Characterization of a new thermostable direct haemolysin produced by a Kanagawa-phenomenon-negative clinical isolate of Vibrio parahaemolyticus

The production of two haemolysins, thermostable direct haemolysin (Vp-TDH) and a Vp-TDH-related haemolysin (Vp-TRH), by clinical isolates of Vibrio parahaemolyticus has previously been reported. Here we describe a third type of haemolysin (named Vp-TDH/I), which is produced by a clinical isolate (strain TH012) that is Kanagawa phenomenon negative. Vp-TDH/I was purified by a series of column chromatographies on DEAE-Sephadex A25, hydroxyapatite, Sepharose 4B and Mono Q. By physicochemical, biological and immunological analyses, Vp-TDH/I was demonstrated to be similar, but not identical, to Vp-TDH and Vp-TRH. The gene encoding Vp-TDH/I was cloned and the deduced amino acid sequence of Vp-TDH/I confirmed that Vp-TDH/I has a sequence different from those of previously known Vp-TDH and Vp-TRH. Not only purified Vp-TDH/I but also live cells of the Vp-TDH/I-producing strain induced fluid accumulation in ligated rabbit intestine. We conclude that this clinical isolate produces a new type of Vp-TDH-related haemolysin, which may be involved in the pathogenesis of this organism.     

Induction of erythroblast increase in rabbit marrow cells by extracts of leukocytes and certain metabolic compounds. IV. Studies on erythropoiesis

Two kinds of extracts of peripheral blood leukocytes induced erythroblast increase and maturation in vitro in rabbit marrow cells. One extract was a secretion or exudate into plasma from intact leukocytes; the other was the supernatant after homogenizing the cells in ethanol. The extracts were less active than the intact leukocytes. The response with the two extracts together was a little greater than with either alone, but less than additive. 11-Ketopregnanolone and 3′5′ cyclic AMP were active inducers. Neither increased the effects of leukocytes or their extracts.     

Studies on adhesion, haemagglutination and other biological properties of Vibrio cholerae O139

Abstract The adhesive capabilities of eight Vibrio cholerae O139 epidemic strains to isolated rabbit intestinal epithelial cells (RIEC) were observed to be high similar to those observed with a Vibrio cholerae O1 strain isolated from patients. Toxin production by the strains, measured by accumulation of fluid in rabbit ileal loop model, was high and the toxin was lethal as the animal expired within 6 h. Culture filtrates of the strains exhibited the presence of vascular permeability factor which produce induration and necrosis in the adult rabbit and guinea pig skin. All the strains showed high to moderate haemagglutinin titres against chicken erythrocytes and produced El Tor-like haemolysin. SDS-PAGE of the outer membrane preparation of the strains showed the presence of major protein component at 38 kDa region. The lethality of the toxin, high adhesive activity, shifting of the major outer membrane protein band and production of thermolabile haemolysin on Wagatsuma agar were the major variations of these epidemic strains from V. cholerae O1 and V. cholerae non-O1 strains isolated previously.     

Production of RNA-dependent haemolysin by Haemophilus pleuropneumoniae

Five strains of Haemophilus pleuropneumoniae, out of eight strains tested, produced extracellular haemolysin(s) when grown in liquid culture in the presence, but not in the absence, of RNA. The haemolysin produced by the neotype strain was unstable, heat labile, and sensitive to degradation by pronase, trypsin, and chymotrypsin; moreover, trypan blue treated haemolysin preparations were less effective at causing erythrocyte lysis than were untreated preparations. Following growth in the absence of RNA, washed suspensions of the neotype strain produced extracellular haemolysin when incubated in the presence of RNA, glucose, and casein acid hydrolysate; extracellular haemolysin could not be detected if the incubation mixture contained chloramphenicol. The haemolysin produced by washed bacterial suspensions was similar to that produced by growing cultures in that it was unstable, heat labile, and sensitive to inactivation by the same complement of enzymes. Erythrocyte lysis induced by either haemolysin preparation was preceded by a prelytic phase, the duration of which was dependent upon haemolysin concentration and the initial temperature of the haemolysin--erythrocyte mixture. It is concluded that the haemolysin(s) produced by the neotype strain of H. pleuropneumoniae is distinct from, but closely related to both streptolysin S and the haemolysin produced by Treponema hyodysenteriae.     

Dysregulated haemolysin promotes bacterial outer membrane vesicles‐induced pyroptotic‐like cell death in zebrafish

Inflammasomes are important innate immune components in mammals. However, the bacterial factors modulating inflammasome activation in fish, and the mechanisms by which they alter fish immune defences, remain to be investigated. In this work, a mutant of the fish pathogen Edwardsiella piscicida (E. piscicida), called 0909I, was shown to overexpress haemolysin, which could induce a robust pyroptotic‐like cell death dependent on caspase‐5‐like activity during infection in fish nonphagocyte cells. E. piscicida haemolysin was found to mainly associate with bacterial outer membrane vesicles (OMVs), which were internalised into the fish cells via a dynamin‐dependent endocytosis and induced pyroptotic‐like cell death. Importantly, bacterial immersion infection of both larvae and adult zebrafish suggested that dysregulated expression of haemolysin alerts the innate immune system and induces intestinal inflammation to restrict bacterial colonisation in vivo. Taken together, these results suggest a critical role of zebrafish innate immunity in monitoring invaded pathogens via detecting the bacterial haemolysin‐associated OMVs and initiating pyroptotic‐like cell death. These new additions to the understanding of haemolysin‐mediated pathogenesis in vivo provide evidence for the existence of noncanonical inflammasome signalling in lower vertebrates.     

Investigation of the haemolytic activity of Proteus mirabilis strains

Young broth cultures of all P. mirabilis strains tested exhibited haemolytic activity. This activity seemed to be strongly cell-associated as only a very small fraction of this activity was found in the cell-free supernatant. The haemolysin was only produced by actively growing cells. Inhibition studies with trypsin and chloramphenicol suggested that the haemolysin is of protein nature. Lecithin and serum of several species had an inhibitory effect on the haemolysin. Besides erythrocytes of various species also VERO cells were affected by the haemolysin. A correlation was found between the haemolytic activity of a strain and its virulence in an experimental mouse model.     

Haemolytic activity in the serum of brown trout, Salmo trutta

Brown trout serum contains a natural, spontaneous, antibody-independent lytic activity and a haemolysin antibody complement-mediated lytic activity against unsensitized and trout antibody-sensitized sheep erythrocytes, respectively. The use of various activators and inactivators of the mammalian complement system demonstrated that trout serum possesses complement or complement-like components similar in activity to those present in the classical and alternative pathways found in mammals. A single injection of trout with sheep erythrocytes stimulated the production of antibody-secreting cells in lymphoid organs and increased the levels of natural haemolysins. A second injection of sheep erythrocytes further raised the haemolysin values and antibody-secreting cell counts. Serum complement from homologous or closely related fish species was more effective for use in the haemolysin and antibody-secreting cell assays than that from heterologous sources, except guinea pig. Based on physico-chemical properties, gel filtration and immunoelectrophoretic studies, natural and induced anti-sheep erythrocyte haemolysins were found to be similar molecules and are possibly high molecular weight IgM antibodies.     

In vitro studies of haemolysis by some staphylococci grown in chemically defined media

Staphylococcus aureus (five strains) and Staph. epidermidis (one strain) have been evaluated for comparative growth and haemolysin titre in both brain heart infusion (BHI) and in developed, nutritionally adequate, chemically defined media (CDMs) varying only in amino acid composition. The ability to show a particular haemolytic profile was strain-dependent and the haemolytic titre (HU50/ml) was both strain- and medium-dependent. Highest titres of both alpha and beta type haemolysins were obtained in BHI. Maximum titres were in general detected in the late exponential phase in both CDMs and BHI. Titres declined during the stationary phase in CDMs. Staphylococcus epidermidis produced a delta-type haemolysis profile on BHI-based blood agars, but only rabbit blood was sensitive in agars based on a developed, chemically defined medium (CDM/A; 13 amino acids) in which all six staphylococci grew. The addition of yeast extract to CDM/A increased alpha haemolysin titre, but suppressed beta haemolysin formation; beta haemolysin was, however, detected in yeast extract/phosphate-buffered saline. Strain Wood 46 degraded haemoglobin, but only in (initially) whole blood; red blood cell-free haemoglobin-rich plates (BHI) were unaffected during growth. A novel haemolytic profile is described for Staph. aureus NCTC 8532 growing on blood agars based on CDM/A and may relate to the production of methaemoglobin during haemolysis.     

An isogenic haemolysin-deficient mutant of Haemophilus ducreyi lacks the ability to produce cytopathic effects on human foreskin fibroblasts

The haemolysin of Haemophilus ducreyi is the newest member of the Proteus/Serratia family of pore-forming toxins. In order to assess the role of the haemolysin in virulence, we constructed an isogenic haemolysin-deficient mutant of H. ducreyi strain 35000. This strain, designated 35000-3, lacks detectable haemolytic activity. We tested H. ducreyi strains 35000 and 35000-3 for their cytopathic activity against human foreskin fibroblasts (HFFs). We observed strong cytopathic activity when strain 35000 was co-cultured with HFFs. In contrast, cytopathic activity was not observed when strain 35000-3 was co-cultured with HFF cells. We also analysed the isogenic pair of H. ducreyi strains for cytopathic activity against HeLa cells and the keratinocyte cell line HaCaT. Strains 35000 and 35000-3 were strongly cytotoxic when co-cultured with HeLa cells. HaCaT monolayers were slightly damaged by cocultivation with strain 35000-3 but this damage was much less than that observed when HaCaT cells were cocultured with strain 35000. These results indicate that the H. ducreyi haemolysin is responsible for the previously observed cytotoxic activity against HFF cells and is partially responsible for the activity observed with HaCaT cells. The haemolysin, however, is not responsible for the activity observed with HeLa cells.     

Effect of a fungal Cu/Zn superoxide dismutase on the cell-mediated immune response in Graffi tumor bearing hamsters

The antibody-dependent cell cytotoxicity (ADCC) of spleen lymphocytes, isolated from hamsters with progressing myeloid Graffi tumor, was studied. The effect of the application of Cu/Zn superoxide dismutase, isolated from the fungal strain Humicola lutea (HL SOD), before and during tumor transplantation on the lymphocyte ADCC was examined. Myeloid Graffi tumor cells as target cells were used. Antibodies from a rabbit hyper-immune anti-tumor Graffi cells serum, or from tumor-bearing hamsters serum were used in the test. The leukocyte adherence inhibition (LAI) in the presence of tumor antigen was examined also during tumor progression. ADCC of the spleen lymphocytes, determined by both, rabbit and hamster anti-tumor antibodies, decreased during tumor progression. The optimum treatment of the animals by HL SOD induced a 20-30% increase of lymphocyte cytotoxicity against myeloid Graffi tumor cells. Cytotoxicity in presence of tumor bearing hamsters serum was twofold lower as compared to that one determined in the presence of rabbit hyper-immune anti-myeloid Graffi tumor cells serum. Leukocyte adherence inhibition (LAI) index in the presence of tumor antigen increased during tumor development in the groups of treated and untreated animals. The LAI indices of HL SOD-treated tumor-bearing hamsters were lower than that of untreated animals with tumors, what can be explained by a higher adherence ability of leukocytes induced by HL SOD treatment (in formula for calculation of LAI index the adherence value is in the denominator). The results show the beneficial effect of HL SOD on the cell-mediated immune response of myeloid Graffi tumor bearing hamsters, what is probably due to the participation of the enzyme in the host's oxidant-antioxidant balance.     

In vitro studies of haemolysis by some staphylococci grown in chemically defined media

Staphylococcus aureus (five strains) and Staph. epidermidis (one strain) have been evaluated for comparative growth and haemolysin titre in both brain heart infusion (BHI) and in developed, nutritionally adequate, chemically defined media (CDMs) varying only in amino acid composition. The ability to show a particular haemolytic profile was strain-dependent and the haemolytic titre (HU₅₀/ml) was both strain- and medium-dependent. Highest titres of both alpha and beta type haemolysins were obtained in BHI. Maximum titres were in general detected in the late exponential phase in both CDMs and BHI. Titres declined during the stationary phase in CDMs. Staphylococcus epidermidis produced a delta-type haemolysis profile on BHI-based blood agars, but only rabbit blood was sensitive in agars based on a developed, chemically defined medium (CDM/A; 13 amino acids) in which all six staphylococci grew. The addition of yeast extract to CDM/A increased alpha haemolysin titre, but suppressed beta haemolysin formation; beta haemolysin was, however, detected in yeast extract/phosphate-buffered saline. Strain Wood 46 degraded haemoglobin, but only in (initially) whole blood; red blood cell-free haemoglobin-rich plates (BHI) were unaffected during growth. A novel haemolytic profile is described for Staph. aureus NCTC 8532 growing on blood agars based on CDM/A and may relate to the production of methaemoglobin during haemolysis.     

Translocation of exogenous platelet-activating factor and its lyso-compound through plasma membranes is a rate-limiting step for their metabolic conversions into alkylacylglycerophosphocholines in rabbit platelets and guinea-pig leukocytes

Platelets and leukocytes are known to degrade platelet-activating factor (PAF), a potential mediator of inflammation, to its lyso-derivative (lyso-PAF) and then convert this to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines. However, little is known about the mechanism of internalization of PAF and lyso-PAF, which is a prerequisite for their metabolism within the cells. In this work, the internalization of PAF and lyso-PAF by rabbit platelet and guinea-pig leukocyte plasma-membranes were examined by the washing method with bovine serum albumin. The rates of translocation of PAF and lyso-PAF across guinea-pig plasma membranes were significantly higher than those across rabbit platelets. In these cells, the translocation of PAF was found to be accelerated indirectly by activation of PAF receptors by a small portion of added PAF. Results suggest that a temperature-dependent diffusion process is involved in the internalization of these phospholipids. In both rabbit platelets and guinea-pig leukocytes, the translocation of PAF and lyso-PAF through the plasma membranes was shown to be rate-limiting for the metabolic conversion of these compounds to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine.     

Enhancement of cytotoxicity of human peripheral blood lymphocytes by interferon

Human lung carcinoma cells persistently infected with mumps virus (Pc-10/MpV) were lysed with human peripheral blood mononuclear leukocytes (PBML) obtained from seropositive donors who had anti-mumps virus-neutralizing antibody in their sera. This cellular cytotoxicity was due not to the cytotoxic T lymphocytes but mainly to the non-T, non-B cells, possibly related to natural killer (NK) cells. Moreover, it was concerned not with antibody against mumps virus antigens but with alpha-interferon (IFN-alpha) produced in the mixture of human PBML and Pc-10/MpV cells, since this cellular cytotoxicity was suppressed by anti-human IFN-alpha rabbit serum. Exogeneous IFN-alpha augmented the cytotoxicity of non-T, non-B cells, not T cells, for the uninfected Pc-10 cells. IFN-gamma that had been induced by heat-killed Listeria monocytogenes in PBML had the same capacity to augment NK activity did IFN-alpha.     

猪链球菌2型溶血素的化学修饰

用DTT、H2O2、DEPC、EDC、NAI、NBS、PCMB、2,3Diacetyl和SA 等9种化学修饰剂,处理猪链球菌2型江苏分离株提纯的溶血素,研究其分子中氨基酸侧链基团与其溶血活性的关系。结果表明,巯基、氨基、羧基和酪氨酸残基等与其溶血活性无关,而色氨酸、组氨酸和精氨酸的化学修饰引起溶血活性的大幅度下降,二硫键的化学修饰引起溶血活性的大幅度增强。显示色氨酸、组氨酸和精氨酸残基是该溶血素的活性必需基团,二硫键的断裂可引起其活性增强。     

Computer-assisted morphometry of the intracapillary leukocyte pool in the rabbit lung

Computer-assisted morphometry was performed to evaluate the number and cell characteristics of capillary and alveolar leukocytes in rabbit lungs. An image-processing system and a programmable spreadsheet program were used, which allowed morphometric analysis of a large reference area. Neutrophils represented the largest intracapillary leukocyte population (2.2×10⁷/ml parenchyma, which corresponds to an approximately 104-fold microvascular enrichment of this cell type related to cell counts calculated for the capillary blood volume). In addition, large numbers of intracapillary lymphocytes (1.7×10⁷/ml parenchyma; 47-fold enrichment) and monocytes (0.3×10⁷/ml parenchyma; 86-fold enrichment) were detected. The total count of pulmonary leukocytes thus approximated the total number of pulmonary endothelial cells; and the total circulating pools of the different leukocytes were surpassed by the corresponding lung capillary pools, 3.2-fold for neutrophils, 1.2-fold for lymphocytes and 4.8-fold for monocytes. In contrast, alveolar cell numbers ranged from 1–2% of the capillary counts for all types of leukocytes. We conclude that the rabbit lung microvasculature harbours large pools of immunocompetent cells, which may contribute to host-defense mechanisms at the gas-exchange area.     

Immunohistochemical study on arachidonate 5-lipoxygenase in porcine leukocytes and other tissues

Arachidonate 5-lipoxygenase is an enzyme that catalyzes the oxygenation of arachidonic acid, producing 5-hydroperoxy acid. This enzymatic reaction initiates the biosynthesis of various bioactive leukotrienes. An antiserum was raised in a rabbit against the purified 5-lipoxygenase of porcine leukocytes, and various types of porcine leukocytes were immunostained by use of the antibody. As examined by light and electron microscopy, neutrophils and eosinophils were positively stained. The 5-lipoxygenase was localized in the cytoplasm but not in the plasma membrane and subcellular organelles of the positively stained cells. In contrast, lymphocytes were unstained. In porcine ileum, the majority of 5-lipoxygenase-positive cells were eosinophils and mast cells resident in the lamina propria mucosae, whereas parenchymal cells were not stained. In porcine lung, certain bronchiolar or bronchial epithelial cells were clearly immunostained, in addition to eosinophils and mast cells found in the interstitium.     

Pathology of Exfoliated Oropharyngeal Epithelial Cells Infected with Wild-Type Adenovirus

Adenovirus disease was diagnosed in 2 hr by staining exfoliated infected oropharyngeal cells with fluorescein-tagged rabbit antisera to adenovirus types 4 and 7. The method was as sensitive as the standard virus isolation procedures, but serological cross reactions were observed. Viral antigens were detected in both the nucleus and cytoplasm of infected cells. Infection was accompanied by the outpouring of large numbers of polymorphonuclear leukocytes and smaller numbers of mononuclear cells. The method provides a model for the study of the cellular response to viral upper respiratory disease.     

Co-ordinate expression of virulence genes during swarm-cell differentiation and population migration of Proteus mirabilis

The uropathogenic Gram-negative bacterium Proteus mirabilis exhibits a form of multicellular behaviour termed swarming, which involves cyclical differentiation of typical vegetative cells into filamentous, multinucleate, hyperflagellate swarm cells capable of rapid and co-ordinated population migration across surfaces. We observed that differentiation into swarm cells was accompanied by substantial increases in the activities of intracellular urease and extracellular haemolysin and metalloprotease, which are believed to be central to the pathogenicity of P. mirabilis. In addition, the ability of P. mirabilis to invade human urothelial cells in vitro was primarily a characteristic of differentiated swarm cells, not vegetative cells. These virulence factor activities fell back as the cells underwent cyclical reversion to the vegetative form (consolidation), in parallel with the diagnostic modulation of flagellin levels on the cell surface. Control cellular alkaline phosphatase activities did not increase during differentiation or consolidation. Non-flagellated, nonmotile transposon insertion mutants were unable to invade urothelial cells and they generated only low-level activities of haemolysin, urease and protease (0-10% of wild type). Motile mutants unable to differentiate into swarm cells were comparably reduced in their haemolytic, ureolytic and invasive phenotypes and generated threefold less protease activity. Mutants that were able to form swarm cells but exhibited various aberrant patterns of swarming migration produced wild-type activities of haemolysin, urease and protease, but their ability to enter urothelial cells was three- to 10-fold lower.(ABSTRACT TRUNCATED AT 250 WORDS)