2.  South  Eastern  European  Immunology  School      

1  –  4  October  2010      

Inter  University  Centre,  Dubrovnik,  Croatia      

   

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Programme Friday, 1 October 17.00 Registration at IUC 18.30 Keynote Lecture:

Seppo Meri: Microbial escape of innate immunity 20.00 Welcome reception at IUC Saturday, 2 October 09.15 Plenary Lecture:

Sabina Rabatic: Innate immunity to respiratory viruses 10.00 Plenary Lecture:

Hannes Stockinger: Yin/Yang of adaptive immunity between cure and destruction

10.45 Coffee break 11.15 Plenary Lecture:

Hans-Willi Mittruecker: Defence against intracellular bacteria 12:00 Plenary Lecture:

Bernhard Fleischer: Regulation of T cell activation: co-stimulation and co-inhibition

12.45 Lunch break (Restaurant MIMOZA) 15.00 Parallel Workshops

1. How to interpret FACS data 2. Defects  of  innate  immunity  and  how  to  detect  them

15:45 Coffee Break 16:30 Parallel Workshops 3. How to measure T cell immunity

4. Complement  diagnostics,  diagnostics  of  autoimmune  diseases  5.  Antibodies in research and therapeutics      

17:15 Free evening for sight seeing

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Sunday, 3 October 09.15 Plenary Lecture:

Moncef Zouali: B cells and autoimmunity 10:00 Plenary Lecture:

Annette Gospos: Autoantigens in diagnostics of human autoimmune diseases

10:45 Coffee break 11.15 Plenary Lecture:

Friedrich Haag: Mechanisms of tolerance 12:00 Plenary Lecture:

Seppo Meri: Complement activation and regulation 12.45 Lunch (Restaurant MIMOZA) 15:00 Parallel Workshops (repeated according to request by participants) 15:45 Coffee break 16.15 Poster session 20.00 Farewell party with dinner at Restaurant Kavana Poliksar, Old Harbour,

Ribarnica Monday, 4 October 09.15 Plenary Lecture:

Hannes Stockinger: Live-cell imaging to open new views for understanding of immune cell function

10.00 Plenary Lecture:

Hans-Joachim Seitz: Opportunities for young researchers from SE Europe in Germany

10:45 Final Discussion 11.00 Farewell, Certificates, Coffee, Sandwiches 12.00 Departure

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Systemic low-grade inflammatory response following oral warfarin administration in rats Sandra Belij1, Djordje Miljkovic2, Marija Slavić3, Vesna Subota4, Aleksandra Popov1, Ivana Mirkov1, Dragan Kataranovski1, Milena Kataranovski1 1Department of Ecology, Institute for Biological Research “Sinisa Stankovic”, Bulevar Despota Stefana 142, Belgrade, Serbia, 2Department of Immunology, Institute for Biological Research “Sinisa Stankovic”, Bulevar Despota Stefana 142, Belgrade, Serbia, 3Department of Physiology, Institute for Biological Research “Sinisa Stankovic”, Bulevar Despota Stefana 142, Belgrade, Serbia, 4Institute for Medical Biochemistry, Military Medical Academy, Crnotravska 17, Belgrade, Serbia Objective: To investigate the existence of systemic inflammatory response to subchronic oral warfarin consummation in rats. Methods: Dark Agouti rats were treated with warfarin in drinking water (3.5 mg/l) for 30 days. Heparinized blood samples were collected (24 hours following 30-day period) and differential leukocyte counts were determined (May Grünwald-Giemsa protocol). Peripheral blood (PB) granulocyte (isolated on density gradient) viability was determined by MTT reduction assay. Oxidative activity (cytochemical NBT reduction), myeloperoxidase (MPO) intracellular content of PB granulocyte (oxidation of O-dianisidine dihydrochloride), levels of nitrites accumulated in medium conditioned by PB granulocytes (Griess assay), mRNA levels for inducible nitric oxide synthase (iNOS) (RT-PCR), plasma levels of IL-6 and TNF-α (ELISA) and superoxide dismutase (SOD) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumption. Changes in prothrombin time (PT) and partial thromboplastin time (PTT), as basic biological warfarin activity were determined as well. Statistical significance was defined by Mann-Whitney U test (functional granulocyte assays) and t-test (SOD activity, gene expression). Results: Significantly increased PT and PTT were noted following WF administration, documenting access of WF to general circulation. No numerical changes in differential leukocyte counts were noted, without direct cell toxicity on PB granulocyte, evaluated by MTT test. Increase of PMA-stimulated neutrophil NBT reduction capacity (neutrophil priming) and significantly increase in MPO intracellular content, levels of nitrites and mRNA levels for iNOS were noted in PB granulocytes following WF administration. Warfarin consummation resulted in no changes in plasma levels of IL-6 and TNF-α, but significant decrease in the SOD activity was detected, suggesting systemic oxidative activity. Conclusion: Presented data demonstrate a potent pro-inflammatory effect of anticoagulant warfarin on granulocytes, including potentiation of their effectors functions. Absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. These findings contribute to the list of biological activities of anticoagulants, other than those affecting hemostasis. This study was supported by the Ministry of Science and Technological Development of the Republic of Serbia, Grant # 143038.

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Hyper IgE in a HIV positive patient Hristomanova S, Grunevska V1, Balabanova M2, Trajkov D, Petlichkovski A, Kirijas M, Djulejic E, Senev A, Spiroski M Institut of Immunobiology and Human Genetics; 1Clinic for Infective Diseases; 2Clinic for Dermatovenerology; Faculty of Medicine, University “St Kiril and Metodij”, Skopje, Republic of Macedonia Introduction Since1999 on the Institute of Immunology and Human Genetics in Skopje, Republic of Macedonia operates laboratory for allergology. It encompasses three UniCap 100 systems and they are for different assignment. Starting from the early beginning from 1999 persistent increase of both the number of total analysis and the number of analysis per patient is seen. Thus we have started with only 41 analyses in 1999 and we have reached total of 8491 analysis last year. Case report: A patient, 63 years old, with loss of weight and frequent stool previously diagnosed and treated as enterocollitis. Also previously treated at the Dermatological Clinic for herpes zoster infection and undiagnosed allergy. Microbiology tests showed presence of Candida and Klebsiella pneumoniae in the patient's sputum which led to testing and confirmation of HIV/AIDS. After that Lues was diagnosed and because the therapy is with penicillin blood sample was sent to our laboratory, for penicillin allergy testing. Results The results showed increased level of total IgE for almost 12 times above normal ranges and also allergy to ampicillin was revealed. Additional laboratory tests showed increased IgA levels with normal IgM and IgG levels. From acute phase proteins the only significant deviation was extreme raise of CRP followed with decreased levels of transferrin, α2  macroglobulin,   ceruloplasmin and albumin.It is important to mention that no skin changes were visible in the patient. Disscusion Highly increased levels of total IgE indicated the possibility for HIE Sy in this patient. HIE is a rare immunodeficiency disorder with an autosomal dominant inheritance pattern, associated with multiple abnormalities.Clinical manifestations of atopic allergy, drug reactions, and increased serum levels of IgE have already been described during the course of human immunodeficiency virus (HIV) infection.However the relationship of such findings to the immunologic derangements found in patients with HIV is not entirely clear.There are similarities between HIV-positive patients and those with primary HIE Sy, but there are also some important differences. Conclusion In conclusion, this is the first case of HIV positive patient with hyper IgE-like Sy in the Republic of Macedonia. We addressed the important laboratory findings and actual theories explaining the association between HIES and HIV/AIDS.

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Cytokine expression levels in high-risk human papillomavirus (hrHPV) positive women Irina Huica, Coralia Bleotu, Anca Botezatu, Iulia V Iancu, Gabriela Anton Institute of Virology “Stefan S. Nicolau”, 285 Mihai Bravu Ave 030304 Bucharest – Romania Tel/Fax: +40-21-324147

High-risk types of human papillomaviruses (hrHPV) are known to causing cervical cancer. As the host immune response plays an important role in determining the regression of a cervical abnormality or persistence and progression to a malignancy, the purpose of our study was to investigate cytokine levels in persistent lesions of the uterine cervix. Materials and Methods: 184 hrHPV DNA positive patients (Roche), age 17-48 years, were monitored between years 2008-2010. HPV positive case patients with cytological interpretation of ASCUS or LGSIL, presenting HPV persistent infection were investigated for cytokine levels detection. The persistent infections were confirmed by the presence of the same HPV DNA type in consecutive testing. The control group consisted of 10 hrHPV-negative women. Using exfoliated cells collected by cervical cytology brush, we assessed pro- and anti-inflammatory cytokine expression [interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor a (TNF-a), and interferon γ (IFN-γ)] in RT-real-time PCR (Taqman). Results. Persistence was significantly greater by oncogenic HPV types 16, 18 and 51 alone or in association with low/medium risk types. By compare with control group, we found that IFN-γ levels was increased in ASCUS and seems to be associated with low viral oncogene expression. The pro-inflammatory cytokines expression levels (TNF-a, IL-6, and IL-10) were higher in LGSIL lesions. While IL-4 levels were higher in LSIL lesions, especially in HPV 16 and 18 positive cases, IL-2 levels were slightly increased in patients presenting ASCUS and HPV 18 positive infection. No correlation with other oncogenic types (31, 33 and 45) was observed. Conclusions. Among HPV-persistent infected women, pro-inflammatory cytokine expression levels are increased and that may be considered as a prognostic marker for oncogenic potential of high-risk HPV.

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Characterization  of  peripheral  regulatory  T  cell  subsets  in  patients    with  posttraumatic  stress  disorder      Mladen  Jergović  (1),  Krešo  Bendelja  (1),  Anđelko  Vidović  (2),  Katja  Gotovac  (1),  Sabina  Rabatić  (1),  Neda  Aberle  (3),  Zlatko  Sudac(3),  Ante  Sabioncello  (1),      1Institute  of  Immunology,  Department  for  Cellular  Immunology,  Zagreb,  Croatia;  2University  Hospital  Dubrava,  Department  of  Psychiatry,  Zagreb,  Croatia;    3Children's  Department,  General  Hospital  Dr  Josip  Bencević,  Slavonski  Brod,  Croatia      Posttraumatic  stress  disorder  (PTSD)  is  associated  with  elevated  risk  for  inflammatory  and  autoimmune  diseases.  Regulatory  T  cells  play  a  major  role  in  maintaining  homeostasis  of  the  immune  system  by  controlling  the  activity  of  effector  cells  including  auto-­‐reactive  T  lymphocytes.    Regarding  the  role  of  regulatory  T  cells  in  inflammatory  and  autoimmune  diseases,  we  hypothesized  that  PTSD  patients  might  exhibit  changes  in  composition  of  regulatory  T  cells  in  peripheral  blood.      For  this  purpose  we  have  analyzed  absolute  numbers  of  T  cells  as  well  CD4  andCD8  subpopulations  in  peripheral  blood  from  PTSD  patients  (N=21)  and  age  matched  healthy  controls  (N=22).  Frozen  PBMC  samples  were  later  analyzed  regarding  regulatory  T  cell  composition.  Natural  regulatory  T  cell  (nTreg)  population  is  characterized  as  CD4+CD25+FOXP3+  .  Additionally,  nTreg  cells  express  other  markers  which  define  different  subpopulations.  Mature  nTreg  express  low  levels  of  CD127  and  HLA-­‐DR  while  terminally  differentiated  nTreg  cells  lose  CD127  and  display  higher  expression  of  FOXP3.  Induced  regulatory  T  cells  (iTreg)  are  characterized  by  low  expression  of  CD127  and  lack  of  HLA-­‐DR  marker.  Their  precursors  pTreg  cells  do  not  express  CD127  or  HLA-­‐DR.  In  addition  to  these  markers  we  have  analyzed  the  expression  of  CD39,  membrane  ectoenzyme  which  hydrolyzes  extracellular  ATP  and  ADP  into  AMP.      Although  PTSD  patients  have  a  lower  absolute  number  of  T  cells  andT  helper  cells  we  found  no  differences  in  Tregs  percentage  and  composition.  These  results  indicate  that  PTSD  patients  might  exhibit  lower  suppression  of  the  immune  response  due  to  lower  number  of  T  helper  cells  but  not  changes  in  T  regs  frequency  and  composition.    

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Quercetin increases sensitivity of bladder cancer cells to cisplatin and hyperthermia Pavle Josipović*, Ivo Karač*, Domagoj Karković*, Branimir Lodeta+ and Nada Orsolić*. Faculty of Science (Roosvelt square 6, Zagreb, Croatia)[*], and General Hospital (Ivana Meštrovića bb, Varaždin, Croatia)[+]. Hyperthermia provokes regression of human cancer. Great differences in heat

sensitivity have been observed between different cell types and tissues. Considering that

hyperthermia has produced only limited results, attention has been focused on searching

for substances able to sensitize tumour cells to the effects of hyperthermia. Flavonoid

quercetine has been recognized as hyperthermic sensitizer in ovarian and uterine

cervical tumours, leukaemia and in prostate carcinoma cells. Quercetine and cisplatin

inhibit Ehrlich ascites tumor growth and mammary carcinoma induced peritoneal

carcinomatosis growth.

We have observed that both quercetine and cisplatin at doses of 1 and 50 µM synergize

with hyperthermia (43ºC), with the effect of reducing the clonogenic activity of T-

24/83 and UMUC-3 and inducing the inhibition cell growth in both cell lines. As

revealed by clonogenic assay and MTT analyses, quercetine and cisplatine have

reduced a number of colonies and increased the cell cytotoxicity in hypertermal

condition, in concentration- depedent manner. In addition, T-24/83 cell line are more

sensitive to quercetine, cisplatine and hypertermal treatment than UMUC-3 cells.

Higher reduction of heat shock proteins (Hsps) expression in the T-24, in relation to

UMUC-3 cell line, promotes the higher induction of apoptosis by quercetine, which

may be a reason for better antiproliferative mechanism in T-24 cell line.

Exact antitumor mechanisms of hyperthermic chemoimmunotheraphy effectiveness still

need to be elucidated. Quercetine and cisplatin can be usefully combined with

hyperthermia in therapy of bladder cancer cells.

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Comparison  of  two  strategies  for  resolution  of  ambiguous  HLA  typing  results    Meri   Kirijas,   Stefan   Saltirovski,   Aleksandar   Senev,   Mirko   Trpevski,   Slavica  Hristomanova,   Olivija   Efinska-­‐Mladenovska,   Aleksandar   Petlichkovski,   Dejan  Trajkov,  Mirko  Spiroski    Institute   of   Immunobiology   and  Human  Genetics,   Faculty   of  Medicine,   St   Cyril   and  Methodius  Universisty,  Skopje,  Republic  of  Macedonia    Introduction:   The   precise   identification   of   HLA   Class   I   and   Class   II   alleles   is  critical   for   successful   transplantation   of   hematopoietic   cells,   development   of  peptide  based  viral  and  cancer  vaccines,  investigation  of  immune  response  and  in  anthropological   studies.   The   DNA   based   techniques   for   HLA   typing   have   been  proved  to  be  powerful  in  resolving  many  serological  ambiguities  and  detecting  of  single   nucleotide   polymorphisms.   However,   there   is   still   not   a   single   technique  that  offers   ambiguity-­‐free  MHC   typing.  Defining   strategies   to   resolve  ambiguities  created   by   HLA   DNA   typing   remains   in   focus   of   interest   for   HLA   DNA   typing  professionals.    

 

Aim:   The   aim   of   this   study   is   to   compare   two   strategies   for   resolution   of  ambiguities  arising  from  the  SSOP  typing  method.  The  first  is  based  on  using  of  a  high  resolution  SSP  typing  method,  and  the  second  is  based  on  statistical  analysis  using  the  HLA  completion  software.      

Material   and   methods:   Two   hundred   and   fourteen   unrelated   random   healthy  Macedonian  volunteers  of  Macedonian  origin  and  nationality,  Christian  Orthodox  religion   and   residents   of   different   regions   of   the   Republic   of   Macedonia   were  included   in   this   study.  Peripheral  blood  was  drawn  after  signing  of   the   informed  consent   and   genomic   DNA   was   extracted   from   the   peripheral   blood   leukocytes  using  the  standard  phenol/chloroform  procedure  and  stored   in  the  anthropology  project   field   of   the  Macedonian   human  DNA  Bank   (hDNAMKD)   until   processing.  Representative   ambiguous   results   were   selected   from   the   database   and  probabilistically   resolved   using   the   software   tool   available   at:  http://microsoft.com/science.  In  addition,  83  typings  using  the  high  resolution  SSP  method  were  performed.  From  the  results  compared,  only  2  were  not  concordant,  and  overall  concordance  of  97,59%  was  achieved.        

Conclusion:  In  conclusion,  the  statistical  method  for  resolution  of  ambiguous  HLA  typings  using  HLA  Completion  tool  is  satisfactory  add  in  anthropological  research.  However,  additional  method  should  be  used  when  mistyping  is  not  an  option,  such  as  setup  of  transplantation.    

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Proteomic approach for indirect quantification of breast cancer antigens in human serum Daria Ler1,2 1 Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, International

University of Sarajevo, 2 Institute of Genetic Engineering and Biotechnology, Sarajevo, Bosnia and

Herzegovina

Objectives: Immune system of patients with cancer gives response to tumor antigens

with the production of antibodies. The observed accumulation of such “auto-antibodies”

in cancer patients suggests them to be of high diagnostic/prognostic value. In this term,

specific ELISA-based approach was optimized to identify and validate new serum

tumor marker ecPKA (extracellular Protein Kinase A), potential candidate for

noninvasive detection of early human breast neoplasm. This auto-antibody ELISA is

thought to identify the cancer antigens by detecting the presence of auto-antibodies

against the tumor protein in human serum.

Method and Materials: The anti-ecPKA auto-antibodies levels were measured by solid

phase enzyme linked immunosorbent assay (ELISA). For this purpose a heterogeneous

group of breast cancer patients (BCP), respectively 120 serum samples, was tested as a

pilot project.

Results:

- Reproducible and robust assay, functional in quantifying specific anti-PKA auto-

antibodies.

- Two relatively high significant changes were obtained, namely between the two

BCP subgroups (adjuvants with HR+ and patients with metastasis), and between

one BCP subgroup (adjuvants with HR+) and healthy donors.

- No significant difference was obtained between the BCP group and the healthy

donors.

Conclusion: Anti-ecPKA auto-antibody ELISA represents a good alternative to the

current antigen determination method, it is functional, easy to perform, cost- and time

saving method but further studies are necessary to indicate the probable utility of this

new assay as a cancer screening tool and to define its promising role in early

diagnosis/prognosis.

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The involvement of IL-17 in the development of zymosan-induced arthritis

in SCID mice

Viktoriya Milanova, Nina Ivanovska and Petya Dimitrova

Department of Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia,

Bulgaria

IL-17 is a key cytokine involved in the regulation of immune response and is

abnormally produced in chronic inflammatory conditions. Recently, it is suggested that

IL-17 can participate in innate immune reactions including activation of dendritic cells,

NK cells and recruitment of neutrophils at inflammatory sites. In the present study we

have investigated the involvement of IL-17 in the development of zymosan-induced

arthritis (ZIA) in SCID and BALB/c mice. ZIA developed earlier and the inflammation

was more severe in BALB/c than in SCID mice. CD69 positive neutrophils appeared to

the synovium of both strains on day 3 and day 7 of ZIA. However, at late stage of

disease the percentage of activated neutrophils was significantly higher in SCID

compared to BALB/c mice. At the same time point SCID mice showed elevated

synovial level of IL-17. We further investigated the ability of IL-17 to sustain the in

vitro differentiation of osteoclasts isolated from SCID and BALB/c mice. Our data

suggest a complex role of IL-17 in zymosan-induced inflammation.

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The role of target tissue in modulating CNS autoimmunity

Momčilović M1, Miljković Ž2, Miljković Đ1, Mostarica Stojković M2 1Institute of Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia 2Institute of Immunology and Microbiology, School of Medicine, University of Belgrade,

Belgrade, Serbia

Objectives: Experimental autoimmune encephalomyelitis (EAE) is an animal model of

multiple sclerosis (MS), inflammatory, demyelinating disease of the CNS. In every

species tested so far some strains are resistant to the induction of EAE. The analysis of

the mechanism underlying EAE resistance can contribute to the understanding of the

EAE and MS pathogenesis. In order to find out the role of the target tissue in CNS

autoimmunity we examined potential differences between cells isolated from spinal

cords (SC) of AO and DA rat strains, EAE resistant and susceptible, respectively.

Methods: EAE was induced in AO and DA rats by immunization with rat spinal cord

homogenate mixed with complete Freund's adjuvant. Inflammatory cells were obtained

from the SC of rats perfused with sterile PBS and separated on the 30%/70% Percoll

gradient. Flow cytometry was used for phenotypization of CNS-infiltrating cells,

detection of apoptotic cells and IFN-gamma and IL-17-producing cells. IFN-gamma and

IL-17 gene expression in CNS infiltrating cells were measured using real time PCR.

Results: Although AO rats did not exhibit any neurologic signs of disease after the

immunization in contrast to severely diseased DA rats, infiltration of immune cells into

SC was evident in both strains. However, the infiltration was less extensive in AO rats,

and the higher percentage of CD4+ T cells was found among cells infiltrating SC of DA

rats. Further, CD4+ T cells were also more activated in DA rats, as judged from the

higher percentage of OX40+ and CD25+ cells within T cells isolated from SC of

immunized animals, and less prone to apoptosis induction. Also, cells infiltrating CNS

of AO rats are less capable to produce IFN-gamma and IL-17, major marker cytokines

of Th1 and Th17 effector cells, respectively.

Conclusion: Our results presented indicate that besides regulatory mechanisms working

in the lymphoid tissues outside the CNS, additional modulating effects exerted within

the CNS parenchyma contribute to the resistance of AO rats to the induction of EAE.

This work was supported by the Serbian Ministry of Science (grants 143029 and

145066)

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KIR  gene  frequencies  in  women  with  infertility  problems    Aleksandar   Petlichkovski,   Dejan   Trajkov,   Slavica   Hristomanova,   Eli   Djulejic,  Mirko   Trpevski,   Olivija   Efinska-­‐Mladenovska,   Meri   Kirijas,   Aleksandar   Senev,  Mirko  Spiroski    Institute   of   Immunobiology   and  Human  Genetics,   Faculty   of  Medicine,   St   Cyril   and  Methodius  University,  Skopje,  Republic  of  Macedonia    Introduction:   Natural   killer   (NK)   cells   are   the   predominant   lymphocyte  population  in  the  decidua.  Being  the  most  abundant  leucocytes,  the  activity  of  NK  cells   is   important   in  different   immuno-­‐pathological   conditions,   such  as   recurrent  spontaneous   abortions,   infertility   and   problems   in   implantation.   The   NK   cells  recognize  HLA  class  I  molecules  on  trophoblasts  trough  killer  immunoglobulin-­‐like  receptors  (KIRs)  found  on  their  surface.  The  KIRs  are  classified  as  either  activating  or  inhibitory,  regarding  the  effect  they  produce  on  NK  cells  upon  interaction  with  corresponding   ligand.   Since   KIR   genes   exhibit   extensive   polymorphism   and  individuals   differ   in   both   the   number   and   kind   (activating   vs.   inhibitory)   of  KIR  genes,  it  is  hypothesized  that  the  KIR  gene  content  might  influence  the  pregnancy  outcome.    The  Aim:  The  aim  of   this  pilot  study   is   to  analyze   the   frequency  of  different  KIR  genes  in  women  with  infertility  problems,  and  compare  them  to  healthy  women.    Material   and   Methods:   Total   of   122   healthy   women   and   29   women   with  reproductive  problems  participated  in  this  study.  After  signing  of  written  consent  DNA   was   isolated   from   peripheral   blood   using   phenol/chloroform  method.   The  genotyping  of  16  KIR  genes  was  performed  using  commercially  available  kit  from  Dynal   Biotech,   (Pel-­‐Freez   Clinical   Systems,   Brown  Deer,  WI,   USA),   based   on   SSP  method.    Results:  All   the   framework   genes   (KIR3DL3,   KIR3DP1,   KIR2DL4   and   KIR3DL2)  were   present   in   all   studied   individuals.   In   the   patient   group,   the  most   prevalent  activating   gene  was   KIR2DS4,   present   in   all   individuals   (100%),  while   the  most  frequent   inhibitory   KIR   genes   were:   KIR2DL1,   KIR2DL3   and   KIR3DL1,   all   with  estimated   frequency   of   80%.  These   findings  were   comparable  with   the   group   of  healthy  control  individuals.    Conclusion:  In  conclusion,  simple  KIR  gene  frequencies  do  not  significantly  differ  among   two   studied   groups.   Further   analysis   of   frequencies   of   corresponding  genotypes  or  in  the  ratio  of  activating/inhibiting  genes  content  in  two  groups  are  needed.    

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Toll-Like Receptors 2, 4, 9 and MyD88 Expression Correlates with Disease Activity in Experimental Autoimmune Encephalomyelitis Savić E1, Popadić D1, Marković M1, Mostarica-Stojković M1 1Institute of Microbiology and Immunology, University of Belgrade, School of Medicine, Belgrade, Republic of Serbia In multiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE), helper T cells (Th), primed in periphery and differentiated into Th1 and Th17 subsets, orchestrate the process of neuronal damage of the central nervous system (CNS). Recently, innate immunity and foremost Toll-like receptors (TLRs) were found crucial for shaping the adaptive response. TLR expression is increased in MS brain lesions. However, the results demonstrating the role of several TLRs (2, 3, 4 and 9) in EAE are controversial. Nevertheless, there is a consensus that mice lacking adaptor protein MyD88 do not develop EAE. Dark Agouti (DA) and Albino Oxford (AO) rats, susceptible and resistant to EAE, respectively, were immunized with rat spinal cord homogenate in complete Freund’s adjuvant. Draining lymph nodes (DLNs) and caudal portion of spinal cord (SC) were taken in total for Real Time PCR analysis of mRNA for TLR2, TLR3, TLR4, TLR9 and MyD88 before onset (3 dpi), with first neurological signs (7 dpi), in the peak of disease (14 dpi) and in recovery phase (21 dpi). Susceptible DA strain had significantly higher TLR2, 4, 9 and MyD88 mRNA expressions in SC in peak of clinical illness compared to EAE-resistant AO rats. Additionally, AO strain exhibited significantly higher TLR3 expression in SC in the induction period. Our data supported the studies in which TLR2, 4, 9 and MyD88 expressions correlated with more severe EAE and TLR3 signaling contributed to milder disease. (Supported by grant No 145066, Ministry of Science and Technological Development, Republic of Serbia)

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Allele  frequency  of  HLA-­DQB1  locus  in  Macedonian  population    Aleksandar   Senev,   Meri   Kirijas,   Mirko   Trpevski,   Slavica   Hristomanova,   Olivija  Efinska-­‐Mladenovska,  Aleksandar  Petlichkovski,  Dejan  Trajkov,  Mirko  Spiroski    Institute   of   Immunobiology   and  Human  Genetics,   Faculty   of  Medicine,   St   Cyril   and  Methodius  Universisty,  Skopje,  Republic  of  Macedonia    Aim.  The  aim  of  this  paper  was  to  genotype  HLA-­‐DQB1  locus  in  healthy  unrelated  

Macedonian  population.    

 

Material   and   Methods.   Reverse   Line   Strip   typing   for   HLA-­‐DQB1   locus   was  

performed  on  a  population  of  217   samples   from  healthy   individuals.  The   results  

were   obtained   as   alleles,   and   as   NMDP   Codes.   Alleles   were   genotyped   and  

expressed   as   high   resolution   with   more   than   four   digits   with   slash   between  

ambiguous   alleles.   NMDP   Codes  were   expressed   as   four   digits   for   unambiguous  

results,   and   with   combination   of   numbers   and   characters   for   the   certain  

ambiguous   combination.   We   did   not   found   any   genotypic   ambiguities   in   HLA-­‐

DQB1.    

 

Results.   We   have   found   33   different   alleles   of   HLA-­‐DQB1   in   Macedonian  

population,  12  of  which  were  unambiguous  with  the  frequency  of  40.55%,  and  21  

were   ambiguous  with   the   frequency  of   58.53%.  The  biggest   frequency   (33.64%)  

was   found   for   05   unambiguous   groups,   and   for   03   ambiguous   groups   (36.40%).  

The  biggest  allele   frequency   in  Macedonian  population   for  HLA-­‐DQB1  was   found  

for  03NX  (030101/0102/09/13)  with  27.88%,   followed  with  0502  with  13.82%,  

and  02MN  (0201/02/03)  with  10.37%.  

 

Conclusion.   Allele   frequency   of   HLA-­‐DQB1   in  Macedonian   population   is   similar  

with  that  found  in  other  European  populations  and  can  be  used  for  anthropology  

and  disease  association  studies.  

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Isolation and characterization of monoclonal antibodies specific for murine

cytomegalovirus m74 gene product

Faruk Skenderi1,2, Tihana Lenac Rovis2, Barbara Adler4, Stipan Jonjic2,3* 1Department of Physiology and Biochemistry, University of Sarajevo Medical Faculty, Bosnia

and Herzegovina, 2Center for Proteomics, University of Rijeka Medical faculty, Croatia, 3Department of Histology and Embryology, University of Rijeka Medical faculty, Croatia, 4Max

von Pettenkofer-Institut, München, Germany

Human cytomegalovirus (CMV) is a frequent cause of morbidity of immuno-

compromised patients and major viral cause of congenital infections. Recent data

suggest possible role of CMV infection in autoimmune disease and some other diseases.

Many aspects of immunobiology and pathogenesis of CMV infection are still

insufficiently defined. Murine cytomegalovirus (MCMV) infection is generally

accepted as a suitable model for human cytomegalovirus (HCMV) infection. Many

studies focus on dissection of MCMV genome, transcriptome and proteome. HCMV

UL74 gene product, glycoprotein O (gO), is believed to play a role in virus entry into

the cell, assembly of viral particles and viral egress. MCMV gene m74 represents a

position homolog of the HCMV UL74. The product of MCMV m74 gene is MCMV

glycoprotein O (gO) whose role in infection is still to be clarified.

We used hybridoma technology to generate mouse monoclonal antibodies specific to

MCMV gO. BALB/c mice were immunized with UV inactivated MCMV virions. After

seroconversion, the mouse spleen was harvested and lymphocytes fused with mouse

myeloma SP2/0 cell. About 800 hybridomas were screened by ELISA using the lysates

of MCMV infected cells. Selected mother wells were additionally screened by FACS.

In total we obtained 105 hybridoma cell lines which were specific for viral proteins. To

select for a gO specific antibody we created BALB-3T3-m74HA transfectant cell line

expressing MCMV m74 gene product.

Our results show that we have generated several MCMV gO specific monoclonal

antibodies. Further characterization by using western blot, immunoprecipitation and

neutralization assay will eventually find suitability of different mAbs as tools to study

biology of this viral glycoprotein and its role in infection.

*Corresponding author. Mailing address: Medical Faculty Rijeka, Department of Histology and Embryology, B.Branchetta 20, 51000 Rijeka, Croatia. Phone: +38551651206. Email: jstipan@medri.hr

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Effects of cardiac resynchronization therapy on reverse left ventricle remodeling A. E. Stanciu, PhD1, Marcel Stanciu, PhD2, R. Vatasescu3, C. Iorgulescu3, M. Dorobantu, PhD3

1. ”Prof. Dr.Al.Trestioreanu” Institute of Oncology, Bucharest, Romania 2. University “Politehnica” of Bucharest, Bucharest, Romania 3. Clinic Emergency Hospital Bucharest, Bucharest, Romania

Background: Remodeling reflects the structural and functional deterioration that occurs in heart failure. Indices of remodeling constitute an important marker of the severity of heart failure, and reverse remodeling is an accepted goal in the treatment of heart failure. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are matrix-degrading enzymes that have been demonstrated to influence left ventricle (LV) properties and serve as targets of potential anti-remodeling agents. Objectives: The purpose of the study was to investigate the effects of CRT on serum levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), MMP-2 and TIMP-2 in patients with CHF. Methods and patients: 27 patients (21M/8F, aged 64 ± 11 years) with CHF (II-IV NYHA functional class) were investigated for immune activation before (T0 – baseline) and 1 week (T1), 3 (T2), 6 (T3), 12 (T4) months after CRT treatment. In all patients, blood specimens were drawn from a peripheral vein. The serum levels of MMP-2, TIMP-2 and NT-proBNP were measured at the same time by an ELISA method. Cardiac function was assessed echocardiographically. Results: In 100% of the patients there was a rapid and significant clinical improvement. There were no HF hospitalizations or deaths in this cohort. There was an immediate and persistent reduction in LV asynchrony, which was associated with an important and progressive improvement in LV systolic function and extensive LV reverse remodeling. There was also a significant reduction of the serum levels of NT-proBNP, MMP-2 and MMP-2/TIMP-2. CRT positively influences extracellular matrix remodeling by decreasing serum levels of MMP-2 and increasing TIMP-2. The MMP-2/TIMP-2 ratio had decreased from 6.84 ± 7.55 before CRT to 1.95 ± 0.64 at 12 months after CRT treatment. There was a good correlation between changes in ejection fraction (EF) and MMP-2/TIMP-2 (r = 0.45, p < 0.05) and septal-to-posterior wall motion delay (SPWMDSax) and MMP-2/TIMP-2 (r = 0.61, p< 0.05) after CRT. Conclusions. At 12 months follow-up, CRT was associated with a discordant change in serum MMP-2 and TIMP-2 levels. These changes in MMP-2/TIMP-2 ratio lead to reverse LV remodeling in patients with CHF.

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Influence   of   hyperthermic   environmental   temperature   on   adenosine  deaminase  (ADA)  activity  in  serum  and  lymphoid  organs    Dejan   Trajkov1,   Dinevska-­‐Kjovkarova   Suzana2,   Aleksandar   Petlichkovski1,   Biljana  Miova2,   Olivija   Efinska-­‐Mladenovska1,   Slavica   Hristomanova1,   Meri   Kirijas1,  Aleksandar  Senev1,  Mirko  Trpevski1,  Mitev  Slavcho2,  Mirko  Spiroski1    1Institute  of  Immunobiology  and  Human  Genetics,  Faculty  of  Medicine,  University  "Ss.  Kiril  and  Metodij",  Skopje,  Republic  of  Macedonia;    2Department   of   Physiology   and   Biochemistry,   Faculty   of   Natural   Sciences,  University  "Ss.  Kiril  and  Metodij",  Skopje,  Republic  of  Macedonia    Introduction:   Adenosine  deaminase   (ADA)   is   a   cytoplasmatic   enzyme   contained  in  different  tissues.  ADA  is  a  catalyser  of  the  irreversible  hydrolytic  deamination  of  adenosine   into   inosine.   The   influence   of   ADA   on   the   cell   mediated   immunity   is  expressed  by  its  participation  in  the  T  lymphocytes  and  monocytes  differentiation  and  proliferation.  Aim:   The   aim   of   this   study   was   to   analyze   the   influence   of   the   hypertermic  environmental   temperature   (35±10C)   on   the   ADA   activity   in   tissues   rich   with  lymphoid  cells  in  Wistar  rats.    Matherial   and  methods:   The   activity   of   ADA  was   examined   in   thymus,   spleen,  Peyer’s   patches,   mesenteric   lymph   nodes   and   serum.   The   experimental   animals  (n=11   male   rates)   were   exposed   (acclimated)   on   hyperthermic   environment  (35±10C)   and   relative   air   humidity   of   20-­‐30%   within   a   period   of   30   days.   The  control  group  of  animals  (n=10)  were  kept  on  room  temperature  (18-­‐220C).  The  ADA  activity  was  measured  according  to  the  modified  method  of  Giusti.  The  results  obtained   were   processed   with   the   Student’s   t-­‐test.   The   values   p<0.05   were  considered  to  be  statistically  significant.  Results:  The  results  obtained  have  shown  that  there  were  different  changes.  The  body   weight   statistically   significant   decrease   from   175.35±21   g   in   the   control  group  to  159.69±11.14  g  in  the  experimental  animals  (p<0.015).  The  spleen  mass  was  reduced  from  375.94±176.56  mg/100  g  to  237.59±74.06  mg/100  g  (p<0.05),  and   the   thymus   mass   from   245.52±51.55   mg/100g   to   204.84±29.92   mg/100   g  (p<0.05).  Decreased  of  relative  mass  of  both  Peyer’s  patches  and  mesenteric  lymph  nodes   was   not   statistically   significant.   Activity   of   ADA   in   the   thymus   was  significantly   increased   from   10.58±1.57   U/g   in   the   control   group   of   rats   to  13.38±2.7   U/g   in   rats   exposed   on   35±10C   (p<0.012),   and   in   the   spleen   it   raised  from   5.17±3.29   U/g   to   7.8±1.97   U/g   (p<0.05).   The   ADA   activity   in   the   other  lymphoid   organs   (Peyer’s   patches   and   mesenteric   lymph   nodes)   and   serum  showed  no   statistically   significant   changes  between   the   two   groups  of   examined  rats.  Conclusion:  It  could  be  concluded  that  the  influence  of  hyperthermic  environment  (3535±10C,   30   days),   provokes   decrease   in   body   weight,   spleen   and   thymus  relative  mass,  increase  of  the  enzyme  activity  of  ADA  in  thymus  and  spleen  of  rats,  but  there  are  no  changes  in  the  other  lymphoid  organs  and  tissues.  

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List  of  participants    Belij,  Sandra,    Institute  für  Biological  Research  “Sinisa  Stankovic”,  Belgrad,  Serbia    mail:  argyopes@gmail.com    Berisha,  Dr.  Naser,  MD University  Clinical  Center, Obstetrics  &  Gynecology    University  of  Prishtina,  Prishtina,  Kosovo  mail:  vivefjolla@yahoo.com    Bijelic,  Dr.  Bibijana,  MD  Clinical  Hospital  Centre  Osijek,  Scientific  Research  Unit,  Osijek,  Croatia    mail:  bibijana007@gmail.com    Bijelic,  Dr.  Nikola,  MD  University  of  J.J.  Strossmayer,  Faculty  of  Medicine,  Osijek,  Croatia    mail:  nbijelic@mefos.hr    Curaj,  Teuta  Universal  Hospital  Center,  Tirana,  Albania    mail:  Lidapergega@yahoo.com    Dervisevic,  Dr.  Amela  MD  Medical  Faculty,  University  of  Sarajevo,  Sarajevo,  Bosnia  and  Herzegovina    mail:  amela1610@yahoo.com    Drace,  Dr.  Zahida  MD    Clinical  Center,  University  of  Sarajevo    Sarajevo,  Bosnia  and  Herzegovina    mail:  zazilija@yahoo.com    Dzafic,  Dr.  Fejzo  MD  University  Clinical  Center  Tuzla,    Tuzla,  Bosnia  and  Herzegovina  mail:  fejzo9@hotmail.com    Gavranovic,  Dr.  Marija  MD  Clinical  Center  of  Montenegro,    Podgorica,  Montenegro  mail:  maja.gavranovic@yahoo.com    Godinjak,  Dr.  Amina  MD  Clinical  Center  University  of  Sarajevo  Clinic  for  Endocrinology,    Sarajevo,  Bosnia  and  Herzogovina  mail:  amina_g1982@hotmail.com    Hristomanova,  Dr.  Slavica  MD  Institut  of  Immunobiology  and  Human  Genetics,  Faculty  of  Medicine,  Skopje,  Macedonia  

mail:  cacidr@yahoo.com      Huica,  Irina  Institute  of  Virology  “Stefan  S.  Nicolau”,    Bucharest,  Romania    mail:  irina.huica@yahoo.com    Jergovic,  Mladen  Institute  of  Immunology,  Zagreb,  Croatia  mail:    mjergovic@imz.hr    Josipovic,  Pavle,    Faculty  of  Sciences,  Department  of  Animal  Physiology,  Zagreb,  Croatia  mail:  pjosipov@biol.pmf.hr    Kapovic,  Dr.  Agneza  Marija  MD  Children’s  Hospital,  Zagreb,  Croatia  mail:  amk_xx@yahoo.com    Karac,  Ivo  Faculty  of  Sciences,  Department  of  Biology,  Zagreb,  Croatia    mail:  ivo_karac@net.hr    Karkovic,  Domagoj    Faculty  of  Sciences,  Department  of  Biology,  Zagreb,  Croatia  mail:  domagoj_85@yahoo.com    Kirijas,  Dr.  Meri  MD  Institute  of  Immunobiology  and  Human  Genetics,  Skopje,  Macedonia        mail:  meri_paneva@yahoo.com    Kurti-­Prifti,  Dr.  Margarita  MD    Laboratory  of  Immunology,  University  Hospital  Center,  Tirana,  Albania  mail:  priftimarita@yahoo.it    Lacevic,  Dr.  Dzenana  MD  Institute  of  Medical  Biochemistry,  Clinical  Centre  of  University,  Sarajevo,  Bosnia  and  Herzegovina  mail:  balerina56@gmail.com      Latifi-­Pupovci,  Prof.ass.  Hatixhe,  MD  Chair  for  Physiology  and  Immunology,  Medical  Faculty,  University  of  Prishtina,  Prishtina,  Kosovo  mail:  hpupovci@uni-­‐pr.edu    Ler,  Daria    Institute  for  Genetic  Engineering  and  Biotechnology,  University  of  Sarajevo,    Sarajevo,  Bosnia  and  Herzegovina  mail:  dariamail@gmail.com    

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Lokaj-­Berisha,  Dr.  Violeta  MD  University  of  Prishtina   ,  Medical  Faculty  Institute  of  Physiology  and  Immunology  Prishtina,  Kosovo  mail:  vivefjolla@yahoo.com    Lumezi,  Dr.  Besa  MD  Medical  Faculty,  University  of  Prishtina,    Department  of  Physiology  and  Immunology  Prishtina,  Kosovo    mail:  besa_gacaferri@yahoo.com    Marinovic  Terzic,  Dr.  Ivana  MD  PhD  Department  of  Immunology  and  Medical  Genetics,  Split  University  Medical  School,  Split,  Croatia  mail:  ivana.marinovic.terzic@mefst.hr    Masic,  Dr.  Admir  MD    Clinic  for  Neurology  diseases,Clinical  Center  of  University  Sarajevo,    Sarajevo,  Bosnia  and  Herzogovina      mail:  masic.a@bih.net.ba    Milanova,  Viktoriya  Department  of  Immunology,  Institute  of  Microbiology,  Bulgarian  Academy  of  Science,  Sofia,  Bulgaria  mail:  v.milanova8@gmail.com    Momcilovic,  Dr.  Miljana  PhD    Institute  for  Biological  Research  “Sinisa  Stankovic”,  Department  of  Immunology  Belgrad,  Serbia  mail:  mommilja@yahoo.com    Okic,  Dr.  Anel  MD  Canton  Hospital  Zenica  Zenica,  Bosnia  and  Herzogovina      mail:  anel_okic@yahoo.com      Pecani,  Dr.  Arbi  MD  University  Hospital  Center,  “Nene  Tereza”  Tirana,  Albania    mail:  arbipecani@gmail.com    Petlichkovski,  Dr.  Aleksandar  MD  Institute  of  Immunobiology  and  Human  Genetics,  Faculty  of  Medicine,    Skopje,  Macedonia  mail:  petlichkovski@yahoo.com    Prljaca-­Zecevic,  Dr.  Lamija  MD  Clinical  Center,  University  of  Sarajevo    Sarajevo,  Bosnia  and  Herzogovina  mail:  zecevicl@hotmail.com          

Savic,  Dr.  Emina,  MD  Institute  of  Microbiology  and  Immunology,  School  of  Medicine,  University  of  Belgrad  Belgrad,  Serbia  mail:  esavic@med.bg.ac.rs    Semanaj,  Valentina  Laboratory  of  Immunology  University  Hospital  Center  “Mother  Teresa”,    Tirana,  Albania      mail:  vsemanaj@yahoo.com    Senev,  Dr.  Aleksandar  MD  Institute  of  Immunobiology  and  Human  Genetics,  Faculty  of  Medicine,    Skopje,  Macedonia  mail:  aleksenev@yahoo.com    Serdarevic,  Dr.  Fadila  MD  Institute  of  Immunology    University  of  Sarajevo,  Bosnia  and  Herzegovina  Mail:  fserdarevic@gmail.com    Shrestha,  Dilip  Department  of  Biophysics  and  Cell  Biology,  University  of  Debrecen,  Hungary  mail:  aagaman@gmail.com    Skenderi,  Dr.  Faruk  MD  Department  of  Physiology  and  Biochemistry,  University  of  Sarajevo,  Medicial  Faculty  Sarajevo,  Bosnia  and  Herzogovina  mail:  faruk.skenderi@mf.unsa.ba    Skific,  Marijana  University  Hospital  Centre,  Dept.  of    Clinical  Transfusiology  and  Cellular  Therapy,  Zagreb,  Croatia  mail:  mskific@kbc-­‐zagreb.hr    Stanciu,    Dr.  Adina  Elena  Prof.  Al.  Trestioreanu  Institute  of  Oncology,  Bucharest,  Romania  mail:  adinaelenastanciu@yahoo.com    Trajkov,  Dr.  Dejan  Institute  of  Immunobiology  and  Human  Genetics,  Faculty  of  Medicine,    Skopje,  Macedonia  mail:  dejantmk@yahoo.com    Zecevic-­Masic,  Dr.  Mina,  MD    Clinic  for  Pulonary  Diseases  and  TB,  Clinical  Centre  of  University  of  Sarajevo,    Sarajevo,  Bosnia  and  Herzegovina    mail:  minaalma@bih.net.ba        

   

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Faculty    Fleischer,  Bernhard,  Prof.,  Bernhard  Nocht  Institute  for  Tropical  Medicine,  D-­‐20359  Hamburg,  Germany,  www.bnitm.de;  and  Institute  of  Immunology,  University  Medical  Centre  Hamburg-­‐Eppendorf,  D-­‐20246  Hamburg,  Germany,    mail:  fleischer@bnitm.de      Gospos,  Annette,  Dr.,  Director,  Division  Medical  Scientific  Information,  Euroimmun  Inc.,  D-­‐23560  Luebeck,  Germany,  www.euroimmun.de;    mail:  a.gospos@euroimmun.de      Haag,  Friedrich,  Prof.,  Institute  of  Immunology,  University  Medical  Centre  Hamburg-­‐Eppendorf,    D-­‐20246  Hamburg,  Germany,  www.uke.de/institute/immunologie;    mail:  haag@uke.de      Meri,  Seppo,  Prof,  Head,  Haartman  InstituteDepartment  of  Bacteriology  and  Immunology,    PO  Box  21,  Haartman  Institute,  FIN-­‐00014  University  of  Helsinki,  Finland,    mail:  seppo.meri@helsinki.fi      Mittruecker,  Hans-­‐Willi,  Prof.  ,  Institute  of  Immunology,  University  Medical  Centre  Hamburg-­‐Eppendorf,    D-­‐20246  Hamburg,  Germany,  www.uke.de/institute/immunologie;    mail:    h.mittruecker@uke.de      Rabatic,  Sabina,  President,  Croatian  Immunological  Society,  Institute  of  Immunology  Rockefeller  str.10,  100000  Zagreb,  Croatia;    mail:  srabatic@imz.hr    

Seitz,  Hans-­‐Joachium,  Prof.,  Southeast-­‐Europe  Cooperation,  University  Medical  Centre  Hamburg-­‐Eppendorf,  Martinistraße  52,  D-­‐20246  Hamburg,  Germany;    mail:  hjseitz@uke.de    Stockinger,  Hannes,  Prof.  ,  Chairman,  Department  of  Molecular  Immunology,  Center  of  Physiology,  Pathophysiology  and  Immunology,  Medical  University  of  Vienna;  Internet:  http://www.meduniwien.ac.at/immunology;    mail:  hannes.stockinger@meduniwien.ac.at    Zouali,  Moncef,  Prof.,  Director  of  Research,  Institut  National  de  la  Santé  et  de  la  Recherche  Médicale  (INSERM),  University  Denis  Diderot,  Paris,  France;    mail:  moncef.zouali@wanadoo.fr