The effect of laparoscopic greater curvature plication on peripheral blood lymphocyte subsets (CD4+, CDS+ T cells) in morbidly obese patients

The effect of laparoscopic greater curvature plication on peripheral blood lymphocyte subsets (CD4+, CDS+ T cells) in morbidly obese patients

GhadaMorshed,aMD; MRCS;   Samah M.  Fathy,b PhD

a) Surgery Department, Faculty  of Medicine, Fayoum, Egypt.

b) Zoology Department, Faculty  of Science, Fayoum  University, Fayoum, Egypt.

Abstract

Background: Bariatric surgery is the only and effective treatment for morbid obesity and it can also improve the obesity-related comorbidities. However, the effect ofbariatric surgery on immune status is still unclear. In our study we investigated  the relationship between surgical weight loss and peripheral blood lymphocyte percentages.

Methods: Morbidly  obese patients (n=20, age range  25-50 years, body mass index [BMIJ range (37-45kgfm2) who had undergone laparoscopic greater curvature plication LGCP (were enrolled in a prospective study to determine the percentages of their peripheral blood T cells (CD4+,  and CD8+ T cells) before and (4 months) postoperatively  using flow cytometry. The data were expressed as the percentage oftotallymphocytes±the standard error ofthe mean.

Results: A decrease in the BMI occurred at 4months postoperatively with loss of weight of

(31.20±1.2%). PreoperativeBMJwas 44.71±4.3 (range 37-45kg fm2)and postoperative EM!was

31.80±1.1(range  24-33kg  fm2).The mean percentage ofCD4+ T lymphocytes  preoperatively was 38.2±1.5 and postoperatively  was 29.3±2.6  p <0.05 (which is statistically significant) so there is postoperative decrease in the percentage ofCD4+T lymphocytes, the mean percentage ofCD8+ T lymphocytes  preoperatively  was 17.3±1.8 and postoperatively  was 9.5±1.7p<0.05 (which is statistically significant) so there is  postoperative decrease in the percentage ofCD8+ T lymphocytes.

Conclusion: This study found that weight loss after LGCP in morbidly obese patients showed attenuated activation of circulating immune cells (decrease in CD4+ T helper cells percentage and decrease in CD8+ killer T cells percentage) and more regulation of chronic inflammation in morbidly obese patients.

Key words: Bariatric surgery, LGCP, morbid obesity, immunity.

Introduction:

Obesity  is a world  wide  health  problem with  several  comorbidities including respiratory diseases,  cardiovascular diseases, gall stones, osteoarthritis and reproductive disorders.!Furthermore, obesity is associated with decreased immunocompetence) Several studies  revealed   increased   incidence of infections and many types of cancers  in obese individuals.3,4

Bariatric surgery is the only effective treatment for  morbid  obesity  and  it also can improve   the  obesity-related comorbidities.5

In   obese    patients    bariatric    surgery    was

also  associated  with  a  decrease   in  overall mortality.6,7

In   obesity   low   grade   inflammation  is the product of the activated innate immune system, with activated tissue based innate immune     cells    and    circulating   immune cells.8,9  Recent studies   revealed the presence of  multiple  leucocyte  subsets   like mast cells and T-cells in adipose tissue which regulate inflammation.!0-13

It was  also  found   recently   that   obesity reduces  thymopoiesis and reduces     immune surveillance. l4

Since 2006, laparoscopic greater curvature

plication  (LGCP)  technique  has  been evaluated to eliminate AGB, VSG associated complications by restriction without gastric stapling  resection  and  without  an  implant used.

The aim of our study was to detect the effect of laparoscopic greater curvature plication on peripheral blood lymphocytes subsets (CD4+ and CD8+ T cells) in morbidly obese patients.

Patient and methods:

This  prospective  study  included  20 patients underwent laparoscopic greater curvature   plication   (LGCP)  to  assess  the effect of LGCP on excess weight loss and the percentages  of their peripheral blood T cells (CD4+ and CD8+ T cells) before  and four months postoperatively using flow cytometry.

The data were expressed as the percentage of total lymphocytes ± the standard error of the mean. From  June 2010 to July   2012 in El Fayoum University  Hospital we used the National Institute of Health's (NIH) inclusion criteria for bariatric surgery (patients with a body mass index >40kg/m2 or BMI over 35kg/ m2 with at least one comorbidity). We took an informed consent from all our patients. The study included twenty patients; 15 (75%) female  patients  aged  between  (25-46years) and 5 (25%) male patients aged between (38-

50 years). Patients were considered clinically obese with a near BMI 44.71±4.3 (37-45kg/ m2), mean age 39.5±9.5   (25-50years).  The outcome  included  loss  of  weight  in  short time, change of BMI and the percentage of peripheral blood T cells (CD4+ and CD8+ T cells)  before and after surgery.

Patients had full history taking, especially

for   family   history   of   similar   condition, BMI, age, social habits of smoking, alcohol consumption,  present medical history of any drug intake especially steroids, salicylic acid and non-steroidal anti-inflammatory drugs (NSAIDs)... etc.  Their  past  history  of  any deep venous thrombosis (DVT), any post surgical morbidities in the abdomen, any current clinical disease  in the abdomen  (e.g hernia, post surgical scarring...,etc) was also recorded.

The      results      of     their      preoperative

laboratory tests (including complete blood count (CBC), blood sugar, T3. T4, TSH liver, kidney  functions,  their  coagulation  profile and of their pre-operative abdominopelvic ultrasound were noted. Upper GIT endoscopy was done preoperatively for all patients to exclude gastritis, pulmonary function tests, ECG and anaesthetic consultation were done for   all  patients  preoperatively.   Fifteen   of the patients were smokers, with no alcohol consumption. Their imaging studies revealed non  cancerous   abdominopelvic   ultrasound and chest x-ray. Their preoperative laboratory tests revealed mild anaemia in 6 patients. We excluded patients who showed unfitness for general anaesthesia and major abdominal surgery, patients who were sweet or alcohol addicts, also patients with end stage  obesity comorbid diseases as advanced diabetes, advanced atherosclerosis, patients with psychological instability, patients with fear of operations, drug addiction and patients with lack of motivation to weight loss.

Surgical procedure:

LGCP procedure was done under general anaesthesia, and started by division of the greater curvature blood supply using the Harmonic scalpel distally till the pylorus and then proximally till the angle of His. Then the stomach was folded into itself over a 32- Fr bougie applying a first row of extramucosal stitches of2-0 vicryl. This row guided another row   created     with   extramucosal   running suture  lines of 2-0 prolene.  Methylene  blue was injected intraoperatively to check for leakage. The patients started eating 10 days post-operatively. Follow up were at 4 months after surgery. Figures(l-4).

Immune  cell  preparation  and  flow cytometry analysis:

Whole blood from all subjects were collected in acid citrate dextrose (ACD). Simultaneous    collections    were   made    in tubes containing potassium-EDTA for total lymphocyte count and allowed determination of absolute  counts.  Evaluation  of CD4  and CD8 lymphocyte counts and subsets were assessed by three-color  immunofluorescence flow   cytometry   using   a   MAbs   panel  to the  desired  cell  surface  proteins  including

fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated  MAb to CD4 and CD8 (Beckman Coulter Electronics, Hialeah, FL). All antibodies were prepared according  to  the  manufacturer's  directions and  then  were  incubated   with   100p,L  of whole blood for 5 min at 25°C before red cell lysis and fixation using Immunoprep reagents and Q-prep equipment (Beckman Coulter), as directed by the manufacturer. Cells were then stored at 4°C for up to 24 h before analysis by flow cytometer (EPICS XL; Beckman Coulter).

Statistical analysis:

Differences between means and the effects of treatments were determined by one-way ANOVA using Tukey's test,  P<0.05 was considered statistically significant.

Results:

The mean operative time was 65.6±10.2 (60-120) miutes. There  were no conversions. Postoperative  complications  were 5% in the form of nausea and vomiting treated within 12 days, no intraoperative  complications.  Mean hospital stay was 8.2±3.5 range (7-12) days, loss of weight was (31.20±1.2%); preoperative

BMI was 44.71±4.3 (range 37- 45kg/m2) and

postoperative  BMI was 31.80±1.1 (range 24-

33kg/m2). The mean percentage  of CD4+ T lymphocytes preoperatively was 38.2±1. 5 and postoperatively was 29.3±2.6 p <0.05 so there is postoperative decrease in the percentage of CD4+ T lymphocytes.  The mean percentage of CD8+T  lymphocytes  preoperatively  was

17.3±1.8   and  postoperatively   was  9.5±1.7

p<0.05 so  there was  postoperative decrease in the  percentage  of CD8+  T lymphocytes.

Figures(5,6).

Figures (1,2): Division ofthe vascular supply of the greater curvature of the stomach.

Figures (3,4): Plicated  stomach.

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Hist                                Region  ID                                                                   %                             Count                                         MniX                                          Mni Y

1                       A    A                                                                         5803                                          58282                                               609                                           2308

E    E                                               OoOO                                                    0                                              ****                           ••••

2                       Bl    B                                                                       34o5                                          20110                                          Oo183                                          4o50

B2   il                                              0013                                                74                                              1.35                                          3o84

B3   B                                                                       2801                                            16398                                          Ool79                                      Oo177

B4  B                                                                      3702                                         21700                                            2o21                                       00130

  Hist                                          Region  ID                                                                            %                                                       Count                                                  MniX

3                              c   c                                                       3406                                                     20148                                                     4050

4                              D    D                                                                                  41.2                                                       24027                                                      2004

Figure (5): Representative  flow cytometry for morbidly obese patient CD4+ & CD8+ Tcells percentages preoperatively.

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  Hist                                Region   ID                                                                  %                                           Count                                       MniX                                      Mn!Y

  Hisr                                           Region  1D                                                                              %                                   Coum                                                 Mn!X

Figure (6): Representative flow cytometry for morbidly obese patient CD4+ & CD8+ T cells percentages four months postoperatively.

Table (1): Patients characteristics.

Data are expressed as mean values± SD (standard deviation).

Discussion:

C D 4 + T 1 y m p h o c y t e s and CD8+ T lymphocytes are types of white blood cells known as lymphocytes  which have markers on the surface known as CD4, CD8. CD4 cells are commonly  known as T-helper cells, they help to detect and fight offbacterial and viral infection. CD8 cells detect and try to fight off infections caused by viruses or diseases such as cancer and are known as killer T cells.

Morbid       obesity       is       characterized by infiltration of adipose tissue with macrophages.l5  This  study  of  morbidly obese patients showed that dietary energy restriction and weight loss reduced CD4+ T cells  percentage  p<0.05,  reduced  CD8+  T cells percentage p<0.05 and this is considered statistically significant  compared  to other studies which did not show change in CD4+ T cells and CD8+ T cells percentages)6,17

Data on the effect of loss of weight post bariatric  surgery  on  inflammation  and immune cells are emerging, suggesting  the reduction  in the circulating  inflammatory markers.l8   Other  study   showed   also  that

immune cell activation can be regulated by acute energy restriction.l9 The mechanism by which weight loss and energy restriction in obesity attenuate proinflammatory activation of immune cells are unclear.

A study of gastric bypass showed a decrease in T lymphocytes CD95 and CD69 expression indicative of reduction of T lymphocytes activation.20   Our study builds on the data of decreased CD4+ T cells and decreased CD8+ T cells that there is attenuated    activation of circulating  immune  cells  post  weight  loss by laparoscopic greater curvature plication (LGCP).

In our study LGCP showed satisfactory weight   loss   like  other   studies,21,22 there were  no  major  complications  compared  to other studies  which showed   leakage22 and bleeding.23 Limitations  of this study include lack of a control group undergoing dietary energy restriction without weight loss.

In summary, this study found that weight loss after LGCP in morbidly obese   patients showed attenuated  activation of circulating immune  cells    (decrease  in  CD4+T  helper

T cells percentage) and more regulation of chronic inflammation in morbidly obese patients.

References:

1- WHO Consultation on Obesity. Obesity: preventing and managing the global epidemic.  WHO  Tech  Rep  Series  no.

894. Geneva, Switzerland:  World Health

Organization, 2000.

2- Marti A, Marcos A, Martinez JA: Obesity and immune function relationships. Obes Rev 2001; 2: 131-140.

3-  Falagas  ME,  Kompoti  M:  Obesity  and

infection.  Lancet   Infect   Dis   2006;   6:

438-446.

4- Renehan AG, Tyson M, Egger M, et al: Body-mass index and incidence of cancer: A systematic review and meta-analysis of prospective observational studies. Lancet

2008; 371: 569-578.

5- Chohan PS, Jackson B, Poplawski S, et al: Bariatric surgery for morbid obesity: Why, who, when, how, where, and then what? Cleve Clin JMed 2002; 69: 897-903.

6- Sjostrom L, Narbro K, Sjostrom CD, et al: Effects of bariatric  surgery  on mortality in Swedish obese subjects. N Engl J Med

2007; 357: 741-752.

7- Adams TD, Gress RE, Smith SC, et al: Long-term mortality after gastric bypass surgery.NEnglJ Med2007;357:753-761.

8- Van Oostrom AJ, van Wijk JP, Sijmonsma

TP, Rabelink TJ, Castro Cabezas M: Increased   expression   of  activation markers  on  monocytes  and  neutrophils in type 2 diabetes. Neth J Med  2004; 62:

320-325.

9- Wellen KE, Hotamisligil GS: Obesity­ induced inflammatory changes in adipose tissue. J Clin Invest2003; 112: 1785-1788.

10-Liu J, Divoux A, Sun J, Zhang J, Clement

K, Glickman JN, SukhovaGK, Wolters PJ, Du J, Gorgun CZ, et al: Genetic deficiency and   pharmacological    stabilization    of mast  cells  reduce  diet-induced   obesity and diabetes in mice. Nat Med  2009; 15:

940-945.

11-Nishimura   S,  Manabe  I,  Nagasaki   M,

M,  Hara  K,  Ueki  K,  Sugiura  S,  et  al: CD8+ effector T cells contribute to macrophage  recruitment  and  adipose tissue  inflammation  in obesity. Nat Med

2009; 15: 914-920.

12-Winer  S, Chan Y, Paltser G, Truong  D, Tsui  H,  Bahrami  J,  Dorfman  R,  Wang Y, Zielenski  J, Mastronardi  F, et al: Normalization of     obesity-associated insulin resistance through immunotherapy. Nat Med 2009; 15: 921-929.

13-Feuerer M, Herrero L, Cipolletta D, Naaz A, Wong J, Nayer A, Lee J, Goldfine AB, Benoist C, Shoelson S, Mathis D: Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters. Nat Med 2009; 15:

930-939.

14-Yang H, Youm YH, Vandanmagsar B, Rood J, Kumar KG, Butler AA, Dixit VD: Obesity  accelerates thymic  aging. Blood

2009; 114: 3803-3812.

15-Wellen KE, Hotamisligil GS: Obesity - induced inflammatory changes in adipose tissue. J Clin Invest 2003; 112: 1785-1788.

16-Viavdot A, Lord RV, Samaras  K: The effects of weight loss and gastric banding on the innate and adaptive immune system in type 2 diabetes and prediabtes. J Clin Endocriad metab 2010; 95: 2845-2850.

17-Merhi Zo, Durkin HG, Feldman I, et al: Effect of baniatric surgery on peripheral blood lymphocytes subsets in women. Surgery obesity  related Dis 2009;  5 (2):

165-710.

18-Kopp     HP,    Kopp     CW,    Festa     A, Krzyzanowska   K,  Kriwanek   S,  Minar E, Roka R, Schemthaner G: Impact of weight   loss   on   inflammatory   proteins and their association with the insulin resistance syndrome in morbidly obese patients.   Arterioscl   Thromb   Vase  Biol

2003; 23: 1042-1047.

19-Fraser  DA, Thoen  J, Reseland  JE, F0rre

0, Kjeldsen-Kragh  J:  Decreased  CD4_ lymphocyte activation and increased interleukin- 4 production in peripheral blood   of   rheumatoid   arthritis   patients after  acute  starvation.   Clin  Rheumatol