Pharmacodynamic and protective properties of a murine lipopolysaccharide-specific monoclonal antibody in experimental Pseudomonas aeruginosa pneumonia in mice.

We employed a Pseudomonas aeruginosa mouse pneumonia model to evaluate the ability of a murine monoclonal antibody (MAb) specific for the O-side chain of P. aeruginosa Fisher Immunotype-1 lipopolysaccharide (LPS) to achieve and sustain therapeutic levels in plasma and lung tissue, reduce bacterial populations in the lung, and prevent pneumonia-associated mortality. An IgG3 MAb (Y1-5A4) administered to mice i.v. over a dose range of 125-1,000 micrograms/mouse produced plasma and lung tissue levels at 2 hr of 61-507 micrograms/ml and 4.3-150 micrograms/g, respectively. The 1,000 micrograms MAb dose reduced bacterial counts in lung tissue (log10 cfu/g +/- S.D.) and blood (log10 cfu/ml +/- S.D.) 20 hr post-treatment (18 hr post-challenge) from 10.00 +/- 0.66 to 7.66 +/- 0.91 (P less than 0.01) and from 4.39 +/- 0.81 to less than 3.0, respectively. Administration of MAb to mice in doses of 125-500 micrograms 2 hr prior to a 3 x 50% lethal bacterial challenge produced significant protection against death, with a calculated 50% protective dose of 167 micrograms. Protection was noted following administration of 1,000 micrograms of MAb up to 6 hr after bacterial challenge (P less than 0.05, compared with untreated control). Histological examination of lung tissue from infected mice revealed less acute inflammation, necrosis, and hemorrhage in MAb-treated compared with untreated control animals and greater localization of Pseudomonas antigen within the phagocytic cells in alveolar space. These findings document the in vivo therapeutic efficacy of an LPS-specific IgG MAb in a murine model of acute P. aeruginosa pneumonia, based in part upon the achievability of effective MAb concentrations in plasma and lung tissue.     

Anti-inflammatory effect of pre-elafin in lipopolysaccharide-induced acute lung inflammation

The aim of the present study was to evaluate the anti-inflammatory activity of pre-elafin, an elastase-specific inhibitor, in lipopolysaccharide (LPS)-induced acute lung inflammation. C57BL/6 mice were pre-treated intranasally with recombinant human pre-elafin or vehicle only. One hour later, they were instilled intranasally with LPS (2 microg/mouse). Animals were sacrificed 6 hours after LPS instillation and bronchoalveolar lavage (BAL) was performed with three 1-ml aliquots of saline. LPS induced a lung inflammation characterised by a 100-fold increase in BAL neutrophils compared to control animals (265.8 +/- 54.5 x 10(3) and 2.4 +/- 1.3 x 10(3) neutrophils/ml, respectively). Pre-elafin dose-dependently reduced the neutrophil influx in the lung alveolar spaces by up to 84%. No elastase activity was detectable in all BAL fluids tested. Pre-elafin also reduced significantly LPS-induced gelatinase activity, as shown by zymography, and BAL macrophage inflammatory protein-2 (MIP-2) and KC levels, two potent neutrophil attractants and activators. Moreover, pre-elafin also significantly reduced mRNA levels of the three members of the IL-1 ligand family, namely IL-1alpha, IL-1beta and IL-1 receptor antagonist (IL-1Ra), type II IL-1 receptor, and TNFalpha as assessed in whole lung tissue by RNase protection assay. Thus, pre-elafin may be considered as a potent anti-inflammatory mediator.     

Rotavirus virus-like particles administered mucosally induce protective immunity.

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.     

IL-10 is an important mediator of the enhanced susceptibility to pneumococcal pneumonia after influenza infection

Secondary pneumococcal pneumonia is a serious complication during and shortly after influenza infection. We established a mouse model to study postinfluenza pneumococcal pneumonia and evaluated the role of IL-10 in host defense against Streptococcus pneumoniae after recovery from influenza infection. C57BL/6 mice were intranasally inoculated with 10 median tissue culture infective doses of influenza A (A/PR/8/34) or PBS (control) on day 0. By day 14 mice had regained their normal body weight and had cleared influenza virus from the lungs, as determined by real-time quantitative PCR. On day 14 after viral infection, mice received 10(4) CFU of S. pneumoniae (serotype 3) intranasally. Mice recovered from influenza infection were highly susceptible to subsequent pneumococcal pneumonia, as reflected by a 100% lethality on day 3 after bacterial infection, whereas control mice showed 17% lethality on day 3 and 83% lethality on day 6 after pneumococcal infection. Furthermore, 1000-fold higher bacterial counts at 48 h after infection with S. pneumoniae and, particularly, 50-fold higher pulmonary levels of IL-10 were observed in influenza-recovered mice than in control mice. Treatment with an anti-IL-10 mAb 1 h before bacterial inoculation resulted in reduced bacterial outgrowth and markedly reduced lethality during secondary bacterial pneumonia compared with those in IgG1 control mice. In conclusion, mild self-limiting influenza A infection renders normal immunocompetent mice highly susceptible to pneumococcal pneumonia. This increased susceptibility to secondary bacterial pneumonia is at least in part caused by excessive IL-10 production and reduced neutrophil function in the lungs.     

Endothelin, PAF and thromboxane A2 in allergic pulmonary hyperreactivity in mice

The role of endothelin, PAF and thromboxane A2 in airway hyperreactivity (AHR) to carbachol induced by ovalbumin sensitization and challenge in Balb/c mice was investigated. Ovalbumin sensitization and challenge induced significant AHR to carbachol in actively sensitized and challenged mice. Treatment of these mice with the PAF antagonist CV-3988 (10 microg kg(-1), i.v.) completely abolished OVA-induced AHR to carbachol. Treatment of sensitized mice with the TxA2 antagonist L-654,664 (1 mg kg(-1), i.v.) partially blocked the induction of AHR in OVA-challenged mice. The intranasal administration of 50 pmol of the ET(A) receptor antagonist BQ-123 had no effect on the PIP but produced a significant reduction at the dose of 100 pmol. The intravenous administration of BQ-123 (100 pmol) reduced the PIP only at the highest doses of carbachol. The ET(B) receptor antagonist BQ-788 administered either via the intranasal or intravenous route had no effect on the PIP at the dose of 100 pmol. Na?ve mice treated with either U-44069 (25 or 100 microg kg(-1), i.v.), endothelin-1 (100 pmol, intranasally) or the ET(B) receptor agonist IRL-1620 (100 pmol, intranasally) showed a marked increase in airway reactivity to carbachol. These results suggest an important role for endothelin, PAF and thromboxane A2 in AHR in mice actively sensitized and challenged with ovalbumin.     

DNA vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: size,dose, route of injection and duration of protection-early protection correlates with Mx expression

Rainbow trout of different sizes (10 and 100g) were injected intramuscularly (i.m.) or intraperitoneally (i.p.) with different doses (range 10 ng-10 microg) of a viral haemorrhagic septicaemia (VHS)-DNA vaccine (pcDNA3vhsG). As controls, fish were injected with the pcDNA3 plasmid alone, or with inactivated VHS virus. Fish were challenged at different times post-vaccination (p.v.) to assess protection. At certain times p.v., serum samples were analysed for neutralising antibody and liver tissue was analysed for Mx mRNA expression. A DNA dose of 0.5 microg injected by the i.m. route induced protection in fish of all sizes in challenges performed either 1 or 4 weeks p.v. This dose also conferred effective protection up to 9 months p.v. in fish >100 g. With lower doses of DNA (0.1 and 0.01 microg) and challenge at 4 weeks p.v., 10 g fish were partially protected but protection was not observed in 100 g fish. Vaccination by the i.p. route induced no or lower levels of protection compared with the i.m. route. Fish vaccinated with 0.5 microg DNA i.m. had no detectable serum neutralising antibody (NAb) at 4 weeks p.v. (with the exception of a single 10 g fish) but antibody was detected at 8 weeks and 6 months p.v. but not at 9 months p.v. However, cohorts of these fish showed effective protection at all timepoints. Lack of detectable levels of NAb (at 9 weeks p.v.) despite partial protection in challenge at 4 weeks p.v. was also observed with 0.01 microg doses of DNA i.m. NAb was detected in sera of fish at 8 weeks after vaccination with 0.1 microg i.m. but not in fish vaccinated with doses of 0.01-0.5 microg i.p. Early protection (1 week p.v.) correlated with elevated Mx gene expression.     

Immune response induced by N-lauroylsarcosine extracted outer-membrane proteins of an isolate of Edwardsiella ictaluri in channel catfish

A virulent isolate of Edwardsiella ictaluri (AL-93-75), the causative agent of enteric septicaemia of catfish (ESC), was used to derive a lipopolysaccharide-reduced N-lauroylsarcosine outer-membrane protein (OMP) fraction vaccine. The OMP fraction was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to whole-cell lysate, purified lipopolysaccharide (LPS) and a crude cell-wall fraction. The OMP fraction contained less than 2% (W/V) LPS. SDS-PAGE showed that whole cell lysates contained 27 proteins from 107 to 14.3 kDa, whereas OMP contained nine proteins from 97 to 14.3 kDa, LPS contained two proteins at 45 and 37 kDa bands and a smear of bands below 14.3 kDa, and cell wall fraction contained 21 proteins from 97 to 8 kDa. Channel catfish, Ictalurus punctatus, were vaccinated with 12.5 microg/100 microl OMP and immunogenicity was confirmed by subsequent Western blots. Blots showed that 97, 80, and 19 kDa proteins were immunogenic. Rapid enzyme-linked immunosorbent assays (ELISA) demonstrated that OMP produced a weak, but observable antibody response by 21 days post injection. OMP concentrations of 3.13, 6.25, 12.5, 25, and 50 microg/100 microl total protein were tested for protective immunity. Marginal protection by relative percent survival (RPS) was only seen for fish injected with 12.5 microg/100 microl with RPSs between 55-67.5%. A booster dose of 12.5 microg/100 microl OMP did not significantly enhance protection.     

Protective action of taurine,given as a pretreatment or as a posttreatment,against endotoxin-induced acute lung inflammation in hamsters

To assess the effect of taurine on lipopolysaccharide (LPS)-induced lung inflammation, oxidative stress and apoptosis, female Golden Syrian hamsters were intratracheally instilled with bacterial LPS (0.02 mg in phosphate buffered saline (PBS) pH 7.4), before or after a 3-day intraperitoneal treatment with a single dose of taurine (50 mg/kg/day in PBS pH 7.4), and bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected at 24 hr after the last treatment. In comparison to BALF samples from animals receiving only PBS pH 7.4, and serving as controls, those of LPS-stimulated animals exhibited a higher count of both total leukocytes and neutrophils and increased expression of tumor necrosis factor receptor 1. In comparison to lungs from control animals, those from LPS-treated animals showed increased cellular apoptosis, lipid peroxidation, decreased glutathione levels, altered activities of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase) and focal inflammation confined to the parenchyma. A treatment with taurine was found to significantly attenuate all these alterations, with the protection being, in all instances, greater when given before rather than after LPS. The present results suggest that taurine is endowed with antiinflammatory and antioxidant properties that are protective in the lung against the deleterious actions of Gram negative bacterial endotoxin.     

Sendai virus infection of mice with protein malnutrition.

Sendai virus pneumonia was produced in BALB/c mice fed protein-deficient diets in an effort to understand the severity of viral pneumonia in infants in developing countries. Animals on the deficient diet became clinically malnourished, and some aspects of cellular immunity were altered. In protein-deprived animals, the 50% lethal dose of intranasally administered Sendai virus was over 1,000-fold lower, pulmonary virus titers were higher, the infection was prolonged, and lung infection was established at a lower inoculum than in normal animals.     

Simvastatin attenuates vascular leak and inflammation in murine inflammatory lung injury

Therapies to limit the life-threatening vascular leak observed in patients with acute lung injury (ALI) are currently lacking. We explored the effect of simvastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor that mediates endothelial cell barrier protection in vitro, in a murine inflammatory model of ALI. C57BL/6J mice were treated with simvastatin (5 or 20 mg/kg body wt via intraperitoneal injection) 24 h before and again concomitantly with intratracheally administered LPS (2 microg/g body wt). Inflammatory indexes [bronchoalveolar lavage (BAL) myeloperoxidase activity and total neutrophil counts assessed at 24 h with histological confirmation] were markedly increased after LPS alone but significantly reduced in mice that also received simvastatin (20 mg/kg; approximately 35-60% reduction). Simvastatin also decreased BAL albumin (approximately 50% reduction) and Evans blue albumin dye extravasation into lung tissue (100%) consistent with barrier protection. Finally, the sustained nature of simvastatin-mediated lung protection was assessed by analysis of simvastatin-induced gene expression (Affymetrix platform). LPS-mediated lung gene expression was significantly modulated by simvastatin within a number of gene ontologies (e.g., inflammation and immune response, NF-kappaB regulation) and with respect to individual genes implicated in the development or severity of ALI (e.g., IL-6, Toll-like receptor 4). Together, these findings confirm significant protection by simvastatin on LPS-induced lung vascular leak and inflammation and implicate a potential role for statins in the management of ALI.     

Phase I evaluation of intranasal trivalent inactivated influenza vaccine with nontoxigenic Escherichia coli enterotoxin and novel biovector as mucosal adjuvants, using adult volunteers

Trivalent influenza virus A/Duck/Singapore (H5N3), A/Panama (H3N2), and B/Guandong vaccine preparations were used in a randomized, controlled, dose-ranging phase I study. The vaccines were prepared from highly purified hemagglutinin and neuraminidase from influenza viruses propagated in embryonated chicken eggs and inactivated with formaldehyde. We assigned 100 participants to six vaccine groups, as follows. Three intranasally vaccinated groups received 7.5-microg doses of hemagglutinin from each virus strain with either 3, 10, or 30 microg of heat-labile Escherichia coli enterotoxin (LTK63) and 990 microg of a supramolecular biovector; one intranasally vaccinated group was given 7.5-microg doses of hemagglutinin with 30 microg of LTK63 without the biovector; and another intranasally vaccinated group received saline solution as a placebo. The final group received an intramuscular vaccine containing 15 microg hemagglutinin from each strain with MF59 adjuvant. The immunogenicity of two intranasal doses, delivered by syringe as drops into both nostrils with an interval of 1 week between, was compared with that of two inoculations by intramuscular delivery 3 weeks apart. The intramuscular and intranasal vaccine formulations were both immunogenic but stimulated different limbs of the immune system. The largest increase in circulating antibodies occurred in response to intramuscular vaccination; the largest mucosal immunoglobulin A (IgA) response occurred in response to mucosal vaccination. Current licensing criteria for influenza vaccines in the European Union were satisfied by serum hemagglutination inhibition responses to A/Panama and B/Guandong hemagglutinins given with MF59 adjuvant by injection and to B/Guandong hemagglutinin given intranasally with the highest dose of LTK63 and the biovector. Geometric mean serum antibody titers by hemagglutination inhibition and microneutralization were significantly higher for each virus strain at 3 and 6 weeks in recipients of the intramuscular vaccine than in recipients of the intranasal vaccine. The immunogenicity of the intranasally delivered experimental vaccine varied by influenza virus strain. Mucosal IgA responses to A/Duck/Singapore (H5N3), A/Panama (H3N2), and B/Guandong were highest in participants given 30 microg LTK63 with the biovector, occurring in 7/15 (47%; P=0.0103), 8/15 (53%; P=0.0362), and 14/15 (93%; P=0.0033) participants, respectively, compared to the placebo group. The addition of the biovector to the vaccine given with 30 microg LTK63 enhanced mucosal IgA responses to A/Duck/Singapore (H5N3) (P=0.0491) and B/Guandong (P=0.0028) but not to A/Panama (H3N2). All vaccines were well tolerated.     

Different sensitivity of rabbit heart and skeletal muscle to endotoxin-induced impairment of mitochondrial function.

The involvement of mitochondrial dysfunction in septic disturbances of tissues is controversial. The aim of this study was to investigate the effects of endotoxin-induced sepsis on the function of heart and skeletal muscle mitochondria. Rabbits were made septic by subcutaneous injection of endotoxin (lipopolysaccharide, LPS) from Escherichia coli at concentrations of 100 or 150 microg LPS.kg(-1) 24 h prior to the experiments. Mitochondrial respiration was measured in saponin-skinned muscle fibers and compared with photometrically detected activities of respiratory chain enzymes as well as with function of perfused hearts. In heart fibers a dosage of 100 microg LPS.kg(-1) caused a significant decrease of state 3-respiration for the substrates pyruvate (-38%), octanoyl-carnitine (-38%) and succinate (-30%) with correspondingly decreased respiratory control indexes (RCI). In addition, endotoxin caused a decreased temporal stability of the rate of state 3-respiration. At least in part these changes can be attributed to a reduced activity of complex I + III (-50%) of the respiratory chain. State 4-respiration rates were not significantly altered. The lowered state 3-respiration in heart mitochondria seems to contribute to the impairment of heart muscle function as detected by an increase of coronary vascular resistance (CVR) in endotoxin-treated hearts. Functional properties of mitochondria from M. Vastus lasteralis were not affected by 100 microg LPS.kg(-1) but a higher dosage of 150 microg LPS.kg(-1) caused decreased RCI for the substrates pyruvate (-29%) and octanoyl-carnitine (-32%). Also the activity of complex I + III was not significantly affected at lower dose of endotoxin but decreased (-42%) after treatment with 150 microg LPS.kg(-1). Results demonstrate the involvement of impaired mitochondria in the pathophysiology of septic organ failure and a tissue specificity of endotoxaemia.     

Gonadotropins and sex hormones modulate interrenal function in soft-shelled turtle

The aim of the current work was to investigate the role of gonadotropins and female sex hormones on interrenal activity in soft-shelled turtles, Lissemys punctata punctata. 1) FSH treatment (3 microg/100 g body wt daily for 10 days) caused interrenal hypertrophy with increased nuclear diameter, raises of acid phosphatase and alkaline phosphatase concentrations, and depletions of cholesterol (except the free fraction) and ascorbic acid levels from the interrenal gland. However, LH treatment (3 microg/100 g body wt daily for 10 days) failed to produce any perceptible change in the interrenal activity. The combined treatments of FSH and LH (3 microg each/100 gm body wt daily for 10 days) produced no further change beyond that of FSH alone. 2) Estrogen treatment with the low dose (25 microg/100 g body wt daily for 10 days) had no effect, but with higher doses (50 microg or 100 microg/100 gm body wt daily for 10 days) is caused interrenal stimulation by inducing the same manifestations to those of FSH. The degree of manifestations was higher with the higher dose (100 microg daily) than that of the moderate dose (50 microg daily). Progesterone treatment with the low dose (25 microg /100 g body wt daily for 10 days) had no significant effect, but with the moderate (50 microg daily) and higher (100 microg daily) doses suppressed interrenal activity by showing the reverse changes to those of estrogen. The degree of manifestations was higher with the higher dose than that of the moderate one. The combined treatments of estrogen and progesterone (100 microg each/100 g body wt daily for 10 days) caused interrenal stimulation but to a lesser extent than that of estrogen alone. The findings are briefly discussed.     

Localized vs. systemic inflammation in guinea pigs: a role for prostaglandins at distinct points of the fever induction pathways?

In guinea pigs, dose-dependent febrile responses were induced by injection of a high (100 microg/kg) or a low (10 microg/kg) dose of bacterial lipopolysaccharide (LPS) into artificial subcutaneously implanted Teflon chambers. Both LPS doses further induced a pronounced formation of prostaglandin E(2) (PGE(2)) at the site of localized subcutaneous inflammation. Administration of diclofenac, a nonselective cyclooxygenase (COX) inhibitor, at different doses (5, 50, 500, or 5,000 microg/kg) attenuated or abrogated LPS-induced fever and inhibited LPS-induced local PGE(2) formation (5 or 500 microg/kg diclofenac). Even the lowest dose of diclofenac (5 microg/kg) attenuated fever in response to 10 microg/kg LPS, but only when administered directly into the subcutaneous chamber, and not into the site contralateral to the chamber. This observation indicated that a localized formation of PGE(2) at the site of inflammation mediated a portion of the febrile response, which was induced by injection of 10 microg/kg LPS into the subcutaneous chamber. Further support for this hypothesis derived from the observation that we failed to detect elevated amounts of COX-2 mRNA in the brain of guinea pigs injected subcutaneously with 10 microg/kg LPS, whereas subcutaneous injections of 100 microg/kg LPS, as well as systemic injections of LPS (intra-arterial or intraperitoneal routes), readily caused expression of the COX-2 gene in the guinea pig brain, as demonstrated by in situ hybridization. Therefore, fever in response to subcutaneous injection of 10 microg/kg LPS may, in part, have been evoked by a neural, rather than a humoral, pathway from the local site of inflammation to the brain.     

Effects of different serotypes of Escherichia coli lipopolysaccharides on body temperature in rats

The effects of Escherichia coli O55:B5, O127:B8, and O111:B4 serotypes' lipopolysaccharides (LPS) on body temperature were investigated in rats. LPSs were injected intraperitoneally at doses of 2, 50, and 250 microg/kg. A multiphasic and no-dose dependent increase in rectal temperature was observed in response to E. coli O55:B5 LPS at all doses, and in response to E. coli O127:B8 LPS at 2 and 50 microg/kg doses. The highest dose of the latter caused a dual change in rectal temperature, in which hypothermia preceded fever. E. coli O111:B4 LPS was either pyrogenic or hypothermic at 2 and 250 microg/kg doses; respectively, whereas a dual response was observed when the 50 microg/kg dose was injected. Although dual responses were observed after administration of all LPSs at 50 microg/kg dose when the body temperature was recorded by biotelemetry, the hypothermia induced by E. coli O55:B5 LPS was significantly smaller. These data suggest that LPSs induce dose and serotype-specific variable changes on body temperature in rats. This variability may be related to the structure of LPSs. The data also indicate that LPS causes hypothermia with or without fever in rats.     

A murine model of sepsis following smoke inhalation injury

Acute lung injury (ALI) by smoke inhalation with subsequent pneumonia and sepsis represents a major cause of morbidity and mortality in burn patients. The aim of the present study was to develop a murine model of ALI and sepsis to enhance the knowledge of mechanistic aspects and pathophysiological changes in patients with these injuries. In deeply anesthetized female C57BL/6 mice, injury was induced by four sets of cotton smoke using an inhalation chamber. Afterward, live Pseudomonas aeruginosa (3.2 × 10⁷ colony-forming units) was administered intranasally. The indicated dose of bacteria was determined based on the results of a dose-response study (n = 47). The following study groups were monitored for survival over 96 h: (1) sham injury group, (2) only smoke inhalation group, (3) only bacteria group, and (4) smoke inhalation plus bacteria group. Each group included 10 mice. The survival rates were 100%, 90%, 30%, and 10%, respectively. The double hit injury was associated with excessive releases of pro-inflammatory cytokines in the plasma, and enhanced neutrophil accumulation, increased lipid peroxidation, and excessive formation of reactive nitrogen species in the lung. In mice receiving only smoke inhalation injury, no systemic cytokine release and increased lung tissue lipid peroxidation were observed. However, smoke alone significantly increased neutrophil accumulation and formation of reactive nitrogen species in lung tissue. In conclusion, bacterial pneumonia is predominantly responsible for mortality and morbidity in this novel murine model of smoke inhalation and pulmonary sepsis. Reactive oxygen and nitrogen species mediate the severity of lung injury.     

Inhibition of TNF-alpha production contributes to the attenuation of LPS-induced hypophagia by pentoxifylline

Cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) are assumed to mediate anorexia during bacterial infections. To improve our understanding of the role that these two cytokines serve in mediating infection during anorexia, we investigated the ability of pentoxifylline (PTX), a potent inhibitor of TNF-alpha production, to block the anorectic effects of the bacterial products lipopolysaccharide (LPS) and muramyl dipeptide (MDP) in rats. Intraperitoneally injected PTX (100 mg/kg body wt) completely eliminated the anorectic effect of intraperitoneally injected LPS (100 microg/kg body wt) and attenuated the anorectic effect of a higher dose of intraperitoneally injected LPS (250 microg/kg body wt). Concurrently, PTX pretreatment suppressed low-dose LPS-induced TNF-alpha production by more than 95% and IL-1beta production 39%, as measured by ELISA. Similarly, high-dose LPS-induced TNF-alpha production was reduced by approximately 90%. PTX administration also attenuated the tolerance that is normally observed with a second injection of LPS. In addition, PTX pretreatment attenuated the hypophagic effect of intraperitoneally injected MDP (2 mg/kg body wt) but had no effect on the anorectic response to intraperitoneally injected recombinant human TNF-alpha (150 ug/kg body wt). The results suggest that suppression of TNF-alpha production is sufficient to attenuate LPS- and MDP-induced anorexia. This is consistent with the hypothesis that TNF-alpha plays a major role in the anorexia associated with bacterial infection.     

小檗碱改善流感病毒性肺炎小鼠肺血管通透性的作用及机制

目的:观察小檗碱对流感病毒感染所致病毒性肺炎小鼠肺血管通透性的影响,并探讨其作用机制。方法:BALB/c小鼠108只随机分为3组,正常组、模型组、小檗碱组,25μL 50LD50病毒液滴鼻建立流感病毒感染的小鼠肺炎模型,感染后1 h,正常组和模型组予以双蒸水灌胃,小檗碱组予药物0.005 g.kg-1d-1腹腔注射;各组均给药2次/d,连续给药5 d。感染后的2 d、4 d、6 d,处死小鼠,肺组织称重以检测肺含水量;1%伊文氏兰5 mL/kg尾静脉注射检测肺血管通透性;Bicinchoninic acid(BCA)法检测肺泡灌洗液(BALF)中蛋白含量;放免法或酶免法测定肺组织中PGE2、PLA2及LT-B4含量。结果:病毒感染后,模型组肺含水量持续升高,肺血管通透性及BALF蛋白含量在感染后第4天开始明显升高,小檗碱降低了肺含水量、肺血管通透性及BALF蛋白含量(P<0.01);模型组肺组织中PGE2、PLA2、LT-B4的含量明显升高,小檗碱不同程度地抑制了PGE2、PLA2、LT-B4的表达。结论:小檗碱通过抑制流感病毒感染后肺组织中PGE2、PLA2、LT-B4的释放,降低了肺血管通透性及肺含水量,对病毒性肺炎中肺水肿的形成,起到一定的治疗作用。     

Evaluation of DNA vaccination of spotted sand bass (Paralabrax maculatofasciatus) with two major outer-membrane protein-encoding genes from Aeromonas veronii

Genes encoding two major outer membrane proteins (OMPs) of the bacterial pathogen Aeromonas veronii, Omp38 and Omp48, were used to construct DNA vaccines. The protective effect of such vaccines against motile aeromonad septicaemia was evaluated in spotted sand bass (Paralabrax maculatofasciatus), an endemic species of the Mexican Northwest Pacific coast and a potential resource for the aquaculture industry. Weak protein expression, as determined by immunoblotting, was observed after transfection of eukaryotic cells with the DNA vaccines. Fish immunized with a single intramuscular injection of 20 microg of the omp38 and omp48 DNA vaccines showed slightly, but significantly elevated serum antibody levels 4 and 6 weeks after vaccination, compared to fish vaccinated with the control plasmid pcDNA3.1. Spotted sand bass vaccinated with the omp38 and omp48 DNA vaccines and challenged with A. veronii by intraperitoneal route recorded a relative percent survival (RPS) between 50 and 60%. Histopathological signs of motile aeromonad septicaemia were observed in around 40% of omp38 and omp48-vaccinated fish and 80% of pcDNA3.1-vaccinated control fish. The results indicate that P. maculatofasciatus vaccinated with a single dose of DNA plasmids encoding the major OMPs from A. veronii shows partial protection against infection and mortality by A. veronii experimental infection.     

Passive immunization against transmissible gastroenteritis virus in piglets by ingestion of milk of sows inoculated with attenuated virus.

Pregnant sows were inoculated with the attenuated strain, TO--163, of swine transmissible gastroenteritis virus. Suckling piglets born from them received challenge inoculation with the virulent virus at 3 days after birth, and examined for ability to prevent infection and the immunoglobulin (Ig) classes of antibody in milk. A pregnant sow was inoculated intramuscularly with a dose of 10(8.0) TCID50 and intranasally with a dose of 10(9.3) TCID50 of attenuated virus. Piglets born from it suffered from diarrhea after challenge inoculation, but none of them died eventually. Their dam was also affected with diarrhea for 4 to 7 days after challenge inoculation of them. Another pregnant sow was inoculated twice with 10(9.3) TCID50 of attenuated virus, first by the intramuscular and secondly by the intranasal route. Of nine piglets born from it, one excreted soft feces after challenge inoculation, but all survived to grow normally. Their dam manifested no clinical symptoms at all after challenge inoculation of them. The higher the titer of virus inoculated into pregnant sows, the higher the neutralizing antibody titer in serum and milk of the sows after farrowing. The puerperal sow which had received two doses of 10(9.3) TCID50 each of attenuated virus by the intramuscular and intranasal route, respectively, presented the highest neutralizing antibody titer of all the inoculated sows. This titer was 2,048 in serum and 14,183 in colostrum immediately after farrowing. In that sow IgG was the main class of immunoglobulins in neutralizing antibody in milk. Even the IgA antibody titer of that sow was higher than that of any other sow which had been administered with virus of low titer. It was 392 and 19 3 and 9 days, respectively, after farrowing.