periapical pathoses of pulpal origin develop in response to microbial irritants in the root canal systems .
bacterial 
 cell wall components react with monocytes , macrophages , other cells of the immune system , as 
 well as with fibroblasts , leading to the production of proinflammatory 
 cytokines , such as il-1 , il-1 , tnf- , il-6 , and il-8 .
irritants may be 
 mechanical or chemical , but are most often bacterial . the interaction between these irritants and host defensive cells results in release of numerous mediators
that , through the root canal system , are capable of initiating immunologic 
 reactions in periapical tissues resulting in the formation on inflammatory periapical lesions .
these reactions include immune response mediated by cells , through the actions of t lymphocytes and cytokines , and hummoral immune 
 response mediated by antibodies , activated by b - lymphocyte products .
tnf- is a monocyte - derived protein that has a wide range of proinflammatory and immunomodulatory effects on a number of different cell populations .
tnf- is a cytokine which stimulates bone 
 resorption , prostaglandin synthesis , and protease production by many cell types , including fibroblasts and osteoblasts .
the local production of pge2 , il1- , and tnf- has been demonstrated in periapical lesions [ 4 , 5 ] .
tnf- is a cytokine initially 
 identified as a causative of hemorrhagic necrosis in certain tumors and was 
 later shown to be the same molecule as cachectin , a serum product earlier known 
 as a mediator of wasting in chronic disease .
in addition to its diverse 
 bioactivities , tnf- is the only molecule other than il-1 that is presently 
 known to have osteoclast - activating function .
although extensive research has 
 been performed in the area of periapical inflammatory mediators , only few studies 
 focused on the role and levels of tnf- in periapical exudate [ 6 , 7 ] .
the aim 
 of this study was to determine the levels of tnf- in periapical exudates and 
 to evaluate possible relationships between this cytokine and radiological 
 findings in the involved teeth .
the patients with 
 noncontributory medical histories and diagnosed with apical periodontitis were 
 selected for the study as follows . 

group 1 consisted of 20 subjects 
 whose single - rooted teeth had the diagnosis of chronic apical periodontitis , 
 with dull or absent pain , and size of the lesion < 1  cm . 

group 2 consisted of 20 subjects 
 whose single - rooted teeth were diagnosed with chronic apical periodontitis , 
 with dull or absent pain , but with the lesion size of > 1  cm.group 3 was control and 
 comprised of 20 subjects whose investigated single - rooted teeth showed symptoms 
 of acute periapical condition accompanied by excruciating pain , necrotic pulp , 
 and radiological thickening of the apical periodontal ligament . 

primary access to the pulp chamber was gained using slow - speed round dental burs , with 
 placement of rubber dam isolation .
after determination of the working length , the 
 necrotic pulp was removed and the root canal was enlarged to iso size 40 .
a sterile paper point was 
 inserted into the root canal up to the instrumentation length and held in place 
 for 1 minute to absorb the exudate for the sample [ 6 , 7 ] .
paper points were immersed into sterile ependorf 
 vials containing phosphate buffered saline ( pbs ) and stored at 70c until 
 further analysis [ 8 , 9 ] .
the volume of the fluid was calculated from a 
 standard curve and expressed as l .
size of the radiolucent area was determined 
 by two independent examiners at the time exudate samples were collected . 

periapical lesions were classified as small , when the longer diameter was 
 shorter than 1  cm , while the remaining lesions were classified as large . in the 
 control group , the radiological thickening of the periodontal ligament was 
 present .
group 1 consisted of 20 subjects 
 whose single - rooted teeth had the diagnosis of chronic apical periodontitis , 
 with dull or absent pain , and size of the lesion < 1  cm . 

group 2 consisted of 20 subjects 
 whose single - rooted teeth were diagnosed with chronic apical periodontitis , 
 with dull or absent pain , but with the lesion size of > 1  cm .
group 3 was control and 
 comprised of 20 subjects whose investigated single - rooted teeth showed symptoms 
 of acute periapical condition accompanied by excruciating pain , necrotic pulp , 
 and radiological thickening of the apical periodontal ligament .
determination of the tnf- levels in supernatant of periapical exudate is 
 based on utilization of monoclonal antibodies specific for tnf- which bind 
 competitively to tnf-. investigation is based on  sandwich 
all subjects were informed of the aims and procedures of research . within 
 the research ,
they are guaranteed in respect of their basic ethical and bioethical principles : 
 personal integrity ( independence , righteousness , well - being , and safety ) as 
 regulated by nrnberg codex and 
 the most recent version of helsinki 
  declaration . only those subjects who gave a written permission in the form of informed 
 consent
the present study demonstrated the presence   of tnf- 
 in all periapical exudate samples .
differences in concentrations of tnf- between 
 control group ( 40 , 57  28 , 15  pg / ml ) , the group with large radiolucent areas 
 ( 2365 , 79     582 , 95  pg / ml ) , as well as between control and the canals associated with 
 small radiolucent areas ( 507 , 66  278 , 97 ) were statistically significant ( p    <        .05 ) . 

there was also statistically significant difference between the samples with 
 large radiolucent areas and small radiolucent areas .
eia data for tnf- were 
 categorized according to radiological diagnosis , using the nonparametric 
 kruskal - wallis   test ( see figure 1 ) .
soluble mediators produced and secreted by various inflammatory , 
 immunologically - active , and structural cells , commonly referred to as 
 cytokines , play the leading role in pathogenesis of infectious disease , acting 
 side - by - side with other inflammatory mediators : kinines , vasoactive amines , 
 components of the complement system , and metabolites of arachidonic acid .
secretion of the cytokines is initiated with the purpose of activating 
 immunological response to irritants and increasing local concentrations of 
 inflammatory cells in order to prevent further colonization of bacteria within 
 the tissues .
enhanced reaction of the host to various antigens results in bone 
 resorption and formation of granulomatous tissue , which are the typical 
 features of periapical lesion . in this investigation
, levels of the proinflammatory cytokine tnf- in 
 periapical exudate were analyzed due to its leading role in pathogenesis of 
 periapical lesions .
these exudates present the inflammatory exudate from the periapical tissues 
 and theoretically contain locally produced and secreted factors 
 .
they 
 resulted from the inflammatory response and contain host mediators related 
 in response to the infection 
 .
highest concentrations of tnf- were detected in periapical lesions with 
 large radiolucent areas , while these levels were significantly lower in the 
 periapical lesions with smaller radiolucent areas . in the control group 
 diagnosed as acute periodontitis , with radiological changes consistent with 
 periodontal ligament thickening , which differs 
 in clinical symptoms from the other two experimental groups , and
is characterized by radioloical changes consistent with 
 periodontal ligament thickening , the levels of the investigated cytokine demonstrated the lowest levels .
these results show 
 different values from the results obtained by ataoglu et al . ; however , 
 these authors compared the levels of two kinds of cytokines : il-1 and tnf-. in 
 the work of ataoglu et al .
, il-1 was detected in all samples of periapical 
 exudates and its levels were significantly higher than tnf- levels ; however , the 
 authors focused their attention only on the tnf- levels in periapical exudates 
 demonstrated in all tissue specimens with significant differences regarding 
 certain experimental groups . a study by kjeldsen et al .  
using elisa 
 technique also showed significantly higher concentrations of tnf- in crevicular 
 fluid in patients with chronic adult periodontitis .
it is likely that chronic 
 periodontal infection may evoke an immune response that may result in the production 
 of slightly higher levels of tnf-. it was proved that tnf- leads to bone resorption through osteoclast 
 activation and stimulation of the secretion of proteolytic enzymes , plasminogen 
 activator ( pa ) , and matrix metalloproteinases ( mmp ) , which are in charge of 
 destroying extracellular matrix of the bone tissue .
.   showed that 
 there is a small fraction of 
 tnf--positive cells within the granuloma with a macrophage - like 
 morphology .
ultrastructural analyses showed that   there are some macrophages which have adjusted the extracellular 
 secretion ; therefore , these macrophages might be the main 
 source of cytokines in the tissue .
wang et al .    showed that both tnf- and il-1 are secreted in 
 the infected rat pulps and periapical lesions .
the cells appeared as soon as 2 
 days following the pulp chamber opening and their numbers increased steadily 
 until the day 30 .
these findings demonstrate the presence of il-1- and tnf--secreting cells in the pulp 
 and periapical tissues immediately following the pulp exposure , which supports 
 the assumption that the abovementioned cytokines play a role in pathogenesis of 
 pulp and periapical pathoses . in the analysis of tnf- levels in healthy , symptomatic , and asymptomatic 
 human pulps ,
the highest 
 concentrations of tnf- , with regard to clinical classification , were found in 
 symptomatic reversible pulpitis , and the difference was statistically 
 significant in comparison with irreversible asymptomatic pulpitis and healthy 
 pulp .
the highest concentrations of tnf- , with regard to histological 
 classification , were found in histologically confirmed moderate inflammation , 
 followed by severe inflammation , mild inflammation , and finally healthy pulp .
conclusion can be drawn that cytokine tnf- could be an objective marker 
 in radiologically confirmed periapical lesions .
the lowest levels were 
 demonstrated in control group , which was characterized by radiological 
 thickening of the periodontal ligament , while somewhat greater concentrations 
 were found in teeth with periapical radiolucencies of < 1  cm diameter ; finally , 
 the greatest concentrations were measured in teeth with radiolucent lesions 
 > 1  cm , which confirms the relationship between tnf- and the emergence and 
 development of periapical lesions .
objective analysis of tnf- levels enables 
 establishment of a relationship between different concentrations of this 
 cytokine and different radiological changes .
 aim . 
 the aim of this study was to determine tumor necrosis factor - alpha 
             ( tnf- ) levels in periapical exudates and to evaluate their relationship with radiological findings .           
 methodology . 
 periapical exudates were collected from root canals of 60 single - rooted teeth using absorbent paper points . 
 
 tnf- 
  levels were determined by enzyme - linked immunosorbent assays . 
 the samples were divided into three groups according to the periapical radiolucent area . 
 
 results . 
 nonparametric kruskal - wallis test revealed significant differences between 
 tnf- concentrations in control group ( 40 , 5728 , 15 pg / ml ) and group with larger radiolucent areas ( 2365 , 79582 , 95 
 pg / ml ) , as well as between control and canals with small radiolucent areas 
 ( 507 , 66278 , 97 ) ( p<.05 ) . 
 conclusions . 
 the levels of 
 tnf-   increase significantly in teeth with periapical pathosis , from smaller to bigger lesions . 
 this research and its results have shown that objective analysis of the 
 tnf- 
  levels enables establishment of a relationship between different concentrations of 
  tnf-   and different radiological changes . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION
5. CONCLUSION