mice. Consistent with a role for macrophages in fibrogenesis, the number of liver macrophages in young and 9-month-old mice increased, while in chronically injured livers of 18-month-old mice, the number of macrophages was reduced, and was less than in the livers of young and 9-month-old injured livers. CONCLUSIONS: Our data indicate that the fibrogenic response to injury varies substantially with age, and moreover that macrophage recruitment and dynamics may be an important component in differential age-associated fibrotic disease.

Chronic aspiration shifts the immune response from adaptive immunity to innate immunity in a murine model of asthma.

OBJECTIVE AND DESIGN: The hypothesis that aspiration of gastric fluid drives the anti-ovalbumin response toward a Th2 reaction even in animals not prone to Th2 responses was evaluated. SUBJECTS: Forty-eight male C57BL/6 mice were used. METHODS: Mice were sensitized and challenged with ovalbumin starting 5 weeks prior to the initiation of weekly aspirations of either gastric fluid or normal saline as a control. Weekly aspiration continued during the course of exposure to ovalbumin. TREATMENT: Aspiration consisted of 50 µl of gastric fluid with 50 µl of 0.9 % normal saline used as a control. Antigen exposure consisted of sensitization to ovalbumin via intraperitoneal injection on days 0 and 14 and challenge on day 21 with aerosolized antigen for 30 min. RESULTS: No evidence of a shift toward a Th2 response as a result of gastric fluid aspiration was seen in the Th1-prone strain utilized, although a profound down-regulation of a broad array of T cell-associated cytokines and chemokines and up-regulation of macrophage-associated markers was observed as a result of aspiration. CONCLUSIONS: These data provide support for the hypothesis that the clinical association between asthma and gastroesophageal reflux disease (GERD) does not involve an exacerbation of asthma by GERD-associated aspiration of gastric fluid, but may cause immune reactions unrelated to the asthma pathology.

Coagulopathy in α-galactosyl transferase knockout pulmonary xenotransplants.

BACKGROUND: After substantial progress on many fronts, one of the remaining barriers still opposing the clinical application of xenotransplantation is a disseminated intravascular coagulopathy (DIC) that is observed in the pre-clinical model of porcine-to-primate transplantation. The onset of DIC is particularly rapid in recipients of pulmonary xenografts, usually occurring within the first days or even hours of reperfusion. METHODS: In this study, we describe the results of two porcine-to-baboon transplants utilizing porcine lungs depleted of macrophages, deficient in the α-1,3-galactosyltransferase gene, and with the expression of human decay-accelerating factor, a complement regulatory protein. RESULTS: In both cases, evidence of DIC was observed within 48 h of reperfusion, with thrombocytopenia and increases in levels of thrombin-antithrombin complex evident in both cases. Depletion of fibrinogen was observed in one graft, whereas elevation of D-dimer levels was observed in the other. One graft, which showed focal lymphocytic infiltrates pre-operatively, failed within 3 h. CONCLUSIONS: The results indicate that further efforts to address the coagulopathy associated with pulmonary xenotransplantation are needed. Further, evidence suggests that resident porcine immune cells can play an important role in the coagulopathy associated with xenotransplantation.

Spontaneous bacterial cell lysis and biofilm formation in the colon of the Cape Dune mole-rat and the laboratory rabbit.

A wide range of techniques, including high-throughput DNA sequencing methods, have been applied to the evaluation of the normal intestinal flora. However, the inability to grow many of those species in culture imposes substantial constraints on the techniques used to evaluate this important community. The presence of biofilms in the normal gut adds further complexity to the issue. In this study, a flow cytometric analysis was used to separate intact bacterial cells, cell debris, and other particulate matter based on bacteria-specific staining and particle size. In addition, an analysis of biofilm formation using fluorescent light microscopy was conducted. Using these approaches, the ratio of bacterial cell debris to intact bacterial cells as a measure of spontaneous lysis of bacterial cells in the gut of the Cape dune mole-rat (Bathyergus suillus) and the laboratory rabbit (Oryctolagus cuniculus) was examined, and the degree of biofilm formation was semi-quantitatively assessed. The results suggest that the degree of spontaneous cell lysis was greater in the appendix than in the cecum in both the mole-rat and the rabbit. Further, the results point toward extensive epithelial-associated biofilm formation in the proximal mole-rat and rabbit large bowel, although the biofilms may be less structured than those found in laboratory rodents and in humans.

Evolution of bacteria in the human gut in response to changing environments: An invisible player in the game of health.

Several factors in Western society, including widespread use of antibiotics, chronic inflammation, and loss of complex eukaryotic symbionts such as helminths, have a dramatic impact on the ecosystem of the gut, affecting the microbiota hosted there. In addition, reductions in dietary fiber are profoundly impactful on the microbiota, causing extensive destruction of the niche space that supports the normally diverse microbial community in the gut. Abundant evidence now supports the view that, following dramatic alterations in the gut ecosystem, microorganisms undergo rapid change via Darwinian evolution. Such evolutionary change creates functionally distinct bacteria that may potentially have properties of pathogens but yet are difficult to distinguish from their benign predecessors.

Short-lived alpha-helical intermediates in the folding of beta-sheet proteins.

Several lines of evidence point strongly toward the importance of highly alpha-helical intermediates in the folding of all globular proteins, regardless of their native structure. However, experimental refolding studies demonstrate no observable alpha-helical intermediate during refolding of some beta-sheet proteins and have dampened enthusiasm for this model of protein folding. In this study, beta-sheet proteins were hypothesized to have potential to form amphiphilic helices at a period of <3.6 residues/turn that matches or exceeds the potential at 3.6 residues/turn. Hypothetically, such potential is the basis for an effective and unidirectional mechanism by which highly alpha-helical intermediates might be rapidly disassembled during folding and potentially accounts for the difficulty in detecting highly alpha-helical intermediates during the folding of some proteins. The presence of this potential was confirmed, indicating that a model entailing ubiquitous formation of alpha-helical intermediates during the folding of globular proteins predicts previously unrecognized features of primary structure. Further, the folding of fatty acid binding protein, a predominantly beta-sheet protein that exhibits no apparent highly alpha-helical intermediate during folding, was dramatically accelerated by 2,2,2-trifluoroethanol, a solvent that stabilizes alpha-helical structure. This observation suggests that formation of an alpha-helix can be a rate-limiting step during folding of a predominantly beta-sheet protein and further supports the role of highly alpha-helical intermediates in the folding of all globular proteins.

Adaptation in a mouse colony monoassociated with Escherichia coli K-12 for more than 1,000 days.

Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo, and an increased tendency for spontaneous lysis in vitro. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.

Gastroesophageal reflux-associated aspiration alters the immune response in asthma.

BACKGROUND: A large number of studies point toward chronic aspiration associated with gastroesophageal reflux disease (GERD) as an important factor involved in the development of asthma, the incidence of which has increased dramatically in industrially developed countries. Recent work suggests that medical intervention aimed at acid blockade is not sufficient to relieve the effects of chronic aspiration on asthma pathology, leaving surgical treatment of the disease as one of the few remaining options. This study examined the effect of chronic aspiration on the airway-associated immune response to allergens using a model of experimentally induced airway hypersensitivity in Balb/c mice. METHODS: The mice received aspiration of gastric fluid on days 1, 8, 15, 22, 29, 36, 43, and 50 and were sensitized to ovalbumin by intraperitoneal (IP) injection on days 33 and 47, challenged with aerosolized ovalbumin on day 54, and killed on day 56. Control mice received sham gastric fluid aspirations, sham induction of airway hypersensitivity, or both. RESULTS: Chronic aspiration of 50 microl murine gastric fluid once per week for 8 weeks had a profound effect on the immune system in the lung, with upregulation of the macrophage/monocyte-associated cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-12 (IL-12) and profound downregulation of a broad array of T-cell-associated cytokines including interleukins 2, 4, 5, 6, 10, 13, and 23, as well as interferon-gamma. The aspiration-induced depression of IL-5 production in particular was found only in mice with airway hypersensitivity and not in control mice without airway hypersensitivity. CONCLUSIONS: The results indicate that chronic aspiration of gastric fluid has a profound effect on the nature of the allergic response to aerosolized allergens, suggesting that the aspiration may be an important factor affecting the pathogenesis of asthma.

Early Immune Response to Acute Gastric Fluid Aspiration in a Rat Model of Lung Transplantation.

OBJECTIVES: Chronic aspiration of gastric fluid contents can decrease long-term survival of pulmonary transplants due to development of obliterative bronchiolitis. However, little is known about the early immune response and the cascade of events involved in the development of obliterative bronchiolitis. MATERIALS AND METHODS: We utilized a rat orthotopic pulmonary transplant model and a single aspiration of either gastric fluid or normal saline to investigate the histologic, cellular, and cytokine changes associated with an acute gastric fluid aspiration event compared with normal saline at 2 and 10 days after aspiration. RESULTS: Our observations included a decrease in pulmonary compliance and increased airway inflammation and acute rejection of the transplanted lung, as well as increases in macrophages, granulocytes, and proinflammatory cytokines such as interleukin 1ß, transforming growth factor ß1 and ß2, and tumor necrosis factor α in bronchoalveolar lavage fluid from the transplanted lung of gastric fluid-aspirated rats compared with normal saline-aspirated rats. CONCLUSIONS: The acute inflammatory response observed in the present study is consistent with changes found in chronic models of aspiration-associated injury and suggests a potentially important role for mast cells in the development of obliterative bronchiolitis.

A mole rat's gut microbiota suggests selective influence of diet on microbial niche space and evolution.

IMPACT STATEMENT: The composition of the microbiota is of critical importance for health and disease, and is receiving increased scientific and medical scrutiny. Of particular interest is the role of changing diets as a function of agriculture and, perhaps to an even greater extent, modern food processing. To probe the connection between diet and the gut's microbial community, the microbiota from a mole rat, a rodent with a relatively unusual diet, was analyzed in detail, and the microbes found were compared with previously identified organisms. The results show evidence of an adaptive radiation of some microbial clades, but relative stability in others. This suggests that the microbiota, like the genome, carries with it housekeeping components as well as other components which can evolve rapidly when the environment changes. This study provides a very broad view of the niche space in the gut and how factors such as diet might influence that niche space.

Immunization enhances the natural antibody repertoire.

The role of immunization in the production of antibodies directed against immunogens is widely appreciated in laboratory animals and in humans. However, the role of immunization in the development of "natural antibodies" has not been investigated. Natural antibodies are those antibodies present without known history of infection or immunization, and react to a wide range of targets, including "cryptic" self-antigens that are exposed upon cell death. In this study, the ability of immunization to elicit the production of natural antibodies in laboratory rats was evaluated. Laboratory rats were immunized with a series of injections using peanut extracts (a common allergen), a high molecular weight protein conjugated to hapten (FITC-KLH), and a carbohydrate conjugated to hapten (DNP-Ficall). Significantly greater binding of antibodies from immunized animals compared to controls was observed to numerous autologous organ extracts (brain, kidney, liver, lung, prostate, and spleen) for both IgM and IgG, although the effect was more pronounced for IgM. These studies suggest that immunization may have at least one unforeseen benefit, enhancing networks of natural antibodies that may be important in such processes as wound repair and tumor surveillance. Such enhancement of natural antibody function may be particularly important in Western society, where decreased exposure to the environment may be associated with a weakened natural antibody repertoire.

The role of oxidative stress, inflammation and acetaminophen exposure from birth to early childhood in the induction of autism.

The wide range of factors associated with the induction of autism is invariably linked with either inflammation or oxidative stress, and sometimes both. The use of acetaminophen in babies and young children may be much more strongly associated with autism than its use during pregnancy, perhaps because of well-known deficiencies in the metabolic breakdown of pharmaceuticals during early development. Thus, one explanation for the increased prevalence of autism is that increased exposure to acetaminophen, exacerbated by inflammation and oxidative stress, is neurotoxic in babies and small children. This view mandates extreme urgency in probing the long-term effects of acetaminophen use in babies and the possibility that many cases of infantile autism may actually be induced by acetaminophen exposure shortly after birth.

Production and Use of Hymenolepis diminuta Cysticercoids as Anti-Inflammatory Therapeutics.

Helminthic therapy has shown considerable promise as a means of alleviating some inflammatory diseases that have proven resistant to pharmaceutical intervention. However, research in the field has been limited by a lack of availability to clinician scientists of a helminth that is relatively benign, non-communicable, affordable, and effectively treats disease. Previous socio-medical studies have found that some individuals self-treating with helminths to alleviate various diseases are using the rat tapeworm (cysticercoid developmental stage of Hymenolepis diminuta; HDC). In this study, we describe the production and use of HDCs in a manner that is based on reports from individuals self-treating with helminths, individuals producing helminths for self-treatment, and physicians monitoring patients that are self-treating. The helminth may fit the criteria needed by clinical scientists for clinical trials, and the methodology is apparently feasible for any medical center to reproduce. It is hoped that future clinical trials using this organism may shed light on the potential for helminthic therapy to alleviate inflammatory diseases. Further, it is hoped that studies with HDCs may provide a stepping stone toward population-wide restoration of the biota of the human body, potentially reversing the inflammatory consequences of biota depletion that currently affect Western society.

Effect of an anti-C5a monoclonal antibody indicates a prominent role for anaphylatoxin in pulmonary xenograft dysfunction.

BACKGROUND: In contrast to renal or cardiac xenografts, the inhibition of complement using cobra venom factor (CVF) accelerates pulmonary xenograft failure. By activating C3/C5 convertase, CVF depletes complement while additionally generating C5a and other anaphylatoxins, to which pulmonary xenografts may be uniquely susceptible. The current study investigates the role of C5a in pulmonary xenograft failure in baboons. METHODS: Left orthotopic pulmonary xenografts using swine lungs expressing human CD46 were performed in baboons receiving: I) no other treatment (n=4), II) immunodepletion (n=5), and III) immunodepletion plus a single dose of mouse anti-human C5a monoclonal antibody (anti-C5a, 0.6 mg/kg administered intravenously) (n=3). The extent to which anti-C5a inhibits baboon C5a was assessed in vitro using a hemolytic reaction involving baboon serum and porcine red blood cells and by ELISA. RESULTS: Baboons in Group III exhibited significantly prolonged xenograft survival (mean=722+/-121 min, P=0.02) compared to baboons in Group I (mean=202+/-24 min) and Group II (mean=276+/-79 min). Furthermore, baboons in Groups I and II experienced pronounced hemodynamic compromise requiring inotropic support whereas those in Group III remained hemodynamically stable throughout experimentation without the need for additional pharmacologic intervention. CONCLUSIONS: These findings indicate that C5a exacerbates pulmonary xenograft injury and compromises recipient hemodynamic status. Moreover, blockade of anaphylatoxins, such as C5a, offers a promising approach for future investigations aimed at preventing pulmonary xenograft injury in baboons.