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PubMed2000

Intracellular trafficking and killing of Streptococcus pneumoniae by human alveolar macrophages are influenced by opsonins.

Human alveolar macrophages (HAM) are the major resident phagocytic cells of the gas-exchanging areas of the lung. Following contact with macrophages, bacteria enter phagosomes, which gradually acquire the characteristics of terminal phagolysosomes, with incorporation of lysosome-associated membrane protein (LAMP). We measured the binding of type 1 Streptococcus pneumoniae to the surface of HAM and then measured subsequent internalization and phagosomal incorporation of LAMP-1 under various opsonic conditions. Opsonization with serum containing immunoglobulin resulted in significantly greater binding of pneumococci to HAM compared with opsonization with immunoglobulin G (IgG)-depleted serum containing complement, which in turn resulted in marginally increased binding over that observed in the absence of opsonization. Internalization of opsonized S. pneumoniae gradually increased to a maximum of 20% of bound bacteria by 120 min of warm incubation, with 20% of internalized pneumococci being localized within LAMP-containing compartments by 80 min. Internalization of opsonized S. pneumoniae by HAM correlated with a reduction of bacterial viability. When inocula were adjusted so that pneumococcal binding under different conditions was equalized, subsequent internalization, trafficking to LAMP-containing compartments, and reduction of bacterial viability were less efficient in the absence of opsonization than that observed following opsonization with adsorbed or IgG-replete adsorbed serum. Once bound to the surface of HAM, pneumococci opsonized with adsorbed serum with or without IgG were internalized, processed, and killed equally well. In conclusion, binding, intracellular trafficking, and killing of S. pneumoniae by HAM are each significantly increased by opsonization with serum containing immunogloblin and/or complement.

Antigens, CD↗

PubMed2003

Evaluation of tetracycline, erythromycin, penicillin and streptomycin for decontaminating koala semen contaminated in vitro with chlamydiae.

Semen from seven koalas was extended in a tris-citrate glucose diluent containing one of four antibiotics at different concentrations and then contaminated with a standard concentration of chlamydiae. These semen preparations were then tested for residual chlamydial viability by an in vitro cell culture assay, and any detrimental effect of the antibiotics on the motility and viability of the sperm was assessed. Penicillin at 25 iu/ml or more, erythromycin at 1000 microg/ml or more and tetracycline at 200 microg/ml or more were highly effective at rendering the chlamydiae non-viable, but streptomycin showed no antichlamydial activity. There was a significant reduction of the motility of spermatozoa extended in diluents containing erythromycin (P < 0.05), but spermatozoa incubated with tetracycline up to concentrations of 200 microg/ml were not affected.

Animals↗

PubMed2000

Effect of IFN-gamma on the killing of S. aureus in human whole blood. Assessment of bacterial viability by CFU determination and by a new method using alamarBlue.

Given the increasing incidence of methicillin resistant Staphylococcus aureus (MRSA) and the recent emergence of MRSA with a reduced susceptibility to vancomycin, alternative approaches to the treatment of infection are of increasing relevance. The purpose of these studies was to evaluate the effect of IFN-gamma on the ability of white blood cells to kill S. aureus and to develop a simpler, higher throughput bacterial killing assay. Using a methicillin sensitive clinical isolate of S. aureus, a clinical isolate of MRSA, and a commercially available strain of MRSA, studies were conducted using a killing assay in which the bacteria were added directly into whole blood. The viability of the bacteria in samples harvested at various time points was then evaluated both by the classic CFU assay and by a new assay using alamarBlue. In the latter method, serially diluted samples and a standard curve containing known concentrations of bacteria were placed on 96-well plates, and alamarBlue was added. Fluorescence readings were taken, and the viability of the bacteria in the samples was calculated using the standard curve. The results of these studies demonstrated that the CFU and alamarBlue methods yielded equivalent detection of bacteria diluted in buffer. For samples incubated in whole blood, however, the alamarBlue method tended to yield lower viabilities than the CFU method due to the emergence of a slower growing subpopulation of S. aureus upon incubation in the blood matrix. A significant increase in bacterial killing was observed upon pretreatment of whole blood for 24 h with 5 or 25 ng/ml IFN-gamma. This increase in killing was detected equivalently by the CFU and alamarBlue methods. In summary, these studies describe a method that allows for the higher throughput analysis of the effects of immunomodulators on bacterial killing.

Blood Bactericidal Activity↗

PubMed2000

Inositol phosphoryl transferases from human pathogenic fungi.

The IPC1 gene from Saccharomyces cerevisiae, which encodes inositolphosphorylceramide (IPC) synthase, was first identified as a novel and essential gene encoding resistance to the natural product antifungal aureobasidin A (AUR1). The formation of IPC in fungi is essential for viability, suggesting inhibitors of IPC1p function would make ideal antifungal drug candidates. Homologs of the AUR1/IPC1 gene were identified from a number of human pathogenic fungi, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Cryptococcus neoformans. Comparison of these genes with other homologous genes from Candida albicans, Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe reveals a conserved structural motif for inositolphosphoryl transferases which is similar to a motif recently described for lipid phosphatases, but with unique characteristics.

Candida↗

PubMed2003

Enhanced pathogenicity of susceptible strains of the Bacteroides fragilis group subjected to low doses of metronidazole.

Different concentrations of metronidazole are used widely to treat protozoan and fungal infections. As an antibacterial drug, metronidazole is mainly used against anaerobes, of which the Bacteroides fragilis group is the most important in terms of the frequency of recovery and antimicrobial resistance patterns. The objective of this study was to investigate (1) in vivo metronidazole-induced modifications in the B. fragilis group reflected by altered virulence, and (2) the interference of metronidazole in cellular viability of these samples when subjected in vitro to human polymorphonuclear leukocytes (PMNs). Strains adapted to low metronidazole concentrations were observed to be more virulent, as demonstrated experimentally in mice by weight loss, quantitative evidence of tissue damage, hemorrhage and anatomopathology of spleen, liver and small intestine samples. A significant increase (P < 0.05) in mean bacterial viability rate of about 2.62-fold was observed for all the drug-adapted strains after contact with human PMNs. However, the level of this phenomenon was quite different among the tested species. These results draw attention to the risk that prolonged therapy, even with low concentrations of metronidazole, may affect the pathogenicity of Bacteroides strains, producing changes in host-bacteria relationships.

Animals↗

PubMed2006

The m-xylene biodegradation capacity of Pseudomonas putida mt-2 is submitted to adaptation to abiotic stresses: evidence from expression profiling of xyl genes.

The effect of archetypal environmental stresses on expression of the catabolic xyl genes of the TOL plasmid pWW0 of the m-xylene degrading strain Pseudomonas putida mt-2 has been investigated. To this end, a subgenomic DNA chip was employed which included structural and regulatory DNA sequences of the TOL pathway along with selected descriptors of specific physiological conditions. Cells were separately exposed to m-xylene under various oxygen tensions, temperatures and nitrogen sources as well as situations of DNA damage, oxidative stress, carbon and iron starvation, respiratory chain damage, and contact with arsenic, but at doses which did not cause a gross effect on growth or cell viability. The incidence of each stress class was categorized through the corresponding descriptors in the chip in respect to the relative output of xyl transcripts. While most of the stresses downregulated the m-xylene biodegradation-related genes, some uncouplers of the respiratory chain (azide) and small doses of arsenate appeared to stimulate their expression. The replacement of NH4+ by NO3- as N source augmented expression of the TOL cistrons also. We subsequently subjected P. putida mt-2 cells to the multiple abiotic stress brought about by exposure to crude tar from the 2002 oil spill of the Prestige tanker, which embraces a complex mixture of hydrocarbons. The resulting expression profile of xyl genes and stress-responding markers over time suggested that adaptation to external insults precedes any significant expression of the catabolic genes. The consequences of this hierarchy of responses for microbial biodegradation in situ are discussed.

Adaptation, Physiological↗

PubMed1990

Determination of viability within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii.

Concern has been raised about the percentage of viable cells within soil rhizobia populations measured by the immunofluorescence direct count method. The purpose of this study was to evaluate a direct viable count technique which is based on the fact that viable bacteria in natural populations undergo cell elongation when they are exposed to a combination of substrate and the inhibitor of DNA gyrase, nalidixic acid. A soil extraction procedure was developed to recover a high proportion of soil bacteria (ca. 10(9)/g of soil) in suspensions with an optical clarity suitable for accurate microscopic enumeration. After incubation for 16 to 20 h at 27 degrees C in the presence of yeast extract (200 mg/liter) and nalidixic acid (10 mg/liter), between 65 and 74% of the bacteria in soil suspension became significantly elongated (greater than or equal to 4.2 microns). In contrast, less than or equal to 0.5% of the same population could be cultured, regardless of the medium composition, nutrient concentration, or incubation conditions. The direct viable count method was combined with immunofluorescence to compare the percent viability and kinetics of appearance of elongated cells within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii. Although the majority of these organisms were viable, as observed by immunofluorescence, we obtained evidence that subpopulations within the soil rhizobia community were in different states of competence to respond to substrate. A consistently low percentage (less than or equal to 30%) of the population of serotype 23 was elongated even after 24 h of incubation and regardless of when the soil was sampled.(ABSTRACT TRUNCATED AT 250 WORDS)

Colony Count, Microbial↗

PubMed1973

Function of T4D structural dihydrofolate reductase in bacteriophage infection.

Various properties of the bacteriophage structural dihydrofolate reductase (DFR) have been examined to determine its function during phage infection. It has been found that a binding site for reduced nicotinamide adenine dinucleotide phosphate (NADPH), most likely on the DFR present in the phage tail plate, is required for phage viability. Attachment of adenosine diphosphoribose, an analogue of NADPH, to this site prevents phage adsorption and injection. This adenosine diphosphoribose inhibition can be competitively reversed by the addition of NADPH or oxidized nicotinamide adenine dinucleotide phosphate. It is suggested that, during phage infection, the host bacterial cell might leak compounds functionally similar to the pyridine nucleotides. These compounds have been shown to nonenzymatically change the conformation of the phage tail plate DFR which is apparently necessary for successful injection.

Adenine Nucleotides↗

PubMed1999

Acid-induced acid tolerance and acidogenicity of non-mutans streptococci.

Acid tolerance and acidogenicity of non-mutans streptococci and their capacity of acid adaptation were studied. The cells of non-mutans streptococci (Streptococcus sanguis [Streptococcus sanguinis], Streptococcus gordonii, Streptococcus oralis and Streptococcus mitis) grown at pH 7.0 showed 0.0088% to 71% viability after acidification at pH 4.0 for 60 min, whereas the cells of mutans streptococci (Streptococcus mutans) were not killed by the acidification. Washed cells of non-mutans streptococci lowered pH to 4.04-4.33 in the presence of glucose, while mutans streptococci cells lowered pH to 3.70. When the growth pH was shifted to 5.5 for 30-90 min, the viability of non-mutans streptococci after the acidification at pH 4.0 for 60 min increased (0.25% to 91%) and the minimum pH values of the cells in the presence of glucose decreased (3.90 4.19). Along with the increase in acid tolerance and acidogenicity, non-mutans streptococci increased activities of H(+)-ATPase and arginine deiminase and amounts of stress proteins cross-reacting with 60 kDa and 70 kDa heat shock proteins (Hsp60 and Hsp70). These results indicate that non-mutans streptococci were capable of increasing their acid tolerance and acidogenicity in response to environmental acidification. Furthermore, it is suggested that the acid adaptation observed in non-mutans streptococci cells could involve the induction of H(+)-ATPase, arginine deiminase and stress protein syntheses. The strains of non-mutans streptococci, which are pioneer bacteria for dental plaque formation and predominant in plaque microbial flora, may play a significant role in shifting the dental plaque environment toward acidic and consequently promoting the colonization of more acid-tolerant and acidogenic bacteria such as mutans streptococci and lactobacilli.

Acids↗

PubMed2000

Enhancement of neonatal innate defense: effects of adding an N-terminal recombinant fragment of bactericidal/permeability-increasing protein on growth and tumor necrosis factor-inducing activity of gram-negative bacteria tested in neonatal cord blood ex vivo.

Innate defense against microbial infection requires the action of neutrophils, which have cytoplasmic granules replete with antibiotic proteins and peptides. Bactericidal/permeability-increasing protein (BPI) is found in the primary granules of adult neutrophils, has a high affinity for lipopolysaccharides (or "endotoxins"), and exerts selective cytotoxic, antiendotoxic, and opsonic activity against gram-negative bacteria. We have previously reported that neutrophils derived from newborn cord blood are deficient in BPI (O. Levy et al., Pediatrics 104:1327-1333, 1999). The relative deficiency in BPI of newborns raised the possibility that supplementing the levels of BPI in plasma might enhance newborn antibacterial defense. Here we determined the effects of addition of recombinant 21-kDa N-terminal BPI fragment (rBPI(21)) on the growth and tumor necrosis factor (TNF)-inducing activity of representative gram-negative clinical isolates. Bacteria were tested in citrated newborn cord blood or adult peripheral blood. Bacterial viability was assessed by plating assay, and TNF-alpha release was measured by enzyme-linked immunosorbent assay. Whereas adult blood limited the growth of all isolates except Klebsiella pneumoniae, cord blood also allowed logarithmic growth of Escherichia coli K1/r and Citrobacter koseri. Bacteria varied in their susceptibility to rBPI(21)'s bactericidal action: E. coli K1/r was relatively susceptible (50% inhibitory concentration [IC(50)], approximately 10 nM), C. koseri was intermediate (IC(50), approximately 1,000 nM), Klebsiella pneumoniae was resistant (IC(50), approximately 10,000 nM), and Enterobacter cloacae and Serratia marcescens were highly resistant (IC(50), >10,000 nM). All isolates were potent inducers of TNF-alpha activity in both adult and newborn cord blood. In contrast to its variable antibacterial activity, rBPI(21) consistently inhibited the TNF-inducing activity of all strains tested (IC(50), 1 to 1,000 nM). The antibacterial effects of rBPI(21) were additive with those of a combination of conventional antibiotics typically used to treat bacteremic newborns (ampicillin and gentamicin). Whereas ampicillin and gentamicin demonstrated little inhibition of bacterially induced TNF release, addition of rBPI(21) either alone or together with ampicillin and gentamicin profoundly inhibited release of this cytokine. Thus, supplementing newborn cord blood with rBPI(21) potently inhibited the TNF-inducing activity of a variety of gram-negative bacterial clinical pathogens and, in some cases, enhanced bactericidal activity. These results suggest that administration of rBPI(21) may be of clinical benefit to neonates suffering from gram-negative bacterial infection and/or endotoxemia.

Adult↗

PubMed2005

Resistance of human periodontal ligament fibroblasts to the cytolethal distending toxin of Actinobacillus actinomycetemcomitans.

BACKGROUND: The cytolethal distending toxin (CDT) of Actinobacillus actinomycetemcomitans is a typical member of this Gram-negative bacterium holotoxin family that targets a wide spectrum of eukarytotic cells, typically causing cell cycle arrest at either the G(1) or G(2)/M phase of the cell cycle. In view of the possible role of the CDT as a prominent A. actinomycetemcomitans virulence factor in periodontal diseases, we have examined the effects of the toxin on primary cultures of human periodontal ligament fibroblasts (HPLF). METHODS: HPLF and an immortalized human gingival epithelial cell line, GMSM-K, were exposed to recombinant A. actinomycetemcomitans CDT. Effects of the toxin on cell proliferation and cell cycle were assessed by a cell viability assay and flow cytometry, respectively. Double-strand DNA damage was detected by pulsed field gel electrophoresis. Binding of the toxin and its individual subunits to HPLF was examined by immunofluorescence microscopy. RESULTS: Viability of HPLF was not reduced following prolonged exposure to the CDT. There was no indication of cell cycle arrest or double-strand DNA damage. GMSM-K cells exhibited morphological alterations and a rapid decrease in cell viability within 6 and 12 hours, respectively, following exposure to the toxin for 5 minutes. These effects were dependent on toxin dose and age of the cultures and occurred more rapidly compared to CDT-treated HeLa cells. CDT-treated GMSM-K cells displayed cell cycle arrest at the S phase of growth and double-strand DNA damage was observed by 6 hours post-intoxication. Holotoxin and the CdtA subunit were detected on the surface of both HPLF and epithelial cells. CONCLUSIONS: These results demonstrate that HPLF are resistant to the cytotoxic effects of the A. actinomycetemcomitans CDT. The mechanism of resistance is not known but may be related to the inability of the toxin to cause DNA damage. The difference in sensitivities of HPLF and oral epithelial cells to the CDT has important implications for the role of this putative microbial virulence factor in periodontal pathogenesis.

Adult↗

PubMed2004

Interactions between high pressure homogenization and antimicrobial activity of lysozyme and lactoperoxidase.

It was the objective of this work to evaluate the effect of high pressure homogenization on the activity of antimicrobial enzymes such as lysozyme and lactoperoxidase against a selected group of Gram positive and Gram negative species inoculated in skim milk. Lactobacillus helveticus, Lactobacillus plantarum and Listeria monocytogenes were the most pressure resistant species while Bacillus subtilis, Pseudomonas putida, Salmonella typhimurium, Staphylococcus aureus, Proteus vulgaris and Salmonella enteritidis were found to be very sensitive to the hyperbaric treatment. The enzyme addition enhanced the instantaneous pressure efficacy on almost all the considered species as indicated by their instantaneous viability loss following the treatment. Moreover, the combination of the enzyme and high pressure homogenization significantly affected the recovery and growth dynamics of several of the considered species. Although L. monocytogenes was slightly sensitive to pressure, the combination of the two stress factors induced a significant viability loss within 3 h and an extension of lag phases in skim milk during incubation at 37 degrees C. The hypothesis formulated in this work is that the interaction of high pressure homogenization and lysozyme or lactoperoxidase is associated to conformational modifications of the two proteins with a consequent enhancement of their activity. This hypothesis is supported by the experimental results also regarding the increased antimicrobial activity against L. plantarum of the previously pressurised lysozyme with respect to that of the native enzyme.

Animals↗

PubMed2003

Influence of surfactant hydrophobicity on the detachment of Escherichia coli O157:H7 from lettuce.

The influence of surfactant hydrophobicity on detachment of Escherichia coli O157:H7 from lettuce was determined. Lettuce pieces inoculated with the pathogen were rinsed with Tween and Span surfactants of different hydrophobicity. Of the Tweens, only Tween 85, the Tween with the lowest hydrophile/lipophile balance (HLB), significantly detached the pathogen from lettuce surface. Span 85 (the surfactant with the lowest HLB studied) exhibited the greatest ability among surfactants tested to detach cells from lettuce. This surfactant removed cells attached to the leaf cuticle but not to the cut edge, and caused no detectable reduction in viability of cells remaining on the lettuce. Treatment with Span 85 did not detach cells when they were allowed to attach in the presence of calcium ions. The combination of NaCl/NaHCO3 (pH 10) and Span 85 did not detach cells possibly due to reduced hydrophobicity of the Span at this pH. This study suggests that surfactants of low HLB disrupt hydrophobic interactions between E. coli O157:H7 and the lettuce surface but cannot cause release of cells adhering to hydrophilic structures such as cut or damaged tissue.

Bacterial Adhesion↗

PubMed1998

Macrolide antibiotic inhibition of translation and 50S ribosomal subunit assembly in methicillin-resistant Staphylococcus aureus cells.

Methicillin-resistant Staphylococcus aureus cells were treated with three macrolide antibiotics to examine the inhibitory effect of the drugs on the growth rate and cell viability. Inhibition of protein synthesis and 50S ribosomal subunit assembly were also examined. The growth rate and cell viability were reduced by each antibiotic in both erythromycin-susceptible and erythromycin-resistant MRSA organisms. Translation and the formation of the 50S ribosomal subunit were inhibited to an equal extent in the erythromycin-susceptible cells, but protein synthesis was affected to a greater extent by each macrolide in the erythromycin-resistant organisms. Clarithromycin was the most inhibitory of the three compounds, followed by erythromycin and azithromycin in relative effectiveness. The use of these compounds against MRSA organisms is discussed.

Anti-Bacterial Agents↗

PubMed2006

Immunization with 3-oxododecanoyl-L-homoserine lactone-protein conjugate protects mice from lethal Pseudomonas aeruginosa lung infection.

Quorum-sensing systems have been reported to play a critical role in the pathogenesis of several bacterial infections. Recent data have demonstrated that Pseudomonas N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-homoserine lactone, 3-oxo-C12-HSL), but not N-butanoyl-L-homoserine lactone (C4-HSL), induces apoptosis in macrophages and neutrophils. In the present study, the effects of active immunization with 3-oxo-C12-HSL-carrier protein conjugate on acute P. aeruginosa lung infection in mice were investigated. Immunization with 3-oxo-C12-HSL-BSA conjugate (subcutaneous, four times, at 2-week intervals) elaborated significant amounts of specific antibody in serum. Control and immunized mice were intranasally challenged with approximately 3 x 10(6) c.f.u. P. aeruginosa PAO1, and survival was then compared. All control mice died by day 2 post bacterial challenge, while 36 % of immunized mice survived to day 4 (P<0.05). Interestingly, bacterial numbers in the lungs did not differ between control and immunized groups, whereas the levels of pulmonary tumour necrosis factor (TNF)-alpha in the immunized mice were significantly lower than those of control mice (P<0.05). Furthermore, the extractable 3-oxo-C12-HSL levels in serum and lung homogenate were also significantly diminished in the immunized mice. Immune serum completely rescued reduction of cell viability by 3-oxo-C12-HSL-mediated apoptosis in macrophages in vitro. These results demonstrated that specific antibody to 3-oxo-C12-HSL plays a protective role in acute P. aeruginosa infection, probably through blocking of host inflammatory responses, without altering lung bacterial burden. The present data identify a promising potential vaccine strategy targeting bacterial quorum-sensing molecules, including autoinducers.

4-Butyrolactone↗

PubMed1967

Diploid hybridization in a heterothallic haploid yeast, Saccharomyces rouxii.

By crossing of a heterothallic haploid yeast, Saccharomyces rouxii, we have succeeded in obtaining diploid hybrids. This paper shows one possible method of breeding heterothallic haploid yeasts for industrial application. S. rouxii is highly salt-tolerant and plays an important role in shoyu and miso fermentation. Therefore, genetic improvements of the properties are of commercial importance. Since newly isolated S. rouxii could neither conjugate nor sporulate on sporulation media commonly used, a suitable medium for conjugation and sporulation of S. rouxii was firstly investigated. A 5% NaCl Shoyu-koji extract agar was found to be most efficient. Next, we tried to get diploid strains by mass culture of two mating types on the conjugation medium, but several phenomena made this difficult: (i) zygotes quickly sporulated before budding; (ii) several zygotes showed terminal budding, but the buds could not grow into diploid cells, suggesting they would be heterocaryon; and (iii) a few zygotes lost their viability. After trying to isolate and cultivate a large number of zygotes in various combinations of crossing by micromanipulation, we fortunately recognized that large cells arose from some combinations. The analysis of ploidy suggested that the large cells would be diploid. Also, they showed sporulation of typical Saccharomyces, i.e., two to four spores in an unconjugated ascus. The diploid strains thus obtained were highly salt-tolerant and stable in liquid medium. Therefore, the procedure presented here would be effective for breeding salt-tolerant S. rouxii.

Conjugation, Genetic↗

PubMed1980

Effect of sodium fluoride on the viability and growth of Streptococcus mutans.

A fluoride-sensitive (FS) strain of Streptococcus mutans and a laboratory-induced fluoride-resistant (FR) offspring were compared for the effects of sodium fluoride on viability and growth. There was a significant fluoride-related loss of viability in resting cell suspensions of the FS strain during a 47-hour exposure to fluoride levels above 75 ppm that was not encountered with the FR strain. The addition of 300 ppmF to actively growing six-hour broth cultures almost totally arrested the growth of the FS strain, while only slightly reducing that of the FR culture. The addition of 600 ppmF immediately terminated FS growth, and greatly reduced the rate and maximum growth of FR cultures.

Cell Division↗

PubMed1988

Functional abolition of monocyte HLA-DR by aldehyde treatment. A novel approach to studies of class II restriction elements in antigen presentation.

The effect of low concentration aldehyde treatment (0.0012 to 0.005%) on the expression of HLA-DR Ag by human monocytes was investigated. This treatment was shown to selectively abolish the expression of HLA-DR determinants defined by a monomorphic mAb (YE2.36) in a rosette assay. The expression of class I MHC Ag and Fc gamma R remained unaffected. As a result, the presentation of the recall Ag tetanus toxoid and streptokinase-streptodornase (SK-SD) to freshly isolated autologous T cells and T cell clones was completely inhibited. Increasing the concentration of aldehyde to 0.05% consistently produced partial restoration of Ag-presenting capacity. Low dose aldehyde treatment did not affect monocyte viability or membrane turnover. Thus, aldehyde-treated monocytes produced a second generation of HLA-DR and expression was almost completely restored to normal after 24 h of culture. The presentation of monocyte class II Ag as alloantigens was also inhibited by low dose aldehyde treatment but inhibition was much less marked when monocytes were aldehyde treated at 2 h rather than at 24 h of culture. This is consistent with the reexpression of HLA-DR which occurred readily in the first 24 h of culture and much less readily thereafter. Low dose aldehyde treatment did not affect Ag uptake and processing. However, monocytes which had been pulsed with Ag, aldehyde-treated to abolish HLA-DR, and then cultured to allow regeneration of HLA-DR could present Ag only when given a second Ag pulse, suggesting that once the association between microbial Ag and HLA-DR had formed the Ag was not then free to reassociate with novel HLA-DR. Low dose aldehyde treatment did not affect monocyte IL-1 production, neither did it inhibit the detection of HLA-DR by soluble mAb in FACS analysis. These results are consistent with the view that low dose aldehyde treatment disrupts the tertiary structure of human Ia molecules such that allostimulatory determinants and restriction elements for exogenous Ag are rendered inaccessible to T lymphocyte receptors and to cell-bound anti-DR mAb in the rosette assay, although DR determinants may remain accessible to soluble mAb.

Aldehydes↗