IMMUNOLOGY ανοσια · 2018-10-26 · CD19+IgM+: 58.1±42.7 μ/ L vs. 117.9±58.94 μ/ L,...

ανοσιαΤΟΜΟΣ 14, ΤΕΥΧΟΣ 3 • 2018

ISSN

2459-3524

anosiaQUARTERLY EDITION OF THE HELLENIC SOCIETY OF IMMUNOLOGY

VOLUME 14, NUMBER 3 • 2018

ΤΡΙΜΗΝΙΑΙΑ ΕΚΔΟΣΗ ΤΗΣ ΕΛΛΗΝΙΚΗΣ ΕΤΑΙΡΕΙΑΣ ΑΝΟΣΟΛΟΓΙΑΣ

′

HELLENIC

SOCIETY OF

IMMUNOLOGY

ΕΛΛΗΝΙΚΗ

ΕΤΑΙΡΕΙΑ

ΑΝOΣOΛOΓΙΑΣ

European Congress of ImmunologyECI 2018

Extended abstracts submitted in the contextof the Hellenic Society of Immunology

TRIMESTRAL EDITION OFTHE HELLENIC SOCIETY OF IMMUNOLOGYAlexandroupoleos 41-45, 115 27 Athens |Tel. + 30 211 4095472 | Fαχ: + 30 211 4095472 |E-mail: [email protected] |http: //www.helsim.gr

HONORARY PRESIDENTS† M. PavlatouE. KaklamaniI. Oikonomidou

BOARD OF H.S.I.President : Al. TsirogianniVice President : P. BouraGeneral Secretary : Al. SarantopoulosSpecific Secretary : As. FylaktouTreasurer : N. KafasiMembers : Chr. Nikolaou Al. Siorenta

EDITORIAL BOARDEditor in Chief : A. SarantopoulosHonorary President : P. Boura

Members :A. Poletaev G. KyriazisI. Theodorou Ch. NikolaouM. Varla-Leftherioti G. NtalekosE. Vlachaki Ch. PapasteriadiI. Gkougkourelas P. SkendrosM. Daniilidis O. TsitsiloniE. Kaklamani K. TseliosM. Kanariou M. ChatzistilianouI. Kountouras

SUBMISSION OF ARTICLES-SUBSCRIPTIONSanosiaA. Sarantopoulos, editor-in-chief2nd AUTH Dpt of Internal Medicine Hippokratio Gen. Hospital of Thessaloniki49 Konstantinoupoleos Str. | 546 42 Thessaloniki, GreeceE-mail: [email protected]

Annual subscription € 30,00

PUBLISHERUNIVERSITY STUDIO PRESSLeonidas A. MichalisArmenopoulou 32, 546 35 ThessalonikiTel. 2310 209637, 2310 209837, Fax 2310 216647E-mail: [email protected]

HELLENIC

SOCIETY OF

IMMUNOLOGY

contents

VOLUME 14 • NUMBER 3ISSN 2459-3524

anosia

EUROPEAN CONGRESS OF IMMUNOLOGY – ECI 2018

Extended abstracts submitted in the context of the Hellenic Society of Immunology

Αnalysis of B cell lymphocyte subpopulations in pre- and post-dialysis

end stage renal disease patientsD-V. Daikidou, A. Fylaktou, M. Stangou, D. Asouchidou,V. Nikolaidou, E. Sampani,

Th. Karampatakis,A. Anastasiou, Ch. Dimitriadis, A. Papagianni ......................................... 6

Study of PD-1/PD-L1 expression in hematological malignanciesA. Dodopoulos, K. Psarra, E. Grigoriou, I. Tsonis, G. Papagiannopoulou, A. Tsirogianni ...... 11

Soluble triggering receptor expressed on myelocytes -1 is strongly

correlated with disease activity in systemic lupus erythematosusA. Gkantaras, I. Gkougkourelas, A. Georgiadou, P. Boura ...................................................... 15

Heavy/Light Chain (HLC) ratio measurements in Intact Immunoglobulin

Multiple Myeloma (IIMM) patients - A single center experienceI. Kakkas, I. Konstantellos, S. Delimpasi, K. Papageorgiou, N. Harhalakis, A. Tsirogianni ..... 20

HLA typing and autoantibodies screening among family

members of celiac disease Greek patientsV. Kitsiou, K.Ampelakiotou, D. Kouniaki, K. Soufleros,Th. Athanassiades, K.Tarassi,

S. Pomoni, E. Synodinou, D. Siampani, I. Charoniti, A. Tsirogianni ....................................... 23

Serum pre-inflammatory cytokines TNF-a and IL-6 have higher

predictive strength compared to metalloproteases and markers

of tumor activity, bone metabolism and cell apoptosis in breast

cancer patients with bone metastasesA. Notopoulos, A. Sarantopoulos, P. Notopoulos, K. Psarras, C. Likartsis, E. Alevroudis,

I. Petrou, Z. Iconomou, G. Meristoudis, E. Zaromytidou, A. Doumas ................................. 26

Impact of rituximab therapy on IgG4 levels in Rheumatoid Arthritis patientsA. Sarantopoulos, E. Farmaki, M.G. Mytilinaiou, A. Gkantaras, A. Sarantopoulou,

P. Boura, A. Tsirogianni ............................................................................................................... 32

Proteomic analysis of a protective for rheumatoid arthritis TRAF-1 SNP.

Further implications on autoimmunity and cancerA. Sarantopoulos, K. Papavasileiou, G. Leonis, V. Melissas, I. Theodorou,

P. Boura, A. Notopoulos ............................................................................................................. 34

TRIMESTRAL EDITION OFTHE HELLENIC SOCIETY OF IMMUNOLOGYAlexandroupoleos 41-45, 115 27 Athens |Tel. + 30 211 4095472 | Fαχ: + 30 211 4095472 |E-mail: [email protected] |http: //www.helsim.gr

HONORARY PRESIDENTS† M. PavlatouE. KaklamaniI. Oikonomidou

BOARD OF H.S.I.President : Al. TsirogianniVice President : P. BouraGeneral Secretary : Al. SarantopoulosSpecific Secretary : As. FylaktouTreasurer : N. KafasiMembers : Chr. Nikolaou Al. Siorenta

EDITORIAL BOARDEditor in Chief : A. SarantopoulosHonorary President : P. Boura

Members :A. Poletaev G. KyriazisI. Theodorou Ch. NikolaouM. Varla-Leftherioti G. NtalekosE. Vlachaki Ch. PapasteriadiI. Gkougkourelas P. SkendrosM. Daniilidis O. TsitsiloniE. Kaklamani K. TseliosM. Kanariou M. ChatzistilianouI. Kountouras

SUBMISSION OF ARTICLES-SUBSCRIPTIONSanosiaA. Sarantopoulos, editor-in-chief2nd AUTH Dpt of Internal Medicine Hippokratio Gen. Hospital of Thessaloniki49 Konstantinoupoleos Str. | 546 42 Thessaloniki, GreeceE-mail: [email protected]

Annual subscription € 30,00

PUBLISHERUNIVERSITY STUDIO PRESSLeonidas A. MichalisArmenopoulou 32, 546 35 ThessalonikiTel. 2310 209637, 2310 209837, Fax 2310 216647E-mail: [email protected]

HELLENIC

SOCIETY OF

IMMUNOLOGYanosiacontents

VOLUME 14 • NUMBER 3ISSN 2459-3524

Epidermal Growth Factor (EGF) as biomarker of renal function

outcome in different forms of primary glomerular diseasesM. Stangou, A. Fylaktou, E. Sampani, C. Nikolaidou, C. Stamos, D. Daikidou,

E. Moraiti, A. Papagianni .......................................................................................................... 38

Interaction between the HLA-Shared Epitope (SE) and smoking

in anti-CCP positive Greek patients with Rheumatoid ArthritisK. Tarassi, E. Mole, V. Kitsiou, D. Kouniaki, Th. Athanassiades, K. Soufleros,

E. Synodinou, A. Bletsa, Ch. Sfontouris, A. Tsirogianni ........................................................... 42

Study of soluble vascular endothelial cell adhesion molecules

sICAM-1 and sVICAM-1 in hemodialysis patientsC. Tsigalou, T. Konstantinidis, A. Tsirogianni, G. Romanidou, M. Aslanidou,

K. Kantartzi, A. Grapsa, M. Panopoulou ................................................................................. 45

Implanted pro-activated silicon scaffolding vaccines in the treatment

of cancer (Εμφυτεύσιμα εμβόλια με τη χρήση προενεργοποιημένων

ικριωμάτων πυριτίου στη θεραπεία του καρκίνου)I. Zerva, Chr. Lanara, E. Stratakis, E. Athanasaki ................................................................. 49

UNIVERSITY STUDIO PRESSE!"#$%&' E(&$)*µ+,&!-, B&./01, & 2%3&+"&!-,ΘΕΣΣΑΛONIKH Aρμενοπούλου 32, 546 35 • ΑΘΗΝΑ Σόλωνος 94, 106 80Τηλεφωνικό Κέντρο: 2310 208731Ε-mail: [email protected] − www.universitystudiopress.gr

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Introduction

Chronic Kidney Disease (CKD) is linked to increased

incidence and severity of microbial infections, impaired

response to vaccination and increased risk of virus as-

sociated cancers1-3. Reduction in the number and dys-

function of lymphoid cells have been described in pa-

tients on chronic hemodialysis, however, it is still un-

clear whether these changes are due to uremia or to

renal replacement treatment itself. Furthermore, it re-

mains undetermined which stages of B cell maturation

are affected4,5.

B lymphocytes consist 10-15% of total peripheral,

50% of spleen and about 10% of bone maρrow lym-

phocytes. Apart from the production of antibodies,

they can act as antigen presenting cells, they can acti-

vate T cells and complement and also they produce

several types of cytokines. All these activities require

expression of different types of receptors on their

surface. Antigen receptors usually consist by im-

munoglobulin bind to signaling molecules. During their

maturation, B cells, pass through the formation of B

lymphocyte precursor, pro-B, pre-B, immature B, tran-

sitional B, mature B, plasmablast and plasma cell. During

these phases, they express different types of receptors.

CD19 and CD45 are expressed during all phases, but

CD5 is expressed on innate B1a cells, CD10 on tran-

sitional B cells, while CD27 molecule on memory B

cells. Also, during the early phases of maturation, they

express receptors to IL-7, which stimulates production

of lymphocytes and during the more advanced stages,

after being exposed to antigen stimulation, they ex-

press receptors to B cell activating factor (BAFF). BAFF

is produced by APC cells and stimulates maturation

of transitional B cells6-9.

The present study aimed at the analysis of B lym-

phocyte subsets in a cohort of pre-dialysis ESRD pa-

tients.

Methods

B cells (CD45+CD19+) and their subsets B1a

(CD19+CD5+), naive (CD19+CD27–), memory

(CD19+CD27+), CD19+ BAFF+ and CD19+IgM+,

were quantified using flow cytometry in the peripheral

blood of 21 pre-dialysis and 11 post-dialysis patients.The

results were compared to healthy control group, of

similar age, race and sex.

Inclusion criteria were: age >18-75 years, End

Stage Renal Disease (ESRD) at pre-dialysis stage. Pa-

tients were excluded when they had active autoim-

mune or chronic inflammatory disease, medical history

of malignancy, corticosteroids or immunosuppresive

treatment for the last 12 months. Furthermore, CRP,

ECI 2018

Analysis of B cell lymphocyte subpopulations

in pre- and post-dialysis end stage renal

disease patients

Daikidou D-V1, Fylaktou A2, Stangou M1,Asouchidou D2, Nikolaidou V2, Sampani E1,

Karampatakis Th2,Anastasiou A2, Dimitriadis Ch1, Papagianni A1

1Department of Nephrology, Aristotle University of Thessaloniki, Hippokration Hospital, Thessaloniki, Greece2Department of Immunology, National Peripheral Histocompatibility Center, Hippokration Hospital, Thessaloniki, Greece

ανοσ′ια 2018 ; 14, 3: 6 – 10

ανοσ′ια 2018 ; 14, 3 7

C3, C4, IgG, IgA, and IgM levels were also evaluated.

The first blood sample for the evaluation of B cell

subpopulations was collected one day before starting

dialysis method and the second one 6 months after

being on hemodialysis (HD) or on Continuous Am-

bulatory Peritoneal Dialysis (CAPD).

Results

Mean Age of the patients was 62.4±12.5 years, M/F

12/9). ESRD patients had reduced lymphocyte count

(1606±655 μ/L vs. 2459±520 μ/L, p<0.001) and B cell

(CD19+) count (82.7±54.9 μ/L vs. 177.6±73.8 μ/L,

p<0.001) compared to controls. Likewise, whereas the

percentages of B cell subsets were not particularly af-

fected, except for B1a subset (CD19+CD5+) which

presented a significant increase (4.1±3.8% vs. 0.7±0.7%

p<0.001) (Table 1), the absolute number of almost all

subsets was significantly smaller in ESRD patients

(CD19+: 81.3±60.4 μ/L vs. 162.1 ±64.5 μ/L, p=0.005,

Naive: 55.6±46.6 μ/L vs. 97.2±46.6 μ/L, p=0.004, Mem-

ory: 27.1±15.6 μ/L vs.83.5±56.8 μ/L, p<0.001,

CD19+BAFF+:69.5±47.5 μ/L vs. 154.7±74.6 μ/L, p<0.001,

CD19+IgM+: 58.1±42.7 μ/L vs. 117.9±58.94 μ/L, p=0.001)

(Table 2, Figure 1).

There were few differences fount in serum CRP,

C3, C4 and Immunoglobulins levels between ESRD

patients and controls, although they did not reach sta-

tistical significance (Figure 2).

Table 1. Differences in the frequencies of B cells subtypes between patients on pre-dialysis stage and controls.

B cells (%) CD45+ CD19+ CD5+ CD27+ CD27- IgM+ BAFF+

Controls 34.5±10.3 7.3±4 0.7±0.7 39.1± 7.5 60.9± 7.5 69.3± 11.3 83.9± 12.5

Patients 22.6± 8.3 5.8±4.3 4.1±3.8 35.3± 15.7 64.8± 15.7 69.9± 11.9 82.3± 8.5

p 0.005 0.38 <0.0001 0.38 0.38 0.91 0.69

Table 2. Differences in the B lymphocyte subpopulations between patients on pre-dialysis stage and controls.

B cells CD45+ CD19+ CD5+ CD27+ CD27- IgM+ BAFF+

Controls 2342± 625 177.6± 74 1.1±1.5 83.5± 57 97.2± 47 117.9± 59 154.7± 75

Patients 1766± 949 82.7±55 4.1±5.5 27.1± 16 55.6± 47 58.1± 43 69.5± 46

p 0.085 <0.0001 0.027 0.001 0.004 0.001 <0.0001

Figure 1. B cell subpopulations (frequency and absolute number) in patients with ESRD, differences from healthy controls.

100,0200

150

100

50

0

75,0

50,0

25,0

0,0

%

Abso

lute

Coun

t

CD19+CD5+% CD19+CD5+CD19+CD27+

CD19+BAFF+CD19+IgM+

NaiveCD19+BAFF+% Naive%CD19+CD27+% CD19+IgM+

Control Group

Patients Group

Control Group

Patients Group

8 ανοσ′ια 2018 ; 14, 3

There was a significant positive correlation be-

tween White Blood Cells (WBCs) with complement

components (C3 and C4) and also, significant correla-

tion between CD45+ cells and C3 levels) (Figure 3).

In 11 patients who had a follow-up 6 months after

starting on renal replacement treatment no differ-

ences were found, apart from the reduction of B1a

percentage (1.0±0.8% vs. 3.0±2.4%, p=0.038), and in-

crease of CD19+IgM+ (66.9±14.7μ/L vs. 74.7±7.4μ/L,

p=0.041) (Figure 4).

Figure 2. Differences between ESRD patients and healthy controls in the serum levels of CRP, C3, C4 and Immunoglobulins.

Control Group

Patients Group

Control Group

Patients Group300

200

100

0CRP C3 C4 IgA IgG IgM

Error Bars: 95% Cl Error Bars: 95% Cl

2.000

1.500

1.000

500

0

Figure 3. Correlation between WBC with C3, WBC with C4 and CD45+ cells with C3 levels in patients with ESRD before starting

on dialysis.

120,0

35,0

30,0

25,0

20,0

15,0

100,0

80,0

60,0

100,0

80,0

60,05000 6000 7000 8000 9000 10000 5000 6000 7000 8000 9000 100001000 2000 3000 4000

WBC CD45 WBC

C3

C3

C4

y=21,26+9,59E-3*x y=69,88+0,01*x y=9,66+1,98E-3*x

R2Linear=0,430 R2Linear=0,449 R2Linear=0,390

Figure 4. Reduction of B1a (CD19+CD5+) cells and increase of CD19+IgM+ cells 6 months after starting on dialysis.

20 120

100

80

60

40

20

0

15

10

5

0

%CD5 + (pre-HD)

p = 0.038 p = 0.04

%CD5 + (post-HD) CD19IgM + (post-HD)CD19IgM + (pre-HD)

*19

17

ανοσ′ια 2018 ; 14, 3 9

Discussion

In our study we estimated levels of B lymphocyte sub-

types in pre-dialysis patients and compared them with

a healthy control group, with similar age, sex and race.

Measurement were repeated for the patients group,

6 months after the initiation of dialysis. We tried to

find if there are any differences between End Stage

Renal Disease (ESRD) patients and controls and also,

we studied the changes of these cell subpopulations af-

ter commencing on a dialysis method. We also com-

pared the cell type concentration with other markers

of systemic inflammation. Our results showed a signif-

icant reduction in total lymphocyte count, and in most

B cell subpopulations in patients with ESRD on pre-

dialysis stage compared to controls, especially in

CD19+CD45+, CD19+CD27+, CD19+IgM+,

CD19+BAFF+, but in the same time there was a signif-

icant increase of B1a cells (CD19+CD5+). These findings

are indicative of an elimination in the population of B

cells being at the advanced stages of maturation, in as-

sociation of a significant increase in the frequency and

number of B cells at the early phases of maturation.

These findings are in accordance with previous reports,

which showed a significant imbalance between imma-

ture and memory B cells in ESRD patients4,10,11.

Deficiencies in immune response, including alter-

ations in B cell numbers and phenotypes and leading

to increased incidence of infectious diseases, malig-

nancies, atherosclerotic lesions and reduced response

to immunization, are well described in CKD pa-

tients3,12. Recent investigators have implicated reduced

number of B lymphocytes with increased incidence of

cardiovascular events in chronic kidney disease pa-

tients and also with increased risk of cardiovascular

and all-cause mortality13.

There is however a disagreement between the in-

vestigators, regarding the B cell subtypes which are af-

fected. Initial studies showed a global depletion of B cell

subtypes, including CD19+CD5+ cells, CD19+CD10+

and CD19+CD27+ cells and suggesting that B-cell lym-

phopenia should be attributed rather to central bone

marrow suppression due to uremic toxins, than to pe-

ripheral repression of B cell maturation4,14.

However, these studies estimated B lymphocyte

numbers and subpopulations in patients with ESRD

on dialysis and not on pre-dialysis patients. Our results

showed that CD5+ B cells were significantly increased

at ESRD, but, when they re-evaluated in the same pa-

tients after initiating dialysis, they were significantly re-

duced, as were the CD19+IgM+ B-cells.

This is a prospective, on-going study and these are

only the preliminary results. We confirmed the reduc-

tion of total lymphocyte number, the reduction in sev-

eral B-cell subpopulations, especially, memory B cells

and B cells expressing BAFF receptor, and we suggested

that there is an increase in immature B1a cells at pre-

dialysis stage. What happens after initiating a dialysis

method and which parameters in chronic kidney dis-

ease are implicated, whether these are the uremic tox-

ins, dialysis membrane, peripheral cytokines, such as IL-

7 of BAFF or apoptosis, remains to be elucidated.

Correspondence address:

Dimitra-Vasilia Daikidou

e-mail: [email protected]

References

1. Dai L, Golembiewska E, Lindholm B, Stenvinkel P. End-Stage

Renal Disease, Inflammation and Cardiovascular Out -

comes. Contrib Nephrol 2017; 191: 32-43.

2. Tecklenborg J, Clayton D, Siebert S, Coley SM. The role of the

immune system in kidney disease. Clin Exp Immunol 2018;

192(2): 142-150.

3. Ishigami J, Matsushita K. Clinical epidemiology of infectious

disease among patients with chronic kidney disease. Clin

Exp Nephrol 2018 Sep 3. doi: 10.1007/s10157-018-1641-8.

[Epub ahead of print].

4. Madeleine VP, Sastry G, Lili S, Pavan G, Reza E, Nosratola DV.

Effect of end-stage renal disease on B-lymphocyte sub -

populations, IL-7, BAFF and BAFF receptor expression.

Nephrol Dial Transplant 2010; 25: 205-212.

5. Smith MJ, Simmons KM, Cambier JC. B cells in type 1 diabetes

mellitus and diabetic kidney disease. Nat Rev Nephrol

2017; 13(11): 712-720.

6. Engels N, Wienands J. Memory control by the B cell antigen

receptor. Immunol Rev 2018; 283(1): 150-160.

7. Heinzel S, Marchingo JM, Horton MB, Hodgkin PD. The re -

gulation of lymphocyte activation and proliferation. Curr

Opin Immunol 2018; 51: 32-38.

8. Kristiansen TA, Vanhee S, Yuan J. The influence of developmental

timing on B cell diversity. Curr Opin Immunol. 2018;51:7-

13. doi: 10.1016/j.coi.2017.12.005. Epub 2017 Dec 19.

9. Khan WN. B cell receptor and BAFF receptor signaling

regulation of B cell homeostasis. J Immunol. 2009 Sep 15;

183(6): 3561-3567.

10. Kim KW, Chung BH, Jeon EJ, Kim BM, Choi BS, Park CW, Kim

YS, Cho SG, Cho ML, Yang CW. B cell-associated immune

profiles in patients with end-stage renal disease (ESRD).

Exp Mol Med 2012; 44(8): 465-472.

11. Pahl MV, Gollapudi S, Sepassi L, Gollapudi P, Elahimehr R, Vaziri

ND. Effect of end-stage renal disease on B-lymphocyte

subpopulations, IL-7, BAFF and BAFF receptor expression.

Nephrol Dial Transplant 2010; 25: 205-12.

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12. Wachter DL, Neureiter D, Câmpean V, Hilgers KF, Büttner-Herold M,

Daniel C, Benz K, Amann K. In-situ analysis of mast cells and

dendritic cells in coronary atherosclerosis in chronic kidney

disease (CKD). Histol Histopathol 2018; 33(8): 871-886.

13. Molina M, Allende LM, Ramos LE, Gutiérrez E, Pleguezuelo DE,

Hernández ER, Ríos F, Fernández C, Praga M, Morales E.

CD19+ B-Cells, a New Biomarker of Mortality in Hemo -

dialysis Patients. Front Immunol 2018; 9: 1221. doi:

10.3389/fimmu.2018.01221. eCollection 2018.

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failure have reduced numbers of memory B cells. Clin

Exp Immunol 2004; 137: 589-594.

Introduction

Patients suffering from malignant hematological dis-

eases, often fail to reach full remission. T lymphocyte

dysfunction, derived from inhibitory receptor signaling,

could be one of the causes. Coinhibitory receptors

are mainly expressed on activated T lymphocytes, to

maintain self-tolerance and prevent excessive tissue

damage during inflammatory reaction1. Programmed

Death protein 1 (PD-1) upregulation on activated lym-

phocytes as a result of ligand (PD-L1) overexpression

in malignant cells, inhibits T cell mediated cytotoxicity

and cytokine secretion2. The aim of this study was to

assess PD-1 and PD-L1 expression on patients’ lym-

phocytes and malignant cells respectively, using Multi-

color Flow Cytometry (MFC).

Patients – Methods

PD-1/PD-L1 expression was assessed on bone marrow

(BM) and peripheral blood (PB) samples from 101 pa-

tients with hematological malignancies (Acute lym-

phoblastic leukemia, ALL: n=7; acute myeloid leukemia,

AML: n=32; myelodysplastic syndromes, MDS: n=15;

multiple myeloma, MM: n=8; non Hodgkin lymphoma,

NHL: n=19; chronic lymphocytic leukemia/lymphoma,

CLL/SLL: n=20). PD-1 expression was evaluated in 11

PB or BM samples of healthy individuals. Cases were

analyzed for PD-1 and PD-L1 expression with a 7- and

5-color panel respectively.

Results

PD-1 is significantly overexpressed on T lymphocytes

from ALL, AML, MDS and NHL patients compared to

control group. PD-1 strongest expression was ob-

served on T lymphocytes from MM patients and PD1

weakest on CLL patients. PD-1 is significantly upregu-

lated on ΝΚ lymphocytes concerning ALL, AML and

ECI 2018

Study of PD-1/PD-L1 expression in hematological

malignancies

A. Dodopoulos1, K. Psarra1, E. Grigoriou1, I. Tsonis2, G.Papagiannopoulou1, A.Tsirogianni1

1Immunology-Histocompatibility Department, Evaggelismos General Hospital, Athens, Greece2Hematology - Lymphoma and BMT Units Dept, Evaggelismos General Hospital, Athens, Greece

ανοσ′ια 2018 ; 14, 3: 11 – 14

ABSTRACT

The aim of this study was to assess PD-1 and PD-L1 expression on patients with hematological malignancies’ lymphocytes and

malignant cells respectively, using Multicolor Flow Cytometry (MFC). PD-1/PD-L1 expression was assessed on bone marrow (BM)

and peripheral blood (PB) samples from 101 patients with hematological malignancies. PD-1 is significantly overexpressed on most

lymphocytes subpopulations from ALL, AML, MDS, MM and NHL patients compared to control group. PD-L1 was overexpressed

on patients’ malignant cells compared to lymphocytes. PD-L1 highest expression was shown on ALL lymphoblasts and the lowest

on malignant plasma cells from MM patients. Therefore Multicolor FC could be of use in identifying and monitoring patients that

might be of benefit from immunotherapy with novel monoclonal antibodies.

Key words: PD-1, PD-L1, hematological malignancies, multicolor FC.

12 ανοσ′ια 2018 ; 14, 3

ανοσ′ια 2018 ; 14, 3 13

MDS patients. PD-1 overexpression was also found in

MDS and MM patients’ B and CD8 T lymphocytes re-

spectively. Positive correlation regarding PD-1 expres-

sion in lymphocyte subpopulations was shown for

NHL (between CD4 and CD8 T cells), MDS (between

NK-B and NK-CD4 T cells) and MM patients (be-

tween NK-B and B-CD4 T cells) (Fig 1, 2). PD-L1 was

overexpressed on patients’ malignant cells compared

to lymphocytes. PD-L1 highest expression was shown

on ALL lymphoblasts and the lowest on malignant

plasma cells from MM patients (Fig 1, 2). Strong cor-

relation (r>0.5, p<0.001) was found between AML pa-

tients’ PD-1 and PD-L1 levels.

Discussion

Our results are in agreement with several studies from

the literature. Tamura et al. (2005) have shown that

both PD-L1, PD-L2 are overexpressed in both

leukemic cell lines and de novo AML patients3. By using

molecular techniques Yang et al. (2014) found that PD-

L1, PD-L2, PD-1 and CTLA-4 mRNAs are significantly

elevated in malignant myeloblasts from AML and MDS

patients4,5. In vivo studies from Si et al. (2012) shown

that PD-1and TIM-3 upregulation on T lymphocytes, is

Figure 1. PD-1 (CD279) and PD-L1 (CD274) expression in BM sample from an AML patient at diagnosis.

Figure 2. (a) PD-1 overexpression on patients’ T lymphocytescompared to healthy individuals. (b) PD-L1 overexpression onpatients’ malignant cells (blue boxplots) compared tolymphocytes (green boxplots).

*

*

*

*

100,00

10,00

132125

66

55

33

3335

39

64

82

141

1,00

10

1

0

,00

control CLL

CLL

NHL

NHL

MM

diagnosis

diagnosis

MDS AML ALL

MM MDS AML ALL

PD1_T_cells

PDL1_malignat_cellsPDL1_lymphocytes

a

b

required for pre-B-ALL progression6. According to

Qorraj et al. (2017), PD-1 overexpression in exhausted

T cells is related to monocyte dysfunction and disease

progression in CLL patients7.

Furthermore, PD-L1 is upregulated in monoclonal

B cells from CLL patients8. Grzywnowicz et. al. (2015)

confirmed that PD-1 overexpression in CLL correlates

with mutational status for IGHV and ZAP-70 genes9.

Andorsky et al. (2011) found that PD-L1 is significantly

upregulated in NHL cell lines (from DLBCL, ALCL pa-

tients) not only in malignant lymphocytes, but also in

immunoregulatory cells like Tregs, histiocytes and

macrophages9. Interesting data from clinical trials stress

out the clinical usage of soluble PD-L1 levels for MM

and DLBCL (Diffuse Large B cell lymphoma) patients10

Regarding MM, Liu et al. (2007) report that PD-L1

is overexpressed in malignant plasma cells from MM pa-

tients, in contrast to asymptomatic monoclonal gam-

mopathies10. Recent studies from Chang et al. (2018) cor-

related PD-1 levels in MM with prognostic factors like

diagnosis’ disease burden and β2 microglobulin levels11.

Our results suggest that PD-1 and PD-L1 are up-

regulated on lymphocytes and malignant cells, respec-

tively. Monoclonal anti-PD1 antibodies (nivolumab,

pembrolizumab) are used in solid tumor cancer pa-

tients’ treatment and are in clinical trials for many

hematological malignancies as well. Multicolor FC

could be of use in identifying and monitoring patients

that might be of benefit from immunotherapy with

novel monoclonal antibodies.


Katherina Psarra


References

1. Rezaeeyan H, Hassani SN, Barati M, et al. J Hematopathol.

2017; 10: 17-24.

2. Tsirigotis P, Savani BN, Nagler A. Programmed death-1 immune

checkpoint blockade in the treatment of hematological

malignancies, Annals of Medicine 2016; 48: 6: 428-439.

3. Bryan LJ, Gordon L.I. Blocking tumor escape in hematologic

malignancies: The anti-PD-1 strategy. Blood Rev. 2015; 29:

25-32.

4. Annibali O, Crescenzi A, Tomarchio, et al. PD-1/PD-L1

checkpoint in hematological malignancies. Leuk. Res. 2018;

67: 45-55.

5. Yang H, Bueso-Ramos C, Di Nardo, et al. Expression of PD-L1,

PD-L2, PD-1 and CTLA4 in myelodysplastic syndromes is

enhanced by treatment with hypomethylating agents.

Leukemia 2014; 6: 1280-1288.

6. Postow MA, Callahan MK, Wolchok JD. Immune check point

blockade in cancer therapy. J. Clin. Oncol. 2015; 33: 1974-

1982.

7. Grzywnowicz M, Karabon L, Karczmarczyk A, et al. The function

of a novel immunophenotype candidate molecule PD-1

in chronic lymphocytic leukemia. Leuk. Lymphoma 2015;

56: 2908-2913.

8. Andorsky DJ, Yamada RE, Said J, et al. Programmed death

ligand 1 is expressed by non-Hodgkin lymphomas and

inhibits the activity of tumor-associated T cells. Clin.

Cancer Res. 2011; 17: 4232-4244.

9. Zhu X, Lang J. Soluble PD-1 and PD-L1: predictive and pro -

gnostic significance in cancer. Oncotarget 2017; 8: 97671-

97682.

10. Liu J, Hamrouni A, Wolowiec D, et al. Plasma cells from multiple

myeloma patients express B7-H1 (PD-L1) and increase

expression after stimulation with IFN-gamma and TLR

ligands via a MyD88-, TRAF6-, and MEK-dependent

pathway. Blood 2007; 1: 296-304.

11. Jelinek T, Mihalyova J, Kascak M, et al. PD-1/PD-L1 inhibitors in

haematological malignancies: update 2017. Immunology

2017; 152: 357-371.

14 ανοσ′ια 2018 ; 14, 3

Introduction

Systemic lupus erythematosus (SLE) is a chronic au-

toimmune disease, whose clinical activity varies over

time, mainly manifesting with flares and remissions1. The

need for early diagnosis, monitoring, stratification and

accurate prognosis of lupus patients has led to the in-

tensive investigation of biomarkers, mainly biological,

biochemical, molecular or immunological variables

whose changes could reflect disease activity and/or re-

spond to treatment challenges2,3. Lupus biomarkers

can be classified into the classic ones, such as the com-

plement levels (C3, C4) and anti-dsDNA antibody

titers, and the modern ones, which consist of a variety

of cytokines, chemokines, adhesion molecules and lym-

phocyte subpopulations, such as the CD4+CD25high-

FOXP3+ T regulatory cells (Tregs)4.

Triggering Receptor Expressed on Myelocytes-1

(TREM-1) was discovered in 2000 as an innate immu-

nity receptor expressed on the surface of macro -

phages and neutrophils5. Its expression is induced by

Toll-like receptor (TLR) activation6. TREM-1 promotes

inflammatory reactions by enhancing the respiratory

burst and the production of pro-inflammatory cy-

tokines7,8. Specific enzymes, called sheddases, cleave

the membrane-bound part ofTREM-1 (mTREM-1) and

release a soluble form in the extracellular space9. The

possible anti-inflammatory actions of sTREM-1 are cur-

ECI 2018

Soluble triggering receptor expressed on

myelocytes -1 is strongly correlated with disease

activity in systemic lupus erythematosus

A. Gkantaras, I. Gkougkourelas, A. Georgiadou, P. Boura

Clinical Immunology Unit, 2nd Internal Medicine Department, Hippokration General Hospital, Aristotle University of Thessaloniki,

Thessaloniki, Greece

ανοσ′ια 2018 ; 14, 3: 15 – 19

ABSTRACT

Background: Soluble Triggering Receptor Expressed on Myelocytes -1 (sTREM-1) is an innate immunity receptor, which participatesin inflammatory reactions. Its serum levels reflect the magnitude of systemic inflammatory response and can discriminate betweeninfectious and non-infectious causes. Its role in systemic lupus erythematosus (SLE) is unknown. In this study we examined sTREM-1 in SLE patients with regard to disease activity.Patients and Methods: Sixteen patients with SLE were enrolled. Diagnosis was based on the revised 1997 American College ofRheumatology criteria. Disease activity was measured with the SLE Disease Activity Index-2000 (SLEDAI-2K). sTREM-1 levels weredetermined by Enzyme Linked Immunosorbent Assay (ELISA) in serum samples. Seventeen age- and sex-matched healthy individualscomprised the control group. Statistical analysis was performed with the SPSS package; p<0.05 was considered significant.Results: Serum sTREM-1 was significantly higher in SLE patients than in normal controls. Its levels were strongly correlated toSLEDAI-2K.Conclusions: Serum sTREM-1 is strongly correlated to disease activity in SLE, probably reflecting the generalized activation of theinnate immunity response in this disease and may be an additional biomarker in SLE.

Key words: Soluble Triggering Receptor Expressed On Myeloid Cells-1 (sTREM-1); Innate Immunity; Systemic Lupus Erythematosus

(SLE); Disease Activity.

rently investigated. Activation of the TREM-1 pathway

has been described in sepsis, pneumonia, chronic ob-

structive pulmonary disease, pancreatitis, gout and

peptic ulcer disease, as well as in systemic autoim-

mune diseases, like rheumatoid arthritis, Adamanti-

ades-Behcet disease and inflammatory bowel dis-

ease10-19. Recently, the plasma sTREM-1 was proposed

as a lupus biomarker since it was significantly corre-

lated with disease activity20,21. Liu et al. also confirmed

that, in lupus patients, sTREM-1 levels were significantly

elevated compared to healthy controls, while higher

sTREM-1 concentrations were associated with higher

anti-dsDNA titers22.

The aim of the present study was to investigate

the possible correlation of sTREM-1 with disease ac-

tivity in lupus, as expressed by the SLE Disease Activity

Index 2000 (SLEDAI-2K). This index was introduced

in 1985 in order to describe and quantify the variability

of disease activity over time among lupus patients and

it was revised in 200223,24.


Study population

Sixteen SLE patients (15 females, mean age 32±11 years,

mean disease duration 5±4 years) and 17 age- and

sex-matched healthy individuals were enrolled. SLE di-

agnosis was based on the revised 1997 American Col-

lege of Rheumatology criteria25. All patients were re-

cruited from the Clinical Immunology Outpatient Clin-

ic of the 2nd Department of Internal Medicine, Hip-

pokration General Hospital of Thessaloniki, over a pe-

riod of 2 years. Exclusion criteria were concomitant

infection, pregnancy and concurrent malignancy, along

with glucocorticoid and/or immunosuppressive treat-

ment for at least 6 months prior to blood sampling.

The control group consisted of 17 age- and gender-

matched healthy subjects (mean age: 26±7 years, 15

women) who fulfilled the same exclusion criteria.The

SLEDAI-2K score was used for the assessment of dis-

ease activity23,24. The Bioethics Committee of the Hos-

pital approved the study protocol and informed con-

sent was obtained from all participants.

Determination of sTREM-1 Serum

Concentration

Peripheral venous blood samples were obtained from

patients and controls upon visit. The samples were

centrifuged at 1000g for 10 minutes and then the plas-

ma was frozen and stored at -70°C. Commercially

available specific enzyme-linked immunosorbent assay

(ELISA) kits (USCN Life Sciences®) were used to

measure the concentration of sTREM-1, following the

manufacturer’s instructions. Each 100 μl serum sample

was transferred to the microstrip wells of the ELISA

plate and subsequently incubated for 2 hours at 37°C.

Then, the first detection reagent was added and the

reaction system was incubated for 1 hour at 37°C, fol-

lowed by 3 washing steps. After the addition of the

detection antibody, the reaction system was incubated

for 30 min at 37°C. Streptavidin-conjugated horserad-

ish peroxidase was used to detect antibody binding,

which was developed using a substrate solution. The

optical density was determined spectrometrically, after

adding sulphoric acid solution. The spectometrical

analysis was conducted with a microplate reader set

at 450±10 nm with a wavelength correction set at

570 nm to subtract background.The measurement of

sTREM-1 concentration was in accordance with a cal-

ibration curve, which used a human sTREM standard.

As read in the manufacturer’s instructions, a four-pa-

rameter logistic curve, fit for each set of samples as-

sayed, was used for the generation of the standard

curve. The lower detectable concentration was 3.8

pg/ml. All the values were within the linear portion of

the standard curve. The inter- and intra-assay coeffi-

cient of variation for the s-TREM tests were <10%.

Statistical Analysis

Results are expressed as mean±SD. Kolmogorov-

Smirnov test was used to test parametric data. Stu-

dent’ s t test was used to compare mean values. The

correlation between sTREM-1and SLEDAI-2K was

tested with the Spearman test.All analyses were per-

formed using SigmaStatSoftware 9 SPSS. A p<0.05

was considered statistically significant.

Results

The demographic, clinical, and serological characteris-

tics of the patients are shown in Table 1. Mean serum

sTREM-1 levels were significantly higher in lupus pa-

tients (43.2 pg/ml, range 10.2-80.1 pg/ml) compared to

healthy controls (5 pg/ml; range 3.3-8.3 pg/ml),p<0.001,

Figure 1. Mean SLEDAI score was 11±5 (range: 0-22).

sTREM-1 levels were positively correlated to SLEDAI-

2K (r=0.51, p=0.04), Figure 2.

16 ανοσ′ια 2018 ; 14, 3

ανοσ′ια 2018 ; 14, 3 17

Discussion

SLE is considered a systematic chronic autoimmune

disease of unknown etiology with various clinical man-

ifestations and a wide range of immunological abnor-

malities26-28. sTREM-1 levels in SLE are significantly low-

er compared to septic patients, but significantly higher

than healthy controls, while no difference was ob-

served between SLE and rheumatoid arthritis (RA)29.

Molad et al. also reported higher concentrations of

sTREM-1 in the serum of SLE patients (mean 1.1±2.8

pg/ml) as compared to healthy controls (mean 0.11±0.3

pg/ml; p<0.0001)20,30. These results are in agreement

with the findings of the present study.

The observed elevated sTREM-1 levels could be

attributed to the fact that SLE is characterized by el-

evated inflammation markers during disease flares3,4.

The involvement of TREM-1 in disease pathogenesis is

in line with experiments in C57BL/6lpr/lpr mice, in which

the inactivation of TLR4, a receptor that interacts with

sTREM-1, not only decreased autoantibody titers, but

also led to remission of lupus nephritis31.

The high expression of membrane and soluble

TREM-1 in systemic autoimmune diseases is activated

after recognition of certain damage-associated mo-

lecular patterns (DAMPs) through pattern recognition

receptors (PRRs), such as TLR4 and TLR920,32. DAMPs,

which are non-self or self-proteins, act as neoepitopes

and proinflammatory mediators, inducing the expres-

sion of TREM-1 and changing the phenotype of CD14+

monocytes33-36.

Furthermore, TREM-1-activated monocytes result

in the production of multiple proinflammatory cy-

tokines, chemokines and other inflammatory media-

tors that play a role in the propagation of tissue dam-

age in SLE37. Additionally, there is evidence that Toll-

like receptors (TLR) and other innate immune recep-

tors are implicated in SLE pathogenesis by enhancing

recognition of self-molecules26,38-40. In turn, TLR sig-

naling enhancesTREM-1 expression8. The human TLR9

also recognizes other DAMPs and seems to be in-

volved in the collapse of immunological tolerance to

endogenous nucleic acids41. It has also been demon-

strated that TLR9, presumably in synergy with TLR4,

induces the expression of TREM-1, leading to the clin-

ical manifestations of anti-glomerular basement mem-

brane antibodies-related nephritis42.

In the present study, there was a significant pos-

itive correlation between levels of sTREM-1 and

SLEDAI-2K. This may be associated with an increased

Table 1.The demographic, clinical, and serological characteristicsof SLE patients

Parameter SLE patients (N=16)

Demographic data Mean ± SD

Age (years) 32 ± 11

Female:Male Ratio 15:1

Disease duration (years) 5 ± 4

SLEDAI 11 ± 5

SLE ACR criteria No (%)

Malar rash 8 (50)

Discoid rash 1 (6.25)

Photosensitivity 7 (43.75)

Oral ulcers 5 (31.25)

Arthritis 10 (62.5)

Serositis 5 (31.25)

Renal disorders 9 (56.25)

Neuropsychiatricdisorders 4(25)

Pancytopenia 3 (18.75)

ANA 16 (100)

Anti-dsDNA 10 (62.5)

Figure 1. Box chart showing the mean values and the distributionof sTREM-1 in SLE patients and normal controls.

100,00

80,00

60,00

40,00

20,00

,00

sTRE

M-1

(pg/

ml)

SLE Patients Healthy Controls

Figure 2. Scatter plot demonstrating correlation of TREM-1 levelswith total SLEDAI-2K score (Spearman correlation).

100908070

60504030201000 2 4 6 8 10 12

SLEDAI

sTRE

M-1

(pg/

ml)

14 16 18 20 22 24

r=0,51p=0,04

18 ανοσ′ια 2018 ; 14, 3

shedding of TREM-1 and/or augmented production of

sTREM-1 due to NF-kB driven gene induction. Mem-

brane-bound TREM-1 expression does not respond

to SLEDAI-2K changes, indicating that the former

mechanism predominates (unpublished data).

Molad et al. reported very increased levels of

sTREM-1 in SLE patients, up to ten times higher than

healthy controls, although there was no significant cor-

relation with disease activity20. These results are con-

troversial, as the range of reported normal values was

rather different from those reported in previous stud-

ies. As stated by the author, there was a great hetero-

geneity in the sample, probably due to national or ge-

netic variation20.

On the contrary and in concordance with our

results, Bassyouni et al. found a significant correlation

of plasma sTREM-1 with SLEDAI (r=0.245, p=0.03).

sTREM-1 was detected in higher levels in patients with

neuropsychiatric lupus and pancytopenia21. Although

the investigators included a large number of individuals

leading to unambiguous results, the study included pa-

tients taking already glucocorticoids and other im-

munosupressants21. Immunomodulating therapy is a

major confounding factor in studies of inflammatory

biomarkers since they are involved in the NF-kB and

other pro-inflammatory pathways.

Our study is the first, to our knowledge, to eval-

uate sTREM-1 in treatment-naive SLE patients. The lim-

itations are the low number of participants, the inclu-

sion of patients with the same genetic background (all

Caucasians) and the lack of longitudinal measurements

to assess the biomarker’ s sensitivity to change.

In conclusion, sTREM-1 may be a promising bio-

marker, reflecting the activation of innate immunity in

SLE. Larger studies are needed to clarify if sTREM-1

could play a role in determining the activity status of

SLE or even herald a flare.

Conflict of interest

None

Acknowledgements

To IKY fellowships of excellence in postgraduate stud-

ies in Greece –Siemens program.


I. Gkougkourelas


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41. Lamphier MS, Sirois CM, Verma A, Golenbock DT, Latz E. TLR9and the recognition of self and non-self nucleic acids. AnnN Y Acad Sci 2006; 1082: 31-43.

42. Du Y, Wu T, Zhou XJ, Davis LS, Mohan C. Blockade of CD354(TREM-1) ameliorates anti-GBM-induced nephritis. In -flammation 2016; 39: 1169-76.

Introduction – Aim

During 2009, Bradwell et al presented a new tech-

nique for intact Immunoglobulin Multiple Myeloma

(IIMM) and Waldenstrom’s Macroglobulinemia (WM)

monitoring1,2. They developed and validated a method

for the separate quantification of the kappa and lamb-

da bounded amounts of circulating IgG, IgA and IgM

(Heavy/Light Chain-HLC assay). This was achieved by

developing antisera with specificity for unique epitopes

present at the junction between the heavy and light

chains constant regions of each immunoglobulin mol-

ecule1,2. This assay allows the quantification of the ab-

solute value of the involved IgGκ, IgGλ, IgAκ, IgAλ, IgMκ

and IgMλ along with their deriving ratios (IgGκ/IgGλ

etc, Heavy/Light Chain ratio, HLC ratio) as presented

in Tables A and B. According to the literature, these

measurements have been proven sensitive and specific

for the monitoring of patients with IIMM and WM3,4.

Additionally, the prognostic significance of HLC meas-

urements for symptomatic IIMM patients (before

treatment initiation) has been investigated. According

to the results of two relatively recent studies (Bradwell

2013, Ludwig 2013), extreme low or high HLC ratios

(<0.01 or >200) were associated with decreased over-

all survival of symptomatic IIMM patients5,6.

The purpose of this study was to investigate ex-

istence of any prognostic significance of HLC meas-

urements for symptomatic IIMM patients (before

treatment initiation), diagnosed and treated in our

Hospital’s Hematology and Lymphoma Department.

Heavy/Light Chain (HLC) ratio measurements in

Intact Immunoglobulin Multiple Myeloma (IIMM)

patients-A single center experience

I. Kakkas1, I. Konstantellos2, S. Delimpasi2, K. Papageorgiou1, N. Harhalakis2, A. Tsirogianni1

1Immunology and Histocompatibility Department, 2Hematology and Lymphoma Department, “Evaggelismos” General Hospital, Athens, Greece

ανοσ′ια 2018 ; 14, 3: 20 – 22

ECI 2018

ABSTRACT

The aim of this study was the investigation for existence of any prognostic significance of HLC measurements for symptomatic

IIMM patients. Forty-one newly diagnosed symptomatic IIMM patients were studied. The isotype of paraprotein was in 31 cases

IgG and in 10 cases IgA. Patient median follow-up was 16 months (range: 6-24). HLC ratio was determined in all patients before

treatment initiation. HLC measurements were performed by using the HevyliteTM assays (The Binding Site Group Ltd, UK) on a

SPA PLUS turbidometer. Statistical analysis was done by using the x2 test. At the time of last evaluation, five patients had died due

to disease progression and their median survival was seven months (range: 2-14). Extreme HLC ratios (<0.01 or >200) emerged

in 14 patients (7/31 IgG and 7/10 IgA, p <0.05). Two out of five deceased patients were IgG and three IgA. Also, four out of the five

deceased patients had extreme HLC ratios (p <0.05). It is noted that all three IgA deceased patients emerged extreme HLC

ratios (p <0.01). It is clear from the above-mentioned that there is a statistically significant correlation between mortality and HLC

ratio extreme values, especially for IgA patients.

Key words: Intact Immunoglobulin Multiple Myeloma (IIMM), Heavy/Light Chain (HLC) ratio, Mortality.

ανοσ′ια 2018 ; 14, 3 2121 ανοσ′ια 2018 ; 14, 3


Forty-one newly diagnosed symptomatic IIMM pa-

tients were studied. Twenty-five of them were men

and 16 women. Their median age was 68 years (range:

43-83). The isotype of paraprotein was in 31 cases IgG

and in 10 cases IgA. Twenty-four patients were ISS

stage I, 13 stage II and four stage III. Patient median fol-

low-up was 16 months (range: 6-24). Patient charac-

teristics are also presented in Table 1.

HLC ratio was determined in all patients before

treatment initiation. HLC measurements were per-

formed by using the HevyliteTM assays (The Binding

Site Group Ltd, UK) on a SPA PLUS turbidometer.

Results

Statistical analysis was done by using the x2 test. At

the time of last evaluation, 36 patients were alive. Five

patients had died due to disease progression and their

median survival was seven months (range: 2-14). Ex-

treme HLC ratios (<0.01 or >200) emerged in 14 pa-

tients (7/31 IgG and 7/10 IgA, p <0.05). Two out of five

deceased patients were IgG and three IgA. Also, four

out of the five deceased patients had extreme HLC

ratios (p <0.05). It is noted that all three IgA deceased

patients emerged extreme HLC ratios (p <0.01). The

aforementioned are also presented in Table 2.

Conclusions

Despite the limited number of patients in our study,

it is clear from the above-mentioned that our results

regarding IIMM patient mortality are in concordance

with those from the previously mentioned studies

(Bradwell 2013, Ludwig 2013). Especially, there is a sta-

tistically significant correlation between IgA isotype of

paraprotein and HLC ratio extreme values (<0.01 or

>200). Also, there is a statistically significant correlation

between mortality and HLC ratio extreme values, es-

pecially for IgA patients.


Ioannis Kakkas


References

1. Bradwell AR, Harding SJ, Fourrier NJ, et al. Assessment of

Monoclonal Gammopathies by Nephelometric Measure -

ment of Individual Immunoglobulin κ/λ Ratios. Clin Chem

2009; 55: 1646-1655.

2. Keren DF. Heavy/Light Chain Analysis of Monoclonal Gam -

mopathies. Clin Chem 2009; 55: 1606-1608.

Table 1. Patient Characteristics

Patients number 41

Gender Male 25 (61%)

Female 16 (39%)

Age (Median/Range) 68 έτη (43-83)

Isotype of Paraprotein IgG 31 (75%)

IgA 10 (25%)

ISS stage I 24 (58%)

II 13 (32%)

III 4 (10%)

Patients follow-up (Median/Range) 16 months (6-24)

Deaths 5 (12%)

Table 2. Extreme HLC ratio (ER) and mortality

ER (+) ER (-)

Patients number 14 (34%) 27 (66%)

IgG patients 7 (22%) 24 (78%)

IgA patients 7 (70%) 3 (30%) p<0.05

Dead patients 4 (80%) 1 (20%) p<0.05

Dead IgG patients 1 (50%) 1 (50%)

Dead IgA patients 3 (100%) 0 (0%) p<0.01

Deaths 5 (12%)

HLC Range Median value

IgGκ 4.23-12.18 g/L 7.76 g/L

IgAκ 0.43-2.36 g/L 1.27 g/L

IgGλ 2.37-5.91 g/L 4.00 g/L

IgAλ 0.40-1.73 g/L 0.87 g/L

HLC ratio Range Median value

IgGκ/IgGλ 1.26-3.20 1.96

IgΑκ/IgΑλ 0.58-2.52 1.40

Table A. Normal control measurements

HLC/HLC ratio Range Median value

IgGκ 5.22-81.6 g/L 49.4 g/L

IgGκ/IgGλ 5.8-1613 52.0

IgGλ 14.3-73.1 g/L 37.7 g/L

IgGκ/IgGλ 0.003-0.28 0.08

IgAκ 3.68-67.4 g/L 38.0 g/L

IgΑκ/IgΑλ 14.0-3675 85.0

IgAλ 2.10-55.6 g/L 18.9 g/L

IgΑκ/IgΑλ 0.001-0.44 0.04

Table B. Measurements of patients with IIMM and WM

22 ανοσ′ια 2018 ; 14, 3

3. Koulieris E, Panayotidis P, Harding, et al. Ratio of involved/unin -

volved immunoglobulin quantification by Heavylite(TM)

assay: clinical and prognostic impact in Multiple Myeloma.

Exp Hematol Oncol 2012; 1: 9.

4. Leleu X, Koulieris E, Maltezas D, et al. Novel M-Component

Based Biomarkers in Waldestrom’s Macroglobulinemia.

Clinical Lymphoma, Myeloma & Leukemia 2011; 11: 164-167.

5. Bradwell A, Harding S, Fourrier N, et al. Prognostic utility of

intact immunoglobulin IgGκ/IgGλ ratios in multiple

myeloma patients. Leukemia 2013; 27: 202-207.

6. Ludwig H, Milosavljevic D, Zojer N, et al. Immunoglobulin

heavy/light chain ratios improve paraprotein detection

and monitoring, identify residual disease and correlate

with survival in multiple myeloma patients. Leukemia 2013;

27: 213-219.

Introduction

Celiac disease is a chronic, immune-mediated, inflam-

matory intestinal disease caused by exposure to di-

etary gluten which leads to enteropathy with small-

bowel mucosal villous atrophy, in genetically predis-

posed individuals1.

Only a few decades ago, CD was considered an

uncommon disorder that affected children mainly in

Europe or in countries where Europeans had emi-

grated. However, in the past 50 years the prevalence

has risen significantly, with an estimate of overall preva-

lence rates increased in most countries in about 1-2%

of the population2.

Celiac disease can occur at any age3 and is char-

acterized by a variety of clinical manifestations (intesti-

nal or extra-intestinal) involving multiple organs. How-

ever, many pts are asymptomatic or have an atypical

form of the disease4,5. A number of clinical entities,

malignancies and autoimmune diseases, are significantly

associated with CD. Since the clinical spectrum is wide

and varying, early diagnosis is very difficult. So, undiag-

nosed pts may have chronic symptoms, severe com-

plications and excess mortality6.

The criteria for CD diagnosis include a combina-

tion of serological testing for IgA tissue Transglutami-

nase antibodies (anti-tTgAbs) and IgA Endomysial an-

tibodies (anti-EmA Abs), genetic testing (HLA-DQ2,-

DQ8), small bowel biopsy as well as response to a

gluten-free diet7.

Testing is indicated for individuals with symptoms,

and for some at-risk groups that show a particularly

high prevalence of CD such as pts with CD-associated

diseases (insulin-dependent diabetes mellitus, autoim-

mume thyroiditis, autoimmume hepatitis, Sjogren syn-

drome etc.) and CD family members8.

HLA typing and autoantibodies screening among

family members of celiac disease Greek patients

V. Kitsiou, K.Ampelakiotou, D. Kouniaki, K. Soufleros,Th. Athanassiades, K.Tarassi,

S. Pomoni, E. Synodinou, D. Siampani, I. Charoniti, A.Tsirogianni

Immunology-Histocompatibility Dept., “Evaggelismos” General Hospital of Athens, Greece

ανοσ′ια 2018 ; 14, 3: 23 – 25

ECI 2018

ABSTRACT

The aim of this study was the assessment of HLA and autoantibodies screening in the determination of familiar celiac disease

(CD) prevalence, in a Greek cohort. Twenty-nine recently diagnosed CD patients (pts) and 101 first-degree family members (FMs),

were studied. HLA-DQA1*/DQB1* typing by high resolution PCR-SSP techniques, anti-tissue transglutaminase (tTG) by ELISA and

anti-endomysial (EMA) autoantibodies (AAbs) by IIF, were performed. At least one CD predisposing HLA allele was typed in 28/29

(96.5%) pts and 77/101 (76.2%) FMs. Among them 86 (66.1%) were DQ2, 12 (9.2%) DQ8 and 7 (5.3%) DQ2/8 double positive. All

pts and 21 FMs were positive in at least one of the tested AAbs (100% tTG and 96% EMA) and all of them but one carried a high

risk HLA allele. The study revealed 21 new CD cases, all possessing both genetic and serological CD markers. In our cohort the

prevalence of CD among first-degree relatives appears higher (20.8%) than in the general population. A screening strategy with

HLA genotyping as well as tTG and EMA AAbs testing, should be strongly recommended for the identification of undiagnosed

FMs of CD pts.

Key words: Celiac Disease, HLA haplotypes, Autoantibodies, Family members.

24 ανοσ′ια 2018 ; 14, 3

The aim of our study was to estimate the preva-

lence of celiac disease and to assess the value of the

determination of HLA-DQ2/DQ8 genotypes com-

bined with serological markers (anti-tTg and anti-EmA

IgA Abs) for the identification of possible celiac disease

among FMs of CD pts, in Greek population; to our

knowledge no previous similar study has been con-

ducted.

Material and methods

In this study 29 CD pts (19 female and 10 male) diag-

nosed by immunological testing and biopsy according

to ESPGHAN criteria, were included. Subsequently,

samples from 101 first-degree FMs (49 female and 52

male) asymptomatic or not presenting typical symp-

toms, were tested. Forty-five (44.5%) were parents, 33

(32.7%) were siblings and 23 (22.8%) were CD pa-

tients’ children.

HLA class II (-DQA1*/-DQB1*) typing was per-

formed initially at low resolution level (two digits) us-

ing PCR-SSOP molecular techniques (Lab Type SSO

commercial kit, One Lambda). HLA-DQ2, HLA-DQ8

and respected HLA-DQA1* alleles were typed at high

resolution level (four digits) by the use of PCR-SSP

molecular techniques (commercial kits by Life Tech-

nologies and Olerup Corp.).

Anti-EmA Abs were assessed by Indirect Im-

munofluorescence (IIF), in a substrate of monkey oe-

sophagus sections (commercial kits, by Inova Diagnos-

tics). The initial dilution used was 1/5 and samples with

a titer over 1/5 were considered positive. For the de-

tection of anti-tTgAbs, enzyme-linked immunosorbent

assay (ELISA) was applied (commercial kits, by Inova

Diagnostics). Sera samples with a value over 25 Units

were considered positive.

Results

At least one CD predisposing HLA allele was typed

in 28/29 (96.5%) pts and 77/101 (76.2%) in FMs. In par-

ticular, 86 individuals (66.1%) were DQ2 positive, 12

(9.2%) DQ8 positive and 7 (5.3%) DQ2/8 double pos-

itive.HLA-DQA1*05:01/DQB1*02:01 haplotype was

present in 70 (53.8%), *02:01/02:02 in 19 (14.6%),

*03:01/03:02 in 17 (13.1%), *03:03/02:02 in 7 (5.4%),

*03:03/03:02 and *03:01/03:05 in 1 (0.8%) individuals

(Figure 1).

All pts and 21 FMs were positive in at least one

of the anti-EmA and anti-tTG Abs, and all of them but

one patient carried a high risk HLA allele, 41 -DQ2

and 8 -DQ8 (Figure 2). However, 56 individuals (43.1%)

without positive Abs expressed a high risk HLA allele.

Our study revealed 21 new CD cases (4 parents,

10 offsprings, 7 siblings) according to ESPGHAN diag-

nostic criteria, all possessing both genetic and sero-

logical CD markers.

In all CD cases (patients and FMs) the most pre-

dominant HLA allele was HLA-DQ2 (82.0%) and par-

ticularly the HLA-DQA1*05:01/DQB1*02:01 haplotype

(70.0%), followed by HLA-DQ8 (16.0%) and the HLA-

DQA1*03:01/DQB1*03:02 haplotype.

Discussion

Celiac Disease is an autoimmune disease with a severe

food intolerance that occurs as a result of a complex

interaction of environmental factors and multiple gene

variants.

The serological diagnostics tools for CD include

the determination of anti-tTg and anti-EmA IgA Abs.

Figure 1. HLA haplotypes’ frequency in CD pts and FMs.

60

50

40

30

20

10

0

HLA-DQA1*05:01/DQB1*02:01

HLA-DQA1*02:01/DQB1*02:02

HLA-DQA1*03:01/DQB1*03:02

HLA-DQA1*03:03/DQB1*02:02

HLA-DQA1*03:03/DQB1*03:02

HLA-DQA1*03:01/DQB1*03:05

Figure 2. Correlation between high risk HLA alleles and auto -antibodies.

PositivetTG, EmA

50 individuals(29 pts & 21 FMs)

41 (82%)HLA-DQ2positive

8 (16%)HLA-DQ8positive

1 (2%)HLA-DQ2/8

negative

ανοσ′ια 2018 ; 14, 3 25

The sensitivity of the tTG-IgA is about 95% while the

specificity is also 95% or higher. Anti-EmA IgA Abs

identification has a variable sensitivity between 75-

90% and specificity up to 100%. Individual tests may

be combined in order to increase the sensitivity. Once

a definitive diagnosis is established, use of these sero-

logic tests is recommended to verify compliance with

the gluten-free diet, which should be evaluated on a

yearly basis or every time pts experience clinical symp-

toms possibly related to gluten exposure1,4,8,9.

Genetic susceptibility is a prerequisite for devel-

oping CD, with HLA class II as the most important

genetic factor identified so far. Approximately 99% of

pts with CD have HLA-DQ2, -DQ8 or both alleles

and only 1% of pts do not express HLA-DQ2/8

genes10. In our study 98% (49/50) of all CD cases ex-

pressed at least one predisposing HLA allele. For the

patient with no HLA-DQ association, mutations of

genes encoding molecules and mediators of immune

reaction, of mucosal barrier-related genes, and of oth-

ers, might be responsible for the development of the

disease7.The HLA–DQ2 and –DQ8 alleles are present

in 25-30% of the general population. However, carrying

these two alleles is important but not sufficient for

CD, indicating the presence of non-HLA-susceptible

genes. Therefore, HLA-DQ2/DQ8 have a high nega-

tive value and are particularly useful to exclude diag-

nosis especially in at-risk groups11.

Evidence for a familial risk in CD has been accu-

mulated from many courses. Epidemiologic data sug-

gest an aggregation in 5-15% of CD relatives and a

striking 83-86% concordance rate among monozygot-

ic twin pairs. In our cohort CD prevalence among

first-degree FMs appears remarkably higher than in

general population and in agreement with literature

(20.8%). These data suggest that in contrast to the

general population, genetic and serological screening

in CD families may play a significant role in the prog-

nosis as well as in the early diagnosis of CD1,2.

The data of this and other similar studies indicate

that a screening strategy with HLA genotyping as well

as anti-tTG and anti-EmA Abs testing, should be

strongly recommended in FMs of CD pts and in other

high risk groups12.This approach would have a favor-

able cost-benefit ratio in the context of prognosis,

early diagnosis and potentially dietary intervention in

atypical CD cases.


Vasiliki Kitsiou


References

1. Szałowska D, Bąk-Romaniszyn L. Family recognition of celiac

disease. Przegląd Gastroenterologiczny 2013; 8(6): 390-395.

2. Freeman HJ. Risk factors in familial forms of celiac disease.

Word Journal of Gastroenterology 2010; 16(15): 1828-1831.

3. Aggarwal S, Lebwohl B, Green P, et al. Screening for celiac disease

in average-risk and high-risk populations. Therapeutic

Advances in Gastroenterology 2012; 5(1): 37-47.

3. Fasano A, Catassi C. Current Approaches to Diagnosis and

Treatment of Celiac Disease: An Evolving Spectrum.

Gastroenterology 2001; 120: 636-651.

4. Pelkowski T, Viera A. Celiac Disease: Diagnosis and Manage -

ment.Am Fam Physician. 2014 Jan 15; 89(2): 99-105.

5. Kelly C, Bai J, Liu E, et al. Advances in Diagnosis and Manage ment

of Celiac Disease. Gastroenterology 2015; 148(6): 1175-1186.

6. Megiorni F, Pizzuti A. HLA-DQA1 and HLA-DQB1 in Celiac

disease predisposition: practical implications of the HLA

molecular typing. Journal of Biomedical Science 2012, 19: 88.

7. Rubio-Tapia A, Hill I, Kelly C, et al. CG Clinical Guidelines:

Diagnosis and Management of Celiac Disease. The

American Journal of Gastroenterology 2013; 108: 656-676.

8. Rubio-Tapia A, Van Dyke C, Lahr B, et al. Predictors of Family Risk

for Celiac Disease: A Population-Based Study. Clinical

Gastroenterology and Hepatology 2008; 6(9): 983-987.

9. Lauret E, Rodrigo L. Celiac Disease and Autoimmune-

Associated Conditions. Biomed Res Int. 2013; 2013: 127589.

10. Ludvigsson J, Bai J, Biagi F, et al. Diagnosis and management of

adult coeliac disease: guidelines from the British Society of

Gastroenterology. Gut. 2014 Aug; 63(8): 1210-28.

11. Husby S, Koletzko S, Korponay-Szabo IR, et al. European Society

for Pediatric Gastroenterology, Hepatology, and Nutrition

Guidelines for the Diagnosis of Coeliac Disease. J Pediatr

Gastroenterol Nutr. 2012 Jan; 54(1): 136-60.

Objective

Taking into account a great number of studies and at-

tempts to formulate widely acceptable criteria, classical

parameters such as histological analysis, grade, TNM

staging and imaging have shown a proven strength in

the daily clinical practice, as well as certain clear con-

straints that arise from their use of mainly morpho-

logical features, rendering them rather rough ap-

proaches that fail to adequately capture the profile

and fluctuations of breast cancer (BC) patients and

do not have the desired accuracy to make decisions

selecting individualized therapeutic strategies.

Serum pre-inflammatory cytokines TNF-a and IL-6

have higher predictive strength compared to

metalloproteases and markers of tumor activity,

bone metabolism and cell apoptosis in breast

cancer patients with bone metastases

A. Notopoulos1, A. Sarantopoulos2, P. Notopoulos3, K. Psarras4, C. Likartsis1,

E. Alevroudis1, I. Petrou, Z. Iconomou, G. Meristoudis1, E. Zaromytidou1, A. Doumas5

1Department of Nuclear Medicine, Hippokration General Hospital of Thessaloniki22nd Department of Internal Medicine, Aristotle University of Thessaloniki, Hippokration General Hospital of Thessaloniki3Neuroinformatics Laboratory, Technological Institute of Central Macedonia42nd Propedeutical Surgery Department, Aristotle University of Thessaloniki, Hippokration General Hospital of Thessaloniki 52nd Department of Nuclear Medicine, Aristotle University of Thessaloniki, AHEPA University General Hospital of Thessaloniki

ανοσ′ια 2018 ; 14, 3: 26 – 31

ECI 2018

ABSTRACT

Objective: To evaluate the predictive strength of 32 serum markers in breast cancer patients with bone metastases (BC+BM)

under treatment.

Methods: The serum level of all markers has been determined in 153 BC+BM patients (a) at their enrollment in the study, (b) one

month later, and (c) after six months. We created a conventional “scan score” based on the number, size and metabolic activity of

BM in the initial bone scintigraphy. Levels of p53, bcl-2, TRAIL, caspase-3, FasL, Fas, MMP-1, MMP-2, TIMP-1, DKK-1, OPG, RANKL,

TRAP-5b, BAP and OPN were determined by an ELISA assay, while CEA, CA 15-3, TPA, CA 27.29, CYFRA 21-1, ICTP, PICP, PINP,

PIIINP, PTHrP, IGF1, CT, OC, TNFα and IL-6 were assayed by radiometric methods. The clinicopathological characteristics and serum

markers were compared among the subgroups identified either on the basis of the scan score (S1, S2 and S3) or of the disease

outcome (A1-recession or stability, A2-deterioration).

Results: S1 score was observed in 81 (52.94%) patients, S2 in 49 (32.03%) patients and S3 in the remaining 23 (15.03%) patients.A1

subgroup included 107 (69.93%) patients and 46 (30.07%) patients belonged to subgroup A2. IL-6, bcl-2 p53, MMP2 and OPG/RANKL

ratio efficiently reflected the extent and severity of the initial skeletal involvement. TNFa, IL-6, p53, MMP2 and TRAP had the higher

predictive strength being significantly lower in all measurements in patients with subsequent disease remission or stabilization.

Conclusion: The pre-inflammatory cytokines IL6 and TNFa help to predict more accurately BC+BM subgroups’ clinical behavior.

Key words: Pre-inflammatory cytokines, Biomarkers in human immune responses, Cancer immunology, Breast cancer.

ανοσ′ια 2018 ; 14, 3 27

The introduction of immunohistochemical (IHC)

methods has provided valuable predictive indicators for

the weighted application of specific therapies and the

discrimination of subgroups in both the TNM stages and

in the groups identified on the basis of receptor expres-

sion. However, tumors with common morphological and

IHC phenotypes may have varying outcomes. The ge-

netic signatures introduced into the clinical practice seem

to use gene groups with multiple pathobiological inter-

dependences and statistically they tend to form clusters

that are clearly correlated with already known param-

eters, thereby diminishing their potency as independent

prognostic or predictive factors.

All the above should be combined to achieve a

relatively satisfactory approach to the question the

clinical oncologists face: whether a certain BC patient

(not a group of patients) will benefit from the treat-

ment they intend to apply.This question has also been

of key importance in BC patients with bone metas-

tases (BM), as on the one hand BMs are responsible

for significant morbidity and decreased quality of pa-

tients’ life and a considerable financial burden on

healthcare systems and on the other hand the pro -

spective is not still desperate as it used to be in the

past, due to the effective combination of anti-absorp-

tion therapy – bisphosphonates (predominantly zole-

dronic acid) or monoclonal antibody against RANKL

(denosumab) – with hormonal therapy (such as ta-

moxifen, other SERMs, aromatase inhibitors) or tar-

geted therapy (such as monoclonal antibody to HER2

receptor or tyrosine kinase inhibitor of EGFR) or

chemotherapy or radiotherapy. Therefore, a rational

choice of optimal treatment with the minimum pos-

sible side effects and a way of timely and reliable as-

sessment or prediction of the outcome is needed.

Thus we proceeded to a prospective study of 32

serum indicators in 153 BC+BM patients at the begin-

ning of treatment, one month and six months later in

order to assess their predictive strength and their abil-

ity to assess the therapeutic efficacy earlier than the

imaging methods that require tumor size changes re-

flecting the accumulation or reduction of thousands

or millions neoplastic cells.

Patients - Methods

The serum level of all markers has been determined in

153 BC patients (a) when they were diagnosed to suffer

from BM and they enrolled in the study, (b) one month

later, and (c) after six months. Their mean age was

60.75±11.65 years, while the average duration of the dis-

ease (since initial diagnosis of BC) was 6.97±5.07 years,

the average height was 163.93±5.64 cm and their aver-

age weight 65.96±7.88 kg. Neither patients with bone

lesions requiring urgent radiation treatment nor patients

with an estimated life expectancy shorter than 6

months participated in the study. Additionally, patients

suffering from chronic renal failure, severe cardiovascular

disease, Paget’s disease or other conditions significantly

affecting bone metabolism or those taking therapies

with such an effect were not included in the study.

For all the enrolled patients, demographic data,

medical history, clinical, laboratory, imaging and biopsy

findings, levels of indicators, their follow-up, medication,

etc. were collected and transferred to a personal data

sheet to allow immediate access to useful data, the

addition or modification of information and the sta-

tistical processing of data electronically. The patients

enrolled in the study followed treatment that most

commonly involved the combination of an anti-ab-

sorbent agent (mainly zoledronic acid) and either ta-

moxifen or herceptin or letrozole.

We created a conventional “scan score” based

on the number, size and metabolic activity of BM in

the initial bone scintigraphy, ranging from S1 (1-2 small

BM with medium activity) to S3 (multiple BM).

Levels of p53, bcl-2, TRAIL, caspase-3, FasL, Fas,

MMP-1, MMP-2, TIMP-1, DKK-1, OPG, RANKL, TRAP-

5b, BAP and OPN were determined by an ELISA as-

say, while CEA, CA 15-3, TPA, CA 27.29, CYFRA 21-1,

ICTP, PICP, PINP, PIIINP, PTHrP, IGF1, CT, OC, TNFα and

IL-6 were assayed by radiometric methods. Blood

samples were collected from each patient, centrifuged

at 2,000x g for 15min, and stored at until assaying. All

samples were tested in duplicate and the intra-assay

reproducibility was satisfactory (2.31%-11.67%). Inter-

assay coefficient of variation (CV) had always been

less than 15%. Immunoassays were performed accord-

ing to the manufacturers’ instructions.

The study protocol has been approved by the

Provincial Ethical Committee and the Regional Health

Authority and is in accordance with the Helsinki Dec-

laration II and Standards of Good Clinical Practice. All

participants signed an approved written consent.

The clinicopathological characteristics and serum

markers were compared among the subgroups iden-

tified either on the basis of the scan score (S1, S2 and

S3) or of the disease outcome (A1-recession or sta-

bility, A2-deterioration).

28 ανοσ′ια 2018 ; 14, 3

Data are shown as mean±SD, unless otherwise

indicated. Basic demographic characteristics and mark-

ers’ serum levels were compared with Student’s t test

for unpaired observations. Both descriptive statistics

and graphs and the results of the statistical tests that

were applied (z-test, t-test, correlation tests, normality

tests, run tests, principal component analysis and factor

analysis), were performed using the Statistical Package

XL STAT version 2013.3.05 of Addinsoft, applied on

the spread sheet data we created with the well known

program Microsoft Office Excel 2010. Additionally, SPSS

version 20 was used. Differences and correlations

were considered statistically significant for p<0.05.

Results

S1 score was observed in 81 (52.94%) patients, S2 in

49 (32.03%) patients and S3 in the remaining 23

(15.03%) patients (Fig. 1). Before entering the study,

97/153 (63.40%) patients underwent hormone therapy,

while various chemotherapy regimens were applied

to 73/153 (47.71%) patients and radiotherapy protocols

were applied to 90/153 (58.82%) patients. Fig.2 shows

the distribution of patients based on the scan score

and the history of hormonal therapy, while Fig. 3 shows


and the history of chemotherapy and Fig. 4 depicts


and the history of irradiation of the breast. As these

habits may exert an impact on the levels of several

indicators, smokers occurred in 37 (24.18%) patients

and systemic coffee intake in 138 (90.20%) patients.

Concerning the receptor expression, 101 (66.01%) pa-

tients were ER+, while 52 (33.99%) were ER-, 78

(53.42%) were PR+ and 66 (46.58%) PR-, while for 7

patients we had no information on their PR status.

As we can see in Table 1 and in the boxplots of

Fig. 5, IL-6, bcl-2, p53, MMP2 and OPG/RANKL ratio

Figure 1. Pie chart of the distribution of BC+BM patients basedon the scan score.

S1

S2

S3

BC+BM

Figure 2. Distribution of BC+BM patients based on the historyof hormonal treatment and the current scan score.

100 123

80

60

40

20

158

33

16

49

32

0

scanscore

Y Nharmonal treatment

num

ber

Figure 3. Distribution of BC+BM patients based on the historyof chemotherapy and the current scan score.

123

80

60

40

20

9 14

2128

4338

0

scanscore

Y N

Figure 4. Distribution of BC+BM patients based on the historyof breast irradiation and the current scan score.

100 123

80

60

40

20

158

31

18

44

37

0

scanscore

Y Nbreast irradiation

num

ber o

f pat

ients

ανοσ′ια 2018 ; 14, 3 29

Figure 5. Boxplots of the 5 biomarkers that best correlate with the scan score.

60,00

300,00 6,00

5,00

4,00

3,00

2,00

1,00

,00

200,00

100,00

1200

1000

800

600

400

200

0

,00

,00

20,00

40,00

60,00

80,00

100,00

40,00

IL6

p53

MM

P2 bcl2

OPG/

RANK

L

20,00

1 2 3 1 2 3 1 2 3,00

scan score scan score scan score

1 2 3scan score

1 2 3scan score

70123

77136

126

97

33

2879

89

9

4890

BiomarkersS1ό S2

pdf

S1ό S3

pdf

S2ό S3

pdf

CEA 0,008 84.596 0,003 102 0,583 70

CA15.3 0,126 86.169 0,048 102 0,584 63.287

TPA 0,201 86.422 0,156 102 0,778 70

CA27.29 0,531 128 0,345 102 0,668 70

CYFRA 21.1 0,239 128 0,381 102 0,987 70

p53 0,002 128 <0,001 102 0,004 70

bcl2 <0,001 128 0,005 30.766 0,448 34.079

TNFa 0,367 128 <0,001 102 0,001 70

IL6 0,014 128 <0,001 102 0,048 70

FasL 0,758 128 0,702 102 0,575 70

Fas 0,679 128 0,284 102 0,193 70

FasL/Fas 0,772 128 0,653 102 0,536 70

TRAIL 0,536 128 0,514 102 0,293 70

MMP1 0,240 128 0,018 102 0,244 70

MMP2 0,003 73.179 0,005 27.157 0,462 70

TIMP1 0,036 128 0,011 102 0,413 55.481

caspase3 0,391 128 0,792 102 0,705 70

Dickkopf 1 0,253 128 0,727 102 0,638 70

Table 1. Comparison of the mean values of 18 biomarkers between the subgroups S1, S2 and S3 of BC+BM patients.

[df=degrees of freedom for the performance of Student t-test]

30 ανοσ′ια 2018 ; 14, 3

efficiently reflected the extent and severity of the initial

skeletal involvement. BAP, PTHrP, FasL, FasL/Fas, TRAIL,

Caspase-3, and Dickkopf-1 had the comparatively

worst performance in discriminating subgroups with

different scan score, as they were measured in prac-

tically similar levels in the 3 subgroups.

A1 subgroup included 107 (69.93%) patients and

46 (30.07%) patients belonged to subgroup A2. Pa-

tients of A1 subgroup had initially (prior to initiation

of treatment) statistically significantly reduced levels

of CA 15-3 TPA, TRAP, and CT, while the levels of the

other markers of bone metabolism and tumor mark-

ers did not differ statistically significantly. Patients of A1

subgroup had statistically significantly reduced levels

of CA 15-3, TPA, CEA, OC, OPG, RANKL, and

OPG/RANKL at both after one and six 6 months,

while the levels of the other markers of bone metab-

olism and tumor markers did not differ statistically sig-

nificantly. Among all the biomarkers, TNFa, IL-6, p53,

MMP2 and TRAP had the higher predictive strength

being significantly lower in all measurements in patients

with subsequent disease remission or stabilization.

Due to the large number of interdependent vari-

ables, we proceeded on the technique of factor analy-

sis which has advantages over the technique of mul-

tiple regression analysis, because it overcomes the

problem of multi-collinearity, due to the inherent cor-

relation and interdependence of some biomarkers,

which obscures the exact effect of any particular bio-

marker. Factor analysis reduces the number of vari-

ables, creating new complex variables (latent variables)

derived from the measured ones (observed or man-

ifest variables), highlighting structures based on the

grouping of variables under consideration, without in-

troducing any a-priori hypothesis concerning both the

number and the identity of the hidden variables. Factor

analysis revealed three new complex variables with

eigenvalues 4.6, 2.4 and 1.8 respectively (Fig. 6). The

eigenvalue represents the total dispersion that is in-

terpreted by each factor. The statistical programs take

into account only factors that have eigenvalues greater

than 1.0. We then used Varimax method to maximize

the variations observed. The finally resulting two prin-

cipal factors explain 34.25% of the variabilty of all pa-

rameters. Table 2 contains the specific load of each

marker on the two principal factors. The 1st complex

variable interprets a percentage of 22.15% of the total

variance of the initial measurements and receives a

major contribution from IL-6, TNFα, bcl-2, p53, and

MMP1. The 2nd complex variable interprets a percent-

age of 12.10% of the total variance of the initial meas-

urements and receives a major contribution from

FasL/Fas, Fas, and CA15-3.

Conclusions

In advanced BC, the time interval needed for scinti-

graphic or radiological evidence of BM resolution limits

its value to guide therapeutic decision making in prac-

tical terms. This sets the challenge forthward and our

study provides further support for the emerging con-

Table 2. Specific load of each marker on the 2 principal

factors.

F1 F2

CEA 0,330 -0,313

CA15-3 0,276 -0,538

TPA 0,233 -0,153

CA27.29 0,205 0,243

CYFRA21-1 0,179 0,001

p53 0,492 0,103

bcl2 0,549 -0,102

TNFA 0,593 -0,074

IL6 0,642 0,111

FasL 0,147 0,076

Fas 0,191 -0,862

FasL / Fas -0,155 0,905

TRAIL -0,102 -0,115

MMP1 0,460 0,450

MMP2 0,583 0,253

TIMP1 0,465 0,017

casp3 0,052 -0,098

DKK1 0,059 0,14

Figure 6. Scree plot of the latent variables and their specificEigenvalue.

5

4,5

Eigenvalue

Cum

ulat

ive va

riabi

lity (

%)

4

3,5

3

2,5

2

1,5

1

0,5

0 0

20

40

60

80

100

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18F19 F20 F21 F22axis

ανοσ′ια 2018 ; 14, 3 31

cept of using biomarkers whose serial measurements

in patients with BC+BM could successfully complement

imaging techniques not only for monitoring purposes,

but also for early and valid prediction of outcome.

Regarding the efficacy of the markers to reflect

the extent and severity of skeletal involvement, TNFa,

p53, TRAP, IGF1 and OPG/RANKL ratio exhibited the

best performance.

In the present study, a set of biomarkers com-

prising IL-6, TNFa, p53, ICTP, TRAP and CA 15-3 had

significantly lower levels in all measurements in patients

with remission or stabilization of the disease. It is very

interesting that the preinflammatory cytokines IL6 and

TNFa had superior performance – comparing to

markers of tumor activity, bone metabolism, cell apop-

tosis and tissue invasion – in predicting more accu-

rately BC+BM subgroups’ clinical behavior.

On the base of the well-established criteria, there

should be built meta-levels further and more accu-

rately refining BC subgroups of similar behavior.


Athanasios KC Notopoulos


Introduction

Rituximab (RTX – anti-CD20 monoclonal antibody)

is an effective therapeutic option in a variety of dis-

eases, including Rheumatoid Arthritis and IgG4-Re-

lated Disease. On IgG4-RD, it has been shown that

apart from B-cell depletion, rituximab induces remis-

sion by reducing IgG4 levels. On RA, even though se-

lective IgG4 reduction has been described, there has

still been no report investigating the inter-association

between IgG4 and other IgG subclass levels, following

rituximab therapy. On this regard, we investigated

whether B-cell depletion in RA is also associated with

a selective reduction of IgG4 and if the alterations de-

tected are analogous to disease remission index

(DAS28) variations.

Patients and Methods

31 RA patients, 25 female and 6 male, with a median

age of 59 years (34-73), and duration of disease 9,5

years (1-30), on standard of care DMARD treatment

and rituximab administration every 6 months for 2

years, were investigated for alterations on disease ac-

tivity along with IgG subclasses levels. All parameters

were assessed at enrollment (T0), and after 6, 12 and

24 months. On this 2-year period all patients had been

periodically receiving rituximab every 6 months.

ECI 2018

Impact of rituximab therapy on IgG4 levels

in Rheumatoid Arthritis patients

A. Sarantopoulos1, E. Farmaki2, M.G. Mytilinaiou3, A. Gkantaras1, A. Sarantopoulou2,

P. Boura3, A. Tsirogianni4

12nd Department of Internal Medicine, Hippokration General Hospital, Aristotle University of Thessaloniki, Greece2First Department of Pediatrics, Hippokration General Hospital, Aristotle University of Thessaloniki, Greece3Clinical Immunology Unit, 2nd Department of Internal Medicine, Hippokration General Hospital, Aristotle University of Thessaloniki, Greece4Department of Immunology and Histocompatibility, 'Evangelismos' General Hospital, Athens, Greece

ανοσ′ια 2018 ; 14, 3: 32 – 33

ABSTRACT

Rheumatoid Arthritis (RA) is an autoimmune disease characterized by T-cellular adaptive immunity predominance in exerting

pathophysiological tissue damage, thru a type IV hypersensitivity reaction. Activation of the humoral axis of the immune response

is considered as an epiphenomenon, contributing constrictively in the manifestation of extra-articular lesions of the disease, thru

a type III hypersensitivity reaction mechanism. On this regard, it has been long ago rationalized that therapies targeting B-cells lead

to remission by annulation of antigen presenting properties of these cells, properties that seem to be of crucial importance in Τ-

cell clonal shaping taking place in the tertiary lymph-node like structures developed in the pannus.

On the other hand, IgG4-related disease (IgG4-RD) has long been considered a B-cell driven entity, even though recent data

suggest implication of CD4+-cytotoxic cells as the keystone player in disease pathophysiology.

Either way, these two entities share few common pathophysiological aspects, rendering quite difficult the attempt to cross-attribute

any pharmacological mechanisms of action, especially when it comes to targeted therapies such as B-cell depletion. Nevertheless,

observation of common pharmacological effects at the level of immune modulation may provide clues for the enrichment of the

pathophysiological model of both diseases.

ανοσ′ια 2018 ; 14, 3 33

After 2 years of rituximab administration, patients

achieved a good response to treatment (EULAR cri-

teria). Igs’ levels were not statistically altered, though

all of them declined (data for IgM and IgA not shown).

Furthermore, from IgG subclasses, only IgG4 levels

statistically declined.

Discussion

On IgG4-RD, levels of IgG4 have been associated with

disease activity and IgG4 variations following RTX

therapy have been assessed as response index. This is

the first time that IgG subclasses variations are co-in-

vestigated in a non-IgG4RD after rituximab adminis-

tration. As in IgG4-RD, in RA, RTX therapy also affects

IgG4 levels. This observation may be interpreted in

two total different ways.

One possible conclusion is that IgG4-RD and RA

share common pathophysiological aspects in which

RTX therapy intervenes in order to achieve disease

remission. This case would be quite improbable if it

had been postulated some years ago. Nevertheless,

the recent characterization of IgG4-RD as a T-cell me-

diated disease may provide a step for this theory to

expand and be further validated.

On the other hand, the established and well-de-

scribed pathophysiological pathways of these two dis-

eases are still far away from sharing common aspects.

In this case, it may be concluded that IgG4 reduction is

a drug-associated characteristic. According to this ra-

tionale, it is not verified that the specific drug feature is

effective in IgG4-RD, since in RA the mechanism of ac-

tion of RTX is totally different and has already been

described. Therefore, down-regulation of IgG4 in both

diseases may be the result of reduced need for home-

ostatic effector mechanisms, as a result of disease re-

mission achieved thru other, cell mediated mechanisms.

Either the case, IgG4 is a parameter that needs

to be further assessed in several diseases’ pathophys-

iology and targeted biological therapies may be a use-

ful tool to this process, apart from being a means for

achieving disease remission. Last but not least, further

study of IgG4 may reveal emerging homeostatic im-

plications of the IgG subclass in diverse diseases.


Αlexandros Sarantopoulos


References

Carruthers MN, Topazian MD, Khosroshahi A, et al. Rituximab for

IgG4-related disease: a prospective, open-label trial. Ann

Rheum Dis. 2015 Jun; 74(6): 1171-7.

Khosroshahi A, Bloch DB, DeshpandeV, Stone JH. Rituximab Therapy

Leads to Rapid Decline of Serum IgG4 Levels and Prompt

Clinical Improvement in IgG4-Related Systemic Disease.

Arthritis Rheum. 2010 Jun; 62(6): 1755-62.

Sarantopoulos A, Gkougkourelas I, Mytilinaiou M, Klonizakis P,

Georgiadou A,Boura P. Rituximab selectively reduces IgG4 levels

in rheumatoid arthritis patients. DOI: 10.1136/annrheumdis-2017-

eular.6690.

Lighaam LC, Rispens T. The Immunobiology of Immunoglobulin

G4. Semin Liver Dis. 2016 Aug; 36(3): 200-15.

Falkenburg WJ, van Schaardenburg D, Ooijevaar-de Heer P, Wolbink

G, RispensT. IgG Subclass Specificity Discriminates Restricted

IgM Rheumatoid Factor Responses From More Mature

Anti-Citrullinated Protein Antibody-Associated or Isotype-

Switched IgA Responses. Arthritis Rheumatol. 2015 Dec;

67(12): 3124-34.

Table 1. DAS28 and IgG class and subclasses variations. Because of non-normal distribution of our sample, the results were

expressed as median and range and statistical analysis was performed by using the Kruskal Wallis tests.

DAS28 IgG IgG1 IgG2 IgG3 IgG4

T0 4,46(2,06-6)* 12,2(5,1-23,1) 7,7(3,59-12,4) 2,7(0,844-5,21) 0,573(0,079-1,85) 0,451(0,032-2,1)*

T6 3,8(2,2-6,44) 11,1(5,78-15) 7,28(3,38-11,2) 2,7(1,23-5,36) 0,445(0,086-1,58) 0,35(0,044-1,06)

T12 4,01(1,68-5,77) 11,1(5,36-19,2) 6,81(3,35-12,2) 2,53(0,876-4,67) 0,423(0,07-1,88) 0,279(0,036-1,24)

T24 3,27(1,5-5,07)* 11,2(4,92-14,5) 6,59(3,34-10,3) 2,42(0,92-4,7) 0,405(0,08-0,823) 0,248(0,025-1,08)*

*p<0.05 *p<0.05

Results

Introduction

Rheumatoid Arthritis (RA) is the most common sys-

temic autoimmune disease, with a respective expanded

genetic research1. Immunogenetic studies have docu-

mented the positive correlation of various gene loci

with incidence and/or disease profile. However, the de-

scription of gene loci negatively related to the incidence

of RA is rarely documented2. Even more seldom is the

functional, proteomic analysis of such an SNP and of

the respective intracellular signaling pathway.

On 2008, a Genome-Wide-Association Study

(GWAS) revealed an association of the TRAF1/C5 loci

with Rheumatoid Arthritis. TRAF (TNF Receptor-As-

sociated Factor) family consists of seven proteins that

are responsible for the intracellular signaling of the

TNFsfR (TNF superfamily receptors). Out of these

proteins, TRAF1 and its genetic variants have been

thoroughly studied in the context of autoimmune dis-

eases and in particular, Rheumatoid Arthritis. On RA,

the most outlined association was that of rs3761847,

a SNP in the intron, non-functional area of the gene.

This association was attributed to intra-gene param-

eters (linkage disequilibrium). Further investigation by

our team on the transcriptional areas of the TRAF1

gene (exons) revealed other SNPs that were as well

associated to RA. Their allocation on exon areas per-

mitted further proteomic study, revealing quaternary

proteomic modifications that could modulate the in-

tracellular TNFR-TRAF1-Nfkb pathway.

Many TNFRsf members are modulatory interme-

diates of cellular maturation and/or proliferation

(Table. 1). Their implication on cell homeostasis has

been well documented, especially in autoimmunity and

cancer. The fact that TRAFs consist a protein network

responsible for the intracellular signaling of the TNFRsf

(Fig. 1), raised the question whether these proteins

and related SNPs may also be implicated in cancer

pathogenesis.

Methodology – Results

On this study, 172 patients and 95 controls were ge-

netically assessed. Patients and controls were provided

by the Rheumatoid Arthritis outpatient clinic of the

Clinical Immunology Unit / 2nd Department of Internal

ECI 2018

Proteomic analysis of a protective for rheumatoid

arthritis TRAF-1 SNP. Further implications

on autoimmunity and cancer

A. Sarantopoulos1, K. Papavasileiou2, G. Leonis3, V. Melissas4, I. Theodorou5,

P. Boura6, A. Notopoulos7

12nd Department of Internal Medicine, Hippokration General Hospital, Aristotle University of Thessaloniki, Greece2Molecular Thermodynamics and Modeling of Materials Laboratory, Institute of Nanosciences and Nanotechnology,

National Center for Scientific Research “Demokritos”, Athens, Greece3Department of Chemistry and Department of Pharmacy, National and Kapodistrian University of Athens, Greece4Department of Chemistry, University of Ioannina, Greece5UF d'Histocompatibilité et Immunogénétique chez Assistance publique- hopitaux de Paris. Faculty of Medicine UPMC

Université Denis Diderot (Paris VII) / University Paris VII Paris 13, Île-de-France, France6Clinical Immunology Unit, 2nd Department of Internal Medicine, Hippokration General Hospital, Aristotle University of Thessaloniki, Greece7Department of Nuclear Medicine, Hippokration General Hospital, Thessaloniki, Greece

ανοσ′ια 2018 ; 14, 3: 34 – 37

ανοσ′ια 2018 ; 14, 3 35

Member Name Ligand Function

TNFRSF1A TNFR1 TNFα, LTα TNF mediated inflammation and apoptosis

TNFRSF1B TNFR2 TNFα, LTα Apoptosis

TNFRSF3 LTβ-R LIGHT, LTβ Lemphopoiesis

TNFRSF4 OX40 OX40L Costimulation

TNFRSF5 CD40 CD40L Costimulation APCs and Β lymphocytes

TNFRSF6 Fas, CD95 FasL Apoptosis

TNFRSF6B Decoy Receptor 3 FasL, TL1A, LIGHT Apoptosis

TNFRSF7 CD27 CD27L Costimulation

TNFRSF8 CD30 CD30L CD8+ T-L downregulation

TNFRSF9 4-1-BB, CD137 4-1-BBL Costimulation

TNFRSF10A Death Receptor 4 TRAIL DC and cancer cell apoptosis

TNFRSF10B Death Receptor 5 TRAIL DC and cancer cell apoptosis

TNFRSF10C Decoy Receptor 1 TRAIL DC and cancer cell apoptosis

TNFRSF10D Decoy Receptor 2 TRAIL DC and cancer cell apoptosis

TNFRSF11A RANK RANKL Osteoclast genesis, induction of antigen-presentation process

TNFRSF11B Osteoprotegerin (OPG) TRAIL, RANKL Osteoclast genesis

TNFRSF12A TWEAK-R TWEAK Inflammation

TNFRSF13B TACI APRIL, BAFF BAFF-R competition

TNFRSF13C BAFF-R BAFF Β-L maturation, plasmablast survival

TNFRSF14 HVEM LIGHT Costimulation

TNFRSF17 BCMA APRIL, BAFF Th2 deviation, Β-L maturation, plasmablast survival

TNFRSF18 GITR GITRL Costimulation, Tregs

TNFRSF19 TAJ Tissue ontogenesis

TNFRSF19L RELT Costimulation

TNFRSF21 Death Receptor 6 Β and Τ-L inhibition

TNFRSF25 Death Receptor 3 TL1A Inflammation

EDAR EDA1 E1 Embryogenesis

Table 1. TNF-Receptor superfamily members, their ligands and function.

TACI: Transmembrane activator and CAML interactor HVEM: Herpes virus entry mediator, BCMA: B- cell maturation antigen, TAJ: Toxicity and JNK inducer,

RELT: Receptor Expressed in Lymphoid Tissues, EDA: Ectodysplasin A

36 ανοσ′ια 2018 ; 14, 3

Figure 1. The Intracellular TRAF associated signaling cascade.

Figure 2. The damaging effect of rs 143265058 on the structure of TRAF-1 protein.

ανοσ′ια 2018 ; 14, 3 37

Medicine / Aristotle University of Thessaloniki / Hip-

pokration Hospital. An immunogenic sequence of the

seven exons of the gene TRAF1 was performed in all

patients and controls.

Among other results, on the position 9:120905076

of exon 7, the registered polymorphism G/A

(rs143265058) was described in the controls group. The

same polymorphism was not confirmed in any of the

patients. Statistical elaboration characterized the finding

as important, supporting further functional proteomic

study of the respective transcript with computing pro-

grams (software). The proteomic study revealed that

the presence of this polymorphism leads to a differen-

tiation of the quaternary structure of TRAF1 protein3

(Fig. 2).

Discussion

The present reference is one of the extremely rare

genetic studies describing a protective gene locus

against rheumatoid arthritis, and a pioneer of its kind

in the use of Applied Informatics in the depiction of

the quaternary structure of the encoded protein. At

the same time, it is one of the few immunogenetic

studies describing the functional proteomics of the

specific encoded protein, plotting on a molecular level

interaction modifications affecting the intracellular sig-

naling pathway of TNFα. This effect on intracellular

signaling pathways may address diverse susceptibility

of the specific and similar SNPs not only to RA, but

to other clinical manifestations implicating this pathway,

such as cancer. On this regard, one of the perspective

aims of this study is to identify if the observed SNPs

on TRAF1 protein may have an additive or depleting

effect on the interaction with its ligands, in particular

with of those participating in intracellular pathways

implicated in cell proliferation. This approach could re-

veal secondary “checkpoints” that might be used to

alter the immune response to the desired direction,

for the treatment of cancer. The most outlined of

TRAF1 ligands implicated in cancer homeostasis and

development are listed in Table 2.


Αlexandros Sarantopoulos


References

1. Kim K, Bang SY, Lee HS, Bae SC. Update on the genetic

architecture of rheumatoid arthritis. Nat Rev Rheumatol.

2017 Jan; 13 (1): 13-24.

2. Sarantopoulos A, Theodorou I, Notopoulos A, Koulas I, Boura P.

First description and functional proteomic analysis of a

protective for Rheumatoid Arthritis gene polymorphism

DOI: 10.1136/annrheumdis-2017-eular.6818.

3. SarantopoulosA. TRAF-1 immunogenetic study in Rheumatoid Arth -

ritis. PhD Thesis, Aristotle University of Thessaloniki, 2016.

(Http://thesis.ekt.gr/thesisBookReader/id/38410#page/1/m

ode/2up).

Products Interactant Other Gene Comments

NP_005649.1 NP_001157.1 BIRC2

NP_005649.1 NP_001156.1 BIRC3

Q13077 Q14790 CASP8

BioGRID:113037 BioGRID:113041 TRAF6

BioGRID:113037 BioGRID:109767 IKBKB Nfκβ

BioGRID:113037 BioGRID:114089 IKBKG

Q13077 P20333 TNFRSF1B TNFR2

BioGRID:113037 BioGRID:113038 TRAF2

BioGRID:113037 BioGRID:114257 TRADD TRAF1-TRADD ancoration with TNFR1

BioGRID:113037

BioGRID:113037

BioGRID:114274

BioGRID:114300

RIPK1

RIPK2casp-1 Interaction (Nfκβ)

Table 2. Indicative list of TRAF1 ligands. They all participate in the TNFR-TRAF-Nfκβ signalling pathway.

Introduction

Glomerular diseases usually present with proteinuria,

microscopic hematuria and renal function impairment.

The above triad of clinical symptoms cannot discrim-

inate between the wide range of different histological

types of glomerular and/or tubular disorders. Renal

biopsy remains the “gold standard” procedure for the

diagnosis and validation of glomerular diseases, how-

ever, it is an invasive method, potentially followed by

side effects, and cannot be routinely performed during

patients follow up. Discovery of urinary biomarkers,

capable to differentiate a disease, reflect severity of

renal damage and predict outcome of renal function,

has been the main scope for many years of research1,2.

One such biomarker is the EGF, a peptide growth fac-

tor, which is produced by tubular epithelial cells and is

excreted in the urine3.

EGF acts as a tubular cell survival factor and plays

an imperative role in regenerating tubular epithelial

cells and restoring barrier function in the healing phase

of renal injury. Thus, it protects epithelial cells during

kidney injury and indeed its expression within the kid-

ney is decreased in several kidney diseases4-7.

Aim of the present study was to assess the uri-

nary excretion of EGF in different forms of chronic

ECI 2018

Epidermal Growth Factor (EGF) as biomarker of

renal function outcome in different forms of

primary glomerular diseases

M. Stangou1, A. Fylaktou2, E. Sampani1, C. Nikolaidou3, C. Stamos3, D. Daikidou1,

E. Moraiti1, A. Papagianni1

1Department of Nephrology, Aristotle University of Thessaloniki, Hippokration Hospital, Thessaloniki, Greece2Immunology and Histocompatibility Center, Hippokration Hospital, Thessaloniki, Greece,3Nephrological laboratory, Hippokration Hospital, Thessaloniki, Greece

ανοσ′ια 2018 ; 14, 3: 38 – 41

ABSTRACT

Introduction and aims: EGF acts through EGF receptor and facilitates regeneration of tubular epithelial cells after injury. We

investigated the role of urinary EGF excretion as biomarker for pathology and renal function outcome in glomerulonephritis.

Methods: EGF urinary levels were estimated in 3 forms of glomerular diseases: (1) IgA nephropathy (IgAN), as chronic glomerulonephritis,

[n=50, 21female, age 39.8yrs(18-65)], (2) pauci immune rapidly progressive glomerulonephritis (RPGN), as acute glomerulonephritis, [n=38,

17 female, age 59.5yrs (25-80)] and, (3) Nephrotic syndrome (NS) due to focal segmental glomerulosclerosis (FSGS) [n=23, 9 female, age

47.5yrs(19-79)] and minimal change disease (MCD) [n=12, 7female, age 45.5yrs (37-62)]. Ten healthy volunteers were used as controls. First

morning urine samples were collected on the day of renal biopsy. Patients were followed up for 7.5±2.1yrs.

Results: EGF urinary levels were: IgAN 0.13±0.2, FSGS 0.19±0.2, MCD 0.7±0.4, RPGN 0.15±0.3, controls 0.14±0.07pg/mgUcr. EGF

urinary levels had significant negative correlation with severity of interstitial fibrosis, r=-0.6, p=0.02, in IgAN, with the percentage

of fibrous crescents, r=-0.6, p=0.01 in RPGN, and finally, with the percentage of global sclerosed glomeruli, r=-0,5, p=0,04, degree

of fibrosis, r=-0.6, p=0.005 and interstitial infiltration, r=-0.6, p=0.01 in patients with NS.

Declining renal function was associated with reduced urinary EGF levels in IgAN (0.04±0.04 vs. 0.2±0.2pg/mgUcr, p=0.01), and

FSGS (0.007±0.004 vs. 0.6±0.04pg/mgUcr, p=0.009), but not in RPGN (0.05±0.1 vs. 0.2±0.4pg/mg cr, p=NS).

Conclusions: Reduction of EGF urinary levels in patients with glomerulonephritis is associated with “chronic” histological changes,

and can predict renal function outcome mainly in patients with chronic forms of glomerulonephritis.

ανοσ′ια 2018 ; 14, 3 39

and acute glomerular diseases, to correlate these levels

with the severity of histologic findings and estimate

the predictive role in the course of different forms of

glomerulonephritis.


One hundred twenty three patients were prospec-

tively studied. All patients were diagnosed with pri-

mary glomerular diseases, IgAN (n=50), Focal Seg-

mental Necrotising Glomerulonephritis (FSGN, n=38),

Focal Segmental Glomerulosclerosis (FSGS, n=23)

amd Minimal Change Nephropathy (MCN, n=12). Di-

agnosis in all patients was based on renal biopsy find-

ings. Patients with systemic immune diseases, such as

Systemic Lupus Erythematosus or Rheumatoid Arthri-

tis, with infections or neoplasias, were excluded from

the study.

First morning urine samples were collected the

day of renal biopsy under sterile conditions, cen-

trifuged at 252 g for 10 min and kept at -70°C, until

used. A sandwich enzyme-linked immunosorbent assay

(ELISA) method was applied for the measurement of

EGF. Thirty healthy individuals were used as controls.

Urine cytokine levels were normalised for urine cre-

atinine (Ucr) concentration and were expressed as

pg/mg Ucr.

Renal biopsies were evaluated for the severity of

tubular atrophy, interstitial fibrosis, and frequency of

crescent formation and global sclerosed glomeruli.

Results

Clinical characteristics from patients are shown in table

1 and levels of urinary EGF in different forms of

glomerulopathies are depicted in figure 1.

Correlations of EGF urinary levels with laboratory

and histological findings at time of renal biopsy-di-

agnosis

In patients with IgAN and FSGS/MCN urinary excre-

tion of EGF had negative correlation with the severity

of TIN fibrosis, p=0.02 and p=0.005, respectively. In

patients with FSNG there was no correlation between

EGF urinary levels and degree of tubulointerstitial lev-

els, but there was significant negative correlation with

the percentage of global sclerosed glomeruli and fi-

brous crescents, p=0.01 and p=0.001, respectively,

(Table 2, Figure 2).

EGF urinary excretion as a predictive factor to renal

function outcome

EGF urinary levels had significant correlations with eGFR

at last follow up. FSGS patients were separated to pro-

gressors and non-progressors, according to their renal

function outcome, EGF urinary levels at time of renal

biopsy were significantly lower to progressors com-

pared to those who retained renal function (figure 4).

Table 1. Clinical characteristics of patients at time of diagnosis.

IgAN FSNG FSGS MCN

n=50 n=38 n=23 n=12

Age(yrs) 45.5±18 54.5 ± 15 47.5±16.9 45.5±9.3

M/F 35/15 16/17 15/8 7/5

Screat (mg/dl) 1.6±0.8 3.8±2.5 2.1±0.9 0.8±0.2

GFR (ml/min

1.73m2)69±36.4 23.6±18 52.4±31.2 107.2±31.6

Upr (g/24hr) 1.3 ±1.2 1.4±0.9 3.2±1.9 4.9±3.3

Urinary EGF

(pg/mg Ucr)0.13±0.2 0.15±0.3 0.2±0.2 0.7±0.4

Table 2. Correlations of EGF urinary levels with severity of

histologic lesions.

Correlation of EGF

with histology IgAN FSNG FSGS/MCN

r p r p r p

Global sclerosis (%) ΝS -0.5 0.01 -0.5 0.04

Fibrous crescents (%) ΝS -0.6 0.001

Int. fibrosis (%) -0.6 0.02 ΝS -0.6 0.005

Int. infiltration ΝS ΝS -0.6 0. 01

Figure 1. EGF urinary excretion in different forms of glomerulardiseases, differences with healthy controls.

EGF Urinary excretion

p=0.0004

p=NS

p=NS

p=NS

pg/m

g Ucr

2

1

0controls IgAN FSNG FSGS MCN

40 ανοσ′ια 2018 ; 14, 3

Discussion

Primary glomerular diseases have different etiologies,

immune reactions, histology, and different outcome of

renal function. Primary glomerular diseases are char-

acterized by the presence of acute and/or chronic le-

sions. Common acute lesions include endothelial hy-

perplasia, focal necrosis, cellular crescent formation, in-

flammatory infiltration, while chronic lesions consist

of focal or global sclerosis, fibrous crescents and tubu-

lointerstitial fibrosis.

IgAN is characterized by the presence of both

acute and chronic lesions, however the vast majority

of patients develop chronic renal pathology and

progress slowly to renal function impairment. FSNG

is the typical form of acute glomerulonephritis, char-

acterized by necrosis and extracapillary proliferation.

MCN and FSGS are the principal causes of nephrotic

Figure 2. Differences in EGF urinary levels, according to different histologic findings.

0.3 0.25 0.5

0.4

0.3

0.2

0.1

0.0

0.20

0.15

0.10

0.05

0.00

0.2

0.1

0.0

pg/m

g Ucr

pg/m

g Ucr

pg/m

g Ucr

IgA nephropathy FSNG FSNGp=0.006 p=0.04

p=0.01

<25% >25% <25% >25% <25% >25%Tubular atrophy (%) Tubular atrophy (%)Global sclerosed glomeruli (%)

Figure 3. Correlation of EGF urinary levels with percentage of global sclerosed glomeruli and degree of tubular atrophy in patientswith nephrotic syndrome (due to FSGS and MCN).

1,501,50

1,00

1,00

0,50

0,50

0,00

0,00

0 20 40 60 0 25 50 75

p=0.04

p=0.005r= -0.6

Urin

ary E

GF (p

g/m

g cr

eat)

EGF U

rinar

y lev

els (

pg/m

g cr

eat)

% of global sclerosed glomeruli tubular atrophyFSGS / MCN

Figure 4. Correlation of EGF urinary levels with renal functionoutcome.

0,40

160,00

120,00

80,00

40,00

0,00 0,50 1,00 1,50

r=0,6p=0,001

r=0,4p=0,011,00

0,50

0,00

0,00 25,00 50,00 75,00

0,30

0,20

0,10

0,00

50,0 100,0 150,0GFR (ml/min/1,73 m2)

Urinary EGF (pg/mg creat)

pg/m

g Uc

r

EGF (

pg/m

g Uc

r)GF

R (m

l/min

)

EGF (

pg/m

g Uc

r)

GFR (ml/min/1,73 m2)

IgAN

FSGS

FSNG

1.5

1.0

0.5

0.0controls non pr progressors

p=0.007

p=0.02

ανοσ′ια 2018 ; 14, 3 41

syndrome, and they also share many histological find-

ings. Podocyte injury and fusion of foot processes, with

or without podocyte hypertrophy and hyperplasia,

and only scarce inflammatory findings are common

findings in both diseases and so, differential diagnosis

between FSGS and MCN is usually difficult7. Descrip-

tion of biomarkers definitely associated with particular

histologic findings should be extremely useful in dis-

tinguishing between FSGS and MCN and predicting

renal function outcome in other types of glomeru-

lonephritis8.

In the present study there was significant reduc-

tion in urinary levels of EGF in FSGS patients, com-

pared to MCN, which can discriminate the two enti-

ties. More interestingly, there was also a negative cor-

relation with the degree of glomerulosclerosis, fibrous

crescent formation and TIN fibrosis. Furthermore, uri-

nary levels of EGF at time of diagnosis could predict

long term renal function outcome of all types of

glomerulonephritis included in the study.

EGF is produced by epithelial cells, it stimulates

the activation of nuclear factor (NF)-kB and cyclin D1

expression in human proximal tubular cells and thus,

participates in the regulation of cell proliferation9.

EGF urinary levels at time of diagnosis, in all four

types of glomerular diseases studied, showed a nega-

tive correlation with chronic histologic findings in renal

biopsy and also they seemed to have a predictive val-

ue in renal function outcome, as EGF excretion was

significantly lower in patients who progressed during

follow up compared with those patients whose renal

function remained stable. It could be a simplified ex-

planation, advocating that given the tubular origin of

EGF, its production will be eliminated in cases of tu-

bular atrophy, seen in advanced stages of renal im-

pairment. However, EGF receptor which is expressed

at tubular epithelial cells, after activation by urinary

proteins, seems to have a central role in this proce-

dure. EGFR is used by EGF and TGF-β, which antago-

nize for the receptor, and also, have opposite results10.

Our results suggest that EGF urinary levels are

able to discriminate between MCN and FSGS, and

also predict renal function outcome in acute and

chronic forms of primary glomerulopathies.


Μaria Stangou


References

1. D'Agati VD, Mengel M. The rise of renal pathology in

nephrology: Structure illuminates function. Am J Kidney

Dis 2013; 61(6): 1016-25.

2. Konvalinka A, Scholey JW, Diamandis EP. Searching for new

biomarkers of renal diseases through proteomics. Clin

Chem 2012; 58(2): 353-65.

3. Worawichawong S, Worawichawong S, Radinahamed P, Muntham

D, Sathirapongsasuti N, Nongnuch A, Assanatham M,

Kitiyakara C. Urine epidermal growth factor, monocyte

Chemoattractant Protein-1 or their ratio as biomarkers

for interstitial fibrosis and tubular atrophy in primary

glomerulonephritis. Kidney Blood Press Res. 2016; 41(6):

97-1007.

4. Lechner J, Malloth NA, Jennings P, Heckl D, Pfaller W, Seppi T.

Opposing roles of EGF in IFN-alpha-induced epithelial

barrier destabilization and tissue repair. Am J Physiol Cell

Physiol. 2007; 293(6): C1843-50.

5. Satirapoj B, Dispan R, Radinahamed P, Kitiyakara C. Urinary

epidermal growth factor, monocyte chemoattractant

protein-1 or their ratio as predictors for rapid loss of renal

function in type 2 diabetic patients with diabetic kidney

disease. BMC Nephrol. 2018 Sep 21; 19(1): 246. doi:

10.1186/s12882-018-1043-x.

6. Gesualdo L, Di Paolo S, Calabro A, Milani S, Maiorano E, Ranieri

E, Pannarale G, Schena FP. Expression of epidermal growth

factor and its receptor in normal and diseased human

kidney: an immunohistochemical and in situ hybridization

study. Kidney Int. 1996; 49(3): 656-65.

7. Maas RJ, Deegens JK, Smeets B, Moeller MJ, Wetzels JF. Minimal

change disease and idiopathic FSGS: manifestations of the

same disease. Nat Rev Nephrol. 2016; 12: 768-776.

8. Königshausen E, Sellin L. Circulating Permeability Factors in

Primary Focal Segmental Glomerulosclerosis: A Review

of Proposed Candidates. Biomed Res Int. 2016; 2016:

3765608.

9. Chen ZD, Xu L, Tang KK, Gong FX, Liu JQ, Ni Y, Jiang LZ, Hong J,

Han F, Li Q, Yang XH, Sun RH, Mo SJ. NF-κB-dependent

transcriptional upregulation of cyclin D1 exerts cytopro -

tection against hypoxic injury upon EGFR activation. Exp

Cell Res. 2016 Sep 10; 347(1): 52-59.

10. Samarakoon R, Dobberfuhl AD, Cooley C, Overstreet JM, Patel

S, Goldschmeding R, et al. Induction of renal fibrotic genes

by TGF-β1 requires EGFR activation, p53 and reactive

oxygen species. Cell Signal. 2013; 25: 2198-2209.

Introduction

Rheumatoid Arthritis (RA) is a chronic inflammatory

disease that predominantly involves synovial joints and

affects up to 1% of adults worldwide1,2. Genetic and

environmental factors interact in the etiology of the

disease3. The strongest genetic association is consid-

ered to be within the HLA (Human Leukocyte Anti-

gen) region, particularly the HLA-DRB1 genes4. Several

HLA-DRB1 alleles encoding the Shared Epitope (SE)

at amino acid position 70 - 74 in the third hypervari-

able region of the DRB1 molecules have been associ-

ated with higher risk for RA developing5. The influence

of HLA genes is largely seen in the RA subset char-

acterized by the presence of anti-CCP (cyclic citrulli-

nated protein) antibodies which are highly specific for

RA and may precede development of RA by several

years and are associated with disease severity and ra-

diographic progression6.

Smoking is the most established environmental

risk factor for anti-CCP positive RA, with the risk in-

creasing in proportion to the number of pack-years.

Indeed, this risk is further increased in subjects carrying

one or two copies of HLA-DRB1*-SE7,8.

The aim of the present case-control study was

the assessment of the association of HLA-DRB1*- SE

in the presence or absence of anti-CCP antibodies in

smokers and non-smokers Greek patients with RA.

Materials and methods

A total of 83 RA patients with mean age 63.14±12.54

years were studied. All patients met at least four of

the American College of Rheumatology criteria for

RA (ACR 1987) at the time of enrollment.The mean

disease duration was 11.62±10.89 years. Three-hundred

sixteen (316) healthy individuals, aged 18-65 years, were

used as controls. The subjects in both group were un-

ECI 2018

Interaction between the HLA-Shared Epitope

(SE) and smoking in anti-CCP positive Greek

patients with Rheumatoid Arthritis

K. Tarassi1, E. Mole2, V. Kitsiou1, D. Kouniaki1, Th. Athanassiades1, K. Soufleros1,

E. Synodinou1, A. Bletsa1, Ch. Sfontouris2, A. Tsirogianni1

1Immunology-Histocompatibility Dept & 2Rheumatology Dept, Evangelismos Hospital, Athens, Greece

ανοσ′ια 2018 ; 14, 3: 42 – 44

ABSTRACT

As genetic and environmental factors involve in of Rheumatoid Arthritis (RA) etiopathogenesis, the aim of the study was the

assessment of association of HLA-DRB1*-SE in the presence/absence of CCP autoimmunity in Greek patients with RA (smokers

and non-smokers). Eighty-three (83) RA patients (41 smokers, 42 have never smoked) were typed for HLA-DRB1* alleles by

molecular techniques (PCR-SSOP and -SSP). In 62 out of 83 (74.7%) anti-CCP antibodies (abs) were detected by ELISA. Conclusions:

a) an increased frequency of HLA-DRB1*01:01, *10:01, *04:05 alleles, as well as the protective role of *04:02, *04:03 alleles, in Greek

patients with RA were confirmed, b) presence of any SE, particularly *10:01 allele, strongly influences the production of anti-CCP

abs and c) interaction between smoking and any SE, particularly *01:01 allele, is associated with anti-CCP (+) RA in Greek patients.

Key words: shared epitope, smoking, rheumatoid arthritis, alleles, anti-CCP.

ανοσ′ια 2018 ; 14, 3 43

related and of Greek origin. Smoking status was as-

sessed by questionnaires. Current and past smokers

were classified as smokers (n=41) and those who had

never smoked were classified as non-smokers (n=42).

Anti-CCP antibodies were detected by ELISA

commercial kits. An antibody titer ≥25U was consid-

ered as positive.

The HLA-DRB1* high-resolution typing was per-

formed by molecular techniques: polymerase chain re-

action using and hybridization with sequence-specific

oligonucleotide probes and/or specific primers (PCR-

SSOP, PCR-SSP). The HLA-DRB1*01:01, *10:01, *04:01,

*04:04, *04:05 and *04:08 were classified as the SE

alleles.

Statistical analysis: Regression analysis was per-

formed to determine the Odds ratio (OR). Propor-

tions were compared by the chi-square (x2) test. P

values less than 0.05 were considered as statistical sig-

nificant.

Results

In 62 out of 83 RA patients (74.7%) positive anti-CCP

antibodies (abs) were detected.

In RA patients and in comparison to the controls,

increased frequency of HLA-DRB1*01:01 (28.9% vs

6.8%, OR=4.4), *10:01 (16.9% vs 2.4%, OR=8.4), *04:01

(3.6% vs 2%, OR=1.8), *04:04 (7.2% vs 1%, OR=7.6) and

*04:05 (15.7% vs 3.7%, OR=4.8), as well as decreased

frequency of *04:02 (1.2% vs 2%, OR=0.6) and *04:03

(4.8% vs 6.8%, OR=0.7) were found (Figure 1).

Among RA patients, 77.1% possess 1SE vs 18.9%

of controls (OR=14.4), whereas 10.8% possess 2SE vs

1% of controls (OR=11.8).Also, 88.7% of anti-CCP (+)

carry 1SE vs 42.9% of anti-CCP (-) patients (OR=10.5,

p= 6x10-5), whereas 2SE possess 12.9% of anti-CCP

(+) vs 4.8% of anti-CCP (-) patients (OR=2.96). Par-

ticularly, DRB1*01:01 (27.4% vs 14.3%, OR=2.3) and

*10:01 (21% vs 4.8%, OR=5.3) alleles were presented

with a higher frequency in anti-CCP (+) compared to

anti-CCP (-) RA patients.

Furthermore, anti-CCP (+) smokers patients in

comparison to anti-CCP (+) non-smokers are pre-

sented with an increased frequency of DRB1*01:01

(41.9% vs 12.9%, OR=4.9). Among the anti-CCP (+)

smokers, 96.8% possess 1SE vs 80.6% of anti-CCP (+)

non-smokers (OR=7.2), whereas 12.9% possess 2SE vs

12.9% of anti-CCP (+) non-smokers (OR=1) (Table 1).

Figure 1. Frequency of HLA-DRB1 SE alleles in Greek RApatients and controls.

CONTROLS*01:01 *10:01 *04:01 *04:02 *04:03 *04:04 *04:05

6.8 2.4 2 1 6.8 1 3.728.9 16.9 3.6 1.2 4.8 7.2 15.7RA

RA - HLA-DRB1 SE (%)

Table 1. Interaction of SE, anti-CCP and smoking in RA patients (S: Smokers, nS: non Smokers, C: Controls).

HLA RA C RA vs C CCP+ CCP-

CCP(+)

vs

CCP(-)

CCP (+)S

vs

CCP(+)nS

% % OR p % % OR p OR p

DR1 28.9 10.8 3.4 6x10-5 33.9 14.3 2.1 NS 2.82 NS

DR4 33.7 16.1 2.6 6x10-4 37.1 23.8 1.9 NS 1.52 NS

DR10 16.9 2.9 6.4 1x10-5 21 4.8 5.3 NS 0.55 NS

DRB1*01:01 28.9 6.8 4.4 1x10-5 27.4 14.3 2.3 NS 4.9 0.02

DRB1*10:01 16.9 2.4 8.4 1x10-6 21 4.8 5.3 NS 0.43 NS

DRB1*04:01 3.6 2 1.8 NS 4.8 0 UD NS 0.48 NS

DRB1*04:02 1.2 1 0.6 NS 1.6 0 UD NS 0 NS

DRB1*04:03 4.8 6.8 0.7 NS 3.2 9.5 0.32 NS 1 NS

DRB1*04:04 7.2 1 7.6 4x10-3 9.6 0 UD NS 2.15 NS

DRB1*04:05 15.7 3.7 4.8 2x10-4 16.1 14.3 1.15 NS 1 NS

DRB1*04:08 2.4 0 UD NS 1.6 4.8 0.33 NS UD NS

1SE 77.1 18.9 14.44 1x10-8 88.7 42.9 10.5 6x10-5 7.2 <0.05

2SE 10.8 1 11.88 9.9x10-5 12.9 4.8 2.96 NS 1 NS

44 ανοσ′ια 2018 ; 14, 3

Discussion

An increased frequency of HLA-DRB1*04:05 (among

DR4 alleles) and especially of DRB1*01:01 and *10:01

alleles, as in other Mediterranean European popula-

tions, and the protective role of *04:02,*04:03 alleles

were confirmed in Greek patients with RA9.

The presence of any DRB1*-SE strongly influ-

enced the production of anti-CCP abs in Greek RA

patients when compared to CCP-negative ones. Par-

ticularly, the presence of DRB1*01:01 and *10:01 alleles

were associated with higher risk for positive anti-CCP

abs compared with negative anti-CCP abs. It has been

postulated that SE motif itself may be directly involved

in RA pathogenesis through citrullinated peptides that

bind to HLA-DRB1*SE molecules for presentation to

CD4+ T-cells.T-cells that recognize citrullinated anti-

gens could subsequently induce B-cells to produce

anti-CCP antibodies10.

The interaction between smoking and any SE,

particularly *01:01 allele, is associated with anti-CCP

positive RA in Greek patients.The association of CCP

positive RA and smoking could be explained by the

observation of citrullinated proteins in the lungs of

smokers, following presentation to T-cells after binding

to HLA-DRB1 molecules encoding the SE11.

Several studies in Caucasians and Asians12 and

more recently in Africans RA patients13 have confirmed

that smoking and/or SE increases the risk of develop-

ing anti-CCP abs, suggesting a strong gene-environ-

mental interaction in disease pathogenesis. Currently,

it was described that anti-CCP positivity in RA is

strongly associated with increasing Rheumatoid Factor

(RF) titer, independent of smoking but it depends on

SE alleles’ positivity14.

As non-HLA genes are also involved in RA

pathogenesis, additional studies of interaction between

other genes (PTPN22, PADI) and environmental fac-

tors such as consumption of alcohol, coffee and tea

are needed in our cohort of Greek RA patients, for a

more comprehensive approach to genetic and envi-

ronmental correlations.


Katerina Tarassi


References

1. Harris EDJ. Rheumatoid Arthritis. New England Journal of

Medicine. 1990; 322(18): 1277-89.

2. Imboden JB. The immunopathogenesis of rheumatoid

arthritis. Annual Review of Pathology. 2009; 4: 417-34.

3. Klareskog L, Padyukov L, Ronnelid J, et al. Genes, environment

and immunity in the development of rheumatoid arthritis.

Current Opinion in Immunology. 2006; 18(6): 650-5.

4. Bowes J, Barton A. Recent advances in the genetics of RA

susceptibility. Rheumatology (Oxford, England). 2008;

47(4): 399-402.

5. Gregersen PK, Silver J, Winchester RJ. The shared epitope

hypothesis. An approach to understanding the molecular

genetics of susceptibility to rheumatoid arthritis. Arthritis

and rheumatism. 1987; 30(11): 1205-13.

6. Berglin E, Padyukov L, Sundin U, et al. A combination of

autoantibodies to cyclic citrullinated peptide (CCP) and

HLA-DRB1 locus antigens is strongly associated with

future onset of rheumatoid arthritis. Arthritis research &

therapy. 2004; 6 (4): R303-8.

7. Kallberg H, Ding B, Padyukov L, et al. Smoking is a major pre -

ventable risk factor for rheumatoid arthritis: estimations of

risks after various exposures to cigarette smoke. Annals of

the Rheumatic Diseases. 2011; 70(3): 508-11.

8. Linn-Rasker SP, van der Helm-van Mil, F A van Gaalen, et al.

Smoking is a risk factor for anti-CCP antibodies only in

rheumatoid arthritis patients who carry HLA-DRB1

shared epitope alleles. Annals of the Rheumatic Diseases.

2006; 65(3): 366-71.

9. Ioannidis JP, Tarassi K, Papadopoulos IA, et al. Shared epitopes and

rheumatoid arthritis: disease associations in Greece and

meta-analysis of Mediterranean European popu ations.

Seminars in arthritis and rheumatism. 2002; 31(6): 361-70.

10. Bax M, van Heemst J, Huizinga TW, Toes RE. Genetics of

rheumatoid arthritis: what have we learned? Immuno -

genetics. 2011; 63(8): 459-66.

11. Makrygiannakis D, Hermansson M, Ulfgren AKl. Smoking

increases peptidylarginine deiminase 2 enzyme ex pression

in human lungs and increases citrullination in BAL cells.

Annals of the rheumatic diseases. 2008; 67(10): 1488-92.

12. Lee YH, Bae SC, Song GG. Gene-environmental interaction

between smoking and shared epitope on the

development of anti-cyclic citrullinated peptide antibodies

in rheumatoid arthritis: a meta-analysis. International

journal of rheumatic diseases. 2014; 17(5): 528-35.

13. Traylor M, Curtis C, Patel H, et al. Genetic and enviromental

risk factors for rheumatoid arthritis in a UK African

anchestry population: the GENRA case-control study.

Rheumatology 2017; 56: 1282-92.

14. Murphy D, Mattey D, Hutchinson D. Anti-citrullinated protein

antibody positive rheumatoid arthritis is primarily deter -

mined by rheumatoid factor titre and the shared epitope

rather than smoking pe se. PLoS ONE 2017: 12(7): 1-11..

Introduction

Nowadays, it is well accepted that hemodialysis (HD)

patients still face with an extremely high rate of mor-

bidity and mortality1. The most noteworthy reason

amongst several others is cardiovascular disease (CVD)

(approximately 50% of deaths)2,3. Several studies have

reported that if we compare end stage renal patients

undergoing hemodialysis with an age age-matched gen-

eral population concerning CVD incidence and death

rates,there is a significant increase in the first group2-4.

In HD patients though, traditional risk factors, namely

lipid disorders, hypertension, diabetes fail to explain the

high frequency of CVD5. It seems that, in these patients,

protein-energy wasting (PEW) and chronic inflamma-

tion increase the atherosclerotic procedure and con-

sequently the CVD incidence6,7.

In particular, recurring contact of mononuclear

cells with the dialyzer membranes, dialysis tubes and

solutions, oxidative and carbonyl stress,increase of in-

flammatory cytokines may lead to chronic inflamma-

tion7. One of the major events in developing athero-

ECI 2018

Study of soluble vascular endothelial

cell adhesion molecules sICAM-1 and sVICAM-1

in hemodialysis patients

C. Tsigalou1, T. Konstantinidis1, A. Tsirogianni2, G. Romanidou3, M. Aslanidou1,

K. Kantartzi3, A. Grapsa1, M. Panopoulou1

1Laboratory of Microbiology, Democritus University of Thrace, Alexandroupolis, Greece2Immunology-Histocompatibility Dept. Evangelismos General Hospital, Athens, Greece, Athens, Greece3Division of Nephrology, Democritus University of Thrace, Alexandroupolis, Greece, Alexandroupolis, Greece

ανοσ′ια 2018 ; 14, 3: 45 – 48

ABSTRACT

Introduction-Aim: Cardiovascular complications are the leading cause of mortality and morbidity in patients with end-stage renal

disease undergoing chronic hemodialysis (ESRD-HD). Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion

molecule-1 (VCAM-1) are involved as markers of the atherosclerotic burden.The aim of the study was to determine the level of

sICAM 1 and sVCAM-1 in patients with ESRD-HD.

Materials and Methods: The study enrolled 60 patients, mean age 64±13 yrs, 39(65%) and 21(35%). The levels of sICAM-1and

sVCAM-1 were measured at the beginning and after 6 months by elisa kit (Bender MedSystem, Austria). Statistical analysis was

performed with OriginPro version 8. Student t-test and one way ANOVA were used for data that followed Gaussian distribution,

while the Kruskal-Wallis tests were used for data that did not follow the Gaussian distribution. For all measurements, a two-tailed

p value ≤0.05 was considered as significant.

Results: The mean value of sICAM-1and sVCAM-1at the start of dialysis was 442.01±215.5ng/mland 11704,83 ±421,4ng/ml respectively.

The levels of sICAM-1 after 6 monthsincreased significantly (442.01±215.5ng/ml vs 835,08 ±339ng/ml p<0.0001), but not the levels of

sVCAM-1(11704,83 ±421,4ng/ml vs 10461,31±314.7ng/ml p=0.058).Moreover,sICAM-1 was elevated significantly in dialysis patients with

co-morbidity of coronary artery disease and diabetes mellitus type II (442.01±215.5ng/ml vs 631,28±134ng/ml =0,089).

Conclusions: A significant increase sICAM-1 at 6 months compared with baseline was observed as evidence for chronic

inflammatory/immune system involvement. The role of sVCAM-1 and sICAM-1 as markers of atherosclerosis in chronic renal

disease may implicate an upregulation of inflammatory/immune response and may facilitate possible therapeuticinterventions.

Key words: sVCAM-1, sICAM-1,hemodialysis, endothelial dysfunction, inflammation.

46 ανοσ′ια 2018 ; 14, 3

sclerosis is the contact and consequent adhesion of

leukocytes to the vascular endothelium bringing about

endothelial dysfunction and microvascular events8.

There are four categories of adhesion molecules: the

integrins, the selectins, the adhesion molecules in the

immunoglobulin superfamily and the cadherins9.

ICAM-1 and VCAM-1 are adhesion molecules ex-

pressed on the surface of vascular endothelial cells as

a way of reacting to proinflammatory cytokines which

are present in the uraemic circulation.

Low grade inflammation and endothelial dysfunc-

tion play aleading role in the emergence, development

and continuation of atherosclerosis10. Therefore, novel

risk factors for CVD as ICAM-1 and VCAM-1 seem to

be pivotal contributers to morbidity and mortality

rates in HD patients according to the above men-

tioned mechanism.

In this study we determined the levels of serum

ICAM-1 and VCAM-1 in patients with End Stage Renal

Disease (ESRD) undergoing HD in two time points

and correlate them and to co-morbidities such as

CVD and Diabetes Melitus.

Materials and Methods

The study enrolled 60 patients with ESRD on main-

tenance HD. The main eligibility criteria were: a. age

between 18 and 80 years b. regular 3 times weekly

sessions for at least six months c. being clinically stable

for the past 3 months. Most of the patients (82%)

were treated with standard bicarbonate dialysis and

only a minority of them (18%) were treated by he-

modiafiltration.The average fractional urea clearance

(Kt/V) in the study cohort was 1.43±0.18.

Blood samples were collected in standard vacu-

tainer tubes with no anticoagulant from all patients

midweek, before dialysis and after overnight fasting

both at the initial enrollment visit and at a 6 months

follow-up visit. The serum thus obtained was frozen

and stored at -20c until assessment.

Serum ICAM-1 levels were determined by an en-

zyme-linked immunosorbent assay by a commercial

available kit (Bender MedSystems, GmbH, Vienna, Aus-

tria).With minimum detectable value of 2.17 ng/ml. The

overall intra- and interassay coefficient of variation has

been calculated to be 9,5% and 12,9%, respectively.

Serum VCAM-1 levels were determined by an

enzyme-linked immunosorbent assay by a commercial

available kit (Bender MedSystems, GmbH, Vienna, Aus-

tria). With minimum detectable value of 0.59 ng/mL.

The overall intra- and interassay coefficient of variation

has been calculated to be 3,1% and 5,2%, respectively.

All samples were tested simultaneously in dupli-

cate. The average value of the 2 time point measure-

ments was used for data analysis.

Statistical analysis was performed with OriginPro

version 8. Student t-test and one way ANOVA were

used for data that followed Gaussian distribution, while

the Kruskal-Wallis tests were used for data that did not

follow the Gaussian distribution. For all measurements,

a two-tailed p value ≤0.05 was considered as significant.

Results

Demographic, clinical and laboratory values of the

study cohort of 60 hemodialysis patients are shown

in Table 1. The mean age was 64±13 yrs, 39(65%)

Table 1. Demographic, clinical and laboratory values of the

study cohort of 60 hemodialysis patients

Variable

Study

cohort

n=60

Age (years) 64 ± 13

BMI (kg/m2) 26 ± 4

Male/female (n) 39/21

Cardiovascular risk factorsComorbidities

Hypertension, n (%) 30 (50%)

Diabetes mellitus, n (%) 11 (18%)

Dyslipidemia, n (%) 10 (17%)

Current smokers, n (%) 7 (12%)

MI, n (%) 6 (10%)

Stroke or TIA, n (%) 8 (13%)

Coronary artery disease, n (%) 22 (37%)

Heart failure, n (%) 15 (25%)

Atrial fibrillation, n (%) 11 (18%)

Revascularization procedure (PCI/CABG), n (%) 5 (8%)

Neoplastic disease, n (%) 4 (7%)

Anemia, n (%) 15 (25%)

Hemodialysis parameters

Kt/V 1.43±0.18

Flux (high) 11 (18%)

Duration of hemodialysis, years 4.6 (2.1-8)

Kidney disease factors/ Causes of kidney disease

Polycystic kidney disease, n (%) 9 (15%)

Glomerular disease, n (%) 19 (32%)

Obstructive renal disease,n (%) 13 (22%)

Other disease, n (%) 19 (20%)

Kidney transplantation, n (%) 7 (12%)

Values are expressed as means 6 standard deviation whereas labo -ratory data refer to the mean of baseline and 6-month measure -ments. BMI, body mass index; CABG, coronary artery bypass grafting,Kt/V, fractional urea clearance, MI, myocardial infarction; PCI, percu -taneous coronary intervention; TIA, transient ischemic attack.

ανοσ′ια 2018 ; 14, 3 47

and 21(35%). The Median duration (IQR) of HD

treatment was 4,6 (2,1 to 8) years. All patients were

of Caucasian ethnic origin.

The mean value of sICAM-1 and sVCAM-1 at the

start of dialysis was 442.01±215.5 ng/ml and 11704,83

±421,4 ng/ml respectively. It was observed that the levels

of sICAM-1 after 6 months increased in a statistically

significant manner (442.01±215.5 ng/ml vs 835,08±339

ng/ml p<0.0001), Figure 1, but the level of sVCAM-1 had

no significant and meaningful increase (11704,83 ±421,4

ng/ml vs 10461,31±314.7 ng/ml p=0,058).

Moreover, the value of sICAM-1 was elevated in

statistically significant manner between dialysis patients

with co-morbidity of coronary artery disease and di-

abetes mellitus type II (442.01±215.5 ng/ml vs

631,28±134 ng/ml = 0,089) Figure 2.

Discussion

It is generally accepted that cardiovascular complications

are the leading cause of morbidity and mortality among

dialysis patients.In a post-hoc analysis of the EVOLVE

trial the 54% of deaths in a cohort of 3883 hemodialysis

patients was attributed to cardiovascular causes11. Fur-

thermore, it is also previously documented that elevated

serum concentrations of soluble adhesion molecules

are present in patients with ESRD under peritoneal

dialysis or HD12. The reasons of this increase appears to

be multifaceted entailing inadequate clearance and en-

hanced synthesis11. It has also been demonstrated that

the release of pro-inflammatory cytokines up-regulate

the expression of sICAM-1 and sVCAM-111.

The present data clearly suggests a significant in-

crease sICAM-1 at 6 months compared with baseline

was observed as evidence for chronic inflammatory/im-

mune system involvement. Additionally, sICAM-1 was

significantly elevated between dialysis patients with co-

morbidity of coronary artery disease and diabetes mel-

litus type II. In diabetic patients,microvascular complica-

tions are very common and there is a solid bond be-

tweenadhesion molecules and diabetic nephropathy8.

Chronic inflammation favors the accumulation of

inflammatory cytokines such as interleukin-6 and tu-

mor necrosis factor-a while these cytokines augment

the production of systemic inflammation markers

namely CRP and vascular inflammation markers such

as sICAM-1 and sVCAM-1 and sE-selectin1,13.

Consequently, pathogenesis and propagation of

atherosclerosis in patients with ESRD undergoing he-

modialysis might be estimated by vascular inflamma-

tion markers and the role of sVCAM-1 and sICAM-1

as markers of atherosclerosis in chronic renal disease

may implicate an upregulation of inflammatory/im-

mune response and may facilitate possible therapeu-

ticinterventions.


Christina Tsigalou


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Figure 1. Serum levels of sICAM-1 in dialysis patients.

1200

1000

800

600

400

200

01st mth

442,01

835,08

6th mth

P<0,0001

aICAM

Figure 2. Serum levels of sVCAM-1 in dialysis patients.

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cytokines in patients with diabetic nephropathy.

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48 ανοσ′ια 2018 ; 14, 3

Η ανάπτυξη νεοεπιτόπων των καρκινικών κυττάρων

μέσω σημειακών μεταλλάξεων, ενθέσεων/απαλοιφών,

αναστροφών κτλ, υπήρξαν στόχος των εμβολίων του

καρκίνου την τελευταία δεκαετία1. Δεδομένου ότι οι νε-

οεπίτοποι διαφεύγουν την κεντρική ανοχή, θα μπορού-

σε να επιτευχθεί η ανάπτυξη Τ ειδικών κυττάρων υψη-

λής συγγένειας με στόχο τη κατασκευή εξατομικευμέ-

νου εμβολίου. Η μετάλλαξη στην οποία θα βασίζεται ο

εμβολιασμός θα μπορεί να ταυτοποιεί πολλαπλά κοινά

νεοαντιγόνα, συμπεριλαμβανομένων των mutRas,

mutP53, mutVHL, mutEGFR, ή mutIDH11, τα οποία θα

μπορούσαν να χρησιμοποιηθούν για τη διάγνωση και

την τροποποίηση των πρωτοκόλλων θεραπευτικού εμ-

βολιασμού. Ωστόσο, η εισαγωγή των τεχνολογιών της

νέας γενιάς αλληλούχησης (NGS) αποκάλυψε ότι οι

ανθρώπινοι καρκίνοι ήταν πολύ πιο περίπλοκοι, καθώς

μεταφέρουν χιλιάδες μεταλλάξεις, κάθε μια από τις

οποίες θα μπορούσε να αποτελέσει πιθανό νεοεπίτοπο

για παρουσίαση από τα τάξης Ι αντιγόνα ιστοσυμβα-

τότητας2,3. Το σύνολο των σωματικών μεταλλάξεων

ενός μεμονωμένου όγκου, αναφέρεται ως «μεταλλαξι-

δίωμα» ("mutanome"), μέσα από το οποίο θα μπορού-

σε να γίνει επιλογή των νεοεπιτόπων.

Οι συνεχείς προσπάθειες επικεντρώνονται στην

επιλογή των καλύτερων νεοαντιγόνων για τη χρήση

τους σε εμβολιασμό, η επιλογή ενός φορέα, η οποία

θα μπορούσε να υποστηρίξει την διέγερση του ανο-

σοποιητικού συστήματος, καθώς και η επιλογή τρόπων

ECI 2018

Εμφυτεύσιμα εμβόλια με τη χρήση

προενεργοποιημένων ικριωμάτων πυριτίου

στη θεραπεία του καρκίνου

Ι. Ζέρβα1, Χ. Λαναρά2, E. Στρατάκης2, Ε. Αθανασάκη1

1Τμήμα Βιολογίας, Πανεπιστήμιο Κρήτης, 2FORTH, Ηράκλειο Κρήτης

ανοσ′ια 2018 ; 14, 3: 49 – 52

ΠΕΡΙΛΗΨΗ

Με την πάροδο των χρόνων, η έλλειψη αποτελεσματικών δραστικών ανοσοθεραπειών για τον καρκίνο έχει οδηγήσει στην ανάπτυξη

πολλών νέων στρατηγικών. Ένα από τα σημαντικότερα προβλήματα είναι η αδυναμία ανάπτυξης ανοσολογικών αποκρίσεων ενάντια

στα καρκινικά αντιγόνα εφόσον αυτά συνήθως αναγνωρίζονται ως αντιγόνα του εαυτού. Έχει αποδειχθεί ότι η εύρεση νέων ογκο-

ειδικών αντιγόνων είναι μια προσέγγιση χρονοβόρα, δαπανηρή και με περιορισμένη αποτελεσματικότητα. Προηγούμενες μελέτες

έχουν δείξει ότι εμφυτεύσιμα μικροδομημένα τρισδιάστατα ικριώματα πυριτίου μπορούν να υποστηρίζουν τη προσκόλληση μα-

κροφάγων, να επιτρέπουν τη φυσική πρόσληψη και παρουσίαση αντιγόνου, οδηγώντας σε παραγωγή ειδικού αντισώματος και

φλεγμονωδών κυτοκινών in vitro, ενώ η in vivo εμφύτευση τους προκαλεί την απαραίτητη φλεγμονώδη αντίδραση στον οργανισμό

συνοδευόμενη από έκκριση ειδικού αντισώματος, και ανάπτυξη Τ- και Β-κυττάρων μνήμης. Στόχος της παρούσας έρευνας είναι η

χρήση της τεχνολογίας των ικριωμάτων πυριτίου στην ανάπτυξη ανοσολογικής απόκρισης ενάντια στα καρκινικά κύτταρα του

ξενιστή. Η μελέτη πραγματοποιήθηκε με τη χρήση του πειραματικού μοντέλου καρκίνου του μαστού σε ΒALB/c ποντίκια. Μετά την

επίτευξη της καρκινογένεσης με 4Τ1 κύτταρα, τα ζώα δέχτηκαν εμφύτευμα με προ-ενεργοποιημένα ικριώματα πυριτίου στα οποία

το αντιγόνο ήταν το κυτταρικό εκχύλισμα ιστοσυμβατών 4Τ1 κυττάρων. Η μέθοδος αυτή επιτρέπει τη φυσική επιλογή των ανοσογόνων

επιτόπων με στόχο την ανάπτυξη ειδικής κυτταρικής και χυμικής απόκρισης ενάντια στον όγκο. Προκαταρκτικά πειράματα με

χρήση αλλογενών κυττάρων όγκου στο παρελθόν έδειξαν ότι η προτεινόμενη τεχνολογία μπορεί πράγματι να οδηγήσει σε ογκο-

ειδική ανοσολογική απόκριση. Ο σκοπός της παρούσας έρευνας είναι η ρύθμιση της ισορροπίας ανοχής/ανοσίας του ξενιστή και

η ανάπτυξη της εξατομικευμένης ειδικής απόκρισης κατά του όγκου.

50 ανοσ′ια 2018 ; 14, 3

χορήγησης, συμπεριλαμβανομένων των ανοσοενισχυ-

τικών και/ή νέων βιοδιασπώμενων υλικών. Επιπλέον, η

ανάγκη της εξατομικευμένης γονιδιωματικής αλληλου-

χίας για την κατασκευή των ειδικών για των ασθενή

φαρμάκων αυξάνει δραματικά τις δαπάνες, καθιστώ-

ντας τέτοιες προσεγγίσεις ασύμφορες για τη πλειο-

ψηφία του πληθυσμού.

Η ανάπτυξη μιας ανοσολογικής απόκρισης έγκει-

ται στην επιτυχή παρουσίαση αντιγόνων από αντιγο-

νοπαρουσιαστικά κύτταρα (APCs). Η παραδοσιακή

άποψη για την αντιγονοπαρουσίαση δηλώνει ότι αντι-

γόνα που συντίθενται ενδοκυτταρικά παρουσιάζονται

από το μείζον σύμπλεγμα ιστοσυμβατότητας (MHC)

μαζί με τάξης Ι μόρια και αναγνωρίζονται από τα ενερ-

γοποιημένα CD8+ κυτταροτοξικά Τ (Tcyt) κύτταρα,

ενώ τα εξωκυττάρια αντιγόνα εσωτερικεύονται σε φα-

γοσώματα ή ενδοσώματα, που στη συνέχεια ωριμά-

ζουν και υποβάλλονται σε σειρά μοριακών αλλαγών,

όπως η οξίνιση και η σύντηξη με οργανίδια που πε-

ριέχουν ένζυμα, ιδίως λυσοσώματα. Τα παραγόμενα

αντιγονικά πεπτίδια φορτώνονται στα τάξης ΙΙ MHC

μόρια και μεταφέρονται στην κυτταρική επιφάνεια των

APCs για να τα παρουσιαστούν στα CD4+ Τ βοηθη-

τικά κύτταρα (Th)5.

Η ενεργοποίηση των Τ βοηθητικών κυττάρων

αποτελεί μια από τις πιο σημαντικές δραστηριότητες

του ανοσοποιητικού συστήματος, δεδομένου ότι με-

σολαβεί τη χυμική ή κυτταρική ανοσία, καθώς και την

ανοχή. Έτσι, τα Τ-βοηθητικά κύτταρα θα διεγείρουν τα

Β κύτταρα για συγκεκριμένη παραγωγή αντισωμάτων

ή Tcyt για τη θανάτωση των κυττάρων-στόχων, ενώ η

έκφραση των αρνητικών δεικτών της επιφάνειας θα

καταστείλει και τα δύο είδη της ανοσίας για να εξα-

σφαλίσει την ομοιόσταση του ανοσοποιητικού συστή-

ματος.

Η χορήγηση του αντιγόνου μαζί με ανοσοενι-

σχυτικό είναι μέχρι τώρα απαραίτητη ώστε να επαχθεί

ανοσία, αντί της ανοχής. Τα ανοσοενισχυτικά ορίζονται

ως χημικές ενώσεις ή μακρομόρια που αυξάνουν την

ανοσολογική απόκριση στο αντιγόνο με ελάχιστη το-

ξικότητα ή μακροχρόνια ανοσία. Οι ουσίες αυτές επι-

τυγχάνουν το στόχο τους μιμούμενες συγκεκριμένα

μοτίβα εξελικτικά διατηρημένων μορίων που ονομά-

ζονται PAMPs, τα οποία περιλαμβάνουν λιποσώματα,

λιποπολυσακχαρίτη (LPS), συστατικά βακτηριακών κυτ-

ταρικών τοιχωμάτων και ενδοκυτταρικά νουκλεϊκά

οξέα όπως δίκλωνο RNA (dsRNA) , μονόκλωνο DNA

(ssDNA) και μη μεθυλιωμένου DNA που περιέχει

CpG. Επειδή το ανοσοποιητικό σύστημα έχει εξελιχθεί

για να αναγνωρίζει αυτά τα ειδικά αντιγονικά τμήματα,

η παρουσία ενός ανοσοενισχυτικού σε συνδυασμό με

το εμβόλιο μπορεί να αυξήσει σημαντικά την έμφυτη

ανοσοαπόκριση προς το αντιγόνο με αύξηση των

δραστηριοτήτων των δενδριτικών κυττάρων (DCs),

λεμφοκυττάρων και μακροφάγων, μιμούμενο έτσι μία

φυσική μόλυνση.

Ανοσοενισχυτικά, όπως άλατα αλουμινίου, πετρε-

λαίου σε νερό γαλακτώματα και λιποσώματα διευκο-

λύνουν την αργή απελευθέρωση αντιγόνου και κατά

συνέπεια την αποδοχή του από τα APCs. Άλλες βοη-

θητικές ουσίες, όπως η monophosphoryl λιπιδίων Α,

CpG, ή πολυ Ι:C, ενεργοποιούν τα APCs μέσω σύνδε-

σης με Toll-like υποδοχείς6. Όλες αυτές οι βοηθητικές

ουσίες προκαλούν φλεγμονή στο σημείο της χορήγη-

σης, αλλά έχουν και δυνατότητες για μακροπρόθεσμες

παρενέργειες και συνεπώς δεν έχουν εγκριθεί για αν-

θρώπινη χρήση. Την πιο διαδεδομένη χρήση ανοσο-

ενισχυτικού στις κλινικές πρακτικές έχουν τα μεταλλικά

άλατα αλουμινίου7, τα οποία θεωρούνται πλέον ασφα-

λή και χρησιμοποιούνται ευρέως σε εμβόλια8.

Η ανάπτυξη αποτελεσματικών και χωρίς παρε-

νέργειες εμβολίων κατά του καρκίνου είναι ένα ανοικτό

ζήτημα στην έρευνα. Ένα από τα σημαντικότερα προ-

βλήματα είναι η αδυναμία των ανοσολογικών αποκρί-

σεων έναντι αντιγόνων που σχετίζονται με όγκους

(ΤΑΑ), που συνήθως αναγνωρίζονται από το ανοσο-

ποιητικό σύστημα ως αυτο-αντιγόνα. Ωστόσο, τα κύτ-

ταρα όγκου εκφράζουν πολλαπλά νεοαντιγόνα, τα

οποία είναι ειδικά για κάθε όγκο και τις περισσότερες

φορές ειδικές για κάθε άτομο. Πλήθος κοινών νεοα-

ντιγόνων έχουν χρησιμοποιηθεί ανεπιτυχώς σε τεχνο-

λογίες εμβολίων. Σε αυτές τις περιπτώσεις, η αποτυχία

του εμβολίου έγκειται στην επιλογή του νεοεπίτοπου,

του φορέα και του ανοσοενισχυτικού.

Εστιαζόμενοι σε αυτή τη κατεύθυνση την τελευ-

ταία πενταετία, μετά από επιτυχή συνδυασμό in vitroκαι in vivo χειρισμών αναπτύχθηκαν βιοϋλικά που επι-

τρέπουν τη φυσική «φόρτωση» του αντιγόνου, την πα-

ρουσίασή του in vitro και την περαιτέρω ενεργοποίηση

της ανοσολογικής απόκρισης in vivo14. Η εμφύτευση

των ικριωμάτων πυριτίου με προσροφημένα, ενεργο-

ποιημένα από το αντιγόνο μακροφάγα ήταν σε θέση

να οδηγήσει σε παραγωγή φλεγμονωδών κυτοκινών

και αντιγονο-ειδικών αντισωμάτων στον ορό, ανιχνεύ-

σιμα ακόμη και 30 ημέρες μετά την εμφύτευση.

Χρησιμοποιώντας αυτή τη τεχνολογία, στόχος

της παρούσας έρευνας ήταν η ανάπτυξη εμφυτεύ-

σιμων ικριωμάτων, όπου το αντιγονικό ερέθισμα θα

ανοσ′ια 2018 ; 14, 3 51

είναι εκχύλισμα ολόκληρων καρκινικών κυττάρων με

σκοπό την ανάπτυξη ειδικής χυμικής και κυτταρικής

ανοσολογικής απόκρισης στο συνολικό «muta -

nome» ενός συγκεκριμένου καρκινικού όγκου.

Αν και οι μελέτες συνεχίζονται, τα μέχρι τώρα απο-

τελέσματα δείχνουν ότι η προτεινόμενη τεχνολογία

μπορεί να διεγείρει το ανοσοποιητικό σύστημα ενάντια

συγγενικών κυττάρων όγκου. Χρησιμοποιώντας τα συγ-

γενικά καρκινικά κύτταρα 4Τ1, τα οποία αντικατοπτρί-

ζουν την πραγματική κατάσταση του προτεινόμενου

εξατομικευμένου εμφυτεύσιμου εμβολιασμού και εφό-

σον καθορίστηκαν οι βέλτιστες συνθήκες καλλιέργειας

για την ανάπτυξη μακροφάγων και επώαση αυτών με

εκχύλισμα κυττάρων όγκου 4Τ1 σε μικροδομημένα

ικριώματα πυριτίου, το σύστημα δοκιμάστηκε για την

ικανότητά του να υποστηρίζει την ανάπτυξη ανοσολο-

γικής απόκρισης με αύξηση αντιγονο-ειδικών Τ κυττα-

ροτοξικών κυττάρων και παραγωγή κυτοκινών.

Αρχικά καθορίστηκαν οι βέλτιστες συνθήκες καλ-

λιέργειας των μακροφάγων σε ικριώματα πυριτίου χα-

μηλής τραχύτητας μετά από επώαση με εκχύλισμα 4T1

κυττάρων με στόχο την in vitro διέγερση των λεμφο-

κυττάρων και στη συνέχεια ενεργοποιημένα ικριώματα

(ικριώματα με προσκολλημένα μακροφάγα που είχαν

ενεργοποιηθεί με εκχύλισμα 4T1 κυττάρων) εμφυτεύ-

τηκαν σε ζώα που έφεραν 4T1 όγκο (Εικόνα 1). Τα ζώα

που δέχτηκαν το εμφύτευμα παρουσίασαν αυξημένα

επίπεδα κυτοκινών που δρουν ενάντια στον όγκο (πα-

ράγοντας νέκρωσης όγκου-α) καθώς και μειωμένα επί-

πεδα κυτοκινών που ευνοούν την ανοσοκαταστολή

του οργανισμού (ιντερλευκίνη 10), καθώς και παραγωγή

αντιγονο-ειδικού αντισώματος. Μια πολύ σημαντική

ένδειξη λειτουργίας του συστήματος ήταν επίσης και

η αύξηση Τ κυτταροτοξικών κυττάρων, καθώς αποτε-

λούν τον κατεξοχήν κυτταρικό πληθυσμό που δρα

ενάντια των καρκινικών κυττάρων.

Η ασφάλεια των εμβολίων είναι ένα σοβαρό θέμα

που απασχολεί την Παγκόσμια Οργάνωση Υγείας18.

Παρά το γεγονός ότι οι νεοεπίτοποι στερούνται πα-

θογένειας και μολυσματικότητας, ανοσοενισχυτικά ή

υλικά-φορείς παρουσιάζουν παρενέργειες, οι οποίες

θα μπορούσαν να αξιολογηθούν μόνο με μαζική χρή-

ση εγείροντας όμως έτσι σοβαρά βιοηθικά ζητήματα.

Με τη χρήση των μη βιοδιασπώμενων εμφυτευμάτων

αποφεύγονται οι παρενέργειες των ανοσοενισχυτικών,

ενώ παρέχεται την απαραίτητη ανοσολογική διέγερση

στον ξενιστή14.

Η παρούσα έρευνα αποσκοπεί στο να επιτρέψει

στα αντιγονοπαρουσιαστικά κύτταρα του παραλήπτη

(APCs) να παρουσιάσουν τα κύτταρα του όγκου του,

εξασφαλίζοντας έτσι καλύτερη αντιγονο-παρουσίαση

για κάθε άτομο. Στην περίπτωση αυτή, η επιλογή του

νεοεπιτόπου θα εκτελεστεί με φυσικό τρόπο από τα

APCs και κατά την εμφύτευση θα μπορούσε να τονώσει

τα Τ-κύτταρα για ανάπτυξη ειδικής ανοσοαπόκρισης.

Στόχος της προτεινόμενης μελέτης είναι η επέ-

κταση και διεθνοποίηση της ήδη υπάρχουσας ευρε-

σιτεχνίας με τίτλο «Εξατομικευμένα εμφυτεύσιμα εμ-

βόλια με αντιγονικά διεγερμένα μονοκύτταρα» (ΟΒΙ

Εικόνα 1. Πραγματοποιήθηκε καρκινογένεση με ένεση καρκινι-κών κυττάρων 4Τ1 και ακολούθως έγινε εμφύτευση ικριώμα-τος με ενεργοποιημένα μακροφάγα.

Πίνακας 1. Αποτελέσματα κυτοκινών, ειδικού αντισώματος και Τ-κυτταροτοξικών κυττάρων στα ζώα που δέχτηκαν

εμφύτευμα με ενεργοποιημένα μακροφάγα σε σύγκριση με ζώα που δε δέχτηκαν εμφύτευμα.

20140100471, 19/9/2014) σε εφαρμογή ενάντια στον

καρκίνο. Το προτεινόμενο έργο είναι πρωτοποριακό

στο χώρο του και η επιτυχία του θα έχει όχι μόνο ση-

μαντικό επιστημονικό αντίκτυπο, αλλά συγχρόνως κοι-

νωνικό και οικονομικό όφελος, εφόσον θα μπορεί να

αντιμετωπίζει σε εξατομικευμένο επίπεδο την μάστιγα

της εποχής μας, τον καρκίνο.

Αλληλογραφία:

Ιωάννα Ζέρβα


Βιβλιογραφία

1. Türeci Ö, Vormehr M, Diken M, Kreiter S, Huber C, Sahin U. Tar geting

the Heterogeneity of Cancer with Individualized Neo -

epitope Vaccines. Clin Cancer Res. 2016; 22(8): 1885-96.

2. Segal NH, Parsons DW, Peggs KS, Velculescu V, Kinzler KW,Vogelstein B, et al. Epitope landscape in breast and color -

ectal cancer. Cancer Res 2008; 68: 889-92.

3. Kreiter S, Vormehr M, van de Roemer N, Diken M, Lower M,Diekmann J, et al. Mutant MHC class II epitopes drive thera -

peutic immune responses to cancer.Nature 2015: 520: 692-6.

4. Meirow Y, Kanterman J, Baniyash M. Paving the road to tumor

development and spreading: Myeloid-Derived Sup pressor

Cells are ruling the fate. Front Immunol. 2015; 6: 523.

5. Burgdorf S, Kurts C. Endocytosis mechanisms and the cell biology

of antigen presentation.Curr Opin Immunol.2008: 20: 89-95.

6. Reed SG, Bertholet S, Coler RN, Friede M. Novel perspectives

for influenza vaccine formulation and administration.

Trends Immunol. 2009; 30: 23-32.

7. Aguilar JC, Rodriguez EG. Vaccine adjuvants revisited. Vaccine

2007; 25: 3752-62.

8. Lindblad EB. Aluminium compounds for use in vaccines.

Immunol Cell Biol 2004; 82: 497–505.

9. Gherardi RK, Coquet M, Cherin P, Belec L, Moretto P, Dreyfus PA,Pellissier JF, Chariot P, Authier FJ. Macrophagic myofasciitis

lesions assess long-term persistence of vaccine-derived

aluminium hydroxide in muscle Brain 2001; 124: 1821-31.

10. Gupta RK. Aluminum Compounds as Vaccine Adjuvants

Vaccine 1995; 13: 1623-25.

11. Gupta RK, Siber GR. Adjuvants for human vaccines—current

status, problems and future prospects Vaccine 1995; 13:

1263-76.

12. Alving CR, Detrick B, Richards RL, Lewis MG, Shafferman A, EddyGA. Laser vaccine adjuvant for cutaneous immunization

Ann N Y Acad Sci 1993; 690: 265-75.

13. Chen X, Kim P, Farinelli B, Doukas A, Yun SH, Gelfand JA,Anderson RR, Wu MX. A novel laser vaccine adjuvant in -

creases the motility of antigen presenting cells. PloS One

2010; 5 e13776.

14. Zerva I, Simitzi C, Siakouli-Galanopoulou A, Ranella A, StratakisE, Fotakis C, Athanassakis I. Implantable vaccines: In vitro

antigen presentation enables in vivo immune response.

Vaccine 2015: 33: 3142-9.

15. Ranella A, Barberoglou M, Bakogianni S, Fotakis C, Stratakis E.Tuning cell adhesion by controlling the roughness and

wettability of 3D mocro/nano silicon structures Acta

Biomaterialia 2010; 6: 2711-2720.

16. Zhang S, Gelain F, Zhao X. Designer self-assembling peptide

nanofiber scaffolds for 3D tissue cell cultures. Sem in

Cancer Biol 2005: 15: 413-20.

17. Van Goethem E, Poincloux R, Gauffre F, Maridonneau-Parini I,Le Cabec V. Matrix architecture dictates three-

dimensional migration modes of human macrophages:

differential involvement of proteases and podosome-like

structures. J Immunol 2010: 184: 1049-61.

18. WHO, 23 JULY 2010, 85th YEAR No. 30, 2010; 85: 285-292.

52 ανοσ′ια 2018 ; 14, 3

ΟΔΗΓΙΕΣ ΓΙΑ ΤΟΥΣ ΣΥΓΓΡΑΦΕΙΣ

Το περιοδικό «Ανοσία» εκδίδεται από την Ελλη-

νική Εταιρεία Ανοσολογίας και αποτελεί το μέσο

προώ θησης της ανοσολογίας στους χώρους διεξαγω-

γής ιατρικής έρευνας και κλινικής πράξης. Στο περιο-

δικό δημοσιεύονται άρθρα σύνταξης, ανασκοπικά

άρθρα, ερευνητικές και άλλες πρωτότυπες εργασίες,

ενδιαφέρουσες περιπτώσεις και γράμματα από και

προς τη σύνταξη.

Υποβολή άρθρων: Τα άρθρα αποστέλλονται

στον εκδότη στη διεύθυνση:

Α. Σαραντόπουλος

Για το περιοδικό ΑΝOΣΙΑ

Τμήμα Κλινικής Ανοσολογίας

Β′ Παθολογική Κλινική ΑΠΘ

Κωνσταντινουπόλεως 49,

546 42 Ιπποκράτειο Γ.Ν.Π.Θ.

Θεσσαλονίκη

Για κάθε τύπο άρθρου ισχύουν ιδιαίτερες οδηγίες:

1. Άρθρα σύνταξης: Συνοπτικά άρθρα που δεν υπερ-

βαίνουν τις 2 σελίδες. Η έκτασή τους δεν πρέπει να

υπερβαίνει τις 500 λέξεις.

2. Άρθρα ανασκοπικά: Πρόκειται για σύνθετη παρου-

σίαση ενός θέματος που περιλαμβάνει την εξέλιξή

του μέχρι σήμερα αλλά κυρίως τις νεότερες απόψεις

και προοπτικές. Το μέγεθος του άρθρου δεν πρέπει

να υπερβαίνει τις 4.000 λέξεις.

3. Ερευνητικές, πρωτότυπες εργασίες: Έχουν κλινικό ή

εργαστηριακό χαρακτήρα ή αποτελούν προϊόν βα-

σικής έρευνας, ενώ η δομή τους πρέπει να περιλαμ-

βάνει περίληψη, περιγραφή υλικού και μεθο δο λογίας,

αποτελέσματα και συζήτηση. Δεν πρέπει να υπερβαί-

νουν τις 3.500-4.000 λέξεις.

4. Ενδιαφέρουσες περιπτώσεις: Παρουσίαση περιστα-

τικών σπάνιων είτε ως προς την κλινικοεργαστη-

ριακή τους πορεία είτε ως προς τη θεραπευτική

τους αντιμετώπιση και εξέλιξη. Τα άρθρα πρέπει να

είναι 1.000-1.500 λέξεων.

5. Γράμματα προς τη σύνταξη: Περιλαμβάνουν α να -

κοινώσεις πρόδρομων αποτελεσμάτων, είτε σχόλια

που αφορούν δημοσιευμένα στο περιοδικό άρθρα.

Δεν θα πρέπει να υπερβαίνουν τις 500 λέξεις.

Σύνταξη χειρογράφων: Τα άρθρα που αποστέλ-

λονται στο περιοδικό πρέπει να είναι γραμμένα στη

νεοελληνική δημοτική. Υποβάλλονται σε σελίδες τύπου

Α4, με διπλό διάστημα και περιθώρια, ενώ οι γραμμα-

τοσειρές που πρέπει να χρησιμοποιούνται είναι οι

Times New Roman και Arial.

Αποτελούν ξεχωριστές ενότητες και πρέπει να υπο-

βάλλονται σε ιδιαίτερη σελίδα:

Α) O τίτλος της εργασίας με τα ονόματα των συγγρα-

φέων, το ίδρυμα από το οποίο προέρχονται και τη

διεύθυνση του συγγραφέα για αλληλογραφία.

Β) Η περίληψη της εργασίας (με την ανάλογη δομή),

η οποία δεν πρέπει να υπερβαίνει τις 200 λέξεις,

μαζί με 3-5 λέξεις-κλειδιά.

Γ) Το κείμενο της εργασίας. Στις ερευνητικές εργασίες

ακολουθείται η σειρά: εισαγωγή, υλικό και μέθοδοι,

αποτελέσματα και συζήτηση. Εάν πρόκειται για κλινική

μελέτη, τότε πρέπει να αναφέρεται ότι πραγματοποι-

ήθηκε σύμφωνα με τη διακήρυξη του Ελσίνκι. Oι φαρ-

μακευτικές ουσίες αναφέρονται με την κοινόχρηστη

και όχι την εμπορική ονομασία τους.

Δ) O τίτλος της εργασίας με τα ονόματα των συγγρα-

φέων, τα αντίστοιχα ιδρύματα απ’ όπου προέρχο-

νται, το κείμενο της περίληψης και τις λέξεις-κλειδιά

στην Αγγλική.

Ε) Oι βιβλιογραφικές παραπομπές.

ΣΤ) Oι υπότιτλοι των εικόνων και οι επεξηγήσεις των

σχημάτων και πινάκων. Κάθε υπότιτλος παρατίθεται

σε ξεχωριστή σελίδα.

Ζ) Oι πίνακες, σχήματα και εικόνες που δυνατό να συ-

νοδεύουν το κείμενο και πρέπει να παρατίθενται σε

ξεχωριστές σελίδες. Oι εικόνες πρέπει να έχουν

ανάλυση 300 dpi τουλάχιστον.

Για την περίπτωση εκτύπωσης έγχρωμων εικόνων,

ο συγγραφέας θα ενημερώνεται από τη Συντακτική

Επιτροπή για τη διαφορά κόστους, την οποία και θα

αναλαμβάνει ο ίδιος να καλύψει.

Βιβλιογραφικές παραπομπές: Το περιοδικό

«Ανοσία» ακολουθεί το σύστημα Vancouver σύμφωνα

με το οποίο οι παραπομπές εμφανίζονται στο κείμενο

με μορφή αριθμών. Εφόσον οι συγγραφείς είναι περισ-

σότεροι από έξι, αναφέρονται οι 3 πρώτοι και ακολου-

θεί η σύντμηση: και συν. ή et al. Oι συντμήσεις των πε-

ριοδικών παρατίθενται σύμφωνα με το Abridged Index

Medicus π.χ.

Μπούρα Π. Ανοσιακή απόκριση. Στο: Τομέας Παθολογίας, Ια-

τρική Σχολή ΑΠΘ, Εσωτερική Παθολογία, Θεσσαλονίκη,

University Studio Press, 2004: 49-53.

Boura P, Papadopoulos S, Tselios K, et al. Intracerebral he mor rhage

in a patient with SLE and catastrophic an tipho spho lipid

syndrome (CAPS): report of a case. Clin Rhe umatol 2005

Aug; 24(4): 420-4.

Όλα τα παραπάνω συγκροτούν το συνολικό κεί-

μενο υποβολής και πρέπει αυτό να αποστέλλεται σε

τρία αντίτυπα στον εκδότη του περιοδικού, μαζί με μια

δισκέτα όπου θα περιέχεται η εργασία σε ηλεκ τρονική

μορφή.

Κάθε υποβολή πρέπει να συνοδεύεται από ενυ-

πόγραφη επιστολή του πρώτου συγγραφέα όπου θα

δηλώνεται ότι η εργασία δεν βρίσκεται σε διαδικασία

κρίσης και δεν έχει δημοσιευθεί από οποιοδήποτε

άλλο περιοδικό και ότι όλοι οι συγγραφείς συμφωνούν

στην πιθανή δημοσίευσή της.

Εφόσον η εργασία είναι τυπικά ολοκληρωμένη

σύμφωνα με τις παραπάνω οδηγίες προωθείται από

την επιτροπή επιμέλειας σύνταξης σε 2 μέλη της συμ-

βουλευτικής επιτροπής. Oι κριτές κάνουν δεκτή ή

απορρίπτουν την εργασία, ενώ διατυπώνουν τις παρα-

τηρήσεις τους οι οποίες επιστρέφονται μαζί με την ερ-

γασία στους συγγραφείς. Στο στάδιο της τελικής

φάσης της εκτύπωσης αποστέλλεται στους συγγρα-

φείς το τελικό δοκίμιο το οποίο επιδέχεται πλέον μόνο

ορθογραφικές διορθώσεις. Στο σημείο αυτό γίνεται και

η παραγγελία των ανατύπων από τους συγγραφείς. Ερ-

γασίες που δημοσιεύονται στο περιοδικό «Ανοσία»

αποτελούν πνευματική του ιδιοκτησία και η αναδημο-

σίευση μέρους ή ολόκληρου του κειμένου απαιτεί την

έγγραφη συγκατάθεση του εκδότη.

IMMUNOLOGY ανοσια · 2018-10-26 · CD19+IgM+: 58.1±42.7 μ/ L vs. 117.9±58.94 μ/ L,...

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