Changes in the peripheral blood lymphocytes after immunization and its effect on the course of experimental inflammatory process in the lungs

It was shown that in the immunization of rabbits with sorbed staphylococcus toxoid and human albumin, at the height of the immune response there occurred an intensification of biosynthesis and destabilization of lysosomal membranes of blood leukocytes, with a simultaneous development of microcirculation disturbances in the lungs. Development of pneumonia against the background of immunization led to a greater destabilization of the lysosomic membranes of leukocytes and to the intensification in the lungs of infiltrative and destructive processes, in comparison with the course of inflammation in the nonimmunized animals. Peculiarities of the antigens used caused some specificity in the course of pneumonia. A relationship between the changes of blood leukocytes and disturbances in the lungs in immunization is supposed.     

Effect of antibiotics on the secretion and percentage yield of lysozyme from human blood monocytes

The effect of various doses of benzylpenicillin, streptomycin, cephaloridine (ceporin) and exogenic lysozyme on stability of lysozyme binding to lysosomal membranes of human blood monocytes was studied with the procedure developed by the authors which included estimation of the secreted and total lysozyme and evaluation of the stability index of the lysosomal membranes (SILM). Investigation of the changes in the blood monocyte membrane stability was conducted with constant and short-term (60 minutes) exposure to the antibiotics followed by their through elimination. It was noted all the antibiotics increased the SILM which was evident of labilization of the monocyte lysosomal membranes with respect to lysozyme. However, the mechanism of labilization with respect to various antibiotics was different judging from the amount of the secreted and total lysozyme. Labilization under the effect of benzylpenicillin and streptomycin was most probably due to changes in the lysozyme functional activity in the presence of these antibiotics and that under the effect of exogenic lysozyme was probably due to penetration into the monocytes and simultaneous impairment of the monocyte secretory activity. Cephaloridine had the labilizing effect by inhibition of the lysozyme intracellular synthesis.     

Role of phospholipids in changes in lysosomal membrane stability in conditions of chronic alcoholic intoxication

Pronounced destabilization of liver lysosomal membranes has been revealed in rats in conditions of 30-day-long alcohol intoxication. Noticeable fractional changes in phospholipid composition of lysosomal membranes have been found. Significant increase in lysophosphatidylethanolamine and lysophosphatidylcholine levels have been observed. Type A2 phospholipase activity was found in lysosomal fractions, with the enzyme activity Ca2+-dependent, optimal at pH 8 and increasing many-fold following alcohol intoxication. The changes in lysosomal membrane phospholipids appear to be related to phospholipase A2 activation.     

Properties of binding sites for chloroquine in liver lysosomal membranes

Chloroquine (CQ) is an antimalarial and antirheumatic drug that accumulates in lysosomes. We purified liver lysosomal membranes of tritosomes from albino mice injected with Triton WR 1339. The membranes were used for the binding assay with CQ in 0.01 M Tris-HCl buffer (pH 7.4). This binding was saturable, with a KD value of 6.2 microM. To understand the nature of CQ affinity, the binding was done under conditions that alter membrane structure and composition. Changes in pH, high ionic strength, and bivalent cations reversibly decreased the binding, while the effect of non-ionic detergents was partially reversed. The cationic detergent Hyamine strongly decreased the binding, and its effect was trypsin and neuraminidase had no effect. The results indicate the existence of binding sites for CQ in liver lysosomal membranes, which were strongly affected by changes of charge in the molecules involved in the binding. The treatment with the enzymes suggests that loss of polar groups of phospholipids increases the affinity of CQ by exposing protein sites located deep in the membrane, or by permiting a closer interaction between the drug and membrane lipids. CQ lysosomotropism and other effects of CQ on the lysosomal apparatus studied by other authors may be due not only to its accumulation inside the acid milieu of the lysosomes, in the same manner as other weak bases, but also to the affinity of CQ for binding sites in the lysosomal membrane.     

Effect of water-soluble antioxidants on the permeability of lysosomal membranes and on the structure of the liver in rats with thermal burns

Experiments on rats were made to study the effect of water-soluble antioxidants on the permeability of lysosomal membranes of liver cells and liver structure under burn. Antioxidants were injected intraperitoneally shortly after burn, whereas examination was performed after one day. It has been discovered that one day after burn there takes place an appreciable destabilization of lysosomal membranes with the release of a lysosomal matrix enzyme, cathepsin D to the cytoplasm. Liver structure had undergone substantial changes by that time. After administration of water-soluble antioxidants lysosomal membranes got stabilized while liver structure manifested but insignificant disorders.     

The Occurrence of Mycoplasmas and Bacteria in Lungs from Sheep in Southern Norway

In order to study the occurrence of mycoplasmas among Norwegian sheep, lungs from a great number of different herds were collected at 3 abattoirs in Southern Norway. The presence of fermenting mycoplasmas and bacteria was examined in both normal and pneumonic lungs to determine whether recovery of these agents could be related to pneumonic changes. Pneumonic lungs demonstrated lesions typical of the condition described as subacute or chronic pneumonia. Mycoplasma ovipneumoniae was found in 87 % of the 126 pneumonic lungs and in 6 % of the 83 normal lungs. Bacteria, mostly Pasteurella haemolytica, were less frequently encountered in the pneumonic lungs, and usually in combination with M. ovipneumoniae. It is concluded that M. ovipneumoniae is widespread among sheep in Southern Norway and can be considered to have etiological significance in subacute or chronic pneumonia, whereas bacteria probably occur mainly as secondary invaders. Changes resulting from moderate invasion by lungworm were found in about half of the lungs, but just as frequently in normal as in pneumonic lungs, and accordingly did not appear to contribute to the pneumonia investigated.     

Effects of some biologically active compounds on phagosome-lysosome fusion in peritoneal macrophages of mice.

Effects of biologically active compounds bilirubin (BR), farmorubicin (FR), and chelerythrine (CR) on phagosome-lysome (P-L) fusion in mouse peritoneal macrophages were studied using fluorescent dye acridine orange as lysosomal labelling and yeast cells as target. It was found that all three compounds tested enhanced P-L fusion. To investigate mechanisms of these effects, changes in fluidity of rat liver lysosomal membranes under influence of BR, FR and CR were studied by measuring fluorescence intensity, lifetime, and polarization of DPH or TMA-DPH incorporated in isolated rat liver lysosomes. In order to characterize the cytoskeleton changes under the action of these biologically active compounds F-actin content in peritoneal macrophages of mice was determined. Our results demonstrate that BR action induces a decrease in DPH and TMA-DPH polarization, FR increases DPH and TMA-DPH polarization, and CR causes only an increase in TMA-DPH polarization in lysosomal membranes. All three compounds tested increase F-actin content in peritoneal macrophages. Thus, the effect of BR on P-L fusion is connected with increasing fluidity of lysosomal membranes and the cytoskeleton changes. The enhancement of P-L fusion under the action of FR and CR can most likely be explained by changes of the cytoskeleton state.     

Acute pneumonia reversibly inhibits hypoxic vasoconstriction in isolated rat lungs.

Pneumonia was induced in rats by instillation of carrageenin (0.5 ml of 0.7% solution) into the trachea. Three or four days after instillation, the lungs were isolated, perfused with blood of healthy rat blood donors, and ventilated with air + 5% CO2 or with various hypoxic gas mixtures. Pulmonary vascular reactivity to acute hypoxic challenges was significantly lower in lungs of rats with pneumonia than in lungs of controls. The relationship between O2 concentration in the inspired gas and Po2 in the blood effluent from the preparation was shifted significantly to lower Po2 in lungs with pneumonia compared to control ones. These changes were not present in rats allowed to recover for 2-3 weeks after carrageenin instillation. We suppose that blunted hypoxic pulmonary vasoconstriction may contribute to hypoxaemia during acute pulmonary inflammation. Decreased Po2 in the blood effluent from the isolated lungs with pneumonia implies significant increase of oxygen consumption by the cells involved in the inflammatory process.     

Effect of metronidazole on the course of experimental anaerobic streptococcal pneumonia

The effect of metronidazole in experimental anaerobic pneumonia in guinea pigs infected with peptostreptococci has been studied. In the untreated animals the prolonged pathological process in the lungs is mainly associated with the suppression of the functional state of the thymus-dependent link of the immune system. Metronidazole suppresses anaerobic flora, stimulates local immune reaction and arrests the development of inflammatory changes in the lungs. The drug eliminates the unbalance in some subpopulations of immunocompetent cells and restores the number of cells regulating immune response to the normal level. The results obtained in this investigation make it possible to recommend the trial of metronidazole in the complex therapy of peptostreptococcal pneumonia.     

Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover.

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.     

Cathepsin D activity in splenocytes in relation to Shigella virulence in an experiment

The overtime study of changes in the activity of cathepsin D, a lysosomal enzyme, in the splenocytes of CBA mice after their infection with virulent and avirulent Shigella strains of the same origin and with the same antigenic structure has been made. As the result of two months of observations, changes in the activity of this enzyme in the cytoplasmic and lysosomal cell fractions have been found to occur in phases. The activity of cathepsin D has been shown to depend on the virulence of Shigella strains used for inoculation. Virulent Shigella strains induce the pronounced and prolonged activation of the enzyme in the lysosomes, as well as in the cytoplasm. The latter phenomenon is probably indicative of the pathological labilization of the lysosomal membranes, induced by the virulent culture. Avirulent Shigella strains induce only the transient activity of the enzyme in the lysosomes without any essential changes in the permeability of their membranes. These data point to the possibility of differentiating virulent and avirulent Shigella strains by the determination of the enzymatic activity of splenocytes in infected animals.     

Arylamidases of rat liver and chemically induced hepatomas. II. Preparation and characterization of monospecific antisera against two distinct arylamidase-active antigens.

Monospecific antisera were prepared against the most prominent arylamidase (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) active antigen in plasma membranes (the plasma membrane arylamidase) and lysomal content (the lysosomal content arylamidase), respectively. Plasma membrane extract and lysosomal content were allowed to react in crossed immunoelectrophoresis against their homologous antisera. The electrophoretic plates were washed extensively, dried and subsequently stained for arylamidase activity.The particular immunoprecipitates were thus identified and could be excised to be used for immunizations. The two resulting antisera precipitated the arylamidase used for immunization, but failed to be monospecific as they precipitated additional antigens. These antisera with restricted specificity against some plasma membrane and lysosomal content antigens, respectively, were used to produce immunoprecipitates intended for new attempts to prepare monospecific antisera by a second cycle of immunizations. A monospecific antiserum against the plasma membrane arylamidase was thus obtained, while a third cycle of immunizations was needed to get a monospecific anti-lysosomal content antiserum. The plasma membrane arylamidase showed ATPase activity also after precipitation with the monospecific antiserum, thus still retaining its characteristics as a multienzyme complex.     

Niemann-Pick disease type B: prenatal diagnosis and enzymatic and chemical studies on fetal brain and liver.

Patients with Niemann-Pick disease type A have a severe neurovisceral disease caused by a deficiency of lysosomal sphingomyelinase activity in all tissues examined. The patients with the type B form have signs and symptoms related to storage of sphingomyelin in the spleen, liver, and lungs, while neurologically they remain normal. They also have a severe deficiency of lysosomal sphingomyelinase activity in all tissues previously examined. Here the brain and liver of a fetus with Niemann-Pick disease type B are examined for enzymatic anc chemical changes. Despite careful analysis, no measurable lysosomal sphingomyelinase could be measured in either organ. Lipid changes were comparable to those observed in fetuses aborted with Niemann-Pick disease type A. The affected child in this family is now age 3 and remains neurologically normal but continues to show organ enlargement and lung infiltration of lipids. It appears that the lack of neurological involvement in type B patients cannot be due to an obvious presence of significant lysosomal sphingomyelinase activity in brain.     

IP-10对流感病毒致肺细胞病变作用的影响

为探讨干扰素y诱导蛋白10（IP-10）在流行性感冒病毒致肺部炎性病变中的作用,用BALB／c小鼠做动物感染模型,对只感染流感病毒和注射IP-10后感染流感病毒小鼠的肺部炎性反应进行了比较。结果显示,注射IP-10后感染流感病毒小鼠的肺部炎性反应明显重于感染对照组。研究结果表明,IP-10对流感病毒造成的肺部炎性病变的严重程度具有重要影响。     

The effect of the immunization of animals with a live plague vaccine on the lysosome count in organ-specific macrophages

Vital fluorochromatic lysosomes of peritoneal, liver, splenic and lung macrophages of white mice, white rates, and guinea-pigs are studied. Reliable differences in a quantity of lysosomal granules of macrophages from various tissues as well as differences between macrophages from the same tissues of different experimental animals are found. At 21 days after animal immunization with live plague vaccine EV the most significant changes in the number of lysosomal granules are revealed in white mice. The number of lysosomes in guinea pigs increased in 1 and 7 days after vaccination, in 14 days their number became normal.     

Phosphomannosyl receptors of lysosomal enzymes in cardiac and skeletal muscles of young and old mice

The endogenous activity and the binding of high-uptake beta-N-acetylglucosaminidase were assayed in the membranes of heart and skeletal muscles of young (2 months) and old (15 months) NMRI-mice (Mus musculus) to evaluate the age-related changes in the phosphomannosyl receptors of lysosomal enzymes in muscular membranes. The total activities of beta-N-acetylglucosaminidase were significantly higher in cardiac and skeletal muscles of old than young mice. The total and the specific (inhibited by mannose-6-phosphate) binding of beta-N-acetylglucosaminidase to the membranes of cardiac muscle, but not to those of skeletal muscle, were higher in old mice than in young ones. The endogenous activity of beta-N-acetylglucosaminidase was significantly higher in the membranes of skeletal muscles of old mice than in those of young mice. The membranes of heart muscles did not show any difference in the endogenous activities. The saturation properties of the binding of beta-N-acetylglucosaminidase to the phosphomannosyl receptors were very similar in the membranes of heart and skeletal muscles of both age groups. We conclude that during aging the number of phosphomannosyl receptors of lysosomal enzymes increases in the membranes of heart muscle while the occupancy of phosphomannosyl receptors with endogenous ligands increases in the membranes of skeletal muscle.     

Influence of membrane physical state on lysosomal potassium ion permeability

Lysosomal permeability to potassium ions is an important property of the organelle. Influence of the membrane physical state on the potassium ion permeability of isolated lysosomes was assessed by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and monitoring the lysosomal proton leakage with p-nitrophenol. The membrane fluidity of lysosomes was modulated by treatment with membrane fluidizer benzyl alcohol and rigidifier cholesteryl hemisuccinate. Changes in the membrane order were examined by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The measurements of membrane potential and proton leakage demonstrated that the permeability of lysosomes to potassium ions increased with rigidification of their membranes by cholesteryl hemisuccinate treatment at 37 degrees C, and decreased with fluidization of their membranes by benzyl alcohol treatment at 2 degrees C. The changes in ion permeability could be recovered by fluidizing the rigidified membranes and rigidifying the fluidized membranes. The results suggest that the physical states of lysosomal membranes play an important role in the regulation of their K(+) permeability.     

Serial histopathological examination of the lungs of mice infected with influenza A virus PR8 strain

Avian influenza H5N1 and pandemic (H1N1) 2009 viruses are known to induce viral pneumonia and subsequent acute respiratory distress syndrome (ARDS) with diffuse alveolar damage (DAD). The mortality rate of ARDS/DAD is extremely high, at approximately 60%, and no effective treatment for ARDS/DAD has been established. We examined serial pathological changes in the lungs of mice infected with influenza virus to determine the progress from viral pneumonia to ARDS/DAD. Mice were intranasally infected with influenza A/Puerto Rico/8/34 (PR8) virus, and their lungs were examined both macro- and micro-pathologically every 2 days. We also evaluated general condition, survival rate, body weight, viral loads in lung, and surfactant proteins in serum. As a result, all infected mice died within 9 days postinfection. At 2 days postinfection, inflammation in alveolar septa, i.e., interstitial pneumonia, was observed around bronchioles. From 4 to 6 days postinfection, interstitial pneumonia with alveolar collapse expanded throughout the lungs. From 6 to 9 days postinfection, DAD with severe alveolar collapse was observed in the lungs of all of dying and dead mice. In contrast, DAD was not observed in the live infected-mice from 2 to 6 days postinfection, despite their poor general condition. In addition, histopathological analysis was performed in mice infected with a dose of PR8 virus which was 50% of the lethal dose for mice in the 20-day observation period. DAD with alveolar collapse was observed in all dead mice. However, in the surviving mice, instead of DAD, glandular metaplasia was broadly observed in their lungs. The present study indicates that DAD with severe alveolar collapse is associated with death in this mouse infection model of influenza virus. Inhibition of the development of DAD with alveolar collapse may decrease the mortality rate in severe viral pneumonia caused by influenza virus infection.