Surgical and pathological outcomes of laparoscopic surgery for transverse colon cancer Purpose Several multi-institutional prospective randomized trials have demonstrated short-term benefits using laparoscopy . Now the laparoscopic approach is accepted as an alternative to open surgery for colon cancer . However , in prior trials , the transverse colon was excluded . Therefore , it has not been determined whether laparoscopy can be used in the setting of transverse colon cancer . This study evaluated the peri-operative clinical outcomes and oncological quality by pathologic outcomes of laparoscopic surgery for transverse colon cancer . Introduction Since its first report -LSB- 1 -RSB- , laparoscopic colon surgery has been controversial with regard to its use for colorectal cancer . Several prospective randomized trials including the COLOR and CLASSIC studies have demonstrated that laparoscopic-assisted surgery for colorectal cancer resulted in a shorter hospital stay , reduced analgesic use , and earlier recovery to bowel movement -LSB- 2 -- 6 -RSB- . Moreover , the COST study established the long-term oncological safety of laparoscopic-assisted surgery for colon cancer , and currently the laparoscopic approach is accepted as an alternative to open surgery for colon cancer -LSB- 7 -RSB- . However , transverse colon cancer was excluded from prior randomized controlled trials . The reasons for exclusion of transverse colon cancer include the following : difficulty in deciding the appropriate operative procedure and extent of lymph node dissection , as well as technical difficulties with the laparoscopic identification , ligation , and lymph node dissection around the middle colic vessels . Therefore , there continues to be debate on whether to use laparoscopic surgery for transverse colon cancer . Materials and methods Between August 2004 and November 2007 , the medical records of all patients who underwent laparoscopic surgery for colorectal cancer were reviewed . Laparoscopic colorectal cancer resection was started August 2004 in our clinic . Pathologic confirmation , colonoscopy , barium enema , computed tomography -LRB- CT -RRB- , ultrasonography , and chest PA were performed for diagnosis in all patients preoperatively . All patients with colorectal adenocarcinoma admitted to our clinic were considered for laparoscopic surgery . Exclusion criteria for laparoscopic surgery were as follows : -LRB- 1 -RRB- patients with colorectal cancer obstruction and failure of stent insertion , -LRB- 2 -RRB- patients with colorectal cancer perforation requiring emergency surgery , -LRB- 3 -RRB- patients with T4 colorectal cancer lesion that could not be resected laparoscopically , and -LRB- 4 -RRB- patients with compromised cardio-pulmonary function in whom pneumoperitoneum under general anesthesia was contraindicated . In this study , transverse colon cancer was defined as lesions between the hepatic flexure and splenic flexure in the colon , requiring ligation of the middle colic vessels at their origin . CT or barium enema was performed in all patients with colon cancer preoperatively for localization of the tumor . If radiological localization was unclear , preoperative colonoscopic Indian ink tattooing or endoscopic clipping was performed . The procedure used for the transverse colon cancer was chosen based on the location of the tumor . A tumor located at the hepatic flexure or within 10 cm distal to hepatic flexure was treated by an extended right hemicolectomy , and a tumor located at the splenic flexure or within 10 cm proximal to the splenic flexure was treated by an extended left hemicolectomy . A tumor located between the above two lesions was treated by a transverse colectomy . An extended right hemicolectomy was defined as lymphadenectomy simultaneously with ligation of ileocolic , right colic , and middle colic vessels at their origins ; an extended left hemicolectomy was defined as lymphadenectomy simultaneously with ligation of left colic and middle colic vessels at their origins . A transverse colectomy was defined as lymphadenectomy simultaneously with ligation of middle colic vessels at their origins . Extra-corporeal anastomosis was performed in all cases of laparoscopic surgery for transverse colon cancer . All patients started a diet after passing flatus . To evaluate the postoperative surgical outcomes and oncological quality of laparoscopic surgery for transverse colon cancer , we compare the age , gender , body mass index -LRB- BMI -RRB- , operating time , blood loss , time to passing flatus , time to start of diet , hospital stay , surgical morbidity , surgical mortality , conversion to open surgery , tumor size , distal resection margin , proximal resection margin , radial margin , and number of harvested lymph nodes between the transverse colon cancer group -LRB- TCC -RRB- and other site colon cancer group -LRB- OSCC -RRB- . In this study , a single surgeon -LRB- YS Lee -RRB- performed all operations . Comparisons between the two groups were made by applying the independent samples t test and χ2 test . Differences were considered to be significant for P value < 0.05 . Results Postoperative clinical outcomes Three hundred and twelve patients underwent colorectal cancer resection in our clinic during these periods . Among them , 44 patients underwent conventional open surgery according to exclusion criteria -LRB- five cases of stent failure or colon obstruction , five cases of emergency operation , 12 cases of far advanced tumor , 12 cases of recurrent cancer operation , seven cases of patient 's refusal , and three cases for old age with high risk for pneumoperitoneum -RRB- . There was no transverse colon cancer in the conventional open surgery group . A total of 268 patients underwent laparoscopic resection for colorectal cancer . Of the 268 patients , 140 patients who underwent laparoscopic resection for rectal cancer were excluded in this study . Finally , 128 patients who underwent laparoscopic resection for colon cancer were enrolled in this study . Of the 128 patients , 34 patients had transverse colon cancer , counting for 10.8 % of total colorectal cancers , and 94 patients had other sites of colon cancer . In the TCC group , extended right hemicolectomy was performed in 18 cases , transverse colectomy was performed in eight cases , and extended left hemicolectomy was performed in eight cases . In the OSCC group , right hemicolectomy was performed in 38 cases , left hemicolectomy was performed in five cases , and anterior resection was performed in 51 cases . There were no statistical differences in age , gender distribution , BMI , operating time , intraoperative blood loss , time to flatus , time to start of diet , and hospital stay between patients with TCC and OSCC -LRB- Table 1 -RRB- . Four patients in the OSCC group had a major complication , one had colon injury , and two had anastomosis leak . A simple laparoscopic closure was performed in the case of colon injury , and two patients with anastomosis leak underwent re-operation . Minor complications that occurred in four cases in the OSCC group -LRB- one case of ileus , two cases of port site minor infection , and one case of urinary retention -RRB- and two cases in the TCC group -LRB- one case of ileus and one case of atelectasis -RRB- were treated successfully conservatively . One case among the TCC group and three cases in the OSCC group were converted to open surgery ; all of these converted cases were due to tumor-related factors , T4 lesion , or huge tumor . There was no surgical mortality in this study . Table 1Clinical characteristics of patients TCC -LRB- N = 34 -RRB- OSCC -LRB- N = 94 -RRB- P valueAge -LRB- years -RRB- 64.1 ± 11.362.5 ± 12.1 NSSex -LRB- M : F -RRB- 15:1946:48 NSBody mass index -LRB- kg/m2 -RRB- 23.5 ± 3.024.0 ± 3.1 NSOperating time -LRB- min -RRB- 211.1 ± 52.2 -LRB- 140 -- 360 -RRB- a220 .4 ± 94.3 -LRB- 60 -- 620 -RRB- aNSBlood loss -LRB- ml -RRB- 100.0 ± 83.8 -LRB- 0 -- 400 -RRB- a114 .2 ± 145.2 -LRB- 0 -- 700 -RRB- aNSTime to pass flatus -LRB- days -RRB- 2.8 ± 0.82.6 ± 1.0 NSDiet start -LRB- days -RRB- 4.2 ± 1.84.2 ± 2.8 NSHospital stay -LRB- days -RRB- 11.4 ± 4.111.2 ± 6.0 NSTCC Transverse colon cancer , OSCC other site colon cancer , NS not significantaValues are ranges . Oncological quality by pathologic outcomes There were no statistical differences in the tumor size , proximal resection margin , distal resection margin , radial margin , and number of harvested lymph nodes between patients with TCC and OSCC -LRB- Table 2 -RRB- . Table 2Pathological outcomes of patients TCC -LRB- N = 34 -RRB- OSCC -LRB- N = 94 -RRB- P valueTumor size -LRB- cm -RRB- 5.2 ± 2.5 -LRB- 0.7 -- 11 -RRB- a4 .6 ± 2.4 -LRB- 0.1 -- 14 -RRB- aNSPRM -LRB- cm -RRB- 19.5 ± 10.2 -LRB- 10.0 -- 25.0 -RRB- a15 .3 ± 11.0 -LRB- 4.2 -- 25.0 -RRB- aNSDRM -LRB- cm -RRB- 13.9 ± 6.9 -LRB- 6.0 -- 25.0 -RRB- a11 .6 ± 5.3 -LRB- 3.0 -- 32.0 -RRB- aNSNo . of lymph nodes24 .4 ± 11.7 -LRB- 3 -- 59 -RRB- a21 .1 ± 8.4 -LRB- 3 -- 59 -RRB- aNSRadial margin -LRB- cm -RRB- 0.9 ± 0.8 -LRB- 0 -- 3.0 -RRB- a1 .0 ± 0.9 -LRB- 0 -- 3.5 -RRB- aNSTCC Transverse colon cancer , OSCC other site colon cancer , DRM distal resection margin , PRM proximal resection margin , NS not significantaValues are ranges . Discussion Since laparoscopic colon resection was first reported in 1991 -LSB- 1 -RSB- , laparoscopic surgery has been widely employed for various benign colorectal disease such as benign mass , diverticular disease , inflammatory bowel disease , rectal prolapse , and now increasingly for colorectal malignant disease . Evidences of the safety and efficacy of the laparoscopic surgery for colorectal cancer had been reported from several prospective randomized controlled studies and meta-analysis of several trials , which favored the laparoscopic surgery for colorectal cancer over conventional open surgery due to its many short-term benefits such as shorter hospital stay , reduced use of analgesics , and earlier recovery of bowel movements -LSB- 2 -- 6 , 8 -- 13 -RSB- . Eventually , long-term oncological safety of laparoscopic colon cancer resection was established , and the laparoscopic approach was accepted as an alternative to open surgery for colon cancer by the COST study -LSB- 7 -RSB- . Transverse colon cancer occurs in about 10 % of cases of colorectal cancer , and it often presents a challenge for the choice of the best surgical procedure based on the location of the tumor and extent of lymph node dissection . There could also be technical difficulties with laparoscopic identification , ligation , and lymph node dissection around the middle colic vessels depending on the surgeon 's experience . Because of these reasons , transverse colon cancer was excluded from almost every prior prospective randomized trial . Therefore , there is continued debate over the validity of laparoscopic surgery for transverse colon cancer . The major controversy about laparoscopic surgery for transverse colon cancer lies on whether or not it is feasible to perform sufficient extent of lymph node dissection around the middle colic artery laparoscopically . As experiences of laparoscopic surgery are accumulating and surgical techniques and instruments are developing , we consider that the extent of laparoscopic lymph node dissection for transverse colon cancer is not less than the extent of conventional lymph node dissection for transverse colon cancer . Some of the difficulties with laparoscopic surgery for transverse colon cancer that need to be resolved include the following : First , precise localization of carcinoma of the transverse colon is important ; this is because the extent of resection and lymph node dissection depends on the location of the tumor in the transverse colon . Diminished tactile guidance can make localization of the tumor in the transverse colon more difficult in small tumor . There are a number of methods used to localize the tumor . Preoperative barium enema is useful for localization of large and advanced tumor , but radiological localization is inconclusive and difficult in early cancer . In such cases , colonoscopic tattooing with Indian ink or placing endoscopic clips is very effective . Using endoscopic clip at the time of preoperative colonoscopy and using fluoroscopy for localization , the clip migration could be the main problem , and using fluoroscopy in the operating theater is troublesome and time consuming . However , in the case of hepatic flexure or splenic flexure colon cancer , by checking X-ray just after placing the endoscopic clip , we could precisely localize both flexure tumors easily . Intraoperative colonoscopy can be used for localization of the tumor , but it is also time consuming , and moreover , it can cause colonic insufflation that makes laparoscopic surgery difficult -LSB- 14 -RSB- . Properly placed tattoos are long lasting and can be placed at the time of diagnostic colonoscopy -LSB- 15 -RSB- . In the event of tattoo failure , one can use intraoperaitve colonoscopy for localization easily . In this study , we performed preoperative colonoscopic tattoo in three cases to localize the mid transverse colon cancer , colonoscopic clipping in three cases of hepatic flexure colon cancer , and barium enema in the other cases . There was no case with non-localization . We think that colonoscopic tattooing is effective in small mid transverse colon cancer , and endoscopic clipping is effective in small hepatic or splenic flexure colon cancer . Second is the laparoscopic identification , ligation , and lymph node dissection around the middle colic vessels . Fujita et al. reported laparoscopic techniques that can be used for identification of middle colic vessel . They suggested that the ventral aspect of the caudal portion of superior mesenteric vein be exposed , and the exposed vessel brought cephalad toward the caudal portion of the pancreas , with identification of the middle colic vessels -LSB- 16 -RSB- . Baca et al. introduced the ` window technique ' for lymphadenectomy with simultaneous resection of the vascular stem -LSB- 17 -RSB- . Ichihara et al. introduced the technique of rotation of mesocolon to identify middle colic artery -LSB- 18 -RSB- . The authors have identified the middle colic vessels using surgical techniques introduced by Fujita in most of the cases , and the in case of slender patients we could directly identify pulsating middle colic artery by stretching the transverse mesocolon by traction of both ends of the transverse colon by the first assistant . Identifying middle colic vessels , excessive traction of the transverse mesocolon by the first assistant may cause tearing of the vein and bleeding . Moreover , the length of the gastrocolic trunk of Henle is relatively short , and attempt to control bleeding from the gastrocolic trunk of Henle can injure the superior mesenteric vein -LSB- 19 -RSB- . It is especially important for a laparoscopic colorectal surgeon to recognize the variable drainage of the right colic and middle colic vein to the gastrocolic trunk of Henle and to have a precise knowledge of superior right colic vein anatomy . Third is the low incidence of transverse colon cancer . Consecutive cases are needed for overcoming the learning curve . Therefore , it might take surgeons longer time to become experienced in the techniques used for laparoscopic transverse colon cancer resection . In this study , there were no significant differences between patients with TCC and OSCC in terms of operating time , blood loss , resection margin , and number of lymph nodes . Schlachta et al. reported that the operating time was longer in the TCC group compared to the OSCC group , and the number of harvested lymph nodes in the TCC group was greater than in the OSCC group because of the extended resection and larger specimens , as well as the need to attend to the middle colic vessels in patients with TCC -LSB- 20 -RSB- . However , in this study , there was no statistical difference in operating time and number of harvested lymph nodes , as well as the surgical outcomes between the two groups . We think that operating time could be similar in the TCC and OSCC groups in experienced hands , and actually , the extent of lymph node dissection in transverse colon cancer is not too wide than that of other site colon cancer , so there were no statistical differences in pathological outcomes between the two groups in this study . There was one case of colon injury proximal to anastomosis caused by electrocautery during the operation in the OSCC group ; this was treated by a laparoscopic simple closure on postoperative day 3 . Three cases of anastomosis leak , one case of right hemicolectomy , and two cases of anterior resection occurred in the OSCC group . A patient with anastomosis leak was diagnosed by clinical symptoms or sign , fecal or purulent discharge from drain , fever , leukocytosis , and peritoneal irritation sign . Radiologic study using a water-soluble dye was not performed in this study . Of the three cases , one patient with a leak of anterior resection was treated conservatively successfully , and two patients with leak cases underwent laparoscopic re-operation . All other minor complications were successfully treated conservatively . Conversion to open surgery occurred in one case in the TCC group and in three cases in the OSCC group ; these differences were not significant . Factors related to the tumor were the cause of conversion to open surgery . One case in the TCC group was converted to open surgery due to T4 lesion in the transverse colon cancer that invaded the anterior wall of the stomach . In the OSCC group , one case with a larger tumor that was hard to handle by laparoscopy , and two cases of sigmoid colon cancer that invaded the uterus were converted to open surgery . However , T4 colon cancer is not a contraindication for laparoscopy if en bloc resection could be performed with the laparoscopy . In those cases converted to open surgery , laparoscopic en bloc resection was impossible . This study has some weak points . First , the number of patients with transverse colon cancer is too small , making up 10 % of the total colorectal cancer resection and 12 % of the total laparoscopic colorectal resection in this study . The second is that although the data in this study were collected prospectively , the data were not derived from a larger case series and this study was not randomized controlled . The third is that the mean follow-up period was too short to evaluate the oncological outcomes -LRB- 15.9 months ; range 1 -- 40 months -RRB- . We think that large-scale prospective controlled trials and long-term analysis are mandatory to overcome these limitations and confirm the oncological safety of laparoscopic transverse colon cancer surgery . We are going to report a long-term analysis after a long-term follow-up involving more cases prospectively . Conclusions The results of this study show no significant differences with regard to surgical outcomes and oncological quality by pathologic outcomes between patients in the OSCC and TCC groups . Further investigations with large-scale prospective studies and long-term analysis of laparoscopic surgery for transverse colon cancer are mandatory to establish the oncological safety of laparoscopic surgery for transverse colon cancer .

O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O

laparoscopy
transverse colon
colon cancer


Altered

phenotype

and

gene

transcription

in

endothelial

cells

induced

by

Plasmodium

falciparum-infected

red

blood

cells

Pathogenic

or

protective

Severe

malaria

is

associated

with

sequestration

of

Plasmodium

falciparum-infected

red

blood

cells

-LRB-

PRBC

-RRB-

in

the

microvasculature

and

elevation

of

intercellular

adhesion

-LRB-

-RRB-

and

TNF

In

vitro

co-culture

of

human

umbilical

vein

endothelial

cells

-LRB-

HUVEC

-RRB-

with

either

PRBC

or

uninfected

RBC

required

the

presence

of

low

level

TNF

-LRB-

pg/ml

-RRB-

for

significant

up-regulation

of

which

may

contribute

to

increased

cytoadhesion

in

vivo

These

effects

were

independent

of

P.

falciparum

erythrocyte

membrane

-LRB-

-RRB-

mediated

adhesion

but

critically

dependent

on

cell

cell

contact

Further

changes

included

increases

in

release

and

soluble

TNF

receptor

shedding

Microarray

analysis

of

HUVEC

transcriptome

following

co-culture

using

a

human

Affymetrix

microarray

chip

showed

significant

differential

regulation

of

genes

which

defined

gene

ontologies

such

as

cell

communication

cell

adhesion

signal

transduction

and

immune

response

Our

data

demonstrate

that

endothelial

cells

have

the

ability

to

mobilise

immune

and

pro-adhesive

responses

when

exposed

to

both

PRBC

and

TNF

In

addition

there

is

also

a

previously

un-described

positive

regulation

by

RBC

and

TNF

and

a

concurrent

negative

regulation

of

a

range

of

genes

involved

in

inflammation

and

cell-death

by

PRBC

and

TNF

We

propose

that

the

balance

between

positive

and

negative

regulation

demonstrated

in

our

study

will

determine

endothelial

pathology

during

a

malaria

infection

Introduction

Pathogenesis

of

severe

malaria

has

stirred

considerable

interest

over

the

years

however

there

are

many

questions

at

the

mechanistic

level

that

remain

unanswered

Severe

malaria

is

characterised

in

part

by

sequestration

of

Plasmodium

falciparum-infected

red

blood

cells

-LRB-

PRBC

-RRB-

at

microvascular

sites

in

the

brain

tissue

leading

ultimately

to

coma

and

subsequent

death

There

is

evidence

that

the

sequestration

of

PRBC

on

the

endothelial

surface

can

result

in

loss

of

integrity

thus

compromising

blood

brain

barrier

function

either

directly

-LRB-

Brown

et

al.

b

-RRB-

by

indirect

mechanisms

including

binding

via

platelets

-LRB-

Wassmer

et

al.

Combes

et

al.

-RRB-

or

by

the

production

of

microparticles

from

endothelial

cells

-LRB-

EC

-RRB-

activated

by

adhering

PRBC

-LRB-

Combes

et

al.

-RRB-

and

also

cytokine-driven

modulation

of

endothelial

cell

metabolism

but

that

this

is

usually

a

small

transient

effect

in

vivo

One

question

is

the

mechanism

underpinning

the

accumulation

of

PRBC

leading

to

micro-vascular

occlusion

as

seen

in

post

mortem

cerebral

malaria

-LRB-

CM

-RRB-

brain

tissue

which

is

believed

to

be

a

progressive

phenomenon

of

PRBC

sequestration

We

suggest

that

the

initial

sequestration

of

PRBC

if

maintained

for

a

prolonged

period

of

time

has

the

ability

to

activate

the

endothelium

to

promote

sequestration

leading

to

deleterious

effects

on

the

host

Our

studies

investigate

the

direct

effects

on

the

endothelium

of

PRBC

retention

for

prolonged

periods

of

time

First

sustained

exposure

of

the

endothelium

to

PRBC

can

result

in

modulation

of

the

endothelium

thus

increasing

its

responsiveness

to

low

levels

of

TNF

-LRB-

TNFlow

-RRB-

This

may

contribute

to

promoting

further

cytoadhesion

in

microvasculature

containing

sequestered

PRBC

This

hypothesis

is

based

on

previous

evidence

of

endothelial

cell

activation

by

abnormal

red

blood

cells

in

various

conditions

including

sickle

cell

disease

diabetes

and

also

malaria

-LRB-

Shiu

and

McIntire

-RRB-

Sickle

cell

RBC

-LRB-

sRBC

-RRB-

diabetic

RBC

-LRB-

dRBC

-RRB-

and

also

PRBCs

can

all

induce

expression

of

adhesion

molecules

such

as

intercellular

adhesion

-LRB-

-RRB-

and

vascular

adhesion

-LRB-

-RRB-

and

E-selectin

-LRB-

Brown

et

al.

Shiu

and

McIntire

Viebig

et

al.

Tripathi

et

al.

-RRB-

In

addition

recent

co-culture

studies

have

demonstrated

PRBC-induced

increases

in

the

activity

of

pro-apoptotic

genes

involved

in

the

caspase

pathway

-LRB-

Pino

et

al.

-RRB-

Other

changes

induced

on

EC

by

sRBCs

include

induction

of

endothelin

and

prostacyclin

production

-LRB-

Shiu

et

al.

-RRB-

and

by

PRBC

include

increased

expression

of

pro-inflammatory

genes

-LRB-

Pino

et

al.

-RRB-

and

release

of

-LRB-

Viebig

et

al.

-RRB-

Serum

levels

of

were

elevated

in

severe

malaria

compared

with

healthy

volunteers

-LRB-

Lyke

et

al.

-RRB-

correlated

with

parasite

count

and

severity

of

disease

at

the

time

of

admission

-LRB-

Burgmann

et

al.

-RRB-

and

there

were

no

apparent

differences

between

febrile

and

a-febrile

volunteers

-LRB-

Hermsen

et

al.

-RRB-

is

an

endothelial-derived

acute

response

activation

marker

Prestored

is

exocytosed

at

a

basal

level

from

Wiebel

palade

bodies

in

ECs

and

is

rapidly

released

in

response

to

various

stimuli

-LRB-

Wolff

et

al.

Utgaard

et

al.

Oynebraten

et

al.

-RRB-

Exposure

of

ECs

to

sRBCs

in

vitro

resulted

in

increased

sensitivity

to

the

pro-inflammatory

cytokine

-LRB-

Shiu

et

al.

-RRB-

We

propose

that

local

increases

in

TNF

levels

occur

in

the

microvasculature

in

early

stages

of

infection

during

shizogony

-LRB-

Bisser

et

al.

-RRB-

In

a

similar

fashion

to

that

observed

with

sRBCs

contact

between

PRBCs

and

the

endothelium

could

potentially

sensitise

the

ECs

to

TNF

There

is

evidence

for

an

important

role

for

TNF

receptors

-LRB-

TNFR

-RRB-

in

CM

pathology

however

most

of

this

comes

from

studies

on

animal

models

Lucas

et

al.

-LRB-

-RRB-

showed

a

close

correlation

between

absence

of

TNFR

II

expression

and

protection

from

CM-associated

brain

damage

in

TNFR

II

knock-out

mice

while

TNFR

II

was

up-regulated

on

brain

endothelium

in

CM-susceptible

mice

-LRB-

Lucas

et

al.

-RRB-

Up-regulation

of

the

soluble

TNFR

I

which

is

the

predominant

type

in

human

serum

was

reported

in

the

serum

of

patients

with

acute

P.

falciparum

infection

-LRB-

Wenisch

et

al.

-RRB-

Interestingly

EC

from

CM-susceptible

mice

had

a

greater

sensitivity

to

TNF

than

that

of

CM-resistant

mice

TNF

induced

up-regulation

of

TNFR

I

and

II

together

with

an

associated

increase

in

and

to

a

greater

extent

in

CM-susceptible

mice

than

in

CM-resistant

mice

-LRB-

Lou

et

al.

-RRB-

In

addition

there

was

an

absence

of

up-regulation

in

TNFR

II

knock-out

mice

suggesting

a

link

between

TNFRs

and

up-regulation

during

malaria

infection

-LRB-

Lucas

et

al.

-RRB-

Our

aim

was

to

investigate

the

ability

of

PRBC

to

modulate

the

endothelium

in

the

presence

and

absence

of

the

inflammatory

cytokine

TNF

in

a

co-culture

system

Firstly

functional

markers

of

EC

modulation

included

endothelial

expression

of

which

has

been

attributed

a

critical

role

in

parasite

adhesion

and

the

release

of

Changes

in

levels

of

TNFR

I

and

II

were

also

investigated

as

a

potential

mechanism

for

any

changes

in

the

sensitivity

of

EC

to

TNF

Second

we

sought

to

assess

the

global

transcriptional

changes

in

ECs

and

elucidate

the

regulation

of

cellular

processes

following

co-culture

under

the

same

conditions

using

a

human

genome

Affymetrix

-LRB-

Affymetrix

Santa

Clara

CA

USA

-RRB-

chip

Our

results

have

led

us

to

propose

a

novel

mechanism

for

the

modulation

of

the

endothelium

during

malaria

infection

that

is

dependent

on

low

level

TNF

and

involves

a

pro-inflammatory

component

but

also

a

concurrent

down-modulation

of

RBC-induced

inflammation

due

to

the

presence

of

the

parasite

within

the

infected

cell

Materials

and

methods

Malarial

parasites

Plasmodium

falciparum

ItG

strain

was

derived

from

the

Brazilian

line

-LRB-

Ockenhouse

et

al.

-RRB-

This

strain

was

used

for

the

PRBCs

in

these

studies

The

ItG

strain

is

a

strong

binder

and

also

binds

to

-LRB-

Gray

et

al.

-RRB-

The

PRBCs

were

cultured

in

supplemented

with

mM

l-glutamine

mM

acid

-LRB-

Hepes

-RRB-

mM

glucose

μg/ml

gentamicin

and

human

serum

at

pH

of

-LRB-

Trager

and

Jensen

-RRB-

All

reagents

were

obtained

from

Sigma

UK

Human

serum

was

isolated

from

whole

blood

obtained

from

the

Royal

Liverpool

Hospital

and

was

approved

by

the

Liverpool

School

of

Tropical

Medicine

Ethical

Committee

Endothelial

cells

Pooled

human

umbilical

vein

endothelial

cells

-LRB-

HUVEC

-RRB-

were

obtained

from

Promocell

-LRB-

Heidelberg

Germany

-RRB-

HUVEC

from

different

batches

were

used

for

each

experiment

at

passages

three

to

five

HUVEC

were

grown

to

confluence

on

gelatin

-LRB-

Sigma

UK

-RRB-

coated

flasks

and

plates

All

co-culture

experiments

were

performed

in

serum-depleted

basal

HUVEC

medium

-LRB-

quiescing

medium

-RRB-

which

consisted

of

-LRB-

Invitrogen

UK

-RRB-

containing

FCS

These

conditions

were

designed

to

increase

the

signal

window

while

maintaining

the

integrity

of

the

HUVEC

monolayer

-LRB-

i.e.

there

was

no

indication

of

cell

apoptosis

or

necrosis

during

the

experiments

-RRB-

For

all

co-culture

studies

ECs

were

co-cultured

with

PRBCs

and

uninfected

RBCs

in

the

absence

and

presence

of

TNFlow

The

sub-optimal

dose

was

verified

in

separate

TNF

dose

response

studies

pg/ml

was

of

the

dose

of

TNF

used

as

standard

for

optimal

induction

of

on

HUVEC

in

our

laboratory

-LRB-

ng/ml

-RRB-

and

had

no

significant

effect

on

expression

-LRB-

data

not

shown

-RRB-

For

the

positive

control

a

high

dose

of

TNF

-LRB-

TNFhigh

-RRB-

was

used

-LRB-

ng/ml

-RRB-

to

stimulate

the

HUVEC

while

medium

alone

served

as

a

negative

control

All

parasite

and

EC

cultures

were

regularly

monitored

for

mycoplasma

using

the

Takara

PCR

mycoplasma

detection

kit

-LRB-

Cambrex

Biosciences

-RRB-

PRBC-EC

co-culture

conditions

HUVEC

grown

to

confluence

in

either

plates

or

flasks

for

functional

studies

and

microarray

studies

respectively

were

co-cultured

with

PRBCs

and

uninfected

RBCs

in

the

absence

and

presence

of

TNFlow

at

°C

The

RBC

suspension

was

adjusted

to

haematocrit

PRBC

at

the

trophozoite

stage

-LRB-

h

post-invasion

-RRB-

were

used

in

all

co-culture

studies

for

the

functional

studies

the

parasites

were

synchronised

using

sorbitol

while

for

the

microarray

studies

the

trophozoites

were

additionally

enriched

using

plasmagel

flotation

In

selected

experiments

the

parasites

were

retrieved

after

the

stained

with

giemsa

and

examined

by

light

microscopy

There

was

no

apparent

rupture

of

RBC

or

change

in

the

parasite

stage

over

this

period

For

all

functional

studies

the

PRBCs

were

at

a

parasitaemia

of

and

co-cultured

with

HUVEC

for

h.

For

microarray

studies

the

PRBCs

were

enriched

on

Plasmagel

to

parasitaemia

ranging

between

and

and

co-cultured

for

h

The

uninfected

RBCs

were

from

the

same

batches

used

for

parasite

culture

and

were

maintained

under

the

same

conditions

as

PRBCs

in

separate

flasks

RNA

expression

microarray

analysis

Following

incubation

the

supernatant

was

aspirated

HUVEC

was

washed

with

cold

and

then

with

M

EDTA

to

remove

the

adherent

RBCs

and

the

cells

harvested

using

Trizol

-LRB-

Invitrogen

UK

-RRB-

RNA

integrity

was

evaluated

by

electrophoresis

on

a

agarose

gel

and

by

spectrophotometry

using

the

absorbance

ratio

at

nm

Five

micrograms

of

each

complementary

biotin-labelled

RNA

was

hybridised

on

the

Affymetrix

GeneChip

Human

Genome

version

-LRB-

Santa

Clara

CA

-RRB-

according

to

the

manufacturer

's

instructions

The

signal

intensity

for

each

feature

on

the

array

was

determined

using

the

percentile

method

provided

by

GCOS

software

-LRB-

Affymetrix

-RRB-

Hybridisations

were

performed

for

four

replicates

of

each

of

the

seven

conditions

-LRB-

control

RBC

PRBC

RBC

TNFlow

PRBC

TNFlow

TNFlow

and

TNFhigh

-RRB-

Data

analysis

was

performed

using

Bioconductor

-LRB-

Gentleman

et

al.

-RRB-

The

microarrays

were

pre-processed

using

a

Robust

Multiarray

Averaging

program

-LRB-

Irizarry

et

al.

-RRB-

The

expression

values

were

calculated

using

the

R

statistical

computing

environment

-LRB-

-RRB-

with

the

affy

package

-LRB-

Gautier

et

al.

-RRB-

The

differential

expression

was

assessed

and

variability

estimated

by

fitting

a

linear

model

to

the

data

that

fully

models

the

systematic

part

of

each

gene

using

the

limma

package

From

the

limma

output

log-fold

changes

from

one

condition

compared

with

another

P-values

corrected

for

a

false

discovery

rate

-LRB-

Hochberg

and

Benjamini

-RRB-

together

with

B-statistic

-LRB-

log-odds

that

a

gene

is

differentially

expressed

-RRB-

can

be

obtained

and

used

to

determine

up

and

down-regulation

of

genes

Due

to

the

complexity

of

the

microarray

design

-LRB-

i.e.

four

replicates

seven

conditions

-RRB-

for

non-hierarchical

clustering

the

transcripts

present

on

the

human

chip

were

filtered

down

to

based

on

their

moderated

F-statistic

at

P-value

These

were

then

clustered

by

similarity

The

unsupervised

clustering

program

enabled

us

to

discriminate

biological

clusters

-LRB-

ArrayMiner

OptimalDesign

-RRB-

For

each

cluster

Gene

Ontology

-LRB-

GO

-RRB-

assignments

were

extracted

using

the

GOstats

package

-LRB-

-RRB-

The

gene

list

of

each

cluster

was

used

to

identify

all

the

unique

GO

terms

in

order

to

determine

whether

genes

that

comprise

a

given

cluster

have

a

common

function

process

or

location

in

the

cell

For

each

GO

term

GOstats

analysed

whether

that

term

was

being

significantly

over-represented

in

the

data

using

a

P-value

cut-off

of

P

protein

expression

HUVEC

grown

to

confluence

in

plates

were

co-cultured

with

PRBC

and

uninfected

RBC

at

a

haematocrit

of

and

a

parasitaemia

of

and

suspended

in

supplemented

with

FCS

in

the

absence

and

presence

of

pg/ml

TNF

-LRB-

TNFlow

-RRB-

for

h

at

°C

Following

the

incubation

period

the

supernatant

was

removed

centrifuged

at

for

min

to

remove

any

RBCs

and

stored

at

°C

The

HUVECs

were

washed

once

with

cold

and

then

with

M

EDTA

to

remove

the

adherent

RBCs

and

subsequently

harvested

by

trypsinisation

for

analysis

by

flow

cytometry

FACS

expression

on

HUVEC

was

determined

by

staining

the

cells

using

a

fluorescein

isothiocyanate

-LRB-

FITC

-RRB-

conjugated

mouse

anti-human

antibody

-LRB-

Serotec

-RRB-

using

standard

staining

protocols

and

the

cells

fixed

in

paraformaldehyde

and

analysed

by

flow

cytometry

expression

was

expressed

as

geometric

mean

of

the

fluorescence

intensity

and

TNF

receptor

expression

The

supernatants

stored

from

the

co-culture

studies

were

analysed

using

a

standard

sandwich

ELISA

kit

-LRB-

IDS

-RRB-

using

a

horse-radish

peroxidise

based

colorimetric

detection

system

to

quantify

released

from

ECs

production

was

expressed

as

a

concentration

in

pg

per

ml

Similarly

soluble

TNFR

I

sTNFR

I

-LRB-

-RRB-

and

soluble

TNFR

II

sTNFR

II

-LRB-

-RRB-

were

detected

using

sTNFR

I

-LRB-

-RRB-

and

sTNFR

II

-LRB-

-RRB-

ELISA

kits

-LRB-

Biosource

-RRB-

TNFR

level

was

expressed

as

TNFR

concentration

in

ng

per

ml

In

order

to

understand

the

kinetics

of

TNFR

expression

on

the

surface

of

ECs

in

response

to

co-culture

with

PRBCs

the

ECs

were

co-cultured

with

PRBCs

and

uninfected

RBCs

for

and

h.

Following

the

incubation

period

HUVECs

were

harvested

and

dual

stained

for

surface

TNFRs

with

monoclonal

anti-human

RII-FITC

-LRB-

-RRB-

and

monoclonal

anti-human

RI-PE

-LRB-

-RRB-

antibodies

-LRB-

R&D

Systems

Europe

-RRB-

The

receptor

expression

was

expressed

as

the

geometric

mean

of

the

fluorescence

intensity

Trypsin

digestion

of

RBC

The

ability

of

PRBCs

to

induce

changes

in

surface

levels

following

trypsinisation

was

determined

PRBCs

and

uninfected

RBCs

were

washed

twice

with

PBS

and

incubated

with

mg/ml

trypsin

in

PBS

for

min

at

room

temperature

with

gentle

mixing

-LRB-

modified

from

Chaiyaroj

et

al.

-RRB-

After

incubation

the

trypsin

was

inhibited

with

FCS

at

a

final

concentration

of

Conditions

for

trypsinisation

were

optimised

to

prevent

cell

lysis

and

the

cells

were

stained

with

giemsa

before

and

after

trypsinisation

to

confirm

RBC

integrity

was

maintained

The

RBCs

were

washed

three

times

in

PBS

and

resuspended

in

supplemented

with

FCS

and

co-cultured

with

HUVEC

as

described

We

also

analysed

adhesion

of

PRBCs

to

microslides

coated

with

μg/ml

Fc-tagged

recombinant

-LRB-

Gray

et

al.

-RRB-

under

laminar

flow

conditions

at

shear

stress

of

Pa

before

and

after

trypsinisation

in

order

to

evaluate

mediated

cytoadhesion

Transwell

experiments

Confluent

HUVECs

were

co-cultured

with

PRBCs

separated

using

a

μm

transwell

filter

-LRB-

Falcon

-RRB-

which

prevents

contact

between

HUVECs

and

RBCs

but

allows

soluble

factors

to

diffuse

through

This

set-up

allowed

us

to

determine

whether

contact

between

RBCs

and

ECs

was

necessary

for

induction

of

expression

on

the

ECs

Results

Up-regulation

of

expression

following

co-culture

with

infected

and

uninfected

RBC

is

expressed

constitutively

at

low

levels

on

the

surface

of

HUVEC

in

culture

Neither

PRBCs

nor

uninfected

RBCs

was

capable

of

inducing

appreciable

expression

over

h

-LRB-

Fig.

-RRB-

However

exposure

to

both

PRBCs

and

TNFlow

simultaneously

resulted

in

significant

up-regulation

of

expression

-LRB-

P

-RRB-

TNFlow

alone

was

incapable

of

inducing

expression

-LRB-

Fig.

-RRB-

Up-regulation

of

release

following

co-culture

with

infected

and

uninfected

RBC

Following

incubation

with

either

PRBCs

or

uninfected

RBCs

for

h

levels

in

the

supernatant

were

increased

over

basal

levels

-LRB-

P

-RRB-

-LRB-

Fig.

-RRB-

When

the

HUVECs

were

concurrently

exposed

to

PRBCs

and

TNFlow

release

was

further

amplified

over

basal

levels

-LRB-

P

-RRB-

Uninfected

RBCs

in

combination

with

TNFlow

induced

similar

levels

of

release

-LRB-

P

-RRB-

-LRB-

Fig.

-RRB-

Up-regulation

of

soluble

TNF

receptor

release

and

down-regulation

of

surface

expression

following

co-culture

with

infected

and

uninfected

RBC

Soluble

forms

of

both

TNFR

I

-LRB-

-RRB-

and

TNFR

II

-LRB-

-RRB-

were

significantly

increased

by

-LRB-

P

-RRB-

following

treatment

with

either

PRBCs

or

uninfected

RBCs

for

h.

Thus

the

phenomenon

of

soluble

receptor

release

from

the

surface

of

ECs

when

co-cultured

with

RBCs

appeared

to

be

independent

of

the

parasite

-LRB-

Fig.

and

b

-RRB-

Surface

levels

of

the

TNFRs

were

monitored

over

h

to

understand

the

dynamics

of

TNFR

expression

Both

TNFR

I

and

TNFR

II

were

significantly

reduced

in

response

to

both

PRBCs

and

uninfected

RBCs

by

-LRB-

P

-RRB-

and

-LRB-

P

-RRB-

respectively

within

min

-LRB-

Fig.

-RRB-

Interestingly

the

levels

of

the

TNFR

I

but

not

TNFR

II

fell

over

time

under

basal

conditions

albeit

at

a

slower

rate

than

co-culture

with

RBCs

This

could

be

a

result

of

serum

depletion

when

complete

medium

was

replaced

with

FCS

for

the

duration

of

the

co-culture

experiment

In

the

case

of

both

receptors

the

observed

down-regulation

in

response

to

RBCs

was

significantly

greater

than

that

seen

under

basal

conditions

Loss

of

adhesion

phenotype

following

trypsinisation

of

PRBC

Following

trypsinisation

PRBCs

did

not

lose

the

ability

to

up-regulate

expression

on

co-culture

with

ECs

-LRB-

Fig.

-RRB-

As

before

both

PRBCs

and

uninfected

RBCs

induced

an

increase

in

surface

levels

of

in

the

presence

of

TNFlow

mediated

adhesion

under

laminar

flow

however

was

abolished

following

trypsin

treatment

of

PRBCs

-LRB-

Fig.

-RRB-

In

additional

experiments

when

the

PRBCs

were

separated

from

the

ECs

in

plates

using

μm

transwell

inserts

there

was

no

stimulation

of

expression

-LRB-

data

not

shown

-RRB-

Thus

the

EC

activation

observed

in

our

system

was

critically

dependent

on

cell

cell

contact

although

it

did

not

involve

specific

cytoadherence

Modulation

of

the

endothelial

cell

transcriptome

following

co-culture

with

infected

and

uninfected

RBC

Using

ArrayMiner

software

-LRB-

OptimalDesign

-RRB-

seven

non-hierarchical

clusters

were

identified

-LRB-

Fig.

-RRB-

As

illustrated

on

the

resulting

heatmap

there

is

minimal

change

in

the

gene

expression

profiles

with

either

PRBCs

or

RBCs

compared

with

controls

-LRB-

Fig.

-RRB-

However

when

HUVECs

were

exposed

to

either

PRBCs

or

RBCs

in

combination

with

TNFlow

differential

expression

was

visible

in

three

out

of

the

seven

clusters

compared

with

control

or

TNFlow

alone

-LRB-

Fig.

and

b

-RRB-

A

comprehensive

analysis

of

the

microarray

data

was

performed

and

has

been

submitted

to

Array

Express

-LRB-

Accession

No

-RRB-

Our

analysis

focuses

on

genes

that

were

differentially

and

significantly

expressed

at

P

compared

with

control

levels

We

have

concentrated

on

over-represented

gene

ontologies

and

cited

genes

that

best

exemplify

these

GO

terms

In

cluster

both

PRBCs

and

uninfected

RBCs

in

combination

with

TNFlow

produced

over-expression

-LRB-

Fig.

and

b

-RRB-

TNFlow

alone

however

produced

a

significant

under-expression

Genes

in

this

cluster

included

the

inflammatory

protease

caspase

the

acute

response

cytokine

inflammatory

cytokines

IFN-γ

and

lymphotoxin

β

adhesion

molecules

E-selectin

and

chemokines

and

In

clusters

and

opposing

effects

were

induced

when

HUVECs

were

exposed

to

either

PRBCs

or

RBCs

in

combination

with

TNFlow

Over-expression

of

genes

was

observed

in

response

to

uninfected

RBCs

and

TNFlow

and

conversely

PRBCs

and

TNFlow

resulted

in

under-expression

of

genes

in

these

clusters

-LRB-

Fig.

and

b

-RRB-

In

cluster

TNFlow

on

its

own

had

no

effect

compared

with

controls

while

in

cluster

TNFlow

alone

produced

a

small

reduction

in

gene

expression

-LRB-

Fig.

-RRB-

Genes

in

cluster

included

the

protease

calpain

toll-like

and

the

junction

protein

desmocollin

while

genes

in

cluster

included

the

junction

protein

decorin

MHC

class

II

transferrin

endothelin

and

endothelin

receptor

type

A

While

it

is

difficult

to

extrapolate

roles

for

these

genes

from

this

in

vitro

analysis

of

the

transcriptome

we

can

make

some

speculations

based

on

recent

studies

For

instance

transferrin

induction

may

be

a

mechanism

for

iron

homeostasis

to

prevent

accumulation

of

iron

in

endothelial

cells

which

can

lead

to

oxidative

damage

of

the

cells

-LRB-

Nanami

et

al.

-RRB-

Endothelin

is

a

known

vasoconstrictor

in

vivo

down-regulation

of

endothelin

and

its

receptor

may

be

a

mechanism

for

maintaining

vascular

tone

and

integrity

during

malaria

infection

-LRB-

Basilico

et

al.

-RRB-

Down-regulation

of

MHC

class

II

may

represent

suppression

of

the

host

immune

response

by

the

parasite

The

genes

in

each

cluster

were

further

analysed

and

categorised

by

GO

-RRB-

terms

using

the

GOstats

package

of

R/Bioconductor

to

understand

their

basic

biological

functions

The

over-represented

GO

terms

defining

genes

in

each

cluster

in

terms

of

biological

process

cellular

component

and

molecular

function

showing

of

the

total

number

of

genes

being

annotated

with

that

term

were

evaluated

Fig.

illustrates

distribution

of

the

over-represented

gene

ontologies

with

respect

to

biological

process

and

cellular

component

in

clusters

and

which

reflect

inflammatory

and

protective

functions

in

the

endothelial

cells

The

full

analysis

for

all

three

clusters

is

detailed

in

Supplementary

Fig

-LRB-

see

on-line

supplementary

data

-RRB-

Whilst

some

GO

terms

fell

below

the

cut-off

it

is

important

to

note

that

they

were

nonetheless

significant

and

have

been

tabulated

for

further

information

-LRB-

Supplementary

Tables

and

see

on-line

supplementary

data

-RRB-

A

wide

range

of

GO

terms

were

over-represented

in

each

cluster

within

categories

of

biological

process

cellular

component

and

molecular

function

with

a

number

of

common

and

parallel

themes

including

cell

communication

signal

transduction

cell

adhesion

organismal

physiological

process

ion

transport

response

to

external

stimuli

and

immune

response

Interestingly

the

genes

in

each

of

these

clusters

were

primarily

cell

membrane-associated

or

related

to

the

extracellular

region

and

the

extracellular

matrix

The

molecular

functions

of

the

genes

in

each

cluster

were

wide

ranging

although

receptor

activity

signal

transducer

activity

ion

channel

and

protein

binding

activities

were

parallel

themes

in

all

three

clusters

The

fold

changes

for

the

genes

were

calculated

for

each

of

the

conditions

for

visualizing

the

changes

in

expression

compared

with

control

levels

Expression

of

and

RNA

was

up-regulated

reaching

and

respectively

over

control

levels

-LRB-

P

-RRB-

when

HUVECs

were

incubated

with

a

combination

of

PRBCs

and

TNFlow

Interestingly

transcription

reached

similar

levels

in

the

presence

of

a

combination

of

uninfected

RBCs

and

TNFlow

Table

illustrates

the

changes

in

and

expression

under

the

different

co-culture

conditions

Table

shows

the

changes

in

expression

of

selected

genes

which

illustrate

specific

biological

processes

of

ECs

that

may

have

a

role

in

mediating

either

pathology

or

protection

and

their

respective

fold

changes

in

the

presence

of

PRBCs

uninfected

RBCs

and

TNFlow

compared

with

controls

Although

the

magnitudes

of

change

are

relatively

small

they

are

highly

reproducible

and

biologically

significant

at

P

over

four

separate

replicates

The

chemokines

-LRB-

Gro-β

-RRB-

-LRB-

Gro-γ

-RRB-

and

E-selectin

were

all

over-expressed

in

the

presence

of

PRBCs

and

TNFlow

with

variable

increases

in

expression

of

and

in

the

presence

of

uninfected

RBCs

and

TNFlow

-LRB-

Table

-RRB-

Gro-β

has

been

shown

to

have

a

role

in

the

adhesion

of

monocytes

to

ECs

-LRB-

Schwartz

et

al.

-RRB-

while

has

similar

properties

to

and

can

be

co-induced

with

-LRB-

Gijsbers

et

al.

-RRB-

E-selectin

was

also

over-expressed

in

response

to

PRBCs

uninfected

RBCs

and

TNFlow

reaching

up

to

over

controls

with

PRBCs

and

TNFlow

Molecules

involved

in

cell

adhesion

such

as

and

were

both

over-expressed

in

PRBCs

uninfected

RBCs

and

TNFlow

with

a

greater

effect

in

PRBCs

-LRB-

Table

-RRB-

-LRB-

fractalkine

-RRB-

has

recently

been

implicated

in

mediating

adhesion

of

PRBCs

to

ECs

-LRB-

Hatabu

et

al.

-RRB-

Other

adhesion

receptors

such

as

desmocollin

and

integrin

β1

were

both

under-expressed

with

PRBCs

and

TNF

and

over-expressed

with

uninfected

RBCs

and

TNF

-LRB-

Table

-RRB-

A

number

of

inflammatory

genes

such

as

MHC

class

I

superoxide

dismutase

-LRB-

SOD

-RRB-

and

lymphotoxin-β

were

all

over-expressed

in

the

presence

of

PRBCs

and

TNFlow

-LRB-

Table

-RRB-

Whilst

both

MHC

class

I

and

SOD

were

also

over-expressed

with

uninfected

RBCs

and

TNFlow

lymphotoxin

β

was

under-expressed

Table

illustrates

differential

expression

of

molecules

such

as

TNFR-associated

factor

-LRB-

TRAF

-RRB-

Fas

associated

factor

-LRB-

-RRB-

prostaglandin

receptor

and

which

all

have

a

role

in

signal

transduction

A

number

of

cell

apoptosis-related

genes

were

differentially

regulated

in

our

co-culture

system

-LRB-

Table

-RRB-

Both

caspase

and

caspase

which

are

believed

to

be

inducers

of

apoptosis

-LRB-

Ho

and

Hawkins

-RRB-

were

under-expressed

in

the

presence

of

PRBCs

and

TNFlow

In

addition

caspase

expression

was

markedly

increased

in

the

presence

of

uninfected

RBCs

and

TNF

Calpains

which

are

inducers

of

cell

death

via

a

caspase-independent

pathway

-LRB-

Utz

and

Anderson

Perrin

and

Huttenlocher

-RRB-

were

also

regulated

in

our

system

We

looked

at

the

expression

of

two

calpains

and

Calpain

was

under-expressed

and

there

was

a

small

increase

in

calpain

expression

in

the

presence

of

PRBCs

and

TNFlow

-LRB-

Fig.

-RRB-

Thus

our

data

does

not

suggest

induction

of

apoptosis

in

fact

there

appears

to

be

a

trend

towards

protection

from

apoptosis

and

this

was

supported

by

our

inability

to

detect

any

positive

staining

with

annexin

V

and

the

absence

of

any

morphological

changes

in

the

ECs

after

co-culture

Discussion

Sequestration

of

PRBCs

at

vascular

sites

is

a

critical

event

in

the

pathogenesis

of

severe

malaria

Here

we

have

investigated

the

effect

of

prolonged

exposure

to

PRBCs

on

the

endothelium

We

have

demonstrated

a

phenomenon

whereby

ECs

are

differentially

modulated

when

co-cultured

with

PRBCs

in

the

presence

of

TNFlow

which

could

occur

at

microvascular

sites

early

in

infection

for

instance

during

schizogony

Unlike

previously

published

work

in

our

system

there

was

no

apparent

induction

of

or

modulation

of

RNA

transcription

by

PRBCs

alone

Significant

de

novo

induction

and

differential

regulation

of

gene

transcription

were

dependent

on

the

presence

of

low

levels

of

TNF

PRBCs

alone

did

modulate

certain

EC

functions

such

as

release

and

TNFR

shedding

from

the

cell

surface

but

release

was

further

enhanced

in

the

presence

of

TNFlow

Up-regulation

of

was

maintained

following

removal

of

surface

expressed

by

trypsin

digestion

but

was

abolished

when

cell

cell

contact

was

prevented

This

demonstrated

that

while

cell-to-cell

contact

was

crucial

for

modulating

EC

function

there

was

no

requirement

for

adhesion

via

specific

receptors

in

this

model

Despite

the

differences

in

the

reported

effects

of

PRBCs

on

ECs

the

unifying

theme

in

all

the

co-culture

studies

is

this

critical

requirement

for

close

apposition

of

the

two

cell

types

in

the

co-culture

system

in

the

induction

of

EC

activation

The

transwell

system

whilst

preventing

cell-to-cell

contact

allowed

the

movement

of

parasite-derived

soluble

factors

Therefore

we

could

exclude

the

role

of

soluble

parasite-derived

factors

in

EC

activation

In

all

cases

when

the

cell

cell

contact

was

compromised

using

a

filter

EC

activation

was

abolished

-LRB-

Randolph

and

Furie

Pino

et

al.

Viebig

et

al.

-RRB-

Thus

the

variable

observations

may

reflect

differences

in

the

respective

co-culture

conditions

for

instance

studies

by

Viebig

et

al.

-LRB-

-RRB-

were

performed

in

complete

HUVEC

culture

medium

and

studies

by

Pino

et

al.

-LRB-

-RRB-

were

performed

in

parasite

culture

media

while

our

studies

were

performed

in

a

serum-depleted

basal

HUVEC

medium

in

order

to

enhance

the

signal

window

for

the

EC

measurements

whilst

maintaining

EC

integrity

It

is

clear

from

our

work

and

that

of

others

that

care

will

need

to

be

taken

in

interpreting

the

results

of

model

co-culture

systems

and

that

further

work

will

be

required

to

develop

ex

vivo

models

of

severe

disease

It

is

possible

that

no

single

model

will

be

able

to

reflect

the

variable

pathology

represented

in

malaria

infection

Our

study

is

unique

in

the

observation

that

concomitant

presence

of

low

level

TNF

is

necessary

for

PRBCs

to

modulate

the

endothelium

namely

HUVECs

Indeed

a

recent

study

also

suggested

that

PRBCs

alone

do

not

have

the

ability

to

induce

in

HUVECs

and

that

this

may

be

a

phenomenon

specific

to

brain-derived

ECs

-LRB-

Tripathi

et

al.

-RRB-

Using

HUVEC

as

an

in

vitro

model

for

studying

PRBC-EC

interactions

in

vivo

we

have

demonstrated

that

close

apposition

or

cell

cell

contact

of

the

PRBCs

with

ECs

is

a

critical

factor

in

mediating

this

activation

Whilst

uninfected

RBCs

also

had

an

effect

on

ECs

in

the

presence

of

low

level

TNF

it

is

noteworthy

that

in

vivo

apposition

of

uninfected

RBCs

on

ECs

only

occurs

secondary

to

cytoadhesion

or

as

a

result

of

vessel

occlusion

The

ability

of

normal

RBCs

to

stick

to

and

modulate

ECs

in

vitro

however

is

not

a

novel

observation

Indeed

normal

RBCs

can

bind

to

ECs

mediated

by

plasma

factors

such

as

fibrinogen

and

fibronectin

-LRB-

Wautier

et

al.

-RRB-

and

the

binding

can

induce

low-grade

expression

in

vitro

-LRB-

Brown

et

al.

-RRB-

RBCs

express

a

wide

range

of

molecules

on

their

surface

which

have

been

implicated

in

mediating

adhesion

to

endothelial

cells

under

different

conditions

These

include

-LRB-

i

-RRB-

-LRB-

-RRB-

which

can

bind

to

endothelial

-LRB-

Walmet

et

al.

-RRB-

-LRB-

ii

-RRB-

the

blood

group

Lutheran

molecule

-LRB-

LU

-RRB-

over-expressed

on

sRBCs

can

bind

to

laminin

present

on

cells

or

in

the

intercellular

space

-LRB-

Eyler

and

Telen

-RRB-

-LRB-

iii

-RRB-

advanced

glycation

end

products

-LRB-

AGEs

-RRB-

present

on

RBCs

bind

to

their

receptor

-LRB-

RAGE

-RRB-

on

endothelium

-LRB-

Wautier

and

Schmidt

-RRB-

activating

endothelial

cells

and

-LRB-

iv

-RRB-

a

molecule

related

to

blood

group

Rhesus

-LRB-

Hermand

et

al.

-RRB-

binds

to

integrins

present

on

leukocytes

-LRB-

-RRB-

and

on

platelets

-LRB-

-RRB-

offering

a

surface

which

can

be

involved

in

thrombosis

-LRB-

reviewed

by

Wautier

and

Wautier

-RRB-

Thus

although

does

not

play

a

role

in

the

induction

of

expression

seen

by

us

it

would

normally

be

required

to

bring

the

PRBCs

and

ECs

together

in

vivo

Contrary

to

our

hypothesis

that

up-regulation

of

TNFRs

in

response

to

PRBCs

contributed

to

the

increased

sensitivity

to

TNF

unexpectedly

we

observed

significant

down-regulation

of

surface

TNFR

expression

and

increased

receptor

shedding

It

is

possible

that

the

enhancement

of

the

effects

of

RBCs

is

at

the

level

of

intracellular

signal

transduction

Rapid

TNFR

shedding

by

cleavage

of

the

extracellular

region

of

the

receptors

as

observed

in

our

studies

is

a

common

response

to

various

stimuli

-LRB-

Aderka

et

al.

-RRB-

and

may

be

a

mechanism

for

down-modulation

of

both

TNF

receptors

This

rapid

release

of

TNFRs

in

response

to

an

insult

to

the

endothelium

or

physical

cell

contact

with

the

endothelium

is

believed

to

be

a

non-specific

pro-inflammatory

response

and

up-regulation

of

soluble

TNFR

I

has

been

correlated

with

parasitaemia

in

the

serum

of

patients

with

acute

P.

falciparum

infection

-LRB-

Wenisch

et

al.

-RRB-

The

TNFR

shedding

seen

in

response

to

PRBCs

or

uninfected

PRBCs

alone

may

be

part

of

a

non-specific

inflammatory

response

of

ECs

to

prolonged

cell

cell

contact

Similarly

release

in

our

system

may

also

represent

a

non-specific

inflammatory

response

to

cell

cell

contact

with

RBCs

as

was

demonstrated

in

a

monocyte/EC

co-culture

model

and

was

further

shown

to

be

independent

of

mediated

binding

-LRB-

Lukacs

et

al.

-RRB-

Similarly

to

our

direct

measurements

of

there

was

no

significant

change

in

EC

gene

transcription

in

response

to

PRBCs

or

RBCs

alone

but

in

combination

with

TNFlow

significant

regulation

of

the

EC

transcriptome

was

induced

Analysis

of

the

transcriptome

revealed

a

wide

diversity

of

gene

ontologies

representing

genes

that

were

significantly

regulated

however

care

must

be

taken

in

interpreting

the

data

since

there

is

a

high

degree

of

redundancy

across

GO

terms

which

was

not

accounted

for

in

this

analysis

for

instance

has

diverse

biological

roles

including

cell

communication

cell

adhesion

signal

transduction

and

immune

response

In

spite

of

such

deficiencies

the

GO

terms

that

were

over-represented

including

cell

communication

cell

adhesion

signal

transduction

ion

transport

and

immune

response

fit

well

with

our

hypothesis

that

the

infected

RBC

is

able

to

modulate

the

host

endothelial

response

From

previous

work

we

had

expected

to

see

a

pro-inflammatory

response

induced

by

PRBCs

-LRB-

cluster

Fig.

-RRB-

Less

predictable

was

the

down-regulation

of

genes

in

the

presence

of

PRBCs

and

TNFlow

-LRB-

clusters

and

Fig.

-RRB-

which

may

represent

a

parasite-specific

adaptive

response

that

favours

parasite

survival

and

development

by

protecting

the

integrity

of

the

host

endothelium

Up-regulation

of

these

genes

under

the

same

conditions

may

result

in

the

induction

of

a

pro-adhesive

effect

that

can

potentially

feed

back

to

reinforce

pathological

sequestration

that

is

typically

seen

in

the

later

stages

of

severe

malaria

In

addition

induction

of

an

immune

response

as

in

cluster

may

confer

a

protective

effect

on

the

host

Thus

we

propose

that

the

balance

between

these

converse

effects

of

PRBCs

and

TNF

on

the

endothelium

may

be

the

critical

factor

in

determining

the

response

by

endothelium

and

the

clinical

outcome

of

a

malaria

infection

in

vivo

The

over-expression

of

genes

in

response

to

uninfected

RBCs

and

TNFlow

in

two

of

the

clusters

whilst

unexpected

is

a

common

phenomenon

in

static

in

vitro

co-culture

studies

For

instance

normal

RBCs

can

interact

with

ECs

to

induce

the

expression

of

a

variety

of

pro-inflammatory

molecules

including

-LRB-

Brown

et

al.

-RRB-

-LRB-

Brown

et

al.

Wautier

et

al.

-RRB-

and

activation

of

NADPH

oxidase

which

mediates

the

generation

of

reactive

oxygen

intermediates

-LRB-

Wautier

et

al.

-RRB-

This

is

a

potential

mechanism

to

induce

expression

of

genes

to

counteract

the

response

to

PRBCs

and

may

reflect

a

partially

protective

host

response

during

a

malaria

infection

balanced

against

inducing

a

pro-adhesive

environment

particularly

early

in

infection

when

systemic

inflammation

is

unlikely

to

be

a

major

contributor

to

pathogenesis

Modulation

of

host

cells

has

been

demonstrated

in

other

co-culture

models

for

instance

tyrosine

phosphorylation

of

scyncitiotrophoblasts

in

a

model

of

placental

malaria

-LRB-

Lucchi

et

al.

-RRB-

and

ecto-phosphorylation

of

following

the

initial

PRBC

attachment

which

is

believed

to

further

enhance

cytoadhesion

-LRB-

Yipp

et

al.

-RRB-

While

co-culture

models

give

useful

insights

into

mechanisms

of

action

of

PRBCs

on

host

cells

in

malaria

pathogenesis

care

must

be

taken

in

the

interpretation

and

extrapolation

of

this

data

to

pathogenesis

in

vivo

since

these

mechanisms

may

not

be

reflected

in

the

in

vivo

situation

In

summary

our

co-culture

model

identifies

a

novel

mechanism

whereby

PRBCs

whilst

mobilizing

a

pro-inflammatory

and

a

seemingly

pro-adhesive

effect

on

the

host

endothelium

have

the

additional

ability

to

suppress

gene

expression

pathways

involved

in

signal

transduction

apoptosis

and

immune

response

as

part

of

a

mechanism

to

support

parasite

survival

through

maintaining

the

overall

integrity

of

the

host

EC

This

fits

well

with

histo-pathological

observations

in

severe

malaria

where

transient

dysfunction

of

the

blood

brain

barrier

is

observed

-LRB-

Brown

et

al.

-RRB-

but

the

overall

health

of

the

endothelium

seems

to

be

maintained

despite

the

presence

of

many

adherent

PRBCs

-LRB-

Medana

and

Turner

-RRB-

Our

suggestion

is

that

the

host

response

to

sequestration

is

complex

and

involves

a

balance

between

pro-

and

anti-inflammatory

pathways

both

mediated

by

apposition

of

the

PRBC

and

interacting

with

other

mechanisms

of

host

response

to

malaria

infection

variably

throughout

an

infection

This

balance

is

critical

to

the

health

of

an

EC

and

the

resulting

pathology

associated

with

severe

malaria

O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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vascular endothelium
co-culture
icam-1
tumour necrosis factor