Officials say 35 Taliban killed in Afghan battle

An Afghan official says a military operation has killed 35 Taliban fighters.

Juma Gul Himat says the two-day battle in southern Uruzgan province was led by Afghan forces and also involved …

Herongate win ; Football

ROYDON .............0 HERONGATE ATHLETIC . . . . 2 (EssexOlympian ProKit League Senior Division Two) HERONGATE returned toleague action after eight weeks away due to snow and waterloggedpitches and returned to winning ways at this tricky away fixture atbottom of the league side Roydon.

On a heavy pitch Herongate settled the better of the two sideswith wide players Hans Summers and Dave King causing problems forthe home defence with their pace.

Crudely …

Stocks Higher After New Home Sales Data

NEW YORK - Wall Street advanced modestly Tuesday, with investors relieved but unimpressed that new home sales declined in May by a smaller amount than expected.

The Commerce Department reported that sales of new homes fell 1.6 percent in May to a seasonally adjusted annual rate of 915,000. It was the fourth decline in the past five months, but a milder decrease than analysts had predicted. In April, new home sales had jumped by 12.5 percent.

The stock market's gains were small, however. Investors were a bit unnerved by the Conference Board's index on consumer confidence, which fell more than anticipated, and still jittery about subprime lending troubles and ahead of …

T.I. performing 2 days before heading to prison

T.I. is taking the stage for his last concert before heading to prison.

The Grammy-winning rapper is set to perform in Atlanta on Sunday night, two days before he begins his prison time on a federal weapons conviction.

He must report by noon Tuesday to the Federal Correction Institution at Forrest City, Ark. His sentence is for a year and a day.

The 28-year-old rapper, whose real name is …

Local & National scoreboard

Todays' odds

College Football

Today

FAVORITE OPEN TODAY O/U UNDERDOG

Bowling Green 3 31/2 571/2 at Miami (Ohio)

at N. Illinois 17 17 461/2 Ball St.

South Florida 1 1 451/2 at Rutgers

Friday

at Cincinnati 91/2 9 531/2 West Virginia

Temple 41/2 4 44 at Akron

Saturday

Southern Miss. 3 3 491/2 at Marshall

Boston College 51/2 4 42 at Virginia

at Illinois 31/2 41/2 48 Northwestern

at Louisville 5 71/2 45 Syracuse

at Ohio St. 13 161/2 361/2 Iowa

at Wisconsin 9 81/2 541/2 Michigan

at Penn St. 21 251/2 511/2 Indiana

Michigan St. +1 21/2 53 at Purdue

at Wake Forest 6 …

Columbia and DePaul

An example of leadership for a pathetic New York university

ONE RARELY LOOKS TO HALLS of academia these days - to the students or faculty who walk them, or the administrators who build them - for guidance or leadership. No wonder.

As a rule, intellectuals in politics do not provide a good mix. Columbia University proved that this week, in inviting the Iranian president Mahmoud Ahmadinejad to their campus.

Notwithstanding some pointed and direct questioning and comments from Columbia president Lee Bollinger, who introduced the Iranian leader at the Sept. 24 event, it was the visitor who got the better of his hosts.

In truth, when it comes to street fighting, …

Honduras defeats Colombia 2-1 in friendly

Hendry Thomas' goal midway through the second half led Honduras over Colombia 2-1 in a friendly on Wednesday.

Before an evenly divided Honduran and Colombian capacity crowd of 18,886 at Lockhart Stadium, Thomas broke a 1-1 tie in the 63rd minute. Thomas was positioned near the far post when he received Amado Guevara's free kick from the right wing and headed in a shot past goalkeeper Agustin Julio.

…

Pill system designed by student

An Aberdeen student has designed a system to help people taketheir medicine

Sean McFleat, a 21-year-old product design student at Gray'sSchool of Art in Aberdeen, wanted to help people who take more thanone type of drug.

His product - called …

China's automakers look to alliances for survival

Chinese automakers are beginning to consolidate to compete more effectively with foreign OEMs entering the market following China's membership in the World Trade Organization (WTO).

Late last month First Automotive Works (FAW) - China's largest automaker - said it would buy 50.98 percent of Tianjin Xiali, a subsidiary of Tianjin Automotive, while Shanghai Automotive Industry Corp. (SAIC) and Yuejin Motors are reportedly negotiating an alliance and Dongfeng Motors is said to be in talks with Beijing Auto.

The FAW-Tianjin deal is the first in what analysts say will be a long stretch of mergers and joint ventures expected during the next several years. These alliances will …

Bear bites camper sleeping inside tent; authorities close 2 campgrounds near US national park

A bear attacked a man sleeping inside a tent early Thursday, leading authorities to close two campgrounds near Yellowstone National Park.

The camper suffered bite and claw marks on his arms while protecting himself. His name and age weren't disclosed. Authorities said he was taken to West Park Hospital in Cody, Wyoming, which refused to release any information.

As a precaution, the U.S. Forest Service closed the Soda Butte campground, where the camper was attacked, and the nearby Chief Joseph campground, both in the …

Dear Abby: ; Readers' ideas turn restless dreams into restful slumber

DEAR ABBY: I would like to comment on the letter from "Once aMechanic ... in Arizona" (Dec. 24), who still dreams about his jobeven though he is now retired. I once worked in the customer servicedepartment of a major retailer. During one Christmas season, arecurring dream would interrupt my sleep each night. In my dream, mybedroom door would open and a stream of customers would file in andline up next to my bed, their arms loaded down with purchases orreturns.

After many frustrating nights of restless sleep, I finallyfigured out a solution. I envisioned a flashing neon sign on myheadboard that read: "Lane Closed." The following night when thecustomers began filing in, I …

Derrick Parker brought dramatic urgency to his role

Derrick Parker brought dramatic urgency to his role

A young bass-baritone, Derrick Parker, making his Chicago debut in Chicago Opera Theater's production of George Frederic Handel's opera "Acis & Galatea" Anatheanum Theater brought to his role of Polyphemus a raging jealous fury with a powerful sonority and with bursting, dramatic urgency.

Interpreting the Baroque opera written by Handel in addition to Parker were Michael Smallwood as Acis; Nathalie Paulin, Jackalyn Short, Damon, Stephen Noon, A Swain and Kelli Harrington as A Nymph.

Many are familiar with Handel's oratorios, but are not acquainted with his many opera's he wrote. "Acis & Galatea" is one of …

Antofagasta says profit rose 2 percent in 2007

Antofagasta PLC, a Britain-based mining company with operations in Chile, said Tuesday full-year profit rose 2 percent after metals prices went up.

Net income for the 12 months through Dec. 31 rose to US$1.38 billion (euro898 million) from US$1.35 billion in the previous year, the company said in a statement. Revenue rose 1.2 percent to US$3.8 billion (euro3.47 billion).

"During 2007 metal prices remained well above historical levels," the company said.

London copper prices averaged 323.3 cents per pound compared with 305.3 cents per pound in the previous year, ranging from a low of under 240 cents to a peak of nearly 380 cents, reflecting changes in market sentiment during the year.

The company said the market for copper was volatile in 2007 but the company expects demand to remain fundamentally sound in the coming year and forsees continued strength China and in regions such as South Asia and the Middle East.

In 2008, copper production is expected to increase by about 8 percent to 463,000 tonnes (510,370 US tons). In 2007 it rose 5.9 percent.

That growth "is expected to help offset the effects of a slowdown in the United States. Supply continues to be affected by disruptions, including labour disputes, energy supply constraints, equipment availability and longer lead times to develop new projects," Antofagasta said.

The molybdenum business was also strong in 2007 as demand outstretched supply. Prices rose to an average US$30 a pound from US$25 at the start of the year. "Demand is expected to remain strong, driven by the continued demand from both the steel and catalyst sectors," the company said in its statement. Molybdenum is used in the construction of high-strength alloys.

The company also said it would seek new business by searching for sources of geothermal energy.

"As countries in the southern cone (of South America), particularly Chile, are facing severe problems relating to shortages of energy supplies and significantly increasing costs, the group has decided to enter the energy exploration business and has formed management and exploration teams to search for geo-thermal prospects," Antofagasta said.

Nepal assembly set to abolish monarchy

Lawmakers prepared Wednesday to declare Nepal the world's newest republic and bring an end to its centuries-old Hindu monarchy, as Nepalis rejoiced in the streets of the capital in anticipation of the announcement.

Across Katmandu, young men marched with red Maoist flags as residents young and old celebrated what many here see as the culmination of a two-year peace process with communist rebels that began after weeks of unrest forced King Gyanendra to restore democracy.

Near the convention center where Nepal's newly elected Constituent Assembly was expected to meet later Wednesday, thousands chanted "Long Live the Republic!" and denounced Gyanendra.

The king remained silent as it became apparent that his days as Nepal's monarch were numbered, and the country's leading politicians have in recent days threatened to remove him from the 1970s-era concrete palace by force if he refuses to go peacefully.

But in an apparent bid to defuse any potential standoff, the country's newly elected assembly will give the outgoing king 15 days to vacate his palace after the republic is declared, said Bimalendra Nidhi of the centrist Nepali Congress, the second largest party in the assembly.

Nidhi made the comments after his party met with the country's former rebels, the Maoists, who hold the most seats in the assembly and are expected to lead the country's new government.

There was no immediate reaction from the palace, which has rarely commented on political developments in Nepal since King Gyanendra was forced to end his royal dictatorship and restore democracy after widespread protests two years ago.

The Maoists then gave up their 10-year fight for a communist Nepal, and the election of the assembly in April marked the culmination of the peace process with the former insurgents.

The assembly is charged with governing Nepal as it rewrites the constitution. On Tuesday, 575 of its members were sworn in.

Another 26 members are to be appointed, and last-minute talks between the political parties on filling those seats and how much power to give to the newly created presidency forced the assembly to postpone its first meeting until late Wednesday afternoon.

When the assembly does finally meet, the political parties have made it clear that their first act will be to declare Nepal a republic and do away with the 239-year-old Shah dynasty, which united Nepal and has reigned ever since.

But getting rid of the Shah dynasty is in many ways the least of the new government's problems, as evidenced by a string of bombings that hit Katmandu on Monday and Tuesday, all apparently aimed at pro-republic politicians and activists.

While the four bombings only wounded two people, they underscored how difficult it will be to fashion lasting peace and bring widespread prosperity to this Himalayan land that was bled for a decade by the Maoist insurgency and is still regularly bloodied by political violence.

Authorities have deployed 10,000 policemen in Katmandu to head off more violence and banned rallies around the palace and the convention center where the assembly is meeting.

The Maoists, meanwhile, say 20,000 volunteers from their youth wing are in Katmandu to help control the celebrations. But that hasn't eased fears of violence. The young Maoists regularly are accused of intimidating, roughing up and sometimes killing opponents.

The Maoists have promised to bring sweeping change to Nepal, a largely impoverished country that in many places more closely resembles medieval Europe than a modern nation.

Once Nepal has been declared a republic no one is certain what will happen, with the Maoists still struggling to form a government and political violence persisting.

If Gyanendra peacefully leaves the palace for good, he is expected to move to the palatial private Katmandu home where he lived before assuming the throne in 2001 following a massacre at the royal palace in which a gunman, allegedly the crown prince, gunned down late King Birendra and much of the royal family before killing himself.

___

Associated Press Writer Matthew Rosenberg contributed to this story.

US stocks tumble as oil falls on economic worries

Wall Street extended last week's slide Monday as investors worried that the quarterly results companies begin releasing this week will signal the economy is in worse shape than feared.

Oil prices helped fuel the pessimism, tumbling 8 percent to a new low for the year as investors bet economic weakness would curb demand. Wall Street normally welcomes falling oil as a boost for consumers who pay less to put gas in their car, but steep drops can touch off deeper fears about the overall economy.

Wall Street is expecting fourth-quarter and full-year earnings will be particularly bleak, especially after several companies warned last week that they are being hit hard by the recession. Aluminum producer Alcoa Inc., which last week announced it would slash production, fell again Monday after an analyst lowered his rating on the stock. Alcoa said after the market closed that it lost $1.19 billion during its fourth quarter as demand for aluminum plunged.

Financial stocks also declined as investors looked to Citigroup Inc. and Morgan Stanley, which could announce a deal as soon as Wednesday to combine their brokerage operations. The potential tie-up underscores the troubles some banks are still having with tattered balance sheets, and a prominent analyst said Citigroup might still need to raise cash.

"I think that the biggest concern right now is the economy and whether this thing is going to get worse or it's going to get better," said Bernie McGinn, chief executive of McGinn Investment Management.

The intensity of the fear that permeated the market and provoked the heavy selling of September, October and November has lessened, McGinn said, but investors are still hesitant to flood back into the market. Monday's decline came on light volume, indicating an absence of buyers, not a rush of sellers. Traders said many investors were sitting on the sidelines until they get a better read on companies' quarterly numbers and, more important, their forecasts for the year.

"The level of anxiety and the level of fear has moderated some, but it sure as heck hasn't turned into optimism," McGinn said.

According to preliminary calculations, the Dow Jones industrial average fell 125.21, or 1.46 percent, to 8,473.97.

Broader stock indicators also declined. The Standard & Poor's 500 index fell 20.09, or 2.26 percent, to 870.26, and the Nasdaq composite index fell 32.80, or 2.09 percent, to 1,538.79.

The Russell 2000 index of smaller companies fell 12.50, or 2.60 percent, to 468.80.

Declining issues outpaced advancers by nearly 4 to 1 on the New York Stock Exchange, where volume came to 1.31 billion shares compared with 1.16 billion traded Friday.

___

On the Net:

New York Stock Exchange: http://www.nyse.com

Nasdaq Stock Market: http://www.nasdaq.com

International Coal Group completes debt offering

International Coal Group netted $188 million from a new debtoffering designed to ease a cash crunch that threatened three minedevelopment projects, according to a document filed with theSecurities and Exchange Commission.

A big chunk of the proceeds went to repay other debts, ICG said.The company paid off a $25 million loan from W.L. Ross & Co. Ross isICG's largest shareholder and the investment company of the company'schairman, Wilbur Ross.

Oil falls below $99 per barrel

NEW YORK (AP) — Oil prices are sliding after the U.S. government reported that crude supplies grew unexpectedly last week.

Benchmark crude fell by 73 cents Thursday morning to $98.63 per barrel in New York. Brent crude fell by 50 cents to $107.06 per barrel in London.

The Energy Information Administration says that U.S. oil supplies rose by 3.9 million barrels last week. Analysts expected supplies to shrink. The weekly report also says that U.S. oil demand has dropped by 7.8 percent from the same time last year. Gasoline demand has declined by 5.6 percent.

No. 6 Texas rolls into 'hostile' Texas Tech

LUBBOCK, Texas (AP) — Tommy Tuberville would love to give Texas Tech fans what they savored when Texas last came to town: bragging rights.

"There's some games you have at every school that your fans have a passion about," said Tuberville, who will coach his first Big 12 game Saturday night against the No. 6 Longhorns. "When I was in all those rival games at Auburn, at Ole Miss, at Miami, that's what you feel like: Do this, and your fans have the opportunity to be able to strut a little bit."

Texas, naturally, has other plans for the Big 12 opener for both teams. And this week has included a lot of chatter about that last visit back in 2008 when the Red Raiders stunned the top-ranked Longhorns 39-33 on a final-second touchdown pass to Michael Crabtree.

This time around, the Longhorns secondary is more experienced and should be ready for the Red Raiders' pass-heavy offense, while a revamped and more complex Texas Tech defense deals with a 'Horns offense that's running more than passing.

Texas safety Scott Christian said interceptions have been a hot conversation topic lately.

"We feel turnovers will be key in a game like this," he said.

Texas Tech receivers know what they're in for, said Austin Zouzalik, who's caught one of Taylor Potts' seven TD passes this season. Potts and his teammates rank 11th in passing offense (334.5 yards per game).

"It's well known all over the country that they are an athletic bunch, a well-coached bunch," Zouzalik said of the Longhorns. "They are always fast, and they are always fundamentally sound. We aren't going to get away with as much with sloppy routes."

It will be the first meeting for Mack Brown and Tuberville as head coaches. In the 1991 Cotton Bowl, Tuberville coached the Miami defense and the Hurricanes beat the Longhorns 46-3. Three years later and with Tuberville in the same job at Texas A&M, the Aggies beat Texas 34-10 in a Southwest Conference clash.

Brown is concerned about penalties (averaging 55 yards a game) and a dearth of turnovers. The Longhorns (2-0) have gotten only one fumble and one interception. They'll be facing Potts, who has not thrown an interception this year.

"We need to stay on the field offensively," said Brown, who is 3-3 in six trips to Lubbock.

And Texas wants to get on the scoreboard sooner.

"We've only scored three points in the first quarter of the first two games," Brown said. "We've scored 41 points in the second quarter."

Texas quarterback Garrett Gilbert is young and inexperienced, Tuberville said, but grew up a lot when he had to replace Colt McCoy in January's 37-21 loss to Alabama in the national title game.

Potts, a senior, has more experience, and he's played against Texas and in other big games.

"I think that's an advantage for us," Tuberville said. "Who makes the least mistakes I think that will be a huge factor between those two guys.

Of course, Tech's defense will be tested on the ground as well as in the air. And after last week, that gives Tuberville some pause: The Red Raiders gave up 433 yards and 23 first downs in their 52-17 win at New Mexico.

"They're going to run it down hill. They're going to play action," Tuberville said. "It'll be interesting to see how we hold our own defensively in terms of executing what we've tried to do the first two weeks, which we've done pretty well at times and sometimes we haven't looked very good."

A win over a "very good" Texas team likely jump Texas Tech into the Top 25, said Tuberville, though he thinks his team is already there.

"You know that really doesn't make any difference when you got to put a game plan together," he said.

What might make a difference is the game is in Lubbock.

"It is hostile," said Christian, the Texas safety. "It seems a little different for me than any other atmosphere, but the game is still played the same way so you just try to tune out the atmosphere."

SAfrica needs to shed inferiority against Brazil

South Africa's players have to overcome the talent of Brazil and their understandable feelings of inferiority if they are to carry the hopes of a nation and reach the Confederations Cup final.

Like any opponent going up against the record five-time World Cup winners, South Africa knows it is as good as beaten if it goes onto the field for Thursday's semifinal match star-struck by former world player of the year Kaka and striker Robinho.

The likes of burly central defender Matthew Booth cannot match the South Americans when it comes to skill, so Bafana Bafana is relying on raucous home support to rouse them to a performance of greater intensity than their opponents can muster.

"We don't want to get carried away putting these guys up on a pedestal," South Africa goalkeeper Rowen Fernandez said. "Yes, they're idols to millions and we respect them all as footballers, but we're going to go out there Thursday and compete on the pitch.

"It's 11 versus 11, obviously, and I'm sure Matthew is dying to get stuck into Robinho and give Kaka a piece of his mind."

Fernandez and Booth are among four players in the South African squad that beat Brazil at the 2000 Sydney Olympics, but an under-23 tournament is quite another thing compared to a full international like Thursday's at Ellis Park.

"Playing for a draw against them isn't going to do us any good," South Africa midfielder Teko Modise said. "They're going to allow us space and time to attack them and to play and we'll look to utilize that. We really want to play our hearts out and attack. We need to score as many goals as we can because we all know they're capable of scoring many.

"When we first came into the camp, we said we want to go to the semis and, now we're here, we also want to go to the final as well. It's possible. The Brazilians are not unbeatable."

But Brazil has racked up 10 goals in group games against World Cup holder Italy, African champion Egypt and the United States, compared to the two South Africa managed in a significantly easier group.

"But by the same token, we might look on the positive side and say they also conceded goals," South Africa striker Katlego Mashego said. "It's a plus for us that they can concede goals. We get them tired, we might sneak in and have one or two chances. It's not impossible. We just have to give our all."

And South Africa could have the right player to take advantage of any slips by a defense that conceded three times in a 4-3 win over Egypt before tightening for 3-0 wins over the United States and Italy.

Bernard Parker made his first mark on the tournament when he stopped his own teammate from scoring a winning goal against Iraq by blocking a shot on the line, but the striker then scored both goals against New Zealand and has hit a tournament-high 10 shots on target.

Spain striker David Villa is way back in second place with seven on-target efforts, albeit with three goals.

Brazil, though, has the statistic that counts.

The South Americans have scored more goals than anyone else, two more than European champion Spain.

With Luis Fabiano, Robinho and Kaka exchanging positions fluidly in attack, Booth and his colleagues could be in for a torrid time.

Even so, Brazil will be without Juan after the key defender was ruled out for the remainder of the tournament with a left thigh injury and the players were not taking anything for granted ahead of a match they are overwhelming favorites to win.

"Tradition doesn't win or lose matches," Luis Fabiano said. "In a semifinal, you have to be careful. To make the final we need to enter the match with the same concentration and motivation that we had against Italy.

"It's going to be a difficult match. They are playing at home and will give their lives against Brazil."

One thing that is guaranteed is that Ellis Park will echo to the near-deafening noise of local supporters blowing their vuvuzela trumpets, something that those unfamiliar with South African football have said can be distracting.

"They are expressing themselves and they have a unique way of getting behind the team," Fernandez said. "The players, the South African ones anyway, are comfortable with it.

"If it works as a psychological benefit for us, I hope the supporters go on Thursday and blow even louder."

The truth is out there, but so are the fantasies

Sen. William Roth (R-Del.) promised earlier in his Senate careerthat he would not run for office after he turned 70. He is now 78,and is gearing up to run next year for his sixth six-year term. Heboasts many triumphs as chairman of the Senate Finance Committee,including 1998 legislation that made it harder for the IRS to collectpenalties from tax-evaders. Thanks to the law, collection ofpenalties was down nearly 80 percent in the last fiscal year.

No wonder Roth got his bill through. His committee turned uphorrendous evidence of IRS abuse. A man testified that agents stormedinto his house, drew a gun on his son, knocked the son down andwatched as his daughter changed clothes. This was riveting stuff. Itmade the network news and was widely reported in print. It confirmedmany people's suspicion that the IRS is a fascist outfit. Roth madeit a dramatic part of his book The Power to Destroy.

There is only one thing wrong with this gripping tale: It isn'ttrue. In depositions for a civil suit, the son has said that heneither saw a gun nor was knocked down. The daughter under oath saysthe agents did not watch her undress.

The ironic thing is that the IRS could not defend itself fromthese charges when they were brought before the committee because itis not a fascist organization. It was observing its own rules ofconfidentiality for the man making reckless accusations. The WallStreet Journal, in a story reporting these developments, quotescritics of the Roth law as saying this "shows the risk of legislatingbased on horror stories."

It also shows how willing people are to believe the worst aboutthe IRS in particular and government agencies in general. The oldhysteria in America shouted, "The communists are coming! Thecommunists are coming!" A more lasting cry in this country has been,"The government is coming! The government is coming!" Naturally, noone likes to pay taxes, and griping about that can settle on thecollectors, rather than the legislators who determine what we pay.Even in this country, which has a lower tax rate than any of themajor industrial nations, we are restive under the necessity. Butdemonizing the civil servants who carry out their jobs is the path tothe Oklahoma City bombing, where government workers were destroyed asif they were not our fellow citizens but some alien creatures lackinghumanity.

If the IRS had been staging a fascist-type raid on the man whotestified, it would not have been accompanied by local police andstate beverage agents, acting under legal warrant. The IRS' actionswere under scrutiny from authorities not willing to be compromised byother people's violence or unlawful conduct. That is why the witness'children could not back up his story of their own brutalization.There were too many eyewitnesses, from too many separate authorities,to make the story stick. Roth's committee could have checked theallegations with local police who accompanied the IRS. But itpreferred the horror story. It would be good for the senator's book.

As it turned out, the IRS had been misled by an untrustworthyinformant in this case. It was in error. But common sense should havemade people hesitate to believe a man who claimed the agency hadchosen such a procedure, and such associates, as a successful way toknock citizens about, flourish guns and leer at a teenager.

Unfortunately, common sense does not go very far when people areout to demonize an enemy. It certainly did not go far with WilliamRoth.

Garry Wills is adjunct professor of history at NorthwesternUniversity and winner of numerous awards, including the PulitzerPrize and the National Humanities Medal.

FBI report: 'Hate crimes fueled by race'

Racial bias fueled the 44.9 percent of 9,730 reported hate crimes for 2001 that were committed mostly by whites, according to a FBI's Uniform Crime Reporting (UCR) report which concluded these acts are on the rise.

According to that document, last year, both state and local law enforcement agencies reported to the FBI 9,730 incidents involving 11,451 separate offenses.

But, of the 9,730 incidents reported, 9,721 were single-bias incidents, or those that involved a singular bias motivation.

Of the 9,721 single-bias incidents, 44.9 percent were motivated by racial bias, according to the report, with 21.6 percent being driven by prejudice against an ethnicity or national origin.

The majority of known hate crime offenders (65.5 percent) were white, 20.4 percent were Black, 8.2 percent were of unknown race, and the remainder were of other races or were members of a group that consisted of offenders of varying races.

The majority of hate crime incidents (30.9 percent) occurred in or on residential properties. Highways, roads, alleys, or streets were the settings for 18.3 percent of the reported incidents, and 10.1 percent took place at schools and colleges. The remaining incidents were distributed among various locations.

Other incidents included: 18.8 percent which resulted from a bias against a particular religion, 14.3 percent involved a bias against sexual-orientation, and 0.4 percent which was motivated by a disability bias.

The document listed 11,451 hate crime offenses reported last year with 67.8 percent of the crimes being against persons, and 31.5 percent of the offenses as crimes against property. Federal officials concluded that intimidation continued to be the most frequently reported hate crime offense committed against individuals.

These acts accounted 55.9 percent of all crimes against persons. At 83.7 percent, the offense of destruction/damage/vandalism of property was the most frequently reported crime hate crime.

However, less than 1 percent (0.6 percent) of hate crimes were crimes against society.

During 2001, there were 12,020 total victims of hate crime. Of that total, 11,998 were victims of single-bias incidents. Of the 11,998 victims of single-bias incidents, 46.2 percent were victims of racial prejudice, 22.0 percent were victims of ethnicity or national origin bias, 17.7 percent were targets of religious intolerance, 13.9 percent were attacked because of sexual orientation and 0.3 percent were victims of a disability bias.

Of the 12,020 total victims of hate crimes, 22 were victims of multiple-biases.

According to the report, 10 of the hate crime victims were murdered in 2001. Five of these homicides were attributed to a bias against an ethnicity or national origin, four involved racial bias and one was driven by bias against a sexual orientation.

Law enforcement agencies reported 9,239 known offenders in connection with the 9,730 incidents reported in 2001.

Last year, 11,987 law enforcement agencies contributed hate crime data to the UCR Program, and approximately 17.6 percent of those agencies submitted reports to the FBI that at least one hate crime occurred in their jurisdictions.

FBI officials say these figures indicate a slight increase over the number of agencies submitting data in 2000.

Jesse optimistic on S. African trade mission

Jesse optimistic on S. African trade mission

In a telephone call-in from Johannesburg, S.A. on his weekly radio broadcast at Dr. King's Workshop, the Rev. Jesse L. Jackson Sr. said he hopes his trip will stimulate trade between Africa and African American businesses in a major way.

Leading a three-nation tour, where he was joined by his wife, Jacqueline, son, Jonathan, and a delegation of business persons from his Rainbow PUSH/Wall Street, LaSalle Street Trade Bureaus, Jackson said the trip is designed to stimulate trade between African American firms and Africa.

He's hoping to bridge the digital divide between the two continents.

Having visited Accra, Ghana, Lagos, Nigeria and Johannesburg and Cape Town in South Africa, Jackson said he has focused on debt relief, the devastating impact of AIDS on that continent where he's trying to mobilize world opinion on this issue, as well as business investment.

Jackson wants to engage in trade partnerships with African nations and their leaders. He's scheduled to return to Chicago Thursday.

Jackson told the audience while many will celebrate Valentine's Day, "this year, we'll have 2 million in jail of whom 55 percent are Black with more than half on non-violent drug charge. These laws are insane on one hand and very profitable on the other.

"We will not stop until we rescue our children and reclaim them and turn these jails into museums," he told a cheering audience.

Jackson said this Saturday at Dr. King Workshop, 930 E. 50th St., he's conducting a voter registration drive because it is the last Saturday for residents to register in order to vote in the March 21 primary.

He said South Africa "paid a big price for the right to vote and now they are voting. We paid a big price for the right to vote, and we're not voting."

Jackson also announced that Rubin "Hurricane" Carter, the boxer who was the subject of the movie "Hurricane," will join him Friday where he'll show the film to Cook County Jail inmates. Carter will also be at Dr. King's Workshop, 10 a.m., Saturday Feb. 19th as well.

Jackson, who recently appeared on WVON's Cliff Kelley show, said the theme of his trip is: "Connecting Telecommunications and Media Markets Through U.S. Minority and African Business Partnerships."

Article Copyright Sengstacke Enterprises, Inc.

Photo (Jesse Jackson Sr.)

Officials OK new Charleston subdivisions

Charleston officials have approved several new subdivisions.

Members of the Municipal Planning Commission also approved arequest to abandon parts of two little-used streets.

Members of the Municipal Planning Commission gave final platapproval to Pison Development, which is building a 22-lot subdivisioncalled The Woodlands off Clark Road. The access road begins outsideof city limits but part of the development lies within cityboundaries.

Commission members also approved Robert Swoope's plan to create afive-lot Heritage Ridge Subdivision at 1978 Smith Road. The projectwas delayed for more than a year while the developer met requirementsof the Charleston Sanitary Board.

Jamestown developer George Neilan won the commission's blessingfor the already built, relocated section of Salem Road, and for hisapplication to dedicate the road as a city street, but withconditions.

Commission member and city Councilwoman Mary Jean Davis said shewants to have a letter of approval from the city Street Departmentbefore council's Planning Committee takes up the two road measures.Davis also stipulated that Neilan post two required bonds for thestreet. City Council has final approval on the matters.

Finally, commission members approved Marathon Petroleum LLC'srequest for the city to abandon Standard Street and part of CreelAvenue.

The two streets lie within the property of Marathon's petroleumproducts river terminal off MacCorkle Avenue, lawyer Nick Casey said.Marathon will pay the city $1 per square foot, or about $25,000, forthe property, he said.

The city's Planning Department supported the street closings forseveral reasons, Planning Director Dan Vriendt said the city would nolonger have to do maintenance or have liability for the streets, andthe closings would help Marathon meet Homeland Security requirements.

8 inmates killed in Syria prison fire

DAMASCUS, Syria (AP) — Syria's state-run news agency says eight prisoners have died of suffocation and burns after an inmate set fire to mattresses.

The fire broke out Monday in the coastal city of Latakia. SANA quoted Latakia police chief Kamal Fleih as saying prisoners were mostly murder and drug convicts.

He said two policemen were wounded while trying to extinguish the fire.

Latakia already has become a flashpoint for violence in recent weeks as Syria faces a wave of antigovernment protests. The city has a potentially volatile mix of different religious groups.

NASCAR's Roush OK after plane crash

NASCAR team owner Jack Roush is OK after he was involved in a plane crash in Wisconsin, Roush Fenway Racing president Geoff Smith tells The Associated Press.

Smith didn't immediately have additional details of the crash. Roush, an aviation buff, was expected to attend the Experimental Aircraft Association's annual AirVenture in Oshkosh, Wis., this week.

The EAA was expected to post a statement on its website Tuesday night.

It is the second close call for Roush, who crashed a plane into a pond in Alabama in 2002 and was rescued by an ex-Marine who lived nearby.

Jackson estate in record deal worth up to $250 mln

Even in death, Michael Jackson is breaking new records.

The King of Pop's estate has signed the biggest recording deal in history: a $200 million guaranteed contract with Sony Music Entertainment for 10 projects over seven years, according to a person familiar with the deal.

The record-breaking contract through 2017 could be worth up to $250 million if certain conditions are met. One of the albums will be of never-before-released Jackson recordings that will come out in November, the person said.

The person spoke on condition of anonymity because the official announcement is expected later Tuesday.

Future projects may also include a video game, a DVD compilation of videos and a rerelease of "Off the Wall," Jackson's fifth studio album, which first came out in 1979, accompanied by some unreleased material. Before his sudden death in June at age 50, the pop star had wanted to reissue the album, people familiar with the deal said.

One of the projects already counted in the contract was the two-disc album that accompanied "This Is It," the film based on footage of concert rehearsals for what was to have been Jackson's comeback at London's O2 arena.

Including the more than 5 million copies of that special release, Jackson has sold some 31 million albums since his death, about two-thirds of them outside the United States.

"During his life, Michael's contracts set the standard for the industry," said John Branca, the co-administrator of the Jackson estate, in a statement prepared for release Tuesday. "By all objective criteria, this agreement with Sony Music demonstrates the lasting power of Michael's music by exceeding all previous industry benchmarks."

Rob Stringer, chairman of Sony Music's Columbia Epic Label Group, said in prepared remarks, "We're dedicated to protecting this icon's legacy and we're thrilled that we can continue to bring his music to the world for the foreseeable future."

The landmark deal is worth more than all other benchmarks, such as the all-encompassing rights deals that concert promoter and ticket-seller Live Nation Entertainment Inc. had previously signed with Madonna at $120 million and Jay-Z for $150 million.

Jackson's deal is even more remarkable because it does not include royalties from merchandise.

The contract shows the value of legacy artists. It also comes at a time of decline for the music industry, with sales down about half from their peak in 2000 mainly due to free file-swapping.

The money will go a long way to settling Jackson's debts, estimated at around $400 million when he died. But the singer whose life was plagued with scandal has had a resurgence in popularity in death.

Distribution rights for "This Is It" were sold to Sony Pictures, another unit of Sony Corp., for $60 million and the movie went on to gross $252 million worldwide, the most of any concert film ever.

Revenue from that, song sales and merchandising agreements brought into the estate revenues of about $100 million, lawyers for the estate's administrators told a Los Angeles Superior Court judge in December, when they sought a percentage as an administration fee.

The Walt Disney Co. even brought back the 17-minute Jackson movie "Captain EO" to its Disneyland theme park in Anaheim last month. The original began running at the park in 1986 but was pulled in 1997.

Jackson's most lasting and valuable asset is the 50 percent stake in Sony/ATV Music Publishing, a company that owns publishing rights to music by The Beatles and numerous other artists, including Elvis Presley and Bob Dylan. Split with Sony Music, the copyright catalog itself is estimated to be worth $2 billion.

The new financial windfall comes even as circumstances around his death remain in legal limbo.

Dr. Conrad Murray faces an involuntary manslaughter charge for allegedly giving Jackson a lethal combination of sedatives. He is due back in a Los Angeles court April 5.

Neutrophil-Bead Collision Assay: Pharmacologically Induced Changes in Membrane Mechanics Regulate the PSGL-1/P-Selectin Adhesion Lifetime

ABSTRACT

Visualization of flowing neutrophils colliding with adherent 1-�m-diameter beads presenting P-selectin allowed the simultaneous measurement of collision efficiency (ε), membrane tethering fraction (f), membrane tether growth dynamics, and PSGL-1/P-selectin binding lifetime. For 1391 collisions analyzed over venous wall shear rates from 25 to 200 s^sup -1^, ε, decreased from 0.17 to 0.004, whereas f increased from 0.15 to 0.70, and the average projected membrane tether length, L^sup m^^sub tether^. increased from 0.35 �m to ~2.0 �m over this shear range. At all shear rates tested, adhesive collisions lacking membrane tethers had average bond lifetimes less than those observed for collisions with tethers. For adhesive collisions that failed to form membrane tethers, the regressed Bell parameters (consistent with single bond Monte Carlo simulation) were zero-stress offrate, k^sub off^(0) = 0.56 s^sup -1^ and reactive compliance, r = 0.10 nm, similar to published atomic force microscopy (AFM) measurements. For all adhesion events (� tethers), the bond lifetime distributions were more similar to those obtained by rolling assay and best simulated by Monte Carlo with the above Bell parameters and an average of 1.48 bonds (n = 1 bond (67%), n = 2 (22%), and n = 3-5 (11%)). For collisions at 100 s^sup -1^, pretreatment of neutrophils with actin depolymerizing agents, latrunculin or cytochalasin D, had no effect on ε, but increased L^sup m^^sub tether^ by 1-74- or 2.65-fold and prolonged the average tether lifetime by 1.41- or 1.65-fold, respectively. Jasplakinolide, an actin polymerizing agent known to cause blebbing, yielded results similar to the depolymerizing agents. Conversely, cholesterol-depletion with methyl-β-cyclodextrin or formaldehyde fixation had no effect on ε but reduced L^sup m^^sub tether^ by 66% or 97% and reduced the average tether lifetime by 30% or 42%, respectively. The neutrophil-bead collision assay combines advantages of atomic force microscopy (small contact zone), aggregometry (discrete interactions), micropipette manipulation (tether visualization), and rolling assays (physiologic flow loading). Membrane tether growth can be enhanced or reduced pharmacologically with consequent effects on PSGL-1/P-selectin lifetimes.

INTRODUCTION

Neutrophils, platelets, and endothelium interact through various bonding mechanisms during inflammation and thrombosis. P-selectin, a dimer presented on activated platelets or activated endothelium, helps to capture flowing neutrophils via P-selectin glycoprotein ligand-1 (PSGL-1) located on neutrophil microvilli. Under flow conditions, the adhesive function of P-selectin/PSGL-1 bonding depends on the sum of discrete chemical interactions in the bond interface as well as the dynamics of macroscopic force transmission to the molecular level via the deformable microvilli. Cellular processes such as membrane tether formation and whole cell deformation affect selectin bond rupture by altering force transmission.

The Bell model is an empirically useful approach for quantifying the force sensitivity of bond lifetimes where k^sub off^ = k^sub off^(0) exp(rF^sub B^/k^sub B^T) and k^sub off^ is the dissociation constant; F^sub B^ is the force on the bond; k^sub off^(0) and r are the experimentally determined unstressed off-rate and reactive compliance, respectively; k^sub B^ is the Boltzmann constant; and T is the temperature. Bell model parameters have been measured for selectin-ligand pairs in a variety of ways, including rolling cells and/or rolling beads (1-5); atomic force microscopy (AFM (6-8)); molecular force probe (9,10); laser trap (11); and dynamic force spectroscopy (DFS (12-14)). Values of k^sub off^(0) determined by extrapolation to zero force for PSGL-1/P-selectin are consistent with those obtained in solution phase using plasmon resonance (15). These different mechanical methods create various force loading profiles to drive bond rupture dynamics (16). At venous wall shear rates of 25-200 s^sup -1^, physiological loading during neutrophil capture is characterized by rapid loading (~1-50 ms) to a peak value (~10 pN to 100s pN) with subsequent force relaxation due to tether pulling (17-19). Experimental challenges include whether only single bond interactions are being evaluated (20), the requirement of estimating forces and geometries on both microscopic and macroscopic length scales, the integrity of purified molecule presentation, and the temporal/spatial resolution of each method. In general, the k^sub off^(0) of L-selectin is considered to be greater than that of P-selectin or E-selectin, consistent with the fact that neutrophils roll on L-selectin at a velocity 7.5-11.5 times faster than they do on either P or E-selectin (21).

The dimeric PSGL-1 molecule is located primarily on the microvilli tips of neutrophils. Microvilli extend ~0.25-0.35 �m from the neutrophil surface as measured by electron microscopy or micropipette aspiration (17), and as calculated based upon comparison of the off-rates of fixed cells and beads (3). Micropipette aspiration techniques have been developed to explore the mechanics of tether formation from neutrophils (17,22,23) and from endothelial cells (24). Membrane tether formation using micropipette manipulation has revealed two regimes of tether growth: microvilli extension, characterized by linear elastic deformation of the neutrophil (F^sub B^ < 34 pN); and membrane tether formation, in which lipid membrane flows freely and at a constant velocity when F^sub B^ is >~45-61 pN (17,19,22). Between 34 and 61 pN, the formation of a membrane tether versus an extended microvillus depends upon the degree of interaction between cytoskeleton and membrane (17). Membrane tether pulling during flow perfusion over adherent patelets or selectin-coated surfaces is visualized as translation of the cell downstream of an adhesion point at a constant velocity well below that of the free-stream velocity (18).

Pharmacological agents targeting actin polymerization can be used to study the relationship between the cytoskeleton function and cell mechanics. Actin polymerization and membrane fluidity in the leukocyte can be altered using actin-polymerizing agents such as jasplakinolide or depolymerizing agents such as cytochalasins or latrunculin (25-27). Cytochalasins depolymerize actin, although their modes of action and specificity are varied (26). Latrunculin A is a more specific actin depolymerizing agent that binds to actin monomer and prevents incorporation into F-actin. Jasplakinolide increases actin polymerization and stabilization by decreasing the amount of sequestered actin and enhancing F-actin nucleation (28-29) with some observed cytotoxicity (30). Neutrophils treated with cytochalasin B or D exhibit a decrease in cortical tension and cytoplasmic viscosity during aspiration into micropipettes (31-33) or tether extraction by micropipette (23). Similar increases in deformability after treatment with cytochalasin B were also observed in eosinophils (27). Perfusion of neutrophils treated with cytochalasin is associated with localization of PSGL-1 to the pointed ends of teardrop-shaped cells (34). Actin depolymerization is also associated with a loss of membrane ruffles, as evidenced by 1), the ability of cytochalasin B to abolish tether formation (35); and 2), the smooth shape of latrunculin A-treated neutrophils under scanning electron microscope (SEM) (36). In contrast, jasplakinolide treatment of neutrophils results in an increase in global rigidity as measured by aspiration into a micropipette (37). However, AFM elasticity studies of jasplakinolide, cytochalasin, or latrunculin-treated neutrophils suggests that all three reagents reduced the local Young's modulus of the plasmalemma, possibly due to the effect that jasplakinolide caused disaggregation but not disassembly of the actin fibers (38). Also, jasplakinolide can cause membrane blebbing (37). In contrast, the membrane cholesterol content of a cell can also be manipulated to study the effects of membrane fluidity on cell adhesion. Methyl-β-cyclodextrin (MβCD) can be used alone or in complexes with cholesterol to deplete or enrich the plasmalemma with cholesterol, respectively (39). Cholesterol depletion in neutrophils has been shown to be associated with resistance to deformation during aspiration (33), as does chemical fixation. Unexpectedly, MβCD has been reported to reduce neutrophil actin polymerization (40).

To study the effect of neutrophil membrane tether formation on selectin bond mechanics, we developed a new method of probing interactions between a flowing cell and a small point source of ligand presented on a 1-�m-diameter adherent bead. The advantages of this approach:

1. Discrete reactions can be probed that have a defined initiation time.

2. The small bead perturbs the laminar flow field to a minimal extent, but provides excellent ligand presentation for bonding analogous to the raised platelet pseudo-nucleus or the endothelial nucleus.

3. Adhesion contact area is exceedingly small, and minimizes bonding to a single tether.

4. Both on-processes (i.e., the collision efficiency) and off-processes (i.e., bond lifetime) are probed simultaneously.

5. There is minimal interference of cell body bonding with the surface of the flow chamber.

6. Physiologically relevant force-loading dynamics are created by hydrodynamic flow.

7. High temporal and spatial resolution allows detection and analysis of membrane tether and bond dynamics.

EXPERIMENTAL PROCEDURES

Materials and neutrophil isolation

Human serum albumin (HSA; Golden West Biologicals, Temecula, CA) and fibrinogen (Enzyme Research Labs, South Bend, IN) were stored according to manufacturers' instructions. Polystyrene, 1.05-�m-diameter microspheres coated with Protein A were obtained from Bangs Labs (Fishers, IN). Labeling the beads with P-selectin was accomplished through the use of a recombinant human P-selectin/Fc chimera (R&D Systems, Minneapolis, MN) where the Fc region is that of human IgG^sub 1^. Jasplakinolide, cytochalasin D, and methyl-β-cyclodextrin (MβCD) were purchased from Sigma-Aldrich (St. Louis, MO). Latrunculin A was from R&D Systems and was a kind gift from Dr. P. Janmey (University of Pennsylvania). A monoclonal antibody against human P-Selectin glycoprotein ligand-1 (R&D Systems) was used in control experiments. Formaldehyde used in neutrophil fixation experiments was obtained from Fisher Scientific (Pittsburgh, PA).

Human blood was obtained via venipuncture from healthy adult donors who had not taken any medications in the prior 10 days. Blood was mixed with anticoagulant sodium citrate (9 parts blood to 1 part citrate). Neutrophils were isolated by centrifugation with a separation medium (Cedarlane Laboratories. Hornby. Ontario) as previously described (18,41). Neutrophils were counted and diluted with a 2% solution of human serum albumin in Hank's Balanced Salt Solution (HBSS; Invitrogen, Carlsbad, CA) with Ca^sup 2+^ to a final concentration of 1-2 � 10^sup 6^ cells/ml. In some cases, neutrophils were treated with jasplakinolide (10 �M), cytochalasin D (15 �M), latrunculin (0.3 �M), or MβCD (10 mM) for 30 min at room temperature before perfusion. In the case of treatment with formaldehyde, cells were fixed for 15 min with a 3% formaldehyde solution. The cells were then washed and resuspended in HBSS with Ca^sup 2+^ and 2% HSA.

Neutrophil-bead collision assay

Microspheres coated with Protein A were labeled with P-selectin by the method described by Rodgers, et al. (42). Beads were incubated with periodic vortexing for 45 min at room temperature with up to 0.2 �g/ml P-selectin-IgG chimera. This yielded a Protein A-saturated surface density of~5.4 � 10^sup 3^ IgG-chimera/�m^sup 2^. After labeling, the microspheres were then washed three times with a solution of 1% HSA in HBSS. In preparation for attachment to the chamber, the beads were then incubated for 4 h at 4�C with 200 �g/ml fibrinogen. Rectangular glass capillaries (Vitrocom, Mountain Lakes, NJ) having a cross-section of 0.2 � 2.0 mm, a length of 7 cm, and a wall thickness of 0.15 mm were used as flow chambers, as described previously (18,41). Chambers were incubated with fibrinogen (100 �g/ml) for 120 min at room temperature. The microspheres were perfused into the chambers, and the bead-coated chambers were incubated at room temperature overnight in order for the beads to have full adhesion to the capillary surface. Bead-coated flow chamber surfaces were then washed and blocked with HBSS with 2% HSA for 5 min before perfusion studies. Fibrinogen caused no β^sub 2^-integrin mediated adhesion since tests employing anti-PSGL-1, calcium-free buffer, or P-selectin-free beads all resulted in zero detectable adhesion (see Results), and resting neutrophils were used that failed to adhere to the chamber walls even at γ^sub w^ = 25 s^sup -1^.

Neutrophils were perfused into the chambers using a syringe pump (Harvard Apparatus, Holliston, MA) at various wall shear rates (γ^sub w^) as calculated via the Navier-Stokes equation for laminar flow of a Newtonian fluid: γ^sub w^ = 6Q/B^sup 2^W, where Q represents the flow rate (cm^sup 3^/s), B is the total plate separation (0.02 cm), and W is the width (0.2 cm). The wall shear stress can be calculated from τ^sub w^ = (6Q/μ)/(B^sup 2^W), where μ is the buffer viscosity (0.01 Poise at room temperature). For differential interference contrast microscopy, a Zeiss Axiovert 135 microscope with a 63 � (NA 1.40) oil immersion objective lens (Plan Apochromat) was used (Carl Zeiss, Jena, Germany). For high-speed imaging, images were captured using a Motion-Corder Analyzer high-speed digital camera (Eastman Kodak, Rochester, NY) at an imaging rate of 240 frames per second (fps). The images were played back at 5 fps or frame-by-frame to videotape for analysis.

During frame-by-frame analysis, adhesive interactions were identified as those collisions that had a visible pause in neutrophil motion lasting for at least one frame, and had velocities that had decreased below the hydrodynamic velocity (Fig. 1, A-D). An adhesion collision efficiency (ε) was calculated from these observations as the number of collisions resulting in an adhesive interaction, divided by the total number of collisions observed. The lifetimes of all adhesive interactions were recorded. These values always greatly exceeded those of simple hydrodynamic collision without bonding. Adhesive neutrophils were defined as tether-forming when they translated in the direction of flow at a velocity below the hydrodynamic stream velocity (18). Membrane tethering fraction (f) was defined as the ratio of tetherforming events to the total number of adhesive interactions. Tether lengths were measured using Scion Image or ImageJ software (National Institutes of Health, Bethesda, MD) from the center of the adhesive bead to the lagging edge of the neutrophil. Approach and release velocities were determined by collecting images of neutrophils as they approached, interacted, and released from a bead at 240 fps.

Estimating force on the P-selectin/PSGL-1 adhesive tether

Kinetic Monte Carlo (MC)

Simulations were performed as previously described (44) using Gillespie's algorithm for stochastic reactions (45). Four possible event types were allowed at a given time-step: 1), breaking a P-selectin/PSGL-1 bond; 2), creating a new bond; 3), growth of the tether; and 4), departure of the neutrophil from the bead after the final bond broke. A bond was defined with respect to the surface densities on a per-protein interface basis such that a total of two bonds could be counted between one PSGL-1 and one P-selectin molecule, since both are dimers. According to the stochastic theory, each of the events has the following probability: 1). a^sub 1^dt = k^sub off^(F.r)ndt; 2),a^sub 2^dt = k^sub on^ X^sub PSGL-1^ X^sub P-sel^dt; 3), a^sub 4^dt = v^sub t^/d^sub t^dt; and 4), a^sub 3^dt = γδ^sub n,0^dt. The contact area (0.071 �m^sup 2^) was assumed to be a circle with the diameter of the tether (d^sub t^, ~ 300 nm; (46)). We assumed that the initial conditions for the simulations consisted of n = 1 or 2 bonds, X^sub PSOL-1^ = 100 free PSGL-1 and X^sub P-sel^ = 770 P-selectins (for ~50 PSGL-1 dimers per microvillus (47,48), for 10-15 thousand dimers on 252 microvilli per cell (48,49): ~5.4 � 10^sup 3^ P-selectin chimeras/�m^sup 2^). For each event simulated, the tether growth velocity was randomly selected from the experimental mean tether growth rale (v^sub t^) distribution obtained at each wall shear rate (γ). The value δ^sub n,0^ is the δ-function and equals 1 when no bonds are present, thus allowing escape of the neutrophil.

RESULTS

Adhesion of neutrophils during collision with P-selectin-coated beads

Using 240-fps imaging, we detected the generation of stress lines, or pulling of the ruffles in the membrane of the neutrophil during adhesion to the bead (Fig. 1 C). This rapid change in morphology represents a direct visualization of the earliest phase of membrane stressing and microvilli extension under flow conditions. Such stress-line features were not present for cells that collided with the bead but failed to form an adhesion to the bead. As the adhesive collision progressed, the formation of membrane tethers began as the neutrophil translated downstream of the bead (Fig. 1 B). Because we were only able to image membrane tethers that grew past a defined minimum discernible length of 0.5 �m (Fig. 1 A), the membrane tether formation that we observed was in the regime of membrane tether formation and not microvillus extension.

We measured the adhesion collision efficiency ε for neutrophil collisions with 1.0-�m P-selectin-coated beads at various shear rates for three separate donors. The collision efficiency decreased from ε = 0.17 to 0.0043 as γ^sub w^ was increased from 25 to 200 s^sup -1^ (Fig. 2 A). The specificity of bonding interaction was verified at 100 s^sup -1^ through the use of anti-PSGL-1 monoclonal antibody, adherent beads containing no P-selectin chimeric IgG, and calcium-free HBSS. In each of these three control cases, no adhesive collisions between neutrophils and beads were observed in over 150 collisions (ε = 0) monitored at each condition (data not shown), compared to ε = 0.079 for P-selectin-coated beads.

The membrane tethering fraction (f), defined as the fraction of adhesive collisions resulting in the formation of an observable membrane tether, was measured at each shear rate (Fig. 2 B). The averages shown in Fig. 2 B were over several donors, with N > 3 donors in all cases except at γ^sub w^ = 200 s^sup -1^, where N = 2 due to low capture efficiency (ε = 0.0043). Neutrophils were least likely to form membrane tethers at the lowest shear rates, where membrane tether formation fraction was 0.15 � 0.08 at γ^sub w^ = 25 s^sup -1^. The tethering fraction increased to a plateau of ~0.62-0.70 at γ^sub w^ ≥75 s^sup -1^. indicating that neutrophil rolling at venous flows is typically accompanied by tether pulling.

We measured the projected membrane tether length, L^sup m^^sub tether^, for each interaction and at each shear rate. For adhesive interactions that formed tethers of length <500 nm (below detection), we assigned a length of 0 �m. For average membrane tethers lengths calculated for all adhesive interactions, L^sup m^^sub tether^ increased from a minimum of 0.35 � 0.9 �m at γ^sub w^ = 25 s^sup -1^ to a maximum of ~2 �m at γ^sub w^ ≥ 75 s^sup -1^ (Fig. 2 C), in agreement with prior estimates obtained by reconciling neutrophil and rigid bead pause time distributions (3).

At all shear rates tested, the longevity of binding events that had tethers exceeded the lifetime of events lacking tethers (p < 0.005) (Fig. 2 D). We found that the average overall lifetime of adhesion decreased with increasing wall shear rate from 1.38 � 0.96 s at 25 s^sup -1^ to 0.14 � 0.11 s at 200 s^sup -1^ (Fig. 2 E). The average lifetime for interactions with and without membrane tether formation exhibited the same trend, decreasing with an increase in wall shear rate (Fig. 2 D). Due to the overall long lifetimes of adhesive bonds at low shear rates, the effect of tether formation at γ^sub w^ < 50 s^sub -1^ was to increase the overall lifetime of the bond by ~1.5-fold. However, at wall shear rates s 100 s^sup -1^, adhesive interactions with membrane tethers experienced lifetimes 3-3.2-fold greater than those without membrane tether formation. These prolongations are consistent with the original predictions of Shao et al. (17). Under all shear conditions, the life of adhesive events greatly exceeded that of nonadhesive hydrodynamic collisions (Fig. 2 E).

From tether lifetime and length data, we calculated a membrane tether growth rate (v^sub tether^) for each membrane-tether-forming interaction. These growth rates were averaged at each shear rate tested, and the resultant velocities were plotted as a function of shear rate (Fig. 3). The tether growth rate increased linearly from an average v^sub tether^ = 1.4-21.3 �m/s over the shear range tested. These results agree with those previously obtained for neutrophil interactions with adherent platelets (18). The critical wall shear rate (x-intercept of Fig. 3) to initiate tether growth was calculated as γ^sub w^ = 24 s^sup -1^ corresponding to a critical force of F^sub crit^ = 24.3 pN (�~10%), slightly lower than prior measurements made using micromanipulated neutrophils (22,23).

Bell model parameters for PSGL-1/P-selectin

In an effort to characterize the performance of the neutrophil-bead collision assay relative to other assays, we analyzed adhesion lifetime distributions to facilitate the comparison to earlier studies that employed pause-time analysis of flow-chamber rolling data or bond-lifetime distributions from AFM or DFS. To calculate Bell model parameters for the interaction between neutrophils and beads coated with P-selectin, apparent k^sub off^ values were calculated at each wall shear rate using the average force calculated for the events (using LL^sup m^^sub tether^ for each event). In reality, the set of events collected at each wall shear rate was heterogeneous, since the applied force depends on the length of the tether for each event. For dissociation of the PSGL-1/P-selectin adhesions following first-order kinetics, the decay in number of attached cells was used to graphically fit the apparent kinetic off-rate at each shear rate analyzed (Fig. 4 A), which agreed with the statistical point estimate (SPE) of k^sub off^ =1/t (44). Despite the observed variation in tether lengths (and thus force loading) across the events measured at each wall shear rate, the regression plots were highly linear (R^sup 2^ = 0.98 or 0.99), indicating that linearity is not necessarily proof that a single class of events is being analyzed. The average forces were then used to regress the Bell parameters from values of k^sub off^ (from the slopes from Fig. 4 A) to obtain the apparent unloaded off-rates and reactive compliance for all data (� tethers) (Fig. 4 B) or events lacking tethers (Fig. 4 C). The k^sub off^ at γ^sub w^ = 200 s^sup -1^ was determined from only 20 events (Fig. 4 B) or eight events (Fig. 4 C) due to the extremely low capture efficiency at this shear rate (approximately four in every 1000 collisions generated an adhesive event). Thus, we also regressed the graph of F^sub B^ versus k^sub off^ with (SPE 2 in Fig. 4, B or C) and without (SPE 1 in Fig. 4, B or C) inclusion of k^sub off^ at 200 s^sup -1^. The weighted Monte Carlo simulation of all shear rates corresponded best with the SPE regression where data at γ^sub w^ = 200 s^sup -1^ was neglected. Cell flattening may also be more pronounced at 200 s^sup -1^, thus reducing force loading at this higher level of flow.

We determined the zero-stress off-rate k^sub off^(0) = 0.67 s^sup -1^ and reactive compliance, r = 0.07 nm (Fig. 4 B and curve lb in Fig. 5 A; ~�< 8%) for all events (� tethers) and for γ^sub w^ = 25-125 s^sup -1^. In comparing these Bell parameters with earlier literature reports (curves 2-14 in Fig. 5), we then evaluated the Bell parameters (by SPE and by MC) for only those events that displayed no detectable membrane tether since the force applied on the adhesion is the same from event to event at a given wall shear rate. These events yielded Bell parameters (k^sub off^(0) = 0.53 s^sup -1^, r = 0.10 nm) that 1), were best modeled by single-bond MC (where almost no additional bonds formed); 2), were very poorly fit by a multiple bonding hypothesis (n = 2 bonds initially); and 3), were quite consistent with literature values shown in curves 2-8 in Fig. 5 and the recent DFS data of Heinrich et al. (14).

An alternate hypothesis was also tested using the entire data set (� tethers) where the MC simulation was performed so that no new bonds could form (k^sub on^ = 0), assuming that there were no new encounter pairs because of tension on the tether tip continually shrinking the contact area. For k^sub on^ = 0 and using the Bell parameters obtained for no tether growth (k^sub off^(0) = 0.53 s^sup -1^, r = 0.10 nm) as the best estimate of single bond kinetics, we then reanalyzed the entire event set (with and without tethering) and determined that the data set was best fit by a distribution of initial bonds for d = 1.1 (Eq. 6) corresponding to an average of 1.48 bonds (n = 1 bond, 67%; n = 2, 22%; and n = 3-5, 11%; see Appendix for details). We note that the data at γ^sub w^ = 200 s^sup -1^ was quite sparse as a consequence of the extremely low capture efficiency at this shear rate and contributed little to the MC determination of the Bell parameters. Using the Bell parameters obtained for no tether growth and k^sub on^ = 0, the initial condition of n = 2 bonds (a fully engaged dimer interaction) allowed generation of a set of pause times (curve 1c of Fig. 5) quite consistent with the data of Park et al. (3), a result also obtained by King et al. (50). This simulation matched curves 12-14 and was then fit with apparent parameters of (k^sub off^)^sub app^ = 0.76 s^sup -1^ and r^sub app^ = 0.05 nm. Figs. 4 and 5 indicate that the neutrophil-bead collision assay allows accurate measurement of lifetime distributions that are consistent with prior measurements and analyses for the PSGL-1/P-selectin interaction.

Chemical treatment of perfused neutrophils

Neutrophils were treated with cytochalasin D (15 �M), latrunculin (0.3 �M), jasplakinolide (10 �M), MβCD (10 mM). or formaldehyde (3% v/v) for 30 min. Latrunculin-treated neutrophils that were perfused at 100 s^sup -1^ formed an unusual teardrop shape (Fig. 1 F). This effect was not observed with any of the other treatments.

None of the treatments significantly altered the primary capture efficiency of the neutrophils with the P-selectin-coated bead (Fig. 6 A), indicating that the reagents had minimal effect on the concentration or activity of PSGL-1 on the neutrophil surface. Only latrunculin treatment caused a slight increase in the fraction of cells that formed membrane tethers (p = 0.024; Fig. 6 B) due to the extreme softness of the cells. Cytochalasin D and jasplakinolide had no significant effect on the membrane tether formation fraction. Cholesterol-depleting MβCD significantly reduced the likelihood that cells would form membrane tethers at γ^sub w^ = 100 s^sup -1^ (p = 0.0007), as did fixation of the cells with formaldehyde (p < 0.0001). All of the treatments significantly altered the average membrane tether length pulled from the cell (Fig. 6 C), with actin-depolymerizing reagents associated with an overall increase in membrane tether length, and cholesterol depletion and fixation with decreased membrane tether lengths. Interestingly, jasplakinolide, which acts to polymerize actin in the cell, had an affect similar to the depolymerizing reagents-likely due to the membrane blebbing observed.

For all chemical treatments (excluding formaldehyde, where no tethers formed), the set of adhesion lifetimes with membrane tether formation was significantly longer than the lifetime without tethers (p < 0.0001) (Fig. 6 D). All of the treatments caused a significant overall change in the adhesion lifetimes (Fig. 6 E), and latrunculin was the only reagent that hardly altered (14.6% prolongation, p = 0.079) the lifetimes of membrane-tether forming adhesions, potentially due to the distinct morphology of tethering involving the entire cell (Fig. 1 F). Too few membrane tethers were pulled from formaldehyde-treated cells to allow analysis (n = 1). Fixation and cholesterol depletion caused a general decrease in overall adhesion lifetime with treatment, whereas the actin-depolymerizing agents increased the lifetime of interactions. Similar to our results for the average tether length, jasplakinolide acted in a similar manner as the depolymerizing agents.

The tether growth rates (v^sub tether^) for membrane-tether-forming interactions of treated neutrophils with P-selectin-coated beads were also analyzed (Fig. 7). These results were compared against the average tether growth rate for untreated cells perfused overheads at 100 s^sup -1^ (average v^sub tether^ = 8.1 � 4.9 �m/s), and Student's t-tests were performed comparing the set of control cell growth rates to those for the treated cells. In all cases except latrunculin (where cells had an unusual morphology), treatment with actin- or cholesterol-affecting reagents caused significant differences between the two samples. Cytochalasin and jasplakinolide both caused tethers to grow faster than untreated cells, whereas cholesterol depletion decreased the tether growth rate relative to untreated cells. In comparing the tether growth rate velocity for all events (solid bar, Fig. 7 A) with the measured lifetimes of those events (Fig. 6 E), it was clear that increases in the tether growth velocity caused prolongation in the PSGL-1/P-selectin lifetime due to decreased forces on the bonds (Fig. 7 B). It would be highly unlikely that the lifetime of the tether attachment to the bead would alter the tether growth rate velocity, a process that is dictated by lipid flow from the neutrophil.

DISCUSSION

Beads coated with P-selectin and adsorbed onto flow chamber surfaces served as discrete sources of ligand for studying the function of PSGL-1 in the native membrane of neutrophils. This new method allowed the simultaneous probing of several distinct processes in tether growth and P-selectin/PSGL-1 bond formation and rupture under hemodynamic venous flow conditions.

A central issue addressed by this technique was whether tether growth shields the bond from force loading, and to what extent. The data in Fig. 2 D comparing the average lifetime of populations with and without tethers at each shear rate suggests, but does not prove, that tether growth results in longer lived bonds. Long-lived bonds allow more time for tether growth and consequently longer tethers. Short-lived bonds provide less time for tether growth and thus result in shorter tethers. We have used five different pharmacological treatments to alter membrane and cell mechanics. The most extreme treatment was formaldehyde fixation, which obliterated tether formation and reduced the bond lifetime to a level identical to that found for events that did not form tethers using unfixed neutrophils (Fig. 6 D). Since the collision efficiency was not reduced with formaldehyde, this reduction in the overall lifetime of all events by formaldehyde was not likely due to a defect in the bond or the level of PSGL-1 on the fixed neutrophil. This result extends the prior understanding of formaldehyde effects (3) by simultaneously measuring both tether length and bond life for each adhesion event. A less extreme method of reducing tether growth was achieved through the use of MβCD to extract cholesterol out of the neutrophil membrane. MβCD reduced the fraction of cells forming tethers and reduced the tether length and the tether growth velocity, but not to the extent achieved with formaldehyde. Consistent with tether length shielding the bond from loading, MβCD reduced the overall lifetime of bonds. Since the collision efficiency was not reduced with MβCD, this reduction in the overall lifetime of all events by MβCD was not likely due to a defect in the bond or the level of PSGL-1 on the surface of cholesterol-depleted neutrophils. MβCD has previously been reported to increase leukocyte rolling velocity (33). On a local scale of the plasmalemma at the base of the tether, neutrophils depleted of cholesterol exhibit the rigidifying effects that have previously been demonstrated on a global whole-cell scale using micropipette aspiration (33).

Conversely, agents that caused longer tethers to form (cytochalasin D, latrunculin, and jasplakinolide) also prolonged the average lifetime of the adhesive interaction (Fig. 6 D). Latrunculin softens the entire cell body and causes an unusual tear-shape morphology in the bead collision assay, which may lead to cell flattening and reduction of force loading at a given shear rate. In contrast, jasplakinolide hardens the overall cell but causes plasmalemma blebbing, which in turn facilitates membrane tether formation (an increase in f) because lipid is already detached from the cytoskeleton and available for flow into the tether. Jasplakinolide serves as an example where whole cell mechanics, i.e., cell hardening as detected by micropipette aspiration (37), does not predict local plasmalemma dynamics during PSGL-1-mediated bonding. In the study of Sheikh et al. (37), it was only the neutrophil core that would resist entry into the pipette; membrane blebs that had formed on the surface of the cells would easily enter. We also observed membrane blebs on the surface of neutrophils treated with this reagent (not shown), which indicated that the lipid bilayer separated from the neutrophil at these locations (51). This separation of bilayer and cytoskeleton reduces the minimum force necessary to pull a tether by eliminating the membrane-cytoskeletal adhesion portion of tether adhesion energy (51), thus rendering the cell locally as less rigid than untreated cells.

Micropipette studies looking at global effects on rigidity due to cytochalasin D (32-34) through measurement of the cortical viscosity previously pointed to depolymerization as a mechanism of neutrophil softening. Other studies looking specifically at tether growth after treatment with either cytochalasin D (23) or latrunculin A (36) have shown that the membrane slip between the lipid bilayer and cytoskeleton is a major contributor to energy dissipation during tether formation. In our study, on average, membrane tethers generated by cells treated with cytochalasin D or latrunculin A had faster tether growth rates (Fig. 7) than untreated cells, and consequently longer bond lifetimes than untreated cells (Fig. 6). These results point to a greater ease of lipid flow into the tether during formation under venous flow conditions. Neutrophils that were fixed or treated with MβCD had opposite effects to those seen with depolymerizing agents, and formed tethers with significantly reduced growth rates compared to untreated cells and consequently had shorter lifetimes (Figs. 6 and 7). A force as low as 25.6 pN at a wall shear rate of 25 s^sup -1^ was detected to cause membrane tether pulling. This force is ~50% of that predicted by micropipette studies, where micromanipulation of the cell may lead to low level cellular activation and consequent slight cortical stiffening.

Since this study focused on both on-rate and off-rate processes, a high saturating level of P-selectin IgG chimera was used in these studies to allow a reliable determination of the collision efficiency as a function of shear rate (Fig. 2 A). Since the contact area is so small at the tip of the tether and the interaction time so short during the actual neutrophil-bead collision (Fig. 2 E), the method resulted in events dominated by single bond interactions (estimated at >65%) and provided Bell parameters that were consistent with those obtained from rolling experiments (curve 1b in Fig. 5). A subset of all events, those without tethers where forces are most uniform and accurately estimated at each γ^sub w^ (curve 1a in Fig. 4 B), provided Bell parameters consistent with single-bond MC and prior AFM and rolling beads experiments (curves 2-8 in Fig. 5). Rebonding processes (k^sub on^ in the MC) did not appear significant in the data sets and this suggests that the contact area at the tip of the tether, when pulled, becomes exceedingly small, possibly approaching the dimensions of an AFM cantilever tip. Future work employing sparsely-coated beads that is focused solely on off-processes (where ε is not determined) should allow the event distribution to be dominated by single bonds.

Catch-bonding has been detected by AFM and in flow chambers for the PSGL-1/P-selectin bond at force loadings in the 5-20 pN range (7). Catch-bonding may be one of several underlying mechanisms to explain the unexpected hydrodynamic-thresholding observed for neutrophil rolling on selectins (35). Platelet translocation on vWF A1 domain also displays thresholding at ~1 dyn/cm^sup 2^ (44) with r > 0 at F^sub bond^ > 36 pN. Neutrophils rolling on selectin-coated surfaces display biphasic force-enhanced adhesion at low shears, and force-decreased adhesion at shear rates above a certain maximum in shear (52). The direct observation of a decrease in off-rate at low shear rates that switches to an increase at higher shear rates provided the first experimental observation of catch-bonding in the P-selectin/PSGL-1 system (7). Catch-bonding has also been reported for L-selectin/PSGL-1 at low forces (8,53). In the current study, we did not look at the force regime below the catch-bond threshold for P-selectin/PSGL-1 (F^sub B^ < 10-20 pN, γ^sub w^ < 15 s^sup -1^), and thus our results are for those interactions in the slip-bond regime only.

The neutrophil-bead collision assay may prove useful in future studies, including: determination of the on-rate as a function of shear rate and ligand density; thresholding studies at low flows where contaminating flow fluctuations would have to be coincident with the bead collision and thus hydrodynamic fluctuation effects are better filtered out of the assay; studies of activated neutrophils to probe VCAM-1 or ICAM-1 binding lifetimes; or the analysis of patient neutrophils where neutrophil membrane mechanics and inflammation are potential cofactors in vaso-occlusive phenomena.

Authors acknowledge helpful conversations with Dr. M. B. Lawrence (University of Virginia).

This work was supported by National Institutes of Health grant No. HL56621 (to S.L.D.). K.E.E. is a recipient of an Ashton Fellowship.

[Reference]

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[Author Affiliation]

K. E. Edmondson,*[dagger] W. S. Denney,*[double dagger] and S. L. Diamond*[dagger][double dagger]'

* Institute for Medicine and Engineering, [dagger] Department of Bioengineering, and [double dagger] Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania

[Author Affiliation]

Submined May 10, 2005, and accepted for publication August 3, 2005.

Address reprint requests to S. L. Diamond, Tel.: 215-573-5702; E-mail: sld(3) seas.upenn.edu.