Pneumolysin activates neutrophil extracellular trap formation.

The primary objective of the current study was to investigate the potential of the pneumococcal toxin, pneumolysin (Ply), to activate neutrophil extracellular trap (NET) formation in vitro. Isolated human blood neutrophils were exposed to recombinant Ply (5-20 ng ml(-1) ) for 30-90 min at 37°C and NET formation measured using the following procedures to detect extracellular DNA: (i) flow cytometry using Vybrant® DyeCycle™ Ruby; (ii) spectrofluorimetry using the fluorophore, Sytox(®) Orange (5 µM); and (iii) NanoDrop(®) technology. These procedures were complemented by fluorescence microscopy using 4', 6-diamino-2-phenylindole (DAPI) (nuclear stain) in combination with anti-citrullinated histone monoclonal antibodies to visualize nets. Exposure of neutrophils to Ply resulted in relatively rapid (detected within 30-60 min), statistically significant (P < 0·05) dose- and time-related increases in the release of cellular DNA impregnated with both citrullinated histone and myeloperoxidase. Microscopy revealed that NETosis appeared to be restricted to a subpopulation of neutrophils, the numbers of NET-forming cells in the control and Ply-treated systems (10 and 20 ng ml(-1) ) were 4·3 (4·2), 14.3 (9·9) and 16·5 (7·5), respectively (n = 4, P < 0·0001 for comparison of the control with both Ply-treated systems). Ply-induced NETosis occurred in the setting of retention of cell viability, and apparent lack of involvement of reactive oxygen species and Toll-like receptor 4. In conclusion, Ply induces vital NETosis in human neutrophils, a process which may either contribute to host defence or worsen disease severity, depending on the intensity of the inflammatory response during pneumococcal infection.

Partial complementation of Sinorhizobium meliloti bacA mutant phenotypes by the Mycobacterium tuberculosis BacA protein.

The Sinorhizobium meliloti BacA ABC transporter protein plays an important role in its nodulating symbiosis with the legume alfalfa (Medicago sativa). The Mycobacterium tuberculosis BacA homolog was found to be important for the maintenance of chronic murine infections, yet its in vivo function is unknown. In the legume plant as well as in the mammalian host, bacteria encounter host antimicrobial peptides (AMPs). We found that the M. tuberculosis BacA protein was able to partially complement the symbiotic defect of an S. meliloti BacA-deficient mutant on alfalfa plants and to protect this mutant in vitro from the antimicrobial activity of a synthetic legume peptide, NCR247, and a recombinant human ß-defensin 2 (HBD2). This finding was also confirmed using an M. tuberculosis insertion mutant. Furthermore, M. tuberculosis BacA-mediated protection of the legume symbiont S. meliloti against legume defensins as well as HBD2 is dependent on its attached ATPase domain. In addition, we show that M. tuberculosis BacA mediates peptide uptake of the truncated bovine AMP, Bac7(1-16). This process required a functional ATPase domain. We therefore suggest that M. tuberculosis BacA is important for the transport of peptides across the cytoplasmic membrane and is part of a complete ABC transporter. Hence, BacA-mediated protection against host AMPs might be important for the maintenance of latent infections.

Development of a multiplexed bead-based immunoassay for the simultaneous detection of antibodies to 17 pneumococcal proteins.

Presently, several pneumococcal proteins are being evaluated as potential vaccine candidates. Here, we gather novel insights in the immunogenicity of PLY, PsaA, PspA, PspC, NanA, Hyl, PpmA, SlrA, Eno, IgA1-protease, PdBD, BVH-3, SP1003, SP1633, SP1651, SP0189 and SP0376. We developed a multiplex bead-based immunoassay (xMAP(®) Technology, Luminex Corporation) to simultaneously quantify antibodies against these 17 pneumococcal proteins in serum. The median fluorescence intensity (MFI) values obtained for human pooled serum with the multiplex assay were between 82% and 111% (median 94%) of those obtained with the singleplex assays. For IgG, the coefficient of variation (CV) in serum ranged from 2% to 9%, for IgA, the CV ranged from 3% to 14% and for IgM, the CV ranged from 11% to 15%. Using this immunoassay, we showed that anti-pneumococcal antibody levels exhibited extensive inter-individual variability in young children suffering from invasive pneumococcal disease. All proteins, including the proteins with, as yet, unknown function, were immunogenic. In conclusion, the multiplex Streptococcus pneumoniae immunoassay based on proteins is reproducible. This assay can be used to monitor anti-S. pneumoniae antibody responses in a material- and time-saving manner.

Microvesicles released from pneumolysin-stimulated lung epithelial cells carry mitochondrial cargo and suppress neutrophil oxidative burst.

Microvesicles (MVs) are cell-derived extracellular vesicles that have emerged as markers and mediators of acute lung injury (ALI). One of the most common pathogens in pneumonia-induced ALI is Streptococcus pneumoniae (Spn), but the role of MVs during Spn lung infection is largely unknown. In the first line of defense against Spn and its major virulence factor, pneumolysin (PLY), are the alveolar epithelial cells (AEC). In this study, we aim to characterize MVs shed from PLY-stimulated AEC and explore their contribution in mediating crosstalk with neutrophils. Using in vitro cell and ex vivo (human lung tissue) models, we demonstrated that Spn in a PLY-dependent manner stimulates AEC to release increased numbers of MVs. Spn infected mice also had higher levels of epithelial-derived MVs in their alveolar compartment compared to control. Furthermore, MVs released from PLY-stimulated AEC contain mitochondrial content and can be taken up by neutrophils. These MVs then suppress the ability of neutrophils to produce reactive oxygen species, a critical host-defense mechanism. Taken together, our results demonstrate that AEC in response to pneumococcal PLY release MVs that carry mitochondrial cargo and suggest that these MVs regulate innate immune responses during lung injury.

Breast cancer patients' clinical outcome measures are associated with Src kinase family member expression.

BACKGROUND: This study determined mRNA expression levels for Src kinase family (SFK) members in breast tissue specimens and assessed protein expression levels of prominent SFK members in invasive breast cancer to establish associations with clinical outcome. Ki67 was investigated to determine association between SFK members and proliferation. METHODS: The mRNA expression levels were assessed for eight SFK members by quantitative real-time PCR. Immunohistochemistry was performed for c-Src, Lyn, Lck and Ki67. RESULTS: mRNA expression was quantified in all tissue samples. SRC and LYN were the most highly expressed in malignant tissue. LCK was more highly expressed in oestrogen receptor (ER)-negative, compared with ER-positive tumours. High cytoplasmic Src kinase protein expression was significantly associated with decreased disease-specific survival. Lyn was not associated with survival at any cellular location. High membrane Lck expression was significantly associated with improved survival. Ki67 expression correlated with tumour grade and nuclear c-Src, but was not associated with survival. CONCLUSIONS: All eight SFK members were expressed in different breast tissues. Src kinase was highest expressed in breast cancer and had a negative impact on disease-specific survival. Membrane expression of Lck was associated with improved clinical outcome. High expression of Src kinase correlated with high proliferation.

Temporal analysis of invasive pneumococcal clones from Scotland illustrates fluctuations in diversity of serotype and genotype in the absence of pneumococcal conjugate vaccine.

In September 2006, the seven-valent pneumococcal conjugate vaccine (PCV7; Prevenar) was introduced into the childhood vaccination schedule in the United Kingdom. We monitored the population of invasive pneumococci in Scotland in the 5 years preceding the introduction of PCV7 by using serogrouping, multilocus sequence typing (MLST), and eBURST analysis. Here, we present a unique analysis of a complete national data set of invasive pneumococci over this time. We observed an increase in invasive pneumococcal disease (IPD) caused by serotypes 1, 4, and 6 and a decrease in serogroup 14-, 19-, and 23-associated disease. Analysis of sequence type (ST) data shows a significant increase in ST306, associated with serotype 1, and a decrease in ST124, associated with serotype 14. There have also been increases in the amounts of IPD caused by ST227 (serotype 1) and ST53 (serotype 8), although these increases were not found to reach significance (P = 0.08 and 0.06, respectively). In the course of the study period preceding the introduction of PCV7, we observed considerable and significant changes in serogroup and clonal distribution over time.

Pneumolysin induces release of matrix metalloproteinase-8 and -9 from human neutrophils.

The research question addressed in the current study was: does the pneumococcal pore-forming toxin, pneumolysin, mobilise matrix metalloproteinase (MMP) -8 and -9 from isolated human blood neutrophils at sublytic concentrations of 5, 10 and 20 ng.mL(-1)? MMPs were measured in the supernatants of unstimulated neutrophils and of cells exposed to pneumolysin and the chemoattractant N-formyl-L-methionyl-l-leucyl-l-phenylalanine (f-MLP; 0.1 microM), individually and in combination, using ELISA procedures, and alterations in cytosolic Ca(2+) concentrations were monitored using a fura-2 acetoxymethyl ester (fura-2/AM)-based spectrofluorimetric method. Treatment of neutrophils with pneumolysin alone caused dose-related release of both MMPs, whereas f-MLP caused modest increases; the combination of both activators was, however, most effective. Pneumolysin/f-MLP-activated release of the MMPs from the cells was paralleled by increases in cytosolic Ca(2+). Exposure of human neutrophils to pneumolysin is accompanied by mobilisation of MMPs, which is potentiated by f-MLP. If operative in vivo, pneumolysin-mediated release of MMPs from neutrophils and other cell types may contribute to the pathogenesis of severe pneumococcal disease.

Dual function of pneumolysin in the early pathogenesis of murine pneumococcal pneumonia.

Streptococcus pneumoniae is one of the most common etiologic agents of community-acquired pneumonia, particularly bacteremic pneumonia. Pneumolysin, a multifunctional cytotoxin, is a putative virulence factor for S. pneumoniae; however, a direct role for pneumolysin in the early pathogenesis of pneumococcal pneumonia has not been confirmed in vivo. We compared the growth of a pneumolysin-deficient (PLY[-]) type 2 S. pneumoniae strain with its isogenic wild-type strain (PLY[+]) after direct endotracheal instillation of bacteria into murine lungs. Compared with PLY(-) bacteria, infection with PLY(+) bacteria produced greater injury to the alveolar-capillary barrier, as assayed by albumin concentrations in alveolar lavage, and substantially greater numbers of PLY(+) bacteria were recovered in alveolar lavages and lung homogenates at 3 and 6 h after infection. The presence of pneumolysin also contributed to the development of bacteremia, which was detected at 3 h after intratracheal instillation of PLY(+) bacteria. The direct effects of pneumolysin on lung injury and on the ability of pneumococci to evade local lung defenses was confirmed by addition of purified recombinant pneumolysin to inocula of PLY(-) pneumococci, which promoted growth of PLY(-) bacteria in the lung to levels comparable to those seen with the PLY(+) strain. We further demonstrated the contributions of both the cytolytic and the complement-activating properties of pneumolysin on enhanced bacterial growth in murine lungs using genetically modified pneumolysin congeners and genetically complement-deficient mice. Thus, pneumolysin facilitates intraalveolar replication of pneumococci, penetration of bacteria from alveoli into the interstitium of the lung, and dissemination of pneumococci into the bloodstream during experimental pneumonia. Moreover, both the cytotoxic and the complement-activating activities of pneumolysin may contribute independently to the acute pulmonary injury and the high rates of bacteremia which characterize pneumococcal pneumonia.

Hyaluronidase augments pneumolysin-mediated injury to human ciliated epithelium.

OBJECTIVES: The main objective of this study was to investigate the effects of pneumococcal hyaluronidase (0.1-10microg/ml), alone and in combination with pneumolysin (50 and 100ng/ml), on human ciliated epithelium. METHODS: Ciliary beat frequency (CBF) and structural integrity of human ciliated respiratory epithelium in vitro were studied using a phototransistor technique and a visual scoring index, respectively. RESULTS: Hyaluronidase per se did not affect either CBF or the structural integrity of the epithelium. However, preincubation of the epithelial strips with hyaluronidase (10microg/ml) for 30min at 37 degrees C significantly potentiated pneumolysin-mediated ciliary slowing and epithelial damage. Hyaluronan, a substrate of hyaluronidase, had no effects on the ciliated respiratory epithelium in concentrations up to 100microg/ml and did not antagonize the injurious effects of pneumolysin on the epithelium. However, preincubation of the epithelial strips with hyaluronan (100microg/ml) was associated with attenuation of the ciliary slowing and epithelial damage induced by incubation of the strips with hyaluronidase (10microg/ml) for 30min at 37 degrees C followed by addition of pneumolysin (50ng/ml). CONCLUSIONS: Although having no direct effects alone, hyaluronidase may contribute to pneumolysin-mediated damage and dysfunction to respiratory epithelium, thereby favoring colonization and subsequently extra-pulmonary dissemination of the pneumococcus.

Role of two-component systems in the virulence of Streptococcus pneumoniae.

Understanding of how the human pathogen Streptococcus pneumoniae perceives and responds to its environment in the host offers insight into the pathogenesis of disease caused by this important bacterium and the potential for improved interventions. A central role in this environmental response is played by two-component systems (TCSs), which both sense the environment and drive the cellular response. Molecular advances in the form of genome sequencing, signature-tagged mutagenesis, differential fluorescence induction and microarray analysis have yielded considerable progress in the study of these systems in S. pneumoniae. These recent advances are discussed here, focusing in particular on the role of TCSs in the virulence of S. pneumoniae.

Carcinogenic and cocarcinogenic effects of inhaled synthetic smog and ferric oxide particles.

The carcinogenic and cocarcinogenic activity of synthetic smog, ferric oxide (Fe2O3) dust, and a mixture of the two air contaminants was determined in a long-term inhalation study with Syrian hamsters. Inhaled Fe2O3 particles definitely enhanced diethylnitrosamine tumorigenicity in the peripheral lung. Synthetic smog did not. When tested at a concentration of 40 ppm methane equivalents or 40 mg/m3, respectively, neither air pollutant by itself appeared carcinogenic. Fe2O3 caused pulmonary fibrosis and synthetic smog caused alveolar bronchiolization in many of the exposed animals.

Secondary disease mortality in rat-mouse radiation chimeras.

Two statistical experimental designs were used to investigate factors involved in 90-day mortality from secondary disease in lethally irradiated mice treated with rat bone marrow. Secondary disease is a graft-versus-host syndrome that has a mortality of about 65-95% in this transplant situation. When the factors--age-of-donor-cells, day-of-cell-injection, dose-of-marrow-cells, sex, and environment--were examined for their main effects and interactions, some combinations of these factors were found to give about 25% 90-day mortality. The experiments indicate that the lowest mortality can be achieved with an injection of 40 million or more cells 1 day after irradiation and an ultraclean environment of unlimited filter-top caging.

Expression of the pneumolysin gene in Escherichia coli: rapid purification and biological properties.

The gene for pneumolysin, the thiol-activated toxin from Streptococcus pneumoniae, has been expressed in Escherichia coli. The recombinant protein has been purified using a rapid, high yield, purification procedure and has been shown to be identical with respect to N-terminal amino-acid sequence, specific activity, effect on human polymorphonuclear phagocytes and effect on human complement to the native toxin purified from the pneumococcus. This provides a large enough source of material to begin investigation of pneumolysin as a candidate for inclusion in a pneumococcal vaccine.

1.2 Angstroms crystal structure of the S. pneumoniae PhtA histidine triad domain a novel zinc binding fold.

The recently described pneumococcal histidine triad protein family has been shown to be highly conserved within the pneumococcus. As part of our structural genomics effort on proteins from Streptococcus pneumoniae, we have expressed, crystallised and solved the structure of PhtA-166-220 at 1.2 Angstroms using remote SAD with zinc. The structure of PhtA-166-220 shows no similarity to any protein structure. The overall fold contains 3beta-strands and a single short alpha-helix. The structure appears to contain a novel zinc binding motif. The remaining 4 histidine triad repeats from PhtA have been modelled based on the crystal structure of the PhtA histidine triad repeat 2. From this modelling work, we speculate that only three of the five histidine triad repeats contain the residues in the correct geometry to allow the binding of a zinc ion.

Self-interaction of pneumolysin, the pore-forming protein toxin of Streptococcus pneumoniae.

The pathogenically important cholesterol-binding pore-forming bacterial "thiol-activated" toxins (TATs) are commonly believed to be monomeric in solution and to undergo a transition on membrane binding mediated by cholesterol to an oligomeric pore. We present evidence, gained through the application of a number of biochemical and biophysical techniques with associated modelling, that the TAT from Streptococcus pneumoniae, pneumolysin, is in fact able to self-associate in solution to form the same oligomeric structures. The weak interaction leading to solution oligomerization is manifested at low concentrations in a dimeric toxin form. The inhibition of toxin self-interaction by derivatization of the single cysteine residue in pneumolysin with the thiol-active agent dithio (bis)nitrobenzoic acid indicates that self-interaction is mediated by the fourth domain of the protein, which has a fold similar to other proteins known to self-associate. This interaction is thought to have implications for the understanding of mechanisms of pore formation and complement activation by pneumolysin.

The molecular mechanism of pneumolysin, a virulence factor from Streptococcus pneumoniae.

Pneumolysin, a member of the thiol-activated cytolysin family of toxins, is a virulence factor from the Gram-positive bacterium Streptococcus pneumoniae. The toxin forms large oligomeric pores in cholesterol-containing membranes of eukaryotic cells. A plethora of biochemical and mutagenesis data have been published on pneumolysin, since its initial characterization in the 1930s. Here we present an homology model of the monomeric and oligomeric forms of pneumolysin based on the recently determined crystal structure of perfringolysin O and electron microscopy data. A feature of the model is a striking electronegative surface on parts of pneumolysin that may reflect its cytosolic location in the bacterial cell. The models provide a molecular basis for understanding the effects of published mutagenesis and biochemical modifications on the toxic activity of pneumolysin. In addition, spectroscopic data are presented that shed new light on pneumolysin activity and have guided us to hypothesise a detailed model of membrane insertion. These data show that the environment of some tryptophan residues changes on insertion and/or pore formation. In particular, spectroscopic analysis of a tryptophan mutant, W433F, suggests it is the residue mainly responsible for the observed effects. Furthermore, there is no change in the secondary structure content when the toxin inserts into membranes. Finally, the basis of the very low activity shown by a pneumolysin molecule from another strain of S. pneumoniae may be due to the movements of a key domain-domain interface. The molecular basis of pneumolysin-induced complement activation may be related to the structural similarity of one of the domains of pneumolysin to Fc, rather than the presumed homology of the toxin to C-reactive protein as previously suggested.

Studies on the structure and mechanism of a bacterial protein toxin by analytical ultracentrifugation and small-angle neutron scattering.

Pneumolysin, an important virulence factor of the human pathogen Streptococcus pneumoniae, is a pore-forming toxin which also possesses the ability to activate the complement system directly. Pneumolysin binds to cholesterol in cell membrane surfaces as a prelude to pore formation, which involves the oligomerization of the protein. Two important aspects of the pore-forming activity of pneumolysin are therefore the effect of the toxin on bilayer membrane structure and the nature of the self-association into oligomers undergone by it. We have used analytical ultracentrifugation (AUC) to investigate oligomerization and small-angle neutron scattering (SANS) to investigate the changes in membrane structure accompanying pore formation. Pneumolysin self-associates in solution to form oligomeric structures apparently similar to those which appear on the membrane coincident with pore formation. It has previously been demonstrated by us using site-specific chemical derivatization of the protein that the self-interaction preceding oligomerization involves its C-terminal domain. The AUC experiments described here involved pneumolysin toxoids harbouring mutations in different domains, and support our previous conclusions that self-interaction via the C-terminal domain leads to oligomerization and that this may be related to the mechanism by which pneumolysin activates the complement system.SANS data at a variety of neutron contrasts were obtained from liposomes used as model cell membranes in the absence of pneumolysin, and following the addition of toxin at a number of concentrations. These experiments were designed to allow visualization of the effect that pneumolysin has on bilayer membrane structure resulting from oligomerization into a pore-forming complex. The structure of the liposomal membrane alone and following addition of pneumolysin was calculated by the fitting of scattering equations directly to the scattering curves. The fitting equations describe scattering from simple three-dimensional scattering volume models for the structures present in the sample, whose dimensions were varied iteratively within the fitting program. The overall trend was a thinning of the liposome surface on toxin attack, which was countered by the formation of localized structures thicker than the liposome bilayer itself, in a manner dependent on pneumolysin concentration. At the neutron contrast match point of the liposomes, pneumolysin oligomers were observed. Inactive toxin appeared to bind to the liposome but not to cause membrane alteration; subsequent activation of pneumolysin in situ brought about changes in liposome structure similar to those seen in the presence of active toxin. We propose that the changes in membrane structure on toxin attack which we have observed are related to the mechanism by which pneumolysin forms pores and provide an important perspective on protein/membrane interactions in general. We discuss these results in the light of published data concerning the interaction of gramicidin with bilayers and the hydrophobic mismatch effect.

Role of interleukin-18 in experimental infections with Streptococcus pneumoniae.

IL-18, a multifunctional cytokine, has been shown to be involved in the immune response to numerous pathogens including several bacterial species. To study its role in infection by the Gram-positive bacterium Streptococcus pneumoniae, wild-type and IL-18 knockout BALB/c mice were compared in murine models of pneumococcal pneumonia, bacteraemia and nasopharyngeal colonization. The influence of IL-18 varied with the infection type, whereby it contributed to increased bacterial loads in pneumonia, reduced levels of colonization and had no effect on levels of bacteraemia following intravenous challenge. Likewise, the influence of IL-18 on pneumonia varied between two infecting pneumococcal strains. Comparison of these results with previous data also suggested that the influence of IL-18 in pneumococcal pneumonia differs with the mouse strain genetic background. Overall, these results demonstrate the complex influence of IL-18 in the response to the pneumococcus.

Normative amplitude-integrated EEG measures in preterm infants.

OBJECTIVE: Assessing qualitative patterns of amplitude-integrated electroencephalography (aEEG) maturation of preterm infants requires personnel with training in interpretation and an investment of time. Quantitative algorithms provide a method for rapidly and reproducibly assessing an aEEG recording independent of provider skill level. Although there are several qualitative and quantitative normative data sets in the literature, this study provides the broadest array of quantitative aEEG measures in a carefully selected and followed cohort of preterm infants with mild or no visible injury on term-equivalent magnetic resonance imaging (MRI) and subsequently normal neurodevelopment at 2 and 7 years of age. STUDY DESIGN: A two-channel aEEG recording was obtained on days 4, 7, 14 and 28 of life for infants born ⩽30 weeks estimated gestational age. Measures of amplitude and continuity, spectral edge frequency, percentage of trace in interburst interval (IBI), IBI length and frequency counts of smooth delta waves, delta brushes and theta bursts were obtained. MRI was obtained at term-equivalent age and neurodevelopmental testing was conducted at 2 and 7 years of corrected age. RESULT: Correlations were found between increasing postmenstrual age (PMA) and decreasing maximum amplitude (R= -0.23, P=0.05), increasing minimum amplitude (R=0.46, P=0.002) and increasing spectral edge frequency (R=0.78, P=4.17 × 10(-14)). Negative correlations were noted between increasing PMA and counts of smooth delta waves (R= -0.39, P=0.001), delta brushes (R= -0.37, P=0.003) and theta bursts (R= -0.61, P=5.66 × 10(-8)). Increasing PMA was also associated with a decreased amount of time spent in the IBI (R= -0.38, P=0.001) and a shorter length of the maximum IBI (R= -0.27, P=0.03). CONCLUSION: This analysis supports a strong correlation between quantitatively determined aEEG measures and PMA, in a cohort of preterm infants with normal term-equivalent age neuroimaging and neurodevelopmental outcomes at 7 years of age, which is both predictable and reproducible. These 'normative' quantitative values support the pattern of maturation previously identified by qualitative analysis.

Antigen presentation by spleen dendritic cells.

It is now recognized that dendritic cells (DC) isolated from mouse spleen play an important role in activating T lymphocytes. These DC, which show many similarities to veiled cells found in the paracortical regions of spleen and lymph node, may be closely related to the epidermal Langerhans cells. It is known that DC are extremely effective allostimulators. We have also found that although DC lack demonstrable phagocytic ability, they are extremely potent at presenting soluble polypeptide antigens to primed T cells. Since T lymphocytes comprise several distinct subsets (particularly cytotoxic, helper, and suppressor) in our most recent studies we have asked whether DC are able to trigger all these different subsets of T cells. We examined the ability of different spleen cell types coupled with the hapten NP to induce antigen-specific T suppressors for a delayed type hypersensitivity (DTH) response. It was found that T suppressors were generated only when hapten was conjugated to a spleen-derived antigen-presenting cell. Further analysis revealed that macrophages but not DC were able to induce defined sets of suppressor cells in vivo (although DC were able to trigger a very powerful DTH response). We also examined the ability of DC to activate T cells which are required to cooperate with B cells in the production of antibody. Even though DC were able to trigger T lymphocytes to produce lymphokines, these activated T cells did not act as helper cells in a standard hapten-carrier system. Possible mechanisms for this dichotomy of DC function are discussed.