Inhibition of Adherence of Actinobacillus pleuropneumoniae to Porcine Respiratory Tract Cells by Monoclonal Antibodies Directed Against LPS and Partial Characterization of the LPS Receptors

Actinobacillus pleuropneumoniae is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia. We have previously identified the lipopolysaccharides (LPS) as the major adhesin of A. pleuropneumoniae involved in adherence to porcine respiratory tract cells. In the present study, adherence of A. pleuropneumoniae to porcine tracheal frozen sections was inhibited by homologous monovalent Fab fragments produced from monoclonal antibodies 5.1 G8F10 and 102-G02 directed, respectively, against the A. pleuropneumoniae serotype 1 or serotype 2 O-antigens. These results confirm the important role played by LPS in adherence of A. pleuropneumoniae and suggest that these adhesins might represent good vaccine candidates. We also investigated the presence of A. pleuropneumoniae receptors in tracheal cell preparations from piglets of four different breeds. Using Far-Western binding assays, we identified proteins recognized by whole cells of A. pleuropneumoniae reference strains for serotype 1 and 2, and local isolates belonging to the same serotypes, and also recognized by extracted LPS from both reference strains. We confirmed the proteinaceous nature of these LPS-binding molecules by their staining with Coomassie brilliant blue, sensitivity to proteinase K digestion, resistance to sodium m-periodate oxidation, and their inability to stain with glycoprotein-specific reagents. Four low-molecular-mass bands (14–17 kDa) seemed to correspond to histones. We also identified proteins at Mr 38,500 that could represent putative receptors for A. pleuropneumoniae LPS in swine respiratory tract cells. Received: 16 April 1999 / Accepted: 1 July 1999     

Priming of neutrophils by lipopolysaccharide for enhanced release of superoxide. Requirement for plasma but not for tumor necrosis factor-alpha

When human neutrophils are incubated with LPS, they become primed for enhanced release of O2- in response to stimulation by FMLP. We investigated two aspects of LPS priming: 1) whether priming depends on secretion of TNF-alpha by monocytes present in neutrophil preparations, and 2) whether plasma is required for priming. Using plasma-Percoll gradients, we isolated neutrophils that contained only 0.1% monocytes. At 37 degrees C, these neutrophils were significantly primed by LPS (100 ng/ml) within 30 min. In contrast, LPS-treated monocytes required 60 min to secrete significant neutrophil-priming activity, the major component of which was TNF-alpha. Further, antibody against TNF-alpha failed to inhibit priming of neutrophils by LPS at 15, 30, and 45 min, and inhibited only 15% at 60 min. The results suggested that TNF-alpha or other factors from monocytes were not essential for priming of neutrophils by LPS. Neutrophils that had been washed free of plasma by centrifugation through 50% Percoll responded only weakly to LPS with respect to priming for enhanced O2- release and increased expression of alkaline phosphatase activity on the cell surface. Priming of washed neutrophils could be restored by adding back plasma (0.1 to 1.0%). This effect of plasma was not blocked by heating the plasma to 56 degrees C but was blocked at 100 degrees C. LPS priming could be blocked by polymyxin B, even in the presence of plasma. Thus, priming required both LPS and plasma. Neutrophils incubated with LPS in the absence of plasma were not primed by subsequent addition of plasma, but were primed by addition of plasma and LPS. Culture supernatants from neutrophils incubated with 20 ng/ml LPS in the absence of plasma failed to prime fresh neutrophils, but supernatants from neutrophils incubated with LPS in the presence of 1% plasma were able to prime fresh neutrophils. These results implied that neutrophils inactivated LPS and that plasma protected LPS from inactivation. Nevertheless, such inactivated LPS retained the ability to gel Limulus lysate at 10 pg/ml, and the ability to prime monocytes at 100 pg/ml. Thus, plasma prevented a neutrophil-specific inactivation of LPS.     

Isolation and Characterization of LPS Mutants of Actinobacillus pleuropneumoniae Serotype 1

The major adhesin of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, has been previously identified as lipopolysaccharide (LPS). The purpose of the present study was to isolate and characterize A. pleuropneumoniae LPS mutants. Screening of LPS mutants was performed with colony dot and sensitivity to novobiocin. One mutant obtained by colony dot (F19) and one mutant selected for its increased sensitivity to novobiocin (33.1) did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 O-antigen compared with the parent strain. Mutants F19 and 33.1 did not express high-molecular-mass LPS bands as determined in silver-stained SDS-PAGE gels. The core-lipid A region of mutant 33.1 and of the parent strain had similar relative mobilities and reacted with serum from a pig experimentally infected with the serotype 1 reference strain of A. pleuropneumoniae, while the same region in mutant F19 showed faster migration and did not react with this serum. Use of piglet tracheal frozen sections indicated that mutant F19 was able to adhere to piglet trachea as well as the parent strain, while mutant 33.1 adhered [half as much as] the parent strain. Finally, both LPS mutants were markedly less virulent in mice than the parent strain. Taken together, our observations support the idea that LPS is an important virulence factor of A. pleuropneumoniae. Received: 23 December 1996 / Accepted: 19 February 1997     

Changes in the ascorbic acid levels of peritoneal lymphocytes and macrophages of mice with endotoxin-induced oxidative stress

Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock. The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg). The intake by cells of this antioxidant present in vitro at different concentrations was also studied. The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30h after LPS injection. The cells were obtained from the peritoneum at 2, 4, 12 and 24h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min. The hepatic AA levels were also studied at 0, 2, 4, 12 and 24h after LPS injection. The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h. The AA levels decreased after this time, in agreement with changes in the AA hepatic levels. The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection. Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro. This takes place mainly at 30–60 min of incubation in cells from controls and at 10 min in cells from treated mice 12–24 h after LPS injection. The incorporation decreased at these times of endotoxic shock, a few hours before death. In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration. These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it.     

Ecabet sodium attenuates reactive oxygen species produced by neutrophils after priming with bacterial lipopolysaccharides

The pathogenic roles of reactive oxygen species (ROS) have been implicated in ulcerative colitis (UC). The aim of this study was to examine the effects of ecabet sodium on ROS produced by human neutrophils, particularly after being primed by bacterial lipopolysaccharides (LPS). Neutrophils were isolated from six healthy volunteers. Each well of a 96‐well microplate received neutrophil suspension (1.0 × 10⁵ cells) and the plates were incubated at 37°C for 30 min with or without E. coli LPS (f.c. 0.001 ng/µL). Ecabet sodium (f.c. 0–5.0 mg/mL) was added before starting or after finishing the incubation. Neutrophils were stimulated by opsonized zymosan (OZ; 1.0 mg/mL) or calcium ionophore (A21837; 0.3 µmol/L) and luminol‐dependent chemiluminescence response was measured using a Lumi Box H‐1000. Ecabet sodium attenuated ROS production at a concentration of 5.0 mg/mL (p < 0.05) in LPS‐primed neutrophils. However, attenuating effects were not significantly different when ecabet sodium was added before or after the incubation with E. coli LPS. Ecabet sodium may have some attenuating effects on ROS produced by human neutrophils even after neutrophils are primed by bacterial LPS. These results may explain, in part, the therapeutic effects of ecabet sodium for UC. Copyright © 2003 John Wiley & Sons, Ltd.     

Electron microscopic observations of the phagocytosis and subsequent fate of Aeromonas salmonicida by Atlantic salmon neutrophils in vitro

Neutrophils isolated from the blood of Atlantic salmon (Salmo salar L.) were studied by electron microscopy at various time intervals after being incubated with opsonised and non-opsonised Aeromonas salmonicida strains, namely, the avirulent MT004 (A-layer negative) and the virulent MT423 (A-layer positive). After 15 min incubation with all four groups of bacteria (virulent, avirulent, opsonised or non-opsonised) a large number of neutrophils showed an elongated shape with the nucleus and all the organelles being located in one pole of the cell. Small vacuoles and clumping of glycogen granules were also observed. Neutrophils devoid of granules were noted after 30 min incubation, the majority containing engulfed bacteria. Degenerate neutrophils were also found in all the groups incubated with bacteria. Phagocytosis of bacteria was observed after 15 min incubation. The number of intracellular bacteria was very low, usually one or two per cell, although some neutrophils incubated with the opsonised avirulent strain MT004 contained a larger number of engulfed bacteria. Ingestion of bacteria was usually accompanied by the formation of phagocytic vacuoles containing an amorphous material of moderate electron-density as well as granule discharge into the vacuole. Both strains (MT004 and MT423), opsonised and non-opsonised, underwent morphological alterations after 3-7 h incubation suggesting that both A. salmonicida strains were killed by the neutrophils.     

Cooled neutrophils become deposited in the microcirculation after infusion: A potential mechanism for microvascular disruption following tissue hypothermia

1. We examined whether changes in rigidity and adhesiveness of neutrophils exposed to cooling and rewarming observed in vitro might impair microvascular perfusion in vivo. Neutrophils from donor rats were fluorescently (calcein-AM) or radioactively (Indium-111) labelled, incubated at 10 or 37 °C in vitro, and infused into recipients. Changes in transit rate and adhesive behaviour within post-capillary venules was quantified in m. extensor digitorum longus (EDL) using intravital microscopy, and tissue distribution determined.

2. There was an increased propensity of cooled cells to undergo adhesion following transfer into the recipient rat. However, cooling had no effect on median transit (354 μm s⁻¹) or rolling (14 μm s⁻¹) velocities during the first 5 min after infusion suggesting that cooling promotes adhesion, but does not delay passage through capillaries. Cooled neutrophils subsequently transformed to stationary adhesion. Their immobilisation was higher than for cells held at 37 °C (P<0.05), and once immobilised they remained firmly adherent to the vessel wall. Cooled, radiolabelled neutrophils showed tissue-specific accumulation after 3 min, but were cleared to the same extent as warmed cells by 20 min.

3. Our data suggest that cooling and rewarming of neutrophils impairs their ability to transit microvessels, reflecting changes in adhesive and mechanical properties observed in vitro, and may contribute to cold-associated circulatory pathology.

Chloroflexus aurantiacus secretes 3-hydroxypropionate,a possible intermediate in the assimilation of CO2 and acetate

Chloroflexus aurantiacus OK-70 fl secreted 3-hydroxypropionate (3HP) during phototrophic growth. The greatest amounts were secreted by cells grown on propionate (0.35 mM 3HP) while the lowest levels were found in autotrophically grown cultures (1.5 M). Large amounts of 2-fluoro,3-hydroxypropionate were formed by autotrophically grown cells exposed to fluoroacetate (FAc). Increased levels of 3HP were observed in these cultures when incubated with acctate. The secretion of 3HP was further stimulated by 0.2 mM KCN, an inhibitor of CO₂ fixation, but only in the presence of acetate. The pathway of 3HP formation was studied by using ¹³C-labelled substrates and NMR. The 3HP formed in the presence of C1-labelled acetate and FAc was labelled at C3 and somewhat less at C2 while with C2-labelled acetate as the tracer 3HP was labelled predominantly at C2. The carboxyl group was derived from CO₂. The 3HP formed by cells grown on propionate and ¹³CO₂ was labelled at all carbon atoms, the label content of C2 and C3 was about 25 and 65% of that of C1 respectively. It is suggested that 3HP is an intermediate in a pathway for acetate assimilation and in a new reductive carboxylic acid cycle for autotrophic CO₂ fixation.Abbreviations 3HP 3-hydroxypropionate - 2F3HP 2,fluoro,3-hydroxypropionate - FAc fluoroacetate - GC gas chromatography - MS mass spectrometry - NMR nuclear magnetic resonance     

Toll-like receptor 4 engagement inhibits adenosine 5'-monophosphate-activated protein kinase activation through a high mobility group box 1 protein-dependent mechanism

Despite the potent antiinflammatory effects of pharmacologically induced adenosine 5'-monophosphate kinase (AMPK) activation on Toll-like receptor 4 (TLR4)-induced cellular activation, there is little evidence that AMPK is activated during inflammatory conditions. In the present studies, we examined mechanisms by which TLR4 engagement may affect the ability of AMPK to become activated in neutrophils and macrophages under in vitro conditions and in the lungs during lipopolysaccharide (LPS)-induced acute lung injury. We found that incubation of neutrophils or macrophages with LPS diminished the ability of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) or hydrogen peroxide (H(2)O(2)) to activate AMPK. Although ratios of AMP to adenosine 5'-triphosphate (ATP) were increased in LPS-treated neutrophils and in the lungs of LPS exposed mice, a condition that should result in AMPK activation, no activation of AMPK was found. Immunocytochemistry and Western blot analysis revealed that nuclear to cytosolic translocation of the proinflammatory mediator high mobility group box 1 protein (HMGB1) correlated with inhibition of AMPK activation in LPS-stimulated macrophages. Moreover, while induced overexpression of HMGB1 resulted in inhibition of AMPK activation, Small interfering RNA (siRNA)-induced knockdown of HMGB1 was associated with enhanced activation of AMPK in macrophages incubated with AICAR. Increased interaction between liver kinase B1 (LKB1), an upstream activator of AMPK, and HMGB1 was found in LPS-stimulated macrophages and in the lungs of mice exposed to LPS. These results suggest that nuclear to cytoplasmic translocation of HMGB1 in TLR4-activated cells potentiates inflammatory responses by binding to LKB1, thereby inhibiting the antiinflammatory effects of AMPK activation.     

Lipopolysaccharide-induced stimulation of CD11b/CD18 expression on neutrophils. Evidence of specific receptor-based response and inhibition by lipid A-based antagonists.

Gram-negative bacterial septicemia is a common clinical syndrome resulting, in part, from the activation of phagocytic leukocytes by LPS. By using flow cytometry, we have characterized LPS-induced expression of the beta 2 integrin CD11b/CD18. After exposure to Salmonella minnesota R595 LPS, expression of neutrophil CD11b/CD18 is rapidly upregulated, beginning within 5 min and achieving a peak fluorescence (typically two- to threefold over base line) by 30 min. The increase in CD11b/CD18 expression was similar in kinetics and magnitude to that produced by FMLP, PMA, and human rTNF-alpha. Concentrations of LPS necessary to stimulate a response were as low as 1 ng/ml of R595 LPS; a maximal response was observed between 30 and 100 ng/ml. The upregulation of CD11b/CD18 due to LPS was not interrupted by protein synthesis inhibitors. A group of glucosamine disaccharide lipid A-like molecules: Rhodobacter sphaeroides lipid A, lipid IVA, KDO2IVA, and deacylated LPS were able to block the stimulatory effect of LPS. This inhibition was specific for the actions of LPS as stimulation of polymorphonuclear leukocytes (PMN) by FMLP, human rTNF alpha, PMA, and rewarming were not altered by the disaccharide inhibitors. PMN which were exposed to the specific disaccharide LPS antagonists and then washed, were refractory to stimulation by LPS. The monosaccharide lipid A precursor lipid X also blocked stimulation of neutrophils by LPS, although with a 100-fold reduction in potency. Unlike the disaccharide inhibitors, PMN exposed to lipid X were still responsive to LPS stimulation after washing. The PMN response to LPS was less sensitive in the absence of serum, although upregulation of CD11b/CD18 could still be seen using higher concentrations of LPS. Monoclonal antibody directed against CD14 (clone 3C10), also specifically inhibited LPS induced PMN CD11b/CD18 expression both in the presence and absence of serum. These findings support the hypothesis that LPS stimulates neutrophils by interacting with specific cellular receptors.     

Evaluation of PCR Based on Gene <Emphasis Type="Italic">apxIVA</Emphasis> Associated with 16S rDNA Sequencing for the Identification of <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> and Related Species

The pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for App diagnosis and characterization. However, in some isolates, conflicting results are found. The present work focus on the characterization of 29 isolates biochemically classified as A. pleuropneumoniae, collected from swine in herds with or without a clinical history of pleuropneumonia. Sixteen isolates were from healthy swine, initially classified as nonserotypable A. pleuropneumoniae; they displayed differences in the molecular characterization patterns of App (genes cpx and apxI, II, and III). Those bacteria that could not be serotyped were submitted to rDNA 16S sequencing. All 29 isolates were analyzed by PCR for the presence of the apxIVA gene. Thirteen isolates (45%) were confirmed to be A. pleuropneumoniae by PCR, nine being from diseased animals (31%) and four from healthy animals (14%) with conclusive serotyping. The rDNA 16S sequencing was used to classify the other 16 isolates in related species other than A. pleuropneumoniae, resulting in eleven A. minor, three A. porcinus, and two Pasteurella sp. Because of conflicting results between biochemical tests and rDNA 16S sequencing, the biochemical characterization was repeated, and the new results were in agreement with the rDNA 16S sequencing data. Biochemical characterization proved to be efficient for the majority of the A. pleuropneumoniae isolates. Nevertheless, conventional tests can render conflicting results, and other methodologies, such as amplification of A. pleuropneumoniae specific apxIVA gene and rDNA 16S sequencing, are very useful for improved classification. We also observed a great variety in rDNA 16S sequences from different A. minor isolates.     

malT knockout mutation invokes a stringent type gene-expression profile in Actinobacillus pleuropneumoniae in bronchoalveolar fluid

Background  

Actinobacillus pleuropneumoniae causes contagious pleuropneumonia, an economically important disease of commercially reared pigs throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses. Since bronchoalveolar fluid (BALF) contains many of the innate immune and other components found in the lungs, we examined the gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after exposure to concentrated BALF for 30 min.     

Natural and endotoxin-induced atresia of preantral and early antral follicles is characterized by DNA internucleosomal cleavage

Preantral follicles (PAF) and early antral follicles (EAF) were isolated from bovine ovaries and classified under a stereomicroscope as atretic or healthy. The atretic follicles were all considered as group I (in vivo atresia), whereas healthy follicles were assigned to five groups (group II, in vivo normal control; group III, in vitro normal control; group IV, in vitro induced atresia; groups V and VI, Lipopolysaccharide (LPS)-induced atresia in vitro). Group I and II follicles were immediately snap-frozen (−70°C) until DNA extraction, whereas group III–VI follicles were incubated (39°C, 5% CO₂, 95% air) for periods up to 72 hr under various conditions. Group III follicles were maintained in complete medium (M199, bovine calf serum, sodium pyruvate, epidermal growth factor, insulin, transferrin, sodium selenite, penicillin, streptomycin, and amphotericin), whereas group IV follicles were incubated in the same medium, but without serum. Group V and VI follicles were maintained in complete medium, but in the presence of LPS (10 or 50 μ/ml, respectively). Results showed that follicles incubated in the absence of serum and those exposed to both doses of LPS became atretic. DNA isolated from all atretic follicles showed fragmentation typical of that described for apoptosis; this was also confirmed by in situ DNA labeling and histology. Atretic follicles did not produce estradiol (P < 0.001), but progesterone values increased with follicle size (P < 0.001) and time of incubation (P < 0.001). We concluded that in the absence of serum or in the presence of LPS, follicles undergo atresia via apoptosis. © 1996 Wiley-Liss, Inc.     

Neutrophil responses to lipopolysaccharide. Effect of adherence on triggering and priming of the respiratory burst.

We studied neutrophil responses to LPS using three methodologic refinements: Teflon bags or serum-coated glass tubes that did not directly trigger neutrophils, LPS-free cytochrome c to measure O2- release, and heat-inactivated serum to inhibit inactivation of LPS by neutrophils. Neutrophils incubated in uncoated glass or plastic tubes adhered to the glass and released O2-, but were not primed for enhanced release of O2- in response to triggering by FMLP. Triggering by the glass or plastic surface did not occur if the neutrophils were stirred to prevent adherence. Adherence to glass or plastic and O2- release were not affected by a mAb (IB4) directed against the beta-chain of the leukocyte adhesion family of surface glycoproteins (CD11/CD18). Neutrophils incubated in glass or plastic did not show enhanced expression of alkaline phosphatase on their surface. When neutrophils were incubated in serum-coated glass tubes or in Teflon bags, there was no O2- release. However, adherence, expression of alkaline phosphatase, and release of O2- were triggered by adding 1 ng/ml LPS plus 1% serum, but not by either LPS or serum alone. In the presence of LPS and serum, O2- release was much higher when the cells were unstirred (adherent) rather than stirred. However, both unstirred and stirred cells expressed a similar elevated level of alkaline phosphatase. LPS-triggered O2- release and adherence were inhibited by antibody IB4. In contrast, priming by LPS for enhanced FMLP-triggered O2- release was greater in stirred cells than in unstirred cells. The antibody enhanced priming of unstirred neutrophils. These results suggested that uncoated glass or plastic triggered O2- release without involvement of leukocyte adhesion glycoproteins. However, neutrophils incubated with LPS and serum expressed alkaline phosphatase and IB4-inhibitable adherence glycoproteins that allowed neutrophils to interact with serum-coated glass or Teflon to trigger O2- release. Priming by LPS for enhanced response to FMLP was suppressed in adherent neutrophils, and this suppression was partly released by IB4. Thus, triggering and priming were reciprocally regulated by neutrophil glycoproteins interacting with surfaces.     

Electronic paramagnetic resonance investigation of the activity of Origanum vulgare L. essential oil on the Listeria monocytogenes membrane

Aims: To evaluate the effect of oregano essential oil on Listeria monocytogenes cytoplasmic membrane. Methods and Results: Nitroxide free‐radical Electron Paramagnetic Resonance was applied on L. monocytogenes after 30 min exposure to oregano essential oil concentrations ranging from 0 to 1·25%. The impact of essential oil on the number of viable cells was evaluated by plate count. Growth dynamics of survivors in BHI and TSB were evaluated by turbidometry. After exposure to essential oil concentrations up to 0·50%, the membrane fluidity was changed and its order increased. When L. monocytogenes was exposed to higher concentrations, membrane order parameters slightly returned to the values of untreated cells. However, when the cells were exposed to EO in the presence of sodium azide, which impairs energy metabolism, the membrane fluidity was progressively enhanced, even at the lowest EO concentration (0·25%). Microbiological analyses confirmed a progressive reduction of viable count, at increasing essential oil concentrations. Both in BHI and TSB, the Lag phase length increased in treated cells with respect to controls, suggesting a cell damage recovery. Conclusions: The combined approach including microbiological and EPR analyses provided relevant information on membrane modification and cell response to essential oils. Significance and Impact of the Study: EPR approach was demonstrated to be an effective and helpful tool to comprehend the modifications exerted by essential oil on the bacterial membrane.     

Cyclic nucleotide metabolism during lymphocyte transformation. II. Comparative studies in the nude Balb/c mouse.

Lymphocytes from the spleens of nude (athymic) Balb/c mice were incubated with LPS and examined for alterations in cyclic nucleotide metabolism. The cGMP concentration increased to 74% above control values within 20 min after the addition of LPS. cAMP concentration appeared to have two peaks, the first at 10 min to 80% above controls. The increase in cGMP can be accounted for by the inhibition of cGMP degradation by cAMP. These changes in cyclic nucleotide metabolism are similar to those observed in the “normal” Balb/c mouse. There are several differences between the cells from the two groups: (i) a delay in the cAMP and cGMP change and (ii) LPS enhanced cAMP inhibition of cGMP degradation in the nude mouse. These differences may be explained by differences in cell populations in the two mice, other than the obvious lack of T cells. Such differences were found on the basis of cell density distribution, where lymphocytes from normal Balb/c mice were found evenly dispersed between two dextran density gradient fractions, d = 1.057 and 1.066 g/ml; whereas, lymphocytes from nude mice were found almost entirely in the d = 1.066 g/ml fraction.     

Neutrophil-induced skeletal muscle damage: a calculated and controlled response following hindlimb unloading and reloading

Neutrophils phagocyte necrotic debris and release cytokines, enzymes, and oxidative factors. In the present study, we investigated the contribution of neutrophils to muscle injury, dysfunction, and recovery using an unloading and reloading model. Mice were submitted to 10 days of hindlimb unloading and were transiently depleted in neutrophils with anti-Ly6G/Ly6C antibody prior to reloading. Leukocyte accumulation and muscle function were assessed immunohistologically and functionally in vitro. In addition, soleus muscles submitted to unloading and reloading were incubated in vitro with LPS (100 microg/ml) to determine whether exogenous stimulus would activate neutrophil response and produce extensive muscle damage. Contractile properties were recorded every hour for 6 h, and muscles were subsequently incubated in procion orange to assess muscle damage. Neutrophil depletion affected neither the loss in muscle force nor the time of recovery in atrophied and reloaded soleus muscles. However, atrophied and reloaded soleus muscles that contained high concentration of neutrophils experienced a 20% greater loss in force than atrophied and reloaded soleus muscles depleted in neutrophils following in vitro incubation with LPS. Procion orange dye also confirmed that neutrophils induced a 2.5-fold increase in muscle membrane damage in the presence of LPS. These results show that neutrophil infiltration during modified mechanical loading is highly regulated and efficiently eliminated, with no significant muscle fiber injury unless the activation state of neutrophils is modified by the presence of LPS.     

Endotoxaemia in rats: role of leukocyte sequestration in rapid pulmonary nitric oxide synthase-2 expression.

Nitric oxide (NO), depending on the amount, time and source of generation may exert both, protective and deleterious actions during endotoxic acute lung injury (ALI). Evaluation of the expression and localization of NOS isoforms in the lung of lipopolysaccharide (LPS)-treated rats may contribute to understanding the role of NO in pathogenesis of ALI. Tissue samples (lung, heart, liver, kidney and spleen) as well as peripheral blood polymorphonuclear cells (PMNs) were collected from control male Wistar rats and LPS - treated animals, 15, 30, 60, 120 and 180 min after LPS injection (2 mg kg(-1) min(-1) for 10 minutes, i.v.). Levels of NOS-2 and NOS-3 mRNA and protein in tissues and PMNs were estimated by RT-PCR, Northern blotting and Western blotting. Additionally, myeloperoxidase (MPO) activity in tissue samples was assayed. NOS-3 mRNA as well as protein were detected in lungs of control animals; pulmonary NOS-3 expression was not influenced by LPS. The induction of NOS-2 mRNA in rat lungs and in PMNs isolated from peripheral blood was observed 15 minutes after LPS challenge. In contrast, increase of NOS-2 mRNA in the heart, kidneys, liver and spleen was observed 2-3 hours after LPS injection. In all tissues rise in NOS-2 mRNA was followed after 1-2 hours by increase of NOS-2 protein. Importantly, progressive leukocyte sequestration in the lung parenchyma that started as early as 15 min after LPS injection was revealed only in the lungs; in other organs no significant changes in MPO activity were detected up to 180 min after LPS injection. In conclusion, infusion of LPS caused much more rapid expression of NOS-2 in lungs as compared to the heart, kidneys, liver and spleen. Early induction of NOS-2 may depend on the LPS-stimulated rapid neutrophil sequestration within lung vasculature and fast induction of NOS-2 in sequestrated neutrophils.     

Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells

Lipopolysaccharide (LPS) is considered as an important virulence factor of Helicobacter pylori, and contributes to infection persistence and disease severity. ADP-l-glycero-d-manno-heptose-6-epimerase is an enzyme essential for LPS synthesis and understanding of its biochemistry is critical for drug development. We cloned one putative ortholog of Escherichia colirfaD, HP0859, from H. pylori 26695. Determination of the native molecular weight of the recombinant HP0859 protein suggests that the protein is likely a hexamer. NADP⁺, instead of NAD⁺, was proved to be the physiological cofactor for HP0859 protein. Circular dichroism spectrum analysis demonstrated that the secondary structure of this protein is significantly altered when the cofactor is removed. We also constructed an HP0859 knockout mutant and examined its phenotypic properties. The HP0859 knockout mutant exhibited a severe truncation of LPS, a decreased growth rate, and a higher susceptibility to novobiocin. Disruption of HP0859 also reduced the adhesive capacity of H. pylori to AGS cells, and the infected cells failed to display the classic hummingbird phenotype. Complementation of the HP0859 knockout mutation restored these phenotypes completely. In conclusion, we demonstrate that HP0859 codes for a protein essential for the LPS inner core biosynthesis in H. pylori and an intact LPS structure contributes to the adherence ability of this bacterium.     

Tracheal submucosal gland serous cells stimulated in vitro with adrenergic and cholinergic agonists A morphometric study

Summary A morphometric analysis was made of alterations in serous cell structure induced by adrenergic and cholinergic agonists. Ferret tracheal rings were exposed for 30 min in vitro to one of the following agonists: phenylephrine, terbutaline, or methacholine (all at 10^–5 M). Controls were incubated similarly in medium containing no drugs or medium containing both the agonist and an excess of the appropriate antagonist (phentolamine, propranolol or atropine, all at 10^–4 M).Electron microscopic observation and stereological analysis of the incubated samples revealed that the volume density of serous cell granules in controls (0.30 ± 0.02, mean ± SE, n = 4) was significantly reduced by phenylephrine (0.19 ± 0.03, n = 4) and methacholine (0.17 ± 0.01, n = 4), but not by terbutaline (0.27 ± 0.04, n = 4). The presence of antagonists in the medium prevented the observed changes (phenylephrine/phentolamine: 0.29 ± 0.03, n = 3 and methacholine/atropine: 0.33 ± 0.06, n = 3). In addition, the volume density of intracellular vacuoles in controls (0.02 ± 0.005, n = 4) was increased in response to methacholine stimulation (0.12 + 0.05, n = 4), but not in response to the other agonists. This effect was blocked by atropine (0.01 ± 0.00, n = 3).We conclude that serous-cell granules are discharged by both alpha-adrenergic and cholinergic, but not beta-adrenergic stimulation. In addition, cholinergic stimulation evokes the formation of intracellular vacuoles, a possible indication of active ion and water transport.