Purification and properties of a protease with elastase activity from Pseudomonas aeruginosa.

The isoelectric points of three proteases (I, II and III), separated from culture supernatants of Pseudomonas aeruginosa strain PAKS-I by isoelectric focusing, were 8.5, 6.6 and 4.5 respectively. Collagenase activity was not detected. More than 75% of the extracellular protease activity of this strain was due to protease II. This enzyme also possessed elastase activity. When purified by ammonium sulphate precipitation, isoelectric focusing and gel chromatography, protease II showed one band on disc electrophoresis and one band on conventional immunoelectrophoresis. The pH optimum, stability and effect of inhibitors and substrate concentration were examined. The molecular weight was 23000 +/- 5000. Protease II was lethal for mice when injected intraperitoneally at a high dose (minimum lethal dose 0.1 mg). Dermonecrosis and subcutaneous haemorrhages were produced in new-born mice upon subcutaneous injection of 10 microgram protease II. A sensitive test for cytotoxicity showed no evidence of cytoplasmic membrane damage to HeLa cells or human diploid embryonic lung fibroblasts by protease II. Morphological changes similar to those produced by trypsin were found.     

A comparison of phenotype and copper distribution in blotchy and brindled mutant mice and in nutritionally copper deficient controls

The murine mottled mutants brindled, Mo br, and blotchy, Mo blo, are valuable animal models for the study of mammalian copper metabolism. In this paper, we present data showing that a nutritionally copper deficient suckling mouse, Cu-, with strong phenotypic similarities to the brindled mutant can be produced by feeding genetically normal dams a copper deficient diet (0.1-0.4 ppm Cu2+) from the day of mating. Comparisons of copper distribution between the Cu- mice and brindled mutants indicate that when a small dose of copper (0.5-0.9 micrograms Cu2+) was administered by intracardiac injection, the copper was abnormally distributed, and that the pattern of tissue distribution was very similar in Cu- mice and brindled mutants 24 h after injection. When, however, a treatment dose (50 micrograms Cu2+) was injected subcutaneously, and tissues assayed 3 d after injection, copper distribution in Cu- mice and brindled mutants was clearly different. Copper deficiency in Cu- suckling mice is entirely derived from maternal effects. Evidence that maternal effects may also influence the survival and phenotype of the brindled and blotchy mutants was obtained by comparing the viability of mutants born to dams carrying mottled mutations on one or both X chromosomes.     

Requirement of endogenous tumor necrosis factor/cachectin for recovery from experimental peritonitis

By intrasplenic immunization we raised a rat mAb (mAb V1q; IgG2a, kappa) with a potent neutralizing activity against natural mouse TNF (1 microgram/ml mAb V1q/100 U/ml TNF). mAb V1q was used to study the role of endogenous TNF in experimental peritonitis induced by sublethal cecal ligation and puncture. mAb V1q persisted for over 5 days in the serum of mice injected with 100 micrograms of the antibody and, therefore, proved useful for in vivo experiments. As little as 20 micrograms mAb V1q/mouse prevented lethal shock of the animals by 400 micrograms LPS/mouse. In sublethal cecal ligation and puncture i.p. injection of mAb V1q directly and up to 8 h after induction of experimental peritonitis lead to death of the animals within 1 to 3 days. The lethal effect of mAb V1q was compensated by injection of recombinant mouse TNF. Similar mAb V1q effects as in immunocompetent mice were shown in severe combined immune deficiency mice deficient of mature functional B and T cells. Taken together, these data suggest that during the early phase of peritonitis endogenous TNF may stimulate nonlymphoid cells such as granulocytes, macrophages, platelets, and fibroblasts to ingest bacteria and to localize inflammation, respectively. These beneficial effects of TNF may determine survival. Thus, our data may have implications for the therapeutic management of a beginning peritonitis.     

Biological activity of a new Soviet natural inducer, double-stranded RNA

Antiviral activity of a two-spiral RNA (ts RNA), a new natural interferon inductor was studied. It was shown that ts RNA extracted from a phage infected E. coli culture was an active inductor of interferon and resistance to infection with the forestspring encephalitis virus experimental animals. In experiments on 10-12 g mice ts RNA administered in a dose of 50 micrograms/mouse 6 hours after the infection induced up to 1280 units/ml of the serum interferon. When the inductor was administered repeatedly, the experimental animals developed hyporeactivity resulting in a marked decrease in interferon production after the 3rd subsequent injection. The most pronounced effect with respect to the forest-spring encephalitis virus was observed when the inductor was administered intraperitoneally in a dose of 20 micrograms/mouse 4 hours before the infection. The protective effect was less pronounced when the inductor was administered 24 and 48 hours before the infection. A two-fold administration of the inductor did not increase the antiviral effect. When the inductor was administered in a dose of 100 micrograms 14 days before the infection, the animals showed an increase in the nonspecific resistance to the infection resulting in a marked antiviral effect.     

Sustained engraftment of mice transplanted with IL-1-primed blood-derived stem cells.

IL-1 is considered the primary mediator of the acute phase response. One of the characteristic manifestations of this response is early neutrophilia that is probably caused by release of mature neutrophils from the bone marrow into the peripheral blood. In the present study, we assessed whether IL-1 had a similar releasing effect on the number of circulating progenitor cells and stem cells. Female BALB/c mice were injected i.p. with increasing (0.1-1.0 micrograms/mouse) concentrations of rhu-IL-1 alpha. IL-1 injection resulted in a marked dose-dependent increase in the number of polymorphonuclear neutrophils, granulocyte-macrophage colony-forming units (CFU-GM), and cells forming spleen colonies (CFU-S day 8 and day 12). The maximal increase was found at 4 to 8 h after injection of 1 micrograms IL-1 per mouse, yielding a mean fivefold elevation in neutrophil count, and a mean 30-fold and 10-fold increase in the number of circulating CFU-GM and CFU-S, respectively. In a subsequent series of experiments, lethally irradiated (8.5 Gy) female recipient animals were transplanted with 5 x 10(5) blood mononuclear cells derived from male IL-1-treated animals. Long-term survival was obtained in 68% of mice transplanted with peripheral blood cells derived from donor animals at 6 h after a single injection of 1 micrograms IL-1. The mean number of circulating CFU-GM in these donor animals was 557/ml blood. At 6 mo after transplantation, greater than 95% of the bone marrow cells were of male origin, as determined using in situ hybridization with a Y-chromosome specific probe. In contrast, long-term survival was reached in less than 10% of mice transplanted with an equal number of blood cells derived from saline-treated controls or donor animals treated with a dose of 0.1 micrograms IL-1. These results indicate that a single injection of IL-1 induces a shift of hematopoietic progenitor cells and marrow repopulating cells into peripheral blood and that these cells can be used to rescue and permanently repopulate the bone marrow of lethally irradiated recipients.     

Potential use of tuftsin in treatment of candida peritonitis in a murine model

A major complication of continuous ambulatory peritoneal dialysis (CAPD) is peritonitis caused by Candida albicans. Increasing the activity of the peritoneal macrophages, the predominant cell type found in the peritoneal cavity, may be a promising treatment for this infection. Tuftsin was found to increase thioglycollate-elicited mouse peritoneal macrophage activity. 2x10(-7) M tuftsin enhanced two-fold cell association with radiolabelled candida, superoxide aniom production, and killing activity. Thus, a model consisting of mice undergoing peritoneal dialysis was developed in order to study the use of tuftsin as a therapeutic drug against peritoneal candidiasis. Administration of tuftsin (50 micrograms/mouse) before candidiasis induction with a lethal dose of candida (7x10(8) candida per mouse) improved mouse survival up to 70%, compared with 10% in the control group. The potential of tuftsin as a treatment for candidiasis was shown when the infection was induced with a sublethal dose of candida. Daily intraperitoneal injections of tuftsin (50 micrograms) to the sublethally infected mice caused a significant decrease in the number of candida recovered from the peritoneal cavity and from the blood (from 700 +/- 190 to 110 +/- 26 CFU/ml and from 100 +/- 26 CFU/ml to 17 +/- 8 CFU/ml, respectively). In addition, a larger number of peritoneal macrophages with greater phagocytic and killing activity were found in the tuftsin-treated mice. The effect of tuftsin may promote its potential use in the therapy of peritonitis in patients undergoing chronic peritoneal dialysis.     

Isolation and characterization of an acidic lethal phospholipase Az from Malayan cobra (Naja naja sputatrix) venom

An acidic, lethal phospholipase Az was purified to electrophoretic homogeneity from the venom of the Malayan cobra (Naja naja sputatrix). The enzyme has an isoelectric point of 5.58, a molecular weight of 12000, and a medium lethal dose (LD50) of 0.86 micrograms/g in mice by intravenous injection. The enzyme also exhibited weak anticoagulant and edema-forming activities. The amino acid composition of the enzyme is similar to those of other cobra venom phospholipases Az.     

A new approach to an influenza live vaccine: modification of the cleavage site of hemagglutinin

A promising approach to reduce the impact of influenza is the use of an attenuated, live virus as a vaccine. Using reverse genetics, we generated a mutant of strain A/WSN/33 with a modified cleavage site within its hemagglutinin, which depends on proteolytic activation by elastase. Unlike the wild-type, which requires trypsin, this mutant is strictly dependent on elastase. Both viruses grow equally well in cell culture. In contrast to the lethal wild-type virus, the mutant is entirely attenuated in mice. At a dose of 10(5) plaque-forming units, it induced complete protection against lethal challenge. This approach allows the conversion of any epidemic strain into a genetically homologous attenuated virus.     

Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide

Enterovirus 71 (EV71) infections could lead to high mortalities and neither vaccine nor therapeutic treatment is available. We investigated vaccination with a synthetic peptide SP70 representing a neutralizing linear VP1 epitope of EV71 strain 41 (subgenogroup B4) and passive transfer of anti-SP70 antibodies to protect suckling Balb/c mice against EV71 infectivity. When the mouse anti-SP70 antisera with a neutralizing antibody titer of 1:32 were passively administered to one-day-old suckling mice which had been challenged with a lethal dose of 1000 TCID(50) per mouse, the neutralizing anti-SP70 antibodies were able to confer 80% in vivo protection. In contrast, suckling mice which did not receive any anti-SP70 antisera did not survive the viral challenge at day 21 postinfection. Histological examination and real-time RT-PCR assays revealed viral infiltration in small intestines of EV71-infected mice. Interestingly, anti-SP70 antibodies play a major role in the inhibition of EV71 replication in vivo and significantly reduced the viral titer. In conclusion, EV71-neutralizing antibodies elicited by the synthetic peptide SP70 were able to confer good in vivo passive protection against homologous and heterologous EV71 strains in suckling Balb/c mice.     

A plasmid encoding Japanese encephalitis virus PrM and E proteins elicits protective immunity in suckling mice

A plasmid encoding Japanese encephalitis virus (JEV) prM and E proteins was constructed, and its efficacy as a candidate vaccine against JEV was evaluated in suckling mice. Groups of 10 BALB/c mice (5-7 days old) were immunized twice via muscular injection with this DNA vaccine, an empty vector or PBS at an interval of 3 weeks, and were challenged with a lethal dose of JEV 3 weeks after the second inoculation. Both cellular and humoral immune responses were examined before the challenge. Two animals from each group were sacrificed to detect the JEV-specific cytotoxic T lymphocyte activity. JEV-specific lactate dehydrogenase release in the DNA vaccine, empty vector and PBS groups was 37.5%, 18% and 8.5% respectively. JEV-specific antibody was detected in 8 of 10 animals in DNA vaccine group with a geometrical mean titer of 1: 28.3. The pooled serum from the same group also showed a neutralizing activity. Six of 8 mice in the DNA vaccine group survived the challenge, with a protection rate of 75%, but all the mice died in the two control groups. These results show that this JEV prM and E DNA vaccine is immunogenic and protective against JEV infection in the mouse model.     

Host-mediated antiviral activity of Lactobacillus casei against cytomegalovirus infection in mice

The protective effect of heat-killed Lactobacillus casei (LC) against murine cytomegalovirus (MCMV) infection was examined. ICR mice treated once with LC 1 day or 2 days before challenge survived lethal infection, but untreated or Lactobacillus fermentum (LF)-treated mice did not. The protective effect was evidenced by an increase in plaque-forming units (PFU) per 50% lethal dose (LD50) and a decrease in titers of infectious viruses replicated in the target organs. This was further confirmed by severity of histopathological damage to the target organs, especially the liver. LC neither inactivated MCMV nor inhibited its replication in mouse embryonic fibroblasts (MEF). The spleen cells from LC-treated mice inhibited its replication in MEF on co-cultivation. Augmentation by LC of splenic natural killer (NK) cell activity correlated with survival of mice from otherwise lethal MCMV infection. Cytotoxic activity of peritoneal cells and level of serum interferon (IFN) were elevated after MCMV infection, but they were not associated with survival of mice nor with treatment of LC. The protective effect of LC was not clear in NK-deficient beige mutant (bgJ/bgJ) mice, when compared with that in their littermate (bgJ/+) mice. Poor protection of bgJ/bgJ mice by LC treatment correlated with failure to induce NK cell activity by LC treatment in the mutant mice. Thus, it is likely that LC protects mice from MCMV infection by augmentation of NK cell activity.     

Biological study of muscle degenerating hemorrhagic factor from the venom of Vipera aspis aspis (aspic viper)

1. A hemorrhagic factor was isolated from Vipera a. aspis venom by gel filtration, ion-exchange chromatography and affinity chromatography. 2. Molecular weight of the purified factor was determined to be 67,000 Da, which was composed of 552 amino acid residues. 3. The minimum hemorrhagic dose (MHD) was measured to be 0.11 micrograms per mouse with subcutaneous injection. The hemorrhagic activity was inhibited by chelating agents and reductant. 4. The hemorrhagic factor possesses proteolytic activity against dimethylcasein. 5. Serum creatine phosphokinase level was rapidly increased within 30 min following injections of this preparation into the mice thighs. 6. Actin, one of the main components of muscle fiber, is apparently digested by the hemorrhagic factor. The pathological findings are further reported in this paper.     

Protection against the mouse-adapted A/FM/1/47 strain of influenza A virus in mice by a monoclonal antibody with cross-neutralizing activity among H1 and H2 strains.

The monoclonal antibody designated C179 was found to neutralize all of the H1 and H2 strains of influenza A virus studied (Y. Okuno, Y. Isegawa, F. Sasao, and S. Ueda, J. Virol. 67:2552-2558, 1993). In the present study, the ability of C179 to protect mice from the lethal effect of the A/FM/1/47 (H1N1) strain was examined. When the mice were injected intraperitoneally with 100 micrograms of C179 per mouse a day before the virus challenge (2.0 x 10(3) focus-forming units per mouse), all of the mice survived. Moreover, significantly higher survival rates were observed in mice receiving 1,000 micrograms of C179 per mouse 2 days after the virus challenge than in those receiving phosphate-buffered saline alone. These results indicate that C179 is effective not only for prevention but also for treatment of mice infected with H1 and H2 strains. The possibility that C179 can be used for passive immunization in humans is discussed.     

Nonenzymatic fibrinolytic reaction to the intravenous administration of small doses of Salmonella endotoxin to rabbits

Intravenous injection of a low dose of Salmonella endotoxin (10 micrograms/kg bw) into rabbits results in an increase in the non-enzymatic fibrinolytic activity of blood at early stages of a pathological process followed by depression of this response at later stages of the pathology. At higher degrees of non-enzymatic fibrinolysis activation the morphological and ultrastructural changes in renal tissues caused by endotoxin injection are the least pronounced. Intravenous injection of heparin after injection of a lethal dose of Salmonella endotoxin (100-200 micrograms/kg) enhances non-enzymatic fibrinolysis activation and decreases the morphological and ultrastructural lesions in renal tissues.     

Precocious decrease of chymotrypsinogen by thyroxine in the pancreas of suckling mice

Chymotrypsinogen activity in mouse pancreas gradually decreased from birth, reaching the adult level on day 20 after birth. In suckling mice the enzyme activity was decreased to 1/9 of the control value by injection of thyroxine. The activity was not affected by insulin, and was slightly increased, rather than decreased, by daily injection of hydrocortisone. The effect of thyroxine seemed to be direct, not due to modification of adrenal function.     

Increase in liver-associated natural killer activity by polyribonucleotides

Several polyribonucleotides are currently in clinical trials for the treatment of cancer or viral diseases. The present report in mice demonstrates that polyinosinic-polycytidylic acid and poly-L-lysine which has been stabilized in carboxymethylcellulose (poly (ICLC) as well as polyadenosinic-polyuridylic acid (poly AU), both potently augment natural killer (NK) activity in the liver, which is often a target organ for the formation of metastases during the progression of human cancer. Following the administration of poly ICLC (10 micrograms/mouse), greater NK activity as measured by lytic units (LU), was observed in the liver (445 LU) than in blood (63 LU) or spleen (20 LU). The high level of NK activity in the liver was in contrast to the low levels observed in untreated mice, and was maintained for at least 9 days post injection. NK activity in the blood and spleen returned to normal levels by day 6. Similar results were obtained with poly AU except that approximately 10-fold more poly AU (100 micrograms/mouse) was required to induce optimal augmentation of NK activity. Further studies demonstrated that the increase in liver-associated NK activity induced by poly ICLC was associated with a 10- to 20-fold increase in liver-associated leukocytes, termed nonparenchymal cells (NPC). Fractionation of the NPC on discontinuous density gradients of Percoll demonstrated that the NK activity mediated by NPC was associated with cells morphologically characterized as large granular lymphocytes (LGL). Further studies demonstrated that the repeated administration of poly ICLC resulted in significantly higher levels of liver-associated NK activity and total liver-associated LGL as compared to a single injection.     

Protection of mice against Japanese encephalitis virus by passive administration with monoclonal antibodies

We have identified and characterized nine antigenic epitopes on the E envelope of Japanese encephalitis virus (JEV) by using mAb. Passive administration of most of the anti-JEV mAb protected mice from i.v. challenge with 1.5 x 10(3) plaque-forming units of JEV, JaGAr-01 strain. Some mAb, which possess high neutralization activity in vitro, showed high protection, and JEV-specific N mAb 503 was found the most protective. Even an injection of 2.5 micrograms/mouse of mAb 503 protected all mice from JEV infection. Furthermore, an injection of about 200 micrograms of mAb 503 on day 5 postinfection protected 82% of the mice, even when JEV was detected in more than 85% of the infected mouse brains. Synergism of protection was observed with mixtures of several mAb directed against different epitopes. Although in a murine macrophage cell line, all of the mAb groups showed antibody-dependent enhancement (ADE) of JEV infectivity in vitro, and only two flavivirus cross-reactive mAb groups showed ADE of dengue virus type 2. The ADE of JEV by mAb seems not to be harmful for in vivo protection experiments, except for two mAb groups: mAb 302 and 201 showed little or no protective activity against JEV infection and, rather, caused early death in infected mice.     

Measurement of virulence of aeromonads using a suckling mouse model of infection

Abstract Virulent and avirulent strains of Aeromonas spp. were identified and virulence quantified using an animal model. Virulence was measured by determining a 50% lethal dose (LD₅₀) 43 h after oral administration of live bacteria. The LD₅₀ of virulent Aeromonas isolates ranged from log₁₀ 7.53 (mean) organisms to log₁₀ 8.88 (mean). Some isolates were avirulent in this model. Detection of cytotoxic activity in culture supernatants correlated with virulence (Fisher exact test, P = 0.0029). There was no correlation between LD₅₀ and the source of the isolate, β-haemolysis or lipopolysaccharide (LPS) banding profile on SDS-PAGE. In this animal model, virulence was multifactorial in that: (i) bacterial multiplication in the gut was associated with fatal infection; (ii) the increase in bacterial numbers in the gut of mice administered a lethal dose of bacteria was accompanied by accumulation of fluid; and (iii) there was evidence of extraintestinal spread of infection. Protection of suckling mice by rabbit antiserum to Aeromonas cell envelopes was observed.     

Effect of heparin on the antiradiation activity of cystamine, decreasing with a reduction in radiation intensity

It was shown that a single injection of heparin (250 units/kg) 15 min and 24 h before irradiation potentiated a slight radioprotective effect of cystamine (dichlorohydrate, 170 mg/kg) which was registered after the administration thereof to mice 30 min before irradiation with an absolutely lethal dose at a dose rate of 0.0025 Gy/c.     

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.