15∼0.3 mL of PBS/mouse) was injected slowly into the area surrounding the nostrils after i.p. injection of 0.25 mL of pentobarbital/ethanol/PBS (0.8 mL/2 mL/8 mL). The following antibodies were used in this study: mouse IgE and IgG Abs from Zymed (San Francisco, CA, USA); rat anti-mouse IgE and IgG Abs from Biosource (Camarillo, CA, USA); HRP-labeled Selleck ACP-196 goat anti-mouse IgE and IgG Abs from Nordic (Tilburg, the Netherlands)

and Cappel (Aurora, OH, USA); FITC-labeled rat anti-mouse CD14 (Sa2–8) Abs from eBioscience (San Diego, CA, USA); and Alexa Fluor 647-conjugated rat anti-mouse CCR3 (83103) Abs, PE-labeled rat anti-mouse CD3 (145–2C11), CD4 (RM4–4), CD11b/Mac-1 (M1/70), Ly-6G (1A8), CD45R/B220 (RA3–6B2), and IgM (R6–60.2) Abs and FITC-labeled rat anti-mouse Ly-6G (1A8), CD3 (145–2C11), CD8 (53–6.7), CD11b/Mac-1 (M1/70), and CD11c (HL3) Abs from PharMingen (San Diego, CA, USA).

Blood samples were taken by cardiac puncture under chloroform anesthesia at various time intervals after i.n. injection of cedar pollen extract-Cry j with or without complete Freund’s adjuvant. The whole blood was incubated in a CO2 incubator at 37°C for 1 hr, stood overnight at 4°C, and then centrifuged at 440 g for 20 min. The supernatant fraction was stored in microtubes at − 20°C prior to use. Mice were anesthetized with chloroform and then bled from the inferior vena cava. After exsanguination, they were decapitated along the line between the upper and lower jaws. The facial

skin was stripped from the head and the nose component separated from the rest of the head along the line of the eyeballs. A segment containing the tip selleck chemicals of the Dehydratase nose and fore-teeth was severed from the rest of the specimen. After removal of the cheek muscles, cheek bones, and back teeth, NALT, which localize bilaterally on the posterior side of the palate, was separated from the rest of the nasal tissue by peeling the palate away. The excised palates were immediately placed into a 60 mm Petri dish containing stainless mesh on ice and 3 mL of ice-cold PBS with 5 mM EDTA. Using a dissection microscope (Nikon; Tokyo, Japan), the NALT was teased gently into the medium with syringe needles to release the cells, which were harvested by using siliconized Pasteur pipettes. Other lymphoid tissues such as submandibular, axillary, inguinal or mesenteric lymph nodes and Peyer’s Patches were removed aseptically; and single-cell suspensions prepared from them as described earlier (14). To evaluate lymphoid organ(s) responsive to i.n. injected allergen, we injected 2% Evans blue in PBS (2 mL/kg) i.n. and allowed it to permeate the neighboring lymphoid organs for 20 min. The mice were then anesthetized with chloroform and bled from the inferior vena cava. After exsanguination, NALT was separated from the rest of the nasal tissue and the skin covering the submandibular lymph nodes excised. The lymphoid organs stained by Evans blue were examined macroscopically.

For the original untreated negative control iDCs, after

cell transfer to a new 24-well plate, one well still remained untreated whereas PD-0332991 ic50 the other well was treated with LPS. After another 24 hr (Day 2), contents of each well were collected for either cell or cytokine assays. After DC treatment with chemokines (Day 1) and subsequent LPS stimulus (Day 2), cell viability was also determined using Trypan blue exclusion. All treatments and controls exhibited at least 90% viable cells (data not shown). Hereafter, any combination of CCL3 and CCL19 at a specific ratio will adhere to the nomenclature: CCL3 + 19 (ratio). To measure the endocytic capacity of DCs upon chemokine or subsequent LPS treatment, DCs were incubated with fluorescently labelled OVA 24 hr after chemokine treatment (Day 1) or 24 hr after subsequent LPS treatment (Day 2) and the amount of OVA

internalized by DCs was determined using flow cytometry. Immature DCs treated with individual chemokines or chemokine combinations exhibited endocytic capacity comparable to untreated iDCs (Fig. 2a). As expected, upon subsequent LPS maturation, iDCs treated only with LPS reduced their selleck chemicals llc endocytic capacity significantly compared with untreated iDCs. However, iDCs pre-treated for 24 hr (Day 1) with individual chemokines or an equal Resveratrol combination of CCL3 + 19 (5 : 5), then subsequently treated with LPS exhibited an endocytic capacity similar to untreated iDCs. Surprisingly, even after subsequent

LPS treatment, iDCs pre-treated with CCL3 + 19 (7 : 3) showed an endocytic capacity 36% higher than untreated iDCs, whereas iDCs pre-treated with CCL3 + 19 (3 : 7) exhibited a 30% lower endocytic capacity than untreated iDCs (Fig. 2a,b). When endocytic capacity (MFIs by flow cytometry) was recalculated, now normalized to the value of endocytic capacity for untreated iDCs on Day 1, iDCs pre-treated with CCL3 + 19 (7 : 3) retained 57% of their endocytic capacity, even after subsequent LPS treatment. Conversely, the normalized endocytic capacity of untreated iDCs or iDCs treated with only LPS was reduced to 44% or 15%, respectively (Fig. 2c). Even though there is no direct evidence explaining why the endocytic capacity of untreated iDCs decreased over time, this natural decrease was presumably attributed to effects of the GM-CSF in the culture media[41-43] and of the cell transfer (Fig. 1)[41] on minimal maturation of these DCs during the 3-day culture in this study.

Here, we present evidence that TCR diversity is an essential aspect of Foxp3+ Treg-cell homeostasis and function. Treg cells with a broader TCR repertoire exhibited sustained survival and expansion in hosts with less diverse Treg cells, which likely reflected their advantage in competition for self peptides and other peptides presented

by MHC class II. Adoptive transfer experiments revealed that the TCR repertoire of Treg-cell populations varied by anatomical location. Functionally, our data strongly suggest that see more TCR diversity is a critical factor for efficient Treg-cell mediated suppression of experimental acute GvHD. If not crossed to a Rag-deficient background, TCR-Tg mice contain functional Treg cells that develop through thymic selection of endogenous, non-clonotypic TCR rearrangements 14, 39, 40. Only in rare exceptions, e.g. in AND- or HA- TCR-Tg mice 41, 42, a limited number of clonotypic thymocytes was shown to develop into Foxp3+ Treg

cells 15, 16, 43. Here, the use of broadly available OT-II TCR-Tg as Treg-cell recipients allowed efficient in vivo expansion of adoptively transferred WT Treg cells with a broader TCR repertoire. Moreover, congenic markers in combination with the eGFP-reporter in the Foxp3 locus assured unambiguous detection of Treg cells after adoptive transfer. To the best of our knowledge, Selleckchem HDAC inhibitor such a robust expansion of adoptively transferred ADP ribosylation factor Treg cells as described here is unprecedented in non-lymphopenic mice. Several studies in humans and mice have implied that TCR diversity is an important feature of Treg cells. A comprehensive study on one single human T-cell repertoire recently concluded that Treg cells were the most diverse T cells 28. The

authors predicted 89 920 TCRα CDR3 sequences in Treg cells (defined as CD4+CD25+) compared with 58 325 in all other naive and transitional CD45RA+ non-Treg cells. This is in line with former data obtained by spectratyping of human Treg-cell CDR3 regions 44, 45. Furthermore, earlier studies using classical sequencing approaches also found at least similar diversity in mouse Treg cells 6, 7. Our study demonstrated that the TCR repertoire of WT mouse Treg cells was indeed very broad, however, at least TCR-Vα8 CDR3 diversity was found to be even higher in WT Foxp3−CD4+ T cells than in Treg cells (Supporting Information Fig. 2). Recent studies suggested that thymic intra- and interclonal competition for limited antigen presented on MHC class II may be an important mechanism to generate Treg cells with a broad TCR spectrum 15, 16, 46. This was specific for natural Treg cells but not for Foxp3−CD4+ T cells and thus led to the conclusion that TCRs from Treg cells may on average have higher affinity for self-peptide-MHC.

In an adoptive therapy model of lymphoma, self-antigen-specific Teff cells were potentiated by even a modest reduction of CTLA4. A subtle reduction of CTLA4 did not curtail Treg-cell suppression. Thus, Teff cells had an exquisite sensitivity to physiological levels of CTLA4 variations. However, both Treg and Teff cells were impacted by anti-CTLA4 antibody blockade. Therefore, whether CTLA4 impacts through Treg cells or Teff cells depends on its expression level. Overall,

the results suggest that the tumor microenvironment represents an “immunoprivileged self” that could be overcome practically and at least partially by RNAi silencing of CTLA4 in Teff cells. A cardinal https://www.selleckchem.com/products/MG132.html capacity of the immune system is to differentiate between “self”, the body’s own tissue, and “non-self”, exemplified by microbial infectious agents. Malignant tumor tissues present a distinct challenge to the immune system as “altered self”. Antigenic proteins from mutated genes in cancer cells, or viral products from transformed tumor cells, may trigger the immune system as tumor-specific

antigens ICG-001 (TSAs) that are not expressed by nonmalignant tissues. However, for the vast majority of tumors, TSA have yet to be identified. Well-studied tumor-associated antigens (TAAs) are in fact self antigens associated with cellular differentiation [1]. The difficulty to identify TSA compels a supposition that cancer cells are largely “self”. The premise of cancer cells as “altered self” would predict the well-recognized association of autoimmune risk with cancer immunotherapy [2]. On the other hand, the “altered self” view could also foretell autoimmunity as a beneficial effector to destroy cancer cells. In other words, although autoimmunity and tumor immunity are often viewed as being on opposite sides of the same coin, they could

also be viewed to be on the same side of the coin, serving as overlapping mechanisms for tumor destruction. Indeed, the remarkable benefits of cancer immunotherapies showed in recent immunotherapy Fenbendazole trials, most notably anti-CTLA4 antibody blockade, often came with the price of autoimmune adverse effects [3]. The intricate tangle of auto-immune toxicity and antitumor immunity substantially affects the benefit/risk ratio calculation in immunotherapies [1]. On the other hand, auto-immunity may serve to benefit clinical management of cancers. Evidence gathered from the clinics treating a variety of cancers with immunotherapies based on IL-2 [4], interferon α-2b [5], or CTLA4 [3, 6] suggests that the therapy-induced autoimmunity, at least in part, may actually mediate the destruction of cancer cells. The clinical observations provoke suggestion of a paradigm shift, to which autoimmunity is not a shunned side effect, but instead an acceptable or even desirable antitumor mechanism [7].

These effectors could arise naturally as the tumours develop, such as the T cells seen in many melanoma patients,2,63,64 or from intentional

immunization with tumour-associated antigens,2–4 or could even be T cells that have been expanded and even genetically modified in vitro and adoptively transferred.65,66 Hence, although we have shown effects of the fusion protein as a single agent, probably enhancing innate responses and the endogenous T-cell response, we hypothesize that the fusion protein LBH589 order would be even more effective in conjunction with immunization schemes. In this context there are a wide variety of innovative approaches for initiating anti-tumour cellular immune responses that show substantial promise (reviewed in refs 1 and 67) as well as recent clinical successes in patients with prostate cancer.68,69 The data presented here represent the first ‘proof of principle’ of the protease-activated cytokine approach using specific

inhibition. Importantly, the tethered cytokine strategy using specific inhibition is a platform technology that could be employed INCB024360 with different immunomodulatory agents to either promote (e.g. IL-12) or inhibit (IFN-β or IL-10) cellular immune responses. This would be particularly useful for cytokines that have potent anti-tumour effects like IL-12 but systemic side-effects limit their usefulness when given systemically.11,70 The scFv format is particularly flexible in this regard. An scFv could be developed against almost any target molecule given the extremely large antibody repertoire in the scFv library and could be made against immunomodulators such as chemokines where the receptor approach is not easily implemented. It is also important to consider that the cytokine environment in the tumour would probably be affected in a cascade fashion as the infiltrating cells change. As a result, it may be possible to alter the balance of cytokines from the generally suppressive environment of the tumour, rich in a variety of immunosuppressive factors, enzymes and cells,1,71–74 to one that is conducive to an ongoing immune response leading to the eradication of

tumours. next The authors would like to thank Drs Edward Messing and Baek Kim for encouragement and helpful suggestions, Dr Robert Rose and Christopher Lane for helpful advice on insect cell expression of proteins, and Drs Barth, Leddy, Courtney, Simon, Valentino and Cohen for comments on the manuscript. This work was made possible by generous gifts from Steven and Alison Krausz and F.C. Blodgett. John Puskas, Denise Skrombolas and Abigail Sedlacek were supported by 5T32AI00728 from the National Institutes of Health. None of the authors involved with this work has any financial interests or any other conflict of interest to disclose. “
“The effects of the soluble forms of the endotoxin receptor molecules sMD-2 and sCD14 on bacterial growth were studied.

One of the most important aspects of subcellular proteomics is the inference of hypothetical protein function based on its subcellular localization. Many proteomic studies in trypanosomatids have focused on specific subcellular compartments or fractions, simply to increase the probability of detecting those proteins and/or improving their functional characterization. The subproteomic studies on specific compartments such as the glycosome, an organelle involved in the first part of glycolytic pathway (62), the buy Small molecule library acidocalcisome, an organelle mediating calcium homoeostasis and pH homoeostasis

(61), the reservosome, a storage organelle (63,64), the flagellum (65), the nucleus (66), plasma membrane (67) and mitochondrion (68) provided an opportunity to improve the functional annotation of hypothetical proteins and search for the candidate drug targets. In addition, glycoproteome (69), GPI-anchored proteome (GPIome) (70) and secreted proteome (secretome) studies (71–74) are of great interest because of their potential role in the interaction with host proteins. Among the many possible post-translational modifications (PTMs), cellular protein phosphorylation is a key mechanism of controlling development in trypanosomatids. The trypanosomatid phosphoproteome (kinome) was recently

characterized (75–77). Studies of other frequent PTMs, such as glycosylation learn more (69), histone acetylation and deamidation (78), have been recently reported. Mass spectrometry coupled with 2-D PAGE has been the most efficient and popular approach to characterize trypanosomatid proteome profiles. With more extensive use of high-throughput proteomic PtdIns(3,4)P2 approaches (79–82), we will soon see the emergence of more complete and diverse proteomic datasets that should complement the transcriptome data and facilitate the unravelling of the pathobiology of trypanosomatids. With genomic data available, transcriptome profiles abundant, and cultivation methods for different life cycle stages well established, trypanosomatids are emerging as ideal model organisms

for metabolomic studies (83). Ultrahigh resolution metabolomic studies in trypanosomatids are offering new tools to identify biomarkers of disease, comprehensively characterize cellular responses to perturbations, and identify novel potential drug targets (84–86). Currently available databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/) (87–89) and Pathway Tools software (90) allow mapping the results onto reconstructed networks. The MetExplore web server (91) offers the tools to link metabolites identified in untargeted metabolomics surveys within the context of genome scale reconstructed metabolic networks. Genome-wide metabolic networks in Leishmania spp.

Of note here, one recent murine study has shown that IL-1 signalling is also essential for Th17 lineage differentiation in mice, and that

IL-6 induces IL-1R expression on T cells. In this report, IL-1r1−/− animals had higher percentages of FoxP3+ T cells compared to wild-type counterparts, and in an EAE model wild-type, but not IL-1r1−/−, FoxP3+ T cells produced IL-17 in the central nervous system (CNS), suggesting a greater similarity in Th17 differentiation and Treg to Th17 conversion between humans and mice than thought previously [79]. Murine Tregs can INCB018424 manufacturer be directed towards the Th17 lineage through receptor–ligand interactions on DC that activate them to produce the appropriate cytokine environment, including (Curdlan-induced) Dectin-1 activation [72] and B7 cross-linking on DC [78]. Conversely, murine Tregs can be protected from IL-6-driven Th17 conversion following exposure to TGF-β and IL-2, as these cytokines in concert reduce surface expression of the IL-6 receptor [75]. As a result, it has been proposed that TGF-β iTregs are more resistant to Th17 conversion in mice than nTregs[75]. This is the only publication that demonstrates a potential difference between nTregs and iTregs in the propensity

to convert to the Th17 lineage and should be accepted only with the caveats that the observed effect cannot be said categorically to be due to inherent differences between nTregs and iTregs and not the result of TGF-β and IL-2 signalling buy Venetoclax per se, and that the concentrations of TGF-β and IL-2 used in iTreg generation in vitro are orders of magnitude higher than those seen in vivo.

Some of these reports have demonstrated that Th17 cells derived from Tregs share common features with Th17 cells generated from naive precursors, Rucaparib order including expression of the chemokine receptor CCR6 [73,76,80]. CCR6 is a chemokine receptor expressed on the surface of Th17 cells, under the control of the Th17 transcription factor receptor-related orphan receptors (ROR)α and RORγt, which directs their migration into sites of inflammation [81]. Interestingly, although ‘converted’ Tregs also express CCR6 (as well as other chemokine receptors in common with Th17 cells [82]), in contrast to Th17 cells they do not express CCL20 [macrophage inflammatory protein (MIP)-3α][81], which is the only known ligand for CCR6 [83]. Th17 cells therefore recruit other Th17 cells and Tregs into sites of inflammation through secretion of CCL20 [81]. Indeed, chronically inflamed tissues in human diseases are characterized by the presence of infiltrating Th17 cells expressing CCR6 [84], and mice are protected from developing EAE if the CCR6–CCL20 interaction is neutralized [81].

Magnetic resonance imaging revealed a mass lesion at the pineal gland accompanied by obstructive hydrocephalus. Following surgery, pathological examinations demonstrated a pleomorphic granular cell astrocytoma. The patient has been free from recurrence for 24 months after surgery without adjuvant therapy. The specimen exhibited nuclear and cytoplasmic pleomorphism. The nuclei varied in size, shape and coarseness. Variability was also observed in the eosinophilic granular bodies, Rosenthal fibers and spindle-shaped Selleckchem BVD-523 tumor cells. GFAP, S-100 and vimentin were immunohistochemically positive. Reticulin network was absent between the tumor cells, and granular cells with ballooned cytoplasm showing positive staining for PAS. Pleomorphic

granular cell astrocytoma is believed to be a form of astrocytoma originating from the pineal gland. Its clinicopathological features resemble those of pleomorphic xanthoastrocytoma. However, it can be differentiated from the latter by the absence of reticulin fibers, absence of basement membrane between adjacent cells, and presence of large numbers of mitochondria. “
“A. Ekonomou, M. Johnson, R. H. Perry, E. K. Perry, R. N. Kalaria, S. L. Minger and C. G. Ballard (2012) Neuropathology and Applied Neurobiology38, 344–353 Increased neural progenitors in individuals with cerebral small vessel disease Aims:

Recent work has highlighted a significant increase of neural stem/progenitor cells after stroke in humans. In this study, we examined neurogenesis in small vessel disease, a key concurrent pathology Pritelivir concentration in Alzheimer’s disease. Methods: We assayed autopsy tissue from 13 vascular dementia patients with small vessel disease and 12 age-matched subjects without cerebrovascular pathology, undertaking immunohistochemistry in the affected brain area and the subventricular zone with a well-characterized battery of antibodies to detect neural stem cells/progenitors and immature (-)-p-Bromotetramisole Oxalate neurones, as well as choline acetyltransferase immunoreactivity. Results: We showed significant increases ranging from 33% to 92% (P < 0.05) in neural progenitor cells around the areas of microvascular

pathology and in the subventricular zone in patients with small vessel disease compared to individuals without cerebrovascular changes, even in patients with severe cerebrovascular disease, as defined by neuropathological assessment. Some of the progenitor cells give rise to immature neurones in the affected areas. These alterations were associated with vascular changes, but were unrelated to the cholinergic deficit observed in the cortex and subventricular zone in these patients, in contrast to other dementias examined such as dementia with Lewy bodies. Conclusions: This study provides evidence for neurogenesis in small vessel disease and may have important implications for the development of new therapies for neurodegenerative diseases. “
“A. H. Hainsworth, R. C. Allsopp, A. Jim, J. F. Potter, J. Lowe, C. J. Talbot and R. J.

Thirty thousands of sorted CD19+ CD25+ or CD19+ CD25− B cells were resuspended in KRG buffer (Krebs-Ringer phosphate buffer) check details with Ca2+, containing 0,1% BSA (Sigma-Aldrich) in a final volume of 30 μl and were placed on the upper well in duplicates. Cells were migrated towards different concentration of CXCL13 (50, 100 and 500 ng/ml), KRG buffer containing 0.1% BSA as a negative control added to the lower wells in a final volume of 30 μl. To determine if the migration was random

(chemokinesis) or directed (chemotaxis), 500 ng/ml of CXCL13 was added to both the upper and lower chamber followed by addition of cells to the upper chamber. Cells were incubated in a humidified atmosphere containing 5% CO2 at 37° for 12 h, thereafter the upper cell suspensions was removed, and the plates with the net were centrifuged at 350 g at 4° for 10 min. The net was discarded followed by an addition of 2 μl trypan blue together with 28 μl formaldehyde (4%). Migrated Ruxolitinib in vitro cells were manually enumerated using a microscope. Expression of homing receptors.  For flow cytometry analyses, 106 spleen cells were placed in 96-well plates and pelleted (3 min, 300 g, 4 °C). To avoid unspecific binding via Fc-receptor interactions, cells were incubated with Fc-block (2.4G2; BD Bioscience) for 8 min at room temperature. All antibodies were diluted in FACS-buffer (PBS containing, 1% FCS, 0.1% sodium azide and 0.5 mm EDTA). The antibodies used were directly conjugated with phycoerythrin

(PE), Pacific blue (PB) and peridinin chlorophyll protein (PerCp). Antibodies used were anti-CD25 (PC61), anti-α4β7 (DATK32), anti-CD62L (MEL-14), anti-CXCR5 (2G8) Coproporphyrinogen III oxidase purchased from BD Bioscience and anti-CD19 (1D3), anti-CXCR4 (2B11) purchased from eBioscience, (San Diego, CA, USA). Cells were stained as previously described, and gating of cells was performed using fluorochrome minus one settings

[13]. All data in the study are presented as levels above the background. Proliferation assay.  Triplicates of sorted CD19+ CD25+ or CD19+ CD25− B cells at a concentration of 2.5 × 105/ml were plated in a volume of 100 μl in round-bottomed 96-well plates and stimulated with either 3 μm CpG-PS, 5 μg/ml E-coli LPS or 0.5 μg/ml of Pam3Cys in a humidified atmosphere containing 5% CO2 at 37° for 48 h and pulsed with 1 μCi 3H-thymidine (Amersham Pharmacia Biotech) for additional 8 h. The cells were harvested onto glass fibre filters (Walluc Oy) and dried, where after incorporated 3H-thymidine was measured using a β-scintillation counter. Statistics.  All statistical analyses have been performed using the Prism software (GraphPad software version 4.0b; La Jolla, CA, USA), and Wilcoxon matched paired test was used when comparing CD25+ to CD25− B-cell subpopulations and Kurskal–Wallis test followed by Dunn’s test for multiple comparisons when comparing more than two cell populations. P < 0.05 was considered as significant. B cells were sorted in to two highly purified populations (>98.

The candidacidal mechanism of 3M-003-activated macrophages was investigated. MMA was used to test for 3M-003 induction of inducible nitric oxide synthase (iNOS) and its effect on candidacidal activity. We found that MMA at 0.2 mM significantly reduced the candidacidal activity of 3M-003 (10 and 100 μg mL−1)-activated macrophages from 40% and 44% to 28%

and 23%, respectively (P<0.05 for both) (Fig. 2b). Moreover, the candidacidal activity of IFN-γ-activated macrophages (51%) was reduced to 36% by 0.2 mM MMA. These findings were reproduced in a second experiment with 3M-003 100 μg mL−1 and IFN-γ 1000 U mL−1. These results indicate that iNOS induced by 3M-003 or IFN-γ can be inhibited by MMA, resulting in decreased killing learn more of C. albicans. Monocytes had low candidacidal activity (0–10%

in various experiments), and treatment with 3M-003 did not significantly Nutlin-3a chemical structure enhance candidacidal activity (maximum, 14%) (Fig. 3a). On the other hand, IFN-γ at 250 U mL−1 significantly (P<0.05) increased monocyte candidacidal activity to 28% (Fig. 3a). IFN-γ concentrations of 500 or 1000 U mL−1 did not prove superior to 250 mL−1. In another experiment where the challenge time was 2 h instead of 4 h, similar results were obtained, for example IFN-γ at 250 U mL−1, but not 3M-003, significantly (P<0.05) increased the candidacidal activity of monocytes compared with the candidacidal activity of monocytes cultured in CTCM. Neutrophils cultured in CTCM had significant candidacidal activity (46%). Treatment of neutrophils with 3M-003 (0.1–10 μg mL−1) did not significantly increase killing of C. albicans (51%) compared with neutrophils treated with CTCM (Fig. 3b). By contrast, neutrophils treated

with IFN-γ (1000 U mL−1) significantly (P<0.01) increased killing of C. albicans (to 82%) compared with killing by control neutrophils (Fig. 3b). Similar data were obtained at E : T of 50 : 1. In another experiment where the E : T ratio was 10 : 1, killing by control (CTCM) neutrophils (25%) was not significantly different from 22% to 32% killing by 3M-003 (1 μg mL−1)-treated neutrophils; however, killing HAS1 by IFN-γ-treated neutrophils was significantly (P<0.01) increased to 54%. When the supernatants from PBMC cultures stimulated by 3M-003 were tested for cytokines by ELISA, high levels of TNF-α and IL-12 were found (Table 1). 3M-003 at 1 μM appeared to be optimal for the production of these proinflammatory cytokines. It can be noted that IL-10 production was increased twofold above the background (Table 1). On the other hand, 3M-003 stimulation of PBMC did not induce IFN-γ production above the background (data not shown). Splenocyte preparations from macerated mouse spleens produced lower amounts of cytokines after stimulation with 3M-003 than did PBMC (data not shown).