Pasteurella haemolytica leukotoxin inhibits mitogen-induced bovine peripheral blood mononuclear cell proliferation in vitro

In this study we demonstrate that partially purified Pasteurella haemolytica leukotoxin inhibits the proliferative response of bovine peripheral blood mononuclear cells (PBMC) to mitogens in vitro. Inhibition of PBMC proliferation did not appear to be due to cell death. Addition of a neutralizing anti-leukotoxin monoclonal antibody restored a normal proliferative response.     

Cytokine profiles following interaction between bovine alveolar macrophages andPasteurella haemolytica

Pasteurella haemolyticais a Gram negative bacterium frequently isolated from the lungs of calves suffering from a fibrinous pneumonic condition known as shipping fever. To understand the pathogenesis of this disease, we investigated the induction of cytokin gene expression in cultures of bovine alveolar macrophages (BAM) stimulated with heat-killedP. haemolytica. Northern blot analysis of total RNA showed thatP. haemolyticainduced early, abundant, and consistent synthesis of IL-1, TNF-α, and IL-8 mRNA. Cytokine mRNAs were detected within 1 hr post-stimulation with heat-killedP. haemolytica. IL-1 and IL-8 mRNA accumulated to high levels with increase in stimulation time, whereas TNF-α mRNA clearly declined by 4 and 8 h post stimulation. IL-1, TNF-α, and IL-8 proteins were also secreted into the culture medium of BAM stimulated with heat-killedP. haemolytica. All three proteins were detected at high levels 8 and 12 h post stimulation withP. haemolytica. BAM cells treated with bovine interferon-α and then stimulated withP. haemolyticaproduced higher amounts of IL-1, IL-8 and TNF-α proteins compared to BAM stimulated withP. haemolyticaalone. These findings demonstrate the powerful and selective induction of cytokine mRNA and protein synthesis in BAM stimulated with heat-killedP. haemolyticaand may explain certain aspects of shipping fever pathogenesis.     

Induction of nitric oxide production by bovine alveolar macrophages in response toPasteurella haemolyticaA1

We assessed the kinetics of inducible nitric oxide synthase (iNOS) mRNA expression and production of nitric oxide (NO) in bovine alveolar macrophages (AMs) stimulated with purified lipopolysaccharide (LPS) fromPasteurella haemolyticastrain 12296. The effect of LPS on iNOS gene expression was dose-dependent and was expressed maximally at 24 h after stimulation with 10 μg/ml of LPS. Production of NO measured as secreted nitrite in supernatants took place in a time and dose-dependent manner with peak production at 24 h after LPS stimulation. Recombinant bovine gamma interferon (rbγIFN) augmented the LPS-induced iNOS gene expression and production of NO. The ability of LPS to induce iNOS gene expression and NO production either alone or in combination with rbγIFN was significantly abrogated by polymyxin B. In addition, the iNOS inhibitor N^G-monomethyl-Larginine (L-NMMA) significantly inhibited LPS and rbγIFN+LPS induced NO production. Our results also demonstrated that NO produced from an exogenous NO donor sodium nitroprusside (SNP), and NO generated from LPS-stimulated AMs (endogenous) caused cytotoxic injury to bovine pulmonary artery endothelial cells in a dose-dependent manner. The cytotoxic injury caused by NO generated from LPS stimulated AMs was inhibited by polymyxin B or L-NMMA. There was a markedly increased concentration of nitrite in the lung lavage fluids of calves followingP. haemolyticainfection. These findings support a role for NO in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.     

Lipopolysaccharide enhances cytolysis and inflammatory cytokine induction in bovine alveolar macrophages exposed to Pasteurella (Mannheimia) haemolytica leukotoxin

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) and lipopolysaccharide (LPS) are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies have characterized in vitro responses of bovine alveolar macrophages (AMs) to Lkt and LPS. Activation of AMs with Lkt or LPS causes induction of proinflammatory cytokines, and Lkt causes cytolysis of AMs at higher concentrations. Since AMs are exposed to both of these bacterial virulence factors during disease, previous studies may have underestimated the possibility of functional interactions between Lkt and LPS. The purpose of this study was to characterize the effect of simultaneous exposure to both Lkt and LPS on AM cytolysis and proinflammatory cytokine expression. Using cellular leakage of lactate dehydrogenase as an indirect measure of cytolysis, we studied AM responses to Lkt alone, LPS alone and Lkt+LPS. We found that 80-200 pg/ml LPS, which does not itself cause cytolysis, synergistically enhanced the cytolysis induced by 2-5 Lkt units (LU)/ml Lkt. Northern blot analysis demonstrated that synergism between Lkt and LPS resulted in increased levels of IL-8 mRNA, and that the kinetic patterns of TNF-alpha and IL-8 mRNA expression induced by Lkt+LPS differed from those induced by each agent separately. Finally, the WEHI 164 (clone 13) bioassay was used to show that Lkt/LPS synergism resulted in enhanced secretion of biologically active TNF-alpha. These results provide direct evidence of synergism between Lkt and LPS in AM cytolysis and inflammatory cytokine expression. Additional studies to characterize the molecular basis of this phenomenon are indicated.     

Identification of major antigenic proteins ofPasteurella piscicida

Two different antigenic protein-coding clones (PPA1 and PPA2) were isolated using anti-Pasteurella piscicidarabbit serum from a genomic DNA library ofP. piscicidastrain KP9038. The PPA1 and PPA2 expressed 7 kDa and 45 kDa proteins inEscherichia coli, respectively, and the molecular sizes of these expressed proteins are the same as these of the major antigenic proteins ofP. piscicida. PPA1 encodes a protein of 83 amino acids residues, which is similar to the bacterial lipoprotein. Comparison of the predicted amino acid sequence of the PPA1-encoded 7 kDa protein ofP. piscicidawith previously reported bacterial lipoprotein sequence data revealed that it shares about 40% amino acid sequence identity. PPA2 has two large open reading frame (ORFs). The larger ORF (encoding 452 amino acid residues) encodes a homolog of DegQ protease, and the smaller ORF (371 amino acid residues) encodes a homolog of DegS protease. The antibodies reacted with the larger ORF-encoded 45 kDa DegQ homolog protein. The DegQ and DegS homolog proteins contain an export signal and a serine protease active site. The structural features of the PPA2-coding locus are similar to those of the loci inE. colifor thedegQanddegSserine protease genes. A sequence in the 3′ non-coding region ofVibrio hollisaethermostable hemolysin gene that is highly homologous with a similar located sequence in thePseudomonas putidap-cresol methylhydroxylase gene is also found in the 3′ non-coding region of thedegShomolog gene of the PPA2.     

CD1d-restricted mouse Vα14 and human Vα24 T cells: lymphocytes of innate immunity

Mouse V α 14 T cells and their human homologs, V α 24 T cells, are prominent subsets of CD1d-restricted T cells. Here we discuss their striking similarities to B-1 B cells andγδ T cells and propose that these immune cells mediate various innate strategies in response to endogenous or exogenous danger signals.     

Association of αvβ3 integrin expression with the metastatic potential and migratory and chemotactic ability of human osteosarcoma cells

Introduction. Expression of adhesion molecules such as α _v β ₃ integrin has been associated with the metastatic potential of tumor cells. The purpose of this study was to determine whether α _v β ₃ expression correlated with the metastatic potential of human osteosarcoma cells. Materials and methods. We developed a series of sublines (LM2–LM7) from human osteosarcoma SAOS parental cells, with progressively increasing potential to form lung metastases in nude mice after intravenous injection. SAOS parental and LM2 cells were poorly metastatic, but LM7 cells resulted in visible metastatic lung nodules by 6–8 weeks. We quantified α _v β ₃ integrin expression using flow cytometry. Results. α _v β ₃ expression correlated with the metastatic potential of the cells, with LM7 cells showing the highest expression. LM7 cell adhesion to vitronectin decreased after treatment with echistatin, a RGD-containing peptide antagonist of α _v β ₃. LM7 cells demonstrated higher chemotactic activity than SAOS cells to a homogenate made from lung tissue. This chemotactic activity was also inhibited by echistatin. These data indicated that α _v β ₃ was critical for the migration of LM7 cells to the lung homogenate. Chemotaxis to a liver homogenate was the same for LM7 and SAOS cells. Migration of LM7 cells through lung endothelial cells was higher than that through liver endothelial cells, and echistatin again inhibited this migration. Conclusions. α _v β ₃ integrin expression may play a role in the metastatic potential of osteosarcoma cells by enhancing the ability of the cells to migrate specifically to the lung. α _v β ₃ integrin may therefore be a potential new target for osteosarcoma.This revised version was published online in August 2005 with a corrected cover date.     

A rat model for combined Trypanosoma cruzi and Pneumocystis carinii infection

Dexamethasone treated rats inoculated with Trypanosoma cruzi developed acute parasitemia. In addition, these animals concomitantly developed severe Pneumocystis carinii pneumonia (PCP) and died after 4 weeks of immunosuppression (100%). However, immunocompetent (untreated) rats inoculated with T. cruzi did not acquire P. carinii and recovered from T. cruzi infection. Rats immunosuppressed, but not inoculated with T. cruzi, developed only PCP and died 5–6 weeks later (93%). In contrast, immunocompetent or immunocompromized IRC mice infected with T. cruzi all died of acute parasitemia in only 8–12 days with no detectable PCP infection. In conclusion, rats immunosuppressed and T. cruzi inoculated can serve as a MOPPS model for a single drug evaluation. In addition, T. cruzi infection independently does not provoke P. carinii pneumonia in this model. Finally, patients with Chagas» disease treated with corticosteroids may be at risk for PCP and should be considered for chemoprophylaxis.     

Determination of the rpoB gene sequences of Bartonella henselae and Bartonella quintana for phylogenic analysis

Using the Genome Walker™ procedure, which allows PCR amplification of genomic DNA using a single gene-specific primer and direct automated sequencing methodology, we obtained the nucleotide sequence of the RNA polymerase β subunit (rpoB) from Bartonella henselae and Bartonella quintana. A phylogenetic tree constructed from these data and other rpoB sequences available in GenBank is, in part, consistent with those previously derived from 16S rRNA gene sequences and confirms the position of Bartonella within the α subdivision of Proteobacteria. In fact, this analysis showed that rpoB data are similar to 16S rRNA data for the α, β and γ subdivisions of Proteobacteria. In contrast, concerning other bacteria included in our study, the topologies of phylogenetic trees were different. Based on the bootstrap values derived from rpoB phylogenic analysis, we believe that this molecule should contribute to better understanding the evolutionary process.     

Comparison of the abilities ofSalmonella typhimurium rpoS,aroAandrpoS aroAstrains to elicit humoral immune responses in BALB/c mice and to cause lethal infection in athymic BALB/c mice

Salmonella typhimurium rpoS and rpoS aroA mutants are effective live vaccines in the murine model of salmonellosis (Coynault et al., Mol. Microbiol. 1996; 22: 149-60). Here, we further investigate the characteristics of these vaccines. The systemic humoral response induced by S. typhimurium rpoS, aroA and rpoS aroA vaccine candidates against S. typhimurium LPS was studied by ELISA. In BALB/c mice, the rpoS aroA strain induced a systemic anti-LPS humoral response similar to that induced by the rpoS and aroA strains. The virulence of aroA and rpoS aroA vaccines in nude (nu/nu) BALB/c mice was also compared. Salmonella typhimurium aroA and rpoS aroA vaccines both produced slowly progressing lethal infections in athymic mice inoculated i.p. but the rpoS aroA strain was more attenuated than the aroA strain, as determined by time to death and bacterial counts in spleens. Finally, a rpoS mutant of Salmonella dublin conferred protection in mice against an oral challenge with a wild-type strain of S. dublin whereas a rpoS mutant of S. typhimurium did not. This suggests that the protection provided by the S. typhimurium rpoS vaccine is serotype-dependent.     

Pseudomonas aeruginosa induces apoptosis in human endothelial cells

Pseudomonas aeruginosa has been shown to enter into human endothelial cells in vitro. To ascertain the effects of bacterial intracellular (IC) infection, endothelial cells were exposed to PAK and PAO-1 strains for 1 h and treated with gentamicin in culture medium for different periods. P. aeruginosa induced a significant production of superoxide and hydrogen peroxide by endothelial cells. Concentrations of IC bacteria were reduced progressively with time and no viable PAO-1 was detected at 24 h after infection. However, IC infection led to killing of 32.2%±2.9 and 51.8%±3.5 of the cells infected with PAK and PAO-1, respectively, as determined by the MTT assay. By three criteria (transmission electron microscopy, DNA electrophoresis and reactivity with annexin V) infected cells exhibited features of apoptosis. Treatment of infected cells with anti-oxidants (catalase, tocopherol and N -acetyl-L-cysteine) significantly decreased the percentage of cell death. In contrast, treatment with aminoguanidine, an inhibitor of inducible NO synthase, increased significantly the killing of PAO-1 infected cells. Based on these results we speculate that in response to P. aeruginosa infection, endothelial cells increase the production of reactive oxygen intermediates to eliminate IC pathogens, but cells do not resist the oxidative stress and die by apoptosis.     

Immunohistochemical assessment of fibronectin and tenascin and their integrin receptors alpha5beta1 and alpha9beta1 in gastric and colorectal cancers with lymph node and liver metastases

The aim of the current study was to assess immunohistochemically and compare the level of expression of tenascin (TN) and fibronectin (FN) and their integrin receptors alpha9beta1 and alpha5beta1 in the primary colorectal and gastric tumors, and in corresponding lymph node and liver metastases from 53 patients. We detected similar high deposition of the studied ECM proteins and their receptors in the stroma of primary tumors and in liver metastases and a lower deposition in lymph node metastases. Cytoplasmic immune reaction for FN and TN was also seen in the tumor cells. A pronounced co-localization of immune deposits for FN and TN and their receptors was found in the stroma of the center and the invasion front (IF) (p<0.0001). A significant decrease of FN immune signal was observed in the IF in primary tumors and liver metastases (p<0.0001). The levels of immunolabeling of FN and TN correlated with the differentiation grade of primary tumors (p<0.0001). In conclusion, we may say that there is heterogeneous deposition of TN, FN and their integrin receptors in the different areas of primary colorectal and gastric tumors and of their metastases. These findings imply that the studied proteins may be involved in cell processes such as growth, adhesion, migration and apoptosis.     

Cloning and molecular analysis of genes encoding two immunodominant antigensofEhrlichia risticii

Ehrlichia risticii, the causative agent of Potomac horse fever, is an obligate intracellular rickettsial organism. To understand the role of 55 and 51 kilodalton immunodominant antigens ofE. risticiiin strain variation, their genes from the 25-D and 90-12 strains were cloned, sequenced, and expressed inE. coli.Sequence analysis revealed that the gene for the 55 kDa antigen was present in a heat shock operon along with the gene for a 10 kDa protein. Homology searches indicated that the 55 kDa antigen and the 10 kDa protein were homologues ofE. coliGroEL and GroES proteins, respectively. There was no nucleotide sequence difference between the genes of the 55 kDa antigen, nor between the entire operons, from both strains ofE. risticii.The sequence-based estimation of the sizes of the putative mature 51 kDa antigens of the 90-12 and 25-D strains were 52.7 kDa and 52.9 kDa, respectively. The 51 kDa antigens from the 90-12 and 25-D strains shared a 98% identity in their deduced amino acid sequences. The difference in some of the amino acids may be responsible for variation in their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where the 51 kDa antigen of the 25-D strain migrates towards a 2 kDa lower molecular weight region. In Western blots, a 155 kDa protein that appeared to be a trimer product of the 51 kDa antigen was identified. The 55 and 51 kDa antigens were overexpressed inE. coliusing a commercial expression system, pRSET A,B,C (Invitrogen Inc., San Diego, CA, U.S.A.). The purified recombinant proteins cross-reacted with antisera toE. canisandE. sennetsu.     

Phagocytic signaling strategies: Fcγreceptor-mediated phagocytosis as a model system

Phagocytosis is a phylogenetically ancient process by which eukaryotic cells engulf insoluble substances whose size exceeds approximately 0.5 μ m. The engulfment process requires the concerted action of several fundamental cellular pathways and is governed by multiple transmembrane signaling events. Here we focus on phagocytosis mediated by a well-studied class of phagocytic receptors that recognize the Fc portion of IgG (Fc_γRs ).     

Recognition and function of Vα14 NKT cells

A novel lymphocyte lineage, V α 14 NKT cells, has recently been identified and appears to be distinct from conventional αβ T cells. V α 14 NKT cells express a single invariant Vα 14 antigen receptor that is essential for their development. They recognize a glycolipid antigen (α -galactosylceramide) or parasitic glycophosphatidylinositols (GPI) in association with a monomorphic class Ib, CD1d, and perform various functions such as Th1 and Th2 cytokine production as well as perforin/granzyme B-mediated cytotoxicity. Although the precise physiological function of V α 14 NKT cells remains to be elucidated, emerging experimental evidence suggests their intriguing regulatory features in the immune system.     

Apoptosis: a possible tactic ofHaemophilus somnusfor evasion of killing by bovine neutrophils?

Haemophilus somnusis an important veterinary pathogen that causes respiratory disease, arthritis, septicaemia and abortion in cattle and sheep. In the present study we investigated the possibility thatH. somnusresists killing by bovine neutrophils, by causing the latter to undergo morphological changes consistent with apoptosis. Both serum-sensitive and serum-resistant strains ofH. somnusenhanced bovine neutrophil chromatin condensation and shape change (i.e. zeiosis)in vitro, suggesting that the cells were undergoing apoptosis. Heat-killed or formalin-killedH. somnushad less effect than viableH. somnus.Chromatin margination of neutrophils was greater whenH. somnuswas opsonized with adult bovine serum, which facilitates phagocytosis of the bacteria.H. somnusculture filtrates did not cause bovine neutrophil chromatin condensation. These findings suggest that direct contact withH. somnusis required for the maximal effect on bovine neutrophils. Apoptosis was confirmed by flow cytometry, using propidium iodide staining to detect DNA fragmentation. These findings suggest thatH. somnuscan evade killing by bovine neutrophils, in part, by inducing these cells to undergo apoptosis.     

Relationships between β-lactamase production and β-lactam susceptibility of environmental strains of Yersinia kristensenii

Strains of Yersinia kristensenii display high susceptibility to carbenicillin (MIC₉₀ < μg/ml) in comparison with the majority of environmental strains of Yersinia closely related to Y. enterocolitica which are resistant to this antibiotic (MIC₉₀ > 256 μg/ml). β-lactamases of 39 strains of Y. kristensenii isolated from foods were analysed by isoelectric focusing and gel electrophoresis of ultrasonically disrupted uninduced cultures. β-lactamase patterns showed the presence of only one of three classes of enzymes of pI 6.7, 7.6 and 8.2, respectively, by strain. One β-lactamase showed electrophoretic mobility different (EM + 2.0 cm/h) from that of all the other enzymes (EM + 1.6 cm/h) belonging to the class of pI 7.6. Induction by cefoxitin revealed the existence of inducible β-lactamases in two out of eight selected strains. The substrate profile of these enzymes, which are probably chromosomally mediated, showed a predominant cephalosporinase activity. None of the type A and B β-lactamases described by Cornelis and Abraham in Y. enterocolitica were found in any of the strains examined. The lack of β-lactamase A (a penicillinase) accounts for the carbenicillin susceptibility of Y. kristensenii strains.     

Evidence for programmed cell death of self-reactive γδ T cell receptor-positive thymocytes

The negative selection of T cells expressing the γδ T cell antigen receptor (γδ T cells) was studied using transgenic mice expressing a γδ receptor with specificity for an H-2T-linked class I major histocompatibility complex molecule from H-2^b mice. The potentially self-reactive γδ thymocytes in H-2^b/d transgenic mice are larger and have lower levels of γδ T cell receptor expression than γδ thymocytes from H-2^d mice. H-2^b/d γδ thymocytes do not respond to H-2^b antigen-presenting cells, and thus are inactive compared to H-2^d γδ thymocytes. However, the H-2^b/d γδ thymocyte population, but not the H-2^d γδ thymocyte population, undergoes a high rate of programmed cell death when placed in overnight culture. These observations constitute the first direct evidence that self-reactive γδ thymocytes undergo programmed cell death. This in vitro programmed cell death of self-reactive γδ thymocytes may reflect the clonal deletion process that results in a depletion of γδ T cells in the peripheral lymphoid organs of adult H-2^b/d mice. We also present evidence that self-reactive γδ T cells, similarly to αβ T cells, undergo a lesser degree of clonal deletion in neonatal mice compared to adult mice.     

Live Bartonella henselae enhances endothelial cell proliferation without direct contact

The proliferation of human umbilical vein endothelial cells (HUVECs) cocultivated with live B. henselae was enhanced in a bacterial dose-dependent manner, and the stimulatory effect was specific to vascular endothelial cells. The inactivation of B. henselae by UV or heat treatment abolished its stimulatory activity, suggesting that live bacteria is necessary for the growth stimulation effect. To investigate the role of direct contact, live B. henselae were separated from HUVECs by a filter membrane (Millicell-CM insert). Even under this condition, an enhanced proliferation of HUVECs was observed. However, no morphological changes in the HUVECs were apparent compared to the B. henselae -infected cells. Furthermore, we isolated a nonpiliated strain of B. henselae that is unable to attach to and enter into endothelial cells. The nonpiliated strain possessed the ability to stimulate the proliferation of cocultivated HUVECs the same as the piliated strain. Moreover, the culture supernatants of B. henselae were also able to induce HUVEC proliferation. Our results indicate that the stimulation of HUVEC proliferation by B. henselae is mediated by soluble factor(s) secreted from the bacteria.