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Dairy stains are protein stains, because they are organic in nature (i.e., from an animal). Fresh stains should be soaked and agitated in cold water before washing. If the stain has dried, scrape or brush off any crusted matter. Soak for up to several hours in cold water with a detergent or an enzyme presoak (which breaks down protein-based stains like egg, grass and blood so your detergent can work more effectively). Launder in warm water.
If the stain remains, soak an additional 30 minutes and rewash. If a colored stain remains, launder with bleach safe for the fabric.
Fruit stains are a dye stain. Launder with detergent in the hottest water safe for the fabric. Do not use soap (bar, flake or detergents containing natural soap), since soap-based products could make the stain permanent. Soak tough stains for 30 minutes in one quart of warm water and one teaspoon of enzyme presoak product.
Removal of old or set stains may require washing with bleach that is safe for that particular fabric. If all the sugars are not removed, a brown stain will appear when the cloth is heated in the dryer or is ironed as the sugar is caramelized. This effect is great for crème brûlée... not so wonderful for your favorite white blouse.
Grass stains are also a dye stain —a combination of protein and other organic matter mixed with chlorophyll, xanthophylls and carotenoids. For stubborn stains like grass, soak in a solution of cool water and a laundry product containing enzymes, like Shout Advanced Ultra Gel Brush, for at least 30 minutes (and several hours for aged stains). Don't use hot water, as it will coagulate the protein and make the stain more difficult to remove. If stains remain, soak an additional 30 minutes, then rewash. (Follow product instructions, and note that some laundry detergents already contain enzymes.)
After soaking, launder in warm water as usual.
Mud stains are a protein stain. Let mud stains dry. Brush to remove the soil. Soak in a solution of 1 quart warm water, 1 teaspoon liquid dish detergent and 1 tablespoon white vinegar for 15 minutes. Rinse with water. Let stand for several minutes.
If the stain persists, sponge it with rubbing alcohol, then rinse with clear water and wash. You may also soak the stain for 30 minutes in an enzyme presoak, and you can add chlorine bleach to the load of laundry if the label says it's safe for the fabric.
Barbecue sauce is a combination stain. Soak in cool water, 1/2 a teaspoon of liquid hand dishwashing detergent and 1 tablespoon vinegar for 30 minutes. Rinse. If stain remains, pre-treat area with a pre-wash stain remover, liquid laundry detergent or a paste of powdered detergent and water. Then wash in warm water. Air dry.
If the stain remains, soak in an enzyme product for anywhere from at least an hour to overnight. Some detergents contain enzymes. Launder in warm water. Do not use hot water or hot air drying or iron the garment until the stain is gone as heat will set it.
A coffee stain is a dye stain, but the cream or milk in the coffee is a protein. Therefore, you will want to treat the stain as both if you like a little cream in your morning cup of joe. If the stain is fresh, immediately rinse with cold water. If you are at home (or once at home), remove the clothing and add laundry detergent directly to the stain and rub together with cold water. Soak in the water for 30 minutes, rubbing detergent together every 5 to 10 minutes. Next, wash clothing on the hottest setting. Ensure stain is out of clothing prior to placing in the dryer. If stain has not been removed, try repeating the steps.
For older stains, apply white vinegar directly to the stain and mix with cold water. Rub stain with a sponge until removed. You may also use baking soda as an alternative solution, applying with a wet cloth to scrub away the stain.
The key to removing stains from white clothing is to attempt to remove the stain immediately. Scoop up or scrape food items off, while blotting up liquid spills from the outside in. Apply a liquid dishwashing soap directly to the stain and rub gently with a light-colored cloth or your fingernail. Do not use a darker colored cloth or terry towel as this may darken the stain. Soak garment in cold water for 10 to 15 minutes prior to washing.
New and older stains may also be removed with the use of corn starch or baking soda, gently scrubbing the powder into the stain with a cold wet cloth. White vinegar and hydrogen peroxide are also great pre-treatment solutions. With any white clothing, it is recommended that you wash separately and pre-soak in 1/2 cup of bleach and cold water.
Like white clothing, it is important that you attempt to remove a fresh stain immediately. Scoop up any food residue while blotting away liquids. Avoid using bar soaps or terry cloth towels on stain. Soak in 1/2 cup white vinegar and cold water prior to washing the clothing on a regular cycle. If it is a tough or set stain, soak garment directly in 1/4 cup of vinegar and 1/4 cup water for 30 minutes prior to washing. Rinse item out prior to washing on a warm cycle to sterilize clothing.
To remove stains from jeans, apply hydrogen peroxide directly onto stain and allow to soak for 30 minutes prior to washing on a regular cycle. Grass stains can be removed by applying petroleum jelly directly to the stain and spreading with your finger. Wash on regular cycle after five minutes.
Remove set and new stains on delicates with the use of a salt-and-lemon-juice solution. Gently rub with the use of a non-invasive cloth or finger, and rinse with very hot water. Never rub garment harshly or with a rough cloth. Allow to dry in the sun. If stain has not been removed, attempt to soak item in two parts warm water, one part detergent and one part oxygen bleaching agent. Wash as recommended per garment instructions.
Another method of removing delicate clothing stains is by hand-washing the garment with the use of borax, baking soda and regular detergent. Lightly scrub the item together with its own fabric. Rinse with cool water and air dry to prevent stain from setting in.
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The house is specially located in front of the beach and the land have 12.000 sq meters (in the near future is going to be a hotel with 20 bangalôs)... The place is amazing for kitesurfers since Atins is being considered one of the best spots for all levels (flat, waves and downwinds)...
There is the possibility to rent only a room as well. Fell free to get in contact, i will be glad to help.
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domain: travel


Binghamton's McGirk's Irish Pub patrons asked to quarantine after positive COVID-19 case
A person who was at McGirk's Irish Pub on July 24 and July 25 has tested positive for COVID-19.
Broome County Health Department officials said anyone who visited the restaurant on Kattelville Road in Binghamton anytime between 4-9:30 p.m. July 24 should self-quarantine until Aug. 7.
Anyone who visited the restaurant between 5:30-11 p.m. July 25 should self-quarantine until Aug. 8.
In a Facebook post Thursday, McGirk's said it will be temporarily closed.
"Upon notification every surface, both inside and outside, were thoroughly cleaned, well cleaned would be a gigantic understatement, actually. We have since taken every recommended precaution to make sure that our establishment is a safe place to visit," the post reads. "We are temporarily closed and ask that you please bear with us during this very challenging time."
Those quarantined are asked to contact their healthcare provider if they develop symptoms.
Testing is also available at Binghamton University. Call 888-364-3065 to make an appointment.


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Saudi Aramco, Sumitomo’s petrochemical facility to be transferred to PetroRabigh
Saudi Aramco and Sumitomo Chemical decided to transfer ownership of a planned $8.5 billion (SAR32 billion) petrochemical facility to their joint venture PetroRabigh , Reuters reported. The new facility, known as Rabigh II, is to be built as an expansion of PetroRabigh’s existing petrochemical plant, increasing output and introducing higher-margin products. The project, located on Saudi Arabia’s Red Sea coast, received a formal go-ahead from the parent firms in 2012; PetroRabigh has said previously it is due to come online in 2016, despite a string of maintenance problems at the existing facility. Ownership of the planned new facility will be transferred from Aramco and Sumitomo to PetroRabigh in the fourth quarter of this year, the company said on Sunday.

domain: news


Main / Simulation / Miso music plectrum ipa
Miso music plectrum ipa
Name: Miso music plectrum ipa
File size: 84mb
Download cracked Miso Music: Plectrum IPA file from the largest cracked App Store, you can also download on your mobile device with AppCake for iOS. Jamstar Acoustics - Learn Guitar · Uberchord - Learn Guitar Chords with Personalized Practice Lessons, Tuner, and The Ultimate Chord · Miso Music: Plectrum. Download consultoresniif.com in AppCrawlr! Author's description. "Strumify is the latest hot music-teaching app for iOS. Miso Music: Plectrum. FREE. Toddler .
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“And six hundred men of the tribe of Dan, armed with weapons of war set forth from Zorah and Eshtaol, and went up and encamped at Kiriathjearim in Judah . … the Danites came to Laish, to a people quiet and unsuspecting, and smote them with the edge of the sword, and burned the city with fire. And there was no deliverer because it was far from Sidon, and they had no dealings with any one … And they rebuilt the city, and dwelt in it. And they named the City Dan, after the name of Dan their ancestor” (Judges 18:11–12, 27–29).
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When it came time for them to create a music video to their new queer love song, indie-pop band Muna decided to pay homage to a cult classic in queer cinema.
On Tuesday (Sept. 7), Muna released their newest single “Silk Chiffon,” featuring their new label-owner Phoebe Bridgers. Described by guitarist Naomi McPherson in a statement as “a song for kids to have their first gay kiss to,” “Silk Chiffon” features the trio and Bridgers singing about queer romance and sex, with the song’s chorus declaring “Silk chiffon/ That’s how it feels, oh, when she’s on me.”
Alongside the song, the band also unveiled its new video for the track, which takes direct inspiration from the 1999 satiric rom-com But I’m a Cheerleader. In the video, lead singer Katie Gavin, dressed in her best high school cheerleading outfit and playing Natasha Lyonne’s titular character, arrives outside of a color-coded conversion therapy camp. She’s greeted by a pink-haired Bridgers, playing one of the camp’s leaders, and her fellow bandmates, Naomi McPherson and Josette Maskin, each shaking their heads, warning her not to enter.
The rest of the video plays out as a loving tribute to the film, with the trio going through the motions and playing the part of converts during the day. But at night, when Bridgers’ cheery-faced counselor isn’t watching, the girls sneak out and head to the nearest gay bar, where they perform a show and live their lives the way they want. “Life’s so fun, life’s so fun/ Don’t need to worry about no one,” they sing on the song’s flirtatious pre-chorus. “She said that I got her if I want/ She’s so soft like silk chiffon.”
“Silk Chiffon” is the first solo release from Muna since the trio signed to Bridgers’ Saddest Factory Records, an imprint of Dead Oceans. The band became the second act to sign to Bridgers’ label in May, following indie singer-songwriter Claud’s signing in October 2020. “We look forward to this lifelong partnership and Phoebe raising our first born children, as was included in the contract,” the band said in a cheeky statement released with the news of their signing. “This will be the last time we speak publicly as we will be giving her our voices in a cursed golden locket (with the exception of our bi-monthly contribution to the Podcast Industrial Complex). And maybe some music.”
Check out Muna’s new video for “Silk Chiffon,” featuring Phoebe Bridgers, below:


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"This was our third visit. Staff is super gracious, and the food is wonderful. The duck salad is lovely—lots of good confit on top of fresh apple and veggies. Not a pairing I would have planned, but definitely works!
This is a great addition to the neighborhood"See more
"This is rather unique place with great cocktails and reasonably priced comfort food. The exception to the reasonably priced statement extends to both the cocktails and the oysters on the half shell, For oysters save your money and go to State of Grace, price is about the same and you know where your oysters come from."See more

domain: food


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Read Frequently Asked Questions
Complimentary Webinar: Predicting Business Events & Maximizing Revenue Potential for Healthcare Enterprises (A Case-study Discussion)
This complimentary webinar will contain a brief overview of the macroeconomic environment and its impact on healthcare enterprises, and the challenges that healthcare entities are facing.
Why Should You Attend:
The drastic changes in the global macroeconomic environment and surging healthcare costs are forcing healthcare enterprises to find new ways to operate more productively, strategically and cost effectively in today's economy.
The first 30 minutes of this presentation will describe the current macroeconomic environment, and the challenges that healthcare companies are undergoing. Following that, the presentation will include a case study as to how a substance abuse program was able to leverage breakthrough techniques to predict revenues with a 95% accuracy range, anticipate and react to future business events, unveil competitors’ strategies, and maximize revenue potential.
The remaining 15 minutes of the presentation will allow participants the opportunity to ask questions and engage in discussions with the expert.
Areas Covered in the Seminar:
- Global macroeconomic challenges & their effects on healthcare companies.
- Case study on substance abuse program.
- Overview of the implementation of breakthrough techniques to anticipate & react to future business outcomes, forecasting revenue figures, improving strategic decision-making, maximizing revenue potential.
Who Will Benefit:
- Senior level healthcare executives, CEOs, COOs, CFOs
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Moe Alsumidaie, is the President and Chief Scientific Officer of Annex Clinical and the preeminent exponent of the application of Business Analytics to Clinical Trials and Healthcare Operations. Having extensive experience in Hospital Management and Phase I-IV Cardiology, Oncology, Immunology, Neurology and Medical Device clinical trials, Moe has worked for Ivy League Institutions and Fortune 500 & 100 enterprises including Stanford Medical Center, Abbott Vascular Devices, Genentech and Roche. Moe holds a BS in Physiology & Neuroscience from UC San Diego, an MBA and a MS in Investment Finance from Northeastern University’s College of Business Administration. At Annex Clinical, Moe's expertise includes financial and business analytics.
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Interpreting FDA's New Guidance Document on Risk-based Monitoring

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- The standard, traditional t-shirt for everyday wear
- Classic, generous, boxy fit
- Male model shown is 6'0" / 183 cm tall and wearing size Medium
- Female model shown is 5'8" / 173 cm tall and wearing size Small
- Heavyweight 5.3 oz / 180 gsm fabric, solid colors are 100% preshrunk cotton, heather grey is 90% cotton/10% polyester, denim heather is 50% cotton/ 50% polyester
- Double-needle hems and neck band for durability
Designed and sold by CoffeeCupLife2
When there is a microchip shortage this little guy is pretty popular. We know this can be a stressful situation so we thought we'd try to put a smile on your face!


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Lisa McGinty, I'm an artist who works mainly in oils. I make my own paint from pigments, and works in a traditional...
Hello, name is Lisa, and I am a artist who lives in South Oxon, who mainly paints in oils. I have taken inspiration...

domain: artistic


text:
Elevated IL-10 has been implicated in reactivation tuberculosis (TB). Since macrophages rather than T cells were reported to be the major source of IL-10 in TB, we analyzed the consequences of a macrophage-specific overexpression of IL-10 in transgenic mice (macIL-10-transgenic) after aerosol infection with Mycobacterium tuberculosis (Mtb). MacIL-10 transgenic mice were more susceptible to chronic Mtb infection than nontransgenic littermates, exhibiting higher bacterial loads in the lung after 12 wk of infection and dying significantly earlier than controls. The differentiation, recruitment, and activation of Th1 cells as well as the induction of IFN-γ-dependent effector genes against Mtb were not affected by macrophage-derived IL-10. However, microarray analysis of pulmonary gene expression revealed patterns characteristic of alternative macrophage activation that were overrepresented in Mtb-infected macIL-10 transgenic mice. Importantly, arginase-1 gene expression and activity were strikingly enhanced in transgenic mice accompanied by a reduced production of reactive nitrogen intermediates. Moreover, IL-10-dependent arginase-1 induction diminished antimycobacterial effector mechanisms in macrophages. Taken together, macrophage-derived IL-10 triggers aspects of alternative macrophage activation and promotes Mtb recrudescence independent of overt effects on anti-TB T cell immunity.
Tuberculosis (TB)5 is one of the most prevalent bacterial infections worldwide and constitutes a leading global health threat (1). Human TB caused by Mycobacterium tuberculosis (Mtb) is responsible for 8 million new cases and ∼2 million deaths annually. Primary TB is thought to depend on a combination of genetic susceptibility of the host and virulence of the infecting Mtb strain, whereas postprimary TB is due to a temporal dysregulation of otherwise intact immune defenses. More detailed elucidation of the balance between protective and inflammatory immune responses to Mtb may accelerate the development of more effective vaccines and therapies targeting both or either form of TB.
The cell-mediated immune response is known to be critical in the host defense against infection with intracellular pathogens such as mycobacteria. Th1 lymphocytes play an important role in granuloma formation by secreting type 1 cytokines, primarily IFN-γ and TNF (2, 3). These cytokines stimulate the antimicrobial activity of infected macrophages, allowing intracellular bacterial killing through reactive nitrogen intermediates (RNI) and LRG-47 (4, 5). IL-12 indirectly promotes an effective cell-mediated immune response by inducing IFN-γ production from T and NK cells. On the other hand, these protective T cell-mediated immune responses also play a causative role in the development of the pathology of this type of chronic disease (6). Hence, down-regulation of the inflammatory response by endogenous factors that limit tissue damage can promote establishment of chronic infection and may also lead to reactivating TB. In fact, increased levels of Th2-type anti-inflammatory cytokines such as IL-4 and IL-10 were detected in patients suffering from postprimary disease (7).
IL-10 is produced by macrophages, T cells, B cells, and a variety of other cell types. The effects of IL-10 on immune responses are mostly inhibitory (8). In macrophages, IL-10 inhibits antimicrobial effector mechanisms, the expression of costimulatory molecules, and the production of proinflammatory cytokines (9, 10). Inhibition of IL-12 production by IL-10 (11) may be one mechanism by which IL-10 blocks the development of protective Th1 immune responses (12). The absence of IL-10 causes overproduction of inflammatory cytokines (13) and the development of chronic inflammatory bowel disease (14). On the other hand, IL-10-deficient (−/−) mice also show increased resistance to intracellular pathogens such as Listeria monocytogenes (15), Leishmania major (16), and Trypanosoma cruzi (17). Therefore, IL-10 is required to prevent immunopathology, but it can also impair protective responses against several pathogens. The immunoregulatory function of IL-10 and the fact that elevated levels of IL-10 have been detected in TB patients (18, 19) suggest a role for IL-10 in susceptibility to TB. However, one report of Mtb infection in IL-10−/− mice found neither increased protective immune responses nor exacerbated immunopathology (20).
By contrast, mice lacking IL-10 were shown to clear Mycobacterium bovis bacillus Calmette-Guérin infection more efficiently (21). Taken together, further investigation of Mtb infection in different IL-10 genetic models differing in the level or source of IL-10 production appear warranted. Indeed, mice overexpressing IL-10 in activated T cells (tIL-10tg mice) were previously reported to be more susceptible to Mtb infection, exhibiting an impaired Th1 development as characterized by decreased numbers of activated T cells in the blood circulation and lung tissue (22). Similarly, another T cell-specific IL-10 transgenic model was also shown to have an increased M. bovis bacillus Calmette-Guérin burden (23). However, because IL-10 is only overexpressed in these mice when T cells become activated, effects of overproduction of IL-10 by innate immune cells could not be studied.
We were interested in analyzing the consequences of a macrophage-specific overexpression of IL-10 during experimental TB for the following reasons: first, macrophages rather than T cells were reported to be the major source of IL-10 in TB patients (18), and second, reactivation disease in patients is typically associated with a strong, rather than diminished, Th1 immune response (24). In this study, infection of mice expressing an epitope-tagged IL-10 under control of the human CD68 promoter specifically in macrophages (macIL-10tg mice) (25) resulted in a very different immunological phenotype than reported for Mtb-infected tIL-10tg mice. Even though macIL-10tg mice were also more susceptible to Mtb infection than nontransgenic littermates, the differentiation, recruitment, and activation of Th1 cells as well as the induction of IFN-γ-dependent effector genes against Mtb were unaffected by macrophage-derived IL-10. Transcriptome analysis revealed that after Mtb infection genes preferentially expressed in macrophages were specifically modulated in the lungs of macIL-10tg mice. Among these, genes typical for alternative macrophage activation, such as Arg1, were significantly induced in the lung transcriptome of Mtb-infected macIL-10tg mice. Taken together, our study revealed that macrophage-derived IL-10 induces alternatively activated macrophages and promotes exacerbation of chronic Mtb infection in the face of intact T cell immunity.
Materials and Methods
Mice and macrophages
Transgenic mice (macIL-10tg mice) specifically expressing IL-10 in macrophages under control of the human CD68 promoter were bred under specific pathogen-free conditions at the Technical University of Munich (25). macIL-10tg mice were on an FVB genetic background, and transgene-negative littermates were used as controls (FVB). Myeolid-specific arginase-1-deficient Tie2cre Arg-1flox/flox mice were housed at St. Jude Children’s Research Hospital (26). In any given experiment, mice were matched for age and sex. For experiments, mice were maintained under barrier conditions in the BSL 3 facility at the Research Center Borstel in individually ventilated cages. All experiments performed were in accordance with the German Animal Protection Law and were approved by the Animal Research Ethics Board of the Ministry of Environment (Kiel, Germany).
Bone marrow-derived macrophages (BMMφ) were obtained after flushing of femora from Tie2cre-pos Arg-1flox/flox mice and cre-negative littermates. To generate BMMφ, bone marrow cells were cultivated in L-929 conditioned medium as source for M-CSF activity for 9 days (27).
Bacteria and infection
Mtb (H37Rv) and Mycobacterium avium (TMC724; originally obtained from Dr. F. Collins, Trudeau Institute, Saranac Lake, NY) were grown in Middlebrook 7H9 broth (Difco) supplemented with Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment medium (Life Technologies), 0.002% glycerol, and 0.05% Tween 80. Mid-log-phase cultures were harvested, aliquoted, and frozen at −80°C. After thawing, viable cell counts were determined by plating serial dilutions of the cultures on Middlebrook 7H10 agar plates followed by incubation at 37°C. All experiments were performed in the BSL 3 laboratories at the Research Center Borstel. Before infection of experimental animals, stock solutions of mycobacteria were diluted in sterile distilled water, and pulmonary infection was performed using an inhalation exposure system (Glas-Col). To infect mice with 100 CFU of Mtb or 105 M. avium per lung, animals were exposed for 40 min to an aerosol generated by nebulizing ∼5.5 ml of a suspension containing 2 × 105 or 1 × 109 live bacteria/ml, respectively. Inoculum size was checked 24 h after infection by determining the bacterial load in the lung of infected mice. Mice were regularly weighed before and after infection. In accordance with the Animal Research Ethics Board of the Ministry of Environment, mice that lost 25% of their original weight during the course of infection had to be sacrificed.
Colony enumeration assays
Bacterial loads in lung, liver, and spleen were evaluated at different time points after infection with mycobacteria to follow the course of infection. Organs from sacrificed animals were removed aseptically, weighed, and homogenized in PBS containing a proteinase inhibitor mixture (Roche Diagnostics) for subsequent quantification of cytokines prepared according to the manufacturer’s instructions. Ten-fold serial dilutions of organ homogenates were plated in duplicates onto Middlebrook 7H10 agar plates containing 10% OADC and incubated at 37°C for 19–21 days. Colonies on plates were enumerated, and results were expressed as log10 CFU per organ.
One lung lobe per mouse was fixed in 4% formalin-PBS, set in paraffin blocks, and sectioned (2–3 μm). For immunohistological detection of NOS2, tissue sections were deparaffinized, placed in 10-mm sodium citrate buffer (pH 6.0), and then pressure-cooked for exactly 1 min. After blocking for 20 min in 1% H2O2 solution, slides were incubated with appropriately diluted polyclonal rabbit anti-mouse inducible NO synthase (NOS2) (BIOMOL) in TBS/10% FCS for 30 min in a humid chamber. Appropriately diluted goat anti-rabbit IgG (bridging Ab) (Dianova) and diluted rabbit anti-goat IgG-peroxidase conjugate (tertiary Ab) (Dianova) were used in sequential incubations of 30 min each. For immunohistological detection of B cells, deparaffinized sections were blocked in H2O2 and sequentially incubated with a monoclonal rat anti-mouse CD45R/B220 Ab appropriately diluted in 10% FCS/PBS, a biotinylated goat anti-rat Ab and streptavidin-HRP (all from BD Biosciences). Development was performed using diaminobenzidine (Sigma-Aldrich) and urea superoxide (Sigma-Aldrich), and hemalum was used to counterstain the slides.
RNA isolation and microarray analysis of pulmonary gene expression
Before and at different time points after aerosol infection with Mtb, weighed lung samples were homogenized in 5 ml of 4 M guanidinium-isothiocyanate buffer, and total RNA was extracted by acid phenol extraction.
Affymetrix GeneChips were used for genome-wide expression analysis of the impact of IL-10 overexpression during pulmonary Mtb infection. Tissue from individual mice was processed as biological duplicates (untreated controls and day 25 after infection) and triplicates (day 42 after infection). Per sample, 3 μg of total lung RNA was processed using the GeneChip Expression 3′ Amplification One-Cycle Target Labeling Kit according to the manufacturer’s instruction. Biotinylated cRNA was hybridized on MOE430A 2.0 GeneChips, which were stained, washed, and scanned following standard procedures. Microarray data have been deposited in the Gene Expression Omnibus database and are accessible through GEO Series accession number GSE14136 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE14316). CEL files were processed for global normalization and generation of expression values using the robust multiarray average algorithm in the R affy package (www.R-project.org). To identify differentially regulated genes, a combination of filtering for absolute (max-min >50) and relative (max/min >2) change across all conditions as well as statistical testing using ANOVA for the variables “infection” and “genotype” was applied. Correction for multiple testing was done using Benjamini-Hochberg (28). The 4188 probe sets passing the filtering criteria were then subjected to hierarchical clustering and visualization procedures using the Spotfire DecisionSite Functional Genomics software. Data mining of differentially regulated gene lists for enrichment of cocitations with cells and tissues was performed using Genomatix Bibliosphere software (Genomatix).
From extracted RNA, cDNA was obtained using murine moloney leukemia virus reverse transcriptase (Invitrogen) and oligo(dT) (12–18-mer; Sigma-Aldrich) as a primer. Quantitative PCR was performed on a LightCycler (Roche Diagnostics) as described previously (29). Data were analyzed using the “Fit Points” and “Standard Curve Method” using hprt as housekeeping gene to calculate the level of gene expression normalized for hprt expression. The following primer sets were used: Hprt, sense, 5′-GCA GTA CAG CCC CAA AAT GG-3′, and antisense, 5′-AAC AAA GTC TGG CCT GTA TCC AA-3′; Tnf, sense, 5′-TCT CAT CAG TTC TAT GGC CC-3′, and antisense, 5′-GGG AGT AGA CAA GGT ACA AC-3′; IL-12p40 (Il12b), sense, 5′-CTG GCC AGT ACA CCT GCC AC-3′, and antisense, 5′-GTG CTT CCA ACG CCA GTT CA-3′; Nos2, sense, 5′-AGC TCC TCC CAG GAC CAC AC-3′, and antisense, 5′-ACG CTG AGT ACC TCA TTG GC-3′; LRG-47 (Irgm1), sense, 5′-AGC CGC GAA GAC AAT AAC TG-3′, and antisense, 5′-CAT TTC CGA TAA GGC TTG G-3′; Arg1, sense, 5′-CAG AAG AAT GGA AGA GTC AG-3′, and antisense, 5′-CAG ATA TGC AGG GAG TCA CC-3′; macrophage mannose receptor (Mmr), sense 5′-CCA CAG CAT TGA GGA GTT TG-3′, and antisense, 5′-ACA GCT CAT CAT TTG GCT CA-3′; Ym1, sense 5′-GGG CAT ACC TTT ATC CTG AG-3′, and antisense, 5′-CCA CTG AAG TCA TCC ATG TC-3′; Fizz, sense, 5′-TCC CAG TGA ATA CTG ATG AGA-3′, and antisense, 5′-CCA CTC TGG ATC TCC CAA GA-3′; and Il4 receptor α (Il4ra), sense, 5′-TGT GAC CTA CAA GGA ACC CA-3′, and antisense, 5′-GCA AAA CAA CGG GAT GCA GA-3′.
Preparation of single-cell suspensions
For Ag-specific restimulation and flow cytometric analysis, single-cell suspensions of mediastinal lymph nodes and lungs were prepared from Mtb-infected mice at different time points. Lymph node cells were isolated by straining through a metal sieve. After depletion of erythrocytes, cells were resuspended in complete IMDM (Invitrogen) supplemented with 10% FCS (Life Technologies), 0.05 mM 2-ME (Sigma-Aldrich), and penicillin and streptomycin (100 U/ml and 100 μg/ml; Invitrogen), counted, and used for additional experiments. For preparation of single-cell suspensions from lungs, mice were anesthetized and injected i.p. with 150 U of heparin (Ratiopharm). Lungs were perfused through the right ventricle with warm PBS. Once lungs appeared white, they were removed and sectioned. Dissected lung tissue was then incubated in collagenase A (0.7 mg/ml; Roche Diagnostics) and DNase (30 μg/ml; Sigma-Aldrich) at 37°C for 2 h. Digested lung tissue was gently disrupted by subsequent passage through a 100-μm pore size nylon cell strainer. Recovered lung cells were counted, diluted in IMDM, and used for additional experiments.
Flow cytometric analysis of surface markers and intracellular cytokines
For flow cytometric analysis of surface markers, cells were washed and incubated with a mixture containing anti-FcγRIII/II mAb (clone 2.4G2), mouse and rat serum to block nonspecific binding to FcR. Cells were then incubated in consecutive steps for 20 min with optimal concentrations of the following Abs, CD4-allophycocyanin, CD3-PerCP, CD44-FITC, CD62L-PE, IAq-PE, CD124-PE (IL-4Rα) (all from BD Biosciences), F4/80-AlexaFluor647 (Caltag Laboratories), and CD206 (MMR) (Serotec). For detection of intracellular IFN-γ, an intracellular cytokine staining kit was used (BD Biosciences). Briefly, single-cell suspensions were prepared at 19 days after infection, and 2 × 106 cells were stimulated with plate-bound anti-CD3/CD28 mAb (clone 2C11 and clone 37/51 at 10 μg/ml, respectively) for 4 h in the presence of GolgiPlug (BD Biosciences). Nonspecific Ab binding was blocked by incubation with a mixture containing anti-FcγRIII/II mAb (clone 2.4G2), mouse and rat serum. Cells were washed and incubated with optimal concentrations of anti-CD4-FITC (BD Biosciences). After staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), and intracellularly accumulated IFN-γ was stained with a PE-labeled anti-IFN-γ mAb (BD Biosciences).
Fluorescence intensity was analyzed on a FACSCalibur (BD Biosciences) gating on propidium iodide-negative macrophages or lymphocytes identified by forward light scatter–side scatter profile, respectively.
IL-10 release and Ag-specific restimulation of CD4+ T cells
To detect IL-10 release in lymph nodes and lungs, 3 × 106 cells from single-cell suspensions of mediastinal lymph nodes and perfused lungs were cultured for 24 h in complete RPMI 1640 (Life Technologies) supplemented with 10% FCS (Life Technologies), 0.05 mM 2-ME (Sigma-Aldrich), and penicillin and streptomycin (100 U/ml and 100 μg/ml; Life Technologies). Supernatants were collected and frozen at −80°C until production of IL-10 was quantified by ELISA. For measuring Ag-specific production of IFN-γ by CD4+ T cells in lymph nodes, single-cell suspension of mediastinal lymph nodes were prepared in IMDM from mice 19 days after infection with Mtb. To enrich CD4+ T cells, single-cell suspensions from lymph nodes were incubated with magnetic CD4 microbeads (Miltenyi Biotec) and separated from other cells on a MACS separation unit (Miltenyi Biotec). Separated CD4+ T cells were collected in IMDM, counted, and diluted. Purity of enriched CD4+ T cells was >95% as determined by flow cytometry. For Ag-specific restimulation, 4 × 105 enriched CD4+ lymph node cells were incubated with 2 × 105 peritoneal macrophages that were pulsed with 25 μg/ml short-term culture filtrate from Mtb (a gift from P. Andersen, Statens Serum Institute, Copenhagen, Denmark) in IMDM. Resident peritoneal macrophages were obtained 1 day before the experiment by peritoneal lavage of uninfected C57BL/6 mice and incubated overnight in 96-well flat-bottom microplates (Nunc) in complete IMDM. After 72 h of restimulation, supernatants were collected and frozen at −80°C until production of IFN-γ was quantified by ELISA.
Quantification of IFN-γ and IL-10 by ELISA
To determine cytokine production after pulmonary infection of experimental animals, supernatants were analyzed in 3-fold serial dilutions using the sandwich ELISA system OptEia (BD Biosciences). After incubation with HRP coupled to avidin and developing with tetramethylbenzidine substrate reagent, the absorbance was read on a microplate reader (Sunrise; Tecan). Using a test wavelength of 450 nm and a reference wavelength of 630 nm, samples were compared with appropriate recombinant cytokine standards. The detection limits for all cytokines were 5 pg/ml.
Arginase activity in lung homogenates
To determine arginase activity in murine tissue, weighed pieces of organs were homogenized in 100 μl of 0.1% Triton X-100 (Sigma-Aldrich) containing a protease inhibitor mixture (Roche). Fifty microliters of 10 mM MnCl2 (Merck) and 50 mM Tris-HCl (Merck) was added to all samples, and the enzyme was activated by heating for 10 min at 55°C. Arginine hydrolysis was conducted by incubating 25 μl of the activated lysate with 25 μl of 0.5 M l-arginine (Merck) at 37°C for 60 min. The reaction was stopped with 400 μl of H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v). As a degree of arginase activity, the urea concentration was measured at 540 nm after addition of 25 μl of α-isonitrosopropiophenone (Sigma-Aldrich; dissolved in 100% ethanol) followed by heating at 95°C for 45 min. One unit of arginase activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol urea/min.
Detection of RNI
To detect RNI in uninfected and infected mice, blood was collected at different time points. Using a commercial nitrate reductase kit (Cayman Chemical) NO3 was converted into NO2. After adding Griess reagents, the content of NO2 was determined by photometric measurement reading the absorbance at 540 nm on a microplate reader (Sunrise; Tecan) as described previously (27).
IFN-γ-induced bacterial growth inhibition in macrophages
To analyze the antibacterial activity of macrophages, BMMφ were cultured 48 h before infection with Mtb in DMEM supplemented with 10% FCS (Biochrom), 2 mM l-glutamine, 1 mM sodium pyruvate, and 10 mM HEPES (all from PAA) in the presence of medium or incubated with 10 ng/ml IL-4 (BD Biosciences) and 10 ng/ml IL-10 (BD Biosciences). One day before infection, cells were incubated with medium or stimulated with 100 U/ml IFN-γ (PeproTech). Cells were inoculated with Mtb H37Rv at a multiplicity of infection of 0.5 for 2 h. To determine bacterial uptake at different time points, macrophages were lysed by addition of 0.5% Triton X-100 (Serva). Lysates were serially diluted in 0.05% Tween 80 (Merck), plated on Middlebook 7H10 agar containing 10% OADC, and incubated at 37°C for 19–21 days. Colonies on plates were enumerated, and results were expressed as log10 CFU per culture.
Quantifiable data are expressed as the means of individual determinations and SDs. Statistical analysis was performed using the unpaired Student’s t test defining different error probabilities (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). ANOVA was performed using the Dunnett multiple comparison test, correcting for multiple testing, which defines different error probabilities between untreated cells and macrophages that have been incubated with IFN-γ (#, p ≤ 0.05; ##, p ≤ 0.01) or IL-4/IL-10/IFN-γ (*, p ≤ 0.05; **, p ≤ 0.01).
macIL-10tg mice are highly susceptible to Mtb infection
To determine the influence of increased levels of macrophage-derived IL-10 on the outcome of experimental TB, mice overexpressing IL-10 under control of the macrophage-specific CD68 promoter were infected with 100 CFU of Mtb via the aerosol route. Whereas FVB control mice were able to control mycobacterial growth in infected lungs at a plateau level of ∼6.9 log10 CFU/lung, bacterial loads in IL-10 transgenic littermates steadily increased reaching significantly higher CFU counts 78 days postinfection (Fig. 1,a). Mtb-infected FVB wild-type mice survived for more than 250 days (Fig. 1 b). In contrast, macIL-10tg animals died significantly earlier, with a mean survival time of ∼100 days.
Activation of CD4+ T cells is not impaired in Mtb-infected macIL-10tg mice
Priming in lymphoid organs and migration of T lymphocytes to the site of infection is indispensable for the induction of defense mechanisms against Mtb. To evaluate whether T cell activation in macIL-10tg mice was impaired in draining lymph nodes and lung tissue, activated CD4+ T cells were quantified by flow cytometry. After infection with Mtb, comparably increasing relative and absolute numbers of activated CD44highCD62LlowCD4+ T cells were observed in lymph nodes of both FVB and macIL-10tg mice (Fig. 2, a and b). Ag-specific restimulation of CD4+ T cells purified from the draining lymph nodes revealed that T cells of macIL-10tg mice were as capable of producing IFN-γ as wild-type littermates (Fig. 2,c). In addition, the relative proportion of activated CD44highCD62LlowCD4+ T cells in the lung was also not affected during the course of Mtb infection in macIL-10tg mice (Fig. 2,d). However, the absolute amount of activated CD4+ T cells in lungs from macIL-10tg mice was found to be enhanced at day 84 after Mtb infection (Fig. 2,e). After polyclonal restimulation, IFN-γ production by CD4+ T cells was comparable in Mtb-infected FVB and macIL-10tg mice (Fig. 2 f). Taken together, macrophage-derived IL-10 did not dramatically affect the induction, migration, and function of T effector cells after Mtb infection. The increased infiltration of activated CD4+ T cells into lungs from macIL-10tg mice may reflect an increased inflammatory reaction to the elevated bacterial load at late time points of Mtb infection.
The expression of NOS2 and LRG-47 (Irgm1) is not impaired in macIL-10tg mice after Mtb infection
IFN-γ-dependent activation of macrophages at the site of infection is key to the induction of the effector molecules NOS2 and LRG-47 (4, 5), and IL-10 has been previously described to affect antimicrobial effector mechanisms (30, 31). Since the expression of T cell-derived IFN-γ was not affected in Mtb-infected macIL-10tg mice, downstream effector mechanisms could have been directly impaired by the macrophage-specific overexpression of IL-10. Quantitative real-time PCR revealed that during Mtb infection transcripts for Nos2 were increasingly induced in lungs from both FVB and macIL-10tg mice to the same extent (Fig. 3,a). In fact, at day 90 postinfection, Nos2 gene expression was found to be even higher in transgenic animals. It should be noted that IL-10 has been shown to induce Nos2 expression in some settings, suggesting that Nos2 is not a canonical target of the IL-10 anti-inflammatory response (32). The kinetics of gene expression of Lrg-47 (Irgm1) was comparable in the lungs of both FVB and macIL-10tg mice peaking on day 21 of Mtb infection (Fig. 3,b). Six weeks after infection, immunohistochemical detection of NOS2 in lung sections verified these findings on the protein level, showing no differences in NOS2 expression between FVB and macIL-10tg mice (Fig. 3 c).
Macrophage-derived IL-10 specifically suppresses IL-12p40 in infected tissue but not in draining lymph nodes
So far, the increased susceptibility of macIL-10tg mice to experimental TB could neither be attributed to an impaired function of IFN-γ-producing T cells nor to defective IFN-γ-dependent downstream mechanisms in macrophages. IL-10 specifically suppresses the production of proinflammatory cytokines in activated macrophages and dendritic cells (9, 33). To evaluate the suppressive effect of macrophage-derived IL-10 on anti-TB effector cytokines during the course of infection with Mtb, we compared the expression of TNF (produced by T cells and macrophages/dendritic cells) and IL-12p40 (produced by macrophages/dendritic cells) in the lungs of FVB wild-type and IL-10tg mice. Until day 90 after infection, increasing amounts of Tnf transcripts were found in the lungs of FVB mice (Fig. 4,a). In the lungs of macIL-10tg mice, however, gene expression was found to be even higher on days 49 and 90 postinfection. In contrast, 49 and 90 days after Mtb infection, gene expression of IL-12p40 (Il12b) was found to be significantly reduced in lungs from macIL-10tg mice when compared with FVB mice (Fig. 4 b).
After Mtb infection, IL-12 is essential for driving protective Th1 immune responses (34). In lymphoid organs, IL-10 inhibits IL-12-driven Th1 development, whereas it has no suppressive effect on already differentiated Th1 cells in infected tissue (35). Because Mtb-infected macIL-10tg mice displayed a normal Th1 development, we speculated that the magnitude of transgenic IL-10 expression would be different in draining lymph nodes and lung tissue. In contrast to dendritic cells, only a few macrophages are present in lymph nodes in experimental TB (36). Accordingly, we found only low levels of IL-10 in lymph nodes from macIL-10tg mice (Fig. 4,c). In contrast, a great amount of IL-10 was secreted in lung tissue from macIL-10tg animals. In FVB mice, IL-10 was below the detection limit in either organ (Fig. 4,c). In line with the low expression of transgenic IL-10 in the lymph nodes of Mtb-infected macIL-10tg mice, IL-12p40 expression was not affected. In contrast, elevated levels of transgenic IL-10 in lung tissue led to reduced expression of IL-12p40 (Fig. 4 d). Taken together, IL-10 secretion by macrophages does not seem to have an overt effect on Th1 differentiation and the expression of Th1 cell effector mechanisms. Macrophage-derived IL-10 rather appears to regulate distinct macrophage functions such as cytokine secretion at the site of infection.
Impact of Mtb infection on pulmonary gene expression in wild-type and macIL-10tg mice
To investigate how macrophage-specific overexpression of IL-10 shapes the host response to Mtb at the transcriptome level, genome-wide expression analysis was performed on tissue RNA isolated from the lungs of FVB and macIL-10tg mice at baseline as well as 25 and 42 days after infection. Excluding variability for the infection itself, only 219 probe sets were different between FVB and macIL-10tg, indicating a comparatively small effect of IL-10 on the transcriptional changes. The effect of IL-10 on the number of up- and down-regulated genes is summarized in Fig. 5 a. Before infection, only 36 probe sets were up-regulated in lungs from macIL-10tg mice compared with FVB animals (data not shown). These included some known IL-10-induced genes (Msr1, Ccr2, Ccr5, Fcgr2b, and Saa3). Only four probe sets, all detecting Igg, gave significantly higher signals in lungs from FVB mice. After infection, at the early time point day 25, of all probe sets induced >3-fold in either lung RNA from wild-type or macIL-10tg mice, only one probe set (C3ar1) had significantly increased expression in macIL-10tg mice, whereas Igh-VJ588 was higher in FVB control animals. Of the 970 probe sets induced by Mtb 42 days after infection, 158 showed higher expression in macIL-10tg, whereas 68 were significantly higher in lungs from wild-type mice. Therefore, the majority of IL-10 effects was apparent at the later stages of infection.
Hierarchical clustering of the probe sets regulated by IL-10 (Fig. 5 b; data are listed in supplemental Table I)6 revealed groups of genes overexpressed in the lungs of macIL-10tg mice already under basal conditions (cluster A) or postinfection (cluster B). These clusters contained known target genes of IL-10 in macrophages like Socs3, Bcl3, Saa3, and Il1rn, consistent with previous observations (32). However, many chemokines (e.g., Ccl2 and Ccl7) and cytokines (Il1b and Il6) were also expressed at higher levels in the lungs of macIL-10tg mice compared with wild-type animals, indicating that the inhibitory effect of IL-10 on proinflammatory gene expression is likely affected by the increased bacterial load in macIL-10tg mice.
Data mining: changes in cellular composition
The presence of various transcripts encoding cell type-specific surface proteins, such as the dendritic cell marker Cd207 in cluster C and the monocyte/macrophage marker Msr1 in cluster B, suggested an impact of IL-10 on the infection-induced changes in the cellular composition of the lung tissue. We used a literature-based data mining tool, Genomatix Bibliosphere, to search for overrepresentation of anatomical terms in lists of genes up-regulated in macIL-10tg or wild-type mice after Mtb infection (Fig. 6 a). Although transcriptional evidence for NK cells, dendritic cells, and T lymphocytes was similar in both genotypes, in the lungs of macIL-10tg mice, a strong enrichment for probe sets associated with macrophages was evident. In contrast, probe sets up-regulated to higher levels in FVB lungs at day 42 were connected to B lymphocytes. Immunohistological analysis also revealed a moderately reduced infiltration of B cells in lungs from macIL-10tg mice (supplemental Fig. 1).6 Taken together, IL-10 appears to regulate leukocyte subset migration to the lung, or local proliferation of the cells, during Mtb infection.
Signs of alternative macrophage activation in the lung transcriptome after Mtb infection
Among the group of genes induced to high levels in infected macIL-10tg mice was Arg1 (Fig. 6,b), encoding the enzyme arginase-1, whose extrahepatic expression is typically associated with IL-4/IL-13-induced alternative macrophage activation, indicating that after Mtb infection IL-10 may promote the development of alternatively activated macrophages. Although Il4 and Il13 mRNA was not detectably expressed in the lung data set, small amounts of IL-4 or IL-13 could promote alternative activation of lung macrophages in macIL-10tg mice. IL-10 may enhance the sensitivity to IL-4 because it increases Il4ra expression on macrophages in vitro (32), an effect that was also observed in the lung data set after Mtb infection (Fig. 6,b). In addition to arginase-1, many other markers have been described to define IL-4Rα-mediated alternatively activated macrophages, especially during pulmonary inflammation such as Fizz1, various chitinases, and MMR (29). Among these genes, only Fizz1 (encoded by Retlna) and the chitinase Chi3l1 were clearly expressed at higher levels in macIL-10tg lungs (Fig. 6 b), whereas the chitinases Ym1 (= Chi3l3) and Ym2 (= Chi3l4) as well as the Mmr (encoded by Mrc1) were down-regulated after infection to similar levels in both genotypes. The differential induction of markers for alternatively activated macrophages in Mtb-infected macIL-10tg mice reflects also some opposite biological effects of IL-10 to IL-4 and IL-13, e.g., the regulation of MMR expression and function (37). In contrast to this rather inhibitory effect, IL-10 has been clearly shown to contribute to alternative macrophage activation during TLR ligation by a strong synergistic induction of arginase-1 (32). Taken together, our genome-wide expression analysis revealed that after infection with Mtb, macrophage-derived IL-10 induces several features of alternatively activated macrophages.
After Mtb-infection, macIL-10tg mice develop alternatively activated macrophages
To verify our findings revealed by genome-wide expression analysis, we next performed quantitative RT-PCR analysis to test the model that macrophage-derived IL-10 promotes alternative macrophage activation after Mtb infection (Fig. 7,a). Six weeks after infection, we found significantly elevated gene expression of Fizz1 and Arg1. In contrast, transcripts for Mmr and the chitinase Ym1 were not elevated. In whole tissue, Il4ra gene expression in macIL-10tg mice was elevated but not significantly different from gene expression in lungs from FVB wild-type mice. To determine the activation state of macrophages in Mtb-infected macIL-10tg mice, we analyzed macrophages from perfused lungs by flow cytometry. Six weeks after infection, IAq, CD80, CD86, and the MMR were expressed to the same extent on propidium iodide-negative F4/80-positive cells from FVB and macIL-10tg mice (Fig. 7 b). Whereas only a subpopulation of macrophages from infected FVB mice expressed the IL-4Rα, all macrophages from macIL-10tg mice were found to be positive for this receptor subunit. Taken together, we found that after Mtb infection IL-10 secretion by macrophages induces an alternatively activated program characterized by an enhanced expression of Fizz1, Arg1, and the Il4ra.
In Mtb-infected macIL-10tg mice, increased arginase-1 activity is concomitant with reduced production of RNI
Classical macrophage activation plays an important role in combating infection with Mtb through IFN-γ-induced expression of effector molecules such as NOS2-dependent RNI (4). This induction may be counterregulated in alternatively activated macrophages. Because in alternatively activated macrophages arginase-1 hydrolyzes l-arginine to urea and l-ornithine, this pathway is also discussed to regulate RNI production in macrophages through depletion of l-arginine as the substrate for NOS2 (39, 40). Therefore, we speculated that in macIL-10tg mice an increased arginase-1 activity modulates the production of RNI, thereby affecting effector mechanisms against Mtb usually exerted by classically activated macrophages. A kinetic quantification of gene expression revealed that Arg1 is not induced in lungs from FVB mice during the course of Mtb infection (Fig. 8,a). In contrast, Arg1 expression in macIL-10tg mice was found to be significantly induced during the late phase of Mtb infection. Accordingly, arginase activity in lungs from macIL-10tg mice was found to be elevated already 49 days postinfection (Fig. 8,b). In contrast, arginase activity was detectable in the lungs of FVB mice but did not increase after infection (Fig. 8,b). Concomitant with the increased arginase-1 activity in infected macIL-10tg mice, RNI were only moderately produced in these animals (Fig. 8,c). In distinction, significantly increasing amounts of RNI were found in sera from Mtb-infected FVB mice (Fig. 8,c). Hence, the increased susceptibility in macIL-10tg mice after infection with Mtb appeared to be caused by an elevated induction of arginase-1 in alternatively activated macrophages, which in turn led to an inefficient production of RNI. To examine whether IL-10-induced arginase-1 expression in macrophages diminishes antimycobacterial effector mechanisms in macrophages, we performed a Mtb growth inhibition assay in arginase-1-deficient BMMφ (Fig. 8 d). Incubation of BMMφ from Tie2cre-neg Arg-1flox/flox arginase-1-competent and Tie2cre-pos Arg-1flox/flox arginase-1-deficient mice lead to a significant reduction of mycobacterial growth 48 and 72 h after infection with Mtb when compared with infected macrophages that have not been activated with IFN-γ. Preincubation with IL-4 and IL-10, however, diminished IFN-γ-dependent mycobacterial growth inhibition in arginase-1-competent BMMφ. In contrast, IL-4/IL-10 treatment of IFN-γ-activated arginase-1-deficient macrophages did not lead to reduced antimycobacterial effector mechanisms, indicating that induction of arginase-1 in macrophages reduces the capability of controlling Mtb growth.
macIL-10tg mice control bacterial growth after M. avium infection
To test our hypothesis that macrophage-derived IL-10 mostly subverts NOS2-dependent effector mechanisms, we infected macIL-10tg mice aerogenically with M. avium, a pathogen that is insensitive to NOS2-derived RNI production (6, 41), and determined bacterial loads in different organs 105 days after infection (Fig. 9). Bacterial loads in macIL-10tg mice were found to be only moderately increased in lung and liver and comparable to FVB mice in spleens. However, infection of NOS2−/− mice with M. avium revealed that bacterial loads rather decrease in the absence of NOS2 (40), corroborating the contention that after infection with RNI-insensitive mycobacteria the suppressive effect of NO on T cells appears (6). Therefore, the inability of macIL-10tg mice to efficiently control bacterial growth after infection with RNI-sensitive and, to some extent, also -insensitive mycobacteria indicates that autocrine IL-10 operates partially by reducing RNI production via arginase-1 induction.
TB is a systemic disease that becomes manifest most prominently in the lung (24). Progressive primary TB occurs when the initial inflammatory focus is unable to contain mycobacterial replication, and the disease spreads to other parts of the lung. Postprimary disease develops when a lesion containing a previously “dormant”, i.e., growth-arrested organism resumes growth after a period of clinical latency. This organism may have been implanted years before during a primary infection that was successfully contained by a protective Th1-type immune response. Reactivation is often a consequence of a perturbed immune status of the host, although the cause of deregulation may be multifactorial.
Elevated levels of the Th2 cytokine IL-10 were detected in individuals with active TB (18, 19). Importantly, macrophages rather than T cells were reported to be the major source of IL-10 in these patients (18). However, whether and how macrophage-derived IL-10 promotes exacerbation of primary infection or reactivation of chronic latent infection is not clear. Our study revealed that in macIL-10tg mice an increased production of IL-10 by macrophages resulted in enhanced Mtb replication, particularly during the chronic phase of infection, and thus bears some resemblance to reactivation TB in humans.
Humans infected with Mtb typically have a strong DTH response, as measured by the purified protein derivative skin test (24). This holds also true for patients suffering from postprimary TB. Thus, despite an apparently adequate initial differentiation of cell-mediated immune responses, Mtb is capable of persisting and can even resume replication within macrophages. In the draining lymph nodes of Mtb-infected macIL-10tg mice, T cells efficiently differentiated into Th1 cells and migrated to the site of infection. Despite the presence of macrophage-derived IL-10 in the lungs of infected transgenic mice, Th1 cells efficiently expressed effector functions and secreted sufficient IFN-γ to induce a classical macrophage activation characterized by expression of NOS2 and LRG-47. However, bacteria survived and successfully proliferated within IFN-γ-activated IL-10tg macrophages. It appears, therefore, that during experimental TB macrophage-derived IL-10 does not affect initiation and maintenance of Th1 immune responses but specifically acts on macrophage functions in a partially dominant manner over IFN-γ at the functional level.
It is important to note that overexpression of IL-10 specifically in T cells resulted in completely different immunosuppressive mechanisms after Mtb infection (22). tIL-10tg mice failed to efficiently prime naive CD4+ T cells in draining lymph nodes, resulting in decreased numbers of activated T cells in the blood circulation and lung tissue; this defective Th1 response eventually led to an increased bacterial burden, due to a failure of classical macrophage activation. In contrast, IL-10 expression in macrophages at the site of infection induces an alternative activation program specifically in macrophages that counteracts concomitant classical macrophage activation. Since T cell-mediated immune responses remain largely unaffected (as is the case in human postprimary TB), the outcome of experimental TB in macIL-10tg mice may therefore better reflect the situation in patients with reactivating TB.
We could link the increased susceptibility of Mtb-infected macIL-10tg mice neither to an impaired function of IFN-γ-producing T cells nor to defective IFN-γ-dependent downstream mechanisms in macrophages. Transcriptome analysis revealed a strong enrichment for probe sets associated with macrophage function in the lungs of Mtb-infected macIL-10tg mice. IL-10 has previously been shown to act primarily on activated macrophages, where IL-10R expression is highest. The IL-10R activates STAT3, and loss of this transcription factor in macrophages mimics loss of IL-10 itself (42). Hence, because STAT3 in myeloid cells is both necessary and sufficient for the effects of IL-10, macrophages are major targets of IL-10 in vivo (42, 43, 44, 45). On a functional level, IL-10 has been shown to negatively regulate macrophage function in a variety of experimental systems (17, 30, 31, 46). In vitro, IL-10 antagonizes the IFN-γ-dependent induction of NOS2 (30, 31). Our finding that macIL-10tg mice fail to efficiently control bacterial growth especially after infection with RNI-sensitive mycobacteria corroborates our contention that the mode of action of autocrine IL-10 proceeds mainly via arginase-1-mediated reduction of RNI production in macrophages. However, we found NOS2 normally expressed in Mtb-infected macIL-10tg mice, whereas the production of RNI was reduced during the course of infection. Hence, other pathways must be affected in macrophages of macIl-10tg mice that lead to an impaired expression of effector molecules such as RNI.
Among the group of genes induced to high levels in Mtb-infected macIL-10tg mice, some were associated with alternative macrophage activation such as Il4ra, Fizz1, and Arg1 (29). Alternative macrophage activation is typically induced by IL-4Rα ligation. Although Il4 and Il13 mRNA was not detectably expressed in the lung data set, small amounts of IL-4 may still drive alternative activation of lung macrophages in macIL-10tg mice. IL-10 may enhance the sensitivity to IL-4 because it increases IL-4 receptor expression on macrophages in vitro (32), an effect that was also observed in the lung data set after Mtb infection. The differential induction of markers for alternatively activated macrophages in Mtb-infected macIL-10tg mice reflects also some opposite biological effects of IL-10 to IL-4 and IL-13, e.g., the regulation of MMR expression and function (37). In contrast to this rather inhibitory effect, IL-10 has been clearly shown to contribute to alternative macrophage activation during TLR ligation by a strong synergistic induction of arginase-1 (32). Hence, during experimental TB, Arg1 expression may be directly induced by IL-10 or indirectly by IL-10-dependent up-regulation of the high-affinity IL-4Rα. Alternative activation of macrophages via the IL-4Rα has been shown to differentially regulate protective and immunopathological immune responses (29). Importantly, analysis of macrophage-specific IL-4Rα-deficient mice clearly demonstrated immunomodulation by macrophages to be a central regulatory mechanism during inflammatory immune responses in vivo (47). Because alternative macrophage activation reduces RNI production (40, 48, 49) and proinflammatory cytokine release (50), protective (39, 51, 52) and pathological (50) immune responses are significantly regulated by these types of macrophages.
In the context of TB and alternatively activated macrophages, one genome-wide expression analysis in IL-4-stimulated murine bone marrow-derived macrophages infected with Mtb has been published so far (51). A specific reanalysis of our lung transcriptome data for differences in the expression of the 15 most highly increased genes in Mtb-infected IL-4-stimulated macrophages revealed that 5 genes were expressed at significantly higher levels in macIL-10tg lungs than in FVB mice after infection (Fcrg2b, Fn1, Cish, Ccl9, and Mrc1), and Cdh1 was increased under basal conditions in macIL-10tg lungs (data not shown). Mmp12 (encoding matrix metalloproteinase-12, MMP-12) and Tfr (encoding the transferrin receptor, TfR) were found to be significantly up-regulated in Mtb-infected alternatively activated macrophages in vitro (51). The induction of the TfR appears to be of major importance in the context of experimental TB because intracellular Mtb exploit the transferrin-transferring receptor complex for iron utilization (53). However, neither our genome-wide expression analysis nor quantitative RT-PCR revealed a differential regulation of Tfr in Mtb-infected macIL-10tg mice in vivo (data not shown). Additionally, Kahnert et al. (51) suggested MMP-12 induced in alternatively activated macrophages to be involved in the pathogenesis of TB. Although our genome-wide expression analysis in Mtb-infected macIL-10tg mice revealed an increased expression of Mmp12 in macIL-10tg mice at later time points of infection, the difference was not significant (data not shown). Moreover, quantitative RT-PCR of Mmp12 did not confirm a difference between wild-type and macIL-10tg mice (data not shown). Taken together, genome-wide expression analysis in Mtb-infected and IL-4-induced alternatively activated macrophages in vitro and in Mtb-infected macIL-10tg mice in vivo revealed an overlapping set of regulated genes, consistent with our interpretation that IL-10 supports a macrophage phenotype in vivo that is phenotypically related to alternatively activated macrophages.
Until recently, little was known about the impact of alternative macrophage activation and arginase-1 on the outcome of Mtb infection. Classical macrophage activation plays a central role in combating infection with Mtb through IFN-γ-induced expression of effector molecules such as the NOS2-dependent production of RNI (4, 54). Importantly, inhibition of endogenous NOS2 reactivates latent infection in Mtb-infected mice, indicating that NOS2-dependent mechanisms in macrophages are crucial for the control of Mtb persisting in immunocompetent hosts (55). The production of RNI might be counterregulated by IL-4Rα- and/or IL-10-dependent mechanisms, leading to arginase-1 expressing alternatively activated macrophages. In fact, RNI production in Mtb-infected alternatively activated macrophages was found to be reduced (51). In line with these findings, we here show the induction of arginase-1 in macrophages subverts IFN-γ-induced antimycobacterial effector mechanisms in macrophages. Since NOS2 shares l-arginine as a substrate with arginase-1, substrate depletion by either enzyme is a key regulatory mechanism in macrophages, and differential expression of NOS2 and arginase-1 is important for regulating macrophage effector functions (40, 49). Very recently, El Kasmi et al. (26) showed that specific deletion of arginase-1 in macrophages reduced lung bacterial loads during Mtb infection, supporting our contention that arginase-1 induction in macrophages undermines NOS2-dependent effector mechanisms against Mtb by depletion of the common substrate l-arginine. In addition, arginase-dependent production of polyamines was shown to promote elevated growth of L. major in macrophages (52) and may also be beneficial for the proliferation of Mtb. Importantly, in human pulmonary TB, high production of IL-10 also parallels arginase activity in PBMC (56). Hence, elevated IL-10-dependent arginase activity during the chronic phase of infection may promote recrudescence of Mtb growth. We do not exclude that macrophage-derived IL-10 suppresses other antimycobacterial effector mechanisms in addition to regulating RNI synthesis. In contrast to infection with Mtb, macIL-10tg mice were indeed able to control bacterial growth after infection with M. avium, which has been shown to be insensitive to the effects of RNI (6, 41). However, because bacterial loads have been described to be further reduced after infection with M. avium in the complete absence of RNI in NOS2−/− mice, it appears that although autocrine IL-10 subverts RNI synthesis in macrophages other suppressive mechanisms may also be induced by IL-10.
We conclude that during Mtb infection macrophage-derived IL-10 drives reactivation during the chronic stage of the disease by acting primarily at the level of the macrophage. Macrophage-derived IL-10 appears to override IFN-γ-dependent classical macrophage activation and effector mechanisms against Mtb by inducing an alternatively activated phenotype. Specifically, Arg1 expression in these macrophages may provide better growth conditions for Mtb. Th2 cytokines that potently drive alternative macrophage activation have been associated with the development of postprimary TB in patients (7, 57). Thus, reactivating TB may, in part, be mediated by a Th2-type immune reaction characterized by an alternative activation state of macrophages that eventually subverts protective immune mechanisms. It is possible that the identification of an increased type 2 cytokine immune response may identify infected individuals at risk to reactivate a latent Mtb infection. Additionally, prophylactic treatment of these individuals with immunomodulating drugs aimed at inhibiting the production of type 2 cytokines or downstream immune mechanisms may prevent or ameliorate reactivation TB.
We thank Alexandra Hölscher, Tanja Sonntag, Susanne Metken, and Manfred Richter for excellent technical assistance; Ilka Monath, Sven Mohr, and Claus Möller for organizing the animal facility and taking care of the mice at the Research Center Borstel; Angela Servatius for management of the macIL-10tg colony and processing the microarray samples at the Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich; members of P. Murray’s laboratory for bone marrow isolation; and Bianca Schneider for help with the Mtb growth inhibition assay.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work is supported by a research grant (“Host defence against infections”) from the Medical University of Lübeck (to C.H.), Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Germany) Grants NIES05T22 (to S.E.), 01KI0784 (to S.E. and C.H.), and NGFN-2 FKZ 01GS0402 (to R.L.), the Deutsche Forschungsgemeinschaft La 1262/3-1 (to R.L.), and National Institutes of Health Grant AI062921 (to P.J.M.).
Abbreviations used in this paper: TB, tuberculosis; BMMφ, bone marrow-derived macrophage; IL-4Rα, IL-4 receptor α; MMP-12, matrix metalloproteinase-12; MMR, macrophage mannose receptor; Mtb, Mycobacterium tuberculosis; OADC, oleic acid-albumin-dextrose-catalase; RNI, reactive nitrogen intermediate; TfR, transferrin receptor.
The online version of this article contains supplemental material.

domain: health


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I have been waiting some time for the Ferguson affair to reach this point. That we would be here, watching a city burn, was inevitable. The only uncertainty was which city would be turned to ash, which ghetto would produce the unrest. Michael Brown’s death sealed that well enough.
Watch this video to get an idea of what’s going wrong here.
Let’s get a few things straight. I don’t care what the ruling was or how much you might think it was unfair. There is no excuse for barbaric behavior like this. Talking heads are prattling on about “understandable anger” and “justifiable rage against oppression.” They are only doing so because the violence did not take place in their hometown. Safe behind their lily-white gated communities, they can theorize about Black Rage and the evils of slavery, 150 years dead. They aren’t suffering, and they don’t seem too broken up that Blacks are bearing the brunt of their own rage. Sometimes I think gated communities are as great a threat to civilization as the fucking ghetto.
Justified violence? This is nothing of the sort. Want to see how justifiable rage works? Go read about Ghandi. At least he didn’t set cities on fire. These people are barbarians, thugs and trash. They are not the civilized people clamoring for an end to police militarization, they are agents of chaos, opportunists taking advantage of the stupidity and misguided self-loathing of the American people. The rioting, the looting, the burning… this is not about Michael Brown any more than the Rodney King riots were spurred on by a love for that individual.
To the people who would still argue with me, I have this message. Read it carefully:
Oh, go on about the militarization of police. Stick a camera on every officer. Fine. I don’t like the agents of the government any more than you do. I’ve been ranting and raving about them for years, but did people listen? No, you told me that government was good and we needed more of it. They needed more power, you said. The expansion of police power concerns me more than most. It concerned me long before this dumbass thug got himself shot.
But no, everyone thought the government was on their side, that it was a good and reasonable actor. Now you know, don’t you? Now watch your cities burn, watch the barbarians inside the gates tear down civilization and the ineffectual but well-armed bureaucrats do their thing, pepper spraying the few peaceful people and ignoring the looters (too dangerous to do your job, eh). I told you this would happen.
Ethnic ghettos + militarized police = Civil War. History is clear on this point. Do you not remember Serbia? Bosnia? The fucking Ottoman Empire? How about the collapse of the Frankish Empire or the breakup of the Soviet Union and its fun in Chechnya. No. Because you were too busy complaining about wealth inequality, $0.77 on the dollar. Videos of men catcalling women. Woe is me. I can’t afford an XBox One.
Ferguson, Missouri. Coming soon to a city near you. My fucking country is infested with dumbasses.

domain: sociology


text:
lives along the Himalayas in Nepal, northern India, southern China, Afghanistan, Bhutan, and Pakistan (CITES,2001; Shrestha,1989; Sathyakumar,1993; Kattel,1991).
usually lives in forests with moderate to steep slopes (Kattel,1991). is found in oak or fir forests (Kattel,1991) and can be found up to the tree line (usually about 4500 m) in mountains. Plants that may be found in its habitat include birch, rhododendron, blue pine, fir, oak, juniper, grass, lichens and shrubs (Kattel,1991; Green,1987).
looks like a small deer with long upper canines that are visible even when the mouth is closed. It's tail is hairless except for a small tuff at the end (Shrestha, 1989), and it has long "hare-like" ears (Sathyakumar,1993). It has an externally visible musk sac that lies between its reproductive organs and umbilicus (Shrestha, 1989). The opening to the sac lies anterior to the urethra (Shrestha, 1989). A musk deer is about 60cm tall and has a shoulder height of about 20cm (Shrestha, 1989).
Musk deer have a caudal gland at the base of their tails (Green,1985-1987).
The mating season is December to January (Shrestha,1989).
The average lifespan of captive bredis 2.4 years. The average lifespan of wild caught captive is about 7 years. The oldest captive Dwarf musk deer (M. berezovskii) from China had a lifespan of 20 years (Sathyakumar,1993).
is a solitary animal with a "bounding gait" (Sathyakumar,1993). The adult males are probably territorial. One interesting aspect of the species is that they usually defecate (but never urinate) at a latrine, which is a spot designated by M chrysogaster as a "toilet". They always squat when urinating or defecating.
They scent mark by rubbing the caudal gland located at the base of their tail against plants. This leaves a greasy smear on the plant (Green,1985-1987).
is a ruminant (Sathyakumar,1993). It can live on poor quality food. In autumn and winter, it mostly eats forbs and the woody plant leaves of trees and shrubs such as oak and gaultheria. In spring and summer, its diet consists mainly of forbs and lichens (Green,1987).
has a shy nature (Sathyakumar,1993), and is easily alarmed (Kattel,1991). It is a solitary animal (Sathyakumar,1993) that is wary of humans.
The musk is used by humans to make soap, perfume (Myers,1999) and indigenous medicine (Sathyakumar,1993).
CITES Appendix I Afghanistan, India, Nepal, Pakistan
Appendix II Bhutan, China
Thepopulation around the Himalayan area has decreased greatly because of poaching animals for their musk and the destruction of natural habitats both by livestock and by local people who collect bamboo for domestic use (Sathyakumar,1993).
One method of conservingis by extracting the musk without killing the musk deer. However, this method takes more time, and is more traumatizing for the musk deer (Shrestha,1989). Musk deer farms are used for this purpose, but these farms have high mortality rates. The main causes of deaths in these farms are trauma, pneumonia and diarrhea (Sathyakumar,1993).
Some problems that may contribute to the high mortality rate are low quality buildings, poor equipment, lack of adequate money, lack of skilled workers, and the remoteness and cold climate of the farm location (Sathyakumar,1993).
In China, there has been reports of success in cutting down mortality rate in Dwarf musk deer farms. In Fozling Farm, Anhui, the survival of young increased from 50% to >90%. In Ma Er Kang Farm, Sichuan, the young survival rate was 74% from 1959 to 1973 for three hundred and thirty-six Dwarf musk deer (Sathyakumar, 1993).
The reasons for keeping musk deer farms are for musk production, research, reintroduction into wild, and reintroduction to existing wild populations (Sathyakumar,1993).
The caudal gland located at the base of the tail secretes a foul smelling viscous yellow substance (Green,1985-1987).
The musk pod is located only in males, and secretes musk (Sathyakumar, 1993). The musk is of highest quality during mating season which is from December to January. During this time, it is a dark red brown granular powder with a strong smell. During November, April, and June, the musk quality is poorer. It has a pasty appearance, its color is creamy white, and it has little smell (Shrestha,1989). The external musk pod is roughly 4.1cm long, 3.3cm wide, and 4.6cm deep (Shrestha,1989).
Gabriel Gam (author), University of Michigan-Ann Arbor, Ondrej Podlaha (editor), University of Michigan-Ann Arbor.
having body symmetry such that the animal can be divided in one plane into two mirror-image halves. Animals with bilateral symmetry have dorsal and ventral sides, as well as anterior and posterior ends. Synapomorphy of the Bilateria.
uses smells or other chemicals to communicate
active at dawn and dusk
a substance used for the diagnosis, cure, mitigation, treatment, or prevention of disease
animals that use metabolically generated heat to regulate body temperature independently of ambient temperature. Endothermy is a synapomorphy of the Mammalia, although it may have arisen in a (now extinct) synapsid ancestor; the fossil record does not distinguish these possibilities. Convergent in birds.
an animal that mainly eats leaves.
forest biomes are dominated by trees, otherwise forest biomes can vary widely in amount of precipitation and seasonality.
An animal that eats mainly plants or parts of plants.
having the capacity to move from one place to another.
This terrestrial biome includes summits of high mountains, either without vegetation or covered by low, tundra-like vegetation.
active during the night
scrub forests develop in areas that experience dry seasons.
reproduction that includes combining the genetic contribution of two individuals, a male and a female
uses touch to communicate
that region of the Earth between 23.5 degrees North and 60 degrees North (between the Tropic of Cancer and the Arctic Circle) and between 23.5 degrees South and 60 degrees South (between the Tropic of Capricorn and the Antarctic Circle).
Living on the ground.
"CITES-listed Species Database: Fauna" (On-line). Accessed November 19, 2001 at http://cites.org/eng/resources/fauna.shtml.
Green, M. 1987. Diet composition and quality in Himalayan musk deer based on fecal analysis. JOURNAL OF WILDLIFE MANAGEMENT, 51: "880-892".
Green, M. 1985-1987. Scent-marking in the Himalayan musk deer (Moschus chrysogaster). JOURNAL OF ZOOLOGY SERIES B: "721-737".
Kattel, B., A. Alldredge. 1991. Capturing and handling of the Himalayan musk deer. WILDLIFE SOCIETY BULLETIN, 19: "397-399".
Myers, P. 1999. "Moschidae" (On-line). Accessed November 19, 2001 at http://animaldiversity.ummz.umich.edu/chordata/mammalia/artiodactyla/moschidae.html.
Sathyakumar, S., S. Prasad, S. Walker. 1993. Status of captive Himalayan forest musk deer Moschus c. chrysogaster in India. INTERNATIONAL ZOO YEARBOOK, 32: "32-38".
Shrestha, M. 1989. Musk deer Moschus chrysogaster: musk extraction from live deer. JOURNAL OF THE BOMBAY NATURAL HISTORY SOCIETY, 86: "438-440".

domain: biology


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domain: sports


text:
What should have remained a local Sanford, Florida story has now reached international status, trumping anything having to do with the U.S. launching a war with Syria. What the UN really wants is for the U.S. to wrap up its civil rights investigation into the Zimmerman/Martin case. They want to make sure our laws are in compliance with “international norms” regarding race-related crimes. Because everyone knows that Zimmerman, a self-proclaimed white Nazi, murdered a poor black child in cold blood simply because he was black.
“‘We call upon the US Government to examine its laws that could have discriminatory impact on African Americans, and to ensure that such laws are in full compliance with the country’s international legal obligations and relevant standards,’ said human rights expert Verene Shepherd, who currently heads the UN Working Group of Experts of People of African Descent… ‘The Trayvon Martin case has highlighted the importance of the need to review those existing laws and policies that can have a discriminatory effect on the basis of race, as African Americans become more vulnerable to such discrimination.’”
A UN special reporter on racism Mutuma Ruteere reiterated the importance of the U.S. finishing its investigation of Zimmerman:
“States are required to take effective measures to review governmental, national and local policies, and to amend, rescind or nullify any laws and regulations which have the effect of creating or perpetuating racial discrimination wherever it exists.”
The UN also quoted a statistic about supposed hate crimes, saying that “According to the 2011 US Department of Justice Hate Crime Statistics, 71.9 per cent of the total number of victims of hate crimes reported to the nation’s law enforcement agencies were victims of an offender’s anti-black bias.”
I think the key word there is “reported.” The ones that are predominantly reported are white-on-black crimes. What we’ve seen recently is that the authorities don’t really view black-on-white crimes as “racially motivated,” so they don’t get reported.
And never mind this other inconvenient statistic: 84% of white victims are killed by whites, and 93% of black victims are killed by blacks. I guess the United Nations doesn’t care about all the gang-related violence in places like Detroit and Chicago that claims thousands of blacks’ lives. They only care about reminding whites of their inherent “privilege” and about how we all enslaved black people earlier in our country’s history.

domain: law


text:
This post contains Amazon affiliate links.
Welcome to the October Pinterest Challenge! Every month my blogging friends and I get together to share projects inspired by Pinterest. This month I’m sharing my fall decorations for a small front porch. I love decorated front porches. Sadly, my porch is quite small and doesn’t have a lot of room for decor So I’m always looking for ideas that can make a big impact in a tight space.
My porch this month was inspired by the small front porch featured by Crating Chicks. I liked how the simple crate adds dimension to the decor, and I love the addition of a lantern!
This month has been a busy one for my family. My daughters joined their school’s marching band this year, which requires a lot of hours. On Saturdays, we go to their marching band competitions, which take all day. I made my porch decor really simple and easy this year, since I didn’t have a lot of time to shop!
On my porch is a plain wood crate that I stained with a dark walnut wood stain.
On the top of the crate I placed a purple potted mum, as well as a craft pumpkin that I painted with turquoise spray paint. I like how new colors are being used in fall decor these days, in addition to the traditional orange and black. Inside the crate I put a yellow potted mum.
Next to the crate I placed a cute “Home Sweet Home” yard sign that I found at a local store. (I like the sign so much that I didn’t want to leave it out in the rain in the yard.) On the other side of the crate I placed a pretty turquoise lantern.
I like how my simple and quick fall front porch decor turned out! The bright colors make me smile when I arrive at my home’s door — I hope it makes others smile, too!
To join next month’s #pinterestchallenge, click here to sign-up: November Pinterest Challenge
Now let’s see what other things Pinterest inspired! Head over and visit the other hosts to see what they crafted, cooked, built, or tried!
Erlene – My Pinterventures • Gail – Purple Hues and Me
Kristie – Teadoddles • Lynne – My Family Thyme
Joanne – Our Unschooling Journey • Jenny – Cookies, Coffee and Crafts
Shirley – Intelligent Domestications • Debbee – Debbee’s Buzz
Beverly – Across The Boulevard • Julie – Sum of their Stories
Michelle – Our Crafty Mom • Marie – The Inspiration Vault
T’onna – Sew Crafty Crochet • Emily – Domestic Deadline
Laurie – My Husband Has Too Many Hobbies • Alexandra – Eye Love Knots
Leslie – Once Upon a Time & Happily Ever After
Roseann – This Autoimmune Life • Toni – Girl, Just DIY
Kelley – Simply Inspired Meals • Lauren – Mom Home Guide
Debra – Shoppe No.5 • Lydia – Lydia’s Flexitarian Kitchen
Susan – Suzer Space • Habiba – Craftify My Love

domain: design


text:
Alan Barr, Professor of Computer Science, and Peter Schröder, Shaler Arthur Hanisch Professor of Computer Science and Applied and Computational Mathematics, have been elected to the first class of the ACM SIGGRAPH Academy. Professor Barr was selected for his contributions to graphics, primarily for extending computer graphics shape modeling to include physically based and teleological modeling. Professor Schröder was recognized for his pioneering work in geometry processing and multiresolution modeling. The ACM SIGGRAPH Academy is an honorary group of individuals who have made substantial contributions to the field of computer graphics and interactive techniques. These are principal leaders of the field, whose efforts have shaped the disciplines and/or industry, and led the research and/or innovation in computer graphics and interactive techniques. [Full list of academy members]
- June 18, 2019 3:00 PM IQI Weekly Seminar - CANCELED Speaker: Anna Pappa Annenberg 107
- June 20, 2019 11:30 AM IQIM Postdoctoral and Graduate Student Seminar Speaker: Aishwarya Kumar Lauritsen 469
- June 20, 2019 4:00 PM DOLCIT Seminar Speaker: Pierre-Luc Bacon Annenberg 213
- June 21, 2019 12:00 PM DOLCIT Seminar Speaker: Adrian Dalca Annenberg 213
- June 21, 2019 1:00 PM Advanced Deep Learning and Tensor Tutorial Annenberg 105
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IST Meeting of the Minds
A day-long research conference featuring talks, laboratory open houses, and poster presentations by distinguished faculty, graduate students, undergraduates, and researchers from JPL showcasing the latest and most exciting work that is underway in CMS.
A day-long event focused on providing startups and companies with a chance for meaningful interactions with undergraduate and graduate students, providing students with an opportunity to find out more about the breadth of applications for computing and mathematical sciences across industries.
Alumni College: Caltech Computes
A day-long event that will explore the ways in which computational thinking is disrupting science and engineering, and creating entirely new disciplines with "CS+X".
Ten Years of DNA Origami
A symposium to honor Paul Rothemund’s contribution to the field.
Caltech's premier interdisciplinary hackathon.
One Entangled Evening
A night of science and entertainment celebrating Feynman’s quantum legacy.
CDS 20th Anniversary
Control and Dynamical Systems celebrates its 20th anniversary.
Southern California Symposium on Network Economics and Game Theory.
CS 25th Anniversary
Computer Science celebrated 25 years of innovative, ground-breaking research.