Search

Objective The Pseudomonas koreensis group bacteria are usually found in soil and are associated with plants. Currently they are poorly described. Here we report on the whole genome sequence of a bacterial isolate from a patient with bronchiectasis that was first identified as P. koreensis, and on its position in the P. koreensis group. Results Strain 16-537536 was isolated from a patient with bronchiectasis from Spain and initially identified by MALDI-TOF as P. koreensis, a member of the Pseudomonas fluorescens complex. However, the average nucleotide identity analysis (ANIb) and whole genome alignments of the draft genome sequence of this strain showed it to be a member of the P. koreensis group of the P. fluorescens complex, but belonging to an undescribed species. In addition, based on ANIb analysis, the P. koreensis group contains several other unnamed species. Several genes for putative virulence factors were identified. The only antibiotic resistance gene present in strain 16-537536 was a class C β-lactamase. The correct identification of bacterial species from patients is of utmost importance in order to understand their pathogenesis and to track the potential spread of pathogens between patients. Whole genome sequence data should be included for the description of new species.

Aim: Genetic characterization of Pandoraea strains recovered from cystic fibrosis patients. Materials & methods: The whole-genome sequence of 12 Pandoraea strains was determined using Illumina technology. The position of the strains within the genus Pandoraea was analyzed using selected partial gene sequences, core genome multi-locus sequence typing and average nucleotide identity analysis. Furthermore, the sequences were annotated. Results: The results show that some strains previously identified as Pandoraea pnomenusa, Pandoraea sputorum, Pandoraea oxalativorans and Pandoraea pulmonicola belong to novel species. The strains did not harbor acquired antibiotic resistance genes but encoded an OXA-type ß-lactamase. Conclusion: The taxonomy of the genus Pandoraea needs to be revised.

Infection of the driveline or pump pocket is a common complication in patients with ventricular assist devices (VADs) and Staphylococcus aureus is the main pathogen causing such infections. Limited evidence is currently available to guide the choice of antibiotic therapy and the duration of treatment in these patients. Patients at the University Medical Center Utrecht who developed a VAD-related S. aureus infection between 2007 and 2016 were retrospectively assessed. Blood culture isolates were typed by whole genome sequencing to differentiate between relapses and reinfections, and to determine whether antibiotic therapy had led to acquisition of resistance mutations. Twenty-eight patients had S. aureus VAD infections. Ten of these patients also suffered S. aureus bacteremia. Discontinuation of antibiotic therapy was followed by relapse in 50% of the patients without prior S. aureus bacteremia and in 80% of patients with bacteremia. Oral cephalexin could ultimately suppress the infection for the duration of follow-up in 8/8 patients without S. aureus bacteremia and in 3/6 patients with S. aureus bacteremia. Clindamycin failed as suppressive therapy in 4/4 patients. Cephalexin appears an adequate choice for antibiotic suppression of VAD infections with methicillin-susceptible S. aureus. In patients without systemic symptoms, it may be justified to attempt to stop therapy after treatment of the acute infection, but antibiotic suppression until heart transplant seems indicated in patients with S. aureus bacteremia.

Infection of the driveline or pump pocket is a common complication in patients with ventricular assist devices (VADs) and Staphylococcus aureus is the main pathogen causing such infections. Limited evidence is currently available to guide the choice of antibiotic therapy and the duration of treatment in these patients. Patients at the University Medical Center Utrecht who developed a VAD‐related S. aureus infection between 2007 and 2016 were retrospectively assessed. Blood culture isolates were typed by whole genome sequencing to differentiate between relapses and reinfections, and to determine whether antibiotic therapy had led to acquisition of resistance mutations. Twenty‐eight patients had S. aureus VAD infections. Ten of these patients also suffered S. aureus bacteremia. Discontinuation of antibiotic therapy was followed by relapse in 50% of the patients without prior S. aureus bacteremia and in 80% of patients with bacteremia. Oral cephalexin could ultimately suppress the infection for the duration of follow‐up in 8/8 patients without S. aureus bacteremia and in 3/6 patients with S. aureus bacteremia. Clindamycin failed as suppressive therapy in 4/4 patients. Cephalexin appears an adequate choice for antibiotic suppression of VAD infections with methicillin‐susceptible S. aureus. In patients without systemic symptoms, it may be justified to attempt to stop therapy after treatment of the acute infection, but antibiotic suppression until heart transplant seems indicated in patients with S. aureus bacteremia.

Oligosaccharide (OS)-protein conjugates are promising candidate vaccinesagainst encapsulated bacteria, such asHaemophilus influenzae,Neisseria meningitidis, andStreptococcus pneumoniae. Although the effects of several variables such as OS chain length and protein carrier have been studied, little is known about the influence of adjuvants on the immunogenicity of OS-protein conjugates. In this study, a minimal protective trisaccharide epitope ofStreptococcus pneumoniaetype 3 conjugated to the cross-reacting material of diphtheria toxin was used for immunization of BALB/c mice in the presence of different adjuvants. Subsequently, half of the mice received a booster immunization with conjugate alone. Independent of the use and type of adjuvant, all mice produced long-lasting anti-polysaccharide type 3 (PS3) antibody levels, which provided full protection against challenge with pneumococcal type 3 bacteria. All adjuvants tested increased the anti-PS3 antibody levels and opsonic capacities as measured by an enzyme-linked immunosorbent assay and an in vitro phagocytosis assay. The use of QuilA or a combination of the adjuvants CpG and dimethyl dioctadecyl ammonium bromide resulted in the highest phagocytic capacities and the highest levels of Th1-related immunoglobulin G (IgG) subclasses. Phagocytic capacity correlated strongly with Th1-associated IgG2a and IgG2b levels, to a lesser extent with Th2-associated IgG1 levels, and weakly with thiocyanate elution as a measure of avidity. Thus, the improved immunogenicity of OS-protein conjugates was most pronounced for Th1-directing adjuvants.

Di-, tri-, and tetrasaccharides, synthesized according to the chemical structure of pneumococcal polysaccharide type 3 (PS3), were coupled to the cross-reactive material (CRM 197 ) of modified diphtheria toxin in different molar carbohydrate/protein ratios using the squarate coupling method. To study protective immunity, female BALB/c mice were subcutaneously immunized twice (with a 3-week interval) using the amount of conjugates corresponding to 2.5 μg of oligosaccharide per mouse. The conjugates evoked PS3 binding immunoglobulin G antibodies that lasted for at least 7 weeks after the booster. Immunogenicity was not influenced by the carbohydrate/protein ratio. All mice with PS3-specific antibodies survived the intraperitoneal challenge with Streptococcus pneumoniae type 3. Therefore, synthetic oligosaccharide-protein conjugates might have potential as vaccines.

T cell maturation in Tcf-1−/− mice deteriorates progressively and halts completely around 6 mo of age. During fetal development thymocyte subpopulations seem normal, although total cell numbers are lower. By 4 to 6 wk of age, obvious blockades in the differentiation of CD4−8− thymocytes are observed at two distinct stages (CD44+25+ and CD44−25−), both of which are normally characterized by extensive proliferation. This lack of thymocyte expansion and/or differentiation was also observed when Tcf-1−/− progenitor cells from the aorta-gonad-mesonephros region (embryonic day 11.5), fetal liver (embryonic day 12.5/14.5), and fetal bone marrow (embryonic day 18.5) were allowed to differentiate in normal thymic lobes (fetal thymic organ cultures) or were injected intrathymically into normal recipients. Despite these apparent defects in thymocyte differentiation and expansion, adult Tcf-1−/− mice are immunocompetent, as they generate virus neutralizing Abs at normal titers. Furthermore, their peripheral T cells have an activated phenotype (increased CD44 and decreased CD62L expression) and proliferate normally in response to Ag or mitogen, suggesting that these cells may have arisen from the early wave of development during embryogenesis and are either long lived or have subsequently been maintained by peripheral expansion. As Tcf-1 is a critical component in the Wnt/β-catenin signaling pathway, these data suggest that Wnt-like factors play a role in the expansion of double-negative thymocytes.

Combined prophylactic treatment with recombinant murine granulocyte colony-stimulating factor (G-CSF) and a suboptimal dose of anti-K1 capsular IgM monoclonal antibody (MAb) significantly enhanced survival in an experimental mouse Escherichia coli O7:K1 peritonitis model compared with untreated animals (67% vs. 11% survival; P < 0.001) and with either treatment alone (67 vs. 29% and 27% survival, respectively; P < 0.01), which suggests synergism between these agents. Enhanced survival by combined treatment was associated with increased neutrophil counts in blood and peritoneal lavage fluid, lower systemic and higher levels of local tumour necrosis factor (TNF) and lower bacterial counts in blood cultures. Mouse neutrophils treated with G-CSF but not infected with E. coli showed enhanced phagocytic and respiratory burst capacity, down-regulation of L-selectin receptors and enhanced expression of Fc RII-III receptors but not of complement receptors.

Two anti-core glycolipid (CGL) IgM monoclonal antibodies (mAbs 8-2 and 26-20), previously shown to display cross-reactivity with heterologous lipopolysaccharide (LPS) in vitro and to provide cross-protectivity against endotoxin challenge in vivo, were evaluated for their potential to protect mice against death from peritonitis caused by heterologous bacterial challenge. Without concurrent antibiotic treatment neither antibody was protective. Compared with a control mAb, prophylactic treatment with mAb 8-2 significantly increased the survival of gentamicin-treated mice challenged with the rough strain Salmonella minnesota Re595. Both mAb 8-2 and a control mAb, in combination with a suboptimal dose of ceftazidime, increased survival following challenge with the clinical isolate Escherichia coli O7:K1. In a model of mucin-enhanced peritonitis, neither mAb was protective against challenge with inocula of E. coli O7:K1, ranging from 10(2) to 10(4) bacteria. We conclude that protection of mice by anti-CGL mAb 8-2 against heterologous challenge is vitally dependent on concurrent treatment with antibiotics and that protection may not be attributable to the anti-CGL specificity of these antibodies.

Surfactant protein A (SP-A) and surfactant protein D (SP-D) are collectins, and both proteins were shown to interact with influenza A virus and alveolar macrophages. However, it is not known whether SP-A and SP-D can serve as opsonins for the phagocytosis of influenza A virus by alveolar macrophages. In the present study, we investigated the opsonic activities of SP-A and SP-D for phagocytosis of fluorescein isothiocyanate (FITC)-labeled influenza A (H3N2) virus by rat alveolar macrophages using flow cytometry. SP-A enhanced the association of the virus with macrophages in a dose-dependent manner, reaching a maximum at an SP-A concentration of 60 microg/ml. An approximate threefold increase in association of influenza A virus with alveolar macrophages in the presence of SP-A over control incubations which contained no SP-A was observed. Half of the total cell-associated fluorescence could be quenched as demonstrated using the extracellular quenching dye trypan blue. These results indicate that SP-A mediates internalization of FITC-labeled influenza A (H3N2) virus by alveolar macrophages. Removal of the carbohydrate moiety of SP-A by N-glycosidase F treatment or cleavage of its sialic acid residues by neuraminidase abolished the enhancement of the phagocytosis of FITC-labeled influenza A virus by alveolar macrophages. Mannan, a mannose homopolysaccharide known to bind to the carbohydrate-binding domain of SP-A, did not affect the SP-A-mediated phagocytosis of FITC-labeled influenza by alveolar macrophages. In contrast, SP-D neither enhanced the association of FITC-labeled influenza A virus with alveolar macrophages nor affected the opsonic activity of SP-A for FITC-labeled influenza A (H3N2) virus at the SP-D concentrations tested. It is concluded that SP-A acts via its sialic acid residues as an opsonin in the phagocytosis of influenza A virus by alveolar macrophages.

To study antibody-mediated protection against Escherichia coli peritonitis in BALB/c mice, monoclonal antibodies (MAbs) were generated against the capsule (K5) and the lipopolysaccharide (O18) of E. coli. Flow cytometric analysis with two selected immunoglobulin M MAbs revealed that bacteria were antigenically heterogeneous. Arbitrarily, three subpopulations in E. coli O18K5 cultures could be distinguished by double immunofluorescence. A subpopulation bound only the anti-K5 MAb, and another subpopulation bound only the anti-O18 MAb. An intermediate subpopulation, however, bound both MAbs. In agreement with this result, combinations of both MAbs enhanced phagocytosis of fluorescein isothiocyanate-labeled bacteria by human polymorphonuclear leukocytes and mouse macrophage J774 cells as well. In protection experiments, combinations of both MAbs, preincubated with 3 50% lethal doses of E. coli O18K5, protected all mice upon intraperitoneal challenge. Relatively high doses of either MAb alone proved to be not fully protective in this infection model. Protection of mice by the combination of MAbs was associated with significantly lower (P < 0.02) tumor necrosis factor levels in serum 90 min after challenge compared with any other treatment group. Similarly, prophylactic administration of MAbs yielded significantly lower (P < 0.01) tumor necrosis factor levels in mice that received the combination of MAbs than in any other treatment group.

Idiotypic cross-reactivity between encephalomyocarditis virus (EMCV) neutralizing monoclonal antibodies UM 21.1 (IgG2b) and UM 21.3 (IgG2a) was detected by neutralization inhibition enzyme immunoassay using polyclonal and monoclonal anti-idiotypic antibodies. One strongly cross-reactive anti-idiotypic monoclonal antibody, designated 21.1A5 (IgG2b), might recognize a recurrent idiotope on EMCV neutralizing antibodies but it did not induce EMCV neutralizing anti-anti-idiotypic antibodies in homologous BALB/c mice.

A recombinantly expressed protein, consisting of cro-β-galactosidase at the N-terminus and amino acid residues 115 to 151 of the E2 membrane of Semliki Forest virus (SFV) at the C-terminus containing two T-helper cell epitopes of SFV, was cross-linked with glutaraldehyde to a noninternal image monoclonal anti-idiotypic antibody (ab2α MAb) able to induce SFV-neutralizing anti-anti-idiotypic (ab3) antibodies in BALB/c mice. This vaccine, which might potentially induce SFV-specific T-helper cell memory, established in BALB/c mice a state of protective immunity against virulent SFV within 10 days of immunization. A steady rise in serum neutralization titre occurred from day 7 to day 28 after primary anti-idiotypic immunization, levelling off thereafter. In primarily immunized mice significant rises of serum neutralization titres, which could be indicative for an operational T-helper cell memory, were not observed after challenge on day 35 with virulent SFV. The results suggest that SFV is neutralized by ab3 antibodies shortly after challenge, preventing, thereby, virus multiplication to levels sufficient to provoke a measurable booster response.

Semliki Forest virus (SFV) infection of mice was used as a model to study the applicability of synthetic peptides containing only linear epitopes as viral vaccines. The identification of linear epitopes with vaccine potential on the E2 membrane protein of SFV was based on the binding of SFV-specific antibodies to a set of overlapping synthetic hexapeptides (Pepscan) representing the whole E2 amino acid sequence. The 14 available E2-specific monoclonal antibodies which were protective in vivo proved to be unsuitable for the identification of linear epitopes because they recognized only conformational epitopes, as indicated by their lack of reactivity with unfolded, reduced E2 protein on immunoblots. Three epitopes were detected with polyclonal anti-SFV serum at amino acid positions 135 to 141, 177 to 185 and 240 to 246 of the E2 protein. Synthetic peptides containing these epitopes were coupled to a carrier protein and tested as a vaccine. Mice immunized with the peptide containing amino acids 240 to 255 of protein E2 were protected against a challenge with virulent SFV but protection of mice immunized with the peptides containing amino acids 126 to 141 or 178 to 186 was only marginally better than that of controls. The prechallenge sera of most peptide-immunized mice reacted with SFV-infected cells but none of these sera neutralized the virus in vitro. However, protection of mice correlated well with SFV-specific antibody titre, suggesting antibody-mediated protection.

Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed DNA vaccines encoding cytotoxic T lymphocytes (CTL) and T helper cell (Th) epitopes of the 38-kDa lipoglycoprotein ofMycobacterium tuberculosisand analyzed and compared their immunogenicities with that of pXJ38, a DNA vaccine encoding the entire 38-kDa protein (X. Zhu, N. Venkataprasad, H. S. Thangaraj, M. Hill, M. Singh, J. Ivanyi, and H. M. Vordermeier, J. Immunol. 158:5921–5926, 1997). Plasmid DNAs encoding a CTL epitope, P3 (pP3), a Th epitope (vTh), or both the Th and the P3 epitopes (pThP3) were prepared and tested in C57BL6/J (H-2b) mice. Our results confirmed that DNA immunization with pXJ38 induces strong CD8+CTL and Th1 responses (high gamma interferon [IFN-γ], low interleukin-4 [IL-4]). Coadministration of plasmid DNAs encoding a Th epitope with those encoding a CTL epitope (vTh+pP3) elicited both antigen-specific CD8+CTL and Th1 responses. High levels of IFN-γ were secreted by spleen cells from all plasmid DNA-vaccinated mice after in vitro stimulation with the recombinant 38-kDa protein. Small or undetectable amounts of IL-4 were observed, which indicates the induction of a Th1-like response. Multiple-epitope vaccination by vTh+pP3 or pThP3 resulted in a broader Th1 response to peptide or epitopes than the single-epitope plasmid DNAs. Antigen-specific immunoglobulin G2a was only detected in sera from mice immunized with the plasmid pXJ38, and not in mice immunized with the epitope-based DNA vaccines. Thus, the absence of an antibody response after immunization with epitope plasmid DNAs and their ability to trigger only a specific cellular immune response may prove to be important advantages for a vaccine against tuberculosis.

Antibiotics may cause an excess release of lipopolysaccharide (LPS) from bacteria and thereby promote the production of tumour necrosis factor (TNF). TNF was measured in the serum of Swiss mice challenged with filtered supernatant of Escherichia coli O7:K1 that had been exposed to various antibiotics in vitro. Expressed as a function of a standardized number of cells remaining after 6 h of exposure to gentamicin, ceftazidime, ciprofloxacin or imipenem, TNF leves associated with antibiotic exposure always exceeded those of controls. However, if differences in the remaining number of bacteria were not taken into account, TNF induction by supernatant of control untreated cultures was greater than that elicited by supernatant from any of the antibiotic-treated cultures. With the exception of imipenem, low-dose antibiotic exposure (0.5 × MIC) invariably induced higher TNF levels than did high-dose exposure (10 × MIC). Considerable antibiotic class- and concentration-related differences were noted. LAL equivalent amounts of LPS released by different antibiotics may diverge in their capacity to induce TNF. Our results do not support the notion that the use of rapidly bactericidal and lytic antibiotics should be avoided.

Flow cytometry revealed that the binding of immunoglobulin M monoclonal antibodies (MAbs) to Escherichia coli O18K5 was modulated by exposure of the bacteria to subinhibitory concentrations of imipenem. The binding of anti-K5 MAb was decreased, while the binding of anti-O18 MAb was increased. In addition, anti-lipid A MAbs bound only to imipenem-treated bacteria. The biological effect of MAb binding was investigated in BALB/c mice by determination of the levels of bacteremia, tumor necrosis factor (TNF) in serum and survival after intraperitoneal challenge with bacteria preincubated with MAb. Neither MAb alone (150 micrograms per animal) proved to be protective against untreated bacteria. Anti-lipid A MAb on its own, in contrast to anti-K5 and anti-O18 MAbs, was not protective against imipenem-treated bacteria. Only combinations which included anti-O18 MAb and anti-K5 MAb exerted in mice enhanced protection against smooth E. coli O18K5 as well as imipenem-treated E. coli O18K5. This was reflected by reduced TNF levels in serum and increased survival. The addition of anti-lipid A MAb to the combination of anti-K5 MAb and anti-O18 MAb reduced serum TNF levels in mice, but not significantly.

In the present study a shared idiotope was found among antibodies against a previously defined linear B-cell epitope of Semliki Forest virus (SFV). The synthetic B-cell epitope, located at amino acid positions 240 to 255 of the E2 membrane protein, was linked to an H-2d-restricted T-helper cell epitope of either SFV or influenza virus. Colinearly synthesized peptides of T-B polarity mixed with adjuvant were used to immunize BALB/c (H-2d) mice. After one booster immunization with either chimaeric peptide high serum antibody titers were measured against both synthetic peptide (240-255) and glutaraldehyde-fixed SFV-infected L cells. Against the synthetic peptide (240-255) a variety of monoclonal antibodies (MAbs) were produced that differed in reactivity with SFV, varied in heavy chain family, isotype, isoelectric point, and idiotype. Against one of the antipeptide MAbs (I02), that strongly reacted with SFV-infected L cells, an antiidiotypic MAb (ab2MAb), designated I02A3, was produced that could be inhibited in its binding to MAb I02 by the synthetic B-cell epitope. Therefore it was concluded that ab2 MAb I02A3 recognizes an idiotope closely associated with the antigen combining site of antipeptide MAb I02. This idiotope was definitively shared by two out of 15 antipeptide MAbs and by SFV-reactive antibodies present in both antipeptide sera and SFV-immune sera.

A synthetic peptide that contains a Semliki Forest virus (SFV) B cell epitope, located at amino acid positions 240 to 255 of the E2 protein, and an SFV T helper (Th) cell epitope, located at positions 137 to 151 of the E2 protein, evoked high titres of SFV-reactive antibodies in H-2d mice. Although the peptide-induced antibodies did not neutralize SFV in vitro, 70 to 100% of the peptide-immunized mice were protected against SFV, even when viral challenge was presented 4 months after immunization. The protection could be transferred by anti-peptide serum, indicating that antibodies were responsible for the protection. When the Th cell epitope of this protective peptide was replaced by an influenza virus Th cell epitope or by another SFV Th cell epitope, the resulting peptides induced lower non-neutralizing SFV-reactive antibody titres and protected a correspondingly lower percentage of mice (50% and 30%, respectively). A peptide with the same Th cell epitope as the best protective peptide but with a less effective SFV B cell epitope protected only 33% of the mice. These results indicate that protection against SFV by a synthetic peptide is primarily dependent on its ability to induce adequate amounts of antibodies with relevant specificity and sufficient affinity; the ability to induce a relevant (SFV-specific) T memory response played only a minor role in protection.

Mapping of T-cell epitopes on the structural proteins of Semliki Forest virus (SFV) was performed by measuring the ability of cloned SFV protein fragments to induce delayed-type hypersensitivity (DTH). The cloned SFV protein fragments were expressed as hybrid proteins with cro-β-galactosidase in Escherichia coli from constructed recombinant plasmids. DTH reactions were measured, as footpad swelling, in BALB/c mice after immunization with whole, UV-inactivated SFV and challenge with the hybrid proteins, and vice versa, using the adjuvant dimethyl dioctadecyl ammoniumbromide to enhance DTH. Only two of the tested hybrid proteins induced DTH, and these DTH reactions were equally strong. The largest DTH-inducing hybrid protein contained the N-terminal 350 amino acids of E2 and part of E3, the smallest contained only the region from amino acid residues 115 to 151 of the E2 membrane protein without any other SFV protein parts. It was concluded that the segment between amino acid residues 115 and 151 of the E2 membrane protein of SFV was responsible for the observed DTH, and thus, contains a T-cell epitope. Sequence homology with known T-cell epitopes on other proteins makes it likely that the DTH-inducing T-cell epitope is located from amino acid residues 120 to 128 of E2.

After subcutaneous infection of mice with Semliki Forest virus, a delayed-type hypersensitivity (DH) could be demonstrated by footpad swelling. Pretreatment with cyclophosphamide resulted in enhanced DH if neutralizing antibodies were undetectable in serum.

The effects of the adjuvant dimethyl dioctadecyl ammonium bromide (DDA) on the immune responses to encephalomyocarditis (EMC) virus were studied in mice. The humoral response, as measured by appearance of neutralizing antibodies, was slightly enhanced in mice immunized by the intraperitoneal route. Intracutaneously, DDA almost did not affect the humoral response but resulted in distinct enhancement of delayed type hypersensitivity (DH), as measured by the footpad swelling test. DH to EMC virus was found to be antigen-specific and could be passively transferred to normal mice with peritoneal exudate cells from immunized mice. Dose-response curves for DH and humoral antibody responses to EMC virus were not concordant. Low doses induced DH on day 6 without measurable circulating antibodies; high doses gave good antibody responses but suboptimal DH reactions. Immunization conferred a state of resistance to infection with virulent EMC virus. Protection seemed more related to DH than to the prevalence of specific antibodies at the time of infection.

This paper describes the minimal requirements to protect mice adoptively against challenge with virulent Semliki Forest virus (SFV). Early immune serum, from donor mice infected with an avirulent strain of SFV, contained mainly neutralizing IgM immunoglobulins. More of these antibodies (1.80 PND50 vs 0.06 PND50) were needed to protect recipient mice against intraperitoneal challenge (10 LD50 of SFV) than against subcutaneous challenge (7 LD50). Adoptive transfer experiments indicated that a minimum of 3 X 10(7) six-day immune spleen cells were also able to protect recipient mice against intraperitoneal challenge with 10 LD50 of SFV. Treatment of these donor cells with cytotoxic antisera and complement revealed both T- and B-lymphocytes were required for optimum adoptive immunity. Surviving recipients of either immune serum or immune spleen cells developed significantly less neutralizing antibodies than control mice. The lower antibody titres in protected mice might be related to either immune serum or immune spleen cell mediated restriction of virus replication; meaning a reduced antigenic stimulus in these mice compared to control mice.

Two encephalomyocarditis virus (EMCV) neutralizing monoclonal antibodies (MAs), recognizing different determinants on EMCV, were both able to protect mice prophylactically and therapeutically against a lethal dose of EMCV.

To investigate the epitope(s) on encephalomyocarditis virus (EMCV) involved in neutralization, two neutralizing monoclonal antibodies (MAs) (MA UM 21.1 and MA UM 21.2) were tested in a competition binding assay (CBA), a mixed neutralization test and an enzyme immunoassay (EIA) with specificity for the detection of idiotypes on MAs. With a CBA in cell culture, using EMCV infected L cell monolayers as binding antigen, strong homologous competition was observed between unlabelled MAs and horse radish peroxidase (HRPO-) labelled MAs but considerable heterologous competition did also occur, especially between the unlabelled MA UM 21.1 and HRPO-labelled MA UM 21.2. In the mixed neutralization test (50% plaque reduction) preincubation with slightly neutralizing or non-neutralizing doses of MA UM 21.2 had no diminishing effect on the neutralizing capacity of MA UM 21.1, the PRT 50 value remains in all cases −10 log titre of 5.8. Furthermore rabbit polyclonal antibodies against the idiotypes of MAs UM 21.1 and UM 21.2 did not cross react in the EIA. In conclusion both MAs recognize different viral determinants as indicated by the results obtained with a CBA, a mixed neutralization test and an EIA for detection of idiotypes on MAs.

The two highly competitive, monoclonal antibodies (MAs), UM 5.1 and UM 8.1, recognize on the E2 glycoprotein of Semliki Forest virus different determinants as indicated by the absence of cross-reactive anti-idiotypic antibodies in rabbit immune sera induced against these highly neutralizing MAs.

The effect of alpha- and beta-adrenoceptor blocking agents on endotoxin-induced release of tumour necrosis factor (TNF), and of interferon in the circulation of Corynebacterium parvum-treated mice was the subject of this study. TNF was quantified after injection of TNF containing heated serum (TNS) into Meth A sarcoma-bearing mice by determining colour, extent, and incidence of haemorrhagic necrosis. The release of TNF was weakly inhibited by the competitive alpha-blocker phentolamine and the beta-blocker propranolol. The non-competitive alpha-blocker phenoxybenzamine inhibited to a higher degree. Endotoxin-induced elicitation of growth-inhibiting principles into TNS was antagonized by propranolol and phenoxybenzamine. Administration of adrenaline before endotoxin inhibited the elicitation of TNF and growth-inhibitory activities, which indicates tachyphylaxis. The release of interferon was effectively inhibited by both alpha-adrenoceptor blockers but not by propranolol. The interferon was heat-labile. The results indicate that endotoxin-induced TNF and interferon are separate factors, elicited in different ways. As both alpha-blockers do not only inhibit reactions at the alpha-adrenergic receptor but also reactions at the serotonin receptor and in the case of phenoxybenzamine also at the choline receptor, it is suggested that endotoxin-induced release of the anti-tumour factors is controlled by reactions mediated by one or more of these receptors. It is suggested that the inhibition of TNF release by propranolol may be due to the membrane-stabilizing activity of this agent.

The lipophilic amines dimethyl dioctadecyl ammonium bromide (DDA) and N,N-dioctadecyl-N′, N′-bis(2-hydroxyethyl)propanediamine (CP-20,961) are compared on their capacities to induce interferon, nonspecific protection to viral infection, and enhancement of delayed-type hypersensitivity (DH). DDA, a well-known adjuvant for the induction of DH is a moderate interferon inducer like CP-20,961. On the other hand, CP-20,961, a known interferon inducer and resembling in structure DDA, is shown to enhance DH to inactivated Semliki Forest virus (SFV). Nonspecific protection to challenge with a lethal dose of either SFV or encephalomyocarditis (EMC) virus was induced on injection of both compounds.

Concanavalin A, endotoxin, poly I: C, and tumour necrosis serum (TNS) were compared for antitumour activity against Meth A sarcoma transplanted in syngeneic BALB/c mice and their capacity to induce tumour necrosis factor (TNF), heat-stable cytostatic factors, and heat-labile interferon in the blood of normal and Corynebacterium parvum-pretreated mice. All the agents induced hyperemia and inhibition of mitosis at 4 h, and by 24 h many tumours had a dark necrotic centre. Subsequent tumour growth was inhibited and in some of the treated mice tumours regressed completely. Poly A: U and normal mouse serum did not induce regression and their effects were less marked in all other respects, suggesting that these events may be linked. The necrotizing effects of concanavalin A and poly I: C are unlikely to be mediated by TNF, because neither agent could mimic endotoxin in eliciting RNase-resistant necrotizing and regressing activity in the serum of mice pretreated with C. parvum. Poly I: C did not induce strong cytostatic activity in the sera of C. parvum-treated mice, and for this and other reasons these factors are unlikely to be responsible for the observed effects. Concanavalin A, endotoxin, and poly I: C induced high levels of serum interferon but purified interferon had only weak antitumour activity in the Meth A system, suggesting that interferon may not be the mediator. From these and other data it is concluded that there is no clear relationship between the capacity of the agents to induce tumour necrosis and their capacity to elicit TNF, cytostatic factors, and interferon.

Vaccines of 6 strains of the genera Corynebacterium and Propionibacterium were tested for induction of hepatosplenomegaly and stimulation of clearance of injected colloidal carbon and viable Listeria monocytogenes. These effects were compared with the ability of these strains to support the endotoxin-induced liberation of tumour necrosis factor, interferon and heat-stable cytostatic factors in mouse circulation. Five strains caused good stimulation of macrophage functions and induced high levels of all factors. One strain was nearly lacking in macrophage stimulatory capacity and also failed to support factor production. These data are discussed in relation to the production mechanisms of the factors.

Lipopolysaccharide (LPS) is the major mediator of gram-negative septic shock. Molecules that bind LPS and neutralize its toxic effects could have important clinical applications. We showed that serum amyloid P component (SAP) neutralizes LPS. A SAP-derived peptide, consisting of amino acids 27 to 39, inhibited LPS-mediated effects in the presence of human blood. In this study, we used a pepscan of overlapping 15-mer peptides and distinguished two additional LPS-binding regions within the SAP molecule, identified in the regions spanning amino acids 61 to 75 and 186 to 200. The corresponding SAP-derived peptides, pep61-75 and pep186-200, inhibited the binding of fluorescein isothiocyanate-labeled LPS to monocytes as efficiently as a bactericidal/permeability-increasing protein (BPI)-derived 15-mer peptide comprising amino acids 85 to 99. The same SAP-derived peptides very potently inhibited LPS-induced priming of phagocytes in human blood. Also, SAP-derived pep186-200 caused a prolonged survival of actinomycin D-sensitized mice treated with LPS to induce septic shock, indicating a potential use of this peptide in the defense against serious gram-negative sepsis in humans.