Magnetic droplet soliton pairs.

We demonstrate magnetic droplet soliton pairs in all-perpendicular spin-torque nano-oscillators (STNOs), where one droplet resides in the STNO free layer (FL) and the other in the reference layer (RL). Typically, theoretical, numerical, and experimental droplet studies have focused on the FL, with any additional dynamics in the RL entirely ignored. Here we show that there is not only significant magnetodynamics in the RL, but the RL itself can host a droplet driven by, and coexisting with, the FL droplet. Both single droplets and pairs are observed experimentally as stepwise changes and sharp peaks in the dc and differential resistance, respectively. While the single FL droplet is highly stable, the coexistence state exhibits high-power broadband microwave noise. Furthermore, micromagnetic simulations reveal that the pair dynamics display periodic, quasi-periodic, and chaotic signatures controlled by applied field and current. The strongly interacting and closely spaced droplet pair offers a unique platform for fundamental studies of highly non-linear soliton pair dynamics.

Detection of MET mRNA in gastric cancer in situ. Comparison with immunohistochemistry and sandwich immunoassays.

Determination of predictive biomarkers by immunohistochemistry (IHC) relies on antibodies with high selectivity. RNA in situ hybridization (RNA ISH) may be used to confirm IHC and may potentially replace it if suitable antibodies are not available or are insufficiently selective to discriminate closely related protein isoforms. We validated RNA ISH as specificity control for IHC and as a potential alternative method for selecting patients for treatment with MET inhibitors. MET, the HGF receptor, is encoded by the MET proto-oncogene that may be activated by mutation or amplification. MET expression and activity were tested in a panel of control cell lines. MET could be detected in formalin fixed paraffin, embedded (FFPE) samples by IHC and RNA ISH, and this was confirmed by sandwich immunoassays of fresh frozen samples. Gastric cancer cell lines with high MET expression and phosphorylation of tyrosine-1349 respond to the MET inhibitor, BAY-853474. High expression and phosphorylation of MET is a predictive biomarker for response to MET inhibitors. We then analyzed MET expression and activity in a matched set of FFPE vs. fresh frozen tumor samples consisting of 20 cases of gastric cancer. Two of 20 clinical samples investigated exhibited high MET expression with RNA ISH and IHC. Both cases were shown by sandwich immunoassays to exhibits strong functional activity. Expression levels and functional activity in these two cases were in a range that predicted response to treatment. Our findings indicate that owing to its high selectivity, RNA ISH can be used to confirm findings obtained by IHC and potentially may replace IHC for certain targets if no suitable antibodies are available. RNA ISH is a valid platform for testing predictive biomarkers for patient selection.

Identification and functional characterization of flgM, a gene encoding the anti-sigma 28 factor in Pseudomonas aeruginosa.

We describe here the functional characterization of the putative flgM gene of Pseudomonas aeruginosa. FlgM of P. aeruginosa is most similar to FlgM of Vibrio parahaemolyticus. A conserved region is present in the C-terminal half of the FlgM of P. aeruginosa and in FlgM homologues of other organisms that includes the sigma(28) binding domain. A role for the flgM gene of P. aeruginosa in motility was demonstrated by its inactivation. The beta-galactosidase activity of a transcriptional fusion of the fliC promoter to lacZ was upregulated in the flgM mutant, suggesting that the activity of FliA, the sigma factor that regulates fliC, was increased. Consistent with these results, an increased amount of flagellin was demonstrated in the flgM mutant of P. aeruginosa strain PAK by Western blot, suggesting that FlgM negatively regulates transcription of fliC by inhibiting the activity of FliA. Direct interaction of the P. aeruginosa FlgM with the alternative sigma factor sigma(28) was demonstrated by utilizing the yeast two-hybrid system. Three putative consensus sigma(54) recognition sites and one sigma(28) site were found in the flgM upstream region. However, analysis of the transcriptional fusion of the flgM promoter to lacZ in different mutant backgrounds showed that the flgM promoter was not entirely dependent on either sigma(28) or sigma(54). A transcript was detected by primer extension that was 8 bp downstream of the consensus sigma(28)-binding site. Thus, a system for the control of flagellin synthesis by FlgM exists in P. aeruginosa that is different from that in the enteric bacteria and seems to be most similar to that of V. cholerae where both sigma(28)-dependent and -independent mechanisms of transcription exist.

The role of different protein components from the Haemophilus ducreyi cytolethal distending toxin in the generation of cell toxicity.

Cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a multicomponent toxin, encoded by an operon consisting of three genes, cdtABC. To investigate the role of the individual products in generation of toxicity, recombinant plasmids were constructed allowing expression of each of the genes individually or in different combinations in Escherichia coli and Vibrio cholerae. Expression of all three genes (cdtABC) was necessary to generate toxicity on cells, and no activity was obtained using combinations in which only one or two of the genes were expressed. Of the individual gene products, the CdtA was shown to exist in two forms with an MW of 23 and 17 kDa, respectively. The CdtB protein alone resulted in DNase activity. CdtC purified from both toxic and non-toxic extracts (from strains expressing cdtCAB and cdtC, respectively) had a molecular weight of about 20 kDa and reacted with a CdtC-specific monoclonal antibody. However, the protein isoelectric point (pI) of CdtC from toxic preparations was about 1.5 pH units more basic than from non-toxic ones. Both forms were immunogenic giving rise to toxin-neutralizing antibodies. Toxicity was reconstructed by combining non-toxic cell sonicates from E. coli, expressing CdtA, CdtB and CdtC proteins individually. Only combinations including all three products gave toxicity, indicating that all are actively involved in the generation of toxic activity on cells. The reconstruction resulted in a 1.5 pH unit shift in the PI of CdtC, making it identical to that of the protein isolated from bacteria expressing cdtABC. The results showed that the CdtB component produces DNase activity, but cell toxicity depends on the involvement of the other two components of CDT and is associated with absorption of all three proteins by HEp-2 cells.

Detection of canine distemper virus nucleoprotein RNA by reverse transcription-PCR using serum, whole blood, and cerebrospinal fluid from dogs with distemper.

Reverse transcription-PCR (RT-PCR) was used to detect canine distemper virus (CDV) nucleoprotein (NP) RNA in serum, whole blood, and cerebrospinal fluid (CSF) samples from 38 dogs with clinically suspected distemper. Results were correlated to clinical findings, anti-CDV neutralizing antibody titers, postmortem findings, and demonstration of CDV NP antigen by immunohistochemistry. The specificity of the RT-PCR was ensured by amplification of RNA from various laboratory CDV strains, restriction enzyme digestion, and Southern blot hybridization. In 29 of 38 dogs, CDV infection was confirmed by postmortem examination and immunohistochemistry. The animals displayed the catarrhal, systemic, and nervous forms of distemper. Seventeen samples (serum, whole blood, or CSF) from dogs with distemper were tested with three sets of primers targeted to different regions of the NP gene of the CDV Onderstepoort strain. Expected amplicons were observed in 82, 53, and 41% of the 17 samples, depending upon the primer pair used. With the most sensitive primer pair (primer pair I), CDV NP RNA was detected in 25 of 29 (86%) serum samples and 14 of 16 (88%) whole blood and CSF samples from dogs with distemper but not in body fluids from immunohistochemically negative dogs. Nucleotide sequence analysis of five RT-PCR amplicons from isolates from the field revealed few silent point mutations. These isolates exhibited greater homology to the Rockborn (97 to 99%) than to the Onderstepoort (95 to 96%) CDV strain. In summary, although the sensitivity of the RT-PCR for detection of CDV is strongly influenced by the location of the selected primers, this nucleic acid detection system represents a highly specific and sensitive method for the antemortem diagnosis of distemper in dogs, regardless of the form of distemper, humoral immune response, and viral antigen distribution.

Dominating interleukin-10 mRNA expression induction in cerebrospinal fluid cells of dogs with natural canine distemper virus induced demyelinating and non-demyelinating CNS lesions.

Canine distemper virus (CDV) infection in dogs is commonly associated with demyelinating leukoencephalitis (DL). Although the mechanism of primary demyelination in distemper remains undetermined recent studies showed a direct virus-induced cytolysis in early non-inflammatory and immune-mediated mechanisms in inflammatory lesions. To further investigate the pathogenesis of this morbillivirus-induced demyelination the expression of a variety of cytokine mRNA species (interleukin (IL)-1beta, IL-2, IL-6, IL-10, IL-12, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, and interferon (IFN)-gamma in cerebrospinal fluid cells of 12 dogs with CDV encephalitis was investigated employing reverse transcription-polymerase chain reaction (RT-PCR) and these findings were correlated to the type of CNS lesions. Neuropathology revealed the whole spectrum of distemper DL lesions from acute to chronic alterations, however, most plaques lacked active demyelination. Three control animals were devoid of any cytokine expression, whereas in distemper animals IL-10 transcripts were found in nine dogs with acute and chronic lesions. IL-6, TNF, and TGF mRNA was found in six, four, and three animals, respectively. IL-12 and IFN-gamma, suggestive of a TH1-like dominated immune response, were detected only in one animal with chronic lesions. Summarized, TNF and IL-6, associated with disease exacerbation, and IL-10 and TGF, indicative of remission, were often observed simultaneously in distemper DL and could not be assigned to a specific disease stage. However IL-10 mRNA remained the most frequently detected cytokine indicating a stage of inactivity in most animals investigated.

Cytokine mRNA expression in whole blood samples from dogs with natural canine distemper virus infection.

Cytokines are soluble polypeptides with many physiological functions and a special role during infection and inflammation. Little is known about cytokine regulation in naturally occurring viral diseases of animals. Especially the role of cytokines in the development and progression of lesions in canine distemper virus (CDV) infection in dogs is largely unknown. Whole blood samples from 14 dogs with CDV infection and three dogs suffering from non-distemper diseases were examined for mRNA of pro-inflammatory cytokines such as interleukin-1beta (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), tumor necrosis factor-alpha (TNF), interferon-gamma (IFN), and the anti-inflammatory transforming growth factor-beta1 (TGF) using reverse transcription polymerase chain reaction (RT-PCR). Blood samples from the three dogs that showed no clinical abnormalities during a pre-vaccination physical examination served as control. CDV infection was confirmed by post-mortem immunohistochemistry for CDV nucleoprotein. The degree of immunoreactivity and the number of virus antigen positive organs were expressed as antigen index. IFN transcripts were not identified in any dog and IL-8 transcripts were present in RNA isolates from all 20 dogs. None of the other cytokines was detected in control animals. IL-1 and IL-6 were each found in one non-distemper dog and TGF transcripts were amplified in two dogs with non-distemper disease. The following transcripts were found in variable numbers in distemper dogs: IL-1 (7/14 dogs), IL-6 (3/14 dogs), IL-12 (3/14 dogs), TNF (8/14 dogs), and TGF (10/14 dogs) with multiple cytokines in ten dogs. No cytokine transcripts were detected in three distemper dogs. There was no obvious correlation between cytokine mRNA expression and respiratory and gastrointestinal tract diseases. In the CNS, demyelination was frequently associated with IL-1, IL-12, TNF and TGF mRNA expression in the blood. IL-6 transcripts were found only in animals with early CNS lesions and TGF was the only detectable cytokine in an animal with chronic demyelination. Lack of detectable cytokine transcripts in whole blood samples was associated with a high antigen index and viremia, indicating that an overwhelming virus infection may suppress cytokine production, possibly due to paralysis of the immune system. Simultaneous occurrence of pro- and anti-inflammatory cytokines in whole blood preparation from most of the dogs with distemper, indicated a complex most likely disease stage dependent orchestrated cytokine expression.

Antibodies specific to surface antigens are not effective in complement-mediated killing of Haemophilus ducreyi.

The bactericidal activity of serum is an important primary host defence against gram-negative bacteria. Little is known regarding such antibodies that are specific to outer membrane (OM) antigens as pili and lipooligosaccharides (LOS) in the bactericidal killing of Haemophilus ducreyi. Presence of serum antibodies with specificity to a 430 kDa protein (polymer of the 24 kDa protein, named fine-tangled pili) and LOS in serum from chancroid patients and healthy individuals were investigated by ELISA. Using a bactericidal assay, we investigated the role of human and rabbit antibodies with the aforementioned specificity. Accessibility of LOS and of OM antigens, as well as the deposition of components of the complement (C) system on the surface of the bacteria, was further investigated by whole-cell ELISA and immunoelectron microscopy. Immunoglobulin G (IgG) antibodies specific to the 430 kDa polymer and to LOS were demonstrated in the majority of sera from chancroid patients and healthy individuals. However, sera from chancroid patients did not significantly enhance the C-mediated killing of H. ducreyi compared with normal human serum (NHS). Similar results were demonstrated using rabbit sera to whole bacteria, specific to the 430 kDa protein and LOS of H. ducreyi. However, using the same assay noncapsulatedH. influenzae was totally killed, as were H. influenzae type b in presence of specific antibodies. This suggests a limited effectiveness of antibodies specific to surface antigens in C-mediated killing of H. ducreyi. LOS was detectable on the surface of H. ducreyi with a specific monoclonal antibody in white-cell ELISA. However, a significant enhancement of LOS detection was demonstrated on washed bacteria. OM antigens of 26, 40, 45 kDa and the major outer membrane protein (MOMP) of 43 kDa were not detectable on the surface of nonwashed and washed bacteria by specific monoclonal antibodies, indicating a lack of accessibility of these antigens on the bacterial surface. However, the C6 to C9 components of C were detected on the bacterial surface, suggesting capacity of forming the membrane attack complex. Altogether, these findings imply that antibodies specific to surface antigens, such as the 430 kDa protein and LOS, are not capable of enhancing killing of bacteria. The demonstrated relative resistance is probably due not to a lack of deposition of the membrane attack complex components, but rather to a blocking of LOS accessibility and OM proteins as potential targets of bactericidal antibodies and C action.

Characterization of mechanisms involved in adherence of Haemophilus ducreyi to eukaryotic cells.

The adherence of Haemophilus ducreyi to eukaryotic cells of various origins was investigated by means of a microassay using radiolabelled bacteria. The influence of physicochemical conditions and of different inhibitors on adherence to HEp-2 cells and human fibroblasts was examined. H. ducreyi strains manifested substantial adherence capacity (range, 11-38% of inoculum) to different cells, not discriminating between human and animal origin. The level of adherence was temperature-dependent, being substantially decreased by incubation at 4 degrees C, but was unaffected in the pH range 4-10. The adherence level was significantly reduced in the presence of sodium chloride or tetramethylurea (a hydrophobic bond-breaking agent). In addition, H. ducreyi bacteria manifested a pronounced capacity for binding Congo red to the surface, in comparison with the low binding ability of H. influenzae type b. This further indicates hydrophobic domains to be accessible on the surface of H. ducreyi. Inhibition studies with bacterial EDTA extract, sialic acid, heparin and heparan sulphate resulted in a significant reduction in adherent bacteria. However, adherence was not inhibited with crude 24 kDa pili material, LOS of H. ducreyi or fibronectin. Neither crude nor purified 24 kDa protein of H. ducreyi bacteria showed any capacity to bind monolayers of HEp-2, HeLa or human fibroblasts cells, as tested by immunoblot using specific polyclonal antibodies. The overall results suggest that adherence of H. ducreyi to eukaryotic cells is not specific to a particular cell type, human or animal. Adherence to HEp-2 cells involves a multiplicity of factors such as ionic and hydrophobic forces, and can be mediated by tissue heparin/heparan sulfate proteoglycans. However, specific binding to HEp-2 cells does not seem to be mediated by the 24 kDa pili protein of H. ducreyi.

GroEL heat shock protein of Haemophilus ducreyi: association with cell surface and capacity to bind to eukaryotic cells.

The Haemophilus ducreyi homolog of GroEL, a 58.5-kDa heat shock protein (Hsp), is a dominant protein produced not only in response to heat stress but also under in vitro growth conditions. Extracellular localization of the 58.5-kDa Hsp was investigated by whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy and in supernatants of washed bacteria by immunoblotting with a Haemophilus ducreyi GroEL-specific mouse monoclonal antibody (BB11). To investigate binding of the Hsp to eukaryotic cells, the 58.5-kDa Hsp was purified by ion-exchange and size exclusion chromatography; incubated with HEp-2 cells, HeLa cells, and human fibroblasts; and then analyzed by immunoblotting. Direct involvement of the 58.5-kDa Hsp in the adherence of H. ducreyi to HEp-2 cells was investigated by using an inhibition assay. An epitope of the 58.5-kDa Hsp was detected by whole-cell ELISA on all of the strains tested, suggesting that it is associated with the cell surface. This was also supported by immunoelectron microscopy results. In supernatants of washed bacteria, the 58.5-kDa Hsp was detected by immunoblotting after 10 h of cultivation. The 58.5-kDa Hsp bound to the eukaryotic cells tested but exerted only limited (about 20%) inhibition of H. ducreyi adherence to HEp-2 cells. These results demonstrate that the 58.5-kDa Hsp of H. ducreyi is associated with the bacterial surface, binds to eukaryotic cells, and partially influences H. ducreyi adherence to HEp-2 cells, indicating possible involvement of the 58.5-kDa Hsp in the attachment of bacteria to host cells and to each other.

Structurally defined epitopes of Haemophilus ducreyi lipooligosaccharides recognized by monoclonal antibodies.

By use of enzyme-linked immunosorbent assay and immunoblotting techniques, the migration patterns and binding epitopes of lipooligosaccharides (LOS) from 10 Haemophilus ducreyi strains were investigated with two monoclonal antibodies (MAbs), MAHD6 and MAHD7, raised against LOS from H. ducreyi ITM 2665. Closely related LOS, with defined structures, from Haemophilus influenzae, Bordetella pertussis, Aeromonas spp., and synthetic glycoproteins were also included in the analyses. The MAbs bound to conserved epitopes of LOS exposed on the surface of H. ducreyi. The MAb MAHD6 reacted with 8 of the 10 LOS from H. ducreyi but with none of the other Haemophilus or Bordetella spp. with structurally defined LOS. It is suggested that MAb MAHD6 binds to a LOS epitope (-DD-Hepp-1-->6-beta-D-Glcp-). This LOS epitope is not present in the hexasaccharide structure of LOS from H. ducreyi ITM 4747 (E. K. H. Schweda, A. C. Sundström, L. M. Eriksson, J. A. Jonasson, and A. A. Lindberg, J. Biol. Chem. 269:12040-12048, 1994). Because MAb MAHD6 reacts with the epitope mentioned above, it also discriminates between the two LOS structures, the hexasaccharide group and the nonasaccharide group, of H. ducreyi strains. MAb MAHD7 recognizes the common conserved inner core region of the LOS because it reacts with all H. ducreyi strains and with LOS with minor components in the inner core epitope structure. Rabbit polyclonal sera raised against the LOS from strains CCUG 4438 and CCUG 7470 were tested with the 10 LOS from the H. ducreyi strains. The antiserum to CCUG 7470 reacted with all H. ducreyi strains as did MAb MAHD7, whereas the antiserum to CCUG 4438 reacted with only its homologous strain and strain ITM 4747. Also, the LOSs of our reference strains CCUG 4438 and CCUG 7470 were structurally analyzed by use of sugar analyses and electrospray ionization-mass spectrometry. The hexasaccharide and nonasaccharide structures obtained from LOS of strains CCUG 4438 and CCUG 7470 were identical to the described LOS structures from H. ducreyi ITM 4747 and ITM 2665, respectively. In conclusion, the MAb MAHD6 recognizes an epitope present in the nonasaccharide LOS group, whereas the MAb MAHD7 recognizes a conserved epitope on LOS of H. ducreyi, which is present in all strains of H. ducreyi tested. Two major groups of oligosaccharides were distinguished by their LOS structures and the reactivity of monoclonal as well as polyclonal antibodies. The majority of H. ducreyi strains possess a nonasaccharide structure of LOS.

Purification and identification of Haemophilus ducreyi cytotoxin by use of a neutralizing monoclonal antibody.

Haemophilus ducreyi produces a cytotoxin responsible for the killing of cultured human epithelial cells. Cytotoxin-neutralizing antibodies were detected in the majority of sera from patients with culture-proven chancroid, and a significantly higher level of such antibodies in patients than in blood donors was noted both in areas where the disease is endemic and those where it is not. We produced neutralizing monoclonal antibodies (MAbs) in mice with a crude osmotic preparation of the cytotoxin. These antibodies, with high capacity to neutralize cytotoxicity, were used for purification and identification of the cytotoxin. Purification was performed by a two-step procedure which included Sephacryl S-200 filtration followed by immunoaffinity chromatography. The purification resulted in poor cytotoxin protein recovery and contamination with MAbs from the affinity column. The results of the gel filtration experiments and immunoblotting indicate that the active cytotoxin consists of a single, small protein with an approximate molecular mass of 20 kDa. Cytotoxins from different strains seem to have the same or similar epitopes. The cytotoxin protein was not detected in preparations from nontoxic strains. The N-terminal amino acid sequence of the 20-kDa band was E-S-N-P-D-P-T-T-Y-P-D-V-E-L-S-P-P-P. This sequence does not resemble that of any currently known bacterial protein.

Comparison of the Etest with agar dilution for antimicrobial susceptibility testing of Haemophilus ducreyi.

Two methods for determining the sensitivity of Haemophilus ducreyi to antimicrobial agents were compared; an agar dilution method and the Etest. The MICs of seven antimicrobial agents; penicillin V, ampicillin, trimethoprim, tetracycline, chloramphenicol, erythromycin and ciprofloxacin were determined for 20 strains of H. ducreyi. The MICs determined with the Etest and with the agar dilution method, showed 86% agreement within one two-fold dilution. The Etest is a good alternative method to the agar dilution technique for antimicrobial susceptibility testing of H. ducreyi. Owing to its simplicity and good reproducibility, the test is well suited for routine use in clinical microbiology laboratories in developing countries.

Localisation and immunological properties of a 24-kDa surface protein of Haemophilus ducreyi.

The cell wall and outer structures of Haemophilus ducreyi bacteria were investigated. The 24-kDa outer protein from two strains was purified with an SDS-PAGE preparative continuous-elution electrophoresis cell. The protein was further characterised by SDS-PAGE and immunoblotting, and the immunological properties were investigated by ELISA. Localisation on the bacterial surface was investigated by immuno-electron-microscopy with a polyclonal antiserum raised against the purified protein. A triple-laminar cell wall typical of gram-negative bacteria, close cellular contact between bacterial cells and outer blebs were seen on thin sections. An additional high mol. wt band of c. 165 kDa was seen when not treated by heating to 100 degrees C. A high density fibrilla-like material was detected on the bacterial cell and in the environment by negative staining and immuno-electron-microscopy with antisera specific for the 24-kDa protein. The surface localisation of the 24-kDa protein was confirmed by an ELISA technique with the specific antiserum and whole bacterial cells as antigen. The presence of antibodies to the 24-kDa protein was demonstrated in antisera to 13 strains of H. ducreyi, indicating antigenic identity or within-species cross-reactivity. Low titres of antibodies to this protein were also detected in 19 antisera raised against different strains of gram-negative bacteria, indicating cross-reactivity with other species. Antibody response to the 24-kDa protein in rabbits immunised subcutaneously with live bacteria resulted in a secondary IgG response. Of 28 sera from patients with culture-verified chancroid, 26 manifested high titres of IgG antibodies to the 24-kDa protein, thus indicating the involvement of this antigen in the disease process in man.

Serum bactericidal activity and phagocytosis in host defence against Haemophilus ducreyi.

Serum bactericidal activity and phagocytic killing are two important mechanisms involved in the host defence against bacteria. Using some in vitro methods, serum bactericidal assay, phagocytic killing by polymorphonuclear leukocytes (PMN) and chemiluminescence, we have evaluated the significance of these mechanisms in the killing of Haemophilus ducreyi bacteria. Furthermore, induction of C3 conversion and deposition of immunoglobulins, C1q and C3, on the surface of bacteria was studied by crossed immunoelectrophoresis and ELISA, respectively. Transmission electron microscopy was employed to study internalization of bacteria by PMN. H. ducreyi and lipooligosaccharide preparations from these bacteria were able to induce conversion of complement factor C3 in normal human serum (NHS). Exposure of bacteria to NHS resulted in deposition of IgG, IgM and complement factors C1q and C3 on the surface of bacteria. H. ducreyi bacteria lost their viability when incubated with fresh but not inactivated normal serum at high concentrations, indicating that the bacteria are sensitive to the complement-dependent bactericidal activity of serum. There were some variations between different strains regarding their susceptibility to the bactericidal activity of NHS, but for eight strains tested, all of the bacteria exposed were not killed in medium containing up to 70% of fresh serum. Complement-mediated opsonophagocytic killing of H. ducreyi by PMN was more effective than complement-dependent bactericidal activity of fresh normal sera. Bacteria treated with heat inactivated immune sera, on the other hand, were as sensitive to the bactericidal effect of PMN as those treated with non-inactivated immune sera, indicating the role of antibodies in opsonophagocytosis. H. ducreyi bacteria were also killed by PMN in the absence of serum antibodies and complement. Using the chemiluminescence assay, H. ducreyi was shown to activate PMN in the absence of serum as well as after opsonization with complement and antibodies. Our results therefore indicate that both opsonic- and non-opsonic mechanisms are involved in the phagocytosis and the subsequent killing of H. ducreyi bacteria. Although both complement and antibodies enhance the ability of phagocytes to kill H. ducreyi, neither component is sufficient for effective killing of H. ducreyi.

Evidence of Haemophilus ducreyi adherence to and cytotoxin destruction of human epithelial cells.

The adherence of ten different Haemophilus ducreyi strains to cultured human epithelial cells and the subsequent destruction of these cells was investigated in vitro using HEp-2 and HeLa cells. Bacterial adherence was measured with two assays, one employing viable bacteria and the other radiolabeled bacteria. In addition, the capacity of H. ducreyi to invade/penetrate the HEp-2 cells was examined. Differential interference contrast and transmission electron microscopy techniques were also used. In both cell lines, all ten strains of H. ducreyi manifested substantial adherence (the rates being 4-20% of the inoculum), irrespective of whether the bacteria were cultivated on solid or liquid media. Bacterial adherence reached a peak after about 2-3 h of incubation, though it was already manifest after only 15 min, a finding suggesting constitutive rather than inducible properties of H. ducreyi adhesins to be involved. The adherence capacity was diminished, but not totally abolished, when bacteria were heat-treated at 100 degrees C for 30 min, indicating the adhesins to be fairly stable. On the other hand, treatment of HEp-2 cells with methanol, glutaraldehyde and emetine dichloride significantly reduced the adherence, indicating viable eukaryotic cells with native surface structures to be involved in bacterial adherence. This capacity of H. ducreyi to adhere to HEp-2 cells was confirmed both by electron microscopy and by differential interference microscopy. Some adherent bacteria were also capable of penetrating epithelial cells, as observed with an invasion assay and confirmed by transmission electron microscopy. Further incubation of the cell monolayers with the ten strains resulted in the cell-death and total damage of monolayers for seven cytotoxin-producing strains, indicating cytotoxin action to be responsible for the destruction of the monolayer. All strains manifested capacity to survive and multiply on the cell monolayer. We propose the first step in the pathogenesis of chancroid to be the adherence of bacteria to epithelial cells, followed by the action of cytotoxin and further bacterial proliferation. This sequence of events is suggested to result in the production of genital ulcers by H. ducreyi organisms.