Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Freshly collected alveolar macrophages (AMs) from non-AUD subjects (n=14) and AUD subjects (n=17) were cultured ex vivo with 5 μg heat-killed S. pneumoniae (Sp); cell culture supernatants were collected after two, 18, and 24 hours had elapsed. Interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in cell culture supernatants. (2A) AUD subjects exhibited higher secretion of IFNγ, but this did not achieve statistical significance (p=0.10). IFNγ quantity increased significantly over time for both subject types (p<0.0001). (2B) No significant differences in secretion of IL-1β were observed between different subject types, however, secretion of IL-1β increased over time (p=0.0003). (2C) Secretion of IL-6 by AMs from subjects with AUDs tended to be higher (p=0.12). Secretion of IL-6 increased significantly over time in each subject group (p<0.0001). (2D) Secretion of TNFα did not differ between groups (p=0.26), but did increase in each group of subjects over time (p<0.0001).

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture, Ex Vivo

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Freshly collected alveolar macrophages (AMs) from non-AUD subjects (n=14) and AUD subjects (n=17) were cultured in the presence of heat-killed S. pneumoniae protein, at doses ranging from 0 μg to 10 μg. Cell culture supernatants were collected at 18 hours. Interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in cell culture supernatants. (3A) IFNγ secretion by AMs rose with exposure to increasing doses of pneumococcal protein (p=0.0002). IFNγ secretion was more elevated in the AUD group (p=0.008). (3B) IL-1β secretion by AMs rose with increasing doses of pneumococcal protein (p=0.02), but was not different between non-AUD and AUD groups. (3C) IL-6 secretion rose with increasing pneumococcal protein doses (p<0.0001), but was not different between non-AUD and AUD groups. (3D) TNFα secretion rose with increasing pneumococcal protein doses (p<0.0001), but was not different between non-AUD and AUD groups.

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Freshly collected alveolar macrophages (AMs) from non-AUD subjects (n=15) and AUD subjects (n=15) were cultured up to 42 hours, with and without the addition of heat-killed S. pneumoniae (Sp, 10μg). In a subset of wells, N-acetylcysteine (NAC) was added after 18 hours of exposure to S. pneumoniae. Cell culture supernatants were collected at the 18 hour and 42 hour time points for analysis of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. (4A) At the 18 hour time point, unstimulated AUD subjects’ AMs secreted more IFNγ (p=0.02) (denoted with asterisk). With pneumococcal protein stimulation, IFNγ secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.002). The addition of NAC at 18 hours during the 42-hour time course was associated with less IFNγ in cell culture supernatants compared to non-NAC treated AMs at 42 hours (p<0.0001 for both non-AUD and AUD subjects). (4B) After pneumococcal protein stimulation, IL-1β secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.0004). Compared to cell culture supernatants at 42 hours without NAC, the addition of NAC at 18 hours during the 42-hour time course was associated with a non-significant rise in supernatant IL-1β among non-AUD subjects (p=0.06), and significantly higher IL-1β in AUD subjects (p=0.009). Cell culture supernatants from pneumococcal protein-stimulated, NAC-treated AMs at 42 hours compared supernatants from pneumococcal protein-stimulated AMs at 18 hours contained IL-1β values that were higher both in non-AUD (p=0.003) and AUD subjects (p=0.01). (4C) At the 18 hour time point, unstimulated AUD subjects’ AMs secreted more IL-6 (p=0.03) (denoted with asterisk). With pneumococcal protein stimulation, IL-6 secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.009). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IL-6 secretion than that by non-NAC treated AMs at 42 hours (p=0.0001 for both non-AUD and AUD subjects). Supernatant IL-6 quantities at the 18 hour time point were significantly higher than those in supernatants from pneumococcal protein-stimulated, NAC-treated AMs in both subject types (p=0.05 for non-AUD, p=0.0001 for AUD). (4D) With pneumococcal protein stimulation, TNFα secretion by AMs rose significantly between the 18 and 42 hour time points (p=0.004). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less TNFα in supernatants than what was measured in non-NAC treated AM culture supernatants at 42 hours (p=0.0001 for both non-AUD and AUD subjects), with values that were significantly less than AM culture supernatants at the 18 hour time point (p=0.02 for non-AUD, and p=0.0006 for AUD). * indicates p≤0.03 between non-AUD and AUD subjects, without pneumococcal stimulation, at 18 hours in culture. # indicates p<0.0001 between NAC and non-NAC treated conditions at 42 hours. & indicates p≤0.05 between non-NAC-treated 18-hour condition and NAC-treated, 42-hour condition.

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from non-AUD subjects (n=8) and AUD subjects (n=8) were cultured in the presence of 5 μg heat-killed S. pneumoniae (Sp); cell culture supernatants were collected at two, 18, and 24 hours. Interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in cell culture supernatants. (5A) For IFNγ, AUD subjects exhibited non-significantly higher secretion (p=0.10). Values increased significantly over time (p<0.0001). (5B) For IL-1β, no significant between-groups differences in secretion were observed (p=0.97), however, secretion increased over time (p=0.002). (5C) For IL-6, secretion values from AUD PBMCs did not differ between groups (p=0.35), but secretion increased over time (p=0.003). (5D) For TNFα, values did not differ between groups (p=0.54), and but did increase in subjects over time (p=0.007).

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from non-AUD subjects (n=8) and AUD subjects (n=8) were cultured in the presence of heat-killed S. pneumoniae (Sp), at doses ranging from 0 μg to 10 μg. Cell culture supernatants were collected at 18 hours, and interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured. (6A) IFNγ secretion over the dose range increased with increasing doses of pneumococcal protein (p=0.04), but values between groups did not differ. (6B) IL-1β secretion increased with increasing pneumococcal protein doses (p=0.004), but were not different between non-AUD and AUD groups. (6C). IL-6 secretion increased with increasing pneumococcal protein doses (p=0.004), but were not different between non-AUD and AUD groups, except at the 0μg (media only) condition (p=0.04, asterisk). (6D) TNFα secretion increased with increasing pneumococcal protein doses (p=0.001), but were not different between non-AUD and AUD groups.

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: The Impact of Alcohol Use Disorders on Pulmonary Immune Cell Inflammatory Responses to Streptococcus pneumoniae

doi: 10.1016/j.alcohol.2018.08.016

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from non-AUD subjects (n=10) and AUD subjects (n=10) were cultured up to 42 hours, with and without the addition of 10 μg heat-killed S. pneumoniae (Sp). In some wells, N-acetylcysteine (NAC) was added after 18 hours in culture. Culture media was collected at the 18 hour and 42 hour time points for analysis of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. (7A) With pneumococcal protein stimulation, IFNγ secretion by PBMCs rose significantly between the 18 and 42 hour time points (p=0.04). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IFNγ secretion than that by non-NAC treated PBMCs (p=0.002 for both non-AUD and AUD subjects), with values that were significantly different than PBMCs at the 18 hour time point in non-AUD subjects only (p=0.02). (7B) With pneumococcal protein stimulation, IL-1β secretion by PBMCs did not rise significantly between the 18 and 42 hour time points (p=0.92). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IL-1β secretion than that by non-NAC treated PBMCs at the 42 hour time point (p=0.002 for non-AUD subjects; p=0.02 for AUD subjects). Values among NAC-treated cells at the 42 hour time point approximated those in untreated cells at the 18 hour time point (p=ns). (7C) With pneumococcal protein stimulation, IL-6 secretion by PBMCs tended to rise between the 18 hour and 42 hour time points, but not significantly (p=0.09). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less IL-6 in cell culture supernatants than that found in supernatants from non-NAC treated PBMCs at the 42 hour time point (p=0.002 for both non-AUD and AUD subjects); values were not significantly different than PBMCs at the 18 hour time point for both subject types. (7D) With pneumococcal protein stimulation, TNFα secretion by PBMCs did not rise significantly between the 18 and 42 hour time points (p=0.44). The addition of NAC at 18 hours during the 42-hour time course was associated with substantially less TNFα in cell culture supernatants than in non-NAC treated PBMCs (p=0.002 for both non-AUD and AUD subjects); TNFα quantity in cell culture supernatant from pneumococcal protein -stimulated, NAC treated cells at 42 hours were significantly less than pneumococcal protein-stimulated PBMCs at the 18 hour time point among non-AUD subjects only (p=0.002). # indicates p≤0.02 between NAC and non-NAC treated conditions. & indicates p≤0.02 between 18-hour condition and NAC-treated, 42-hour condition.

Article Snippet: In some experiments, heat-killed Streptococcus pneumoniae (Strain JY2008, ATCC, Manassas, VA) was used in specific quantities over delineated time points.

Techniques: Cell Culture

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Best practices in assessing cardiac protein O -GlcNAcylation by immunoblot

doi: 10.1152/ajpheart.00104.2023

Figure Lengend Snippet: List of all antibodies used in this study including vendors, catalog numbers, dilutions, and concentrations

Article Snippet: HGAC85 , Novus Biologicals , NB300-614 , 1:1,000 , N/A.

Techniques: Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The common R71 H -G230 A -R293 Q ( HAQ ) human TMEM173 is a null allele

doi: 10.4049/jimmunol.1601585

Figure Lengend Snippet: A–C. Various types of cells from the C57BL/6, HAQ, and MPYS−/− mice were lysed in RIPA buffer and probed for indicated Abs as in Figure 1 (n=3). D–E. BMDC or BMDM from the C57BL/6, mHAQ, and MPYS−/− mice were treated with IL-4 (40ng/ml) or IFNγ (40ng/ml) or mock (PBS) overnight. Cells were lysed in RIPA buffer and probed for indicated Abs as in Figure 1 (n=3). F–G. BMDC from C57BL/6, MPYS−/− or mHAQ were stimulated with HKSP (107c.f.u/ml), LPS (20ng/ml), imiquimod (4ng/ml) or CpG-ODN2395 (8ng/ml) for 5hrs. TNFα (F) and IFNβ (G) were measured in the cell supernatant (n=3). Graph present means ± SEM from three independent experiments.

Article Snippet: Alternatively, BMDC were activated with Heat kill streptococcus pneumonia (HKSP) (10 8 c.f.u/ml) (Invivogen, cat# tlrl-hksp), LPS from Salmonella (25ng/ml) (Sigma, cat# L7261), Imiquimod (4ng/ml) (Invivogen, cat# tlrl-imqs) or CpG-ODN2395 (8ng/ml) (Invivogen, cat# tlrl-2395).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The scavenger receptor CD36 downmodulates the early inflammatory response while enhancing bacterial phagocytosis during pneumococcal pneumonia.

doi: 10.4049/jimmunol.1202270

Figure Lengend Snippet: FIGURE 1. AMs and respiratory epithelial cells express CD36, and S. pneumoniae induces pulmonary CD36 expression. (A and B) FACS staining for CD36 on MH-S (A) and MLE-12 (B) cells. Black fill depicts CD36-stained cells, and open fill represents isotype control Ab. (C) CD36 IHC was conducted from uninfected lungs of WT and CD362/2 mice. The negative control represents Ag-retrieved lungs without incubation with Ab. Original magnification 3 100. (D) WT mice (n = 6 per time point) were intranasally inoculated with 105 CFU S. pneumoniae, and after indicated time points CD36 lung transcript levels were evaluated. (E–J) WT AMs (E, G, H) or respiratory epithelial cells (F, I, J) were treated with heat-killed S. pneumoniae for indicated time points, and CD36 RT-PCR (E, F) and CD36 FACS (G–J) were conducted. (K, L) WT mice (n = 4 per time point) were intranasally inoculated with 105 CFU S. pneumoniae, and CD36 expression was evaluated on AMs from BAL after indicated time points. Depicted is the percentage of CD45+/F4/80+/CD11c+/ CD11b2 cells (AMs) positive for CD36. Representative FACS plots are shown. Data presented are mean 6 SEM, and data are representative of duplicate experiments. *p , 0.05 versus time point 0.

Article Snippet: Cell treatments and cytokine and chemokine ELISAs Cells were plated at 106/ml in 96-well dishes in RPMI supplemented with 3% heat-inactivated FCS and 1% pen/strep and stimulated with 23 107/ml heat-killed S. pneumoniae (ATCC 6303) or 10 mg/ml S. aureus or S. pneumoniae LTA or 1 mg/ml LPS for 6 h. For CD36-blocking experiments, cells were pretreated with CD36-blocking Ab (clone JC63.1) or isotype control for 30 min, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments involving PC-BSA, cells were pretreated with PCBSA or BSA for 1 h, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments studying the effects of EO6 on cytokine synthesis, heat-killed S. pneumoniae (ATCC 6303) or E. coli were incubated with 25 mg/ml EO6 or isotype control Ab for 1 h prior to addition to cells at 2 3 107/ml for 6 h. Following cell treatments, supernatant was harvested and stored at220 ̊C until assays were performed.

Techniques: Expressing, Staining, Control, Negative Control, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The scavenger receptor CD36 downmodulates the early inflammatory response while enhancing bacterial phagocytosis during pneumococcal pneumonia.

doi: 10.4049/jimmunol.1202270

Figure Lengend Snippet: FIGURE 2. CD36 suppresses S. pneumoniae–medi- ated inflammation in vitro in AMs and respiratory ep- ithelial cells. (A, B) WT primary respiratory epithelial cells (A) or AMs (B) seeded at 106/ml were treated with 2 3 107 CFU/ml S. pneumoniae for 6 h with and without pretreatment with a CD36 blocking or isotype control Ab, and levels of the indicated cytokines were evaluated in the supernatant, using ELISA. (C) WT or CD362/2 AMs were treated with 2 3 107 CFU/ml heat-killed S. pneumoniae for 6 h, and levels of TNF-a and MIP-2 were evaluated in the supernatant. (D) WT or CD362/2 AMs were treated with 2 3 107 CFU/ml heat-killed S. pneumoniae or 10 mg/ml S. aureus LTA for 6 h, and levels of TNF-a were evaluated in the supernatant. All data presented are mean 6 SEM and representative of duplicate experiments. *p , 0.05 versus isotype at the identical concentration (A, B) or WT.

Article Snippet: Cell treatments and cytokine and chemokine ELISAs Cells were plated at 106/ml in 96-well dishes in RPMI supplemented with 3% heat-inactivated FCS and 1% pen/strep and stimulated with 23 107/ml heat-killed S. pneumoniae (ATCC 6303) or 10 mg/ml S. aureus or S. pneumoniae LTA or 1 mg/ml LPS for 6 h. For CD36-blocking experiments, cells were pretreated with CD36-blocking Ab (clone JC63.1) or isotype control for 30 min, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments involving PC-BSA, cells were pretreated with PCBSA or BSA for 1 h, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments studying the effects of EO6 on cytokine synthesis, heat-killed S. pneumoniae (ATCC 6303) or E. coli were incubated with 25 mg/ml EO6 or isotype control Ab for 1 h prior to addition to cells at 2 3 107/ml for 6 h. Following cell treatments, supernatant was harvested and stored at220 ̊C until assays were performed.

Techniques: In Vitro, Blocking Assay, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The scavenger receptor CD36 downmodulates the early inflammatory response while enhancing bacterial phagocytosis during pneumococcal pneumonia.

doi: 10.4049/jimmunol.1202270

Figure Lengend Snippet: FIGURE 3. S. pneumoniae binds to CD36 via its PC, which suppresses inflammation. (A) WT BMDMs seeded at 1.6 3 106/ml were incubated with S. pneumoniae or E. coli (MOI 100) that were pretreated with either isotype (KLH) or EO6 Ab for 2 h at 4˚C, and bacterial binding was assessed using FACS. (B) WT or CD362/2 BMDMs seeded at 1.6 3 106/ml were incubated with FITC-labeled S. pneumoniae that had been pretreated with EO6 Ab (MOI 100) for 2 h at 4˚C, and bacterial binding was assessed using FACS. (C) WTAMs seeded at 106/ml were pretreated with either isotype or an EO6 Ab and incubated with 2 3 107 CFU/ml heat-killed S. pneumoniae or E. coli for 6 h, and levels of MIP-2 were evaluated in the su- pernatant. (D) WT or CD362/2 AMs seeded at 106/ml were pretreated with either PC-BSA or BSA and incu- bated with 2 3 107 CFU/ml heat-killed S. pneumoniae for 6 h, and levels of TNF-a were evaluated in the super- natant. (E) WT and CD362/2 BMDMs were incubated with heat-killed S. pneumoniae, 10 mg/ml S. pneumoniae LTA (R6 strain), or 1 mg/ml LPS for 6 h, and levels of MIP-2 were evaluated in the supernatant. All data pre- sented are mean 6 SEM and representative of duplicate experiments (A, B, E). *p , 0.05 versus isotype (A, C) or S. pneumoniae (B).

Article Snippet: Cell treatments and cytokine and chemokine ELISAs Cells were plated at 106/ml in 96-well dishes in RPMI supplemented with 3% heat-inactivated FCS and 1% pen/strep and stimulated with 23 107/ml heat-killed S. pneumoniae (ATCC 6303) or 10 mg/ml S. aureus or S. pneumoniae LTA or 1 mg/ml LPS for 6 h. For CD36-blocking experiments, cells were pretreated with CD36-blocking Ab (clone JC63.1) or isotype control for 30 min, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments involving PC-BSA, cells were pretreated with PCBSA or BSA for 1 h, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments studying the effects of EO6 on cytokine synthesis, heat-killed S. pneumoniae (ATCC 6303) or E. coli were incubated with 25 mg/ml EO6 or isotype control Ab for 1 h prior to addition to cells at 2 3 107/ml for 6 h. Following cell treatments, supernatant was harvested and stored at220 ̊C until assays were performed.

Techniques: Incubation, Binding Assay, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The scavenger receptor CD36 downmodulates the early inflammatory response while enhancing bacterial phagocytosis during pneumococcal pneumonia.

doi: 10.4049/jimmunol.1202270

Figure Lengend Snippet: FIGURE 4. CD36 suppresses early S. pneumoniae– mediated inflammation in vivo. (A–F) WT and CD362/2

Article Snippet: Cell treatments and cytokine and chemokine ELISAs Cells were plated at 106/ml in 96-well dishes in RPMI supplemented with 3% heat-inactivated FCS and 1% pen/strep and stimulated with 23 107/ml heat-killed S. pneumoniae (ATCC 6303) or 10 mg/ml S. aureus or S. pneumoniae LTA or 1 mg/ml LPS for 6 h. For CD36-blocking experiments, cells were pretreated with CD36-blocking Ab (clone JC63.1) or isotype control for 30 min, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments involving PC-BSA, cells were pretreated with PCBSA or BSA for 1 h, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments studying the effects of EO6 on cytokine synthesis, heat-killed S. pneumoniae (ATCC 6303) or E. coli were incubated with 25 mg/ml EO6 or isotype control Ab for 1 h prior to addition to cells at 2 3 107/ml for 6 h. Following cell treatments, supernatant was harvested and stored at220 ̊C until assays were performed.

Techniques: In Vivo

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The scavenger receptor CD36 downmodulates the early inflammatory response while enhancing bacterial phagocytosis during pneumococcal pneumonia.

doi: 10.4049/jimmunol.1202270

Figure Lengend Snippet: FIGURE 5. CD36 contributes to S. pneumoniae clearance in vivo but has no effects on mortality. (A) WT and CD362/2 mice (n = 7) were intranasally in- oculated with 105 CFU S. pneumoniae, and at 44 h post infection pulmonary bacterial load was evaluated. (B) WT or CD362/2 AMs seeded at 1.6 3 106/ml were incubated with FITC-labeled S. pneumoniae (MOI 100), and phagocytosis was assessed using FACS. (C and D) WT and CD362/2 mice (n = 12–13) were in- tranasally inoculated with (C) 105 or (D) 5 3 103 CFU S. pneumoniae, and survival was monitored for 10 d. Data in (A) and (B) are mean 6 SEM. *p , 0.05 versus WT.

Article Snippet: Cell treatments and cytokine and chemokine ELISAs Cells were plated at 106/ml in 96-well dishes in RPMI supplemented with 3% heat-inactivated FCS and 1% pen/strep and stimulated with 23 107/ml heat-killed S. pneumoniae (ATCC 6303) or 10 mg/ml S. aureus or S. pneumoniae LTA or 1 mg/ml LPS for 6 h. For CD36-blocking experiments, cells were pretreated with CD36-blocking Ab (clone JC63.1) or isotype control for 30 min, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments involving PC-BSA, cells were pretreated with PCBSA or BSA for 1 h, prior to stimulation with S. pneumoniae (ATCC 6303) for 6 h. For experiments studying the effects of EO6 on cytokine synthesis, heat-killed S. pneumoniae (ATCC 6303) or E. coli were incubated with 25 mg/ml EO6 or isotype control Ab for 1 h prior to addition to cells at 2 3 107/ml for 6 h. Following cell treatments, supernatant was harvested and stored at220 ̊C until assays were performed.

Techniques: In Vivo, Infection, Incubation, Labeling