text:
I've never cheated on my wife but Saturday night as soon as she goes to sleep is Tubegalore time , midgets ,first time anal ,mom tubes ,BBC ........you name it ,I can't help it.
amateur.you already cheat upfront, so youre first.and yes, cheatin at every single oppurtunity, they think youll do it anyway, and you only live once, and what we live for if not having sex as long as we can?it gets boring after 2 months.after 6 months, first murder thoughts are created.after 1 year, you cant stand her smell anymore.something like that.
didnt have you down as god squad cypwhat ever works brother
Yes doing HIT style bible bashing for maximum gains
Performing miracles in the gym
Same! Married 5 years. Solid girl, parties with me all over the world, keeps up, even makes her own money as a nurse practitioner. This was key for me having a girl that doesn't have to work at all BUT wants to. Not sure why this is such a turn on for me but it is.Sex is decent. After a few bottles of wine it's a 9 but on average a 7.
Lot of retards on Getbig think that women actually want to marry THEM. Ha! When the right woman comes along you will know and it will be the best thing that ever happened to you. Sure, I look at other chicks, sure I jerk off thinking about them but the wife is the one that puts up with your bad moods and washes your dirty socks and still loves you.My advice, don't go thinking you're all that because you probably aren't and if you can't stomach the ups and downs then marriage isn't for you, it's strictly for the adults in this world.
This x10!I have been divorced and got married again this past weekend.Amazes me how so many getbiggers act like all the woman are out to marry them.
Hey congrats on getting married!When are you divorcing?
Don't get married. Ever.
Do you 'don't get married ever' studs ever have long term relationships now, or do you just use and abuse the hoe's?
This x10!I have been divorced and got married again this past weekend.Amazes me how so many getbiggers act like all the woman are out to marry them.A few young 20 's gym rats asked me how they could meet and date "professional woman" like I did.My wife is a VP of a major bank and very smart.I laughed and told 'em they couldn't pull up with their bros in the back seat and take them to someplace besides Burger King for a dinner date.
Congrats Howard!
For making it to the 3 day anniversary?


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Visiting Suriname's Interior On Your Own
by Mattfromtexas on February 16, 2011
One of the main things we wanted to do in Suriname was to go up the Suriname River, which cuts down the belly of the country, into the rain forest where runaway slaves long ago set up settlements to escape their Dutch captors.
These settlements, now villages, continue to maintain ancient African traditions. It is a place where women still prefer to wash dishes and clothes at the river; the language, Saramaccan, is a spoken language; and the only foreigners you'll meet are American Peace Corps volunteers, a clear sign that you've seriously veered off the beaten path.
The idea that African villages exist in Latin America was mind-blowing to us. During our time in Suriname, we hoped to learn more about these Maroon villages and to see for ourselves what life is like on the river.
Researching the Interior proved to be somewhat challenging as the Lonely Planet section on Suriname is disappointingly terse and so few travelers ever make it that way. We inquired with local hostel owners about Interior tour options and were shocked to learn that the cheapest tours were still several hundred dollars per person--hardly a deal for a backpacker and hardly reflective of real costs.
Screw the tour operators! We decided to do it ourselves and were only momentarily discouraged when our new friend warned us: "You can't go there by yourself. There is nothing but jungle and water!" Oh, yeah?
The following is our how-to guide on touring the Interior. We tell you how to secure transportation from the capital to Atjoni (via minivan or "Wagi") and then upriver to the various Maroon villages between Atjoni and Djumu (via motorized dugout canoe). We also cover places to stay and other general advice.
Catching a ride to Atjoni (also called Pokigron) is not easy. There's no bus station or schedule, or buses for that matter. Basically, the way it works is this. At 7am, a handful of minivans line up along Saramacca Straat near the intersection of Prinsen Straat. It is important to note that there are literally hundreds of vans on this street, each going to a different part of the country. They all look exactly the same and generally aren't labeled, so you will have to ask which vans are going to Atjoni. Prices are negotiable, but you will generally pay about SRD$50-70 per person depending on the number of people in your van and the quality of the vehicle. Even if your van is full, the driver will not leave until 9am. The trip to Atjoni takes about four hours. The road is paved half of the way and then switches to gravel.
Here are a few Wagi drivers you can contact to secure your trip to Atjoni.
Kobi - 829-6601 or 868-7710
Jesi - 716-3599
Kwakoe - 823-9406
Kip - 857-4167
Huur - 817-7551
Ali - 820-1047
Salong - 852-6112
Steven - 816-0346
Hendo - 818-8953
Rance - 828-0768
Lloyd - 810-2270
Ewald - 810-3902
When you arrive in Atjoni, you will find a sleepy riverside village with a tiny restaurant (of sorts), a general store, and a dozen or so motorized dugout canoes ready to take passengers upriver. It is a good idea (but not essential) to try and secure a boatman before you arrive in Atjoni. Prices vary according to distance and the amount of luggage you have. Everyone knows everyone else here, so don't feel shy about asking someone to help you find your boatman.
Here are a few boatmen you can contact with their estimated rates per person.
Servicing the Lower Suriname River (Less than 1 Hour from Atjoni)
Asegede - Kajapaati and surrounding area - Tel: 858-1057 / SRD$20-30
Saba - Gangeston and surrounding area - Tel: 868-1178 / SRD$20-30
Marcado - Abenastone and surrounding area - Tel: 829-6647 / SRD$20-30
Servicing the Middle Suriname River (2 to 3 Hours from Atjoni)
Tjado - Tjaikonde and surrounding area - Tel: 864-5945 / SRD$50-70
Bamos - Tjaikonde and surrounding area - Tel: 854-3156 / SRD$50-70
Tudie - Tutu and surrounding area - Tel: 886-5334 / SRD$50-70
Ramoe - Bendikwai and surrounding area - Tel: 886-3644 / SRD$50-70
Belanki - Pikiseei and surrounding area - Tel: 811-1784 / SRD$50-70
Joel - Futunakaba and surrounding area - Tel: 811-1784 / SRD$50-70
Toma - Botopasi and surrounding area - Tel: 814-6338 / SRD$50-70
*Lanti Boat - Malobi and surrounding area - Tel: 711-1153 / SRD$20
*Lanti Boat - Botopasi and surrounding area - Tel: 820-2609 / SRD$20
*Lanti Boat - Kambalua and surrounding area - Tel: 821-4843 / SRD$20
Servicing the Upper Suriname River (3+ Hours from Atjoni)
Afante - Semoisie and surrounding area - Tel: 813-2966 / SRD$50-80
Batoe - Semoisie and surrounding area - Tel: 716-9624 / SRD$50-80
Edson - Semoisie and surrounding area - Tel: 818-5313 / SRD$50-80
Maku - Semoisie and surrounding area - Tel: 812-1057 / SRD$50-80
Mardona - Semoisie and surrounding area - Tel: 829 1940 / SRD$50-80
*Lanti Boat - Semoisie and surrounding area - Tel: 710-8092 - 40 SRD
Reuben - Asindohopo and surrounding area - Tel: 811-9366 / SRD$50-80
Lover - GaanSeei and surrounding area - Tel: 817 3117 / SRD$50-80
Chris - GaanSeei and surrounding area - Tel: 812-4476 / SRD$50-80
*Lanti boats are government-subsidized boats that run on a specific daily schedule, usually marked by white flags. They are generally slower and charge extra if you have lots of bags. Make arrangements for pick-up ahead of time so that you don't miss the only boat leaving that day.
We spent our time in Botopasi, Pikin Slee, and Bendikwai, but after meeting some of the locals and some awesome Peace Corps volunteers, we realized that there are many more places to explore further upriver. Here is a list of the villages, their distance from Atjoni and the details on accommodations. Prices truly range by village and the camp itself.
Less Than 1 Hour from Atjoni
JawJaw - Camp Isadou - Tel: 870-3344
JawJaw - Djamaica Camp - Tel: 863-6172 (ask for Eto)
Gunzi - Tei Wei Camp - No Phone
Anaula - Anaula Nature Resort - No Phone
1 to 2 Hours from Atjoni
Menimi - Camp Minimi - Tel: 815-2726 / 884-4134
Botopasi - Hotel Botopasi - No Phone
Botopasi - Lil' Botopasi - Tel: 812-1033 (ask for Rew)
Futunakaba - KotoHati Camp - Tel: 811-1784 (ask for Joel)
Pikinseei - Camp Paseni - No Phone
Danpaati - Danpaati River Lodge - Tel: 047-1113
2 to 3 Hours from Atjoni
Tio Boto - Tio Boto Tourist & Children's Camp - Tel: 861-7805
3 to 4 Hours from Atjoni
PemPei - PemPei Paati - Tel: 812-1050 (ask for Chapeau)
PemPei - Kumalu - Tel: 822-4932 (ask for Papi)
4+ Hours from Atjoni
Djumu - Awaadon Camp - No Phone
Here are a couple of things to keep in mind when you visit Suriname's Interior on your own.
* The influence of Catholic and Evangelical Christian missionaries has caused some Maroon villages to maintain less of the traditions practiced by their African ancestors. As a result, the religious practices, celebrations, cuisine, infrastructure and general feeling of each village can vary greatly.
* Although you do not need a tour operator to go upriver, unaccompanied tours of the villages is not recommended and is probably prohibited. Check with your hotel/camp first before venturing off.
* Bring Cash! No credit cards are accepted anywhere!!!
* A hammock almost always guarantees you will have a place to sleep.
* Bring bug spray.
* The rivers are infested with piranhas. Swim at your own risk!
* For those of you who want to fish for peacock bass, note that they generally cannot be found in the river as they have all migrated downriver to the reservoir. However, piranhas and other Amazonian species can be caught just about anywhere. I was also told that the fishing is much better the further up the river you go.
* A few of the villages have airstrips. With the right planning, you can travel upriver by boat and then fly back to Paramaribo. This is the most ideal option.
Tran and I would like to thank our Peace Corps peeps for helping us compile this information and for giving us some of the best memories of our entire trip. Great times. Epic party. Please hit us up next time you're in Texas!

subset: commoncrawl


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Original Research ARTICLE
High-Dose Cyclophosphamide Administration Orchestrates Phenotypic and Functional Alterations of Immature Dendritic Cells and Regulates Th Cell Polarization
- 1Department of Occupational Health and Environmental Health, School of Public Health, Hebei Medical University, Shijiazhuang, China
- 2Department of Occupational Health and Environmental Health, School of Public Health, Zhengzhou University, Zhengzhou, China
- 3Hebei Key Laboratory of Environment and Human Health, Hebei Medical University, Shijiazhuang, China
- 4Department of Statistics, Hebei General Hospital, Shijiazhuang, China
High-dose cyclophosphamide (CTX) inhibits the immune response. Dendritic cells (DCs) are professional antigen presenting cells (APCs) with a crucial role in initiating immune responses and sustaining immune tolerance. The relative contribution of DCs to immunosuppression induced by high-dose CTX is not well-documented. In this study, we employed the CTX-induced immunosuppressive rat model to examine alterations in DCs. We generated and cultured monocyte-derived immature DCs (imDCs) in vitro and explored their capacity of antigen uptake, T cell priming, cytokine production, and surface marker expression following high-dose CTX. Subsequently, we co-cultured CTX-treated imDCs with Th cells to determine Th cell polarization, and further explored the Toll-like receptor/Myeloid differentiation primary response 88/Mitogen-activated protein kinase (TLR/MyD88/MAPK) pathway. Our results show reduced cell number and surface maker alterations in splenic CD103+ DCs of CTX-treated immunosuppressed rats. In vitro, high-dose CTX weakened the antigen uptake capacity and enhanced the T cell priming capacity of imDCs, in addition to triggering imDC surface marker alterations. TLR, MyD88, and MAPK expression levels, involved in mediating Th cell polarization, were also significantly elevated. Our collective findings indicate that high-dose CTX administration potentiates phenotypic and functional alterations of imDC. Such changes may contribute to the regulation of Th polarization.
Recent years have witnessed a rapid rise in immunosuppressive diseases, due to factors such as increased environmental pollution, ecological imbalance, and the diversity of new chemicals. Research on immunosuppressants is therefore particularly important and urgently needed. Cyclophosphamide (CTX), the most widely used immunosuppressant, is hydrolyzed into phosphoramide mustard that affects all stages of immunity by inhibiting or killing immune cells directly (Agus et al., 1986; Kurauchi et al., 2017). However, the mechanism remains unclear, and the majority of past CTX-relevant investigations appear to have overlooked the effect of CTX on dendritic cells (DCs). DCs are critical antigen-presenting cells (APCs) that can initiate immune responses and induce immune tolerance, as well as determine the direction of Th polarization (Esterhazy et al., 2016; de Lastic et al., 2016). Therefore, a systemic study of the role of CTX on DCs is required.
The majority of DCs in vivo exist in an immature state, designated as immature DC (imDC), and exhibit high antigen uptake capacity (Wilson et al., 2004). ImDCs can recognize multiple pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), such as lipopolysaccharide (LPS), GpG-DNA, peptidoglycan, lipoprotein, and mycobacterial cell wall components (Wilbers et al., 2016; Qian and Cao, 2018). In addition, only imDCs can mediate immune tolerance via the induction of T cell apoptosis and regulatory T (Treg) cell formation (Dudek et al., 2013; McGovern et al., 2017; Waisman et al., 2017). Following recognition of PAMPs, imDCs elevate their antigen presentation ability and undergo maturation by increasing the expression of MHC-like and costimulatory molecules. Meanwhile, mature DCs (mDCs) have the ability to initiate specific immune responses and regulate helper T (Th) cell polarization (Chow et al., 2016; Eisenbarth, 2019).
CTX is inactive in vitro, and is hydrolyzed into aldehyde phosphoramide by hepatic cytochrome P450 enzymes including CYP2B6, CYP3A4, and CYP3A5 in vivo, resulting in the formation of the toxic chemical phosphoramide mustard (Ekhart et al., 2008; Cartin-Ceba et al., 2016). Therefore, current studies have predominantly examined the effects of CTX in vivo (Salem et al., 2009; Salem et al., 2010; Weir et al., 2014). However, the results derived through this approach may be affected by both the in vivo environment and the cytokine milieu. Recent studies indicating that the expression of P450 family members including CYP1A1 and CYP1B1, could be elevated in bone marrow-derived DCs in response to PM2.5 (Castañeda et al., 2018) and aflatoxin (AF) B1 (Mehrzad et al., 2018), suggests that DCs also have metabolic capacity in vitro. Low-dose CTX enhances the anti-tumor immune responses by augmenting DC-mediated antigen presentation and cytokine secretion, which could increase the expansion of Th1 cells and attenuate the suppressor function of Tregs (Radojcic et al., 2010; Son et al., 2015). In addition, an increase in IL-17-producing CD4+ T cells, indicative of Th17 polarization, has also been observed following low-dose CTX (Madondo et al., 2016). However, few studies have focused on the effects of high-dose CTX administration on DCs, and the subsequent DC-mediated Th polarization.
In the present study, we aimed to examine the potential impact of high-dose CTX on DCs. Firstly, we examined the effect of CTX on CD103+ DCs in an experimental immunosuppressive rat model. Secondly, we focused on the peripheral blood-derived imDC-associated: i) functions; ii) surface marker expression; and iii) the polarization patterns of Th cells in vitro. Finally, we evaluated the expression patterns of Toll-like receptor/Myeloid differentiation primary response 88/Mitogen-activated protein kinase (TLR/MyD88/MAPK) proteins, with a view to elucidate the mechanisms underlying the DC-mediated regulation of Th cell polarization in response to high-dose CTX.
Materials and Methods
Experimental Animals
Male Sprague-Dawley (SD) rats (6–8 weeks, 180–220 g) were purchased from the Laboratory Animal Center of Henan Province (Zhengzhou, China). All rats were maintained in a specific pathogen-free (SPF) laboratory animal facility of Zhengzhou University. Cages, bedding, and food were sterilized by autoclaving. All experimental procedures were performed in strict accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals (NIH Publication no. 8203) and approved by the Institutional Animal Care and Use Committee of Zhengzhou University.
Human Subjects
Whole blood products (buffy coats) from healthy, anonymous donors were purchased from the Henan Red Cross Blood Center (Zhengzhou, China), after obtaining informed consent. All experimental procedures were performed in strict accordance with the ethical requirements of biomedical research issued by the international and national organization, and approved by the Ethics Committee of Zhengzhou University.
CTX Treatment In Vivo
CTX (Sigma-Aldrich, St. Louis, USA) was dissolved in phosphate buffered saline (PBS) and gavaged at a dose of 15 mg/kg to the rats for 28 days. The control group was gavaged with identical volumes of PBS in the same manner. Rats were anesthetized with excessive carbon dioxide.
CTX Treatment In Vitro
In DC viability measurements, propidium iodide (PI) staining was used to exclude dead cells from analysis. After 24 h of CTX treatment, DCs (5 × 105) were collected via centrifugation (300 × g, 5 min) and resuspended with 20 μl PI solution. The ratio of living to total acquired cells was used to calculate cell viability. CV75, the CTX concentration that resulted in 75% DC viability (25% cytotoxicity), was calculated by log-linear interpolation.
Generation of imDCs
Peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll-Paque method (GE Healthcare Life Sciences, Piscataway, NJ) from buffy coats. CD14+ monocytes were isolated from PBMCs using MidiMACS Technology with CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Next, CD14+ monocytes were cultured at 1 × 106 cells/ml in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) in the presence of GM-CSF and IL-4 (50 ng/ml and 35 ng/ml; R&D Systems, Minneapolis, MN, USA) at 37°C and 5% CO2 for 7 days. On day 3, half of the medium was removed from culture and replenished with the same volume of fresh medium containing twofold concentrations of GM-CSF and IL-4. On day 5, the same step was repeated. On day 7, the imDCs were ready for experimental use.
Flow Cytometric Analysis of Th Cells
Detection of Th cells in the peripheral blood of rats was performed according to the literature (Lei et al., 2018).
Histological Analysis and CD103+DCs Immunofluorescence
The spleen samples were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned for staining with hematoxylin and eosin (H&E) staining to assess the degree of immunosuppression. Immunofluorescence (IF) was performed as follows. The same sections of spleen were fixed in 10% neutral formalin and embedded in paraffin. Next, paraffin sections were deparaffinized, rehydrated in xylene and ethanol, and treated with 3% H2O2 for 10 min. After heating in citrate butter for 20 min, sections were blocked with 10% goat serum in Tris-buffered saline (TBS) for 1 h at room temperature. Subsequently, sections were incubated overnight at 4°C with rabbit anti-rat CD103 (dilution 1:200; Abcam). After washing with PBS, sections were incubated with fluorescein isothiocyanate (FITC) goat anti-mouse IgG (dilution 1:400, Boster Biological Technology, Wuhan, China) for 1 h. 4′,6-diamidino-2-phenylindole (DAPI) was added for 10 min followed by three washes with PBS. Each slice was randomly selected from five visual fields, and Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, Maryland, USA) used to analyze the positive cells in individual images.
Magnetic Separation of Splenic CD103+ DCs
A portion of the rat spleen was minced and incubated in 5 ml RPMI-1640 (Gibco) with 2 mg/ml collagenase D (Roche Diagnostics GmbH, Mannheim, Germany) for 25 min at 37°C, followed by the addition of 10 mM ethylenediaminetetraacetic acid (EDTA) and incubation for 5 min. After digestion, splenic cells were dispersed by gentle pipetting, filtered through a 75 μm cell strainer, and used to prepare a single cell suspension. Cells were isolated using anti-CD103 microbeads (Miltenyi Biotec) and the MidiMACS Starting Kit (Miltenyi Biotec). The labeled cells were identified as CD103+ DCs.
Flow Cytometric Analysis of CD103+ DCs and imDC Surface Markers
Isolated CD103+ DCs (1 × 106) were subjected to flow cytometric analysis. Mouse-anti-rat CD54-Alexa Fluor®488 (R&D Systems), CD86-phycoerythrin (PE)-Vio770 (Miltenyi Biotec), CD80-PE (eBioscience, San Diego, CA, USA), CD11b/c-allophycocyanin (APC; Biolegend, San Diego, CA, USA), RT1B (MHC- II)-FITC (BD Biosciences, San Jose, CA, USA), CD1d-APC (eBioscience), and the respective matched isotype controls were employed.
The following markers were used for the staining of imDCs (1 × 106): mouse-anti-human CD80-FITC, CD54-PE, CD86-peridinin chlorophyll (PerCP)-Cy™5.5, MHC-II-APC, CD1a-FITC, CD83-PE, CD11c-PerCP-Cy™5.5, and the respective matched isotype controls (BD Biosciences). All samples were washed, resuspended in 2% PFA, and subjected to flow cytometric analysis (Accuri C6; BD Biosciences).
Antigen Uptake Capacity of imDCs
To assess pinocytosis capacity, imDCs (1 × 105) were incubated with 1 mg/ml FITC-labeled dextran (Sigma-Aldrich) for 1 h. A control sample was simultaneously incubated on ice. All samples were kept on ice until flow cytometric analysis was performed. Uptake of FITC-dextran was determined by measuring the mean fluorescence intensity (MFI) of FITC by flow cytometry. Dead cells were excluded from analysis based on PI fluorescence (BD Biosciences).
Mixed Lymphocyte Reaction (MLR) to Examine the T Cell Priming Capacity of imDCs
To assess the T cell priming capacity of CTX-treated imDCs, a one-way MLR was performed. CD4+ T cells (2 × 106) isolated from PBMCs using CD4 MicroBeads (Miltenyi Biotec), were labeled with 10 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA, USA) for 20 min at 37°C and 5% CO2. Subsequently, CD4+ T cells were suspended in RPMI-1640 medium containing 10% FBS at a density of 5 × 105 cells/ml, before the addition of the CFSE (1:1,000 dilution) staining. Immature DCs were centrifuged (300 × g, 5 min) and resuspended in RPMI-1640 medium at a density of 2.5 × 105 cells/ml. Finally, CFSE-labeled CD4+ T cells (100 µl) were incubated with 100 µl CTX-treated imDCs in RPMI-1640 medium for 7 days at 37°C and 5% CO2. The proliferation of CD4+ T cells was determined by measuring CFSE fluorescence by flow cytometry.
Enzyme-Linked Immunosorbent Assay (ELISA) to Measure Cytokine Production by imDCs
To measure cytokine production by CTX-treated imDCs (1 × 106), acellular supernatants were collected and analyzed for IL-1β, IL-4, IL-6, IL-12p70, transforming growth factor (TGF)-β1, and IFN-γ levels using commercial ELISA kits, according to the manufacturer's protocol (Abconal, Wuhan, CN, USA). Plates were read using an ELISA plate reader (Bio-Tek ELx808, Winooski, VT, USA) at 450 nm.
Quantitative Analysis of imDC Gene Expression
Genetic sequences for the following genes: TLR2, TLR4, TLR9, MyD88, T-bet, GATA-3, FoxP3, and RORγt were obtained from GenBank (National Center for Biotechnology Information, NCBI; Table S1) and the primers were custom-made (Sangon Biotec, Shanghai, China). Isolation of total RNA was performed aseptically on CTX-treated imDCs (1 × 106; for TLR2, TLR4, TLR9, and MyD88) and CD4+ T cells co-cultured with CTX-treated imDCs for 24 h at a CD4+ T cells to imDCs ratio of 2:1 (for T-bet, GATA-3, FoxP3, and RORγt), using the TRI Reagent procedure (Sigma-Aldrich), according to the manufacturer's protocol. Reverse transcription was performed in a total reaction volume of 20 µl, containing 2 µg total RNA, 10 μM random primers, 2.5 nM dNTPs, 0.1 M dithiothreitol (DTT), 20 U RNaseOUT (Invitrogen Waltham, MA, USA), and RNase-free water, at 37°C for 15 min and 85°C for 5 s. Real-time quantitative (RT-q) PCR was performed in a total volume of 25 µl containing 12.5 µl SYBR Premix Ex Taq II, 1.0 µl each of forward and reverse primers (10 µM), 2 µl cDNA, and 8.5 µl dH2O, using the PrimeScriptTM RT reagent Kit (Takara, Dalian, China). The optimized thermal cycling conditions were as follows: 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 60 s. RT-qPCR was performed using a StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA).
Western Blotting to Measure ERK, JNK, and p38 Kinase Activation
CTX-treated imDCs (1 × 106) were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Haimen, China) containing protease inhibitors (Beyotime) and phosphatase inhibitors (Roche, Basel, Switzerland). Lysates were separated on 10% SDS polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% milk for 1 h and incubated overnight at 4°C with primary anti-ERK, anti-phospho-ERK, anti-JNK, anti-phospho-JNK, anti-p38, anti-phospho-p38, and anti-β-Actin antibodies (dilution 1:1,000, Cell Signaling Technology, Boston, MA, USA), followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Cell Signaling Technology) for 30 min at 37°C. Immunoreactive bands were visualized using an enhanced chemiluminescence system (ECL; GE Healthcare, Madrid, Spain).
Statistical Analysis
All data were analyzed using SPSS version 17.0 software (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as mean ± standard error of the mean (SEM). To determine differences between variables, one-way analysis of variance followed by a pair-wise comparison with Student-Newman-Keuls test was performed. P values <0.05 were considered statistically significant, unless otherwise indicated.
High-Dose CTX Caused Immunosuppression in Rats
First, we established a CTX-induced immunosuppressive rat model to accurately determine the effects of high-dose CTX on DCs in vivo. Light microscopy (using a Nikon Eclipse Ci instrument) revealed spleen nodule structures disappeared, red and white pulp structures were disordered, and a large amount of fibrosis was evident in the connective tissues in the CTX-treated group (Figure 1A). Similar changes in the spleen have been previously demonstrated (Duggina et al., 2015; Lapointe et al., 2016). Next, we demonstrated that, compared to the control group, the body weight, spleen, and thymus coefficients were markedly reduced in the CTX-treated group (P < 0.05; Table S2). The percentages of CD3+, CD4+, and CD8+ T cells in peripheral blood were significant reduced in CTX-treated rats, relative to the control group (P < 0.05; Figures 1B–E). Evidences of those were clearly indicative of immunosuppression in our rat model.
Figure 1 High-dose CTX caused immunosuppression in rats. (A) H&E staining of the CTX-induced pathological changes in the spleen of rats (200×). (B) High-dose CTX induced a reduction in the percentage of CD3+, CD4+, and CD8+ T cells in the peripheral blood of rats. The quantitative results of CD3+ (C), CD4+ (D), and CD8+ (E) T cells in the peripheral blood. n = 6. Error bars indicate SEM. *P < 0.05, compared with the control group. All values represent the mean ± SEM.
High-Dose CTX Altered the Surface Markers of Splenic CD103+ DC and Polarization of Peripheral Blood Th Cells of Rats
We further examined splenic CD103+ DCs using flow cytometry and immunofluorescence. The number of CD103+ DCs isolated from the spleen was significantly reduced in CTX-treated rats relative to controls (P < 0.05; Figures 2A–C). CD103+ DCs surface markers were compared in the spleen of CTX-treated and untreated rats. The results showed that the median fluorescence intensity (MFI) values of CD11c and CD54 were increased and those of CD1d, CD86, and MHC-II were decreased in the spleen of the CTX-treated group (P < 0.05; Figure 2D). These results clearly demonstrated that high-dose CTX lowered CD103+ DC numbers, in addition to altering the expression of surface markers on this cell subset in the rat model. In addition, we detected the Th cells polarization in the peripheral blood of rats by flow cytometry. Our results revealed a significant increase in Th1, Th2, and Treg cell numbers, accompanied by a decrease in Th17 cells in peripheral blood of the CTX-treated rats, compared to the control group (P < 0.05; Figures 2E–J).
Figure 2 High-dose CTX altered the surface markers of splenic CD103+ DC and polarization of peripheral blood Th cells of rats. Splenic CD103+ DCs was determined by flow cytometry (A) and immunofluorescence (B). (C) The quantitative results of splenic CD103+ DC. (D) Flow cytometric analysis of splenic CD103+DC surface markers. (E) Th cell polarization in the peripheral blood of rats. The quantitative results of Th1 (G), Th2 (H), Th17 (I), and Treg (J) T cells in the peripheral blood of rats. n = 6. Error bars indicate the SEM. *P < 0.05, compared with the control group. All values represent the mean ± SEM.
High-Dose CTX Altered Antigen Uptake and the CD4+ T Cell Priming Capacity of imDC, As Well As Their Surface Marker Expression Profile In Vitro
We used a transcriptomic approach to detect the genetic alterations experienced by CTX-treated imDCs in vitro and found that 3,456 genes were up-regulated (Figure S1A). We found that the functions of the up-regulated genes were closely linked to metabolic processes (Figure S1B). Subsequently, in order to understand the mode of action of the up-regulated genes, we adopted the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. KEGG analysis revealed the involvement of xenobiotic metabolism pathways and their regulation by cytochrome P450 (Figure S1C). Moreover, cytochrome P450 enzymes were included in those pathways (Table S3). These results demonstrated that imDCs may metabolize CTX in vitro by up-regulating the level of cytochrome P450 enzymes.
To test the toxic effect of CTX on DCs in vitro, imDCs were treated with CTX for 24 h. According to our flow cytometry analyses, MFI values for the markers CD11c, CD54, and CD86 were increased, while those for CD1a, CD80, and CD83 were decreased in the CTX-treated samples, compared to controls (P < 0.05; Figure 3A). Furthermore, CTX-treated imDCs showed lower antigen uptake (P < 0.05; Figures 3B, C) and higher CD4+ T cell priming capacity, compared to untreated control samples (P < 0.05; Figures 3D, E). The results collectively demonstrated that high-dose CTX induced alterations in imDC surface marker expression profile, and a reduction in imDC antigen uptake capacity, while enhancing their ability to prime CD4+ T cells.
Figure 3 Alterations in the surface marker expression profile, antigen uptake and CD4+ T cell priming capacity of CTX-treated imDCs in vitro. (A) The quantitative results of imDC surface marker expression. (B) Detection of antigen uptake capacity of imDCs cultured at 37°C for 1 h in the presence of FITC-dextran by flow cytometry. (C) The quantitative result of antigen uptake capacity of CTX-treated imDCs. (D) The antigen presentation capacity of CTX-treated imDCs assessed by inducing CFSE-labeled CD4+ T cell proliferation in an MLR assay. (E) The quantitative result of antigen presentation capacity of CTX-treated imDCs. Dead cells were excluded from analysis based on PI fluorescence. Error bars indicate the SEM. *P < 0.05, compared with the control group. All values represent the mean ± SEM of three independent experiments.
High-Dose CTX Induced imDC-Mediated Th Cell Polarization
ImDCs have the unique ability to regulate immune responses through the activation and polarization of naïve T cells. Accordingly, we assessed T-bet, GATA-3, RORγt, and Foxp3 expression levels in Th cells co-cultured with CTX-treated imDCs by RT-qPCR. In the CTX-treated group, T-bet, GATA-3, and Foxp3 levels were significantly increased along with a concomitant reduction in RORγt expression, compared to the control group (P < 0.05; Figures 4A–D). ELISAs were further performed to evaluate the concentrations of IFN-γ, IL-4, IL-17, IL-6, and TGF-β in Th cells/CTX-treated imDC co-culture supernatants. Our data showed an increase in IFN-γ, IL-4, and TGF-β levels, accompanied by a decreased in IL-6 and IL-17 levels in the CTX-treated group, compared to the controls (P < 0.05; Figures 4E–I). Based on these findings, we concluded that in response to CTX, imDCs acted to significantly enhance Th1, Th2, and Treg cell-mediated immunity, while attenuating Th17 immunity, consistent with our in vivo data obtained from the immunosuppressive rat model.
Figure 4 High-dose CTX induced imDC-mediated Th cell polarization. (A–D) RT-qPCR analysis of T-bet, GATA-3, RORγt, and Foxp3 mRNA levels in Th cells co-cultured with CTX-treated imDCs for 24 h. (E–I) ELISA detection of IFN-γ, IL-4, IL-17, IL-6, and TGF-β in Th cell/CTX-treated imDC co-culture supernatants. Error bars indicate the SEM. *P < 0.05, compared with the control group. All values represent the mean ± SEM of three independent experiments.
Enhanced Expression of TLR, MyD88, and MAPK Pathway-Associated Signaling Components in imDCs, in Response to CTX
To establish the potential mechanism of Th cell polarization by imDCs, we conducted an RT-qPCR analysis of TLR2, TLR4, TLR9, and MyD88 mRNA. In addition, we performed western blotting to determine ERK, JNK, and p38 protein expression. Notably, TLR2, TLR4, TLR9, and MyD88 transcripts (P < 0.05; Figures 5A–D) and p-p38, p-JNK, and p-ERK protein levels (P < 0.05; Figures 5E–H) were significantly upregulated in the CTX-treated group.
Figure 5 Enhanced expression of TLRs, MyD88, and MAPK pathway-associated signaling components in imDCs in response to CTX. (A–D) RT-qPCR analysis of TLR2, TLR4, TLR9, and MyD88 mRNA expression in imDCs. (E–H) Western blotting analysis of p38, p-p38, JNK, p-JNK, ERK, and p-ERK protein expression in imDCs. Error bars indicate the SEM. *P < 0.05, compared with the control group. All values represent the mean ± SEM of three independent experiments.
In the present study, observation of morphology, organ coefficient, and proportion of T cells confirmed that the CTX-induced immunosuppressive rat model were established. In the rat model, we explored the splenic CD103+ DC numbers and found that a decrease in the number of splenic CD103+ DCs in response to CTX, indicating that CTX induced the apoptosis of CD103+ DCs. Subsequently, we found the expression patterns of antigen-presenting and co-stimulatory molecules on the surface of splenic CD103+ DCs were altered. These molecules have been identified as key factors in the induction of Th cell differentiation in the context of immunological diseases (Walsh and Mills, 2013; Cybulsky et al., 2016). Furthermore, high-dose CTX induces a reduction in the polarization of Th1/Th2 cells, consequently leading to the inhibition of the immune response (Wang et al., 2016). In the present study, we not only demonstrated the above phenomenon, but also detected the increase in Treg cell numbers accompanied by a drop in Th17 cells. There results suggested that splenic CD103+ DCs and Th polarization were affected by CTX in the rat model. Numerous studies have proved that chemicals and biomaterials can alter the phenotype and function of DCs and subsequently regulate Th cell polarization (Jeon et al., 2015; Park et al., 2015; Pattarini et al., 2017). Therefore, we give rise to the hypothesis that Th cell polarization in CTX-treated rats may be due to the effects exerted on DCs by high doses of CTX.
It is well-documented that CTX exerts its immunosuppressive effect once it is metabolized to phosphoramide mustard by the cytochrome P450 enzyme in the liver (Cartin-Ceba et al., 2017). The transcriptomic approach was used to detect the alterations of metabolic genes experienced by imDCs following CTX treatment in vitro. Our results showed that imDCs can metabolize CTX in vitro by up-regulating the expression cytochrome P450-associated genes. Subsequently, we detected the multiple surface markers of imDCs following CTX treatment in vitro, including the costimulatory proteins CD80 and CD86, the cell adhesion molecules CD11c and CD54, the activation marker CD83, and the antigen-presenting molecule MHC-II. Our results showed that the expression of those surface markers altered, as in the rat model. Those markers are in association with the antigen uptake and CD4+ T cell priming capacity of DCs (Diana et al., 2007). In addition, we assessed the antigen uptake and CD4+ T cell priming capacity of imDCs, which are the vital functions of DCs. The results demonstrated that CTX effectively reduced the antigen uptake capability of imDCs, in accordance with the action of other immunosuppressants (Holt and Thomas, 1997; Paolo et al., 2003). CTX enhanced the ability of imDCs to perform CD4+ T cell priming, which was in accordance with the findings of Holt (Holt and Thomas, 1997), who demonstrated that dexamethasone was ineffective in blocking the presentation of antigen to T cells in the MLR. Taken together, our data revealed that CTX altered the surface makers, as well as antigen uptake and CD4+ T cell priming capacity of imDCs, which were closely associated to polarization of Th cells.
DCs also provide key signals for polarization of Th cell subsets. T-bet, GATA-3, RORγt, and Foxp3 are the key transforming growth factors of Th1, Th2, Th17, and Treg cells, respectively (Yu et al., 2015). Furthermore, TGF-β can induce Tregs and also contribute to Th17 formation in the presence of IL-6 (Geginat et al., 2016; Ansa-Addo et al., 2017). We assessed T-bet, GATA-3, RORγt, and Foxp3 expression levels in Th cells co-cultured with CTX-treated imDCs. Our results showed that the polarization of Th cells altered: enhanced Th1 and Th2 responses, which were analogous to T-bet and GATA-3 expression. The Th17 response, represented by IL-17 and RORγt expression, decreased following CTX treatment. The Treg response increased in the CTX group. Moreover, increased TGF-β and lowered IL-6 levels promoted the differentiation of the Treg response and reduced the differentiation of the Th 17 response. In view of the finding that Th polarization is induced through MAPK signaling (Adler et al., 2007; Maria et al., 2009), we further evaluated several proteins associated with this pathway (such as p38, JNK, and ERK) in CTX-treated imDCs. Phosphorylation of these proteins was enhanced in CTX-treated imDCs, supporting the theory that CTX exerted its effect on Th cell polarization via the modulation of the MAPK pathway. Studies have found that DCs stimulate the Th1 response via IL-12 p70 secretion in a p38 MAPK phosphorylation-dependent manner (Jarnicki et al., 2008), Th2 cell polarization occurs via ERK phosphorylation (Agrawal et al., 2003; Kaisar et al., 2018). Moreover, p38 MAPK is proposed to be involved in the regulation of Th17 cells and Tregs (Cannon et al., 2013; Lu et al., 2014). We need to further explore the differential role of MAPK signaling of imDC in the polarization of Th cells.
In summary, CTX induced immunosuppression by reducing DC numbers and altering their surface marker expression profiles in our rat model. Additionally, CTX enhances antigen uptake, prime CD4+ T cells capacity and surface markers of imDCs in vitro, and altered DC-mediated Th cell polarization via the TLR/MyD88/MAPK pathway. It provides insights into the mechanisms implicated in the high-dose CTX treatment of imDCs may therefore be of great value in the management of autoimmune disease and transplantation.
Data Availability Statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.
Ethics Statement
The studies involving human participants were reviewed and approved by Ethics Committee of Zhengzhou University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Zhengzhou University. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
Author Contributions
Conceptualization: LB and WY. Data curation: DW. Formal analysis: JW. Funding acquisition: WY. Methodology: CH, YZ, and YL. Project administration: WY. Writing—original draft: LB. All authors have read and agreed to the published version of the manuscript.
This work was supported by the Natural Science Foundation of China (81273039).
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to acknowledge the Zhengzhou University Department of Public Health for granting permission for the use of laboratory instruments and providing scientific and technical assistance.
Supplementary Material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2020.00775/full#supplementary-material
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Keywords: cyclophosphamide, immature dendritic cells, antigen uptake capacity, T cell priming, Th polarization
Citation: Bao L, Hao C, Wang J, Wang D, Zhao Y, Li Y and Yao W (2020) High-Dose Cyclophosphamide Administration Orchestrates Phenotypic and Functional Alterations of Immature Dendritic Cells and Regulates Th Cell Polarization. Front. Pharmacol. 11:775. doi: 10.3389/fphar.2020.00775
Received: 03 April 2020; Accepted: 11 May 2020;
Published: 25 May 2020.
Edited by:Jie Xu, Fudan University, China
Copyright © 2020 Bao, Hao, Wang, Wang, Zhao, Li and Yao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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We asked a dozen successful amateur and pro portraitists for tips on better family photos. Our interviews turned up a surprising number of common strategies for succeeding with this classic subject. Here's how they suggest you capture your family's personality in pictures. *Keep It Real*Small reminders of daily life are more precious than posed images. The family pictures that will mean the most to you (and others) will be the most candid. Try to capture family members interacting with each other and the world around them. Keep from directing or posing people—it may vex your subjects and reduce your chances for good photos. (Above Photo) Pro Sue Barr chose a Coney Island location for this family portrait. It lent color to the background and coaxed lively expressions from the kids.Sue Barr
Get Down Low – one key to many natural baby shots is to get down on their level. I've spent quite a bit of time over the last week or two lying on the floor next to Xavier. It's something I think we both enjoy anyway but it's also a great place to use your camera. Getting down low and getting in close (see below) does present some challenges in terms of focal length (I'm using a 24-105 zoom – usually at the widest focal length) but it means you end up with shots that feel like you've entered the babies world rather than you're looking down on it from above.
Some photographers also include high resolution photograph files in their packages. These photographers allow their clients limited rights to reproduce the images for their personal use, while retaining the copyright. Not all photographers release files and those who do will most likely charge a premium for them, since releasing files sometimes means giving up any after wedding print or album sales for the most part. In the cases where photographers release the high resolution images they are usually supplied on CD, DVD or USB Stick however, this depends on the individual companies and it's recommended the client(s) checks what is included with the photographers wedding photography package before signing any contracts.
Let the photographer connect with the baby. In order for your baby to feel comfortable, you should avoid crowding your little one. It'll be tempting to run over whenever your baby becomes restless or cries, but you should do your best to let the photographer comfort the baby so the baby doesn't feel startled by too many changes. Always remain in the room though during your baby's shoot.
Darlene Hildebrandt is an educator who teaches aspiring amateurs and hobbyists how to improve their skills through free articles on her website Digital Photo Mentor, online photography classes, and travel tours to exotic places like Peru, Thailand, India, Cuba, Morocco, Bhutan, Vietnam and more. To help you improve and learn she has two email mini-courses. Sign up for her free beginner OR portrait photography email mini-course here. Or get both, no charge!
A cheaper alternative to Canon's 100D is the 1200D. While the specs don't quite stand up to some of the newer cameras on the market, it's the definition of a solid entry level DSLR. It has an 18MP sensor, 3fps continuous shooting speed, RAW and JPEG file formats, and you can load up a beginners guide through its smartphone app to help you get the hang of things. It also shoots 1080p HD video.
Wedding is very crucial part in human life that's why people are more concern about it to remain its pleasure for whole in their life. There are so many moments happening during the wedding ceremony which won't get back without clicking photo on time or keeping any documentary on it. As human nature, we can't keep more things in our mind for long time because we are facing lots of things every day and that erase our backstory by gathering new story, after a certain period we can't recall our best moments like the time it has happened. So the wedding photography is very essential for a newly couple to keep their eye wink for all the time in their good book.
When you shoot a mommy-to-be in her own home she's comfortable to let go in a way that she may not be otherwise. This is a fantastic reason to hold maternity sessions at home. This is not however why I do it. As a photographer I feel like it's my responsibility to help my subjects feel comfortable and capable of giving me their best, no matter where we're shooting. I pride myself on my ability to do this. To me, it's a HUGE part of my success as a photographer (Uh oh. I feel a post in this regard coming on… check back soon). I shoot maternity sessions at home primarily because it's wonderful for a couple to have a capture that truly shows what their life was like at the time their baby came to be a part of their family. I'm a second generation photographer so I've got some really lovely shots of my sweet momma awaiting my arrival. My favorite however is a simple picture of her standing by my bassinet with her hand on her tummy. I CHERISH it.
We would be honored to help tell the story of your family, & capture the new-found love you have for your little one, or little one to be. Pictures last forever & are a priceless investment that captures unique moments that might never be repeated again. Call now to book your session at our Vancouver and Burnaby maternity & newborn photography studio.
That's it! No expectations other than fun. Then prepare yourself. Bring along props, get mom to bring one of their favourite toys or books. I usually have a hand puppet and bubbles in my camera bag along with my gear. If the kids don't want to sit and smile don't force them. Then them run around and be kids for a while and shoot that. Play with them, make it fun. Then they may cooperate and sit for a bit a few minutes later.
I know that many readers feel that they don't have the time for shooting in RAW (due to extra processing) but a wedding is one time that it can be particularly useful as it gives so much more flexibility to manipulate shots after taking them. Weddings can present photographers with tricky lighting which result in the need to manipulate exposure and white balance after the fact – RAW will help with this considerably.
As you start looking through photographers' portfolios, pay attention to the lighting quality in the pictures. Are they dark and moody? Bright and romantic? Each photographer has a certain style, and there will most likely be one that you mesh with better than others. You may also notice while you're looking through portfolios that some photographers refer to themselves "natural light photographers" which just means that they only use whatever light is available, rather than using a flash or other lighting setups. If you are getting married somewhere dark without a lot of windows, you will want to make sure that the photographer knows how to create light when it isn't there. Every lighting situation is different for photographers, so make sure you're looking at photos that have similar characteristics to your venue. An outdoor wedding in the middle of the day with bright sun requires a very different skill than a New Year's Eve reception in a dark church. (Also, a hot tip: ask to see some reception shots, since many of us don't include those in our portfolios. Because if you are having an art gallery reception with no light, you want to make sure we can handle it. If all the reception photos are a little blurry and have been converted to black and white, then low-light situations are probably not that photographer's forte. If you see lots of backlighting and what appears to be bursts of light behind the dancing, it means that that photographer has the ability to create standalone lighting in situations where the light is nonexistent.)
"AMAZING! Stop looking and just book with him. Garrett is very professional and genuinely cares about his clients. He has amazing prices and really is willing to work with you. I was hesitant about hiring a professional photographer but I am so glad I did because the photos look amazing. If I ever need a photographer I will definitely be hiring him again."
Wear solid colors to accentuate your belly. When selecting your outfit for your maternity shoot, consider wearing solid colors that will highlight the shape of your belly. Large patterns can distract or conceal your baby bump.[6] If you wish to wear a pattern with swirls, stripes, or florals, make sure you are happy with how the pattern accentuates your belly.
A summertime family photo shoot offers beautiful landscapes for your photos and warmer weather. The season reflects a laid back vibe that can be mirrored throughout your family's outfit selections. Take advantage of light cottons, pretty sheers, denim and slip into your sandals for a casual look. If your family is taking a summer vacation, use this time to take your family portraits. No matter where you may be during summertime, you can take advantage of the following summer color arrangements and styling tips to guide your selections.
Winter engagement photos are the perfect opportunity to get into the holiday spirit—after all, you have a lot to celebrate! To pull off a festive photo shoot, consider metallic embellishments when choosing what to wear. Monochromatic embellished pieces (like the gold sequin top above) will give your engagement photos just enough sparkle without becoming distracting. Have your fiancé wear a dress shirt in a crisp solid color to complement your glittery outfit.
Why take engagement photos? The best reason is it's a great opportunity to work with your wedding photographer for the first time and get comfortable in front of their lens and with their style. (Most photographers also include an engagement session in their wedding photography packages.) You can also submit a shot to your local paper with your engagement announcement, use them creatively in your save-the-dates and wedding day décor, or give framed prints as gifts to your families. Read on for the basics to help you make the most of your premarital photo session.


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In our opinion, commodity markets coming off of long-term highs or lows typically present traders with an extraordinary prospect. However, it is important to realize that just because a commodity seems "cheap" doesn't mean that it can't go lower. Likewise, while we would never advocate buying (or being bullish with options) a commodity at an all time high, it is always possible that prices can continue higher but generally speaking options in such an environment are over-priced. As a result, they come with magnificently low odds of success.
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You also can limit your exposure to risk on stock positions you already have. Let's say you own stock in a company but are worried about short-term volatility wiping out your investment gains. To hedge against losses, you can buy a "put" option that gives you the right to sell a particular number of shares at a predetermined price. If the share price does indeed tank, the option limits your losses, and the gains from selling help offset some of the financial hurt.
To reiterate, buying options in times of low volatility could prove to be advantageous should the volatility increase sharply. On the other hand, a lack of deviation in the price of the underlying asset will produce lower market volatility and even cheaper option premiums. Once again, pricing is relative and dynamic; "cheap" doesn't mean that it can't get "cheaper".
Just as there are several ways to skin a cat, there are an unlimited number of option trading strategies available in the futures markets. The method that you choose should be based on your personality, risk capital and risk aversion. Plainly, if you don't have an aggressive personality and a high tolerance for pain, you probably shouldn't be employing a futures and options trading strategy that involves elevated risks. Doing so will often results in panic liquidation of trades at inopportune times as well as other unsound emotional decisions.
* Sell orders are subject to an activity assessment fee (from $0.01 to $0.03 per $1,000 of principal). Trades are limited to online domestic equities and options and must be used within two years. Options trades are limited to 20 contracts per trade. Offer valid for new and existing Fidelity customers opening or adding net new assets to an eligible Fidelity IRA or brokerage account. Deposits of $50,000-$99,999 will receive 300 free trades, and deposits of $100,000 or more will receive 500 free trades. Account balance of $50,000 of net new assets must be maintained for at least nine months; otherwise, normal commission schedule rates may be retroactively applied to any free trade executions. See Fidelity.com/ATP500free for further details and full offer terms. Fidelity reserves the right to modify these terms and conditions or terminate this offer at any time. Other terms and conditions, or eligibility criteria may apply.
Commodity options provide a flexible and effective way to trade in the futures markets. Further, options on futures offer investors the ability to capitalize on leverage while still giving them the ability to manage risk. For example, through the combination of long and short call and put options in the commodity markets, an investor can design a trading strategy that fits their needs and expectations; such an arrangement is referred to as an option spread. Keep in mind that the possibilities are endless and will ultimately be determined by a trader's objectives, time horizon, market sentiment, and risk tolerance.
Speculation is a wager on future price direction. A speculator might think the price of a stock will go up, perhaps based on fundamental analysis or technical analysis. A speculator might buy the stock or buy a call option on the stock. Speculating with a call option—instead of buying the stock outright—is attractive to some traders since options provide leverage. An out-of-the-money call option may only cost a few dollars or even cents compared to the full price of a $100 stock.
With this strategy, the trader's risk can either be conservative or risky depending on their preference (which is a definite plus). For iron condors, the position of the trade is non-directional, which means the asset (like a stock) can either go up or down - so, there is profit potential for a fairly wide range. To use this kind of strategy, sell a put and buy another put at a lower strike price (essentially, a put spread), and combine it by buying a call and selling a call at a higher strike price (a call spread). These calls and puts are short.
The price you pay for an option, called the premium, has two components: intrinsic value and time value. Intrinsic value is the difference between the strike price and the share price, if the stock price is above the strike. Time value is whatever is left, and factors in how volatile the stock is, the time to expiration and interest rates, among other elements. For example, suppose you have a $100 call option while the stock costs $110. Let's assume the option's premium is $15. The intrinsic value is $10 ($110 minus $100), while time value is $5.
The purchase of a call option is a long position, a bet that the underlying futures price will move higher. For example, if one expects corn futures to move higher, they might buy a corn call option. The purchase of a put option is a short position, a bet that the underlying futures price will move lower. For example, if one expects soybean futures to move lower, they might buy a soybean put option.
Now, think of a put option as an insurance policy. If you own your home, you are likely familiar with purchasing homeowner's insurance. A homeowner buys a homeowner's policy to protect their home from damage. They pay an amount called the premium, for some amount of time, let's say a year. The policy has a face value and gives the insurance holder protection in the event the home is damaged.
However, options are not the same thing as stocks because they do not represent ownership in a company. And, although futures use contracts just like options do, options are considered lower risk due to the fact that you can withdraw (or walk away from) an options contract at any point. The price of the option (its premium) is thus a percentage of the underlying asset or security.


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Byron Nelson (1912-2005) Signed 10x8 Golf Photograph
Byron Nelson autographed photograph,ORIGINAL 10" x 8" BLACK AND WHITE PHOTOGRAPH DEPICTING BYRON NELSON IN FULL LENGTH GOLF POSE ON COMPLETION OF A DRIVE. BEAUTIFULLY AUTOGRAPHED IN BLACK INK TO THE CLEAR PORTION OF THE IMAGE BY BYRON NELSON, excellent condition.John Byron Nelson, Jr. (February 4, 1912 - September 26, 2006) was an American professional golfer between 1935 and 1946, one of the greats not only in his day but over the history of the sport. Although he won many tournaments in the course of his relatively brief career, he is mostly remembered today for having won 11 consecutive tournaments and 18 total tournaments in 1945.
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