First Report on the Streptococcusgallolyticus (S. bovis Biotype I) DSM 13808 Exopolysaccharide Structure.

Streptococcus gallolyticus subspecies gallolyticus, known as Streptococcus bovis biotype I, is a facultative pathogen causing bacteraemia, infective endocarditis and sepsis that has been linked with colorectal cancer (CRC), but this correlation is still unclear. Bacterial surface structures, such as the major sugar antigens exposed to the outside of the microorganism, are potential virulence factors. One of the primary sugar antigens loosely attached to the cell surface is the biofilm component, exopolysaccharide (EPS). EPSs of S. bovis are poorly characterized molecules. Until now, only one S. macedonicus Sc136 EPS structure was known to the entire S. bovis group. The S. gallolyticus DSM 13808 EPS was investigated by chemical analysis, mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The hexasaccharide repeating unit of the EPS, containing four Glc, two Rha residues and one phosphate group, has been described " →6)-α-d-Glcp-(1→3)-ß-l-Rhap-(1→4)-ß-d-Glcp-(1→3)-[ß-d-Glcp-(1→2)]-α-l-Rhap-(1→2)-α-d-Glcp-(1→P→".

A New Look at the Enterobacterial Common Antigen Forms Obtained during Rough Lipopolysaccharides Purification.

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-ß-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide - endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC®HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.

Lipopolysaccharide-Linked Enterobacterial Common Antigen (ECALPS) Occurs in Rough Strains of Escherichia coli R1, R2, and R4.

Enterobacterial common antigen (ECA) is a conserved surface antigen characteristic for Enterobacteriaceae. It is consisting of trisaccharide repeating unit, →3)-α-d-Fucp4NAc-(1→4)-ß-d-ManpNAcA-(1→4)-α-d-GlcpNAc-(1→, where prevailing forms include ECA linked to phosphatidylglycerol (ECAPG) and cyclic ECA (ECACYC). Lipopolysaccharide (LPS)-associated form (ECALPS) has been proved to date only for rough Shigella sonnei phase II. Depending on the structure organization, ECA constitutes surface antigen (ECAPG and ECALPS) or maintains the outer membrane permeability barrier (ECACYC). The existence of LPS was hypothesized in the 1960-80s on the basis of serological observations. Only a few Escherichia coli strains (i.e., R1, R2, R3, R4, and K-12) have led to the generation of anti-ECA antibodies upon immunization, excluding ECAPG as an immunogen and conjecturing ECALPS as the only immunogenic form. Here, we presented a structural survey of ECALPS in E. coli R1, R2, R3, and R4 to correlate previous serological observations with the presence of ECALPS. The low yields of ECALPS were identified in the R1, R2, and R4 strains, where ECA occupied outer core residues of LPS that used to be substituted by O-specific polysaccharide in the case of smooth LPS. Previously published observations and hypotheses regarding the immunogenicity and biosynthesis of ECALPS were discussed and correlated with presented herein structural data.

Structural Studies of the Lipopolysaccharide Isolated from Plesiomonas shigelloides O22:H3 (CNCTC 90/89).

Plesiomonas shigelloides is a Gram-negative, rod-shaped bacterium which causes foodborne intestinal infections, including gastroenteritis. It is one of the most frequent causes of travellers' diarrhoea. Lipopolysaccharide (LPS, endotoxin), an important virulence factor of the species, is in most cases characterised by a smooth character, demonstrated by the presence of all regions, such as lipid A, core oligosaccharide, and O-specific polysaccharide, where the latter part determines O-serotype. P. shigelloides LPS is still a poorly characterised virulence factor considering a "translation" of the particular O-serotype into chemical structure. To date, LPS structure has only been elucidated for 15 strains out of 102 O-serotypes. Structures of the new O-specific polysaccharide and core oligosaccharide of P. shigelloides from the Czechoslovak National Collection of Type Cultures CNCTC 90/89 LPS (O22), investigated by chemical analysis, mass spectrometry, and 1H,13C nuclear magnetic resonance (NMR) spectroscopy, have now been reported. The pentasaccharide repeating unit of the O-specific polysaccharide is built of one d-QuipNAc and is rich in four d-GalpNAcAN residues. Moreover, the new core oligosaccharide shares common features of other P. shigelloides endotoxins, i.e., the lack of phosphate groups and the presence of uronic acids.

Changes in the lipopolysaccharide of Proteus mirabilis 9B-m (O11a) clinical strain in response to planktonic or biofilm type of growth.

The impact of planktonic and biofilm lifestyles of the clinical isolate Proteus mirabilis 9B-m on its lipopolysaccharide (O-polysaccharide, core region, and lipid A) was evaluated. Proteus mirabilis bacteria are able to form biofilm and lipopolysaccharide is one of the factors involved in the biofilm formation. Lipopolysaccharide was isolated from planktonic and biofilm cells of the investigated strain and analyzed by SDS-PAGE with silver staining, Western blotting and ELISA, as well as NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry techniques. Chemical and NMR spectroscopic analyses revealed that the structure of the O-polysaccharide of P. mirabilis 9B-m strain did not depend on the form of cell growth, but the full-length chains of the O-antigen were reduced when bacteria grew in biofilm. The study also revealed structural modifications of the core region in the lipopolysaccharide of biofilm-associated cells-peaks assigned to compounds absent in cells from the planktonic culture and not previously detected in any of the known Proteus core oligosaccharides. No differences in the lipid A structure were observed. In summary, our study demonstrated for the first time that changes in the lifestyle of P. mirabilis bacteria leads to the modifications of their important virulence factor-lipopolysaccharide.

The New Structure of Core Oligosaccharide Presented by Proteus penneri 40A and 41 Lipopolysaccharides.

The new type of core oligosaccharide in Proteus penneri 40A and 41 lipopolysaccharides has been investigated by ¹H and 13C NMR spectroscopy, electrospray ionization mass spectrometry and chemical methods. Core oligosaccharides of both strains were chosen for structural analysis based on the reactivity of LPSs with serum against P. penneri 40A core oligosaccharide-diphtheria toxoid conjugate. Structural analyses revealed that P. penneri 40A and 41 LPSs possess an identical core oligosaccharide.

The Complete Structure of the Core Oligosaccharide from Edwardsiella tarda EIB 202 Lipopolysaccharide.

The chemical structure and genomics of the lipopolysaccharide (LPS) core oligosaccharide of pathogenic Edwardsiella tarda strain EIB 202 were studied for the first time. The complete gene assignment for all LPS core biosynthesis gene functions was acquired. The complete structure of core oligosaccharide was investigated by ¹H and 13C nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry MSn, and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. The following structure of the undecasaccharide was established: The heterogeneous appearance of the core oligosaccharide structure was due to the partial lack of ß-d-Galp and the replacement of α-d-GlcpNAcGly by α-d-GlcpNGly. The glycine location was identified by mass spectrometry.

First evidence for a covalent linkage between enterobacterial common antigen and lipopolysaccharide in Shigella sonnei phase II ECALPS.

Enterobacterial common antigen (ECA) is expressed by Gram-negative bacteria belonging to Enterobacteriaceae, including emerging drug-resistant pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus spp. Recent studies have indicated the importance of ECA for cell envelope integrity, flagellum expression, and resistance of enteric bacteria to acetic acid and bile salts. ECA, a heteropolysaccharide built from the trisaccharide repeating unit, →3)-α-D-Fucp4NAc-(1→4)-ß-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→, occurs as a cyclic form (ECA(CYC)), a phosphatidylglycerol (PG)-linked form (ECA(PG)), and an endotoxin/lipopolysaccharide (LPS)-associated form (ECA(LPS)). Since the discovery of ECA in 1962, the structures of ECA(PG) and ECA(CYC) have been completely elucidated. However, no direct evidence has been presented to support a covalent linkage between ECA and LPS; only serological indications of co-association have been reported. This is paradoxical, given that ECA was first identified based on the capacity of immunogenic ECA(LPS) to elicit antibodies cross-reactive with enterobacteria. Using a simple isolation protocol supported by serological tracking of ECA epitopes and NMR spectroscopy and mass spectrometry, we have succeeded in the first detection, isolation, and complete structural analysis of poly- and oligosaccharides of Shigella sonnei phase II ECA(LPS). ECA(LPS) consists of the core oligosaccharide substituted with one to four repeating units of ECA at the position occupied by the O-antigen in the case of smooth S. sonnei phase I. These data represent the first structural evidence for the existence of ECA(LPS) in the half-century since it was first discovered and provide insights that could prove helpful in further structural analyses and screening of ECA(LPS) among Enterobacteriaceae species.

[Bordetella pertussis lipooligosaccharide-derived neoglycoconjugates - new components of pertussis vaccine]. / Neoglikokoniugaty lipooligosacharydu Bordetella pertussis-nowe potencjalne skladniki szczepionki przeciwkrztuscowej.

Pertussis is a contagious respiratory tract disease caused by the Gram-negative bacterium Bordetella pertussis. Despite widespread vaccination, in recent years the pertussis incidence has increased. The whole-cell pertussis vaccine has been very effective but reactogenic. Therefore the improved vaccines contain only a few isolated and inactivated antigens of B. pertussis. However, a waning of the acellular vaccine-induced immunity indicates that these vaccines lack some important protective B. pertussis antigens. The vaccine containing an inactivated pertussis toxin induces the production of toxin-neutralizing antibodies, but it does not lead to destruction of bacteria. Since many virulence factors are involved in the pathogenesis of pertussis, beside the toxin-neutralizing activity, the direct bactericidal activity is essential in anti-pertussis immunity. Lipooligosaccharide is the main surface component of B. pertussis. It is a target for bactericidal antibodies during natural infection. The endotoxic activity of LOS makes it unacceptable for acellular vaccines against B. pertussis. However, the non-toxic moiety of the B. pertussis LOS-derived oligosaccharide coupled to a carrier protein forms an immunogenic glycoconjugate which has a potential application as a new component of a pertussis vaccine. In this paper, we present a review of current research and reasons for the increased pertussis incidence. The epidemiologic situation of pertussis in the past decades showing the ineffectiveness of contemporary, acellular pertussis vaccines is also discussed. The immune processes elicited by natural infection with B. pertussis were compared to the vaccine-induced immunity. The important role of bactericidal antibodies against lipooligosaccharide was indicated in effective immune defense. In a number of research papers the immunogenicity and protective properties of glycoconjugates containing the oligosaccharide component of B. pertussis have been described, and its application as a new component of a pertussis vaccine have been implied.

[The structure and significance of enterobacterial common antigen (ECA)]. / Struktura i znaczenie wspólnego enterobakteryjnego antygenu (ECA).

The enterobacterial common antigen (ECA) is a carbohydrate-derived cell surface antigen present in all Gram-negative bacteria belonging to Enterobacteriaceae family. Biosynthetic pathways shared by ECA and LPS (endotoxin) suggest close connections between these antigens. ECA occurs in three different forms: a phosphatidyl-linked linear polysaccharide anchored on the cell surface (ECAPG), a cyclic form built of 4-6 repeating units localized in the periplasm (ECACYC) and as a linear polysaccharide covalently linked to LPS core oligosaccharide (ECALPS). Regardless of ECA form, poly- and oligosaccharides of ECA consist of the biological trisaccharide repeating units: →3)-α-d-Fucp4NAc-(1→4)-ß-d-ManpNAcA-(1→4)-α-d-GlcpNAc-(1→, where Fucp4NAc refers to 4-acetamido-2,4-dideoxygalactose, ManpNAcA to N-acetyl-mannosaminuronic acid and GlcpNAc to N-acetylglucosamine. ECAPG and ECALPS consisting of one unit with Fucp4NAc as a terminal sugar were also identified. The number of the studies shows its occurrence in all members of enteric bacteria with a few exceptions such as Erwinia chrysanthemi. The presence of ECA was also shown for such genera as Plesiomonas [4] and Yersinia [36], previously belonging to the Vibrionaceae and Pasteurellaceae families, respectively. It was one of the reasons to include these two taxa in the Enterobacteriaceae family. The function of ECA is not fully understood, but it was reported that its occurrence is important in resistance of bacterial cells to environmental conditions, such as bile salts in the human digestive tract. The immunogenicity of ECA seems very interesting in the fact that only sparse rough Gram-negative strains, such as Shigella sonnei phase II, Escherichia coli R1, R2, R4, K-12, and Yersinia enterocolitica O:3 are able to induce the production of specific anti-ECA antibodies. It is the effect of the ECALPS, and the evidence for the existence of such covalent linkage was provided by structural analysis of S. sonnei surface antigens.

Core oligosaccharide of Plesiomonas shigelloides PCM 2231 (Serotype O17) lipopolysaccharide--structural and serological analysis.

The herein presented complete structure of the core oligosaccharide of lipopolysaccharide (LPS) P. shigelloides Polish Collection of Microorganisms (PCM) 2231 (serotype O17) was investigated by (1)H, (13)C NMR spectroscopy, mass spectrometry, chemical analyses and serological methods. The core oligosaccharide is composed of an undecasaccharide, which represents the second core type identified for P. shigelloides serotype O17 LPS. This structure is similar to that of the core oligosaccharide of P. shigelloides strains 302-73 (serotype O1) and 7-63 (serotype O17) and differs from these only by one sugar residue. Serological screening of 55 strains of P. shigelloides with the use of serum against identified core oligosaccharide conjugated with bovine serum albumin (BSA) indicated the presence of similar structures in the LPS core region of 28 O-serotypes. This observation suggests that the core oligosaccharide structure present in strain PCM 2231 could be the most common type among P. shigelloides lipopolysaccharides.

New functional ligands for ficolin-3 among lipopolysaccharides of Hafnia alvei.

Ficolin-1 (M), ficolin-2 (L), ficolin-3 (H) and mannan-binding lectin (MBL) activate the complement system and have opsonic activity. The specificity of ficolin-3 is poorly characterized and currently limited to a few ligands only. We present new specific targets for human ficolin-3, identified among lipopolysaccharides (LPSs, endotoxin) of Hafnia alvei. The interaction was restricted to LPSs of four strains: 23, Polish Collection of Microorganisms (PCM) 1200, PCM 1203 and PCM 1205 and limited to their O-specific polysaccharides (O-specific PSs) composed of different numbers of oligosaccharide (OS) repeating units (RUs). Moreover, these LPS/ficolin-3 complexes activated the lectin pathway of complement in a C4b-deposition assay in a calcium- and magnesium-dependent way. A neoglycoconjugate of the O-specific PS fraction of H. alvei 1200 LPS with bovine serum albumin (BSA) was prepared and used as a tool for the determination of ficolin-3 concentration and activity in serum. To confirm a structure of the O-specific PS 1200 selected for the conjugate preparation, structural analysis was performed on a series of O-specific PSs released by the mild acid hydrolysis of the LPS. The isolated O-specific PSs, showing the different length distributions, were devoid of a major part of the core OS region and had Hep-Kdo disaccharide at a reducing end. The neoglycoconjugate was a highly selective tool for the determination of ficolin-3 concentration and activity in serum (lectin pathway activation in the C4b deposition assay) and was not affected by MBL, ficolin-1 and ficolin-2 or natural antibodies.

Structure and Immunogenicity of the Bordetella pertussis LOS-Derived Oligosaccharides in the Endosomal-Like Pre-Processing Mice Model.

Glycoproteins are processed endosomally prior to presentation to T cells and subsequent induction of specific antibodies. The sugar part of glycoconjugate may be degraded while the type of the process depends on the features of the particular structure. The generated carbohydrate epitopes may differ from native structures and influence immunogenicity of the antigens. We have devised a model of endosomal-like pre-processing of Bordetella pertussis 186 oligosaccharides (OSs) to verify how it affects the immunogenicity of their conjugates. The glycoconjugates of structurally defined forms of the dodecasaccharide OS were synthesized and their immunogenicity was assessed using immunochemical methods. The structural features of the oligosaccharides and their sensitivity to deamination were analyzed by NMR spectroscopy. The distal trisaccharide-comprising pentasaccharide conjugated to a protein was the most effective in inducing immune response against the B. pertussis 186 LOS and the immune response to the complete OS conjugates was significantly lower. This could be explained by the loss of the distal trisaccharide during the in-cell deamination process suggesting that the native structure is not optimal for a vaccine antigen. Consequently, our research has shown that designing of new glycoconjugate vaccines requires the antigen structures to be verified in context of possible endosomal reactions beforehand.

Structural analysis of the lipid A isolated from Hafnia alvei 32 and PCM 1192 lipopolysaccharides.

Hafnia alvei, a Gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. The majority of clinical symptoms of diseases caused by this bacterium have a lipopolysaccharide (LPS, endotoxin)-related origin. The lipid A structure affects the biological activity of endotoxins predominantly. Thus, the structure of H. alvei lipid A was analyzed for the first time. The major form, asymmetrically hexa-acylated lipid A built of beta-D-GlcpN4P-(1-->6)-alpha-D-GlcpN1P substituted with (R)-14:0(3-OH) at N-2 and O-3, 14:0(3-(R)-O-12:0) at N-2', and 14:0(3-(R)-O-14:0) at O-3', was identified by ESI-MS(n) and MALDI-time-of-flight (TOF) MS. Comparative analysis performed by MS suggested that LPSs of H. alvei 32, PCM 1192, PCM 1206, and PCM 1207 share the identified structure of lipid A. LPSs of H. alvei are yet another example of enterobacterial endotoxins having the Escherichia coli-type structure of lipid A. The presence of hepta-acylated forms of H. alvei lipid A resulted from the addition of palmitate (16:0) substituting 14:0(3-OH) at N-2 of the alpha-GlcpN residue. All the studied strains of H. alvei have an ability to modify their lipid A structure by palmitoylation.

Structures of two novel, serologically nonrelated core oligosaccharides of Yokenella regensburgei lipopolysaccharides differing only by a single hexose substitution.

Immunochemical analysis of the Yokenella regensburgei lipopolysaccharides (LPS) indicated the presence of the core oligosaccharide-related immunotypes among the investigated strains. The structure of the core oligosaccharide segment of the Y. regensburgei LPS has been investigated using chemical methods, mass spectrometry, and (1)H, (13)C NMR spectroscopy. It was concluded that the core oligosaccharides of the strains PCM 2476 and PCM 2477 are composed of an undecasaccharide. The combined data revealed two immunotypes of the core oligosaccharide recognized by antibodies against the whole bacterial cells. The structural differences between the core oligosaccharides are limited to the outermost terminal hexopyranose residue. In the core oligosaccharide of the strain PCM 2476, it was identified as alpha-d-Glcp and in that of the strain PCM 2477 as alpha-d-Galp. This subtle difference between the glycoforms of the LPS core appeared to be essential for formation of the epitopes recognized by the specific antibodies directed against the Y. regensburgei whole bacterial cells. The oligosaccharides are not substituted by phosphate groups. Instead, the carboxyl groups of Kdo and galacturonic acid residues present in the core provide the negative charges. The undecasaccharides represent a novel core type of bacterial LPS, which is characteristic for Y. regensburgei.

De­O­acylated lipooligosaccharide of E. coli B reduces the number of metastatic foci via downregulation of myeloid cell activity.

Lipopolysaccharides are the main surface antigens and virulence factors of gram­negative bacteria. Removal of four ester­bound fatty acid residues from hexaacyl lipid A of Escherichia coli lipooligosaccharide (LOS) resulted in the de­O­acylated derivative E. coli LOS­OH (LOS­OH). This procedure caused a significant reduction in the toxicity of this compound compared to the native molecule. We investigated the effect of such a structural LOS modification on its biological activity using in vitro assays with monocytic cells of the RAW264.7 line, dendritic cells of the JAWS II line, bone marrow­derived dendritic cells (BM­DCs), and spleen cells. Furthermore, in in vivo experiments with a melanoma B16 metastasis model, the anti­metastatic activity of the compounds and spleen cell reactivity mediated by them representing a systemic response were analyzed. The results revealed that LOS­OH demonstrated weaker ability than LOS to stimulate and polarize an immune response both in vitro and in vivo. It induced lower cytokine production by cells of myeloid lines. Multiple applications of LOS­OH into mice injected intravenously with B16 cells significantly (P<0.05; P<0.01) reduced the number of metastatic foci in the lungs, presumably via silencing of myeloid cell reactivity as well as the inability to stimulate lymphoid cells both directly and indirectly. These findings suggest that LOS­OH maintained in the body of metastasis­bearing mice appears to modulate or downregulate the innate response, leading to the inability of blood myeloid cells to support the migration of melanoma cells to lung tissue.

Two Kdo-heptose regions identified in Hafnia alvei 32 lipopolysaccharide: the complete core structure and serological screening of different Hafnia O serotypes.

Hafnia alvei, a gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. Various 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-containing fragments different from known structures of core oligosaccharides were previously found among fractions obtained by mild acid hydrolysis of some H. alvei lipopolysaccharides (LPSs). However, the positions of these segments in the LPS structure were not known. Analysis of de-N,O-acylated LPS by nuclear magnetic resonance spectroscopy and mass spectrometry allowed the determination of the location of a Kdo-containing trisaccharide in the structure of H. alvei PCM 32 LPS. It was established that the trisaccharide {L-alpha-D-Hepp-(1-->4)-[alpha-D-Galp6OAc-(1-->7)]-alpha-Kdop-(2-->} is an integral part of the outer-core oligosaccharide of H. alvei 32 LPS. The very labile ketosidic linkage between -->4,7)-alpha-Kdop and -->2)-Glcp in the core oligosaccharide was identified. Screening for this Kdo-containing trisaccharide was performed on the group of 37 O serotypes of H. alvei LPSs using monospecific antibodies recognizing the structure. It was established that this trisaccharide is a characteristic component of the outer-core oligosaccharides of H. alvei 2, 32, 600, 1192, 1206, and 1211 LPSs. The weaker cross-reactions with LPSs of strains 974, 1188, 1198, 1204, and 1214 suggest the presence of similar structures in these LPSs, as well. Thus, we have identified new examples of endotoxins among those elucidated so far. This type of core oligosaccharide deviates from the classical scheme by the presence of the structural Kdo-containing motif in the outer-core region.

Bacteriophage preparation inhibition of reactive oxygen species generation by endotoxin-stimulated polymorphonuclear leukocytes.

It has been known that administration of antibiotics may lead to excessive release of bacterial endotoxins and complicate clinical course of patients with Gram-negative infections. This concern may also apply to phages. Endotoxin may in turn activate neutrophils to produce reactive oxygen species (ROS) that are believed to play an important role in the pathogenesis of multiple organ dysfunction in the course of sepsis. We showed that a purified T4 phage preparation with low-endotoxin content could significantly diminish the luminol-dependent chemiluminescence (CL) of peripheral blood polymorphonuclear leukocytes (PMNs) both stimulated by lipopolysaccharides (LPSs) isolated from different Escherichia coli strains. This effect was also observed for live bacteria used for PMNs stimulation and was independent of bacterial susceptibility for T4-mediated lysis. Our data suggest, that phage-mediated inhibition of LPS- or bacteria-stimulated ROS production by PMNs may be attributed not only to phage-PMNs interactions, but also to phage-LPS interactions and bacterial lysis (in case of homologous phage). Interestingly, the T4 preparation did not influence ROS formation by PMNs stimulated with PMA. This suggests that the observed phenomena are also dependent upon the nature of activator. Bacteriophage-mediated inhibition of ROS formation by cells exposed to endotoxin provides new evidence for possible interactions between phages and mammalian cells. It helps in understanding the role of phages in our environment and may also be of important clinical significance.

Lactoferrin-monophosphoryl lipid A complex enhances immunity of mice to Plesiomonas shigelloides CNCTC 138/92.

Our previous study showed the efficacy of lactoferrin-monophosphoryl lipid A isolated from Hafnia alvei LPS complex (LF-MPL(H.a.)) as an adjuvant in stimulation of humoral and cellular immune response in mice to conventional antigens and a lower pyrogenicity of the complex as compared with MPL(H.a.) alone. In the present investigation we demonstrated that LF-MPL(H.a.) complex enhanced the immunity of BALB/c mice immunized with Plesiomonas shigelloides CNCTC 138/92 bacterial vaccine, against P. shigelloides infection. The adjuvant effect was evidenced by a significant increase of the antigen-specific serum IgG, IgG(2a), and IgG(1) and elevation of antigen-specific serum IgA concentrations. In addition, application of the adjuvant facilitated better clearance of the bacteria in spleens and livers of infected mice when compared with MPL(H.a.) alone. These features of the new adjuvant may predispose it for vaccination protocols in humans.