NK-cell and T-cell functions in patients with breast cancer: effects of surgery and adjuvant chemo- and radiotherapy.

Breast cancer is globally the most common malignancy in women. Her2-targeted monoclonal antibodies are established treatment modalities, and vaccines are in late-stage clinical testing in patients with breast cancer and known to promote tumour-killing through mechanisms like antibody-dependent cellular cytotoxicity. It is therefore increasingly important to study immunological consequences of conventional treatment strategies. In this study, functional tests and four-colour flow cytometry were used to detect natural killer (NK)-cell functions and receptors as well as T-cell signal transduction molecules and intracellular cytokines in preoperative breast cancer patients, and patients who had received adjuvant radiotherapy or adjuvant combined chemo-radiotherapy as well as in age-matched healthy controls. The absolute number of NK cells, the density of NK receptors as well as in vitro quantitation of functional NK cytotoxicity were significantly higher in preoperative patients than the post-treatments group and controls. A similar pattern was seen with regard to T-cell signalling molecules, and preoperative patients produced significantly higher amounts of cytokines in NK and T cells compared to other groups. The results indicate that functions of NK and T cells are well preserved before surgery but decrease following adjuvant therapy, which may speak in favour of early rather than late use of immunotherapeutic agents such as trastuzumab that may depend on intact immune effector functions.

Weakening effect of cell permeabilizers on gram-negative bacteria causing biodeterioration.

Gram-negative bacteria play an important role in the formation and stabilization of biofilm structures on stone surfaces. Therefore, the control of growth of gram-negative bacteria offers a way to diminish biodeterioration of stone materials. The effect of potential permeabilizers on the outer membrane (OM) properties of gram-negative bacteria was investigated and further characterized. In addition, efficacy of the agents in enhancing the activity of a biocide (benzalkonium chloride) was assessed. EDTA, polyethylenimine (PEI), and succimer (meso-2,3-dimercaptosuccinic) were shown to be efficient permeabilizers of the members of Pseudomonas and Stenotrophomonas genera, as indicated by an increase in the uptake of a hydrophobic probe (1-N-phenylnaphthylamine) and sensitization to hydrophobic antibiotics. Visualization of Pseudomonas cells treated with EDTA or PEI by atomic force microscopy revealed damage in the outer membrane structure. PEI especially increased the surface area and bulges of the cells. Topographic images of EDTA-treated cells were compatible with events assigned for the effect of EDTA on outer membranes, i.e., release of lipopolysaccharide and disintegration of OM structure. In addition, the effect of EDTA treatment was visualized in phase-contrast images as large areas with varying hydrophilicity on cell surfaces. In liquid culture tests, EDTA and PEI supplementation enhanced the activity of benzalkonium chloride toward the target strains. Use of permeabilizers in biocide formulations would enable the use of decreased concentrations of the active biocide ingredient, thereby providing environmentally friendlier products.

Effect of EDTA on Salmonella enterica serovar Typhimurium involves a component not assignable to lipopolysaccharide release.

The effect of EDTA on Salmonella enterica serovar Typhimurium was studied in different growth phases with cells grown with or without Ca(2+) and Mg(2+) supplementation. EDTA affected the outer membrane much more strongly in the early exponential phase than in the mid- or late exponential phase, as indicated by uptake of 1-N-phenylnaphthylamine (a nonpolar hydrophobic probe, M(r) 219), and detergent (SDS) susceptibility. This effect was, however, not paralleled by LPS release (determined by measuring LPS-specific fatty acids or 14C-labelled LPS in cell-free supernatants, per a standardized cell density), which remained unchanged as a function of the growth curve. The conclusion from these results is that in the early exponential phase the effect of EDTA in S. enterica involves a component that is independent of LPS release.

Characterisation of aerobic gram-negative bacteria from subgingival sites of dogs--potential bite wound pathogens.

Ninety-eight aerobic, gram-negative bacterial isolates from subgingival samples from family-owned dogs with naturally occurring periodontitis were characterised phenotypically by conventional biochemical testing, by cellular fatty acid profiling and by the use of commercial identification systems. The majority (48, 81%) of the fermentative isolates but only 18% of the non-fermenters were identified by conventional biochemical testing alone. With additional cellular fatty acid profiling, another 7 (12%) fermentative and 23 (59%) non-fermentative isolates were identified to genus or group level. Cellular fatty acid analysis was essential for the identification of most non-fermenters, many of which are difficult to identify due to a paucity of positive reactions in routine biochemical tests. Commercial identification systems were less useful and did not contribute to further identification of these problematic isolates. This study underlines the difficulties encountered in the identification of canine oral bacteria--a group of potential bite wound pathogens--and presents schemes for microbiology laboratories to characterise such isolates.

Chitosan disrupts the barrier properties of the outer membrane of gram-negative bacteria.

The mode of antimicrobial action of chitosan (polymeric beta-1,4-N-acetylglucosamine) on gram-negative bacteria was studied with special emphasis on its ability to bind to and weaken the barrier function of the outer membrane (OM). Chitosan (250 ppm) at pH 5.3 induced significant uptake of the hydrophobic probe 1-N-phenylnaphthylamine (NPN) in Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium. The effect was reduced (E. coli, salmonellae) or abolished (P. aeruginosa) by MgCl2. No NPN uptake was observed during exposure of the salmonellae to chitosan at pH 7.2. Chitosan also sensitized P. aeruginosa and the salmonellae to the lytic effect of sodium dodecyl sulfate (SDS); such sensitization was not blocked by MgCl2 and was reversible by washing chitosan-treated cells prior to SDS exposure. Chemical and electrophoretic analyses of cell-free supernatants of chitosan-treated cell suspensions showed that interaction of chitosan with E. coli and the salmonellae involved no release of lipopolysaccharide (LPS) or other membrane lipids. However, chitosan rendered E. coli more sensitive to the inhibitory action of dyes and bile acids used in selective media. Highly cationic mutants of S. typhimurium were more resistant to chitosan than the parent strains. Electron microscopy showed that chitosan caused extensive cell surface alterations and covered the OM with vesicular structures. Chitosan thus appeared to bind to the outer membrane, explaining the loss of the barrier function. This property makes chitosan a potentially useful indirect antimicrobial for food protection.

Permeability barrier of the gram-negative bacterial outer membrane with special reference to nisin.

The effect of nisin pretreatment on organic acid-induced permeability increase in strains of Escherichia coli, Pseudomonas aeruginosa, P. marginalis, and Salmonella enterica sv. Typhimurium was investigated, using assays based on the uptake of a fluorescent dye 1-N-phenylnaphthylamine (NPN) and on the bacterial susceptibility to detergent-induced bacteriolysis. The outer membrane of bacteria which had been pretreated with nisin was shown to be less stable against 1 mM EDTA, as indicated by their significantly higher NPN uptake levels as compared to untreated bacteria. Upon challenge with a tenfold lower concentration of EDTA (0.1 mM) some nisin-treated strains (Typhimurium, P. marginalis) exhibited, however, NPN uptake levels which were lower than those seen in control bacteria, suggesting that nisin had stabilized their outer membrane. Nisin pretreatment also decreased the NPN uptake induced by citric or lactic acid or both in E. coli, P. marginalis, and Typhimurium, whereas in P. aeruginosa the pretreatment resulted in increased NPN uptake in response to citric and lactic acid. These results suggest that, with the exception of P. aeruginosa, nisin could protect bacteria from the outer membrane-disrupting effect caused by the acids. P. aeruginosa was, however, shown to be protected against bacteriolysis induced by the detergents sodium dodecylsulfate and Triton X-100. With a pair of isogenic mutants of Typhimurium differing in their cell surface charge it was shown that the NPN uptake response to I mM EDTA of the abnormally cationic strain was not significantly affected by nisin, whereas in the normal anionic strain nisin strongly strengthened the uptake. Our hypothesis based on these findings is that the normally anionic cell surface of Gram-negative bacteria has a tendency to bind the cationic nisin. The binding of nisin to the surface does not proceed to the cytoplasmic membrane, but in the outer membrane the bound nisin actually stabilizes its structure through electrostatic interactions. With the exception of EDTA, the organic acids at pH 4 did not cause leakage of cell contents from Typhimurium, indicating that these acids do not permeabilize the outer membrane to an extent required for cytoplasmic pore formation by nisin.

Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane.

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.

Fluorometric assessment of gram-negative bacterial permeabilization.

Uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram-negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram-negative bacteria.

Biochemical and genetic characterization of a Prevotella intermedia/nigrescens-like organism.

Thirty-three previously non-typable faintly pigmented Gram-negative anaerobic bacterial isolates, biochemically most closely related to Prevotella intermedia and Prevotella nigrescens, were analysed for enzymic reactions, cellular fatty acid (CFA) composition, electrophoretic mobility of malate and glutamate dehydrogenases, hybridization with P. intermedia and P. nigrescens species-specific oligonucleotide probes and, for genetic heterogeneity, by arbitrarily primed PCR (AP-PCR). P. intermedia ATCC 25611T and P. nigrescens ATCC 33563T were run in parallel for comparison. Twenty-nine isolates originated from the normal oral flora of 18 subjects (including five mother-child pairs), and four isolates from various infections. Except for a negative lipase reaction, enzymic profiles of the test isolates were similar to those of P. intermedia and P. nigrescens. Clustering of CFAs, electrophoretic mobility patterns, hybridization with DNA probes for P. intermedia and P. nigrescens, and AP-PCR band patterns of the test isolates differed from those of the type strains of P. intermedia and P. nigrescens, suggesting the existence, in humans, of a new anaerobic species of pigmented, moderately saccharolytic, indole-positive Gram-negative rods.

Phosphate groups in lipopolysaccharides of Salmonella typhimurium rfaP mutants.

Lipopolysaccharides (LPS) of Salmonella typhimurium rfaP mutants and of a galE strain as a control were subjected to analysis by 31P-NMR in order to assess the location of phosphate groups. This was done to obtain direct proof for our earlier finding by chemical analysis that phosphate was lacking in the core oligosaccharide part of the mutant LPS, whereas the core oligosaccharide normally contains several phosphate groups. Such phosphate deficiency has been associated with the increased susceptibility of the rfaP mutants to hydrophobic antibiotics and detergents. Analysis of the de-O-acylated LPS derivatives of S. typhimurium rfaP strains SH7770, SH8551, and SH8572 by 31P-NMR revealed an almost total lack of phosphate groups in the core oligosaccharide part, the LPS phosphates being largely accounted for by the two monophosphate monoesters of lipid A, linked to positions C-1 and C-4' of the lipid A backbone. Core oligosaccharide-linked phosphates were detected in minor proportions only, indicating the presence of some normally phosphorylated core oligosaccharide, due to the inherently leaky nature of the mutation.

Comparative efficacy of calcipotriol (MC903) cream and betamethasone 17-valerate cream in the treatment of chronic plaque psoriasis. A randomized, double-blind, parallel group multicentre study. Calcipotriol Study Group.

The efficacy, safety and tolerability of calcipotriol cream was compared with betamethasone 17-valerate cream in the treatment of plaque-type psoriasis in a multicentre double-blind, parallel group study. Patients with stable mild-to-moderate chronic disease were randomized to treatment with either calcipotriol, 50 micrograms/g, in a cream formulation (210 patients) or betamethasone 17-valerate cream, 1 mg/g (211 patients). After a wash-out period of 2 weeks, the treatment was applied twice daily, without occlusion, for 8 weeks or to complete clearing. The severity of psoriasis was assessed using the PASI at baseline and after 4 and 8 weeks treatment. The mean percentage reduction of PASI from baseline to end of treatment was 47.8% in the calcipotriol group and 45.4% in the betamethasone group. The reduction from baseline was highly significant in both groups, but the difference between the groups was not significant. There was a difference in the reduction in thickness of the lesions in favour of calcipotriol. The investigator's as well as the patient's overall assessment of treatment response at end of treatment showed no difference between the two treatment groups. Treatment-related adverse events were more frequent with calcipotriol than betamethasone. Lesional/perilesional irritation was reported in 16% and 9% (P = 0.03), and facial irritation in 10% and 0.5% (P < 0.001), respectively. No change was found in serum levels of calcium. Calcipotriol in a cream formulation was effective, safe, well-tolerated, and equal in effect to betamethasone valerate cream.

The peripheral resistance in arteries of legs is inversely proportional to the severity of chronic venous insufficiency.

There are conflicting reports in the literature about the existence of arteriovenous shunting in legs with chronic venous insufficiency. Using duplex scanning, we have earlier shown that there is lowered peripheral resistance in the arteries of legs with venous ulcer together with premature venous filling in angiography. In the present study we investigated the peripheral resistance in the arteries of 16 legs with chronic venous insufficiency without present ulcer. We compared the results with those obtained from 12 healthy legs and from 18 legs with venous ulcer. There was a highly significant inverse correlation between the severity of chronic venous insufficiency and the peripheral resistance in the popliteal, the dorsal pedal and the posterior tibial arteries (p < 0.001). These results suggest that there is arteriovenous shunting in legs with chronic venous insufficiency and that this phenomenon correlates with the degree of chronic venous insufficiency.

Biological characterization of Campylobacter fetus lipopolysaccharides.

Lipopolysaccharides (LPS) of three strains of Campylobacter fetus (subspp. fetus and venerealis, and serotypes A and B), a bacterium of veterinary importance but also a cause of various infections in humans, were assessed for their ability to induce mitogenicity, gelation of Limulus amebocyte lysate, lethal toxicity in mice, and pyrogenicity in rabbits. All C. fetus LPS exhibited activities lower than those of Salmonella typhimurium LPS. LPS of C. fetus subsp. fetus serotype A had the lowest activity in all assays. Since the majority of C. fetus subsp. fetus isolates from humans are serotype A, the lower biological activities of this LPS may aid the pathogenesis of such strains. The lower activities of C. fetus LPS compared with those of S. typhimurium LPS may reflect the presence of longer fatty acid chains in the lipid A of C. fetus LPS, whereas interstrain differences in C. fetus LPS bioactivities may be related to some property influenced by composition of the saccharide moiety.

Propionibacterium acnes isolated from sternal osteitis in a patient with SAPHO syndrome.

A woman presented with palmar pustulosis and deep chest pain in association with osteitic lesions in the lower part of the sternum. Propionibacterium acnes was isolated and grew in pure culture from 6 surgically obtained bone specimens. The patient received clindamycin treatment for 6 months. Synovitis in both her wrists persisted and, based on a clinical suspicion of seronegative rheumatoid arthritis, she was treated with intramuscular gold and methotrexate with no apparent benefit. Subsequently, she was diagnosed with SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, and osteomyelitis). Our patient provides further data on the potential association between P. acnes and SAPHO syndrome.

The secG deletion mutation of Escherichia coli is suppressed by expression of a novel regulatory gene of Bacillus subtilis.

SecG, a membrane component of E. coli protein translocase, stimulates the translocation of proteins across the cell membrane through the cycle of topology inversion, which is coupled to the membrane-insertion and deinsertion cycle of SecA [Nishiyama et al. (1996) Cell 85, 71-81]. A gene of B. subtilis able to suppress the cold-sensitive phenotype of the secG deletion mutant of E. coli was cloned and found to encode a novel regulatory protein, ScgR. Similarity search revealed homology with known proteins such as GlnR of B. subtilis. Plasmid-encoded ScgR stimulated protein translocation in the deletion mutant. ScgR increased the proportion of cardiolipin at the expense of phosphatidylglycerol, but did not affect the composition of other lipid components of the cell, suggesting that the increased cardiolipin level compensates for the SecG function and thereby stimulates protein translocation.

Characterization of lipopolysaccharides of polymyxin-resistant and polymyxin-sensitive Klebsiella pneumoniae O3.

Lipopolysaccharides isolated from the polymyxin-resistant Klebsiella pneumoniae O3 mutant OM-5 and its polymyxin-sensitive parent LEN-1 were analyzed for chemical composition, and their lipid A portions were structurally characterized. The lipopolysaccharide of OM-5 contained approximately five times more 4-amino-4-deoxy-L-arabinopyranose than that of LEN-1. Other saccharide and phosphate components exhibited no significant differences. Structural characterization, including analyses by phosphorus magnetic resonance spectroscopy and by fast atom bombardment mass spectrometry, revealed a novel type of lipid A. In the OM-5 lipopolysaccharide, both phosphates of lipid A were almost totally present as phosphodiesters with 4-amino-4-deoxy-L-arabinopyranose. In the sensitive-type LEN-1 lipid A, the extent of this substitution was much lower, especially in the glycosidically linked phosphate. Phosphate in these K. pneumoniae lipopolysaccharides was almost exclusively found in lipid A. These results show that cationic substituents of phosphates of lipid A play a decisive role in determining polymyxin reactivity. OM-5 was also found to contain a large proportion of heptaacyl lipid A, which represented only a small fraction of lipid A in LEN-1.

Structures of the O-specific polysaccharide chains of Pectinatus cerevisiiphilus and Pectinatus frisingensis lipopolysaccharides.

Mild acid hydrolysis of the smooth-type lipopolysaccharide (LPS) of Pectinatus frisingensis afforded no polysaccharide but monomeric 6-deoxy-L-altrose (L-6dAlt) which was identified by anion-exchange chromatography in borate buffer, GLC/MS, 1H-NMR spectroscopy, and optical rotation. LPS was degraded with alkali under reductive conditions to give a completely O-deacylated polysaccharide, which was studied by methylation analysis, 1H-NMR and 13C-NMR spectroscopy, including sequential, selective spin-decoupling, two-dimensional correlation spectroscopy (COSY), COSY with relayed coherence transfer, two-dimensional heteronuclear 13C, 1H-COSY, one-dimensional NOE and two-dimensional rotating-frame NOE spectroscopy. It was found that the O-specific polysaccharide chain of P. frisingensis LPS is a homopolymer of 6-deoxy-L-altrofuranose built up of tetrasaccharide-repeating units having the following structure: [sequence: see text] Similarly, mild acid degradation of smooth-type LPS of Pectinatus cerevisiiphilus resulted in depolymerisation of the polysaccharide chain to give a disaccharide consisting of D-glucose and D-fucose. Study of the disaccharide by methylation analysis and alkali-degraded LPS by one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectroscopy showed that the O-specific polysaccharide of P. cerevisiiphilus has the following structure: -->2)-beta-D-Fucf-(1-->2)-alpha-D-Glcp-(1-->.

Lipopolysaccharides of polymyxin B-resistant mutants of Escherichia coli are extensively substituted by 2-aminoethyl pyrophosphate and contain aminoarabinose in lipid A.

Lipopolysaccharides (LPS) of two polymyxin-resistant (pmr) mutants and the corresponding parent strain of Escherichia coli were chemically analysed for composition and subjected to 31P-NMR (nuclear magnetic resonance) for assessment of phosphate substitution. Whereas the saccharide portions, fatty acids, and phosphate contents were similar in wild-type and pmr LPS, the latter contained two- to threefold higher amounts of 2-aminoethanol. The pmr LPS also contained 4-amino-4-deoxy-L-arabinopyranose (L-Arap4N), which is normally not a component of E. coli LPS. This aminopentose has been assigned to be linked to the 4'-phosphate of lipid A. Comparative 31P-NMR analysis of the de-O-acylated LPS of the wild-type and pmr strains revealed that phosphate groups of the pmr LPS were mainly (71-79%) diphosphate diesters, which accounted for only 20% in the wild-type LPS. Diphosphate monoesters were virtually nonexistent in the pmr LPS, whereas they accounted for 42% of all phosphates in wild-type LPS. In the lipid A of the pmr strains, the 4'-phosphate was to a significant degree (35%) substituted by L-Arap4N, whereas in the wild-type LPS the L-ArapN was absent. In the pmr lipid A, 2-aminoethanol was completely substituting the glycosidic pyrophosphate but not the glycosidic monophosphate, forming a diphosphate diester linkage at this position in 40% of lipid A molecules. In the wild-type LPS the glycosidic position of lipid A carried mostly unsubstituted monophosphate and pyrophosphate. Thus the polymyxin resistance was shown to be associated, along with the esterification of the lipid A 4'-monophosphate by aminoarabinose, with extensive esterification of diphosphates in LPS by 2-aminoethanol.

Biochemical characterization of Campylobacter fetus lipopolysaccharides.

Lipopolysaccharides (LPS) of five strains of the human and animal pathogen Campylobacter fetus were electrophoretically and chemically characterized. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that all the strains produced smooth-form LPS with O side chains of relatively constant chain length. Upon extraction, LPS partitioned into both the water and phenol phases of phenol-water extracts, which showed that two chemical species of LPS were present in each C. fetus strain. Constituents common to all the LPS, though differing in molar ratios, were L-rhamnose, L-fucose, D-mannose, D-glucose, D-galactose, L-glycero-D-manno-heptose, and D-glycero-D-manno-heptose. L-Acofriose (3-O-methyl-L-rhamnose) was present in only two of the C. fetus strains. On the basis of these differences, it was possible to distinguish between LPS from strains of different serotypes and biotypes. Furthermore, chemical analysis indicated that the phenol phase LPS had a lower level of substitution by certain neutral sugars than did water phase LPS. N-Acetylneuraminic (sialic) acid and D-galactosamine were present in all the C. fetus LPS. Constituents normally found in the core and lipid A regions of LPS, 3-deoxy-D-manno-2-octulosonic acid, D-glucosamine, ethanolamine and its phosphorylated derivatives, and fatty acids [14:0, 16:0 14:0(3-OH), and 16:0(3-OH)] were detected. Unlike Campylobacter jejuni, in which 2,3-diamino-2,3-dideoxy-D-glucose occurs as a constituent of the lipid A backbone, this amino sugar was absent from C. fetus LPS, indicating major structural differences in the lipid A's of these species.