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Lost in a skier's dream
Matador Staff
Mar 15, 2017
In times like these, in which man dominates nature so heavily, untouchedness, wilderness and loneliness are more and more rare. In times like these, real adventures threaten to be replaced by virtual experiences in front of computer and TV displays. But they still exist – the magical places. A place with this extraordinary charm is the village of Bakhmaro in Georgia. Well hidden at 2,000 meters altitude in the small Caucasus. Due to a 15 kilometer long pass road and the lack of heavy machinery and snowblowers, this road cannot be cleared of snow from October to May. And that in the midst of a region where snowfalls of incredible proportions are pretty usual.
Shot by WhiteHearts.
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Blog Archives
Vegan Gourmand's Vegan Whole Beans
I just love Mexican whole pinto beans. You know the kind you get at those real authentic Mexican restaurants?
Well, come to find out they are not all Vegan. Some use an animal stock, either chicken or beef. This recipe has the same great taste as the authentic ones but they are totally Vegan.
Vegan Gourmand's Vegan Whole Beans
2 cups dry Pinto beans rinsed well and checked for rocks
32 oz filtered water
1 med. sweet onion coarsely chopped
1 tsp ground cumin
6 Lg garlic cloves pressed or finely chopped
1 tsp salt
½ teaspoon smoked paprika
Combine all ingredients in an electric pressure cooker and cook on high pressure for 40 min.
Then release pressure and stir.
Start pressure cooker again, cook on high pressure for 10 min. more.
Release pressure stir and enjoy.
Note: I sometimes add leftover salsa to my beans before cooking them, as I did in the batch of beans pictured above.
Vegan Gourmand's Quick Mole Sauce
I love mole but most all the authentic moles have 20 or more ingredients and some of them are hard to find and are not Vegan. Well here is a quick mole recipe you can make with just 11 ingredients.
Vegan Gourmand's Quick Mole Sauce
16 ounce can of tomato sauce
2 Tbs chili powder
1/2 tsp onion powder
1/4 tsp garlic powder
1 tsp cumin powder
1 Tbs cocoa powder
2 tsp of creamy peanut butter
4 tsp brown sugar
1 Lg pinches of ground cinnamon
1 pinches of salt
1 cup water
In a med saucepan add tomato sauce, chili powder, onion powder, garlic powder, cumin powder, coco powder, peanut butter, brown sugar, cinnamon, salt & water. Mix well with a whisk until all are combined.
Turn saucepan heat to medium.
Once mole is hot, simmer over med-low heat for 20-30 minutes or until the tomato sauce flavor is gone and the flavors have matured to that mole flavor. Stir frequently to keep from sticking.
Optional: If you would like a thicker sauce you can do the following: In a separate bowl, whisk together the corn starch and a little cold water and then add it to the saucepan, stir until it is fully incorporated into the mole.
Taste and add more salt if desired.
Remove from heat and it is ready to be used over enchiladas or any favorite dish.
Vegan Cheezy Broccoli & Cauliflower Bake
This is a great side dish for any meal.
There are lots of veggies relaxing in a silky, cheezy sauce.
2 cups broccoli, cut into bite-size pieces or larger if you prefer
2 cups cauliflower, cut into bite-size pieces or larger if you prefer
2 cups cashew milk (or other plant milk, I like the creaminess of the cashew milk)
1⁄4 cup flour
1 teaspoon of Onion powder
1 teaspoon Garlic powder
2 tablespoons nutritional yeast flakes
1 teaspoon salt
1⁄4 teaspoon nutmeg
1 cup of the new Daiya Cutting Board Collection Cheddar Style Shreds vegan cheese (it melts way better and gets stringier than the original does, its great on pizza)
Bread Crumb Topping Mixture
2/3 cup Italian seasoned bread crumbs
4 teaspoon extra virgin olive oil
½ teaspoon salt
½ teaspoon garlic powder
Preheat oven to 400°F.
Prepare the bread crumb topping mixture by combining & mixing all bread crumb ingredients together, then set aside.
Cook broccoli and cauliflower in boiling water for 3 minutes, drain and set aside.
Blend in a blender the milk, garlic & onion powder, flour, nutritional yeast flakes, salt, and nutmeg until blended well.
Cook blender mixture, stirring constantly over medium heat in a saucepan until thickened, add Daiya Cheese, lower heat and stir until all the cheese is melted & incorporated. Stir until the sauce is smooth, turn off heat.
Add broccoli and cauliflower to the saucepan and stir until all the veggies are completely covered in sauce.
Pour mixture into a 9 x 9-inch casserole dish.
Sprinkle bread crumb mixture over top.
Bake until bread crumbs are toasted about 10-15 minutes.
Vegan Corn Chowder Recipe
With the cold weather covering almost 2/3 of the country, I thought a hot warm soup recipe was in order. I took the basic Forks Over Knives Recipe for Corn Chowder and I changed up a few things to make this warm creamy comfort food. I hope you all enjoy it as much as I did.
1 small onion, cut into ¼-inch dice (about 1 cup)
6 garlic cloves, minced (about 1 tablespoon)
6 to 7 cups Vegetable Stock
6 cups fresh or frozen corn kernels
1 large russet potato, peeled and cut into ¼-inch dice (about 3 cups)
1 medium red bell pepper, cored, seeded, and cut into ¼-inch dice (about 1 cup)
1 teaspoon finely chopped fresh or dried parsley
1 teaspoon finely chopped fresh or dried thyme
Salt, smoked paprika and freshly ground black pepper to taste
- In a large soup pot or Dutch oven, place the onion, garlic, and 1½ cups of the Vegetable Stock. Cover the pot and bring to a boil over high heat. Reduce the heat to low and simmer, covered, until the onion is translucent, about 10 minutes.
- Add the corn, potato, and 4½ cups of the remaining stock. Bring to a boil over medium heat. Reduce the heat and simmer until the potato is soft, 10 to 15 minutes.
- Transfer 1/2 of the mixture to a blender and blend until smooth. Return to the pot.
- Add the bell pepper, parsley, and thyme. Bring to a simmer and cook for another 10 minutes, until the flavors have blended and the pepper is tender.
- Add salt and pepper to taste. Dish up into soup bowls and sprinkle some smoked paprika onto the soup before serving to give it a slight smokey flavor. Serve hot.
Storage: Cool soup completely and transfer to an airtight container. Store in the refrigerator for 4 to 5 days or in the freezer for up to 1 month.
Vegan 2017 Documentary – Is now out!
The short documentary "Vegan 2017 – the film" is now out.
It gives a year in review for 2017.
Please give it a watch and be encouraged! It is 47 minutes in length.
Edward Alberola Quote
This quote by Edward Alberola is speaking so much truth that I had to share it.
November is World Vegan Month!
Happy World Vegan Month Everyone!!
Here is how you can celebrate
Here is a quote from The Vegan Society page where they give you 10 ways to celebrate.
"People around the world choose to mark World Vegan Month in an array of different ways. You could host a vegan lunch at work or a vegan dinner party with friends; commit to taking part in vegan outreach in your local community; share your favorite vegan recipes on social media or even challenge your friends, family or work colleagues to go vegan for 30 days by taking our Vegan Pledge
However you choose to mark World Vegan Month, be sure to share it far and wide using the #WorldVeganMonth and #WorldVeganDay hashtags on social media and tag us on Facebook and Twitter using the handle @TheVeganSociety, we would love to see what you get up to!"
2017 Veggie Award Results Are Out!
The 2017 Veggie Award results are now out. Here are some of the winners.
Restaurant Of The Year: Virtuous Pie Portland Oregon
Company Of The Year: Elmhurst Dairy New York City
Person Of The Year: Pamela Anderson – Actress
Product Of The Year: Violife
Book Of The Year: Mercy For Animals
Cookbook Of The Year: The Vegan Air Fryer
Non-Profit Of The Year: PCRM
Fav Fine Dining Restaurant: Candle 79 New York City
Fav Casual Restaurant: Gracias Madre Los Angeles & San Francisco
Fav Restaurant Chain: Veggie Grill
Fav Online Store: Vegan Essentials
Fav Vegan City: Portland Oregon
Fav Celebrity: Ellen DeGeneres
Fav Cookie: Lenny & Larry's
Fav Ice Cream: Ben & Jerry's
Fav Milk: Almond Breeze
Fav Chocolate: Endangered Species
Fav Staple Cheese: Daiya
Fav Artisanal Cheese: Miyoko's Kitchen
Fav Yogurt: Kite Hill
Fav Energy Bar: Larabar
Fav Vegan Snack: Earth Balance Vegan Cheddar Flavor Squares
Fav Candy: Justin's Dark Chocolate Peanut Butter Cups
Fav Protein Powder: Vega One
Fav Condiment: Vegenaise
2017 VegNews Veggie Awards, its time to vote!
That's right folks it's that time of year again where you get to vote for your favorite Vegan products, restaurants, people, and organizations.
It only takes a few minutes and it's a great way to pay homage to your favorites, not to mention the opportunity to win some great prizes.
Click on the banner above to get started or click here.
How Your Body Transforms On A Vegan Diet Shown Week By Week
Here is a great video showing how your body changes week by week on a Vegan diet.

subset: commoncrawl


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There's a movie by that name, right? Two Weddings and a Funeral? No, it's Four Weddings and a Funeral. But still, how fascinating to live as a movie, or part of one. Perhaps that statement is more poignant and profound than meets the eye. Moving our attention deeper, down to our heart, breathing into our belly, we glimpse our life in the script of a movie. We gain a whisper of knowing that our life is, in many ways, just a scene on the screen.
When I sit down to write this monthly connection, sometimes I have a clear idea, sometimes there is an inkling of a thought, and sometimes, like today, those ideas and thoughts dissolve into just this moment.
Earlier this morning I sat quietly in sweet stillness with my journal, with my iPhone, with my colored pencils, and a short sip of espresso. (I am beginning a 5-day intermittent fast that allows for one bit of caffeine per day.) Following a thread from my junk email, I happened upon this quote from Peter Brook.
"it takes real skill to stay silent.
a large part of our excessive, unnecessary manifestations come from a terror
that if we are not somehow signaling all the time that we exist,
we will in fact no longer be there…"
Feeling deeply into the above quote, I see, feel and taste the idea of belonging and my fears of not belonging. I see a large part of excessive social media, phone calls, zoom interfacing, conversation comes from a terror that if I am not sharing, reporting, looking and responding to ….. (FB posts, emails, phone calls, the newest free course, insert your favorite) – that I see you, I feel you, I resonate with you – I will in fact no longer belong, be connected to you. Money activates these centers too. Bookmark this point.
Staying silent, being home in my heart, the realization of belonging is ever present and ever bright. But this year, with the urgency and demands of life on that screen, I confess to have lost faith and touch with being and belonging – even as the truth of connection slaps me in my face daily or each minute. Two weddings and a funeral are, in fact, my year. My father passed away in April. My niece married in June. My nephew married in November. In between those events I add … son joining our firm Colman Knight, purchase and ongoing renovation of cottage, devotion to my mother's well-being, attention to my father's estate affairs, a teacher's physical decline, another teacher's mental decline, just to name a few. My opportunities for silence, space and stillness evaporated. Or, truer, my ability to seek refuge in the real skill of staying silent is weak. My terror of not belonging reaches out, beyond "me", to you, and others. I am reminded this way is not sustainable, ever.
Today, I wonder into these questions and invite you to do the same, if there is a spark of resonance.
What is it like to just be me?
How do my days unfold in chronic wonder?
Flexing in my totality of goodness, who am I with you? Who are we together?
How do all of the "we's" love? Express? Create? Contribute?
What's worth our attention? And how shall we engage action?
How does money weave with these wonderings?
Let's begin again, and again and again.
Juicy surprises are in the works for Somatic Finance. Glimpse and savor the sections below to learn more. We will experience more funerals and more weddings. Our life is a movie. The precious part is we are the directors, writers, actors and producers. Are you producing a comedy, tragedy, drama or mystery?
A sweet and deep bow of gratitude for you and your presence here. I am connected. I belong. And so do you. Let's choose to remember always.


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Thursday, 21 February 2013
Morning Textures
Moffat Beach
© Sunshine Coast Daily Photo - Australia
Another City Daily Photo
2 comments
- Karen said...
You know I wouldn't have noticed if you hadn't mentioned the colour of the sand. The image looks really great to me.
- 21 February 2013 at 11:39
- Jim said...
Beautiful shot.
Sydney - City and Suburbs
- 21 February 2013 at 13:20
Subscribe to: Post Comments (Atom)
© Sunshine Coast Daily Photo
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I've been stamping since I got invited to a Stampin' Up party back in 1994; just as I was about to move from Virginia. I had no intention of getting into yet another craft!! My intent on attending was to purchase a birthday gift for the woman who was hosting the party. Well, that went out the window and here I am today, surrounded by all kinds of stamping "stuff" and a bedroom which I lovingly call my "studio". I have grown and grown – thanks in part to tutorials and challenges on Split Coast Stampers - as well as SpeedyTV, and I am still growing.
You can find Vicky's Cool Whip Clouds tutorial here on SpeedyTV. While you're at it, why not check out her blog here.


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Research Project
Grant-in-Aid for Scientific Research on Priority Areas (A)
The Grants-in-Aid for Scientific Research on Priority Areas No. 09281104 was issued from 1997 to 2000. During the period, we organized a research group with scientists who are considered to be most active in the research field. Atherosclerosis is correlated with a variety of vascular disorders such as coronary heart disease. We therefore placed our goal on elucidation of the molecular mechanism of atherosclerosis and exploration of novel therapeutic strategies. To conduct investigation, the members were divided into 3 subgroups, i.e. Group 1, 2, and 3 with different subject of study as follows. We also formed a Central Committee to coordinate and facilitate the research activity of the subgroups.The Study Group 3 studied endothelial dysfunction and blood cells. There has been accumulating evidence that atherosclerosis is initiated by endothelial dysfunction due to a variety of biological insults. We therefore formed this subgroup to explore the key molecules involved in those events. We discovered novel scavenger receptors such as LOX-1 and SRPSOX. Furthermore, we revealed that adipose tissues produce cytokines abundantly. Among those cytokines, adiponectin has been shown to play an important role in atherogenesis and insulin resistance.In conclusion, we have made a successful progress in atherosclerosis research, which will be translated into clinical application in the near future.
Published: 1997-03-31
Modified: 2016-04-21
User Guide
Terms of Use
Attribution of KAKENHI


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Naradaparivrajaka Upanishad
Die Naradaparivrajaka Upanishad (IAST: Nāradaparivrājaka Upaniṣad) ist ein mittelalterlicher Sanskrittext mit 9 Kapiteln, der dem Atharva Veda zugeordnet wird. Sie gehört zu den kleineren upanishaden und wird auch zu den Sannyasa Upanishaden gezählt.
Die Upanishad beginnt mit einer Geschichte von Nārada, der im mythischen Naimisha - Wald meditiert. Er ist von anderen Weisen begleitet, die ihn bitten, ihnen den Weg zur Moksha zu zeigen.
Nārada erwiedert, ein Mann solle zuerst die samskaraas vollständig durchführen, die Studien des Brahmacharya abschliessen, dann ein Haushalter oder Grihastha füt 25 Jahre sein, ein Vanaprastha oder zurückgezogener Einsiedler für weitere 25 Jahre, sich dann vollständig zurückziehen, wenn er keine Verhaftungen mehr hat, und ruhig und ohne Feindschaft gegen irgendjemanden sein.
Die Upanishad behandelt verschiedene Arten des Pranava und in Kapitel 8 die vier Zustände des Wachens, Träumens, Tiefschlafs und des reinen Bewusstseins des Turiya.
Textauszug - Aiyar
.... Then Narada asked Parameshthi (Brahma) to enlighten him, who had surrendered himself to Him, about samsara-taraka (or that taraka or Pranava which lifts one out of samsara).
Assenting to which, Brahma begafn thus : " Omkara that is Brahman is the vyashti (individual) and the samashti (cosmic). What is the individual ? What is the cosmic ?
Brahma-pranava is of three kinds, samhara- (destructive) pranava, srshti- (creative) pranava, and ubhayatmaka (belonging to both) pranava, as being of two forms, internal and external. (It is also eight :) Antah- pranava, Vyavaharika-pranava, bahya-pranava, arsha-pranava, ubhayatmaka or virat-pranava, samhara-pranava, brahma-pranava, and ardhamatra pranava. Om is Brahman.
Know that the mantra of the one-syllabled Om is Pranava. It has the eight differences of akara, ukara, makara, ardhamatra, nada, bindu, kala, and s akti. Know it is not four (alone) . Akara is associated with ten thousand limbs ; ukara, with one thousand limbs ; makara with one hundred limbs ; ardha matra is of the nature of endless limbs. That which is saguna (associated with gunas) is virat- (preservation) pranava; that which is nirguna (not associated with gunas) is samhara- (or de struction) pranava; that which is associated with gunas and is not so associated, is utpatti- (or origination) pranava. Pluta (the elongated accent) is virat : plutapluta is samhara. The virat- pranava is of the form of sixteen matras and is above the thirty- six tattvas.
The sixteen matras are thus : Akara is the first matra; ukara is the second; inakarais the third; ardhamatra is the fourth ; nfida is the fifth ; bindu is the sixth ; kala is the seventh ; kalatita is the eighth ; s anti is the ninth ; s antyatita is the tenth ; unman! is the eleventh ; manonmani is the twelfth ; purltati is the thirteenth ; tanumadhyama is the fourteenth ; pati is the fifteenth ; para is the sixteenth. Then (again) having sixty-four raatras and their division into the two, Prakrti and Purusha and resolving themselves into the onehundredand twenty-eight differ ences of matras, it becomes saguna and nirguna. Though Brahma- pranava is one only, it is the substratum of all, the support
of the whole universe, of the form of all aksharas (letters), time, Vedas, and S iva. This Omkara should be sought after, that is mentioned in the Vedas of the nature of the Upanishads. Know that this Otnkara is the Atma that is indestructible during the three periods of time, past, present, and future, able to confer sal- ( vation and eulogized by v Brah ma-sound (Vedas).
Having experi enced this one Om as immortal and ageless, and having brought about the Brahma-nature in this body, become convinced that your Atma, associated with the three bodies, is Parabrahman. Through Yis va and others (viz., Taijasa, Prajfia, and Turya) in order, the realisation of Parabrahman should be attained, since Atma is of four kinds through his identification with, and the enjoying of, the gross as well as the enjoyer of the gross, the subtle as well as the enjoyer of the subtle, and through his identification (with the third body) enjoying bliss in the fourth. He has four feet.
The one presiding over the waking state is gross; and since he is the enjoyer of Vis va (the universe), he becomes the sthula-prajfia (gross consciousness). He has nineteen 1 facets and eight parts. He is pervading everywhere and the Lord. He is the enjoyer of the gross and is the chaturatma called Vis va. He alone is the Purusha called Vais vanara. He alone is Vis vajit (the conqueror of the universe). This is the first foot. When this Lord at tains the dreaming condition, he is the sukshma-prajna (subtle consciousness).
O conqueror of all, he is the one having eight limbs, and there is none else. He is the enjoyer of the subtle and is chaturatma, named Taijasa and the protector of elements. He alone is the Hiranyagarbha, presiding over the gross (or subtle matter rather). He is said to form the second foot. Sushupti (or the dreamless sleep) is that state where one sleeps without any desire and where one sees not any dreams. The one identified with this dreamless sleep is Prajnana-ghana, is blissful, of the nature of eternal bliss and the Atma in all creatures; yet he is enjoyer of bliss, has chetas (consciousness) as his (one) foot, is all-pervading, indestructible,
The nineteen are the five organs of sense, the five organs of action, the five pranas, and the four of the mind.
chaturatma and the Lord, and is named Prajna, the third foot. He alone is the Lord of all, the knower of all, the subtle- thoughted, the latent one, and the cause of all creation. He alone is the origin and the destruction. These three (states) are obstacles to all creatures obtaining (the fina 1 !) peace. As is svapna, so is sushupti, it (also) being said to be illusory. The chaturatma, the fourth, as he is Sat, Chit and Ekarasa (the one essence), ends as the fourth and follows (upon the heels of each of the above states), is the knower of the means of vikalpa- jfiana and is the anujnata (the one following knower). Having known them, and known as maya the three vikalpas of sushupti, svapna and antara (the inner), even in this state, is he not (to be known as) Sat-Chit-Ekarasa ?
This shall be expressed as differentiated thus : It is not even the gross prajna ; nor is it the very subtle prajna; nor is it prajna itself (of the causal body) : muni neither is it the trifling prajna ; nor is it the non- prajfia; nor is it the dual prajna; nor is it the internal prajna, though it is without prajna; it is Prajnana-ghana. It can never be known by the organs ; nor it can be known by the reason; it cannot be grasped by the organs of action. It can not be proved. It cannot be reached by thought. It cannot be proved by analogy.
It can be realised by Self-realisation alone. It is with the waking state, etc. It is the auspicious, with changes, without a second. Such a one is thought to be Turya. This alone is Brahman, Brahma-pranava. This should be known. There is no other Turya. To the aspirants after salvation, it is the support, like the sun everywhere ; it is the Self-light. As it alone is Brahman, this Brahma-Akas is shining always. Thus is the Upanishad."
Narada asked : " Who is Brahma-swarupa ? " To which Brahma replied thus : " Brahma-swarupa is thus : Those who know that he (Brahman) is one and I am another are only pas us (animals). The real pas us (animals) are no animals. The wise man who knows Brahman thus (as himself, and himself as Brahman) escapes out of the mouth of death. There is no other path to salvation.
- Thirty minor Upanishads,Narayanasvami K. Aiyar, 1914
- Olivelle, Patrick (1992). The Samnyasa Upanisads. Oxford University Press. ISBN 978-0195070453.


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Perandor (3.5e Deity)
From D&D Wiki
Perandor, the Dark Shadowmage[edit]
|Greater Deity
|The Darkskull
|Home Plane:
|Shadowrealms of Living Spells
|Chaotic Neutral
|Magic, War, Power, Necromancy
|Clergy Alignments:
|Chaotic Non-Good
|Spell, Deathbound, War, Magic
|Favored Weapon:
|Staff of the Magi
|This page needs an image. If you are an artist, or know of any image that would fit this page, please upload a picture and add it.
Perandor is a dread necromancer and tainted scholar, relying on his magical powers to keep him in check. He commands his followers to reanimate a corpse once a month, or lose all their divine favor. However, he reduces the cost for animating corpses by 5 HD's worth of onyx gems.
Clergy and Temples[edit]
Perandor has hidden citadels all over the world, usually in secluded places. His clerics and dread necromancers usually cloak the area with spells of misdirection, so that no adventurers will seek them.
Back to Main Page → 3.5e Homebrew → Deities → Greater


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Get Your Stocking Stuffed by The 4 Kings of Kelt
Who's ready for some Yuletide Music with a Celtic Flair? Just re-issued — our 1997 A Celtic Christmas album featuring a sweet yet zesty blend of Scots pipes, Irish whistle, Blues guitar, Gospel keyboard – check it out here. ~~~ ~~~ ~~~ … Continue reading
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Search results
Ten Counties on WBC Scout List as Statewide Numbers Begin to Decrease
Ashtabula, Fulton, Geauga, Henry, Huron, Lucas, Sandusky, Wayne, Williams and Wood. A total of 27 counties ... the majority of monitoring counties. Despite the overall decrease in WBC numbers, ten counties are ... Monitoring for WBC moths will continue in many counties until the end of August. Figure 1. Average Western ...
Western Bean Cutworm Numbers Continue to Increase
scouting for egg masses (Figure 2). These counties include Fulton, Geauga, Henry, Huron, Lucas, Sandusky, ... counties across Ohio. A total of 26 counties monitored 88 traps, resulting in 636 WBC adults (7.2 statewide ... average moths per trap) (Figure 1). Counties that are averaging more than 7 moths per week should begin ...
4-H Club Resources for COVID 19 Changes
in-person 4-H club meetings, activities and other events may be suspended at any time if local, state, ... in person if Ohio 4-H Planning Guide for In-Person Meetings & Events guidelines can be followed. ... The Ohio 4-H Planning Guide for In-Person Meetings & Events guidelines provide expectations for ...
August 28 Deadline for Coronavirus Food Assistance Program is Fast Approaching
https://www.farmers.gov/cfap, including an on-line application as well as a link to find your local county FSA office. More ... prices quickly reversed course, and what seemed to be improving prices did not materialize in on-farm ... outbreak. Sign-up began at your local Farm Service Agency (FSA) office on Tuesday, May 26 and continues ...
August 28 Deadline for Coronavirus Food Assistance Program Fast Approaching
on-line application as well as a link to find your local county Farm Service Agency office. More detailed ... quickly reversed course, and what seemed to be improving prices did not materialize in on-farm milk ... began at your local FSA (Farm Service Agency) office on Tuesday, May 26 and continues through August 28, ...
Record Negative Producer Price Differential Slams June Milk Checks, Why and How?
Class III prices made an amazing recovery in June, rising from May's bitterly low $12.14/ cwt to the ... rule change. A change in how Class I price is calculated went into effect in 2019, driven by ... things happen when there is a large spread between Class III and Class IV prices and prices rise ...
Drought Conditions Expand but Some Relief Ensues
soil moisture and lowered stream flows. If you are seeing drought impacts in your area, consider ... likely to bring some continued minor improvement to drought conditions throughout Ohio. Figure 3: ...
COVID-19 Updates for the AEDE Community
best we can, as well as support your work, and the success of our students. Visit the CFAES Return to ... virtual and in-person classes to ensure student, staff, and faculty safety and provide the best learning ... learning experience, regardless of delivery method, many of AEDE's classes will be virtual or hybrid for ...
Calcium, Magnesium, and Sulfur
excessive uptake (i.e., luxury consumption) of K and improve Mg uptake. This can sometimes result in fields ...
Milk Prices, Costs of Nutrients, Margins and Comparison of Feedstuffs Prices
University Milk prices In the last issue, the Class III futures for May and June were at $12.22 and ... $17.52/cwt, respectively. The Class III component price for May closed just slightly lower than predicted at ... was $4.53/lb, leading to a higher Class III milk price. The Class III future for July is nearly double ...


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|Name of Core Components
|Country of Origin
1. ALPHA Inverter Co., Ltd. was conceived in 2000. As the first professional AC-drive producer in China, we have created an average annual rate of growth of over 50%. Nowadays, our company has become one of the TOP 3 manufacturers within Chinese inverter industry.
2. The modern-day ALPHA has a workforce of 316 employees, over 70 percent of which are senior technicians. Among them, 45 employees have received a PhD or master's degree. Moreover, our board of directors persists in devoting 10% of annual sales to the R&D field. So far, we have obtained 15 patents for utility models and appearance design, as well as 25 software copyrights. We are recognized as the National High-tech Enterprise by the Chinese Ministry of Science and Technology ... << More


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A British singer songwriter – Grammy-nominated for her work on Beyoncé's 'Lemonade', notably for 'Freedom' featuring Kendrick Lamar – is holding an event next month to help other young female songwriters break into the industry.
Carla Marie Williams – who's also worked with Britney Spears, Sean Paul and Craig David – is founder of Girls I rate, a female-led organisation linked with artists like Ray Blk, Lily Allen and Ms Banks, working to empower women.
Recent research carried out by the PRS Foundation (PRS) found that just 16% of the UK's composers and songwriters identify as women; Carla's weekend-long event, taking place in London on 10th and 11th November, hopes to address this.
"I want to give girls across the world as many opportunities as possible that I didn't have growing up," she said. "I want to encourage more women to pursue their dreams and help increase the imbalance of female songwriters and producers at PRS."
The two-day Girls I Rate Arts Academy Songwriting Weekender – open to female songwriters aged between 16-30 – will also be joined by songwriters Tanya Lacey and Simon Smith, who've worked with the likes of Rihanna, Emelie Sande, Will.i.am, Paloma Faith, and jungle / drum 'n' bass pioneer Shy FX.
The 15 successful programme applicants will be equipped with the fundamental practices, techniques and skills required to work as songwriters, and the weekender will be rounded off with a mixer hosted by Rinse FM and 1Xtra's Nadia Jae – a chance for the girls to network and have their songs played to A&Rs, publishers, managers and other industry representatives.
Apply for the weekend event via the Girls I Rate website.
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Traditionally weddings tend to take place in the brides home town or local area. But in recent years it has become more and more popular to get married further away from home. For whatever reason you may have, getting married away from home can be exciting and who doesn't love to travel especially on the happiest day of their lives.
Why get married away from home?
If you're a fan of tradition and your home comforts then getting married away from your local area isn't likely to appeal to you too much. But for those of us who love to travel it can sound like the perfect wedding.
Just imagine incorporating travel into the best day of your life. Spending time with family and friends while discovering a whole new area. This doesn't necessarily mean abroad either. There are some beautiful areas in the UK if you don't fancy a plane ride to get to your destination.
Where to get married?
Choosing the place to say I do is something that only you can decide on. Would you like something traditionally British like this location on Bijou Weddings? Maybe a luxurious tropical retreat in the Bahamas? Or you might choose an Italian city wedding among the cobbled streets. The opportunities on the location really are endless!
How easy is it to plan a wedding away from home?
Planning a wedding in a location miles away from you can sound really difficult and confusing. But there are so many companies and package deals now that this just isn't the case. Planning a wedding from afar has never been easier.
Depending on the type of outfits you would like to wear the only things you would really need to travel with are your dress, the bridesmaids dresses and the mens suits. Everything else can be bought locally or ordered in ahead of time. If you're more of a casual dress couple and have no intention of a fancy dress and suits then you'll have even less to think about. You might even have envisioned your wedding to include you, your partner and your guests all barefoot on the beach in beach wear.
Should we use a wedding planner or not?
If you thought that planning your own wedding without the help of a wedding planner would save you some money you might be mistaken. Of course you will have to pay a wedding planner to plan your wedding but they might have contacts that you don't have. You wedding planner may be able to get you things at cost price and actually end up saving you money.
Considering using a wedding planner is definitely worth a thought or two. Even more so if you are not a naturally organised person yourself. If you struggle to organise a night out with friends then you should probably go ahead and just book a wedding planner. Having said that if you have any friends or family that you know love to plan parties it may be worth asking a massive favour of them.
This is a collaborative post.


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In desperation on the last day before Christmas I visited a local artist who had supplied beautiful presents before, one of which was sadly destroyed in a recent storm. I didn't find what I was looking for till he pointed to a sculpture hidden in the back of his studio. I liked it immediately. The artist had called this pear "Adam's Temptation".
I liked it even more when I heard the backstory. The temptation had obviously been to much for some and he was asked to remove the piece from the major gallery in Matakana.
However you look at it
this raises some interesting issues.
One is that of censorship of art.
The other is the problem some New Zealanders obviously still living in Victorian times are having with nudity. How is it that the naked body – nature itself – can be so offensive even if some are less beautiful than others. The uproar and possible prosecution of naked swimmers or sun bathers especially in the presence of children. What are we teaching our children if we make them feel that naked bodies including their own are something naughty.
And don't get me started on banning breastfeeding mothers to the toilet because a little bit of nipple might be exposed to the public. It makes me angry.
How do these sick, prudish, moral guardians survive in the age of film, television and the internet. They need serious help before they can be let anyway near our children.Tweet ##NZPOL


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We live in Minnesota. In 1995 the city bought property from my father to construct a levee to satisfy the Army Corp of Engineers which split the property in 2 pieces. The city signed an Easement to build access which crosses the levee but never did it because my father passed away. The property was in the step mother's name until she deeded it over to me and my siblings. A conservancy is buying the property that we can not get to and the city is compensating us for breach of contract for providing the access road for over 20 years. Is this money we will be receiving taxable?
why would you think it is not taxable?
the sales price to the Conservatory, less the cost basis is profit and taxable
the city compensation is straight up income.
I could argue as follows --- (a) the father's property was condemned/sold to the city for a sum plus some work on the part of the city to make the remainder property useful ( via an access corridor). His sales price and therefore the tax of gain included recognition of this contractual performance. Thus the transaction inits entirety was taxed. (b) the father died and the beneficiary ( mother ) got a step-down to FMV; (c) the children took over the property through gift but could not use the property because city did not allow access -- thus their property essentially was not available to them i.e the whole property was un-useable -- their right to enjoy the property was constrained. Now when the city pays them for non-use of their property in-lieu of rectifying the defect, ( for the harm they caused for years ), this is not income -- it is payback for sufferance. No ? Perhaps you need to see a lawyer . To me treating me this compensation ( for damage ) as income would be harm on top of harm -- No ?


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APRIL 10, 2014: In the Sunday, March 23, 2014 edition of the Boston Globe, conservative opinion columnist Jeff Jacoby penned an op-ed titled "Public pensions are eating taxpayers alive".
By Kathy McCabeGlobe Staff April 10, 2014Winchester selectmen have scaled back a plan to make retirees pay half the cost of their health insurance premiums, a plan that ignited a backlash from retired town workers.The Board of Selectmen now proposes increasing premiums to 30 percent for people who retired before 2004, and phasing in the plan over an 18-month period, starting July 1. Later retirees would still pay 50 percent of their premiums, but it would be phased in over two years, according to the proposal.
MARCH 27, 2014: On behalf of our members, the Association offers the deepest condolences to the families of Boston Fire Lt. Edward J. Walsh and Firefighter Michael R. Kennedy, as well as to the Boston Fire Department. The two members of Boston Fire Fighters Local 718 were tragically lost on Wednesday, while fighting a nine-alarm fire on Beacon Street in Boston's Back Bay.
MARCH 6, 2014: State and municipal retirees, receiving their health insurance through the state's Group Insurance Commission (GIC), received welcome good news on Wednesday – health insurance premiums for FY15 will remain largely unchanged.
We've been receiving calls and emails from Boston members who receive an accidental (work related) disability pension. They were surprised to see that the federal income tax statement they recently received from the Boston Retirement Board, known as Form 1099-R, was totally different from the forms they received in previous years.
FEBRUARY 21, 2014: Heeding calls from retiree advocates (including our Association) and Democratic Leaders, President Obama has dropped a provision from his federal budget proposal that would have used the chained CPI to calculate Social Security COLAs.
By REID J. EPSTEIN and EDWARD-ISAAC DOVERE | 2/20/14 1:34 PM EST Updated: 2/21/14 5:36 AM EST
President Barack Obama is done pretending that he'll get any new budget cooperation from House Republicans.
His proposed 2015 budget, due to be released next month, will stick to the economic strategy the White House has laid out without the compromise suggestion he floated last year, White House officials said Thursday.
FEBRUARY 19, 2014: Annual open enrollment for the state's Group Insurance Commission (GIC) is set to run from April 9 – May 7, 2014. If you receive your insurance through the GIC this is your opportunity to make changes to your insurance coverage for the coming fiscal year.
Argue Before State's Highest Court
February 5, 2014: For more than 6 years, we've been reporting on the activities of the Middleborough Retiree Insurance Group (MRIG) which organized back in '07. "Our Association has a tradition of working with local retiree groups, particularly on the issues of municipal health insurance and local pension COLAs (Cost-of-Living Adjustments)," according to President Valeri. "That relationship includes Middleboro.
Social Security & Medicare Central Issues
FEBRUARY 1, 2014: This past week Association President Frank Valeri and Legislative Liaison Shawn Duhamel met with members of the Massachusetts' Congressional Delegation in Washington, DC.
Valeri and Duhamel were in the nation's capital to attend the annual legislative conference of the National Conference of Public Employee Retirement Systems (NCPERS), to which the Association belongs. They met with the offices of all nine members of Congress, as well as Senior Senator Elizabeth Warren.
By Beth HealyGlobe Staff January 22, 2014
The Massachusetts state pension fund posted a 15.2 percent investment gain for 2013, as strong markets helped the fund's managers add $7.9 billion to the retirement accounts of public employees.
US stocks provided the biggest boost to the fund, climbing 33.7 percent during the year, followed by investments in private equity, which rose 21.1 percent.
Includes 3% COLA & Funding Schedule Changes
JANUARY 22, 2014: Within the final budget proposal of his eight-year tenure as governor, Deval Patrick chose to include two provisions of critical long term importance to public retirees and surviving spouses.
As he has consistently done in the past, the governor has recommended a full 3% cost-of-living adjustment (COLA) for state and teacher retirees who retired before July 1, 2013. Like 2013, the new COLA will be paid on a base of $13,000.
JANUARY 2, 2014: The Joint Committee on Public Service is off to an aggressive start early in 2014 with a public hearing scheduled for Tuesday, January 7th at 11:00 AM.
Tuesday's hearing will focus on legislation impacting group classification of active employees. As members know, Massachusetts' public retirement is organized into four groups (1-4), which are set in statute by the legislature.
DECEMBER 10, 2013: The Association's Ralph White has been reelected to a new 3-year term on the State Retirement Board. White topped the ticket in a landslide, winning by more than 11,000 votes.
He received 79% of the total votes cast by the members of the State Retirement System. White has served on the Board since 1996.
DECEMBER 4, 2013: A federal bankruptcy judge has granted approval for the City of Detroit to move forward with its bankruptcy protection plan – a plan that will include cuts to the pensions of existing city retirees and future benefits of active employees.
NOVEMBER 21, 2013: An article in today's DCDB Daily Free E-Newsletter says that Massachusetts Treasurer and Receiver General Steve Grossman, Chairman of the Pension Reserves Investment Management (PRIM) Board, manager of the state's more than $54 billion Pension Reserve Investment Trust (PRIT), has lodged support for mission-based investing focused on climate change.
NOVEMBER 12, 2013: Hundreds of public employees from across all walks of state and local government made their concerns known at a packed public hearing on retiree healthcare reform on October 31st.
The hearing, on H59, was held before the Joint Committee on Public Service. Chaired by Senator William Brownsberger (D-Belmont) and Rep. Aaron Michlewitz (D-North End), the Public Service Committee is now conducting a thorough review of the bill.
No Increase For Medicare Part B Premium
OCTOBER 30, 2013: Today, the Social Security Administration announced that the Consumer Price Index (CPI) for the previous 12 months (September '12 to Oct '13) is 1.5%. The announcement follows recent reports by the Federal Reserve detailing historically low inflation.
OCTOBER 28, 2013: Arguably the biggest issue to come before the legislature during the current two-year session reenters center stage on Beacon Hill this week, when H59, the much watched Retiree Healthcare Reform proposal, receives a public hearing.
OCTOBER 18, 2013: On Tuesday, the Joint Committee on Public Service will conduct a public hearing that includes legislation filed by the Association relative to disability retirement.The bills, filed on our behalf by Representative Tacky Chan (D-Quincy), propose to improve the ordinary disability benefit for future retirees, as well as alter the oversight of disability retirees.
OCTOBER 1, 2013: Today marks the nationwide kickoff of the new health insurance exchanges, the main component of the federal Affordable Care Act (ACA), commonly called Obamacare. Between now and December 15, individuals can sign up for one of the health insurance plans being offered through their state's exchange, with insurance coverage
SEPTEMBER 13, 2013: Yesterday, the 18-member Executive Board of the Mass. Association of Contributory Retirement Systems (MACRS) unanimously voted to oppose legislation that would require the State's Pension Fund Board (PRIM Board) to divest all holdings in the Fund, which include fossil fuel such as oil, gas and coal.
SEPTEMBER 11, 2013: Last night, the Haverhill City Council tabled a request by the Haverhill Retirement Board that would have raised the cost-of-living base for its retirees.
The initiative, voted 3-2 by the Retirement Board, would have increased the base amount, which is currently $12,000, by $1,000 per year for each of the next three years, capping at $15,000 in 2016.
SEPTEMBER 10, 2013: Beginning this December, our Association will have a new President.
In something of a surprise to many members, at last Friday's Annual Meeting and biannual election, President Ralph White announced that he was not seeking election for a new two-year term.
SEPTEMBER 9, 2013: Although no longer President of the Association, Ralph White will remain active in the public retirement arena.
His first chore is to win reelection to the State Board of Retirement for a new three-year term beginning in January. The election is now underway.
Since 1968 the Retired State, County and Municipal Employees Association has been the leading voice for Massachusetts public retirees and their families. Join with our 52,000 members as we continue the fight.
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2 Corinthians 4:2 " But we have renounced the hidden things of shame, not walking in craftiness nor handling the word of God deceitfully, but by manifestation of the truth commending ourselves to every man's conscience in the sight of God."
A dishonest, deceitful disposition is, under given circumstances, cunningly leaving a better impression of oneself by lack of truth, honesty and trustworthiness; to objectively get what one wants by manipulating the truth. It's false humility. Continue Reading


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Some smaller misc items to share this week:
- Finally! Luxury inter-city bus company MegaBus is coming to the Texas Triangle, with service from Houston to Dallas, Austin, San Antonio, Galveston, and New Orleans. They pick up right at Polk and Travis downtown. I do think they're making one mistake, though. On the Dallas route, they need a pickup-dropoff point on the far north side (maybe The Woodlands mall or Greenspoint mall?), and on the Austin and San Antonio routes, they need one on the far west side (maybe Katy Mills mall or City Centre?). People living on that side of town don't want to come all the way downtown to just turn around and go the opposite direction, whether picking up or dropping off.
- On the NYT list of 32 innovations that will change your tomorrow, Houston's biggest need is definitely #6, widespread adaptive cruise control. Simulations indicate that once 25% of cars have it, congestion could be reduced 20-40%!! That is an incredible improvement, and imagine what it might be once most cars have it?
Heck, there may even be a national cost-benefit case for the feds requiring it just like they do for the CAFE standards (in terms of time and gas saved). It's possible to imagine that Houston may have far less traffic congestion by the beginning of the next decade even without much in the way of substantial capacity improvements, which, sadly, may very well be the case with TXDoT's budget straight-jacket.
- Texas dominates the Forbes list of best cities for jobs. Houston is #2 behind Austin.
Instead of government, the big drivers of growth now appear to be three basic sectors: energy, technology and, most welcome all, manufacturing. Energy-rich Texas cities dominate our list — the state has added some 200,000 generally high-paying oil and gas jobs over the past decade — but Texas is also leading in industrial job growth, technology and services. In first place in our ranking of the 65 largest metropolitan areas is Austin, which has logged strong growth in manufacturing, technology-related employment and business services. Houston places second, Fort Worth fourth, and Dallas-Plano-Irving sixth. Another energy capital, Oklahoma City, ranks 10th, while resurgent New Orleans-Metairie places 13th among the largest metro areas.
- Houston is the no. 3 city in the U.S. with 25 Fortune 500 companies.
- Houston ranks #8 on cities for new grads.
- Thank God Houston doesn't have to deal with this sort of land-use crap… (hat tip to John)
Finally, embedded below is a pretty cool history of Houston annexation video. I laughed out loud so hard at the 1836 map note "the freeway network is for reference purposes only – it did not exist at the time." Revised Houston history: "the Allen Brothers discovered the confluence of 3 bayous and a mysterious network of roads created by an ancient civilization (or aliens?), and decided it seemed like a good place to found a city… even though they recognized the long-term inadequacy of the 290 corridor…"


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Little is known about the immune response to noroviruses. To elucidate the immunobiology of norovirus infection in humans, 15 volunteers were challenged with Snow Mountain virus (SMV), a genogroup 2 norovirus. We assessed the cellular and humoral immune response and infection by analyzing stool, serum, saliva, and peripheral blood mononuclear cell (PBMC) responses pre- and postchallenge. In contrast to Norwalk virus (NV), SMV infection was not dependent upon blood group secretor status. Nine of 15 volunteers were infected and showed a ≥4-fold increase over the prechallenge anti-SMV serum immunoglobulin G (IgG) titer, mostly subclass IgG1. Although serum IgG elicited by SMV infection was cross-reactive with Hawaii virus (HV), another genogroup 2 norovirus, salivary IgA was less cross-reactive. Neither SMV-elicited serum IgG nor salivary IgA cross-reacted with NV, a genogroup 1 norovirus. Significant increases in serum gamma interferon (IFN-γ) and IL-2, but not IL-6 or IL-10, were noted on day 2 postchallenge. For the majority of volunteers, both infected and uninfected, PBMCs stimulated with norovirus virus-like particles secreted IFN-γ and other Th1 cytokines, suggesting previous norovirus exposure in most volunteers. Like the IgG antibodies, the SMV-activated T cells were cross-reactive with HV but not NV. IFN-γ production was dependent upon CD4+ cells, consistent with a predominant, but not exclusive, Th1 response. To our knowledge, this is the first report characterizing T-cell and cytokine responses following live norovirus challenge.
Norovirus infection is common among individuals throughout the world, regardless of economic status, resulting in significant morbidity and loss of productivity. Recent reports of consecutive norovirus outbreaks on cruise ships, involving hundreds of passengers and crew, garnered substantial media attention and dramatically illustrate the potentially explosive community spread of these viruses (8). Although the severity of the disease is usually moderate and short term, an effective vaccine would be advantageous to specific segments of the population, such as food handlers, care providers, and military personnel. The major obstacles to norovirus vaccine development are the great degree of antigenic heterogeneity within the family, the possibility that immunity to noroviruses may be short-lived, and the lack of understanding of the correlates of protective immunity. An effective vaccine should also provide protective immunity against an ever-growing list of norovirus strains. Evidence from outbreak investigations and human challenge studies suggests that these viruses are highly infectious, yet the biology and immunology of norovirus infection remain poorly understood (32).
Noroviruses are members of the Caliciviridae family of single-stranded, positive-sense RNA viruses. The majority of the human noroviruses are classified into genogroup 1 (G1) and G2 (47) Each of these genogroups is further divided into genetic clusters. Norwalk virus (NV), Snow Mountain virus (SMV), and Hawaii virus (HV) are the prototype strains of genetic clusters G1.1, G2.2, and G2.1 and are the causative agents of an estimated 5, 8, and 7% of norovirus outbreaks, respectively (13). Strains within a genetic cluster typically show ≥80% amino acid identity in the major capsid protein sequence. Strains within the same genogroup show ≥60% identity, and strains in different genogroups show ≤50% identity. This high degree of genetic variability translates into a high degree of antigenic variability within the noroviruses.
Early human challenge studies demonstrated that some volunteers remained uninfected even when challenged with high doses of NV (30, 35), suggesting that some individuals may be genetically resistant to NV infection. Recently, we demonstrated that the FUT2 gene that encodes an α1,2-fucosyltransferase responsible for the histo-blood group antigen (BGA) H type 1 is a susceptibility allele for NV infection (32). Volunteers who were homozygous for the inactive FUT2 allele were resistant to infection at all dose levels studied. Other BGAs displayed weak modulating effects on NV interaction and pathogenesis. We and other groups have shown that NV virus-like particles (VLPs) bind to H type 1-related BGAs secreted in saliva (14, 20, 32) and membrane bound on mucosal cells (34) and also to synthetic H type 1-related BGA (20, 24). This H type 1-dependent in vitro binding pattern is so far restricted to NV, as other strains of noroviruses demonstrate different BGA binding affinities. Salivary binding assays have suggested that SMV VLP binding is associated with expression of the B BGA, while HV VLPs did not bind to any saliva sample (20). These data suggest that noroviruses use multiple methods for attachment to cells and hence for infection. It is unclear whether the FUT2 allele determines susceptibility to all norovirus infections.
Short-term immunity to NV has been observed in previous human challenge studies (48). However, this immunity did not necessarily extend to heterologous virus challenge, as volunteers who became ill upon challenge with NV also became ill upon subsequent challenge with HV at the same rate as volunteers not previously infected with NV (48). Further rechallenge studies have shown that long-term immunity is not conferred by a single NV challenge, as some volunteers who were susceptible to an initial NV infection were susceptible to infection in subsequent challenges 27 to 42 months later (41).
Previously, in both volunteer studies and outbreak investigations, immunity to noroviruses has almost exclusively been studied by monitoring serum immunoglobulin M (IgM) and IgG responses to virus (35). IgM was shown to be a marker of recent NV infection (5). In many studies, a fourfold increase in anti-norovirus serum IgG titer has been considered indicative of norovirus infection (17). Attempts to study fecal IgA in NV infection have been unsuccessful in associating secretory IgA (sIgA) with infection outcome (40). However, our recent study utilizing saliva samples as a source of sIgA showed a correlation between protection from NV infection and the presence of a memory sIgA response in uninfected susceptible volunteers (32). Importantly, some susceptible volunteers who were not infected did not have strong salivary IgA responses, indicating that non-sIgA mechanisms are also involved in immunity to noroviruses.
In addition to antibody-producing B cells, T cells play an important role in the control of viral pathogens. The cytokines secreted by antigen-specific CD4+ helper T cells guide the adaptive immune response. T helper 1 (Th1) and Th2 responses can be activated by both the same and different epitopes within the same pathogen, but one response type will often dominate over the other. A Th2-favored immune response is primarily characterized by interleukin 4 (IL-4) and IL-10 secretion by CD4+ cells, resulting in production of IgG2 and other anti-inflammatory factors. A Th1-favored response is primarily characterized by secretion of gamma interferon (IFN-γ) by CD4+ cells, resulting in macrophage activation, B-cell differentiation to IgG1 synthesis, and support for cytotoxic T lymphocytes (CTLs). CTLs are important in the control of human immunodeficiency virus (HIV) (4), Epstein-Barr virus (42), influenza virus (26), lymphocytic choriomeningitis virus (52), and numerous other viruses. To date, cellular immunity has not been studied following norovirus infection, but we expect effector T cells to play a fundamental role in viral clearance and immunity.
In this study, we demonstrated that SMV infection was independent of secretor status and BGA expression and that the serum IgG, salivary IgA, and effector T cells elicited following infection were cross-reactive between strains within a genogroup but not between strains in different genogroups. This is the first study to examine the association between mucosal expression of BGAs and infection with a G2 norovirus in humans and to study T-cell activation after challenge with any live norovirus.
Study design and subjects.A human challenge study was conducted to examine the infectivity of SMV. Fifteen healthy adult volunteers were admitted to the University of North Carolina General Clinical Research Center (GCRC) for collection of prechallenge samples and dosing. SMV inoculum was prepared from stool filtrates and safety tested as reported for Norwalk virus (10), with additional testing for other noroviruses, culturable human enteric viruses, hepatitis A virus, HIV, coronavirus, astrovirus, adenovirus, rotavirus, cytopathic viruses, endotoxins, common enteric pathogens (bacteria and protozoa), mycobacteria, and Clostridium difficile toxin. Each volunteer received one dose of SMV (11) ranging from 10 to 105 PCR detectable units, as defined by endpoint titration reverse transcription (RT)-PCR (37). Dose levels were randomized, and no single dose level was administered in sufficient numbers to validate statistical analysis of clinical outcomes by dose. The study population was 47% male, 73% white, and 27% other races, with an average age of 31 years (range 21 to 54). Volunteers remained in the GCRC for the first 5 days postchallenge and returned for follow-up visits on days 8, 14, and 21 postchallenge. During the first 7 days, all volunteer stools and vomit were collected and stored at 4 or −20°C. Sera and saliva were collected daily during the first 5 days postchallenge and at each follow-up visit. Samples were stored at −20°C. PBMCs were harvested from whole blood collected prechallenge and on days 8 and 21 postchallenge using a Ficoll-Pacque (Pharmacia-Amersham) gradient. The cells were cryopreserved in 90% fetal calf serum-10% dimethyl sulfoxide until they were used. All study protocols and methods were approved by the University of North Carolina School of Medicine Committee for Protection of Human Subjects. All volunteers participated after providing informed consent.
Diagnosis of SMV infection.Volunteers were classified as infected if SMV RNA was detected in any postchallenge stool sample by RT-PCR. All fecal specimens were examined by RT-PCR using G2-specific primers located in the RNA-dependent RNA polymerase gene (SR33/SR48) (1).
Measurement of anti-norovirus serum IgG.All sera were tested for anti-norovirus IgG by ELISA using recombinant SMV, HV, or NV VLPs as the antigen (20) and anti-human IgG (whole molecule)-alkaline phosphatase (Sigma Chemicals) as the detector antibody. The VLPs used in this study were composed of the open reading frame 2 (ORF2) major capsid protein from each strain expressed in the VEE vector system and purified as reported previously (3, 20). Seroconversion was defined as a ≥4-fold increase over the prechallenge titer in postchallenge samples collected on either day 14 or 21, depending on patient sample availability. By day 14 postchallenge, all nine of the infected volunteers had a ≥4-fold increase over the prechallenge (day zero) anti-SMV serum IgG titer. Titers remained at ≥4-fold above day zero levels for the duration of the study. For IgG subclass determination, anti-IgG1-biotin or anti-IgG2-biotin antibodies, followed by avidin-alkaline phosphatase (all from Sigma Chemicals), were used as detector antibodies.
Histo-blood group antigen expression.Blood type was inferred from red blood cell (RBC) (Ortho Clinical Diagnostics) hemagglutination by serum samples. Secretor and Lewis phenotypes were determined from saliva by ELISA using anti-Lewisa, anti-Lewisb, anti-A, and anti-B antibodies (Ortho Clinical Diagnostics) (20). The secretor phenotype was confirmed by the FUT2 genotype (32).
Measurement of anti-norovirus and total salivary IgA.Saliva samples were collected, processed, and assayed for anti-norovirus and total IgA by ELISA, as described previously (32). Of the 15 sample sets collected, 12 were included in the analysis. Three sample sets were eliminated from analysis because of poor sample quality (total IgA < 50 μg/ml) (32). Conversion was defined as a ≥4-fold increase over the prechallenge titer in postchallenge samples collected on either day 14 or 21, depending on patient sample availability.
VLP stimulation of PBMCs.Cryopreserved peripheral blood mononuclear cells (PBMCs) were thawed, washed with medium (RPMI 1640, 10% fetal calf serum, 1% l-glutamine, 100 U of penicillin-streptomycin/ml), and dispensed into 96-well plates at 5 × 105 cells/well with either 10 ng of phorbol myristate acetate/ml-0.5 μg of ionomycin/ml, medium only, or 1 μg of SMV, HV, or NV VLPs/ml. The optimum cell number, stimulation time, and VLP concentration were determined empirically. The cells were stimulated for 48 h at 37°C in a 5% CO2 incubator in duplicate or triplicate wells, depending on the number of available cells. Cell culture supernatants were collected and stored at −20°C until they were used.
Measurement of cytokine secretion.Culture supernatants were simultaneously analyzed for secreted Th1 cytokines (IFN-γ, tumor necrosis factor alpha [TNF-α], and IL-2) and Th2 cytokines (IL-4, IL-5, and IL-10) using the Cytometric Bead Array (CBA) assay (BD Biosciences) as recommended by the manufacturer, with the exception that reagents were used at half the recommended volumes. This change did not affect performance (P = 0.92; Mann-Whitney two-tail test). Inclusion of duplicate assay tubes did not significantly affect the results (P = 0.4065; Mann-Whitney two-tail test). Fluorescent beads were counted on a BD Biosciences FacsCalibur flow cytometer, and data were analyzed using Cell Quest Software (BD Biosciences). In additional samples, IFN-γ secretion was measured in duplicate by ELISA (BD Biosciences). The lower limit of detection was 1 pg/ml for the CBA assay and 4 pg/ml for the IFN-γ ELISA. Samples below these limits were assigned a value equal to half the lower limit of detection for analysis purposes (43). For all samples, the medium-stimulated background was subtracted.
Depletion of CD4+ or CD8+ cells.PBMC samples were depleted of CD4+ or CD8+ cells using antibody-coupled magnetic beads (Dynabeads; Dynal Biotech) according to the manufacturer's instructions. Depletion (≥90%) was monitored by fluorescence-activated cell sorter analysis of the samples using anti-CD4-fluorescein isothiocyanate and anti-CD8-phycoerythrin (Sigma Chemicals).
Statistical analysis.The Mann-Whitney two-tail test was used to compare the median responses between groups. The Fisher exact test (Epi Info 2000; Centers for Disease Control and Prevention) was used to test the association among prechallenge IgG, blood type, sIgA, secretor and Lewis phenotypes, and risk of infection.
Infectivity of SMV.Fifteen volunteers were dosed with SMV and were classified as infected if viral RNA was detected in any postchallenge stool sample by RT-PCR (10). Nine (60%) of the volunteers became infected (Table 1). Of these, 7 (78%) experienced symptoms of illness. Since NV infection has been shown to be dependent upon expression of the BGA H type 1 (32), we examined the effect of BGA expression on SMV infection. Expression of BGA on RBCs (blood type) was determined by RBC hemagglutination. Mucosal-cell expression of BGA (secreted BGA) was determined by ELISA of saliva samples, as reported previously (32). In contrast to NV infection, volunteers from every blood type, secretor phenotype, and Lewis type became infected. SMV infection was not associated with blood type (P = 0.608), secretor phenotype (P = 0.525), or Lewis phenotype (P = 0.505) (Table 1). These data likely indicate that NV and SMV strains use different mechanisms for infection, independent of the secretor phenotype.
Serum IgG response to SMV challenge.Seroconversion, a ≥4-fold increase over the prechallenge serum IgG titer, has been used to define norovirus infection in challenge studies and outbreak investigations (18, 11). By day 14 postchallenge, all nine of the infected volunteers had a ≥4-fold increase over the prechallenge (day zero) anti-SMV serum IgG titer, verifying the association between virus excretion and seroconversion. There was no significant difference between the titers of prechallenge SMV-reactive IgG in infected and uninfected volunteers (P = 0.215) (Fig. 1), suggesting that the prechallenge anti-SMV IgG titer is not predictive of the risk of SMV infection. The infected volunteers had postchallenge (day 14 or 21) anti-SMV IgG titers that were significantly higher than prechallenge titers (P = 0.0004), with a median 23.8-fold increase (range, 4.9- to 91.8-fold). The uninfected volunteers did not have a significant rise from the prechallenge titer, with a median change in the anti-SMV IgG titer of 1.5-fold (range, 0.8- to 2.0-fold) (Fig. 1 and data not shown). Seroconversion was highly predictive of infection (P = 0.0002).
Cross-reactivity of serum IgG after SMV challenge.Serum samples collected during outbreak investigations have shown that some norovirus strains share cross-reactive epitopes (18, 19). This observation may in part explain our results indicating that the prechallenge anti-SMV IgG titer was not predictive of SMV infection. To investigate the cross-reactivity of serum IgG generated after SMV challenge, prechallenge and postchallenge (day 14 or 21) serum samples from each volunteer were also tested for anti-HV and anti-NV IgG by ELISA (Fig. 1). As described above for anti-SMV IgG, there were no significant differences between prechallenge titers of anti-HV IgG (P = 0.8572) or anti-NV IgG (P = 0.8572) in infected and uninfected volunteers. After challenge, infected volunteers had postchallenge titers of anti-HV IgG (P = 0.0173) that were significantly higher than prechallenge titers (Fig. 1). Three (33%) of the SMV-infected volunteers had ≥4-fold increases, and an additional two volunteers had a ≥3-fold increase in anti-HV IgG. One volunteer produced a response with greater reactivity to HV than SMV. In contrast to the response to HV antigen, SMV infection did not result in a significant increase in anti-NV IgG (P = 0.90), suggesting that cross-reactive IgG responses are genogroup specific. Among the uninfected volunteers, significant increases over the prechallenge titers were not detected in anti-HV IgG (P = 0.5755) or anti-NV IgG (P = 0.69) titers. None (zero of six) of the uninfected volunteers seroconverted to HV. One (one of six) uninfected volunteer did produce antibody to NV (Fig. 1). The group difference between pre- and postchallenge NV IgG titers was not significant. These data suggest four important points: (i) SMV infection, not just exposure, is essential for activation of an anti-SMV serum IgG response, even in volunteers with prechallenge anti-SMV-reactive IgG; (ii) IgG elicited during an SMV infection is cross-reactive with VLPs from strains within the genogroup but not across genogroups; (iii) SMV and HV share some cross-reactive epitopes, and prechallenge IgG measurements reflect the sum of cross-reactive epitopes among the noroviruses each volunteer has previously encountered and should not be considered strain-specific IgG; and (iv) occasionally, a heterotypic IgG response may dominate following norovirus challenge, reminiscent of a response influenced by exposure history.
Salivary IgA response to SMV challenge.Norovirus infection is likely limited to the gut; thus, mucosal antibodies may be important in protection from infection (32). To measure the mucosal immune response to SMV challenge, salivary samples from 12 volunteers were assayed for anti-SMV and total IgA by ELISA, and the ratio of the two measurements was used for comparisons. As with serum IgG, there was no significant difference between the prechallenge salivary mucosal IgA ratios in infected and uninfected volunteers (P = 0.4237). However, infected volunteers had anti-SMV sIgA titers on day 14 or 21 postchallenge (P = 0.0308) that were significantly higher than prechallenge titers and had a median 6-fold increase (range, 1.5- to 36.8-fold) (Table 2). Four out of six (67%) infected volunteers had a ≥4-fold increase in anti-SMV sIgA titer. Uninfected volunteers did not have a significant rise in anti-SMV sIgA titer (median, 1.4-fold increase; range, 0.64- to 2.0-fold) (Table 2). As with serum IgG, a ≥4-fold increase in anti-SMV sIgA by day 14 or 21 postchallenge was associated with infection (P = 0.0278).
Cross-reactivity of salivary IgA after SMV challenge.To investigate the cross-reactivity of salivary IgA generated after SMV challenge, prechallenge and convalescent-phase saliva samples from five infected volunteers and the one uninfected volunteer who seroconverted to NV were measured for anti-HV and anti-NV sIgA, and the ratio of anti-norovirus sIgA to total sIgA was determined (Table 2). One infected volunteer had a ≥4-fold increase in salivary IgA reactive to HV. None of the infected volunteers had a ≥4-fold increase in salivary IgA reactive to NV. A ≥3-fold increase in anti-HV and anti-NV sIgA was observed in another infected volunteer. Fewer volunteers had a ≥4-fold increase in HV-reactive sIgA compared to IgG. This result may reflect the higher titer of antibody found in serum versus saliva, as opposed to suggesting that sIgA is more strain specific than IgG.
SMV challenge elicits a Th1 immune response.The cellular immune response to norovirus challenge has not been studied previously. However, the role of T cells in some human viral infections has been described (27). To begin to characterize the cellular immune response elicited by SMV challenge, PBMCs were collected from whole blood prechallenge and on days 8 and 21 postchallenge and cryopreserved for in vitro VLP stimulation followed by analysis of secreted cytokines. Eleven volunteers had at least partial PBMC sample sets. Three infected and two uninfected volunteers had complete sample sets. PBMCs from these five volunteers were stimulated with 1 μg of SMV VLPs/ml for 48 h, and the clarified tissue culture supernatants were assayed for Th1 cytokines (IFN-γ, TNF-α, and IL-2) and Th2 cytokines (IL-10, IL-5, and IL-4) using the flow cytometry-based CBA assay (Fig. 2). Cytokine responses were compared by group using the Mann-Whitney two-tail test. In both the infected and uninfected volunteers tested, the greatest cytokine response to SMV VLP stimulation was increased IFN-γ secretion (P = 0.0121, comparing median concentrations of secreted IFN-γ from prechallenge PBMCs to those from peak [day 8 or 21] postchallenge PBMCs). Each volunteer demonstrated a rise in IFN-γ secretion (median increase, 20.0-fold [range, 6.1- to 6,003-fold]). Smaller, but significant, rises above the prechallenge titer were also detected for IL-2 (P = 0.0121), with a median increase of 5.5-fold (range, 4.2- to 276.8-fold), and IL-5 (P = 0.0121), with a median increase of 4.2-fold (range, 4.1- to 16-fold). The timing (day 8 or 21) of peak cytokine up-regulation varied between different volunteers regardless of their infection status. Significant changes were not detected between prechallenge and postchallenge PBMC secretion of TNF-α, IL-10, or IL-4, although some individual responses were elevated (Fig. 2). The lack of a TNF-α response was somewhat surprising, since TNF-α and IFN-γ are often correlated (44), and it may reflect the small sample size of our population. Increased IL-5 is a marker for a Th2 response. The response shown here is statistically significant, even though the absolute concentration of IL-5 detected was low (median, 3.7 pg/ml), especially in comparison to IFN-γ secretion (median, 3,002 pg/ml), as described for rhinovirus-stimulated PBMCs (6). These data indicate a predominant, but not exclusive, Th1 cellular immune response to SMV challenge.
The enhanced secretion of IFN-γ in SMV-stimulated PBMCs from both infected and uninfected volunteers suggested that measurement of IFN-γ levels could provide a quantitative indication of cellular immune response in all SMV-challenged volunteers. Thus, secreted IFN-γ was measured by ELISA for the 11 volunteers with at least two PBMC collections available for comparison (Fig. 3 and data not shown). Four out of six uninfected and all seven infected volunteers had PBMCs collected prechallenge. All four (100%) of the uninfected and six of the seven (86%) infected volunteers had prechallenge SMV-reactive cells that secreted IFN-γ in response to in vitro SMV stimulation, suggesting previous norovirus exposure and immunological memory in 91% of the challenged volunteers. There was no significant difference between prechallenge IFN-γ secretion levels in the infected (median, 73 pg/ml) and uninfected (median, 264 pg/ml) volunteers (P = 0.1738). Among the seven infected volunteers, a significant change from prechallenge IFN-γ secretion was detected in postchallenge PBMC samples (P = 0.0105), with a median increase of 33.2-fold (range, 0.61- to 3,025-fold) (Fig. 3). Five of the seven infected volunteers (71%) had a ≥4-fold increase over prechallenge IFN-γ secretion in postchallenge PBMC samples. Among the four uninfected volunteers, two (50%) had a ≥4-fold increase over prechallenge IFN-γ secretion, with a median increase of 3.0-fold (range, 0.02- to 14-fold) (Fig. 3). As noted for the five volunteer samples analyzed by CBA, peak up-regulation of cytokine secretion (day 8 or 21) between different volunteers varied regardless of their infection status.
To further address the nature of the SMV-induced immune response, prechallenge and postchallenge serum samples were tested for IFN-γ, IL-2, IL-6, and IL-10 by ELISA. Significant increases in both IFN-γ (median increase, 10.9-fold; range, 1.0- to 966-fold [P = 0.0117]) and IL-2 (median increase, 4.8-fold; range, 1.0- to 141.6-fold [P = 0.0093]) concentrations in serum were detected in acute-phase (day-2) serum samples collected from infected volunteers. Significant changes in cytokines were not detected in IL-6 or IL-10 in sera from infected volunteers or in any cytokine in serum samples from uninfected volunteers. Furthermore, prechallenge and postchallenge serum samples from infected volunteers were tested for anti-SMV IgG1 and IgG2. In agreement with previous studies of anti-norovirus IgG subclass distribution (2, 21), most of the detectable response was an increase in IgG1 (median increase, 3.99-fold; range, 0.16- to 19.5-fold). Little change in anti-SMV IgG2 titer was detected (median increase, 1.03-fold; range, 0.98- to 6.4-fold). These acute-phase serum cytokine and IgG subclass data support the PBMC-secreted cytokine data, indicating that the SMV-induced immune response is weighted toward a Th1 response.
Five of the seven infected volunteers (71%) had a ≥4-fold increase over prechallenge IFN-γ secretion in postchallenge PBMC samples. Suppression of IFN-γ responses by other cytokines has been reported in some HIV-infected individuals (16). To determine if additional cytokines could explain the lack of response in the two infected IFN-γ nonresponders (data not shown), culture supernatants from the infected nonresponders were assayed for additional cytokines using the CBA assay (data not shown). One volunteer was found to be producing low levels of the Th1 cytokines IFN-γ (4.9-fold increase over the prechallenge titer), TNF-α (4.2-fold increase), and IL-2 (2.2-fold increase). No changes in Th2 cytokines were detected, indicating that infection in this volunteer did elicit a weak Th1-type immune response. In the other infected nonresponder, IL-10 was the only cytokine response detected (4.9-fold increase), identifying this volunteer as the only infected IFN-γ nonresponder. Among the three uninfected nonresponders, no additional cytokines were detected by CBA analysis (data not shown), supporting previous results suggesting multiple mechanisms for resistance to norovirus infection (32).
SMV challenge elicits a cross-reactive T-cell response.Analogous to our studies of antibody epitopes cross-reactive between norovirus strains, the cross-reactivity of the Th1 cellular response elicited by SMV challenge was investigated. PBMCs from the two uninfected and three infected volunteers (as seen in Fig. 2) who responded to SMV challenge with increased IFN-γ secretion were stimulated separately with HV and NV VLPs, and the culture supernatants were assayed for IFN-γ, TNF-α, IL-2, IL-10, IL-5, and IL-4 using the CBA assay (Fig. 4). After stimulation with HV, postchallenge PBMCs from the infected and uninfected volunteers secreted significantly more IFN-γ than prechallenge PBMCs (P = 0.0214), with a median increase of 10.7-fold (range, 3.2- to 2,086-fold), and IL-2 (P = 0.0121), with a median 3.8-fold increase (range, 2.2- to 180.6-fold) (Fig. 4A). Significant differences were not detected in the other cytokines, indicating that the Th1 response mounted to SMV was cross-reactive with HV (Fig. 4A). In contrast, after NV VLP stimulation, no significant changes were detected between prechallenge and postchallenge PBMCs in the ability to secrete IFN-γ, IL-2, TNF-α, IL-10, or IL-4. There was significantly increased secretion of IL-5 in response to NV VLP stimulation (P = 0.0477), but the concentrations detected were low, as noted for SMV stimulation (Fig. 4B).
To further study the cross-reactivity of the Th1 response generated after SMV challenge, culture supernatants from additional PBMCs stimulated with HV or NV VLPs were assayed for secreted IFN-γ by ELISA (Fig. 3). Ten volunteers had PBMCs available for the cross-reactivity studies. Those volunteers who did not respond to SMV did not respond to HV or NV after SMV challenge (data not shown). Volunteers who had a significant increase in SMV-induced IFN-γ secretion post-SMV challenge also had a significant increase in response to HV stimulation (P = 0.0131), but not to NV stimulation (P = 0.631). Among the six infected volunteers, three (50%) had a ≥4-fold increase in IFN-γ secretion after HV stimulation, with a median increase of 61-fold (range, 0.17- to 183-fold) (Fig. 3). There was a median increase of 1.6-fold (range, 0.06- to 5.37-fold) among the four uninfected volunteers, and one volunteer had a ≥4-fold increase in IFN-γ secretion after HV stimulation (Fig. 3). None of the uninfected volunteers had a ≥4-fold increase in IFN-γ secretion after NV VLP stimulation. The median change was 0.58-fold (range, 0.11- to 1.3-fold) (Fig. 3). As identified with antibody responses, some SMV-induced Th1 cells cross-react with HV but not NV, indicating that strains of the same genogroup share common epitopes that may elicit cross-protection.
IFN-γ secretion is dependent upon CD4+ cells.Both CD4+ and CD8+ T cells secrete IFN-γ (44). Since our method of antigen presentation relies on VLP uptake, processing, and presentation by antigen-presenting cells within the PBMCs, a bias toward detection of CD4+ cells may be expected, although other means of presentation could also occur (44). To identify the cellular source of the Th1 cytokines produced in response to SMV challenge, PBMCs collected on day 8 or 21 postchallenge from five volunteers (three infected and two uninfected) were depleted of either CD4+ or CD8+ cells via antibody-coupled magnetic beads (15) and stimulated with SMV, and the culture supernatant was assayed for secreted IFN-γ (Fig. 5). Cell depletion (≥90%) was monitored by fluorescence-activated cell sorter analysis of depleted and nondepleted cultures (data not shown). Depletion of CD8+ cells did not have a significant effect on IFN-γ secretion (P = 0.6745). However, it is noteworthy that in the two uninfected volunteers (SM07 and SM08 in Fig. 5), depletion of CD8+ cells led to increased IFN-γ secretion after stimulation, suggesting that in these two uninfected volunteers, CD8+ cells may be down-regulating the IFN-γ response in vitro (16), possibly providing a balanced immune response to a neutralized virus. In contrast, depletion of CD4+ cells before SMV stimulation resulted in a significant decrease in IFN-γ secretion after stimulation in all of the volunteers tested (P = 0.0214). IFN-γ secretion decreased an average of 93% (range, 81.5 to 97%) when CD4+ cells were absent from the PBMC culture during stimulation (Fig. 5), identifying CD4+ cells as the primary source of IFN-γ secretion in PBMCs stimulated in vitro with norovirus VLPs.
Human challenge studies have indicated that a subset of individuals may be genetically resistant to noroviruses (30, 35). The characterization of the BGA H type 1 as a ligand that binds NV VLPs (34) led to the observation that individuals who do not express H type 1 (the secretor-negative phenotype) do not become infected with NV even when challenged at very high doses (32), establishing FUT2 as a susceptibility gene for NV and suggesting that H type 1 may be a receptor for virus entry. Further work with other norovirus strains has demonstrated that the noroviruses can use a wide range of molecules in the histo-blood group antigen family as ligands (20, 22). In contrast to NV, SMV infection was not associated with the presence of a functional FUT2 gene or with expression of any of the H type 1-related molecules, as one of three secretor-negative volunteers became infected with SMV. The effect of the BGA genes on SMV infection will need to be further studied in subsequent human challenge studies with larger groups with varied antigen expression. The small number of subjects characterized here is not sufficient to identify genes with weak penetrance, as was noted for the effect of B antigen expression and NV infection (32, 23). Moreover, our data indicate that saliva-VLP binding cannot predict norovirus susceptibility as strictly as suggested (22) and that virus binding and entry mechanisms may be considerably more complex.
In these studies, we used VLPs as the source of norovirus antigen.These VLPs are composed of the major capsid protein expressed from ORF2 of SMV, HV, or NV. While morphologically and antigenically very similar to native virions (29), they do not represent the entire norovirus strain genome, and thus, any immune response measured represents a partial response. Furthermore, the cross-reactivities of antibodies and T cells measured here are dependent upon several factors, including the degree of genetic relatedness between strains and the major histocompatibility complex (MHC) haplotype of each volunteer, as some MHC haplotypes may preferentially present peptides that are not conserved between strains from different genetic clusters (9, 36).
All of the SMV-infected volunteers and none of the uninfected volunteers produced a significant serum IgG response to SMV, indicating that SMV infection, not just exposure, is essential for activation of an anti-SMV serum IgG response, as reported for NV (32). Furthermore, the IgG elicited during an SMV infection cross-reacts with another strain within G2 but not with a G1 strain, supporting previous findings (28, 39). While these results provide a description of the antigenic relatedness of these viral strains to each other and an assessment of general norovirus exposure in the past, interpretation of their relevance to vaccine design and outbreak investigations is complicated. Studies have used the presence of a norovirus VLP-reactive IgG titer as an indication of strain exposure. However, the cross-strain reactivity of anti-norovirus IgG shown here makes it difficult to determine the infection strain history, as proposed earlier (12, 32). This point is particularly important in outbreak investigations, where, in our experience, more subjects are willing to donate serum samples than are willing to donate stool samples for diagnosis. The data presented here suggest that strain identification by serum IgG titer increase would be imprecise. A comprehensive panel of VLPs and refined diagnostic tests will be needed before IgG can be used to unravel prechallenge histories and current infections with norovirus.
Furthermore, data indicating that a subset of uninfected SMV- and NV-challenged volunteers mount a cellular immune response and a mucosal IgA response, respectively, after norovirus challenge support earlier work suggesting that some volunteers are uninfected due to protective immunity (32). None of these "protected" volunteers produced IgG reactive with the challenge strain. It is possible that a pathogen limited to the gut mucosal surface without a systemic distribution may not be significantly affected by serum IgG. Elegant studies of rotavirus infection in gnotobiotic piglets have shown that sIgA, not neutralizing IgG, correlated with protection from rotavirus reinfection (49-51). Concordant with these findings, NV-challenged susceptible volunteers who produced sIgA within the first 5 days after viral challenge were less likely to become infected than susceptible volunteers who did not produce sIgA, indicating that memory sIgA may be a component of protective immunity to NV infection (32). However, this early sIgA response was not detected in all of the susceptible uninfected volunteers. Thus, other mechanisms must participate in protection against norovirus infection as well. Although the numbers are small, among the SMV-challenged uninfected volunteers, an early (day 1 to 5) sIgA response was not detected, supporting earlier evidence that multiple mechanisms of protection from noroviruses exist (32).
This is the first reported characterization of the Th1 and Th2 cellular immune responses to any live norovirus challenge. PBMCs were stimulated in vitro with intact VLPs (43) from three norovirus strains. This approach allowed antigen-presenting cells to internalize native conformation protein, digest it, and present it in the context of syngeneic MHC class II, as would occur in vivo during challenge, resulting in norovirus-specific CD4+-T-cell activation (44). Comparison of Th1 and Th2 cytokines produced after SMV stimulation of PBMCs indicated that the Th response to SMV was weighted toward a Th1 response, with significant increases in secretion of IFN-γ and IL-2 and serum IgG1 production, similar to results seen in a subset of NV VLP-challenged volunteers (46). These cytokines enhance macrophage activation and production of IgG subclasses that favor opsonization and support CD8+ cytotoxic T cells (27). IFN-γ was up-regulated postchallenge in most of the volunteers, regardless of infection. Both infected and uninfected volunteers could be classified as responders or nonresponders based on a ≥4-fold increase in IFN-γ secretion postchallenge. The elapsed time between challenge and the first postchallenge PBMC sample (8 days) may have been too great to allow identification of a difference in the timing of activation of the Th1 cells between the infected and uninfected volunteers. Furthermore, the T cells examined here were collected from blood, while memory T cells to a mucosal pathogen would be located primarily at the site of initial antigen encounter, the gut-associated mucosa (25). Analyses of cytokines and IgG subclasses in daily serum samples support the hypothesis of a preferential but not exclusive Th1 cytokine response following SMV challenge. Increases in both IFN-γ and IL-2 were detected during the initial 48 h postchallenge, and the IgG response was IgG1, with little change in the IgG2 titer in serum samples from the infected volunteers. Furthermore, the only Th2 cytokine in which significant increases were detected after SMV challenge was IL-5, associated with enhanced sIgA production. sIgA has been shown to be associated with protection from NV infection among susceptible volunteers (32). Identification of IL-5 secretion by postchallenge PBMCs in response to SMV and NV in vitro stimulation supports the hypothesis that the mucosal immune response plays an important role in norovirus infection. Additional human challenge studies will be needed to decipher the roles of sIgA and the Th2 immune response in protection from norovirus. Future studies should include measurement of gut cytokine profiles and evaluation of PBMCs collected soon after infection to accurately determine the importance of Th2 responses to infection.
Although these experiments do not address SMV protective immunity, the Th1 response identified in a subset of uninfected volunteers suggests that activation of a Th1 cellular immune response may be associated with protection from infection after challenge in some SMV-susceptible volunteers. Supporting a role for the Th1 response in mediating SMV infection outcome, the only infected volunteer without an increase in IFN-γ secretion after SMV challenge produced IL-10 (data not shown), suggesting that the Th1 response elicited by SMV in the other infected volunteers was being suppressed. This volunteer had high prechallenge titers of HV-reactive serum IgG and salivary IgA and Th1 cytokine secretion. These high titers decreased throughout the course of the study, suggesting that this volunteer had experienced a recent infection with HV or a closely related strain of norovirus before entering the SMV study. SMV-reactive IgG developed normally over time and in magnitude, but the volunteer did not develop a salivary IgA or Th1 immune response to SMV after infection. Although speculative, these data provide suggestive evidence that previous viral infection history may affect the immune response to a current norovirus challenge, as has been shown for dengue serotypes (38) and between lymphocytic choriomeningitis and vaccinia virus infections (31). Clearly, further studies are needed to identify the components of protective immunity and the effect of previous exposure history on infection outcome.
No change in T-cell cytokine secretion, serum IgG, or salivary IgA was detected in any of the uninfected volunteers who did not respond to SMV challenge with enhanced IFN-γ secretion (three volunteers). Lack of activation of an adaptive immune response, as seen in the other three uninfected volunteers who received at least the same dose level, suggests that these volunteers were protected by another mechanism, such as innate immunity or inherent genetic resistance to the virus (lack of receptors), or that the SMV ORF2 capsid may not be as antigenic for the HLA haplotype of these volunteers, hampering our ability to detect an immune response (9, 36).
Importantly, the cellular response elicited by SMV challenge was cross-strain reactive with HV but not NV, suggesting that the antigenic relatedness of the strains we examined mirrored the genetic relatedness. These data are in agreement with previous studies of strain-cross-reactive Th1-mediated responses in HIV (7), dengue virus (33), and influenza virus (26). Additional G1 and G2 VLPs are needed to assess the T-cell cross-reactivity between genoclusters and genogroups, given the high degree of genetic heterogeneity among norovirus strains. In addition to the capsid protein, ORF3 and nonstructural proteins should be examined as a source of cross-reactive T-cell antigen, as anti-influenza A virus (26) and anti-cytomegalovirus (45) CTLs recognized internal antigens and were subtype cross-reactive.
The data provided here indicate that antibody and cellular immune responses generated after SMV challenge are cross-reactive within genogroups. Production of new inocula of additional norovirus strains representing different genetic clusters and further human challenge studies are needed to define the antigenic relationships among strains and the components of protective immunity to each and to determine if specific heterotypic immune responses are cross-protective.
We thank Rebecca Calderon at the U.S. Environmental Protection Agency (EPA) and the staff of the University of North Carolina GCRC for their contributions to the human challenge studies, Shay Fout at the U.S. EPA for his support of the salivary assay development, and Erin-Joi Collins McNeal for technical assistance.
This work was supported by a grant from the U.S. EPA (STAR grant R826139) and a Cooperative Agreement with the U.S. EPA (R-82936501) and grants from the National Institutes of Health (AI056351, AI23946, GM63228, GM67143, and RR00046 to the University of North Carolina GCRC), the North Carolina Biotechnology Center (2000-ARG-0040), and Glaxo Wellcome and by INSERM (Institut National de la Santé et de la Recherche Médicale).
- Received 29 July 2004.
- Accepted 12 October 2004.
- Copyright © 2005 American Society for Microbiology


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Runs In The Family [Beat Tape] [2018]
Soooo I decided to drop a beat tape yesterday. This music is my way of saying Happy Birthday to my Dad Kwame Mugodo and celebrating the love we share for music. Freestyle to it. Dance to it. Do what you do. If it wasn't for him, introducing this beautiful thing that I love to me at an early age, I really don't know where I'd be. So thanks Pops. I owe you my life. Runs In The Family [beat tape] available on for free download.
Track List:
- Sunshine
- India
- Potato
- Let Me See
- Beanie
Produced, mixed and mastered by KDD Pro
The Janet Tape [2018]
Confession – I've always had a semi obsession with Janet Jackson. Since today is her birthday, I decided to drop a little tape (4 songs) that I produced from sample of her songs Living In A World, Come Back To Me, Together Again (Remix) and Warmth. It ended up being pretty dope! I was also blessed to have a feature by The Means on Soul Searching. Take a listen…
Track List:
- Natural (Come Back To Me)
- Soul Searching ft The Means (Together Again Remix)
- Obstacles (Livin In A World)
- Recipe (Warmth)
Produced, mixed and mastered by KDD Pro
Hell If I Know (Single) [2017]
This song was created at the request of Paul von Stoetzel who is the director of a few of my videos and a good friends. He asked me to write as a wedding gift to him and his future wife who played my zombie buddy in my Grim video. I made the beat, wrote the lyrics over the weekend and sent him a rough draft that Monday. He loved it. Within a week or two we were shooting the video and it premiered on the City Pages that Halloween (his favorite holiday) The moral to the song is: Love can be heaven or hell – choose your own adventure.
Produced, mixed and mastered by KDD Pro
CaliSota 1.5 [2017]
Just put out a new playlist – CaliSota 1.5 (born in California, raised in Minnesota) and it's available now on #Spotify and #Itunes Featuring: Jae Havoc, Soul Reflect and NoFuckAround Gang Producers: Jae Havoc, A1 Sound Productions and KDD Pro.
Track List:
- Viewtiful Joe ft Jae Havoc (Produced by Jae Havoc)*
- FWM (Produced by KDD Pro)
- Digital ft Soul Reflect (Produced by KDD Pro)
- Hypnosis (Produced by KDD Pro)
- Hypnotized (Produced by KDD Pro)
- 0% No FuckAround ft No FuckAround Gang (Produced by A1 Productions)+
- Calculated (Produced by KDD Pro)
All songs mixed and mastered by KDD Pro excluding * and +. * by Jae Havoc. + by A1 Productions and Big Jess.
CaliSota [2017]
California and Minnesota are both my homes and biggest artistic influences. Intertwining the funk of Bootsie Collins and Too Short, with the Rock/Soulful edge of Prince and The Time. This is what the marriage of these two States created!!
Track List:
- What I Love (Produced by KDD Pro)
- Let Me Know (Produced by Breez for 4 Pro Audio)*
- The Rain (Produced by KDD Pro)
- Numb Part 2 ft Soul Reflect (Produced by KDD Pro)
- R.I.F. (Produced by KDD Pro)
- Center Stage (Produced by Web Beats)
- Black Lie (Produced by KDD Pro)
All songs mixed and mastered by KDD Pro excluding * and +. * mastered by Breez.
In a world where women have accepted the idea of selling sex for survival and call it empowerment, where dope fiends are considered heroes and drug dealers are knights in shining, armor there is an antithesis: Kut Throat!!! Kut Throat is a raw, honest expression of the world through the jaded eyes of Katana Da Don – a female writer/producer/performer originally from California and now residing in Minnesota. Producing all songs excluding SNOW (Eycee Slim) Katana paints an eerie picture of what the future will be if the ideology being portrayed in the media and other social outlets don't mature. In recognition of this shallow, materialistic entity that has consumed American culture, Katana Da Don fights fire with fire in Kut Throat and chooses to beat them rather than join them!!!