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Stands for drum kits were initially fairly light weight, because they were designed to hold one cymbal, or a snare drum, or later on two rack toms. However, it wasn't until the 1980's that drum companies beefed up the hardware making it possible to have a steady base of support for multiple cymbal arms, etc.
For a while stands keep getting heavier and bigger, finally adding counter-weight booms and getting big enough to jack up your car for a tire change. Nowadays, that "race" has backed off a bit and drum kit stands are available in a variety of sizes (and weights), with clamps and attachments for all sorts of gear. So you do not necessarily need a separate stand for each cymbal or other item with those options available nowadays.
Drum racks had been "home made" versions until recent years. I remember seeing one from the 1930's. They are now available from several major companies in a variety of shapes and materials (aluminum, steel, PVC, other composites) with all sorts of extensions, add ons, clamps, arms you name it. It can probably be configured now from commercially available parts.
Okay, So Why Choose One Over The Other
I have used both racks and traditional stands. I have even used a combination of by having spanner tubes connected to cymbals stands (which was the best of both worlds for me). It worked great for my touring needs because all of the cymbals, toms, microphones, etc. were on the spanner tubes and the supports were actually heavy duty cymbal stands. That made it easy to set up because I didn't have to juggle a one legged post while trying to clamp on a fairly heavy (because of all of the attachment clamps) spanner tube. As just the 28 clamps on the tubes made them very heavy.
About Using Racks
If you seen the photos of Pasha's drum kit, then you realize that without using rack system components that he would require an incredible number of specialty stands. There is no question to me that using the racks are a must for him.
Racks can eliminate a lot of floor clutter if you use a lot of gear that is now on (or else would be) on separate stands. It is now possible to have everything, including your snare drum, and hi-hat, attached to a rack set-up. If you don't use a rug to mark the floor positions of the components of your drum kit, then a rack might help you with positioning the components of your drum kit the same way each time. It might not happen to you, but I have experienced times when the tubes of the rack interfered with the spot I wanted to place a cymbal or a tom. So it takes a lot of time and trial & error to get it all "just right". That also means that a rack system might not be as easy to modify when you find yourself on a stage with less than desirable space.
About Using Stands
Stands can be adjusted and moved "on the spot" without a lot of hassle in situations like a small stage. It is also possible to significantly reduce the number of stands by using clamps and attachments. Stands are a more flexible option if you tend to change your drum kit configuration for different performances.
Whether you choose to use a rack or traditional stands, they must be transported to the next performance. So if you do a lot of one niters, either way could mean schlepping a lot of weight around if you have a large drum kit. I don't believe there is much of a weight or load volume difference.
I did not want 400 pounds of drum stands to transport so here are some of my personal set-up decisions that might give you some ideas.
I have reduced my hardware load considerably during the past five years. The attachments and clamps available now have made that possible.
I don't usually use a really big set, so this is what works for me. On my left side I have one single braced stand that holds a cymbal, a mounted tambourine, and a rack tom. On my bass drum, the mounts hold two rack toms and a cymbal. On my right side, a second single-braced stand holds three cymbals, a cowbell and a jam block. The other hardware left to pack is my throne, bass drum pedals, snare stand and hi-hat stand.
I purchased an SKB Hardware case (that looks much like a hard shell golf club flight case) that is 48" x 18" and has tilt-back wheels. I do not break down the tiers on my stands. I just remove the cymbals rods. I did not disassemble my hi-hat or snare stand. I simply collapse the legs. I can lay the still-assembled stands into the SKB case for transport. When I set up my kit, it is just a matter of placing the cymbals rods back into the ratchets and opening up the tripod legs.
I think racks look really cool with some kits, although when they get really bulky it kind of hides the beautiful finishes on some drum kits. But, I think too many floor stands can look just as cluttered also. Maybe that's just the reality of using a really large drum kit.
I have used racks, stands, and the combination of both. I had my logic and reasons for that. Nowadays my playing involves using various components, depending on the performances requirements. Since I change things quite often I'm back to using stands. If I had a larger kit and was playing the exact same set-up each performance, I'd probably get my rack gear back out.
So, like so many aspects of drumming, it comes down to your personal preference of comfort, stability, convenience, portability, and even the desired "look". hey, it's all about you.
Ken is uses stands by Pacific Drums and Gibraltar Drum Rack. Ken is also an active Drum Forum member at Drum Solo Artist where he is answering drum related questions, and helping drummers with tips and advices. Submitted by: Article Directory

subset: commoncrawl


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Cannabis companies ramp up hiring
Welcome to
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Happy Friday everyone,
We've got a quick edition for you all today. This week, I got an exclusive on a new report from Vangst, Leaflink, and Flowhub about the cannabis industry's outlook for 2020 and beyond, my colleague John Lynch scored an interview with Magic Johnson about his new CBD partnership, and more.Hope you all have enjoyable weekends!
JeremyHere's what we wrote about this week:
Cannabis companies that weathered the pandemic are staffing up to prepare for huge shifts to the industry. A top recruiting firm shares the 3 skills that will be key to landing a job.Cannabis companies say they plan on ramping up hiring, according to a new report from Vangst, Leaflink, and Flowhub.Advertisement
For companies that have weathered the pandemic, sales, marketing, and IT roles will be a priority, as the industry shifts toward e-commerce rather than in-store sales.
Magic Johnson on his new partnership with a CBD retailer and the start of the NBA bubble
This one's from my colleague, John Lynch, who scored an interview with Magic Johson about his new partnership with Uncle Bud's Hemp.Johnson talked about why he's making moves into the CBD-and-hemp space, the NBA bubble, and his thoughts on the rise in COVID-19 cases in California.Advertisement
Executive moves
- Greenlane Holdings has made a slew of changes to its executive team. William Mote, previously the CFO of Basic Fun, joins as CFO, and William Bine, previously an SVP at Crocs, Inc, joins as COO, among other appointments.
- Briana Dougherty Chester has joined cannabis PR shop Mattio Communications as a partner in Los Angeles. She joins the company after a stint at MedMen and Cove PBC.
Deals, launches, and policy moves
- The US House of Representatives approved an appropriations amendment by a vote of 254-163 to protect state-legal cannabis businesses from federal interference. Specifically, the amendment would prevent the federal government from using any funds to prosecute individuals or businesses operating cannabis businesses in compliance with state law.
- Jushi Holdings is upsizing its debt financing to $33.3 million from an initial close of $15.25 million.
- Canadian Securities Exchange-listed iAnthus Capital is recapitalizing in a move that will mostly wipe out shareholder value, reports The Deep Dive. The two scenarios laid out would leave shareholders with either 2.75% equity in the cannabis company or none at all.
What we're reading
Fake businesses are fooling real banks into processing online drug purchases (Wall Street Journal)
As new pot licenses delayed indefinitely, global accounting firm pockets $7M from state to rank applicants (Chicago Sun-Times)SEC complaint: 400 investors defrauded of $25M for supposed marijuana farm, CBD extraction facility (Hemp Industry Daily)Advertisement
Critics of "Big Weed" fret over retail cannabis slotting fees (WeedWeek)
- Lebanon's prime minister and his entire cabinet have resigned following last week's deadly explosion in Beirut
- Karnataka is seeking ₹4,000 crore from the Central government to fund its flood relief measures
- Sachin Pilot says "his fight was ideological and not personal"
- Best yoga mats


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NORTHPORT, Ala. (WIAT) — The the American Red cross is asking donors to give blood at a special blood drive for Neuroblastoma awareness. The blood drive is also in memory of Jaden Elijah Smith.
He was diagnosed with stage four Neuroblastoma at the age of 10. The disease forms solid tumors in the adrenal glands, abdomen, neck, chest, or pelvis.
The sixth grader passed away four months after his diagnosis. The blood drive will be July 7th from noon until 7 p.m.
It will be at the City of Northport Civic center. That's located on Mcfarland Boulevard in Northport.
- Miss Hoover giving back to community with family portraits for the 'Front Porch Project'
- Alabama photo contest opens Aug. 3
- Alabama Department of Public Health releases COVID-19 guidance for schools
- Second stimulus checks: Progress slow on virus relief bill with $1,200 payments
- Central AL Forecast: Just a few showers today, mainly dry trend continues


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Issue 30 | Fall/Winter 2014


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B7-DC cross-linking restores antigen uptake and augments antigen-presenting cell function by matured dendritic cells
Edited by Philippa Marrack, National Jewish Medical and Research Center, Denver, CO (received for review February 19, 2005)
This article has a Retraction. Please see:
Dendritic cells (DCs) are classified in two states: immature DCs (iDCs), which perform sentinel functions, sampling for antigen and danger signals, and mature DCs (mDCs), which exhibit enhanced antigen-presenting functions but are no longer capable of acquiring antigen. We now describe DCs with a different activation phenotype: cells having the strong antigen-presenting functions of mDCs and the antigen-acquiring functions of iDCs. We have described an antibody that binds the costimulatory molecule B7-DC and activates DCs. The resulting phenotype is distinct from iDCs or mDCs matured by using Toll-like receptor (TLR) agonists. Ability to take up antigen increases, while expression of B71/2 costimulatory and MHC molecules remains unchanged. DCs matured with TLR agonists and then superactivated through B7-DC exhibit a previously unrecognized phenotype. These DCs recover the ability to take up antigen, which is normally lost after treatment with TLR-3 and TLR-9 agonists, yet retain the high expression of costimulatory and MHC molecules and strong antigen-presenting functions of mDCs. Immunization using TLR agonists and B7-DC XAb (cross-linking antibody) together as adjuvant resulted in substantially increased cytolytic T cell responses, particularly when minimal peptide antigens were used. By stimulating DCs with two distinct activation signals, a previously unrecognized phenotype exhibiting augmented antigen-presenting functions was obtained.
Dendritic cells (DCs) are heterogenous populations of cells that play important roles in initiating immune responses by their ability to actively acquire antigen and process and present peptides in the context of MHC class I and II molecules to naïve CD8 and CD4 T cells (1–4). Mature DCs (mDCs) express an array of costimulatory molecules, including B7.1, B7.2, CD40, and the recently identified B7-DC/PD-L2. B7-DC/PD-L2 acts as a ligand for PD.1, a receptor expressed on activated T cells. This interaction has been reported to activate or suppress T cell responses (5–8). The basis for these different outcomes has not been clearly established.
DCs express a number of Toll-like receptors (TLRs), thereby bridging innate and adaptive immunity (9). When resting DCs encounter TLR agonists, they become activated, increasing their ability to acquire antigen transiently, and then they undergo maturation, characterized by a loss in ability to acquire antigen and an increase in the expression of MHC antigen-presenting molecules and costimulatory molecules (10–13).
Previously, we reported that cross-linking B7-DC with the antibody sHIgM12 results in activation of DCs, enhancing their ability to stimulate and present antigen to naive T cells (14). Because B7-DC cross-linking antibody (B7-DC XAb) induces increased DC longevity under stressed conditions, enhances processing of exogenous antigen by the MHC class I pathway, and induces cytokine production by DCs in culture, we concluded that ligation of B7-DC with IgM antibody activates a reverse signaling pathway that can profoundly influence DC function (15).
The antibody-induced changes in DCs can have important biological consequences. Systemic treatment of mice bearing melanoma lung metastases results in a strong treatment effect, eliminating the tumor nodules in half of the mice and significantly reducing tumor burden in the remaining animals (16). In a model of allergic asthma, treatment of presensitized animals with B7-DC XAb completely blocked the development of airway inflammatory disease (17).
Activation of DCs, using either TLR or TNF family receptor agonists, has been studied extensively and apparently has common outcomes (18–20). The activated DCs undergo a maturation process that includes loss of their innate ability to acquire exogenous antigens from the environment and induces up-regulation of costimulatory molecules that play important roles in the activation of naïve T cells. In contrast, activation of DCs by cross-linking B7-DC did not result in appreciable up-regulation of the costimulatory molecule CD86, indicating that the antibody-induced activation profile differs substantially from the activation states induced with TLR or TNF family receptor agonists (14, 16). Here, we document key differences in these activation responses and demonstrate that activation after the cross-linking of cell surface B7-DC and activation by treatment with TLR agonists can have synergistic effects on the ability of mice to respond to peptide antigens.
Materials and Methods
Mice and Reagents. C57BL6/J mice were from The Jackson Laboratory. OT-I and OT-II transgenic strains of mice were bred at the Mayo Clinic according to Institutional Animal Care and Use Committee guidelines. Chicken albumin was obtained from Sigma-Aldrich. Fluorescein-coupled ovalbumin (OVA) was obtained from Molecular Probes. The SIINFEKL peptide was synthesized at the Mayo Clinic Protein Core Facility. Phycoerythryin-coupled anti-CD11c (HL-3), anti-B7.1 (CD80), anti-B7.2 (CD86), and anti-IAb were obtained from BD Pharmingen. Purified antibodies against murine B7-DC/PD-L2 (TY-25) and murine CD40 (1C10) and isotype control (eBR2a) were from eBioscience (San Diego). Human monoclonal control antibody (sHIgM39) and the B7-DC sHIgM12 were purified from human sera as described in ref. 14. The B7-DC XAb, sHIgM12, was discovered in the serum of a human patient with Waldenstrom's macroglobulinemia. In this study, antibody purified from patient serum was used to activate DCs. However, we have subsequently determined the structure of this antibody and constructed synthetic genes to produce a monoclonal form. To date, the monoclonal version of antibody induces the same activation profile of DCs described using the serum-derived antibodies. Therefore, we are confident that the biological changes reported below can be attributed to the activity of the sHIgM12 antibody derived from patient serum. A previously described oligodeoxynucleotide, ODN 1826 (21, 22), containing two CpG motifs (set in italics: TCC ATG ACG TTC CTG ACG TT) with phosphorothioate modification was synthesized by the Mayo Clinic Molecular Biology Core, and polyinosinic-polycytidylic acid (poly I:C) was obtained from Calbiochem.
Immunization Regimen. Immunizations were all carried out as described in ref. 22 with slight modifications. Experiments were performed with groups of three mice. Mice received four daily injections of 100 μg of CpG-ODN 1826 in PBS administered s.c. in the flank on days 1, 2, 3, and 4. Ten micrograms of control IgM antibody sHIgM39 or B7-DC XAb sHIgM12 was administered i.v. on days -1, 0, and +1. Some groups of mice received both the CpG-ODN and sHIgM12 treatments. Mice were injected at a proximal site in the flank with 50 μg of soluble OVA protein or the minimal Kb-restricted peptide antigen peptide SIINFEKL mixed with 140 μg of the class II-restricted peptide mixture PADRE (23) emulsified in incomplete Freund's adjuvant (IFA) in a total volume of 100 μl. On day 7, the draining lymph nodes were harvested and assayed directly for cytotoxicity.
In Vitro Cytotoxicity Assay. The 4-h cytotoxicity assay was carried out as described in ref. 24. Briefly, EL4 and EG7 cells were labeled with 250 μCi (1 Ci = 37 GBq) of sodium [Cr51] chromate (Amersham Pharmacia) and resuspended to a concentration of 2 × 104 cells per ml in RPMI medium 1640/10% FCS. Target cells were added to 96-well plates (2 × 103 cells per 100 μl) in triplicate. Draining lymph node cells from the appropriate groups of mice were resuspended to a concentration of 4 × 106 cells per ml and added to a 96-well plate in 100 μl in triplicate. Effector and target cells were coincubated at 37°C for 4 h. Specific lysis was calculated by using the formula [(experimental release - spontaneous release)/(maximum release - spontaneous release)] × 100, where the experimental, spontaneous, and maximal release is an average of triplicates. Averages and standard errors were calculated and plotted by using sigmastat.
Flow Cytometry. DCs were labeled with fluorescently tagged antibodies as described in ref. 14. Briefly, cells were washed with FACS buffer (1% BSA in Hanks' balanced salt solution with 0.02% sodium azide) and centrifuged into a 96-well plate (Nunc). The indicated Abs were added to the wells for a 30-min incubation on ice. After three washes, cells were fixed with 2% paraformaldehyde and analyzed on a FACSCalibur (BD Biosciences Pharmingen). Data were analyzed by using cellquest software (BD Biosciences Pharmingen).
Generation of Bone Marrow-Derived Immature DCs (iDCs) and mDCs. DCs were generated in culture from mouse bone marrow by using an established protocol (25). Briefly, bone marrow was isolated from the long bones of the hind legs. Erythrocytes were lysed by treatment with ammonium chloride/potassium bicarbonate/EDTA at 37°C. The remaining cells were plated at a density of 1 × 106 cells per ml in six-well plates (BD Biosciences) in RPMI medium 1640 containing 10 ng/ml murine granulocyte–macrophage colony-stimulating factor (GM-CSF) and 1 ng/ml murine IL-4 (PeproTech, Rocky Hill, NJ). The cells were incubated at 37°C with 5% CO2. After 2 days of culture, the cells were gently washed and replaced with RPMI medium 10 containing the same concentration of GM-CSF and IL-4. For maturation of DCs, 5-day culture was treated for an additional 24 h with 10 μg/ml CpG or poly I:C. DC maturation status was verified by flow cytometry by staining with CD80-, CD86-, and MHC class II-specific Abs.
Isolation of DCs from Lymph Nodes. DCs were isolated from the lymph node as described in ref. 26. Briefly, the tissue was cut into small pieces and incubated with RPMI medium 1640 containing 2 mg/ml collagenase, 100 μg/ml DNase (Sigma-Aldrich), and 2% FCS for 20 min at 37°C. Subsequently, 0.031 M EDTA (pH 7.5) was added for 5 min to break T cell/DC contact. The cells were subjected to erythrocyte lysis by using ammonium chloride/potassium bicarbonate/EDTA at 37°C, counted, and used for flow cytometry.
Antigen Uptake Assay. iDCs or mDCs, generated as described above, were pulsed with 1 mg/ml OVA-coupled FITC on day 6 of culture and assayed by flow cytometry 24 h later. The cultures were treated with activating antibodies (B7-DC XAb or anti-CD40 antibody) or their respective isotype control antibodies on day 4, 5, or 6 of culture as described below in individual experiments. Antibody treatments had no effect on the number of cells recovered from the cultures.
T Cell Stimulation Assay. Responder splenocyte populations from naive OT-I (27) or OT-II (28) mice were harvested and plated in triplicate after lysis of erythrocytes. Responder cells (3 × 105) were stimulated in vitro for 3 days with titrated doses of chicken albumin-pulsed DCs from various treatment groups. The plated cells were pulsed with [3H]thymidine 18 h before harvest and counted with a Packard cell harvester and counter.
In Vivo Antigen Uptake. All groups of mice were injected with 1 mg/ml FITC-coupled chicken albumin intradermally. Ten micrograms of control sHIgM39 antibody, B7-DC XAb sHIgM12, and antibody against CD40 were all administered i.v. In blockade experiments of B7-DC in vivo, 10 μg of purified TY-25 antibody was administered locally at a site adjacent to the antigen injection site.
Previously, we reported that activation of bone marrow-derived iDCs with B7-DC XAb enhances processing of exogenous antigen by the MHC class I antigen-presenting pathway (15). We examined whether enhanced acquisition of exogenous antigen was a key step in the antibody-induced increases in antigen processing. Six-day bone marrow-derived iDCs were incubated with FITC-conjugated chicken OVA (FITC-OVA) in the presence of the B7-DC XAb sHIgM12 or isotype control antibody sHIgM39. The amount of fluorescence incorporated into DCs (CD11c+ cells) after 24 h of incubation with labeled protein was used as an indicator of antigen uptake. As shown in Fig. 1A , CD11c+ DCs treated with B7-DC XAb fluoresced more brightly than did DCs treated with the isotype-matched control antibody, indicating enhanced antigen uptake. Similar results were obtained by using FITC-conjugated human serum albumin and FITC-conjugated BSA (not shown), demonstrating that a variety of proteins are taken up more readily by DCs activated with B7-DC XAb.
Whereas TLR agonists also enhance antigen uptake by DCs within minutes of TLR engagement, the ability to take up antigen is quickly lost as the DCs undergo a maturation process that includes changes in the cytoskeleton (29). We investigated the effects of B7-DC cross-linking on the activity of DCs that have been matured by using TLR-3 and TLR-9 agonists. DCs stimulated with poly I:C (TLR-3 agonist) or CpG-ODN (TLR-9 agonist) on day 5 lost their ability to acquire soluble FITC-tagged antigen added to the cultures on day 6 (Fig. 1 B and C ). However, it was quite surprising to find that treatment of these TLR agonist-matured DCs on day 6 with B7-DC XAb restored the ability of these matured cells to acquire soluble antigen (Fig. 1 D and E ). Next, we explored whether the timing of exposure to B7-DC XAb relative to a day-5 maturation signal with CpG-ODN altered the ability of DCs to take up tagged protein. B7-DC XAb or isotype control antibody was added to cultures 1 day before CpG-ODN stimulation (day 4) or at the same time as CpG-ODN stimulation (day 5) of DCs. The ability of these cells to take up antigen added on day 6 was assessed. CpG-matured DCs treated with B7-DC XAb displayed enhanced ability to take up labeled protein, irrespective of whether they were treated with the antibody on day 4, 5, or 6 (data are not shown for cells treated on days 4 or 5 with B7-DC XAb because they are essentially identical to data shown for treatment on day 6 in Fig. 1).
Increased expression of the costimulatory molecules CD80, CD86, and class II antigen-presenting molecules is a well known characteristic of mDCs (1, 13). Monocytic DCs treated with CpG-ODN or anti-CD40 antibodies on day 5 of culture strongly up-regulated CD80, CD86, and IAb expression. In the experiments shown in Fig. 2, A, B, and C illustrate the effects of TLR engagement on the up-regulation of CD80, CD86, and IAb for DCs treated on day 6 of culture with IgM isotype control antibody. In D, E, and F, the same analysis is shown for DCs treated on day 6 with B7-DC XAb. Once again, inclusion of the TLR-9 agonist CpG-ODN in the cultures resulted in up-regulation of the maturation markers. The levels of CD80, CD86, and IAb expression obtained with CpG-ODN treatment were comparable to levels achieved by activating the cells with anti-CD40 antibody assessed in a separate experiment (Fig. 2 G, H, and I ). These expression patterns confirm our previous conclusion that activation of iDCs with B7-DC XAb has little effect on the expression of CD80, CD86, or MHC class II molecules. Remarkably, DCs matured after incubation with TLR-9 agonists and subsequently treated with B7-DC XAb (or isotype control antibody) retained their high levels of all three markers. Essentially, identical findings were found when DCs were first matured with the TLR-3 agonist poly I:C and then superactivated with B7-DC XAb (data not shown).
We next investigated the ability of mDCs coactivated with B7-DC XAb to present antigens to T lymphocytes. We had shown previously that treatment of iDCs with B7-DC XAb strongly up-regulates the cells' ability to activate naïve MHC class I- and class II-restricted T cells (14). In contrast, mDCs matured before exposure to soluble protein antigen are not effective antigen-presenting cells (APCs) to naïve class I- and class II-restricted antigen-specific T cells (Fig. 3 A and B ), presumably because mDCs do not take up the exogenous antigen. Interestingly, treatment of TLR-matured DCs with B7-DC XAb restored the ability of the DCs to present antigen and activate naïve T cells (Fig. 3 C and D ). The ability to function as potent APCs was not solely determined by their reacquired ability to take up antigen, because cells treated with isotype control IgM antibody and cells receiving a combination treatment of the TLR agonist poly I:C and B7-DC XAb took up comparable amounts of FITC-conjugated protein antigen (Fig. 3E ) but displayed very different abilities to activate antigen-specific naïve T cells (Fig. 3 F and G ).
Preliminary results indicated that only a small percentage (<10%) of DCs residing in draining lymph nodes take up FITC-conjugated protein antigens within 24 h of antigen challenge. However, systemic treatment of animals with 10 μg of B7-DC XAb administered 24 h before and at the same time as the intradermal challenge with antigen resulted in robust acquisition of the labeled antigen by 32% of CD11c+ cells in the draining lymph node (Fig. 4A ). The enhancing effect of B7-DC cross-linking appeared to be circumscribed to the antigen-draining lymph nodes because antigen was not taken up by cells in the contralateral node (Fig. 4B ), and treatment with an isotype control IgM antibody did not induce strong antigen acquisition by CD11c+ cells in the draining lymph nodes (Fig. 4C ). Systemic treatment of mice with anti-CD40 antibody induced a level of antigen acquisition by resident lymph node cells that was similar (34%) to the levels induced with B7-DC XAb (Fig. 4D ). We have shown previously that monomers of the B7-DC XAb sHIgM12 or IgG antibodies with known specificity for B7-DC block the ability of the intact IgM antibody to activate iDCs (14). Here, we demonstrate that the induction of antigen uptake in vivo by systemic treatment with B7-DC XAb is blocked by a local treatment with the B7-DC-specific IgG antibody TY-25; <1% of the CD11c cells in this treatment group acquired antigen (Fig. 4 D and E ). The blocking effect is not caused by inactivation of DCs, because lymph node cells from mice receiving systemic treatments with anti-CD40 antibody acquired antigen efficiently (31%), even after being locally treated with TY-25 IgG (Fig. 4F ). These experiments extend our findings regarding the effects of B7-DC cross-linking of DCs grown in vitro to DCs residing in draining lymph nodes and confirm the specificity of the sHIgM12 antibody for B7-DC when administered in vivo.
The ability of B7-DC XAb to restore antigen acquisition by mDCs expressing high levels of the costimulatory molecule CD80, CD86, and the class II antigen-presenting molecule IAb suggested the possibility that combined treatment of animals with TLR agonists and B7-DC XAb might have additive or synergistic effects. To test this hypothesis, animals were challenged s.c. with OVA antigen in IFA and evaluated for cytotoxic T lymphocyte (CTL) development in draining lymph nodes. Cytolytic activity was evaluated by using freshly isolated lymph node cells 7 days after administration of OVA:IFA. Animals treated systemically with the IgM control antibody sHIgM39 exhibited no measurable cytolytic activity against the EL4-OVA transfectant EG7 (Fig. 5A ). Whereas treatment of animals with the TLR-9 agonist CpG-ODN or the B7-DC cross-linker sHIgM12 induced CTL activity in day-7 lymph nodes (19:1 effector-to-target ratio for half-maximal killing), a combined regimen of both DC activators induced a 4-fold stronger response (half-maximal killing at 4.5:1 effector-to-target ratio) (Fig. 5A ). None of the lymph node cells was lytic when cultured with the EL4 parental cell line that does not express OVA-derived antigens (Fig. 5B ). We conclude, therefore, that a combined regimen using both a TLR-9 agonist and B7-DC cross-linking is more effective for activating CTLs than either treatment alone.
In a parallel study, we assessed the ability of the individual DC activators to stimulate a response against simple peptide antigens. The MHC class I-restricted minimal peptide SIINFEKL derived from OVA was mixed with the helper class II-restricted peptide PADRE in IFA and evaluated for its ability to generate a CTL response in the draining lymph nodes. The peptide IFA mixture alone was not immunogenic (Fig. 5C ). Treatment with CpG-ODN induced a very weak OVA-specific CTL response, as did treatment with B7-DC XAb. In contrast, animals that received a combined treatment of peptide:IFA with CpG-ODN and sHIgM12 antibody developed robust CTL responses that were >10-fold greater by day 7 after immunization than the responses observed by using single adjuvant immunization schemes. Again, OVA-negative EL4 targets were not lysed by the induced antigen-specific CTLs (Fig. 5D ). These experiments using defined peptide antigens provide clear evidence of synergy in the ability of the TLR agonist CpG-ODN and the B7-DC XAb to potentiate a CTL response and may have significant application in vaccination strategies.
Previous studies have indicated that the combined treatment of DCs with TLR agonists and anti-CD40 can have synergistic effects on DC function (30). To determine whether superactivation of TLR agonist mDCs through engagement of CD40 might also restore antigen uptake by the mDCs, cultures matured on day 5 by stimulation with CpG-ODN were treated with anti-CD40 or isotype control antibodies on day 6 and assessed for their ability to take up FITC-OVA. Superactivation of mDCs with CD40 restored antigen acquisition in mDCs but not to the level achieved with B7-DC XAb treatment (Fig. 1F ). In contrast, restimulation of DCs matured with TLR-3 or TLR-9 agonists with fresh TLR-3 or TLR-9 agonist had no effect on antigen uptake using the same experimental approach (data not shown). Therefore, we find that superactivation of mDCs with either B7-DC XAb or anti-CD40 antibody can restore the ability of these professional APCs to take up antigen.
DCs regulate acquired immunity through the presentation of exogenous antigen to MHC class I- and class II-restricted T cells. Both iDCs and mDCs display diverse sets of receptors. Although many of these receptors tie into common signaling pathways, each has its own distinctive influence on the elicited intracellular responses. Therefore, it is not surprising that activation of DCs by engaging more than one receptor could have additive or synergistic effects. Previous studies have demonstrated synergies induced by engaging TLR-7 and CD40 (30). DCs activated simultaneously through both DC-expressed receptors displayed 10-fold increases in their ability to stimulate CD8+ T cell responses over DCs activated through each individual receptor alone. Simultaneous activation of TLR-3 and TLR-9 induced synergistic levels of nitric oxide, IL-12, TNF-α, and IL-6 production (31). Enhanced expression of TLR-2 and TLR-9 after engagement of TLR-3 and -4 with LPS provides a basis for appreciating how sequential treatment of DCs with activators might alter the course of induced responses (32).
Here, we have described a combination of stimuli through TLR and the costimulatory molecule B7-DC that activate mDC, restoring their ability to acquire antigen and enhancing their ability to rapidly activate cytotoxic T cells from naïve precursors. Both intact protein antigens and defined minimal peptide antigens were effective immunogens when this combined activation scheme was used. Because peptide antigen elicited robust CTL responsiveness when administered together with CpG-ODN and sHIgM12 antibody, enhanced antigen processing does not appear to be an essential step in the development of the robust T cell response to these antigens. We also have shown that mDCs that have been activated with B7-DC XAb and that have acquired equivalent amounts of FITC-tagged protein antigen as iDCs (Fig. 3E ) have enhanced ability to activate naïve T cells after treatment with B7-DC XAb (Fig. 3 F and G ). This finding implies that changes in DC function other than increased antigen uptake and enhanced antigen processing are elicited by the combined treatments of TLR stimulation and B7-DC cross-linking.
The recent recognition that engagement of CD40 can synergize with other DC-activating signals to enhance cellular functions (30) is further supported by our observation that treatment of mDCs with anti-DC40 antibody also restores the ability of these professional APCs to acquire exogenous antigen. The fact that engagement of CD40 or B7-DC with antibodies can induce similar effects suggests overlap in the downstream signaling pathways activated by these treatments. However, the two treatments are distinct in that anti-CD40 antibodies themselves are capable of inducing maturation of DCs, whereas B7-DC XAbs are not. Also, we find that B7-DC cross-linking is more effective at restoring antigen uptake in mDCs relative to engagement of CD40 with antibodies.
We have observed substantial increases in expression of the costimulatory molecules 4–1BB-ligand and SLAM (F. Blocki, S.R., B. Ciric, V. Van Keulen, K. L. Heckman, E. Kwon, and L.R.P., unpublished data). Using the Affymetrix chip platform, we have also recently identified gene sets that are strongly up-regulated in DCs in response to CpG-ODN and to treatment with the B7-DC XAb sHIgM12. Although there is some overlap in the observed expression profiles, there are also substantial differences (F. Blocki, V. Van Keulen, K. L. Heckman, E. Kwon, and L.R.P., unpublished data). It will be interesting to determine whether activation of DCs with a combination of TLR agonists and B7-DC cross-linking induces a unique combination of costimulatory molecules or other immune modulators that results in the augmented antigen-presenting functions of these professional APCs.
It has been reported that endocytosis involves the Rho family GTPases Rac1 and Cdc42. The importance of these proteins in this function has been shown by using two different strategies. In mDCs, endocytosis has been induced by either expressing a constitutively active Cdc42 and Rac1 or by using SopE from Salmonella, an activator of the Rho family of GTPases (33). However, in an independent study, although Rac1 was shown to be involved in endocytosis, an active form of Rac1 did not activate endocytosis in mDCs (34). We have found that ligation of B7-DC resulted in a 3-fold increase in Cdc42 message, whereas there was only a marginal increase upon treatment with poly I:C or CpG (F. Blocki and L.R.P., unpublished data). It is possible that this increase in Cdc42 in itself or in combination with as-yet-unknown proteins could be involved in reactivation of endocytosis in mDCs. In another recent report, it was demonstrated that activation of endocytosis could be achieved in mDCs by exposure to CCR7 ligands (namely, CCL19 and CCL21). This induced activity was abolished upon inhibition of Rac1 and Cdc42 (35). We have found a 3-fold increase in message for CCR7 upon cross-linking B7-DC (F. Blocki, S.R, B. Ciric, V. Van Keulen, K. L. Heckman, E. Kwon, and L.R.P., unpublished data). However, increased expression of CCR7 alone cannot account for the observed increases in pinocytotic activity, because DCs activated with TLR agonists also up-regulate CCR7. Yet stimulation through TLR3 or TLR9 did not induce uptake of exogenous antigen. Therefore, other factors must contribute to the activation of endocytosis in mDCs. It will be of interest to determine whether treatment of DCs with a combination of TLR-9 and B7-DC cross-linking activation signals induces a distinctive set of genes not represented in cells treated with individual activation signals or whether the biological interactions described in this study will be attributable to the interposition of gene products induced separately by the two stimuli.
This work was supported in part by National Institutes of Health Grants P50CA91956, R01CA103921, R01CA80782, R01CA096859, and R01CA104996.
↵ * To whom correspondence should be addressed. E-mail: pease.larry{at}mayo.edu.
Author contributions: S.R., E.C., and L.R.P. designed research; S.R. performed research; S.R. and L.R.P. analyzed data; and S.R., E.C., and L.R.P. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: DC, dendritic cell; iDC, immature DC; mDC, mature DC; TLR, Toll-like receptor; APC, antigen-presenting cell; OVA, ovalbumin; XAb, cross-linking antibody; poly I:C, polyinosinic-polycytidylic acid; IFA, incomplete Freund's adjuvant; CTL, cytotoxic T lymphocyte.
- Copyright © 2005, The National Academy of Sciences
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The gift that keeps on giving
Denise Harris, Clifford E. Lee Nature Sanctuary, AB (Photo by NCC)
Denise Harris is a dedicated Conservation Volunteer who actively gives her time to assist with meaningful stewardship projects across Alberta.
Each spring, I look forward to the arrival of our backyard neighbours, the Canada geese, for I know that spring has once again arrived. For me, spring symbolizes new beginnings filled with hope as new life emerges from beneath the snow and ice, buds burst, and knowing that wildlife's next generation will soon arrive. But when the lockdown for COVID-19 was announced, my yearning to head out and immerse myself in the wonders of volunteering in nature with the Nature Conservancy of Canada (NCC) was redirected. My focus and my priorities were quickly realigned.
For the first time in my lifetime, our species was being "inconvenienced" by a probable pandemic. It brought the truth of Al Gore's "An Inconvenient Truth" presentation to the forefront of my thoughts. Having been raised on a farm without the "convenience" of modern-day technology, I was taught to either entertain myself or do chores. It never bothered me to stay home during the pandemic because my childhood norm was based in social distancing. As time inched forward, however, thoughts of not being able to volunteer sowed the seeds of my malcontent. What do you mean, I can't remove human-made structures, waste, barbed-wire fences and invasive species? I can't plant native species, clear trails or do citizen science?
Thankfully, from my observations of Canada geese behaviour, I had learned that they don't dwell upon negative events. Why? Perhaps because they have their priorities in order or perhaps they know that distraction can result in loss of life or mate, an injury, loss of territory and/or their offspring. They express negative energy by twitching their tail and ruffling their feathers. They vent it out by screaming and/or thrashing their wings on the water, or by diving under the water to cool off — all non-violent ways of letting go. Geese that have experience parenting rarely challenge situations where they have no control; they simply, you might say, turn the other cheek and peacefully move off.
Canada geese (Photo by Denise Harris)
It is their adaptive behaviour that made me realize that with a slight modification, I too could continue to support nature. My daily walks support this concept. The threats posed to wildlife from human garbage in their wetland habitats could be safely removed with a mask and gloves. Two toads that appeared in our window well still required rescue and relocation to a safer, sustainable habitat. Injured waterfowl still needed assessing for possible rescue. Humans still wanted to learn about wildlife species and the way they behave.
In recognizing this, hopefully next time I will skip the malcontent and serenely move forward.
I miss volunteering, but now that NCC's conservation sites are open to the public again* and I can get outdoors and experience nature, inspiration and empowerment continue to rise. You see, a few years back I got a slap in the face; a wakeup call you might say. I wondered, if I only had a couple of years to live, how did I want to be remembered? This triggered a great deal of inner reflection. So, with my priorities realigned, combined with my passion for all things wild and my faith in nature's adaptability, tenacious and resilient behaviours, I believe that our collaboration can and is creating a positive influence on the health and well-being for all species, including our own.
Here's to the hope of seeing you soon, in the wild spaces.
*Most NCC properties are now open to the public. Please respect physical distancing when visiting our natural areas, and consult our website for site-specific updates. Learn more >


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The Mason County Sheriff's Office rescued an injured hiker that fell over 300 feet under the High Steel Bridge over the weekend. The 25 year-old woman suffered head injuries as she slid down the canyon wall below the bridge Saturday.
Breathing but unresponsive when rescuer reached her, she was airlifted to Harborview Medical Center in undisclosed condition.
Detective William Adam tells us on Saturday, June 13, 2015, at approximately 4:14 PM, MACECOM Dispatch received a 9-1-1 call that a female hiking with a group had slid down almost 300 feet to the water below the High Steel Bridge. The Mason County Sheriff's Office Regional Special Operations Rescue Team and Mason County Fire Districts 4, 6, 9 & 11 all responded.
Mason County Sheriff Corporal Tim Ripp and Mason County Fire District 4 Assistant Chief Greg Seals risked their lives repelling 420 feet to the water below and found 25 year old Liz Nansen from Shelton, half in the water and unresponsive. Corporal Ripp pulled Liz from the water and conducted an evaluation while Asst. Chief Seals prepared a stokes litter basket. Liz was then hoisted up to the top of the High Steel Bridge where she was transported and finally air lifted to Harborview Medical Center in Seattle.
This is the second heroic repel and life saving rescue within a month. On May 24, 2015, case # 15-07046, William H. Shaffer, 20, enlisted US Navy stationed in Bremerton, was rescued in the same fashion.
The High Steel Bridge is located off of US Forest Service Road #2340 and is in the Olympic National Forest approximately 25 miles north, northwest from Shelton. The High Steel Bridge was built by the Simpson Logging Company in 1929 as a railway bridge and it stands 420 feet above the south fork of the Skokomish River, which makes it the highest bridge in WA State.
According to the US Forest Service, the entire area under and around the High Steel Bridge has a no trespassing closure order where hiking is prohibited. They have posted signs, but people continue to hike the steep slopes and areas that are extremely dangerous. Some private websites boast that the hike is to be taken at one's own risk, but the US Forest Service and the Mason County Sheriff's Office believe that these websites disregard the law. The area is not to be hiked at all because of the extreme danger as proven within the last month.
UPDATE: Patient airlifted to Harborview Hospital. A press release will be completed on Monday with full details. pic.twitter.com/a1POinSg5a
— Mason County Sheriff (@MasonCoSheriff) June 14, 2015


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|Original language
|Energy & Fuels
|Issue number
|Pages (from-to)
|Number of pages
|Publication status
|Published - 2016
Cite this
Pedersen, M. N., Jensen, P. A., Hjuler , K., Nielsen , M., & Dam-Johansen, K. (2016). Agglomeration and Deposition Behaviour of Solid Recovered Fuel. Energy & Fuels, 30(10), 7858–7866. https://doi.org/10.1021/acs.energyfuels.6b00839


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All OMRON products are capitalized in this manual. The word .. purpose and a comparison between the CQM1H and the CQM1. Section 2. 1. V4+. Voltage pin, input 4. 2. V Common pin, input 4. 3. V3+. Voltage pin, input 3. 4. V Common pin, input 3. 5. V2+. Voltage pin, input 2. 6. V Common . View and Download Omron CQM1 operation manual online. Programmable Controllers. CQM1 Controller pdf manual download. Also for: Sysmac cqm1.
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The E3C-MA11 retains the peak value when the mode selector is set to SET so that the axes of the fiber heads can be easily adjusted to agree with each other. Set the command code C manal the X1 value. H, L, and LL comparison set values and the scaled conversion data. Do not tighten any screw of the terminals to a torque exceeding 0.
Open the compartment on the upper left of the CPU Unit and carefully draw out the battery. Section Changing Displays The following diagram shows which keys to press in order to change Program- ming Console displays. The process value data will be set to FFFF during initialization; do not read the data as the process value at this time.
This indicator cannot be used to detect the disconnection of 9. If the error persists, replace the CPU Unit.
Using Operation Control Commands 2.
OMRON CQM1 Operation Manual
If the buzzer sounds when an attempt is made to clear an error message, eliminate the cause of the error, and then clear the error message refer to cq1m CQM1 Pro- gramming Manual for troubleshooting information.
M3 optimum tightening torque: The Power Supply Unit does not occupy any words. Page 10 Application Precautions cq1 machine to fail to meet the Standard. When there is no item to set, set C7 into the output word and then use as the write data. M3 Optimum tightening torque: Repeat the the IOTC —— instruc- setting.
Section Commands and Responses Response If the output word is set to the correct command code, the sign of the scaling value and the scaling value will be set vqm1 the input word alternately in the fol- lowing format. Word n Data output Page OFF if this error occurs. Functions Forced Zero Zero-shift This manual also for: Section Comparison 2.
LSS Capabilities Section 1, 2, Page Scaling Value Teach Transmission Cables Slave as a terminator. Duplicate Expansion Master Unit. The following display will appear if the selected instruction has not been assigned to another function code. Page Timing signal The Unit requires time to process input and to set data to its input word corresponding to the input.
OMRON CQM1 OPERATION MANUAL Pdf Download.
The size of terminal screw differs for the Master and Slave. DM Write a value in the following ranges to DM according to the sensor to be used.
Page Glossary instruction A direction given in the program that tells the PC of the action to be carried out, and the data to be used in carrying out the action.
The teaching indicator red will be lit.
When the axes agree, the indicator on the light projection fiber head will be lit. Wiring and Connections Section The dielectric strength of the capacitor generally needs to be between and V.
Indicates lists of one sort or another, such as procedures, checklists, etc. Bring up the initial display.


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