A novel murine anti-human Fas mAb which mitigates lymphadenopathy without hepatotoxicity

Defects in Fas-mediated apoptosis are implicated in autoimmune diseases including rheumatoid arthritis (RA). Although induction of Fas-mediated apoptosis could have therapeutic effects on these diseases, it might cause deleterious effects in liver as Fas ligand or an agonistic anti-murine Fas antibody Jo2 causes severe hepatic injury in mice. We report here on the interesting characteristics of the newly obtained anti-Fas mAb, HFE7A, which cross-reacts with the Fas molecules of various species ranging from human to mouse and mitigates autoimmune symptoms without hepatotoxicity in mice. The administration of HFE7A to mice induced apoptosis in the thymocytes, although administration of HFE7A to mice or to marmosets did not induce any sign of hepatitis. The effect of HFE7A on liver is different from that of anti-murine Fas antibody Jo2, which causes acute and lethal hepatic injury to mice. Administration of HFE7A reduced lymphadenopathy and abnormal T cells in MRL-gld/gld mice. HFE7A induced apoptosis in synovial cells prepared from RA patients. Surprisingly, HFE7A protected mice from fulminant hepatitis induced by Jo2. Therefore, HFE7A is a potential therapeutic antibody not only for autoimmune diseases including RA but also for fulminant hepatitis.     

Ultrastructural changes in the air–blood barrier in mice after intratracheal instillations of Asian sand dust and gold nanoparticles

The purpose of this study was to investigate a possible translocation pathway of intratracheally instilled gold nanoparticles after the induction of acute pulmonary injury by Asian sand dust. ICR mice were intratracheally instilled with 800 μg Asian sand particles (CJ-2 particles) 24 h before instillation of 50-nm gold nanoparticles. Lungs from mice treated with Asian sand particles and gold nanoparticles showed an acute focal inflammation with an increased expression of proinflammatory cytokines (IL-6 and TNF-α) and oxidative stress markers (Cu/Zn SOD and iNOS) in alveolar macrophages, type I alveolar epithelial cells, and endothelial cells at the alveolar walls. Electron microscopy revealed a destruction of the alveolar walls with an increased number of endocytic vesicles in the cytoplasm of both type I epithelial cells and endothelial cells; gold nanoparticles were demonstrated in these endocytic vesicles. These findings suggest that translocation of the exposed nanoparticles may be enhanced in the lung tissues with acute inflammatory changes.     

Amelioration of systemic autoimmune disease by the stimulation of apoptosis-promoting receptor Fas with anti-Fas mAb

Fas antigen (Fas) is a cell surface receptor molecule that mediates apoptosis-inducing signals into activated and/or autoreactive peripheral T and B cells by stimulation with Fas ligand or agonistic anti-Fas mAb. The i.p. administration of the hamster anti-mouse Fas mAb RK-8, which induced apoptosis both in vivo and in vitro, did not kill adult mice, whereas those given another hamster anti-mouse Fas mAb Jo2 rapidly die of fulminant hepatitis with hemorrhage. Here, we report that MRL-gld/gld mice thoroughly recovered and/or were prevented from glomerulonephritis, arthritis, sialadenitis, vasculitis and lymphoadenopathy after receiving a single administration of the agonistic anti-mouse Fas mAb RK-8. The serum levels of autoantibodies were decreased after the administration. All the therapeutic effects of RK-8 persisted for >6 months. These findings suggest that the systemic administration of agonistic anti-Fas mAb without fulminant hepatitis- inducing activity is a useful therapeutic strategy for treating systemic autoimmune disease.      

Fas receptor‐mediated apoptosis: a clinical application?

Fas is a membrane protein belonging to the death receptor family. Cross-linking of Fas by its ligand, FasL, or agonistic anti-Fas antibodies, induces apoptosis of cells expressing Fas on the membrane by triggering a cascade of caspases. Since many different tumours express Fas on their membrane, targeting Fas-mediated apoptosis by anti-Fas antibodies may be a promising anticancer therapy. Unfortunately, not all Fas-expressing cells are sensitive to Fas-mediated apoptosis. This has resulted in the discovery of many different inhibition mechanisms of Fas-mediated apoptosis. In addition, mutations in the Fas or p53 gene can also influence the sensitivity for Fas-mediated apoptosis. However, the role of wild-type p53 in Fas expression is still controversial. Because several different cytotoxic drugs are able to induce Fas membrane expression, combination therapy of anticancer drugs with anti-Fas antibodies or FasL is conceivable as an anticancer strategy. The efficiency of the induction of Fas-mediated apoptosis by anti-Fas antibodies, FasL-expressing cells or recombinant FasL (rFasL) in tumours has been demonstrated in vivo in solid tumours implanted in mice. Unfortunately, systemic treatment with anti-Fas antibodies or rFasL causes severe damage to the liver, so most preclinical studies are now focusing on circumvention of this problem by local administration of FasL, or on the use of inducible FasL-expressing vectors as gene therapy.     

Stimulation of Fas signaling down-regulates activity of neutrophils from major trauma patients with SIRS

Posttrauma apoptosis resistance of neutrophils (PMN) is related to overshooting immune responses, systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). Recently, we have shown that the apoptosis resistance in circulating PMN from severely injured patients which is known to be mediated by high serum levels of pro-inflammatory cytokines can be overcome by the activation of Fas death receptor. Here, we aimed to study whether stimulation of surface Fas leads to the inactivation of hyperactivated PMN from critically ill patients with SIRS. PMN from 23 multiple trauma patients (mean injury severity score (ISS) 34 ± 1.9) were isolated at day 1 after admission to the trauma center. PMN from 17 volunteer blood donors served as controls. Neutrophil activity has been determined after ex vivo short (1 h) and long-term (4 h) stimulation of freshly isolated PMN with immobilized agonistic anti-Fas antibodies. We found neutrophil chemotactic migration in response to IL-8, phagocytosis and oxidative burst to be significantly inhibited in control cells already after short-term (1 h) Fas stimulation. In contrast, inactivation of trauma PMN by agonistic anti-Fas antibodies was found to be efficient only after long-term (4 h) incubation of cells with agonistic antibodies. Thus, in trauma PMN down-regulation of neutrophil activity seems to be delayed when compared to cells isolated from healthy controls, suggesting impaired susceptibility for Fas stimulation in these cells. Interestingly, whereas Fas-mediated inhibition of phagocytosis and oxidative burst could be prevented by the broad range caspase inhibitor t-butoxycarbonyl-aspartyl(O-methyl)-fluoromethyl ketone (BocD-fmk), the chemotactic activity in response to IL-8 was unaffected. In conclusion, we demonstrate that stimulation of neutrophil Fas does not only initiate apoptosis but also induces inhibition of neutrophil functions, partially by non-apoptotic signaling.     

The role of aquaporin-1 (AQP1) expression in a murine model of lipopolysaccharide-induced acute lung injury

A murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) was used to evaluate whether aquaporin-1 (AQP1) is involved in lung inflammation and lung edema formation. Swiss strain mice (n = 122) had LPS (5 mg/kg) instilled intratracheally (IT), and were then treated with either 0.9 % saline or dexamethasone (5 mg/kg/day). Mice were euthanized at 2 days and 7 days after treatment. Inflammatory cytokines (TNF-alpha, IL-6), protein concentration in bronchoalveolar lavage (BAL) fluid, lung wet-to-dry weight ratio, histology, immunohistochemistry, and AQP1 Western blot were performed. Lung wet-to-dry weight ratio and lung vascular permeability were also measured in the AQP1 knockout mice (n = 9) that received IT LPS (5 mg/kg) at 2 days. Intratracheal instillation of LPS produced a severe lung injury at 2 days, characterized by elevation of TNF-alpha, IL-6 in the BAL fluid, and by histological changes consistent with increased lung vascular permeability and neutrophil infiltration. AQP1-immunoreactivity in the pulmonary capillary endothelium was reduced at 2 days and 7 days. Administration of dexamethasone improved LPS-induced ALI and retained expression of AQP1. However, depletion of AQP1 did not affect lung edema formation, lung vascular permeability, or lung histology. The results suggest that although AQP1 expression is decreased after lung injury, depletion of AQP1 does not alter lung inflammation and lung edema induced by LPS.     

Didecyldimethylammonium chloride induces pulmonary inflammation and fibrosis in mice

Didecyldimethylammonium chloride (DDAC) is used worldwide as a germicide, in antiseptics, and as a wood preservative, and can cause adverse pulmonary disease in humans. However, the pulmonary toxicity of DDAC has not yet been thoroughly investigated. Mice were intratracheally instilled with DDAC to the lung and the bronchoalveolar lavage (BAL) fluid and lung tissues were collected to assess dose- and time-related pulmonary injury. Exposure to 1500 μg/kg of DDAC caused severe morbidity with pulmonary congestive oedema. When the BAL fluid from survivors was examined on day 3 after treatment, exposure to 150 μg/kg of DDAC caused weakly induced inflammation, and exposure to 15 μg/kg did not cause any visible effects. Next, we observed pulmonary changes that occurred up to day 20 after 150 μg/kg of DDAC exposure. Pulmonary inflammation peaked on day 7 and was confirmed by expression of interleukin-6, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and regulated upon activation, normal T-cell expressed and secreted in the BAL fluid; these changes were accompanied by altered gene expression of their chemokine (C–C motif) receptor (Ccr) 1, Ccr2, Ccr3, and Ccr5. Cytotoxicity evoked by DDAC was related to the inflammatory changes and was confirmed by an in vitro study using isolated mouse lung fibroblasts. The inflammatory phase was accompanied or followed by pulmonary remodeling, i.e., fibrosis, which was evident in the mRNA expression of type I procollagen. These results suggest that administering DDAC by intratracheal instillation causes pulmonary injury in mice, and occupational exposure to DDAC might be a potential hazard to human health.     

Functional Characterization of Recombinant Rat Macrophage Inflammatory Protein-1α and mRNA Expression in Pulmonary Inflammation

Chemokines are important inflammatory mediators that function by activating and recruiting leukocytes to an inflamed tissue. We have recently cDNA cloned the rat chemokine macrophage inflammatory protein–1 (MIP-1) (1). In the present study, we characterize the biological function of recombinant MIP-1 protein and describe expression of its mRNA both in vitro and in a rat model of lung inflammation. In vitro rat rMIP-1 protein was chemotactic for both polymorphonuclear leukocytes (PMNs) and macrophages with maximal activity at 50 nM for both cell types. In in vivo studies, we found that intratracheal instillation of 1 and 5 g of rMIP-1 resulted in a significant (P < 0.05) influx of cells, primarily monocytes/macrophages, into the airspace of the lungs after 6 h. Mean numbers of lavagable PMNs were not elevated significantly (P < 0.05) for either dose of MIP-1. As a model of inflammation, rats were intratracheally instilled with 0.1 mg/kg bacterial lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) was performed 3 h later. Instillation of LPS resulted in an acute neutrophilia, but no significant change in lavagable macrophages. BAL cells from control animals (saline instilled) displayed no basal mRNA expression of either MIP-1 or MIP-2 (positive control). In contrast, both MIP-1 and MIP-2 mRNA levels increased markedly in BAL cells from rats instilled with LPS. The rat alveolar macrophage cell line (NR8383) also showed increased MIP-1 mRNA levels in response to LPS (10 g/ml) with a maximal increase after 6–8 h. The induction of MIP-1 mRNA expression by LPS in NR8383 cells was attenuated by cotreatment with the antioxidants N-acetylcysteine and dimethylsulfoxide, suggesting that the induction of MIP-1 mRNA by LPS is mediated via the generation of reactive oxygen species. We conclude that MIP-1 is a potent chemoattractant for macrophages in vivo, and its mRNA expression in macrophages and BAL cells in response to inflammatory stimuli suggests a fundamental role in acute pulmonary inflammation.     

Antiinflammatory effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on zymosan-induced arthritis in mice

Objective We investigated the antiinflammatory properties of a derived fraction of tetranortriterpenoids (TNTP) obtained from the seeds of Carapa guianensis Aublet. Material and methods Zymosan-induced arthritis and pleurisy in Swiss and C57/Bl6 mice (n = 10 per group). Western blot analysis was performed to analyze nuclear factor-κB (NFκB) translocation in mice peritoneal macrophages stimulated in vitro with zymosan (500 μg/ml). ELISA was performed to evaluate cytokine levels in knee joints. Values of p ≤ 0.05 were regarded as significant. Results Zymosan intra-articular (i. a.) injection (500μg/ cavity) induced a significant increase in knee joint diameter within 6 h, peaked within 24 h and remained above control values for 20 days. Orally-given (p. o.) TNTP (100–200 mg/ kg) inhibited zymosan-induced increase in knee joint diameter and protein extravazation into synovial cavity within 6 h. TNTP (100–200 mg/kg, p. o.) also inhibited total leukocyte influx into the synovial space and tissue, as well as into the mice pleural cavity, due to neutrophil impairment 6 h after zymosan stimulation. The increase in TNF-α, IL-1β and CXCL8/IL-8 levels that were detected in knee synovial extracts obtained from zymosan-stimulated mice was also inhibited by TNTP (100 mg/kg, p. o.). Moreover, the incubation of mice peritoneal macrophages with TNTP (100 μg/ml) inhibited zymosan (500 μg/ml)-induced NFκB translocation into the nucleus 6 h after stimulation. Conclusion Taken together, these results indicate that TNTP present an important antiinflammatory effect, inhibiting zymosan-induced arthritis in mice via the impairment of TNF-α, IL-1β and CXCL8/IL-8 generation, as well as NFκB signaling pathway. Received 7 October 2005; returned for revision 16 January 2006; accepted by M. Parnham 22 May 2006     

The Role of Endogenous Histamine on the Pathogenesis of the Lipopolysaccharide (LPS)-Induced, Acute Lung Injury: A Pilot Study

Abstract —Histamine is widely distributed in the lungs and increases capillary permeability and P-selectin expression. To observe the role of histamine in acute lung injury (ALI), we measured the histamine and protein concentrations and cell numbers in the bronchoalveolar lavage (BAL) of LPS-induced ALI in rats. We instilled LPS (3 mg/kg) intratracheally, in conjunction with the intravenous histamine receptor antagonists (mepyramine, a H1-receptor antagonist, or ranitidine, a H2-receptor antagonist). LPS increased protein concentration and neutrophil numbers in the BAL as well as myeloperoxidase (MPO) activity in lungs after 6 h. LPS also increased histamine concentration in BAL after 2 h. Mepyramine and ranitidine attenuated the increased histamine concentrations. Total cell number in the BAL and MPO activity in the lungs were significantly decreased and neutrophil numbers and protein concentration in the BAL seemed to decrease with the administration of ranitidine at 6 h. In conclusion, endogenous histamine might be involved in the recruitment of neutrophils and protein leaks in LPS-induced ALI via the H2 receptors.     

Telopeptides of type II collagen upregulate proteinases and damage cartilage but are less effective than highly active fibronectin fragments

Objective and Design:  We hypothesize that N-telopeptide (NT) and C-telopeptides (CT) of type II collagen can enhance proteinases and cause cartilage damage and have compared damaging activities to an extensively characterized potent fibronectin fragment (Fn-f). Materials:  NT and CT peptides were synthesized. Methods:  Interaction of labeled peptides with chondrocytes was studied by fluorescence microscopy. Effects on the metalloproteinases (MMPs) MMP-3 and MMP-13 and on ADAMTS-5 were analyzed by western blotting. Cartilage damage was assayed by loss of proteoglycan (PG) from cultured explants. Results:  NT and CT peptides penetrated cartilage, bound to chondrocytes and enhanced proteinase release and cartilage PG depletion. Peptides had detectable activity at 0.3 μM (1 μg/ml) and were comparable at 30 μM (100 μg/ml) to 1 μM Fn-f (29 μg/ml). However, while the Fn-f enhanced IL-1β and TNF-α, the NT and CT peptides did not. Conclusions:  Collagen peptides containing NT and CT regions were less active on a molar basis than Fn-fs but were still potent damaging agents. Since collagen fragments are found in OA cartilage at μg/ml, they have the potential to play a role in physiologic cartilage damage. Received 7 May 2008; returned for revision 9 June 2008; received from final revision 8 July 2008; accepted by J. A. Battista 31 July 2008     

Impact of non-di-(2-ethylhexyl)phthalate cardiopulmonary bypass tubes on inflammatory cytokines and coagulation-fibrinolysis systems during cardiopulmonary bypass

Di-(2-ethylhexyl)phthalate (DEHP), an excellent plasticizer for poly(vinyl chloride) (PVC), is a known endocrine-disrupting chemical. This study was designed to investigate whether a new non-DEHP bilayer tube reduced the release of DEHP, suppressed inflammatory cytokines, and altered coagulation-fibrinolysis systems. Sixteen patients undergoing coronary artery bypass grafting (CABG) were randomly assigned to the non-DEHP bilayer group (group B, n = 8), or the noncoated PVC group (group N, n = 8). The level of DEHP in the blood was measured before and after cardiopulmonary bypass (CPB). The levels of interleukin-6 (IL-6), D-dimer, and thrombin-antithrombin complex (TAT) were also measured at six points during and after CPB. DEHP was significantly lower in group B (472 ± 141 ng/ml) after CPB compared with group N (2094 ± 1046 ng/ml). The IL-6 level was significantly lower in group B (151 ± 131 pg/ml) than group N (206 ± 224 pg/ml) 180 min after protamine administration. The D-dimer level was significantly lower in group B 60 min after protamine administration (6.2 ± 2.4 μg/ml in group B vs 10.4 ± 4.5 μg/ml in group N) and 180 min after protamine administration (4.4 ± 0.7 μg/ml in group B vs 7.3 ± 2.7 μg/ml in group N). Group B had a tendency toward reduced postoperative bleeding compared with group N at any time. The bilayer tube was superior to the noncoated tube in terms of the inhibition of DEHP release, inflammatory cytokines, and the fibrinolysis system.     

In vivo analysis of Fas antigen-mediated apoptosis: effects of agonistic anti-mouse Fas mAb on thymus, spleen and liver

Fas antigen (Fas/CD95) is a cell surface receptor protein that mediates apoptosis-inducing signals. To analyze the function of Fas in vivo, we examined the effects of agonistic anti-Fas antibodies in mice. The i.p. administration of the hamster anti-mouse Fas mAb, RK-8, which induced apoptosis both in vivo and in vitro, did not kill adult mice, whereas those given the another hamster anti-mouse Fas mAb, Jo2, rapidly died of fulminant hepatitis with hemorrhage. Histological analyses of mice given RK-8 indicated severe damage of the thymus, and moderate damage of the spleen and liver. Most of the thymocytes and some hepatocytes underwent apoptosis within 1 day of administration. Flow cytometry revealed that CD4+ T cells were more sensitive to Fas-mediated apoptosis than CD8+ T cells. At day 7 after administration, the thymus was atrophied. These in vivo effects of RK-8 were transient; the thymus was regenerated, and the liver and spleen were apparently normal 1 month after injection. The administration of RK-8 into newborn mice caused severe damage of the liver and thymus. Most of the hepatocytes died and jaundice was induced. The newborn mice died within 1 week. Most hepatocytes of newborn mice may be more sensitive to apoptosis- inducing signals through Fas than those of adult mice. These results indicated that functional Fas, which introduces the death signal in vivo, is expressed on thymocytes, CD4+ splenocytes, and some adult and most newborn mouse hepatocytes.      

Urinary Excretion and Bactericidal Activity of Intravenous Ciprofloxacin Compared with Oral Ciprofloxacin

 Twelve healthy volunteers participated in a randomized crossover study to compare urinary concentrations, serum parameters, and urinary bactericidal activity of ciprofloxacin after single intravenous (i.v.) doses of 200 mg and 400 mg and an oral (p.o.) dose of 500 mg. The median serum concentrations at 1 h after administration were 1 μg/ml, 4.3 μg/ml, and 2.2 μg/ml, respectively. Between the first collection period (0–2 h) and the last collection period (38–48 h), the median urinary concentrations decreased from 394 μg/ml, 675 μg/ml, and 585 μg/ml, respectively, to 0.3 μg/ml, 0.6 μg/ml, and 1 μg/ml, respectively. The urinary concentrations after the 400 mg i.v. and the 500 mg p.o. doses were not statistically different but were significantly higher than those after the 200 mg i.v. dose. The urinary bactericidal titers (UBTs), defined as the highest urinary dilution bactericidal for the organism tested, were determined against Escherichia coli (ATCC 25922) and eight uropathogens up to 48 h after administration of ciprofloxacin. The UBTs after the 400 mg i.v. and the 500 mg p.o. doses were similar and were significantly higher (P<0.05) than those following the 200 mg i.v. dose. After 400 mg i.v. and 500 mg p.o., median UBTs of ≥1 : 4 were present up to 48 h for all strains for which the MIC was ≤0.5 μg/ml, except for one nalidixic-acid resistant Escherichia coli strain for which the MIC was 0.25 μg/ml. Species for which the MIC is ≥1 μg/ml showed median UBTs of ≥1 : 4 for 8–16 h. Median UBTs of ≥1 : 4 were present up to 8 and 12 h for both Pseudomonas strains tested. A once-daily dosage of 400 mg i.v. or 500 mg p.o. might be sufficient for treatment of urinary tract infections caused by highly susceptible pathogens. A twice-daily dosing scheme seems to be preferable for complicated infections caused by pathogens with intermediate susceptibilty (MIC≥1 μg/ml) or for empiric therapy.     

HIV-1-infected myelomonocytic cells are resistant to Fas-mediated apoptosis: effect of tumor necrosis factor-α on their Fas expression and apoptosis

To get insight into the involvement of tumor necrosis factor-α (TNF-α) and Fas (CD95) ligand in apoptosis (programmed cell death) of monocyte/macrophages in HIV-1-infected individuals, various T cell and myelomonocytic cell lines, including the HIV-1-infected clones OM-10.1 and U1 cells, were cultured in the presence of either TNF-α alone, anti-Fas agonist monoclonal antibody (Fas-mAb) alone, or their combinations. TNF-α moderately decreased the viability of myelomonocytic cell lines in a dose-dependent fashion (1–100 ng/ml). Unlike HIV-1-infected T cell lines, the viability of OM-10.1 and U1 cells was not affected by the treatment with Fas-mAb alone at concentrations up to 1,000 ng/ml. However, the viability of OM-10.1 cells further decreased with increasing concentrations of Fas-mAb when exposed simultaneously to TNF-α, suggesting that TNF-α sensitizes the cells to Fas-mAb-induced cell death. FACScan analysis and DNA gel electrophoresis revealed that the cell death was due to apoptosis. Such an effect of Fas-mAb was not identified in U1 cells. TNF-α but not Fas-mAb activated latent HIV-1 in OM-10.1 and U1 cells. Although all myelo-monocytic cell lines expressed Fas on their cell surface, TNF-α significantly up-regulated the expression of Fas in only OM-10.1 cells. These results indicate that, unlike T cells, HIV-1-infected myelomonocytic cells are generally resistant to the Fas-mediated apoptosis. However, they would become sensitive to the apoptosis if the expression of Fas could be up-regulated by TNF-α or other factors. Received: 11 November 1996     

Effect of Dynorphin A (1-17) on Proliferation and IL-2, IL-4, IFN-γ Production by Peripheral Blood Mononuclears

Dynorphin A (1-17) in concentrations of 10⁻⁸–10⁻⁹ M inhibits phytohemagglutinin (2.5 μg/ml)-induced proliferative response of mononuclear fraction lymphocytes. In mitogen-stimulated cultures, 10⁻⁸ M dynorphin A (1-17) stimulates the production of IL-4, inhibits the production of IL-2, and does not modify the production of IFN-γ. Nonselective κ-receptor antagonist naloxone and selective antagonist binaltorphimine hydrochloride abolish the inhibitory effects of both dynorphin A concentrations on the lymphocyte proliferative response. On the other hand, evaluation of the effect of κ-receptor blockade on the production of IL-2 and IL-4 showed that this effect depends on peptide concentration and antagonist type. Hence, the results attest to an important role of κ-receptors in modulation of functional activity of immune cells.     

T cells in hypersensitivity pneumonitis: effects of in vivo depletion of T cells in a mouse model.

C57BL/6 mice were given intranasal instillation of optimal doses of the actinomycete Faeni rectivirgula 150 micrograms/mouse 3 days/wk), an important offending agent causing hypersensitivity pneumonitis. This instillation was associated with a very significant increase in the lung weight of the mice and also a large increase (10-fold) in the number of cells recovered from the bronchoalveolar lavage (BAL) of instilled mice. Also, this instillation was associated with a very significant fibrosis at 4 and 8 wk (2-fold increase in hydroxyproline levels in the lungs). We determined the effect of depleting certain T-cell subsets on the progression of this inflammatory disease. Elimination of the L3T4 subset did not significantly affect the increase in the lung index, the lung cellular influx, or its profile. Fibrosis was also unaffected by this depletion of L3T4+ cells. Similarly, depletion of Lyt2+ (CD8+) cells did not lead to significant changes in these disease parameters. Depletion of all T cells (Thyl+) was also ineffective at modifying the number of infiltrating cells and the lung index score. However, identification of cell types in BAL showed that mice depleted of Thyl+ cells had a cellular influx that was almost exclusively neutrophilic throughout the instillation period, whereas control mice developed only a transient neutrophilic response to F. rectivirgula instillation, which was replaced by a recruitment of mononuclear cells, mostly macrophages. Also, depletion of Thyl+ cells before and during F. rectivirgula challenge had no effect on tumor necrosis factor-alpha levels in the BAL of treated mice (63 +/- 13 U/ml in anti-Thy1. 2 antibodies treated versus 52 +/- 10 U/ml in the BAL of control mice given F. rectivirgula).(ABSTRACT TRUNCATED AT 250 WORDS)     

In Vitro Susceptibility to Gemifloxacin and Trovafloxacin of Streptococcus pneumoniae Strains Exhibiting Decreased Susceptibility to Ciprofloxacin

 The in vitro susceptibility to trovafloxacin and gemifloxacin of Streptococcus pneumoniae strains exhibiting decreased susceptibility to ciprofloxacin (MIC ≥2 μg/ml; 30 strains with intermediate resistance [MIC 2 μg/ml] and 43 strains with complete resistance [MIC ≥4 μg/ml]) was determined. Seventy-three strains collected in a surveillance study carried out from May 1996 to April 1997 in Spain (prior to commercialisation of trovafloxacin and gemifloxacin) from patients with respiratory tract infections were tested. The antibacterial activity of gemifloxacin was affected to a lesser extent than that of trovafloxacin by the increase in the MIC of ciprofloxacin, with gemifloxacin showing significantly (P≤0.001) better antibacterial activity than trovafloxacin in all ciprofloxacin MIC categories (MIC50/MIC90 values of 0.015/0.03, 0.015/0.06, 0.03/0.06 and 0.12/0.25 μg/ml for gemifloxacin vs 0.12/0.12, 0.12/1, 0.25/0.5 and 2/4 μg/ml for trovafloxacin in the 2, 4, 8 and ≥16 μg/ml ciprofloxacin MIC categories, respectively). Nine (12.3%) of these 73 strains exhibited decreased susceptibility to trovafloxacin (≥2 μg/ml), whereas all strains were inhibited by 0.25 μg/ml of gemifloxacin.     

Comparative Study of Trans‐Oral and Trans‐Tracheal Intratracheal Instillations in a Murine Model of Acute Lung Injury

Animal model is of importance to further elucidate the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). We envisioned a possibility that there might be the differences in lipopolysaccharide (LPS)‐induced acute lung inflammation by the trans‐oral and trans‐tracheal intratracheal instillations. We compared the LPS‐induced early inflammatory responses by these two methods. The evaluative system included bronchoalveolar lavage (BAL) fluid biochemical analysis and differential cell counting, lung wet/dry weight ratio and lung histology. In vitro studies were performed on human bronchial epithelial cell line NCI‐H292 and alveolar Type II epithelial cell line A549 stimulated with LPS. Both interleukin (IL)‐8 release in the BAL fluid and IL‐8 secretions from NCI‐H292 and A549 cells were measured. We found that the trans‐tracheal intratracheal instillation promoted the LPS‐induced cell injury, neutrophil infiltration, and pulmonary edema compared to the trans‐oral one. The LPS‐induced pathological changes by the trans‐oral intratracheal instillation were characterized by pulmonary interstitial edema, but the trans‐tracheal intratracheal instillation was exudative pulmonary edema. More IL‐8 is produced from A549 cells than from NCI‐H292 cells under the treatment of LPS. The increased IL‐8 release in the BAL fluid and enhanced inflammatory responses caused by LPS may be due to more LPS delivered into the alveolar spaces by the trans‐tracheal intratracheal instillation compared to the trans‐oral one. The trans‐tracheal intratracheal instillation is proved to be more suitable to establish the murine model of ALI than the trans‐oral one and helpful to further elucidate the pathogenesis of ALI/ARDS. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Evaluation of Ceftazidime Concentration Released in Agar from an E Test Strip

 The aim of this study was to evaluate, using high-performance liquid chromatography, the concentration of ceftazidime in agar released from an E test strip, sampling at the edge of the strip at different points (1, 2, 4, 8, 16, 32, 64, and 128 μg/ml) at 6, 15, and 24 h after its deposition on uninoculated plates. From 6 to 24 h, the ceftazidime concentration in agar increased at the graduations 1, 2, and 4 μg/ml (+140, +82, and +58%, respectively), remained fairly constant at 8 μg/ml (–1.9%), and decreased at 16, 32, 64, and 128 μg/ml (–25, –44, –36, and –58%, respectively). In the 6–24 h range, the ceftazidime concentrations between 16 and 1 μg/ml were ±1 serial dilution of the values reported on the strip, confirming the accuracy of the E test in agar.