POOPERATIVNA SLABOST IN BRUHANJE NELAGODJE IN STISKA OGROŽATA USPEH OPERATIVNEGA POSEGA PREPRECEVANJE Ena sama intravenska injekcija po 4mg pri uvajanju v anestezijo lW ,m ZDRAVLJENJE Ena sama intravenska injekcija po 4mg ondansetron Glaxo Podrobnejše informacije dobite pri: Glaxo Export Limited Podružnica Ljubljana, Cesta v Mestni log 55, 6111 5 Ljubljana, p.p. 17, Slovenija Telefon: (38661) 1231070, 1232097, 1232319 Telefax: (38661) 1232597 RADIOLOGY AND ONCOLOGY Radiology and Oncology is a journal devoted to publication of original contributions in diagnostic and interventional radiology, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinical and experimental oncology, radiophysics and radiation protection. Editor in chief Tomaž Benulic Ljubljana, Slovenia Associate editors Gregor Serša Ljubljana, Slovenia Viljem Kovac Ljubljana, Slovenia Editorial board Marija Auersperg Ljubljana, Slovenia Matija Bistrovic Zagreb, Croatia Haris Boko Zagreb, Croatia Malte Clausen Kiel, Germany Christoph Clemm MUnchen, Germany Mario Corsi Udine, Italy Christian Dittric/i Vienna, Austria Ivan Drinkovic Zagreb, Croatia Gillian Duchesne London, Great Britain Bela Pomet Budapest, Hungary Tullio Giraldi Udine, Italy Andrija Hebrang Zagreb, Croatia Duraa Horvat Zagreb, Croatia Laszlo Horvath Pecs, Hungary Berta Jereb Ljubljana, Slovenia Vladimir Jevtic Ljubljana, Slovenia H. Dieter Kogelnik Salzburg, Austria Ivan Lovasic Rijeka, Croatia Marijan Lovrencic Zagreb, Croatia Luka Milas Houston, USA Maja Osmak Zagreb, Croatia Branko Palcic Vancouver, Canada Jurica Papa Zagreb, Croatia Dušan Pavcnik Ljubljana, Slovenia Stojan Plesnicar Ljubljana, Slovenia Ervin B. Podgoršak Montreal, Canada Miran Porenta Ljubljana, Slovenia Jan C. Roos Amsterdam, The Netherlands Horst Sack Essen, Germany Slavko Šimunic Zagreb, Croatia Lojze Šmid Ljubljana, Slovenia Andrea V ero nesi Gorizia, Italy Publishers Slovenian Medica! Society -Section oj Radiology, Croatian Medica/ Association -Croatian Society oj Radiology Affiliated with Societas Radiologorum Hungarorum Friuli-Venezia Giulia regional groups of S.I.R.M. (Italian Society of Medica! 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Indexed and abstracted by BIOMEDICINA SLOVENICA CHEMICAL ABSTRACTS EXCERPTA MEDICAIELECTRONIC PUBLISHING DIVISION CONTENTS DIAGNOSTIC AND INTERVENTIONAL RADIOLOGY Pseudotumor of the mediastinum -Case report Matic T, Švalba-Novak V, Kraus !, Rubini{: M 5 Transcatheter occlusion of 1>atent ductus arteriosus in adults Pavcnik D, Berden P, Koželj M 9 NUCLEAR MEDICINE Simple quantitative analysis of Ga-67 lung scintigraphy -normal values Huic D, Dodig D, Jurašinovic Ž, Težak S, Poropat M, lvicevic A, Bambir M, Medvedec M ONCOLOGY Residual carcinoma of the uterine cervix after low-dose preoperative intracavitary irradiation Fras AP 15 Outcome of pneumonectomy for primary non-small celi lung cancer -A ten-year experience Nan-Yung Hsu, Chie-Yi Chen, Chung-Ping Hsu 19 Tumor necrosis factor-a (TNF-a): Biological activities and mechanisms of action Novakovi{: S, Jezeršek B 25 Determination of the breast volume after breast conservating surgery Demange L, Tisnes J 44 Trilateral retinoblastoma Bolonaki !, Lydaki E, Stiakaki E, Kalmantis T, Kalmanti M 47 Quantitative analysis of terminal blood network in human spina! cord and progressive radiation myelopathy Vodvaflca P, Malinslcy J, Tichy L 50 RADIOPHYSICS Edge-enhancement performance of the histogram shifting filter Fallone BG, Crooks I 58 Parameterization of megavoltage transmission curves used in shielding calculations Podgorsak MB, Ho AK, Balog JP, Sibata CH 65 BOOK REVIEW Radiation therapy in paediatric oncology Horvdth G REPORT ESTRO teaching course on basic clinical radiobiology Sarl6s G 71 NOTICES 72 AUTHOR INDEX and SUBJECT INDEX, 1994 The publication of the journal is subsidized by the MinistJy of Science and Technology of the Republic Slovenia. Contributions of lnstitutions: Fundacija doc. dr. J. Cholewa, Ljubljana; Inštitut za diagnosticno in intervencijsko radiologijo, KC Ljubljana; Klinika za otorinolaringologijo in maksilofacialno kirurgijo, KC Ljubljana; Klinicki zavod za dijagnosticlw interventnu radiologiju, KBC Rebro, Zagreb; Onkološki inštitut, Ljubljana Radio/ Oncol 1995; 29: 5-8. Pseudotumor of the mediastinum -Case report Tomislav Matic, Velinka Švalba-Novak, Ivan Kraus, Milivoj R.ubinic Department of Interna! Medicine, Clinical Hospital Center Rijeka, Rijeka, Croatia Varices of the azygos and hemiazygos veins secondary to portal hypertension should be considered in differential diagnosis in every mediastinal pseudotumor. We are presenting a very rare case of a Jemale patient with a pseudotumor of the upper mediastinum resulting from thrombosis of the portal vein and portal hypertension. After a splenorenal shunt, chest X-ray showed that the shadow of the mediastinal pseudotumor almost disappeared. Key words: mediastinal neoplasms; hypertension, portal Introduction Portal hypertension represents a group of path­ologic clinical rnanifestations associated with hypertension in the portal venous system or, more precisely, with increased portocaval gra­dient. Increased portal vein pressure is caused by an obstruction of normal intrahepatic or prehepatic blood flow. The only exception is the so-called idiopathic portal hypertension, where the obstruction can be detected neither within the !iver nor extrahepatically, with ali the signs of portal hypertension. Splenomegaly, collateral circulation, bleeding, etc., can deve­lop as a consequence of portal hypertension. Varices are most cornrnonly localized in the esophagus or the stomach ( esophagogastric va­rices) and other localizations are quite uncom­rnon. Rarely, varices of the azygos and the Corespondencc to: Tomislav Matic, M.D., Dcpart­ment of Interna] Medicine, Clinical Hospital Center Rijeka, Krešimirova 42. 51000 Rijeka, Croatia. herniazygos veins, irnitating a tumor of the rnediastinum, may develop.1-7 We are present­ing the case of a young fernale patient with a portal vein thrombosis, portal hypertension and a pseudotumor of the mediastinum. Case report Patient C. N., female, born in 1964, a tourist frorn Lecco (ltaly), was admitted to the Inten­sive Care Unit, Department of Interna! Medici­ne, Clinical Hospital Center Rijeka, on August 17, 1989 because of melena. She had no report­ed illnesses until 1986 when she was hospitalized for an artificial septic abortion, with probable pylephlebitis (inflamation of portal vein) and portal vein thrombosis that remained undisco­vered. Severa! months later, due to persistent abdominal pain she unclerwent gastroenterolo­gical examination and varices of the esophagus and portal vein thrornbosis were detected. In 1987, one year after the detection of the portal vein thrombosis and esophageal varices, blee­ UDC: 616.27-002. 155:616.149-008.341.1 ding frorn the esophageal varices was evident ;\1/atil' T et a/. for the first tirne, and she was hospitalized in Milan. During the next two years she experien­ced four bleedings from the esophageal varices. The last bleeding was evidenced in March of 1989, i.e., five months before she was admitted to our Department. During her last hospitaliza­tion in March of 1989, a chest X-ray revealed a lung shadow in the superior right mediasti­num, which was considered to be a tumor of the superior mediastinum. Already then, surgi­cal derivation of the portal circulatory system was considered. This possibility was then rejec­ted due to the tumor of the upper mediastinum. As stated earlier, the patient was admitted to our Department on August 17, 1989, due to melena initially not profuse. One day before being admitted to our Department, she passed three profuse, fluid, tarry stools. On admission the patient was pale, tachycardic 110/min., blood pressure 15/8 kPa. The hepar was not papable and the spleen was enlarged by 3 cm below the left costal margin. Laboratory fin­dings showed severe anemia, decreased fibrino­gen and thrombocits, decreased total proteins and serum albumin. An urgent esophagogastro­duodenoscopy was immediately performed re­vealing very large non bleeding esophagogastric varices. Bleeding started the same night, and a Sangstaken-Blackmore tube was placed, vaso­pressin and ranitidine were administered paren­terally. She also received a bloocl transfusion. After severa! tarry stools, it appeared that thc bleeding stooped. Unfortunately not for long. Melena and hematemesis persisted and the pa­tient almost expired due to exsanguination. This bleeding was caused by rupture of the esophageal balloon due to maximal air insuffla­tion necessary to maintain the hemostasis effec­tive. The Sangstaken-Blackmore tube was rein­troduced, and with new transfusions applied to both hands, resuscitation was achived with great clifficulty. At this stage it was clear that conservative treatment is not effective. Even the smallest displacement of the tube would provoke new bleedings. An emergency shunt was the only solution that could save the patient. Ultrasono­graphy of the abdomen and a chest X-ray were performed. Ultrasonography of the abdomen showed a normal sized !iver, with no dilatation of intrahe­patic biliary ducts. In the body and tale of the pancreas the lienal vein 11 mm in diameter, was visualized. Only the initial part of the portal vein was demonstrated. The spleen was enlarg­ed, 18 X 5 x 8 cm. There was ascites in the abdomen. Chest X-ray revealed a polycyclic shadow in the upper right mediastinum. Our explanation for this finding would be that it represents a vascular tumor, i.e., dilatation-varices of the azygos and hemiazygos veins (Figure 1). As the bleeding persisted ancl the patient's showed no improvement, we clecided to per­form an emergency shunt. The patient under­went surgery on August 19, 1989, when a splenorenal shunt and a splenectomy were effected. Surgical treatment. We performed extend left subcostal laparotomy. Over 1 liter of ascitic fluid was evacuatecl from the abclominal cavity. The stomach was very distended, filled with coagulated bloocl. Lavage was effected by a nasogastric tube. Numerous varices were found along the lesser and greater curvature of the stomach and the abdominal esophagus, with many peritoneal collaterals. The lymph stasis was pronounced. Inspection and palpation of the !iver revealed normal findings. Spleen was merkedly enlarged, cyanotic, bard in palpation. Figure l. Polycyclic shadow in thc uppcr right mcdia­stinum. Pseudotumor of the mediastinum Other abdominal organs were unremarkable. After preparation of the vascular hylum of the spleen, splenectomy was performed. The lienal vein was mobilized away from the pancreas. The left renal vein was accessed and partially clampeb; venotomy was effected, and an ana­stomosis between the lienal and renal veins was performed. After clamps were removed, a sta­ble functional shunt was obtained. Hemostasis was evaluated, drainage of the spleen cavity effected, and the abdominal wall closed by layers. The postoperative course was complicated by repeated hematemesis. Esophagogastroduode­noscopy revealed considerably smaller esopha­geal varices, and the bleeding was caused by erosions of the gastric mucosa. After treatment with blood transfusion and parenterally admini­stered ranitidine, the bleeding eventually stopp­ed, with considerable improvement in red celi count. The patient stayed at the Department of Surgery during August 19-31, 1989, when she was returned to the Department of Interna! Medicine. To confirm our hypothesis that this was a case of a vascular pseudotumor and not a true tumor of the upper mediastinum, we performed another chest X-ray before the patient was discharged, i.e., nearly a month after the ope­ration. It revealed that the polycyclic shadow in the upper right mediastinum considerable reduced (Figure 2). The patient was released from hospital on September 18, 1989 in a good Figure 2. Polycyclic shadow in the upper right media­stinum almost disappeared after a splenorenal shunt. general condition, with a satisfactory blood count and with normal values of fibrinogen and thrombocytes. During the follow-up, esophago­gastroduodenoscopy evidenced no varices of the esophagus. The shunt was well functioning. The chest X-ray was clear with no traces of polycyclic shadows in the right upper mediasti­num. Four years since the operation the patient feels well, has experienced no difficulties and continued to work. Discussion In a portal vein thrombosis there is a total obstruction of the portal venous blood flow through the !iver. This causes the reverse of blood flow toward natura! anastomoses that connect the portal system with the inferior and superior vena cava system. In a portal vein obstruction due to thrombosis, portal hyperten­sion rapidly develops. In !iver cirrhosis, portal hypertension develops slowly allowing natura! anastomoses to gradually redirect the additional blood from the portal system toward the vena cava, and they gradually dilate. The hepatic portal circulation in cirrhosis is never comple­tely interrupted. It is partially preserved dep­ending on the extent of the cirrhotic process. In portal vein thrombosis, natura! anastomos­es must adapt quickly to the newly developed conditions, i.e., they must be able to transport the complete portal blood flow in the opposite direction of its natura! flow. In this case, we shall mention only two important natura! ana­stomoses. One is through the esophageal veins, connected on one side to the Ieft gastric vein pertaining to the portal system, and on the other side to the azygos and hemiazygos veins that enter the superior vena cava. The other, less known routes, are the small retroperitoneal veins, the so-called Retzius veins, that represent the anastomoses between the superior and infe­rior mesenteric vein on one side and the azygos and hemiazygos vein on the other. In portal thrombosis, these anastomoses transport a large volume of blood to the azygos and hemiazygos vein, which are located in the Matic T et al. retroperitoneal space, extending from the abdo­minal to the thoracic cavity each on one side of the vertebral column, and joining before entering the superior vena cava. Their wall is thin, unresisting to pressure and dilates une­venly forming varices. They become tortuous and elongated, folding in places and developing rope-like vascular masses, giving the impression of a tumor. Such vascular tumors can be located in the abdomen or more often in the thorax. In the abdomen they are placed retroperitoneal­ly, and in the thorax in the posterior mediasti­num more often in the inferior that the superior part. According to French authors they are called pseudotumorous varices of the mediasti­num. 8 We have presented an interesting case of a female patient whose chest X-ray revealed a polycyclic shadow in the superior mediastinum, which could easily be misdiagnosed. If the patient had not bled from the varices, she could have been subjected to an invasive diagnostic procedure for the pseudotumor, with eventual catastrophic consequences. This case represents a good example why, in differential diagnosis of a mediastinal tumor, the possibility of a pseudotumor should be considered when portal hypertension is present. References l. Campbell HE, Baruch RJ. Aneurysm hemiazygos vein associated with portal hypertension. Amer J Roentgenol Radium Ther Nucl Med 1960; 83: 1024­26. 2. Castellino RA, Blank N, Adams DF. Dilated azy­gos and hemiazygos veins presented as paraverte­bral intrathoracic masses. N Engl J Med 1986; 278: 1087-91. 3. Jonsson K, Rain RL. Pseudotumoral esophageal variccs associated with portal hypertension. Radio­logy 1970; 97: 593-97. 4. Leigh TF, Abbott OA, Rogers JV, Gay BB. Ve­nous aneurysm of the mediastinum. Radiology 1954; 63: 696-705. 5. Moult PJA, Waite DW, Dick P. Posterior mediasti­nal venous masses in patients with portal hyperten­sion. Gut 1975; 16: 57-61. 6. Steinberg I. Dilatation of the hemiazygos veins in superior vena caval occlusion simulating mediasti­nal tumor. Amer J Roentgenol Radium Ther Nucl Med 1962; 87: 248-57. 7. Doyle FH, Read AE, Evans KT. The mediastinum in portal hypertension. Clin Radio! 1961; 21: 114­16. 8. Henrion J, Lebrec D, Nahum H, Benhamou J-P. Varices pseudo-tumorales du mediastin en cas d'hy­pertension portale. Gastroenterol Ciin Biol 1979; 3: 453-56. Radio! Oncol [995; 29: 9-11. Transcatheter ocdusion of patent ductus arteriosus in adults Dušan Pavcnik, 1 Pavle Berden, 1 Mirta Koželj2 1 Institute for Radiology, Clinical Medica! Centre, Ljubljana, 2Department of Cardiovascular Disease, Clinical Medica! Centre, Ljubljana, Slovenia Patent ductus arteriosus (PDA) may escape clinical detection and persist into adulthood. Far the purpose of PDA occlusion a new transcatheter technique with double Rashkind umbrella was developed. We present two successful closures of PDA in adult patients with this device. The two years follow-up findings testify to the favourable clinical course, with disappearance of the continous murmur and a normal colour flow echocardiographic examination. Key words: ductus arteriosus, patent therapy; catheterisation Introduction Isolated patent ductus arteriosus (PDA) occurs in 1 in 2000 live, fullterm birth, accounting for approximately 5 % to 10 % of ali types of congenital heart defects. PDA is very common in premature infants, and infants born at high altitude have an increase incidence. There is female preponderance with a ration ranging 1 from 2 : 1 to 3 : l. The clinical significance of the PDA is deter­mined by magnitude of the shunting through the PDA. The direction of the flow will depend upon relative pulmonary and systemic vascular resistance. Normally, the pulmonary vascular resistance drops quickly after birth. Therefore, blood flows from the aorta into pulmonary Correspondence to: Pavcnik Dušan, M.D., Ph.D., Professor of Radiology, Institute for Radiology, Clini­cal Medica! Centre, Zaloška 7, 61000 Ljubljana, Slo­venia. UDC: 616.131.3-007.22-089.819.l arteries. 2 PDA is closed either surgically3 or by percutaneous transcatheter teclmique. First suc­cessful percutanous closure of PDA was rcpor­ted by Postman in 1963.4 Since then numerous tcchniques have been developcd over the years, but only the Rashkind double umbrella closure of PDA has bcen generally accepted (Figure 1). 5.6 Case reports Case 1 A 18-year-old woman prescnted with dyspnea on effort. A clinical diagnosis of PDA had been made at the age of 5, howevcr, her parents refused surgical correction at that tirne. Chest radiograph showed increasccl pulmonary vascu­larity ancl a prominent aortic arch. Carcliac catheterisation showecl conical shapecl PDA 10 mm long ancl 5 mm in cliameter without pul­monary hypertension. After cliagnostic cathete­risation, a 0.035 inch, 300 cm long Amplatz Pavcnik D et al. \Jl/ A Figure l. Rashkind PDA occludcr system in position in the ductus arteriosus. PA = pulmonary artery, A = aorta. guide wire was introduced from the pulmonary aftery through the PDA into the thoracic aorta. The delivery catheter was advanced into the thoracic aorta and PDA was closed by 12 mm Rashkind double umbrela (Figure 2). During the 2-years follow-up, patient has been free of symptoms. Case 2 A 30-years old woman was admitted to the hospital due to accidentally diagnosed PDA. She was symptoms free. On auscultation, a continous machinery murmur in the 2nd in 3rd left interspace was heard. She had no signs of heart failure. The 12-lead ECG showed normal sinus rhythm. The chest x-ray film showed no abnormalities. However, the transthoracic ec­hocardiogram in modified parasternal short axis view of the base of the heart revealed colour flow Doppler signal streaming into the main pulmonary artery. A continuous flow into the pulmonary artery was recorded by continuous were Doppler examination. Both findings are Figure 2A. Lateral angiogram showing typical funnel shaped ductus (arrow). PA = pulmonary artery, A = aorta. Figure 2B. Lateral angiogram showing device in posi­tion (arrow) with hingc point at the pulmonary artery (PA) end, 24 hrs) 3 () o pulmonary edema 2 o o bronchoplcural fistula l l 100 Cardiac causes (29 .4 % ) arrhythmia 7 o o heart herniation 2 () o myocardial infarction l l 100 Othcrs (35.3 % ) hemorrhagc 2 o o chylothorax 2 o o stroke 2 l 50 pulmonary embolus l 1 100 renal failurc 1 I* 100 wound infection 2 o o recurrcnt nervc palsy 2 o o one of thcsc four patients had rcnal failurc. Table 2. Diffcrcnces in opcrativc mortality for various critcria. No. of No. of Critcria o/c) P valuc clcaths patients Agc (ycars) stage (Figure 2). The overall survival rate was 27 % after 3 ycars. Patients with pathological stage I. stage II. stage llla, anc! stage I!Ib diseases showcd 3-year survival ratcs of 60.0 % . 31.3'1/o, 18.2%. and 0%, respectively. The median survival times of paticnts with patholo­gical stagc L stage II, stage IIIa. ancl stage lllb diseases were 3.82 years, 2.66 years. 1.46 years and 0.93 ycars. respcctivcly. Among the surviving 94 paticnts, 13 patients (13.8 % ) died due to pneumonia without evi­dence of disease bcfore dcath. and 40 ( 42.6 % ) cliecl due to distant mctastasis. with bone as the most common metastatic site during the follow-up period. o, O 8 C ·;; 'l: ::, (/) C: o t o a. o Scx Male 8/93 Fcmalc 0/12 Side of rcscction Right 4/36 Lcft 4/69 Staging 1 + II 0/31 Illa 8/63 Pathology Squamous celi ca. 7/84 Adcnocarcinoma 1/16 Prcop. FEVl 2':2.0 L 5/44 <2.0L 3/23 Prcdictecl postop. FEVI 2':l.0 L 8/65 <1.0 L 0/2 Figure 1. Ovcrall actuarial survival od 94 paticnls who 8.6 NS undcrwcnt pncumoncctomy for primary 11011-small celi O.O lung canccr. 11. 1 NS 5.8 O.O NS O> 08 C: 12.7 06 (/) 8.3 NS o t 04 6.3 11.4 NS 0.2 13.0 12 18 24 30 36 42 48 54 60 12.3 NS Survival (months) O.O Figure 2. Actuarial survival or 94 paticnts who undcr­ wcnt pncumoncctomy for primary non-smali celi lung ficancc. canccr by pathological stagc. FEVl: forceu cxpiratory Yoltune in I sccond. NS: no signi­ J-lsu N Y et al. Discussion Most surgeons agree that pneumonectomy, for patients with non-small celi Jung cancer, should be used only for extensive tumors that cannot be removed by lobectomy, or sleeve lobecto­my .1 4• 15 In this study, the patients who under­went pneumonectomy showed a predicted po­stoperative FEVl equal to or greater than o.S L (the preoperative FEVl multiplied by the per­cent of perfusion of the uninvolved Jung). In an experimental clinical study, Adams et al showed the importance of cardiopulrnonary re­serve as a determinant of risk.16 Ventilation perfusion studies, using Xenon-133 gas and Tc-99 rn MAA showed good reliability for calcu­ 1718 lating predicted Jung function.· These pre­dictors are very useful clinically because regio­nal and overall pulmonary functions remain 19 stable after pneurnonectorny.At the tirne of operation, if a (bi-) lobectomy or a sleeve resection was deterrnined to be insufficient for removing the local disease, a pneumonectorny was perforrned. We traced the medica! records of the patients who had under­gone pneumonectomy and found that the proce­dure was performed if the following criteria were present: (1) tumor encroaching to the pulmonary aftery, (2) tumor invasion into the hilar area, (3) tumor invasion into the main bronchus, and (4) tumor invasion into the inter­lobar fissure. Our study showed an operative rnortality rate of 7.6 % , which is in accordance with the results of Ginsberg et al ( 6.2 % ) , 7 Pate! et al (8.6%),11, and Putnam et al (8%).20 Various risk factors of pneurnonectomy have been stu­died. Didolkar et al found that advanced age was associated with an increased mortality rate in pulmonary resection for lung cancer,21 how­ever, this factor was not suggested in the study by Patel et al11 or in our study. This may be due to a patient group with a limited number subjects aged 70 or above; 14.3 % in our study and 16 % in the study of Patel et al. 11 Our report, unlike that of Weiss, 10 did not find that wornen fare better after resection. This may be due to the fact that the number of male patients (93 patients) greatly surpassed the number of fernale patients (12 patients) in our study. The side of resection was not related to rnortality, al22 al23 although Higgins et and Harrnon et suggested that right pneumonectorny is more hazardous in terrns of bronchopleural fistula and ernpyema. In our study, although ali eight operative mortalities occurred in pathological stage IIIa, there was no statistical significance when compared to stages I and II. This may be due to a greater percentage of patients (60 % ) in pathological stage IIIa. We found that no single variable enables the clinician to predict operative moratlity after pneumonecto­rny, and patients should not be excluded from pneumonectomy on the basis of any single criterion. The complications after pneumonectomy were variable, and some were associated with mortality. In our study, although ali patients received prophylactic antibiotics, pneumonia developed in 6 patients. Among them, four patients died due to respiratory failure. Cardiac arrhythmia was the most cornmon complication, occurring in 7 patients (35 % ) , but was not associated with mortality. Wahi et al reported that patients who developed atrial arrhythmia had a longer stay in the intensive care unit and a longer postoperative hospital stay.4 The cause of the abnormal rhythm is unknown. Mediasti­nal shift, hypoxia, abnonnal pH of the blood, as well as other factors, have been implicatecl but none have been proven. Shields suggested prophylactic digitalization for older patients un­clergoing pneumonectomy.24 We found that the most significant factor for determining long term survival of patients un­dergoing pneumonectomy is the pathological stage. Patients categorizecl as pathological stage I or stage II showed a better 3-year survival rate than those with stage IIIa (60 % or 31.3 % vs 18.2 % ). We did not stratify the survival tirne of the various subtypes of pathological stage IIIa by T and N status. Wilkins and co-workers found a significant correlation for survival tirne ancl lymph node status in pneumonectomy­treatecl patients. Patients with uninvolved nodes had a better 5-year survival rate than those with Outcome of pneumonectomy far primary 11011-small celi lung cancer -A ten-year experience 23 involved nodes (42.1 % vs 16.3%).3 Putnam et al found that patients classified as pathological stage I, stage II, stage IIIa showed 3-year survival rates of 45 % , 47 % , and 24 % , respec­tively. In addition, no patient in pathological stage III with a percent predicted FVC of 64 % or less survived more than 1.3 years, compared with a 26 % survival rate after 3 years for other pathological stage III patients (P = 0.009).20 Further multivariate studies may be needed to clarify the significant factors that determine the survival of the pneumonectomy-treated patient. The fate o patients after pneumonectomy has seldom been reported. Ogilvie and coworkers studied patients with lung cancer 10 years after pneumonectomy, and found that patients had a poorer ventilatory capacity and a higher inci­dence of cor pulmonale than those patients with lung cancer 1 year after pneumonectomy.25 In our study, we found that 13.6 % of the patients died due to pneumonia and/or respira­tory failure during a mean follow-up tirne of 39 :months. A management strategy that includes prevention of pulmonary infection, respiratory exercise, prohibition of smoking and regular • expectoration should be considered during fol­low-up. The benefit of adjuvant therapy for the various stages of patients after pneumonectomy remains to be verified. References 1. Paulson DL, Reisch JS. Long term survival aftcr rcscction for bronchogcnic carcinoma. Ann Surg 1976; 184: 324-32. 2. Kirsh MM, Rotman H, Bovc E, ct al. Major pulmonary rescction for bronchogcnic carcinoma in the cldcrly. Ann Thorac Surg 1976; 22: 369-73. 3. Wilkins EW Jr, Scannell JG, Craver JG. Four decades of experience with resection for broncho­genic carcinoma at the Massachusetts General Hospital. .1 Thorac Cardiovasc Surg 1978; 76: 364-8. 4. Wahi R, McMurtrey MJ, DeCaro LF, et al. Determinants of perioperative morbidity and mor­tality after pneumonectomy. Ann Thorac Surg 1989; 48: 33-7. 5. Graham EA, Singer JJ. Successful removal of an entire Jung for carcinoma of the bronchus. JAMA 1993; 101: 1371-4. 6. Churchill ED. Sweet RH, Soutter L, et. al. The surgical management of carcinoma of the Jung . .1 Tlzorac Surg 1950; 20: 349-65. 7. Ginsberg RJ, Hill LD, Eagan RT, et. al. Modern thirty-day operative mortality for surgical resec­tion in lung cancer. .1 Thorac Cardiovasc Surg 1983; 86: 654-8. 8. Kihman LJ, Meyer JA, Ikins PM, et al. Random versus predictable risk of mortality after thoraco­tomy for lung cancer. .1 Thorac Cardiovasc Surg 1986; 91: 551-4. 9. Keagy BA, Schorlemmer GR, Murray GF, et al. Correlation of preopcrative pulmonary function testing with clinical course in patients after pneu­monectomy. Ann Thorac Surg 1983; 36: 253-7. 10. Weiss W. Operative mortality and five-ycar survi­val rates in patients with bronchogenic carcinoma. Chest 1974; 66: 483-7 . 11. Pate! RL, Townsend EE. Fountain SW. Elective pneumonectomy: factors associated with morbid­ity and operative mortality. Ann Thorac Surg 1992; 54: 84-8. 12. Page A, Nakhle G, Mercier C, et al. Surgical ti"eatment of bronchogenic carcinoma: the impor­tance of staging in evaluating late survival. Cancer .1 Surg 1987; 30: 96-9. 13. Mountain CF. A new international staging system for Jung cancer. Clzest 1986; 89 (suppl): 225S-33S. 14. Pcters RM. How big and when? Ann Thorac Surg 1987; 44: 338-9. 15. Johnston MR. Selccting patients with Jung cancer for surgical therapy. Seminar.1· in Oncology 1988; 15(3): 246-54. 16. Adams WE. Perkins JF, Harrison RW, et al. The significance of cardiopulmonary reserve in the late results of pneumonectomy for carcinoma of the lung. Dis Clzest 1957; 32: 280-8. 17. Ali MK, Mountain CF, Ewer MS, et al. Predicting loss of pulmonary function after pulmonary resec­tion for bronchogenic carcinoma. Chest 1980; 77: 337-42. 18. Bria WF, Kanarek DJ, Kazemi M. Prediction of postoperative pulmonary function following tho­racic operations . .1 Thorac Cardiovasc Surg 1983; 86: 186-92. 19. Ali MK, Ewer MS. Atallah MR. et al. Regional and overall pulmonary function changes in Jung cancer. .1 Tlzorac Cardiovasc Surg 1983; 86: 1-8. 20. Putnam .JB, Lammermeier DE, Colon R, et. al. Predicted pulmonary function and survival after pneumonectomy for primary lung carcinoma. Ann Thorac Surg 1990; 49: 909-15. 21. Didolkar MS. Moore RH. Takita H. Evaluation of the risk in pulmonary resection for bronchoge­nic carcinoma. Am .1 Surg 1974; 127: 700-3. Hsu N Y et al. 22. Higgins GA, Beebe GW. Bronchogenic carcino­ma; factors in survival. Arch Surg 1967; 49: 539-49. 24. 23. Harmon H, Fergus S, Cole FH. Pneumonectomy: review of 351 cases. Ann Surg 1976; 183(6): 719­22. 25. Shields TW. Pulmonary resection. In: Shields TW (ed.). General thoracic surgcry (3rd ed.). Lea & Fcbriger, Philadelphia, London, 1989, pp 368-9. Ogilvie C, Harris LH, Meecham J, ct al. Ten years aftcr pncumoncctomy for carcinoma. Brit Med 11963; 27: 1111-5. Radio! Oncol 1995; 29: 25-43. Tumor necrosis factor-a (TNF-a): Biological activities and mechanisms of action Srdjan Novakovic and Barbara Jezeršek Institute of Oncology, Department of Tumor Biology, Ljubljana, Slovenža Tumor necrosis factor-a!cachectin (in further text TNF-a) was originally defined for its ability to cause hemorrhagic necrosis of different types of tumors. ln the meantime, it has become clear that TNF-a is a multifunctional immunoregulatory cytokine with a broacl spectrum of activities upon hematopoetic and nonhematopoetic cel/s. Some of the pleiotropic activities of TNF-a are growth inhibition of some tumor cells; stimulation of human fibroblast, B cel! ancl thymocyte proliferation; activation of phagocytic and endothelial cells; incluction of prostaglandin synthesis as well as regulation of oncogenes, transcription factors and major histocompatibility complex antigen expres­sion. The effect of TNF-a (antiproliferative or stimulative) depends upon the type of target cells, presence of TNF receptors, TNF-a concentration in tissues or upon presence of other mediators capable of affecting the activities of this cytokine. In this paper we are reviewing biological as well as physico-chemical properties of the cytokine, its production and some mechanisms of TNF-o. action. Key words: tumor necrosis factor · TNF history More than 200 years ago some physicians noti­ced tumor reduction in patients with bacterial inflammations; a logical conclusion was that bacteria or their products somehow retard the growth of tumors. 1 This finding encouraged the physicians of the 19th century to treat patients with solid tumors by means of a direct introduction of microorganisms into the tumors. Such a therapy gave different outcomes ranging Correspondence to: Srdjan Novakovic, PhD, Institute of Oncology, Department of Tumor Biology, Zaloška 2, 61105 Ljubljana, Slovenia. Fax: + 386 61 131 41 80. from complete disappearance of tumors or par­tial reduction of tumor burden to complete failure of the therapy.2 The high infectiveness of microorganisms (Streptococcus pyogenes) and consequently a serious risk for bacterial infection in patients was the motive that forced dr. William B. Coley to treat his patients with the toxins from bacterial cultures instead of using microorganisms themselves. For the pre­paration of bacterial toxins he chose Streptococ­cus pyogenes and Bacillus prodigious (now cal­led Serratia marcescens). With the above stated therapy Coley achieved noteworthy results ( dis­appearance or partial reduction of tumors) that are described in his articles frorn 1894, 1896 4 and 1898. 3· · 5 On the account of higher efficacy UDC: C15.277.3.015.4 and safety of Coley's toxins (as they were called Novakovic S and Jezeršek B later) for the treatment of cancer patients this method found acceptance and became commer­cially attractive. Coley's toxins were produced and· used for treatment until the end of 1920 when they gave up their place to radiotherapy. However, the Coley's toxins were not forgot­ 9 ten. In the years 1931,6 1932,7 19358 · and 193610 various researchers executed quite a few experiments on tumors in mice using filtrates of Gram negative bacterial cultures as antitu­mor agents. Ali these experiments share a com­mon fact: tumors with good vascular supply became centrally necrotic at the beginning of therapy and afterwards, necrosis spread out to the external parts of tumors. Bacterial toxins were injected intratumorally -i.e. locally in ali cited experiments. In the year 1943 Shearll reported about experiments with tumor model Sarcoma-37 (Sa-37) where he injected tumor­bearing mice intraperitoneally (i.p.) with poly­saccharide isolated from S. marcescens. Such kind of systemic treatment caused necrosis in the centre of tumors and even more, systemi­cally treated tumors, in which necrosis spread out to periphery, later on completely disappea­red (like in the case when they were treated locally). Ali the above stated experiments were a good starting point for Carswell and co-wor­kers. In the year 1975 they published an article in which they, for the first tirne, used the appellation "tumor necrosis factor" and specu­ lated about its production and its effects. 12 Namely, the very same researchers established that animals, infected with Bacillus Calmette Guerin (BCG) and afterwards treated intrave­nously with endotoxin, produce "with endoto­xin induced serum factor" which causes hemor­rhagical necrosis of tumors. This serum factor responsible for necrosis of tumors was named simply "tumor necrosis factor" (TNF). TNF displayed cytotoxic activity against numerous tumor cells, yet not against normal mouse fibro­blasts (later it turned out to be growth factor for normal fibroblasts). On the basis of these findings Carswell and co-workers described TNF as an agent with selective antiproliferative effect only on tumor cells. In the same article authors also presumed that macrophages are the main producers and bacterial ·endotoxins the main inducers of TNF. .· .Both presumptions later turned out to be cdrrect. It 'is w6ll known today, that T:NF-a (i.e. the agent which Cars­well and co-worke 0 rs·namedTNF) can.Qe produ­ced not only 1:>y.::;inacrophiclges but alsQ by other types of cells,. and that 'the:re are other factors beside endotoxins wh\ch can serve as inducers of TNF-a production. TNF-a is not the only tumor necrosis factor known nowadays, sihce another substance with similar activities, was discovered already in 1968. Ruddle and Waksman13 and also Granger and Wiliams, 14 independently one from anot­her, described a substance, produced by lym­phocytes, with powerful cytotoxic effect on syngenic embrional fibroblasts or on L929 cells. Ruddle and Waksman named this substance "cytotoxic factor", while Granger and Wiliams called it "lymphotoxin". Because of the simila­rity in the amino acid sequence (35% homolo­gy)15 and in their activities (ranging from the cytotoxicity against L929 cells to the ability to produce necrosis of some sorts of sarcomas in vivo), 16 lymphotoxin was classified among tu­mor necrosis factors and called TNF-., while Carswell's TNF was renamed into TNF-a. Today, it is well known that TNF-a can be synthesised not only by macrophages but also by other cells while TNF-. is produced exclusi­vely by T lymphocytes. Yet, the mechanisms of stimulation of TNF-a production in macrop­ hages are completely different from the mecha­nisms of stimulation in other cells. 17 Production of TNF-a TNF-a production is a multistep process which includes the induction of gene transcription and the amplification of TNF-a mRNA, the synthe­sis of prohormone (233 aminoacids-AA), the activation of prohormone with cleavage of the molecule to 157 AA (molecular weight approxi­mately about 17 kDa), and the secretion of TNF-a.18 TNF-a: Biological activities and mechanisms of action Inducers of TNF-a production Substances that trigger TNF-a production can be divided according to their source into extra­cellular ( exogenous) and intracellular ( endoge­nous) inducers. One of well known and most often used extracellular inducers of TNF-a pro­duction is bacterial lipopolysaccharide (LPS), with its active part -lipid A, which is responsi­ble for most of the biological properties of LPS.19· 20 The second (by frequence of its use) extracellular inducer is muramyldipeptide ­MDP (or its structural analogues) that can be employed, either alone or in combination with other agents (LPS, IFN-y), to stimulate the production of TNF-a in macrophages. Besides, a higher degree of transcription of TNF-a gene in vitro was ascertained in the cells exposed to radiation, viruses, bacteria, parasites, as well as to their products. 2 1-26 Some tumor cells27 and plant polysaccharides28 are also known to act as extracellular inducers. On the other hand, interferons (IFN-a and IFN-y), growth factors (GM-CSF), interleukin­2 (IL-2) and TNF-a itself are the most frequen­ 35 tly applied intracellular inclucers.29­ Inhibitors of TNF-a Excessive procluction of TNF-a can be very harmful for the organism. As a consequence ­the production and activities of TNF-a as well as of other cytokines in the organism are strictly controlled. The control is realisecl mostly through the supervision of gene transcription and translation, or through the production of substances that block cytokine's effects (by bin­cling either to the cytokine molecule or to cytokine's specific membrane receptors). Mechanisms of action of the inhibitors, which clirectly influence transcription ancl translation of TNF-a, are stili quite unclear. However, the researchers are alreacly acquaintecl with the fact that individual cytokines, as for example inter­leukins (IL-4, IL-6), transforming growth factor B (TGF-B), prostaglanclins and corticosteroids, can operate as inhibitors in these processes. 36·40 The second group of TNF-a inhibitors, which block the activities of the very cytokine, are proteins that were discoverecl in urine samples of febrile patients. One of these proteins is callecl uromoclulin; i.e. a glycoprotein with molecular weight of 85 kDa. It operates by bincling to TNF-a molecules ancl thus preventing the attac­hment of these molecules to receptors.4 1. 42 The second specific protein, isolatecl from urine samples, has molecular weight of 40-60 kDa; this one also inhibits activities of TNF-a by means of bincling to its molecule and blocking 44 of its attachment to receptors.43· Beside the inhibitors that were isolatecl from urine another group of proteins with similar effects was described by Scuderi ancl co-wor­kers45 in serum. The authors assumed that : these proteins were plasma a-globulins. They •:supposed that the blockacle of activities of se­creted TNF-a was carriecl out through the bin­' cling .of a-globulins to TNF-a molecule which preve\1ted the binding of TNF-a to celi recep­ tors. A more thorough biochemical analysis of TNF receptors demonstrated that proteins (iso­ latecl from urine ancl serum) which inhibit acti­ vities of TNF are nothing but the soluble form of TNF receptors. In this light, uromoclulin was connected with TNF R75 (TNF receptor with molecular weight of 75 kDa), while the other inhibitory protein isolatecl from urine was iden­ tifiecl (accorcling to its molecular weight) as a substance related to TNF R55 (TNF receptor with molecular weight of 55 kDa). Moreover, there is some evidence of exi­ stence of substances capable of blocking the activities of TNF cytotoxic proclucts. Among the most thoroughly examined substances of this character belong manganese superoxide dismutase (MnSOD) ancl metaloproteins (e.g. metalothionein).46· 47 MnSOD prevents the pro­ duction of oxygen free raclicals in mitochondria while metaloproteins bincl these free radicals ancl thus neutralise their effect on celi structu­ res. The best known synthetic TNF inhibitors originate from the group of serine protease inhibitors. Serine proteases play an important Novakovic S and Jezeršek B role in cleavage of prohormone molecules and production of biologically active form of TNF-a. The inhibitors of these proteases inter­ fere with serine protease activity and conse­ quently block the secretion of biologically active form of TNF-a.48 The control of TNF-a production and its activities is a complex process that most likely includes (beside above cited mechanisms of regulation of transcription, translation and se­ cretion, as well as beside the direct effect on the very cytokine) other manners of supervi­ sion. Ali these mechanisms and factors inter­ weave and work together as an entirety. TNF-a producers Quite a few years ago Beutler and co-workers40 established that monocytes and macrophages are the basic producers of TNF-a. Today, it is known that also other cells are capable of TNF-c( production. Among the most important producers we classify NK cells,49 T lymphocy­tes,50 some non-hematogenous cells like for example muscle cells, endothelial cells and mi­croglia,21. 51-53 as well as some tumor cells: cells of colorectal adenocarcinoma, Hodgkin's lym­ 56 phoma, ovarian carcinoma, breast carcinoma.54­ Production of TNF-a in monocytes/macropha­ges As it was already mentioned before LPS, i.e. the endotoxin isolated from celi walls of Gram negative bacteria, is the best known extracellu­lar inducer of transcription of TNF-a gene and of TNF-a synthesis in leukocytes.40 That ex­plains why the LPS's effect on monocyte/ma­crophage population and its stimulation of lym­phocytes to produce TNF-a are most thoroug­hly examined. Despite of that, exact molecular mechanisms of signal transduction and of initia­tion of TNF-a gene transcription remain unclea­red. The researchers presume, that the process of lymphocyte stimulation (to produce TNF-a) is somehow connected to the activity of phos­pholipase A2 and afterwards to the metabolism 31 of arachidonic acid and cAMP activity.22· Namely, Motrri and co-workers3 l ascertained that LPS statistically significantly increases the activity of membrane-bound phospholipase A2. On the other hancl, the inhibitors of phospho­lipase A2 statistically significantly reduce TNF-a mRNA production in monocytes stimu­lated with LPS. Unfortunately, all these fin­clings are not sufficient for exact conclusions about molecular mechanisms involved in regu­lation of transcription, translation and secretion of TNF-a. Not until these mechanism are un­clerstood, the possibility to influence effectively the quantity of cytokine procluced and its co­operation in differcnt activities in organism will be given. Physical and chemical properties of TNF-a TNF-a. is a glycoprotcin whose sequence con­sists of 156-157 amino acicl residues ( 156 AA mouse TNF-a, 157 human and rabbit TNF-a); its molecular weight is 16-18 kDa (16-18 kDa mouse, 17 kDa human, and 18 kDa rabbit TNF-a). These molecular weights are the ones of TNF-a after electrophoresis in sodium-clode­cylsulfate-polyacryl-amicle gel (SDS-PAGE), when the active form clecomposes into mono­mers. Molecular weight of unaltered protein (as for example after gel filtration) ranges from 34 kDa (rabbit) to 45 kDa (human).57 The above statecl facts lead us toward conclusion that the active form of TNF-a is its trimeric form, which bas also been provecl by means of 59 ultracentrifugation and crystallography. 58· Trimeric form of TNF-a is susceptible to temperature changes, since the increase of tem­perature causes decomposition of trimmer form. The isoelectric point (pI) of human TNF­a is somewhere between 5 ancl 6, of mouse TNF-a between 3 and 5 and of rabbit TNF-a 5.57 about Besicles, TNF-a is susceptible to trypsin and chimotrypsin action, as well as to the action of some other proteases (V8),57 for it looses its biological activities when being exposed to these proteases. On the other band, TNF-a retains its biological activities when ex­posed to low pH or organic solvents. 29 TNF-a: Biological aclivilies and mechanisms of aclion TNF-a exists in two different forms, i.e. as a free (soluble) cytokine or bound to cellular membranes ( of monocytes for example ). Mem­brane-bound TNF-a was at first supposecl to be the excretecl cytokine that reversely attachecl itself to its own membrane receptors.60 Hafsli ancl co-workers61 provecl that this statement is incorrect. Namely, they ascertainecl by means of immunofluorescent microscopy that the num­ber of membrane-bound TNF-a molecules re­mains unaltered regarclless of the presence of aclclecl soluble TNF-.. As a matter of fact, if membrane-bouncl TNF-a was the soluble form reversely attachecl to its own receptors, then, in the presence of TNF-., there woulcl be less TNF-G molecules bouncl to membrane, because of the competition for free receptors. Mem­brane-bouncl TNF-G acts cytotoxically as a transmembrane protein; the exact mechanism of action is stili unclear, yct it is known to kili target cells in clirect celi-to-celi contact. Biological properties and actions of TNF-G TNF-G is classifiecl together witb TNF-., inter­ferons, interleukins ancl growth factors into the group of hormone-like substances called cytoki­nes. lts name "tumor necrosis factor" originates from its first known biological property, i.e. from its ability to cause necrosis of tumors. 12 In the year 1984 Pennica and co-workers62 succeeded in cloning of TNF-a cDNA, thus enabling the synthesis of reeombinant TNF-G (rTNF-a). Since physical and chemical proper­ties, as well as biological activities of rTNF-a are identical to the ones of native TNF-a and because of low cost procluction of rTNF-a this acceleratecl experimental work with the cytoki­ne. Today it is known that biological activities of this very cytokine are not confinecl to cyto­static/cytotoxic effect upon tumor celi s only, but that TNF-u co-operates in many other processes (Table 1). TNF-a has (beside the above cited activities) also quite a few sicle-effects (just like the other cytokines do). The most frequent side-effects are fever, anorexia, diarrhea, nausea ancl hypo­ tension. 63.68 The citecl activities rank among the most important ones of this cytokine, yet the list of TNF-a effects is by no means at the end. A complete register of processes, in which TNF-a ta kes part clirectly or inclirectly, is very clifficult to elaborate, since this pluripotent cytokine is secretecl by clifferent cells (blood, liver, spleen, kiclney, muscle cells and cells of central nervous system) ancl participates in a large number of processes in the organism. Also, the pathogene­sis of cerebral malaria ancl the clestruction of tissues in inflammation are connectecl to hyper­ 69· 70 procluction of TNF-u. Table l. Short rcvicw of biological activitics of TNF-a. Antitumor activities CII modulatcs MHC I and II antigcnic cxprcssion (thus it opcratcs as an immunomodulator);71 · 72 CII incluccs transcription of cnzymc inhibitors;73 • incluccs cliffcrcntiation of tumor cclls;74 CII participatcs in cytotoxic activitics of monocytcs and macrophagcs (mcmbranc-bound or solublc TNF­ 76 a);75. • stimulatcs IL-1, IL-6 and IL-8 procluction in ma­crophagcs/monocytcs; 77 · 79 CII stimulatcs proslaglanclinc E2 (PGE2) production in macrophagcs/monocytcs; 77 CII induccs synthcsis of IL-2 rcccptors on T lymphocy­tcs;80 • activatcs NK cclls;81 • incluccs synthcsis and cxprcssion of transforming growth factor (TGF-a) and of rcccptors for cpidcr­mal growth factor (EGFR) on human pancrcatic canccr celi linc.82 Other activities • opcratcs as a mccliator of inflammation ancl ccllular immunc rcsponsc;83 ·85 • participatcs in rcgulation of ccllular ancl physiologi­cal proccsscs: in cliffcrcntiation of cclls, in rcgula­ 88 tion of slccp (i.c. incluccr of slccp);67 · 68• 86 · • influcnccs ccllular mctabolism;89 • opcratcs as a growth factor (stimulatcs fibro­blasts) ;9° ·92 • has antiviral, antibactcrial and antiparasitic cf­fccts;93 • opcratcs as a modulator for ncurons in hypothala­mus (rcgulatcs growth ancl thcir functions);94 • has radioprotcctivc cffcct (most probably by mcans of stimulating thc stromal cclls in bone marrow to proclucc growth factors CSF, that are known to 9596 stimulatc hcmatopoicsis).· Novakovi(; S and Jezeršek B Antitumor activities of TNF-a Carswell discovered TNF-a as a factor capable of causing hemorrhagical necrosis of tumors in mice.12 La ter, Carswell and different resear­chers found out that TNF-a acted cytostatically and cytotoxically on various tumor cells, while having no effect on growth of normal human celi lines. 97· 98 On the basis of these facts they concluded that TNF-a acts directly cytostati­cally/cytotoxically on tumor cells, and that this very cytokine possesses a distinctively selective antiproliferative potential only against tumors cells. Further studies disproved the above con­clusion, since TNF-a acts either antiproliferati­vely on tumor and normal cells or stimulates the growth of both types of cells. The mode of its action depends upon growth conditions in the celi culture and upon the quantity of cyto­ 991 00 As kine added.82· · an example we would like to mention the study of Palombella and Vilcek, 100 where rTNF-a had a cytotoxic effect on 3T3 fibroblasts in non-confluent layers, while promoting growth of the very same fibroblasts in confluent layers. In the same study resear­chers noticed, that the higher the dose of TNF-a the more intensive was the DNA synthesis. 99 The above mentioned in vitro studies82 ·· 100 thus do not support the presumption about selective antiproliferative function of TNF-a ( only against tumor cells), but prove that the mode of its activities in vitro depends upon external conditions, as for example confluency of celi layer, composition of growth medium or presence of other cytokines. Activities of TNF-a in in vivo systems are even more complicated. Carswell and co-wor­kers12 observed a pronounced in vivo antitumor effect of TNF-a (then only TNF) on transplan­ted sarcomas in mice. But they were surprised to see that TNF-a was completely inefficacious as an cytostatic/cytotoxic agent on Meth-A sar­coma celi line in vitro. This fact led towards conclusion that TNF-a in vivo performs its antitumor activities also indirectly and that this indirect action depends upon the treated orga­nism to a great extent. Experiments with tumor models in nude mice confirmed that the antitu­ mor effect of TNF-a virtually depended upon the treated organism (i.e. upon host), actually upon its immune system. Namely, the treatment of experimental tumors in nude mice with TNF-a was a complete failure and even appli­cation of high doses of the cytokine did not result in hemorrhagical necrosis of tumors.101 Indirect antitumor action of TNF-a is brought about mostly by activation of host immune system. The cytokine activates macrophages so that they become tumoricidal (in less than 20 minutes triggers the synthesis of mRNA for TNF and IL-1 in these cells). At the very same tiine TNF-a activates T lymphocytes, NK cells and neutrophils,81· 102-104 exerts an effect upon stromal cells of the host organism and triggets the synthesis of growth factors,95· 96 as well as the production of other cytokines that partici­pate in antitumor activities (IL-1, IL-6 and TGF)77·79 (see Table 1). Besides, TNF-a exerts an effect upon endo­thelial cells where it induces the synthesis of severa! adhesion molecules ( which are located on the cell surface) and modulates the coagula­tion properties of celi surface, thereby increa­sing vascular permeability. The effect upon endothelial cells of tumor blood vessels is po­tentiated if the vessels are newly formed. Most probably these changes result from the direct and indirect actions of TNF-a, which lead to intravascular thrombosis and complete destruc­ 107 tion of tumor blood vessels. 105· Obstruction of blood vessels represents an important mode of TNF-a antitumor activities, especially against non-immunogenic tumors. Poorly immunogenic tumors are quite resistant to the action of TNF-a and destruction of tumor tissue is brought about mostly by means of destroying of blood vessels, which leads to reduced tumor blood supply and afterwards to the phenomenon of central necrosis.101• 108 Immunogenic tumors (e.g. with methylcho­lanthrene induced sarcomas) are more suscepti­ble to TNF-a activities, since such tumors can be completely destroyed with very low doses of this cytokine (injected intratumorally). High susceptibility of immunogenic tumors to TNF-a activities suggests that the immunogenicity of a TNF-a: Biological activities and mechanisms of' action tumor is an important factor in antitumor action of this cytokine and that there are also other mechanisms ( above stated), beside the effect on tumor blood vessels, which participate in affecting of immunogenic tumors. Mechanisms of cytotoxic activity of TNF-a For most of the cytokines it is true that their effect upon target cells is conditioned with the presence of specific receptors on target celi membranes, with binding of the cytokine to these receptors and with transduction of the signal into the interior of the cells. Certain authors assume that TNF-a -receptor complex is transferred into the interior of the cell (by endocytosis), where it disintegrates in lysosomes, excessive material is then excreted, while TNF-a participates in different cellular 109 · 110 processes. Besides, Hasegawa and Bonavida11J noticed that the cytocidal activity of TNF-a depended upon the presence of substances capable of inducing formation of pores in cell membrane, i.e. perforins. This led towards conclusion that TNF-a enters cells directly through membrane pores without binding to receptors (nonspecifi­cally). The mechanisms of action of TNF-a after its direct entrance into the cytoplasm differ from the mechanisms of action after its specific entrance (which is brought about by ,binding of TNF-a to receptors). Direct entrance of TNF-a into the celi generally results in a strong cytotoxic effect in contrast to a broader spectrum of activities of TNF-a after specific binding to celi receptors. Also, Smith and co­workers112 proved that the activities of this cytokine in the cell interior depend upon the mode of cytokine's entrance and that the cell membrane represents some kind of selective barrier of TNF-a activities. When TNF-a was introduced into the cytoplasm of normal ma­crophages directly, the researchers expected these cells to become stimulated (as in the feedback loop of TNF-a effect upon macropha­ges), yet the only outcome was a strong cytoto­xic activitt: On the other hand, the !atest studies report that internalisation of TNF is not obligatory for the activation of intracellular processes. Name­ly, binding of TNF to receptors causes segrega­tion of receptors and trimerization of intracellu­lar parts of receptors, which is sufficient for the 1 18 activation of signaling pathways.113· The cytotoxic action of TNF-a results in "programmed cell death" -apoptosis or/and necrosis of target cells. The "nucleus depen­dent" mechanism of action is supposed to be brought about by means of stimu\ation of gene transcription and of synthesis of different pro­teins. It is well known that TNF-a activates protooncogenes c-fos and c-jun and that the products of these protooncogenes operate as activation factors for promotors of numerous other genes: e.g. for synthesis of various endo­nucleases, MHC I antigenes, TGF-a, and EGFR.119 Apoptosis is a consequence of TNF-a action upon microfilaments (TNF-a causes the disinte­gration of microfilaments) and/or upon the ac­tivation of endonucleases which "cut" ( cleave) the cell chromatin (DNA) to short fragments of approximately 200 base pairs.75· 98 This chro­matin cleavage proceeds nonspecifically at in­ternucleosomal loci. Cells, damaged this way, are no longer capable of repairing the damage and instead of entering the S phase of cell cycle they pass o ver to apoptosis (Figure 1). Necrosis is the result of production of free radicals, which have an effect on mitochondria and on cell structures different form mitochon­dria (i.e. cytoske\eton), as well as a result of inhibition of mitochondrial functions. 101. 120 This theory is relatively old and it is supported by the fact that antioxidants, as for example mito­chondrial enzyme MnSOD, are capable of pro­tecting the cells from TNF-a effects. 121. 122 Ano­ther evidence that speaks for the above mentio­ned theory are experiments of Yamauchi and co-workers.123 Namely, they quantified the cel­lular production of OH-and found out that the amount of this powerful oxidant depends upon the duration of cell exposure to TNF-a. Such kind of dependence was observed only with cells that were susceptible to TNF-a, while Novakovic S and Jezeršek B there were no signs of increased production of OH-in cells resistant to TNF-a action. On the other hand, Wong and co-workers121 and later on Okamoto with co-workers122 pro­ved that the group of proteins induced by TNF-a includes also MnSOD. They observed a statistically significant increase of MnSOD concentrations following the transplantation of TNF-a gene into tumorigenic mouse fibroblasts which are originally susceptible to TNF-a. Ce!ls, changed genetically in such a way, were now able to produce both endogenous TNF-a, as well as MnSOD and thus became resistant to TNF-a. This means that TNF-a operates autoregulatively and that the mechanisms of its action interrelate and integrate with each other. Thus, the basic effect of TNF-a in process of necrosis is the stimulation of production of free oxygen radicals that later on affect cellular structures. Additional proofs speaking for this theory came from experimental results which indicated that the inhibition of celi respiration is a consequence of generation and activities of free radicals (after TNF-a treatment of cells). Further confirmations are results of studies where previous treatment with antioxidants protected cells from cytotoxic activity of TNF-a. 12 0. 121 . 123. 124 TNF-a as a growth ft1ctor With regard to ali biological activities of TNF-a it is difficult to define which one is primary: its antitumor activity, its role in inflammation pro­cesses or its activity as a growth factor. Sugar­man and co-workers97 were among the first to describe TNF-a effects upon different normal and malignant celi lines. They established that TNF-a statistically significantly reduced the celi number of only seven out of twenty-two malig­nant celi lines (after exposure tirne of 72 h) while having a stimulative effect on growth of some normal celi lines ( different human fibro­blast celi lines). On the other hand, Piacibello and co-wor­kers125 ascertained that TNF-a not only has a stimulative effect on growth of normal celi lines, but also promotes growth of some malig­nant cell lines. Namely, they found out that low doses of TNF-a in the presence of GM-CSF (granulocyte-macrophage colony stimulating factor) stimulate the growth of human normal, as well as leukemic stem cells whereas higher doses of TNF-a in the presence of G-CSF (granulocyte colony stimulating factor) inhibit the growth of only normal stem cells. These observations were confirmed by other authors who demonstrated that low doses of TNF-a stimulate growth of, while high doses have an antiproliferative effect on, certain tumor celi lines. 101 In normal human fibroblast cell lines TNF-a operates as growth factor and in most of the cases there is a dose dependence: the higher concentrations the better the cell growth.97 It is important to stress again that beside the dose there are different external factors like confluence of celi cultures, compo­sition of growth mediums, phases of celi cycle and presence of growth factors or cytokines, which influence the mode of TNF-a action. JOO, 126 The TNF-a activity as a growth factor is, just like its antitumor activity, direct and indirect. The direct action comprises activation of genes responsible for synthesis of proteins that direct the celi from GO to G 1 phase of celi cycle thus increasing the number of celi divisions and accelerating the proliferation of cells. 127 The indirect action includes stimulation of cells to produce other growth factors and specific recep­tors for growth factors. Direct and indirect mode of TNF-a action intertwine and it is impossible to fix their limits. TNF-a as an immunomodulatory agent TNF-a in its immunomodulatory role has an effect upon T and B lymphocytes, affects the . expression of MHC class I and II antigens, and stimulates macrophages as well as other cells to produce cytokines.7 1· 128 Effect on T and B lymphocytes TNF-a affects T and B lymphocytes predomi­nantly as a mitogenic factor. Vine and co-wor­ TNF-a: Biological activities and mechanisms of action kers127 established that this cytokine accelerates transition of T lymphocytes from GO to G 1 phase of celi cycle and thus stimulates multipli­fication of these cells while not stimulating the production of IL-2 (i.e. one of the elementary products of T lymphocytes) at the same tirne. The effect upon immune system executed by stimulation of B lymphocytes is even more indirect, since TNF-a is capable of promoting the proliferation of these cells, yet only in the presence of IL-2. Because it is well known that TNF-a affects predominantly the division of T and B lymphocytes and co-operates only indi­rectly in the induction of cytokine synthesis in lymphocytes, there still remains a question about its immunomodulatory role in activation of T and B lymphocytes. It is quite interesting that all lymphocytes after treatment with TNF-a demonstrate a statistically significant increase of receptors for TNF-a. Effect on NK cells The effect of TNF-a on NK cells represents a special pattern of autocrine stimulation. Ban­croft and co-workers129 treated immunodefi­cient mice (suppressed B and T lymphocytes) with dead bacteria of Listeria monocytogenes species, isolated spleen cells of these animals and measured their production of IFN-y. They ascertained that IFN-y production depended upon the dose of injected bacteria or upon the number of activated macrophages, respectively. Since the macrophages do not produce IFN-y (or at least this has not been demonstrated yet), the researchers concluded that NK cells are being stimulated with macrophages or their products, respectively. Because IFN-y is known to be a powerful stimulator of TNF-a produc­tion in macrophages Bancroft and co-workers assumed that it is TNF-a that stimulates NK cells to produce IFN-y: l. bacteria activate macrophages and trigger synthesis of TNF-a in these cells; 2. TNF-a acts upon NK cells and IFN-y production; 3. IFN-y in a feedback loop triggers additional synthesis of TNF-a and the cycle repeats. Effect on macrophages The effect of TNF-a on macrophages represents a classical example of autocrine activity. TNF-a, which is itself a product of macrophages, binds in a feedback loop to specific celi receptors and stimulates these cells. 1 30 It is well known that activated macrophages act cytostatically/cytoto­xically on tumor cells that are susceptible to TNF-a. This fact leads towards conclusion that the antitumor activity of macrophages is brought about by producing TNF-a which then affects tumor cells. However, this is not the only form of cytotoxic activity of activated macrophages. These cells can act cytotoxically also by means of other products as for example IL-1, hydrogen peroxide (H202) and nitric oxide (NO). The combined treatment with TNF-a and NO has a synergistic cytotoxic effect 1 32 on tumor cells. 131• Especially effective pro­duction of NO and TNF-a in macrophages is achieved after stimulation with IFN-y and IL-2, whereas the stimulation of macrophages with IFN-y and MDP or its structural analogues primarily increases the TNF-a production. 132• 133 Receptors for TNF-a In the year 1990 different researchers cloned the cDNA for two types of cel! surface receptors for TNF (TNF-a and TNF-.).70• 134 Both types of receptors are present on the cell surface of most of the cell lines, yet in a different mutual percent relation. 135• 136 These two types of recep­tors, named TNF R-1 (55 kDa) and TNF R-2 (75 kDa), consist of an extracellular and an intracellular part. The extracellular part of TNF R-1 comprises 182 amino acids and the extracel­lular part of R-2 235 amino acids. The intracel­lular parts of receptors are larger -the one of R-1 includes 221 amino acids and the one of R-2 439 amino acids. Since the cloning of two distinct receptors for TNF (each of which binds TNF-a and TNF-.), the past few years have witnessed the rapid emergence of two superfamilies, of which TNFs and their receptors are only representatives. To Novakovic S and Jezeršek B date 12 receptors have been identified with which we can associate eight TNF-related pro­teins. 137 According to the amino acid sequence the two types of receptors differ from each other to a great extent. Human TNF receptors de­monstrate an amino acid homology of only 27% and most of the homologous amino acids (70%) are placed in the extracellular parts of receptors. Thus the structure of intracellular parts differs considerably between the two types of receptors, which indicates that TNF is con­nected to different functions or different proces­ses, respectively (according to the type of recep­tor to which TNF binds). It is interesting that there is a higher resemblancc in the structure of human TNF R-1 and mouse TNF R-1 (homo­logy of 64% ), which is also trne for human TNF R-2 and mouse TNF R-2 (homology of 62% ). Thc resemblance bctween human and rnouse rcceptors appears mostly in the intracellular parts of receptors (homology of 73% between 138 human and mouse TNF R-2). 136· On the basis of cited facts we can conclude that the activity of TNF is relatively species unspecific and that the intracellular proccsses in which TNF co-operates ( after binding to receptors) are quite similar in different animal species. Human TNF R-1 and TNF R-2 thus demons­trate only a slight resemblance in the amino acid sequence of their extracellular parts and differ almost completely in the structure of their intracellular parts. Besides, the intracellu­lar parts ( according to their amino acid sequen­ce) are not even similar to any of the known protcins. 138 The recapitulation of the above statements is that TNF binds to the two types of receptors, induces different processes in the celi (in dependence upon the type of receptor to which it binds) and that the details of TNF activities inside the celi remain to be explained. Thoma and co-workers139 blocked TNF R-1 with antagonistic monoclonal antibodies and in this way prevented tbe cytostatic/cytotoxic ef­fect of TNF-a on different celi lines. From tbese results thcy rnade an inference that TNF acts cytotoxically trought binding to TNF R-1 receptors. Brouckaert and co-workers140 demonstrated a good antitumor effect of recombinant mouse TNF-a (rMuTNF-a) on B16 BL6 melanoma in C57Bl/6 micc, whereas tbe therapy with recom­binant human TNF-a (rHuTNF-a) turned out to be unsuccessful. They also ascertained that 50% more experimental animals died owing to toxic side effects of rMuTNF-a when compared to the number of Iethal outcomes after rHuTNF-a therapy. On the other band, when the mice were pretreated with galactosamine, also rHuTNF-a demonstrated some degree of antitumor activity, but also the toxic side effects were more pronounced. These results Iead to­wards conclusion that the species specific acti­vity of TNF-a (if it is present) can be neutralised and that such activity does not depend only upon the binding to receptors but also upon other factors. In January 1993 Nature published an article about TNF-a activities after its binding to diffe­rent receptors. 14 1 Authors of this article cited some results which are identical to the ones mentioned before. Namely, they confirmed that the cytotoxic effect on tumor cells results from binding of TNF-a to TNF R-1 and described also an interesting example of species specific activity of human and mouse TNF-a; human TNF-a demonstrated an antitumor effect in experimental mice but caused no toxic side effects. The presence of antitumor effect and the absence of toxic side effects of human TNF-a in mice were explained with its ability to bind only to TNF R-1 but not to TNF R-2. Mouse TNF-a, on the other band, bound to both types of receptors on human celi lines and did not act species specifically. 141 Contrary to the above stated results, from whicb could be concluded that the antitumor activity of TNF-a is a consequence of its binding to and operating by means of TNF R-1, other authors established that mouse fibroblasts (TA l cells) are insensitive to human TNF-a but very sensitive to mouse TNF-a. Since human TNF-a binds only to TNF R-1 on mouse cells, while mouse TNF-a binds to both types of receptors, there is a Iogical inference that the cytotoxic effect upon these cells was mediated TNF-u: Biological actil'ili!'s and m!'Clwnisms of action by TNF R-2 receptors. The seconcl confirma­tion, that the cytotoxic effect of TNF-a can also be mediatecl by TNF R-2, is the fact that HeLa cells, which are otherwisc completely insensitive to the antiproliferative activity of human TNF-cx and have only TNF R-1, become very sensitive after insertion of a gene coding for TNF R-2 into their DNA.119 The existence of two types of receptors for TNF-cx is supposecl to be impor­tant also for its activity as a growth factor. In the year 1991 Tartaglia ancl co-workers142 ascer­tained that mouse TNF-cx accelerated celi clivi­sion of mouse thymus celi line ancl of mouse T Iymphocyte cell line (CT-6) while human TNF-cx hacl no effect on the proliferation of cells em­ployecl in the experiment. Since human TNF-a bincls only to TNF R-1 on mouse cells, the authors substituted TNF-cx with agonistic polyc­lonal antiboclies against TNF R-2 ancl TNF R-l. Agonistic antiboclies against TNF R-2 stimula­teci celi c!ivision of both celi lines whereas agonistic antiboclies against TNF R-1 were inef­ficacious. A Iogical conclusion is that TNF R-2 most probably mecliate the stimulation of celi clivision. The researchers of Hoffman-La Roche135 in­cubatecl human monocytes from peripheral bloocl with TNF-cx ancl various inhibitors of TNF-a bincling}o receptors: i.e. with antiboclies against TNF-cx, or with recombinant receptor proteins,_or _wit_h specific neutralising antibodies against TNF R-1 and TNF R-2. In contrast to the above citecl clata they established that the stimulation of celi division by TNF-et is meclia­tecl through both types of receptors, yet the mechanisms of stimulation cliffer ancl clepencl upon the type of receptor. The antiviral effect of TNF-cx is supposecl to be mecliatecl by TNF R-1. Namely, Wong, Tartaglia and co-workers143 stimulatecl the anti­viral activity by means of using agonistic antibo­dies against TNF R-1. The acldition of antibo­dies against TNF R-2 had no antiviral effect. Kalthoff and co-workers144 demonstrated that TNF-cx actions, mediated through binding to clifferent receptors, are not completely explai­ ned yet. They ascertainecl that binding of TNF-et or agonistic antibodies to TNF R-1 of human malignant pancreatic celi lines (HPAF, Capan 2) causes a rapid transcription of TNF R-2 gene and that TNF R-2 operates as a specific receptor through which TNF-cx media­tes transcription of TGF-cx gene. In the very same study thc authors state that binding of agonistic antibodies to TNF R-1 triggered trans­cription of EGF receptor gene. On the basis of cited data it is quite difficult to draw universal conclusions about activities of TNF-cx mediated either by one or by the other type of receptors. Common to ali known reports are the facts that the species specific activity (if such activity exists) is expressed by means of both types of receptors and that the effect of TNF-cx can be substituted by binding of specific agonistic antibodies to receptors. We can also conclude that TNF R-1 and TNF R-2 (after binding of TNF-et to either of the recep­tors) mediate or stimulate, respectively, diffe­rent processes in cells. However, quite often the transduction of signals for intracellular pro­cesses is realised through simultaneous binding of TNF-cx to both types of receptors. Biochemical mechanisms of TNF-cx actions Biochemical processes that follow the entrance of TNF-cx into the celi or the segregation of reccptors are stili not known: neither the pro­teins which bind complementarily to the c01n­plex TNF-cx -TNF-R after internalization, nor the exact procedure of its further action on cellular organelles or celi processes, respective­ly. 138 Variety of its activities inside the celi (Table 1) indicates that TNF-a is invovlved in different chemical processes which represent adclitional complications at creating a general sheme of biochemical mechanisms of TNF-cx action. The cytotoxic effect of TNF-cx is one of its most thoroughly examined activities. Since such an effect reflects either as apoptosis or as necrosis of cells there are at least two different biochernical mechanisms of cytotoxic activity. The first one is orientated directly to celi nu­cleus, whereas the other mechanism affects Novakovic S and Jezeršek B o o cellular organelles or cytoskeleton, respectively. ™ R5. R1)' However, regardless of the fact that there are () ' different mechanisms by which TNF-a acts upon the cells, it is known that during its R-.F intracellular action this cytokine activates pro­ . AlP I / / tooncogenes c-fos and c-jun (i.e. immediate early genes) and stimulates the synthesis of [i'KAl .(MoSJD)) '"' various proteins which direct the cell towards ",._ either apoptosis or necrosis. Most probably also the growth factor-like ENDONUC.BL . 0-Ml»CALS DA7AGE ,.:.,. activity of TNF-a is realised through activation of protooncogenes and stimulation of synthesis of proteins responsible for transition of cells into S phase of celi cycle. The activation of protooncogenes c-fos and c-jun is mediated through nuclear factor kappa B (NF xB), which is itself supposed to be activated by protein kinase C. Biochemical mechanisms of TNF-a action can also be triggered through activation of phospholipase A2 (Figure 1). Namely, the cells following the addition of TNF-a produce ara­chidonic acid and prostaglandins, 145-147 which indicates that activation of phospholipase A2 is also involved in signal transduction. Besides, Palombella and Vilce,k100 suceeded in blocking both the cytotoxic and the mitogenic activity of TNF-a by means of inhibiting phospholipase A2 with dexamethasone. Undoubtedly TNF-a is a cytokine with a very broad spectrum of effects, owing to which we could hardly expect a simple explanation for its biochemical mechanisms of intracellular activi­ties. Figure 1 represents the authors' globa! idea of TNF-a activities based on more or less known data from the literature. TNF-a and other cytokines The role of TNF-a in the defence of organism against foreign or own antigens and the mode of TNF-a action quite often depend upon the presence and activity of other cytokines. When interacting in such a way the cytokines either stimulate or inhibit mutually their activities. The stimulative activity includes stimulation of cytokine as well as of cytokine receptor synthe- Figure l. Biochcmical mecha.nisms of TNF-a (TNF) activities at the cellular leve!. Binding of TNF to receptors [R55 (Rl), R75 (R2)]; putative passage of TNF through the celi membrane (CM) into the interior of the celi ( endocytosis); degradation of TNF-receptor -TNF complex (R-TNF) with lysosomal enzymes (L); generation of adenosine-triphosphate (ATP); activa­tion of G proteins (Gp); activation of phospholipase A2 (PLA2); production of second messengers (SM) as for example cyclic adenosine-monophosphate (cAMP), arachidonic acid, diacylglycerol and inositol­phosphate; activation of protein kinases (PK); arachi­donic acid is either excreted from the celi or represents a substrate for PLA2 -products of which are prosta­glandins (PG); activation of protein kinases A and C, which results with generation of "DNA binding pro­teins" (DNA-Bp) like for example "nuclear factor xB" (NF-xB); NF-xB operates in the nucleus (N) and activates "immediate early genes" (IE genes) as c-myc, c-fos and c-jun, which are further responsible for activation of early gcnes (E genes); the next step is transcription of genes that are normally being transcri­becl in Gl phase of cen cycle; proclucts of these genes cletermine whether the cen continues to S phase or is clirectecl into apoptosis (which is supposecl to be a consequence of enclonucleases' activities); in the syn­thesis of enclonucleases ancl "DNA bincling proteins" as well as in most of energy-clepenclent celi processes participate Ca2+ ions; TNF affects celi organelles as mitochonclria (M) where it stimulates synthesis of oxygen free radicals ancl other toxic products; TNF also triggers production of protective proteins (which protect the celi from its activities) e.g. manganese superoxide clismi1tase (MnSOD). sis. On the other hand, the inhibitory action ( of cytokines upon other cytokines) is realised through inhibition of transcription, translation and/or secretion of cytokines or their specific receptors. Besides, the cytokines can compete for the very same receptors on the cellular TNF-a: Biological activities and mechanisms of action membrane ( e.g. TNF-a and TNF-.) and in respect to the efficiency of their binding to receptors direct cellular processes. Cytokines thus represent some sort of polypeptide hormo­nes at the cellular leve! which through interreac­tions transduce different signals to the cells. The best known functional dependence of TNF-a upon other cytokines is its cytostatic/cy­totoxic activity against tumor cells in combina­tion with interleukins and interferons. Namely, in the year 1985 Sugarman and co-workers97 ascertained that the antiproliferative effect of TNF-a on tumor cells in vitro can be potentia­ted with IFN-y. Similar results were obtained by Serša and co-workers148 · 149 on human adeno­carcinoma and human malignant melanoma celi lines where IFN-a enhanced the antitumor ef­fect of TNF-a. Enhanced in vitro antitumor effect was also observed by other authors after treatment of tumor cells with TNF-a and IL-1. 150 In vivo experiments demonstrated that com­bined treatment with TNF-a and IFN-y not only has a direct antitumor effect but also the indirect one which is carried out through affec­ting of the immune system (synthesis and acti­vities of other cytokines, potentiation of cytoki­ne's own antiproliferative activity). Young and Wright151 established that low doses of IFN-y and TNF-a reduce the number of suppressor T lymphocytes and decrease the concentration of growth factors. Thus these two cytokines are capable of affecting the growth of primary or residual tumors and the development of meta­stases. The antiproliferative effect of TNF-a and IFN-a on leukemic cells in patients with chronic myelogenous leukemia (CML) repre­sents a special pattern of co-operation between the two cytokines. Namely, Moritz and co-wor­kers 152 ascertained that IFN-a treatment of leukemic patients gives very promising results but only until the development of resistance to IFN-a. However, when the patiens were pre­treated with TNF-a, the resistance to IFN-a did not appear and further successful therapy with IFN-a was enabled. A complete stop of tumor growth in mice (5 different types of subcutaneous. tumors) was described by Winkelhake and co.workers153 fol­ lowing combined treatment with TNF-o. and IL-2. It is quite interesting that in the very same experiment monotherapy with a single cytokine was inefficacious and that the efficacy of com­ bined treatment depended predominantly upon the concentration of TNF-a. Maximal effect of the combination was achieved when maximal sublethal doses of TNF-a were used, whereas the concentration of IL-2 even 90% lower than maximal sublethal dose clicl not affect the effi­ cacy of treatment. Beside the fact that combi­ necl therapy inhibitecl the growth of subcuta­ neous tumors, the very same therapy preventecl completely the clevelopment of Jung metastases if only it was startecl early enough. The fact that efficacy of combinecl therapy with TNF-a ancl IL-2 depencls also upon other factors, like for example immunogenicity of tumors, general condition of immune system and scheduling of cytokine application was de­ monstratecl by Agah ancl co-workers.154 Aclcli­ tive effect of combinecl therapy (TNF-a ancl IL-2) on the clevelopment of Jung metastases (inclucecl by methylcholanthrene) was observecl when the researchers treatecl animals first with · IL-2 ancl later with TNF-a. Reverse orcler of cytokine application was less effective. On the -other hancl, the antitumor activity of combinecl therapy with TNF-a ancl IL-2 was extremely low when the very same experiment was repea­tecl using mice with suppressecl immune system. Besicle the antiproliferative effect also the growth factor-like activity of cliffercnt combina­ tions with TNF-a ancl other cytokines represen­ tecl an interesting challcnge for the researchers. Some of thcm establishecl that TNF-a together with growth factors in vitro effectivclly affects bone marrow cells where on one hancl it stimu­ lates the growth of stem cells by means of inclucing synthesis of growth factors and, on the other hand inhibits the growth of some leuke­ mic celi lincs. 155· 156 TNF-a acts synergistically togcther with TGF-. on differentiation of hu­ man leukemic cells (i.c. stimulates the cliffercn­ tiation process) which results in a recluction of their malignant potential. 157 When speaking about combined activity of TNF-a with other cytokines we must also em­ Novakovi<' S ml{/ Jezeriiek 13 phasise its pluripotent role in affecting the cells. Mechanisms of common action of TNF-a with other cytokines are complicated and quite often unclear. In general, combination of TNF-a with interleukins or interferons synergistically inhi­bits tumor growth; with TGF-. it acts synergi­stically on the process of celi differentiation and has in Iower doses, combined with growth fac­tors a synergistic effect on celi proliferation. Future perspectives Cloning of human TNF-a gene was accepted by many researchers as a great step towards the discovery of universal medication for diffe­rent malignant diseases. Such expectations were a logical consequence of numerous reports con­firming the fact that TNF-a demonstrates a distinctive antitumor activity in vitro and in some cases an even more pronounced antitumor effect in tumor models in vivo. Unfortunately, the results of experiments with tumor models never gained an approval in clinical praxis. The problems which accompanied TNF-a applica­tions in clinical conditions were not a result of its insufficient antitumor activity but derived from severe dose-Iimiting toxicity ( elevated body temperature, anorexia, diarrhoea, nausea and hypotension). Nevertheless, ten years of experiments with TNF-a in clinics are far from a complete failure. The researchers are seeking after such a mode of TNF-a application that would retain its antitumor activity while minimi­zing the toxic side effects. One of the possibili­ties is a synthesis of new analogues of TNF-a, which according to incomplete knowledge of the role of certain molecular domains represents quite a difficult task. Namely, the cytokine molecule should be changed in the domain responsible for dose-limiting toxicity, while pre­serving its structure responsible for cytostatic/ cytotoxic effect on tumor cells. Besides, the efforts are being made to create analogues capable of a longer retention at the tumor site, which would limit the effects of cytokine to tumor cells only. In this prospect, there is an idea of synthesising TNF-a chimeric proteins with specific affinity for certain antigens on the surface of tumor cells. The second possibility of local treatment with TNF-a is isolation per­fusion with high doses of TNF-a. Problems arising from such kind of therapy derive from incompetence to control and retain completely the cytokine within the treated organ. Quite often TNF-a "escapes" from the artificial circu­lation Ioop which results in serious adverse effects. The third but most prospective point of view of clinical uses of TNF-a and its analogues is a gene therapy. Gene therapy represents a kind of systemic treatment where genetically engineerecl cells (i.e. with TNF-a gene transfec­ted cells) produce a controllable amount of enclogenous cytokine. 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Synergy between transforming growth factor-. and tumor ncerosis factor-a in thc induction of monocytic differentia­tion of human Icukemic celi Iines. Blood 1990; 75: 626-32. Radio! Oncol 1995; 29: 44-6. Determination of the breast volume after breast conservating surgery Liliane Demange1 and Jean Tisnes2 1 Department of Radiotherapy and Oncology, Institut Jean-Godinot, Reims Cedex 2 Societe de Fabrication d'Instruments de Mesure, Massy Cedex, France A mathematical description has been developed to estimate the difference in vohune between the breasts after breast conservating surgery. This method can help the physician to evaluate the cosmetic results after treatment. It is a simple method for the radiotherapist who routinely uses a simulator. Key words: mastectomy; mammography-methods; breast conservating therapy, cosmetic evaluation, breast volume Introduction The patient with a diagnosis of early breast cancer can now be successfully treated by con­servative techniques. Studies results have de­monstrated that for small tumors (Tl and small T2) lumpectomy followed by radiotherapy is a valid alternative to mastectomy. 1 Today, the physician tries not only to cure the patient but to achieve the best possible cosmetic results.2 In the breast the cosmetic outcome is generally evaluated by the difference in size between the two breasts, teleangiectasia and fibrosis. Measurement of the size of the breast is somewhat subjective. We describe in this paper a more objective method of measuring breast size. Correspondence to: Liliane Demange MD, Institut Jean Godinot, 1, rue du General Koenig, 51056 Reims Cedex, France. UDC: 618.19-006.6-089.87:618.19-073.75 Method Theory Breast is a region bounded by the chest wall on one side and by the skin on the other. This could be schematized by regarding the breast as a segment of a sphere, with the pectoral muscle being a second segment of a sphere within the first. The volume of interest is repre­sented by the difference between the two sphe­rical domes. The problem can now be resolved mathema­tically. The common base of the two segments is a circle of radius R. The outer segment is bounded by the skin (height H); the inner segment is defined by the fascia of the pectoral muscle (height h) from the chest wall. The volume of the breast (v) can be assimilated to the difference in volume of these two spherical domes mathematically expressed as: V = . (H3 -h.) + i R2 (H -h) Determination of the breast volume after breast conservating surgery However, it appears more suitable to the geometry encountered in most of the patients to consider the base of the segments as elliptical (Figure la, lb ). The maximum and minimum radii need to be defined. The maximum radius is in the transverse axis (R) and the minimum in the cranio caudal axis (r). We can now use the ratio (r/R) to modify the above equation Figure la. Representation of thc base of the breast as an ellipse of which the grcat radius R is half the width of the breast and small radius r is half its hcight. V = ...!:. . (H3 -h3) + . R2 (H h) R6 2 V = volume of the breast. R = half the maximum transverse "width" of the breast. r = half the maximum cranio caudal "length" of the breast. H = elevation of the breast. !\ h = elevation of the chest wall. We have used this formula to calculate the volume of the breast in this study. Measurement oj the geometrical dimensions oj the breast With the patient in the supine position, using a conformator (or a CT-scan if available), we drew the contours of the thorax including the breast at two different Ievels. The first contour CMed is taken at the leve! of the two nipples, the second at the leve! of the inframammary fold. The cranio caudal dia­meter (2 r) of the breast is measured directly on the patient as shown on the picture. The most medial limit (A) and the most laterni limit (B) of the breast tissue are located clinically on the patient and are indicated by the conforma­tor on the median contour (CMcd)-The "width" (2R) of the breast is equal to the distance AB. The height H of the external segment of a sphere (to which the breast can be assimilated) is the maximal distance (h) between the median Figure lb. Representation of thc volume of the breast as being the diffcrence between two domed segments of heights H and h and of which the base is an ellipscs of radiuses R and r. and the AB line. can be easily measured on the drawing. The height of the inner segment of a sphere All the measurements required for the calcu­( to which the chest wall is approximated) can lation of the volume of the breast are then only be estimated, as the conformator cannot available. Demange L and Tisnes .1 Application We have measured the vohune of the breast of 22 women who had been treated by lumpec­tomy for breast cancer prior to radiation. Table l compares the volumes of the treated breast and of the other breast: 1) the volume of the other breast ranges from 169 cm3 to 846 cm3 , with an average of 435 cm3 . 2) the volume of the treated breast ranges from 163 cm3 to 799 cm3 , with an average of 405 cm3 . 3) the difference between the volume of the Table 1. Comparison of the volumes of the treated breast and of the other breast. of Difference in volume N ° Volume (cm3) cases between the two breasts Treated brcast Other breast 3 cm % 166 169 3 1,8 Acknowledgment 2 [63 190 27 14,2 3 226 244 18 7,4 The authors wish to thank Janet Armstrong for 4 5 230 200 251 275 21 75 8,4 27,3 her help in the translation, Michel Demange 6 256 280 24 8,6 for the graphical work and Jeannine Prieur for 7 272 288 16 5,5 typing the manuscript. 8 254 304 50 16,4 9 298 319 21 6,6 10 309 348 39 11,2 11 388 397 9 2,3 12 13 380 432 411 448 31 16 7,5 3,6 References 14 450 463 13 2,8 15 490 540 50 9,3 1. Veronesi V, Saccozi R, Banfia Del V. Comparing l6 546 572 26 4,5 radical mastectomy with quadrantectomy, axillary dissection and radiotherapy in patients with small 17 562 578 16 2,7 cancers of the brcast. N Engl J Med 1981; 305; 18 498 608 110 18 6-11. 19 604 628 24 3,8 20 647 660 [3 2 2. Limbergen E, Schueren E, Tongelen K. Cosmetic evaluation of breast conservating treatment for 21 742 763 21 2,75 mammary cancer. 1. Proposal of qualitative scoring 22 799 846 47 5,5 system. Radiother Oncol 1989; 16; 159-244. treated breast and the volume of the other breast ranges from 2 % to 27 % , due mainly to variation of the difference H-h between treated breast and other breast. The same method could be used to measure the difference in vohune between the breast as it appears just after radiotherapy and as it becomes after months or years. The study of a greater number of cases should enable us to establish a correlation bet­ween the calculated difference of volume and the qualitative assessment (minor, marked or major difference), and to derive an objective assessment of one of the factors of the aesthetic result of a conservative treatment of breast cancer. Radio/ Onco/ 1995; 29: 47-9. Terilateral retinoblastoma Irene Bolonaki, Evangelia Lydaki, Eftichia Stiakaki, l'hemistocles Kalmantis, Maria Kalmanti Pediatric Hematology-Oncology, University of Crete Medica/ Schoo/, University Hospital of !rak.lio, lraklio, Crete, Greece A case of trilateral retinoblastoma in a 5 year old girl is described. The patient was diagnosed at the age of 7 months as having a trilateral retinoblastoma and treated with enucleation of the right eye and radiation therapy. Fifty two months later she started vomiting, showed gait disturbances and ataxia. A computed tomography (CT) scan revealed a pineal mass and chemotherapy with radiation therapy were initiated. Despite a temporary improvement she died from progressive CNS disease 4 months later. Key words: eye neoplasms-surgery; retinoblastoma; pinealoma Introduction Trilateral retinoblastoma is a syndrome that includes an intracranial tumor and bilateral retinoblastoma. 1 The location of the tumor is usually in the pineal gland. First trilateral reti­noblastoma was described in 1977. The pineal neoplasm displays histologic features similar to retinoblastoma and propably represents a mary tumor rather than a metastatic focus.2 With the recognition and location of retinobla­stoma in the pineal gland new diagnostic and therapeutic procedures have been suggested but the poor prognosis has not been improved. Corrcspondcncc to: Prof. M. Kalmanti MD, Dcpart­mcnt of Pcdiatric Hcmatology-Oncology, Univcrsity General Hospital of Hcraklion, Crctc, 1352 Grcccc. UDC: 617 .735-006.484:616.83 I .45 Case report The patient was seen at the age of 7 months for evaluation of an abnormal light reflex in both eyes. The family history was insignificant for bilateral retinoblastoma. A computed tomo­graphy (CT) scan showed enhancing lesions in both eyes with no evidence of orbita! or intra­cranial extension. The patient underwent enuc­leation of the right eye followed by radiation to the left eye. Histology of the enucleated eye did not reveal evidence of extraocular extension and optic nerve invasion was not present. The left eye was treated with 4100 cGy in 20 frac­tions with 6 m V photons. The patient remained stable until 5 years of age, when she was seen for vomiting, ataxia and gait disturbances. CT scan (Figure 1) of the head revealed 4 cm enhancing mass in the pineal region with severe hydrocephalus, find­ing confinned by nuclear magnetic resonance (Figure 2) too. There was no evidence of tumor Bolonaki I et al. Figure l. Computed Tomography (CT) scan. Mass in the pineal region with severe hydrocephalus. in either orbit. A trilateral retinoblastoma was diagnosed and a ventriculoperitoneal (VP) shunt was placed. Patient received craniospinal irradiation (3000 cGy) in 15 fractions and irra­diation to the pineal with 4000cGy. Following radiation therapy she was initiated on chemo­therapy consisting of: Prednizone 300 mgr/m2 , Vincristine 1,5 mgr/m2 , Cis-4, diaminodichloro­platine (Cis-Platin) 60mgr/m2 , Cis-chloreathyl­nitrosurea (CCNU) 75 mgr/m2 . Repeated com­puted tomography (CT) scan, one month later, showed decreased of the pineal mass up to 60 % and no signs of hydrocephalus. Three weeks later the patient developed ata­xia, difficulty in walking and progressive qua­dreplexia. Four days later she died from respi­ratory failure. Discussion Trilateral retinoblastoma denotes a solitary midline intracranial tumor in association with bilateral retinoblastoma.1• 2 The location of the tumor is usually in the pineal gland (the third eye) and may originate from photoreceptor-like Figure 2. Nuclcar magnetic resonance. Tumor in the pineal region (4 x3,8 x 5,2cm). cells in ocular tumors and from vestigial photo­ 3 receptor cells of the pineal organ.2• This clinical entity was first described in 19774 while in 1982 the term trilateral retinobla­stoma has been establihsed. 2 No more than 40 cases of trilateral retinoblastoma have been 7• 11 described so far.5­ In almost ali cases reported there, bilateral retinoblastoma appears early such as 7 ,2 rnonths as in our case, with a 68 % positive family history in most of the cases described. 5 The mean age of diagnosis of trilateral tumor was reported to be long, with an interval of 33 months, and almost ali patients presented with signs an'd symptoms of increased intracranial pressure.8.In the case we describe, the patient starteQ having refractory vomiting and ataxia of a ·šhort duration. In initial reports patients did not receive any therapy and ali died from CNS and spina! metastases with median of 1-5 months.9 Since the initiation of chemotherapy in CNS tumors and especially in meduloblastomas which com­prise another primitive predominately midline neuroectodermal tumors, same approach has 49 Terilateral retinoblastoma been applied in trilateral retinoblastomas al­ 10, 12 so. Despite intensive chemotherapy and radio­therapy it seems that this tumor has a propen­sity to seed the cerebrospinal fluid. 12· 13 Aggres­sive chemotherapy and radiation therapy seems to be indicated4 with both modalities increasing the overall survival.5-7• 14 With the rising awareness of the trilateral retinoblastoma syndrome and increasing availa­bility of sensitive central nervous system imag­ing procedures, the incidence of this entity is increasing. Follow up of the CNS in patients with bilate­ral retinoblastomas as well as aggressive and uniform therapy in these tumors are necessary to improve the bad prognosis. References l. Bader JL, Miller RW, Meadows AT, Zimmerman LE, Champion LA, Voule PA. Trilateral retino­blastoma. Lancet 1980; 2: 582-3. 2. Bader JL, Meadows AT, Zimmerman LE, Rorke LB, Voute DA, Champion LA, Miller RW. Bila­teral retinoblastoma with ectopic intracranial reti­noblastoma: Trilateral retinoblastoma. Cancer Ge­net Cytogenet 1982; 5: 203-13. 3. Scott MH, Richard JM. Retinoblastoma in the stale of Oklahoma: a clinocopathologic review. Oklahoma state Medica/ Association 1993; 86: 111-8. 4. Jakobiec FA, Tso MO, Zimmerman LE, Danis P. Retinoblastoma and endocranial malignaney. Cancer 1977; 39: 2048-58. 5. Holladay DA, Holladay A, Montebello JF, Red­mond KP. Clinical Presentation, Treament and Outcome of Trilateral Retinoblastoma. Cancer 1991; 67: 710-5. 6. Blach LE, McCormick B, Abramson DH, Ells­worth RM. Trilateral retinoblastoma incidence and outcome: a decade of experience. lnt J of Radiation Oncology Biology Physics 1994; 29: 7. De Potter P, Shields CL, Sahields JA. Clinical variations of trilateral retinoblastoma: a report of 13 cases. J Ped Opthamol Strah 1994; 31: 26--31. 8. Pesin SR, Shields JA: Seven cases of trilateral retinoblastoma. Am J Opluhalmol l989; 107: 121­6. 9. Kingston JE, Plowman PW, Hungerford JL. Ecto­pic intracranial retinoblastoma in childhood. Br J Opthalmol 1985; 69: 742-8. 10. Zelter PM, Gonzalez G, Schwartz L, Gallo G, Schwartzman E, Dame! A, Muriel FS. Treatment of retinoblastoma: Results obtained from a pro­spective study of 51 patients. Cancer 1988; 61: 153-60. 11. Mukhe1jee M, Gothi R, Doda SS, Aggarwal SK. Trilateral retinoblastoma Am J Reoentgenol 1991; 157: 198-9. 12. Johnson DL, Chandra R, Fisher WS, Hammock MK, McKeown CA. Trilateral retinoblastoma: Ocular and pineal retinoblastoma. J Neurosurg 1985; 63: 367-70. 13. Marks LB, Bentel G, Sherouse GW, Spencer DP, Light K. Cramospiral irradiation for trilateral re­tinoblastoma following ocular irradiation. Med Dosimetry 1993; 18: 125-8. 14. Nelson SC, Friedman HS, Oakes WS, Halperin EC, Tien R, Fuller GN, Hockenberger B, Scroggs NW, Moncino M, Kurtzberg, et al. Successful therapy for trilateral retinoblastoma. Am J Optha­mol 1992; 114: 23-9. 729-33. Radio! Onco/ 1995: 29: 50-7. Quantitative analysis of terminal blood network in human spinal cord and progressive radiation myelopathy Pavel Vodvarka, 1 Jiri Malinsky, 2 Ladislav Tichy2 1 II. Radiotherapy Clinic of Faculty Hospital Ostrava, Paskov, 2 Department of Histology and Embryology, Medica! Faculty of Palacky University, Olomouc, Czech Republic A quantitative analysis of terminal blood network in human spina! cord was done. The relative area of capillars in the gray matter is higher than in the white one and it increases from posterior to anterior spinal cord horns. The capillar area of the gray matter is larger in thoracic segments than in cervical ones. In the white matter, the largest capillar area was found in the anterior columns, the lowest in the deep par/s of posterior columns. Cervical white matter has the lowest relative capillar area whereas lumbar white matter is of the largest one. The results are compared to findings in the irradiated spina! cord. The more radiosensitive parts have been shown of having the lowest values of the relative capillar area. Possible explanation is discussed. Key words: Spina! cord-blood supply; capillaries; myelitis; radiotherapy -adverse effects; quantitative analysis Introduction Progressive radiation myelopathy (PRM) -late reaction of the spina! cord -is a very tragic seq uel of radiation treatment. Unfortunatelly, it usually arises in patients with malignancy controlled long after radiotherapy was comple­ted. PRM was described at first by Ahlbom1 in 1941. The diagnosis of PRM can be stated when the following criteria proposed by Pallis et al.2 are fulfiled: 1 The spina! cord must be included Corrcspondcncc to: Pavel Voclvarka, II. Radiothcrapy Clinic of Faculty Hospital Ostrava, Nadražnf 1, 73921 Paskov, Czcch Rcpublic. UDC: 6l6.832-002-031.81 Supportcd by grant IGA MZCR 2240-2 in the field irradiated,2 the main neurological lesion must be within the segments of the cord exposed to radiation,3 the cord compression from metastasis as the cause of the neurological disorder must be excluded. PRM is one of four clinical fonns of cord radiation injury according to Reagan et al:3 -transient myelopathy -signs of lower neurones -acutely developing quadru-/paraplegia -progressive radiation myelopathy. The PRM mechanism has not been known up to now. Didactically, three theories could be described: vascular, glial, and immunologi­cal, according to the most injured cells in vo­lume irradiated. This injury had been suggested as a crucial in PRM pathogenesis. Because of Quanlitative an11/ysis of terminal hlood network in human spina/ cord the mutual functional dependence of cell compartments and because the radiation hits ali of them ranclomly, the resulted racliation reaction is to be surely considered as a very complex event. Up to now, the correct relation­ship between radiation close, Iength of corcl irradiated and possible cord radiation (which coulcl be very clear as being radiothera­pist's daily bread) is not known. The cervical spina! corcl has been found to be more radiosensitive than the thoracic one and the lumbar cord most radioresistant. There­fore, cases of human lumbar radiation myelo­pathy are rather rare in comparison to the cervical and thoracic ones. +-7 The systematic experimental study of human PRM is not possible, therefore, we can judge a course of events by results of animal experi­ments. The best and the most comprehensive study of radiation effects on an animal spina! cord is Kogel's careful study8 . His experiment conclusion is, that early lesions are caused by death of oligodendroglial cells (responsible for myelinisation) and Iater lesions are caused by the injury of vessels. Lower radiation doses cause later damage -primarily vascular as well. After higher closes, the earlier oligodendroglial damage appears. The vascular component is clamaged with both, lower and higher doses. Therefore, we suggested the results of the vascular spina! cord network study coukl be a suitable substratum for an explanation of different radiosensitivity of various parts of the cord. The spina! cord vascular supply in a human is described in 11 detail both, macro-ancl microscopically.9­ In literature there are papers dealing with quantitative evaluation of capillary network of various brain regions, mainly in the cortex.n Ll Papers describing human spina! cord vascular supply do report in general much more dense capillary network in the gray matter than in the white one. 10 The aim of the study is to fine! differences in size and the density of capillary in white and gray matters between various regions in one segment ancl between segments of the cervical, thoracic ancl lumbar spina] cord. Materials and methods The spina! cord was stuclied on the spina! cords of three adult men (age of 20, 25 and 26 years). The material was gainecl by necropsy. After the spina! channel was openecl, the whole cord was taken out ancl accorcling to spina! roots it was divicled in particular segments. Tissues were fixed by 10 % neutral phosphate formo! ancl embeded in parafin. Slices of 15 µm were stained by Iucid methods (hematoxylin-eo­sin, cresylum violet and methocl by Cluver-Bar­rery), the impregnation by the Bodian's method and the PAS histochemical reaction was carried out as well. The best methocl for capillary quantitave study seems to be the methocl where slices were stainecl by the PAS reaction because capillary basic membranes are best shown. By the quantitative evaluation of blood capil­lary, paraffin slices have been screenecl with the help of the projection microscope Pictoval (Zeiss Jena) by the magnification of 200 times on a paper and outlines of thin praecapillaries and the thinnest sections of capillaries have been clrawn. Arteriolae and larger vessels have not been taken in account. A quantitative ana­lysis has been carried out on such outlines with the semiautomatic device MOP AM 03 fy Kontron. Surface, circumference, diameter and shape factor of terminal parts of bloocl network have been measured. The total areas of gray ancl white matters in the examinecl spina! corcl regions have been measured as well. The total number of capillaries in regions has been recal­culatecl for the relative numbers show the den­sity of capillaries in the area unit of 1111111 2 • Three spina[ corcls segments -C7, Th5 and L3-were evaluated in each spina! corcl in this way. An arrangement of blood vessels network corresponds to literature data. The difference of the density of capillary network in gray and white matters is remarkable. However, without thc quantitative evaluation, there is no chance to judge any other clifference. For the purpose of an evaluation of various regions in white and gray matters, the white matter area has been clividecl into eight regions: Vodvdlka P et al. -region l. anterior columns -region 2. -ventral parts of lateral columns -region 3. and 4. latern! parts of latern! columns region 5. -dorsolateral parts of laterni co­lumns encompassing pyramidal tracts -region 6. -deep perigriseal parts of latern! columns -region 7. fasciculus cuneatus of posterior columns region 8. -fasciculus gracilis of posterior columns. Gray matter bas been evaluated according to the classification Rexed's laminas, however, nine zonas would be too detailed for our purpo­se. Therefore, gray matter has been divided into four levels marked with letters A, B, C, D. The central gray region, responding to the Rexed's zona X, was evaluated separately and it is marked with letter E. Blood capillaries are put in histological prepa­rates either on transverse or longitudinal ( or moreless obligue) sections. From the point of view of a quantitative analysis, there are two inhomogeneous groups, which were evaluated separately. But for the purpose of density mea­surement, they were evaluated together. Results Dimensions of blood capillaries in the gray mat/er By the evaluation of dimensions and shapes of blood capillaries, the following parameters were measured: the area of capillary section, the circumstance of capillary section, the maximal diameter and the shape factor. The information value of area and circumstance parameters were too low, therefore, results of maximal diameters and shape factors are only given here. To emphasize a difference between ventral and dorsal regions of the gray matter, the results of measurement for levels A and B ( they respond to Rexed's laminas 1-VI) and levels C and D (Rexed's laminas VII-IX) are given together. The range of average values describes parame- Table l. Variation range of average values of diame­ters of blood eapillaries, which werc transverally eut in examined regions of the gray matter in segments given. Segment Area Variation range of blood capillaries diameters (um) C7 A+B 13.8-15.6 C+D 14.5-19.2 Th5 A+B 15.0-17.7 C+D 15.5-18.1 L3 A+B 14.3-19.8 C+D 16.3-18.9 Table 2. Variation range of average values of lengths of blood capillaries, which were longitudinally cut in examined regions of the gray matter in segments given. Segment Area Variation range ofblood capillaries diameters (um) C7 A+B 27.8-53.4 C+D 34.1-44.2 Th5 A+B 36.7-52.9 C+D 32.1-41.7 L3 A+B 35.8-56.5 C+D 46.5-54.8 ters which are better observed than the average values themselves; therefore, those ranges are shown in tables. The transversal sections of capillaries are somewhat larger in ventral parts of the gray matter than in white one in all examined segments (Table 1). The difference of gray matter capillary di­mensions between segments are minimal, how­ever, minimal values are in cervical segment C7, and a bit higher in thoracic segment Th5 and also in the lumbar one L3. Shape factor values are not too variable and transversely cut capillaries are in a range of 0.79-0.89. It means that the shape of capillaries is moderately oval, not too different from the regularly spherical one. Capillary on the longitudinal section were evaluated separately, because the maximal di­mension does not conespond to the maximal diameter, but to the length of it, shown in the section (Table 2). The differences between them are also not remarkable; even the length is somewhat longer in all dorsal segments of the gray matter. There are no remarkable dif­ Quantitative analysis of terminal blood network in human spina/ corcl ferences between various segments, however, Blood capillaries density per area unit in both dorsal and ventral parts of the gray matter of the lumbar segment the length is longer than in the other parts. Dimensions of blood capillary in the white matter Substantial differences between diameters of capillaries in the transversal section and bet­ween capillary lengths in the longitudinal sec­tion in various parts of the white matter funiculi were not found. The diameters of capillaries were ranged from 13.5 µm to 24.0 .trn and were mostly less than comparable values in the gray matter. The values of the shape factor were rather homogenous: 0.80-0.88. Capillary lengths on longitudinal sections we­re, on the contrary, higher than in the same situation in the gray matter. Values measured in the various areas were ranged from 34.6 µm to 102.4 .trn. These values were nearly two times higher than those of the gray matter. The cause of it is the radia! entrance of vessels to the white matter from the vasocorona. The capillary network in the gray matter is much richer than in the white matter. The average values of capillary numbers per 1 mm 2 are given in Table 3 for the white matter and in Table 4 for the gray matter. Graphically, the values are shown in Figures 1, 2 and 3. In the cervica\ segment C7, a higher number of white matter capillaries is found in the ante­rior funiculus and in the ventral part of the laterni funiculus. The highest values in the gray matter are found in the region of the vcntral columna, then in thc lateral one and in thc dorsal part of the columna posterior. Extremcly high numbers of blood capillaries were found in the central gray of ali segments. This pattern is logical because the central gray is a region of entrance of aa. sulcocommissurales and their abundant branching into smaller arteriae sup­plying both, ventral and clorsal areas of the gray mattcr. In the thoracic segment Th5, the white matter capillary clensity is similar to the one in the cervical segment. The highest number is founcl Table 3. Average values of blood capillaries number per unit of arca I mm 2 in various rcgions of thc whitc mattcr of thrcc cxamined segments of thc spina! cord. (Dcscription indications 1-7 scc in thc tcxt). Region of thc whitc matter examined C7 Th5 L3 lndication Funiculus 1 Funiculus anterior 33.66 41.65 36.83 2 32.18 29.64 32.33 3 28.10 24.59 32.86 4 Funiculus lateralis 23.95 26.88 29.87 5 25.13 22.31 44.01 6 28.34 28.32 44.54 Funiculus postcrior 7 Fasciculus gracilis 25.91 26.93 32.58 8 Fasciculus ceneatus 24.57 26.90 28.34 Table 4. Averagc values of blood capillaries number pcr unit of area l mm 2 in various regions of thc gray matter of threc examined segmcnts of the spina! cord. Rcgion ofthe gray matter C7 Th5 L3 examined A 67.01 66.41 64.89 B 54.59 88.69 68.06 C 61.50 l04.08 74.82 D 68.93 98.00 75.91 E 133.63 112.31 125.09 VodvdNw P et a/. 20 30 4 3 Figure 1. Density of blood capillaries per area of 1 mm2 in various regions of gray and white matters in the seventh cervical (C7) segment. 20 30 4 40 so 3 60 70 100 Figure 2. Density of blood capillaries per area of 1 mm2 in various regions of gray and white matters in the fifth thoracic (Th5) segment. 20 30 4 40 3 Figure 3. Density of blood capillaries per area of 1 mm3 in various regions of gray and white matters in the third lumbar (L3) segment. in the anterior funiculus. This number is higher than tlrnt in the cervical and lumbar segments. In the gray matter, thc capillary density is significantly higher than in the other two seg­ments except of the dorsal part of columna grisca posterior, where the density is similar. In thc lumbar segment L3, the density of capillaries is higher in both, gray and white matters. The dorsolateral and central parts of laterni funiculus have the highest density of ali white matter areas studied. Density values in fasciculus gracilis and in the laterni part of the lateral funiculus were similar to the ones in the other two segments. In the gray matter of the lumbar segment a very high capillary density was found and accordingly it is divided into two parts (with Rexed' s lamina V as the border), the ventral one having a higher density value. In ali segments studied, the richest blood supply has been found in the ventral areas of the gray matter and the highest density is in the thoracic segment, next in the lumbar and relatively small number of capillaries in the cervical segment. In the white matter, no signi­ficant difference has been found between cervi­cal and thoracic segments. On the contrary, the lumbar segment has higher values than the previous ones. Re/ative area of the blood network By the next criterion, evaluating the grade of vascularisation of various areas, the parameter of the relative area of the blood network was used. The parameter is the value expressing a proportion of ali capillary areas in the whole studied area. Results obtained for the gray matter (Table 5) confirm the previous observation. In ali studied regions, the relative area of blood net­work increased from dorsal (A) to ventral (D) sites. The minimum value is in the cervical segment and the maximum one in the lumbar segment. The same measurement proved the maximal proportion of the blood network in the anterior funiculus and the minimal one in the posterior Quantitative analysis of terminal blood network in human spina/ corcl funiculus (Table 6). By comparison of seg­ments, the highest grade of vascularisation was found in the white matter of the lumbar seg­ment and the minimal in the cervical one. Discussion The given results have revealed a quantitative morphological evaluation of spina! cord capilla­ries and the blood vessel network is a suitable method for studying the differences in blood supply of various regions. Measurements were carried out post mortem, therefore, absolute values do not respond to those in vivo values. We suggest postmortal changes as being propor­tional and thus the mutual comparison, which was the main goal of the paper, is possible and gives a real picture. The gained results have confirmed a marked difference between the rich vascularisation of the gray matter and relatively not so rich in the white one. In the gray matter significant diffe­rences have been found between ventral and dorsal columns and the grade of vascularisation is increasing to the ventral parts of anterior columns. In the white matter, the vascularisa­tion grade is usually somewhat higher in ventral funiculi. By the quantitative measurement signi­ficant diferences have been shown in various spina! cord segments as well. In the gray matter the vascularisation grade is increasing from the cervical to the lumbar ancl to the thoracic segment. The relative area of the vascularisa­tion in the white matter is increasing from the cervical to the lumbar segment. The lumbar segment has significantly higher bloocl supply of the white matter than cervical ancl thoracic segments. The papers clealing with the spina! corcl raclio­sensitivity have claimecl that the cervical corcl 5 7 14 is more sensitive than the thoracic one. -. More prominent histopatological changes are usually founcl in the white_ matter than in the gray one. 1+-16 By lower racliation the close chan­ges are usually clescribecl in clorsal funiculi of the white matter, presumably in cleeper parts. Further preclillect regions are posterolateral su­perficial regions. 16 In the early staclium of late changes, the clecreasing of oligoclenclroglial cells has been noticecl. During the late phase, vascu­lar changes (progressive changes like wall thic­kening, complete fibrose capillaries oblitera­tion) become more prominent. 15 These changes coulcl result in the necrosis of the white matter ancl the gray one as well. It seems that neurones are preservecl intact very long. 16 This is surely the staclium when bloocl supply is sufficient enough to fulfil at least a minimum function. By higher racliation doses ancl by the late re­action, the coagulative necrosis is a preclomi­nant pattern. Table 5. Averagc valucs of relativc area of bloocl capillarics in pcr cent of thc total arca cxamincd ( = 100 °/4,) in various rcgions of thc gray mattcr of thrcc givcn scgmcnts of thc spina! corcl. Region of the gray C7 Th5 L3 mattcr cxaminecl A 0.94 1.74 1.54 B 1.20 2.15 2.10 C 1.30 2.18 2.51 D l.51 2.40 2.63 E 2.53 l.97 2.17 l00 % ) Table 6. Avcragc valucs of rclative arca of bloocl capillarics in pcr cent of thc total arca cxamincd ( in various funiculi of thc whitc mattcr of thrcc givcn scgmcnts of thc spina! corcl. Examinccl fasciculus C7 Th5 L3 of the whitc matter Fasciculus anterior 0.79 0.88 l.21 Fasciculus lateralis 0.69 0.79 0.99 Fasciculus posterior 0.57 0.65 0.94 Vodv1Hka Pet al. The different radiosensitivity of various spina! cord segments ( cervical and thoracic) and diffe­rent histopatological findings following the irra­diation (more prominent consequences in the white matter and their predilect location in posterior and lateral funiculi) seem to be depen­dent on different blood supply patterns of these regions. The less is the grade of vascularisation the more radiosensitive the locations. It is con­tradictory because we know that oxygen sensi­tizes the effect of the radiation (in nondirect ionisation effect).17· 18 In this case, one has to keep in mind we are dealing with a normal health tissue that was irradiated, but not the primarily hypoxic tumor. The hyperbaric oxy­gen irradiation experiments did not prave the spina! cord to be more radiosensitive.19-21 Nor­mally, tissues are properly supplied with oxy­gen; that means tissues with minimum vascula­risation do not suffer from hypoxia. Where vascular network is richer, the higher need of oxygen is expected, for example by a functional demand. Such a rich capillar network could also have better capacity for the reparative process in tissues irradiated because it sufficien­tly provides them with oxygen and other sub­stancies to keep them on going process. We know from our clinical practice that some late reactions are positively affected in the hyperba­ric oxygen. On the other hand, some patholo­gical situations are known with the vascular network impairment. The risk of the late radia­tion reaction in the central nervous system is higher by this way22· 23. We have to suggest the multidimensional system; the current status of it is a result of many mutually dependent processes. The ner­vous system is schematically composed of neu­rones, glial cells and vessels. Each component is a system of cells by itself including many kinds of highly specialized cells. At the same tirne each system is dependent on the proper function of the other complicated system. Mor­phologically and functionally, different hetero­genous neurones without repopulation capacity are in general dependent on the very hetero­genous group of glial cells that can repopu­late24· 25 and both previous systems are depen- 38 MV) and is a result of selective attenuation by the pair production Parameterization of megavoltage transmission cwTes used in shielding calculations interaction of higher energy photons in the first few centimeters of shielding material. The in­accuracy introduced by fitting Eq. (1) to a transmission curve with a shoulder is acceptable since the effect occurs primarily tor beam ener­gies higher than those currently used clinically. It is possible to achieve a better fit to these high energy transmission curves by introducing extra terms into Eq. (1) to account for the shoulder region. The point of this analysis, however, is to de vise an analytic expression which can be used to interpolate between ener­gies. lf more than one parameter were used to describe the transmission curves, the simple analytic expression given by Eq. (2) relating the slope of a transmission curve to beam energy would not be valid. Table 1 gives a listing of the value of a tor photon beams of severa! nominal energies inter­acting in concrete, iron, and lead. The values of a determined tor energies duplicated in NCRP *49 and *51 are slightly different; those shown in Table 1 are the higher of the two values as determined from either report. The difference is most likely a result of the definition of the photon beam energy used in the two reports. In any case the discrepancies are small and can be ignored. Figures 1 (a-c) are plots of a as a function of beam energy for concrete, iron, and lead, respectively. As expec­ted the value of a decreases with increasing Table 1. Valucs of thc parameter a obtaincd from a fit of Eq. (1) to thc mcgavoltagc transmission curvcs in NCRP #49 and #51. Thc crror in thc parameter resulting from the fit is approximatcly constant at 0.5%. Beam Energy Concrete Iron Lead (MV) (cm) (cm) (cm) 1.0 -[6.0340 -5.0740 -2.5003 2.0 -20.7205 -7.1715 -3.7617 3.0 -24.5509 -8.2177 -4.6826 4.0 -29.1533 -9.0735 -5.2512 6.0 -34.6469 -9.8341 -5.5817 8.0 -38.0341 -10.3140 10.0 -40.0698 -10.5116 -5.2341 20.0 -45.5178 -4.8200 31.0 -l l.1280 38.0 -47.9993 86.0 -4.2154 energy tor concrete and iron, reflecting the more penetrating power of higher energy pho­ton beams. For lead, however, a first decreases, reaches a minimum around 6 MV, and then increases tor higher energies. This behavior is a result of the increase in the probability for -15 -20 -25 -30 "' -35 -40 -45 -50 -4 -5 -6 -7 -8 "' -9 -10 -11 -12 -2 -3 "' -4 -5 -6 o Figure 1. Plots of the parameter a, detcrmincd by fitting Eq. (1) to the megavoltage x-ray beam transmis­sion curves for (a) standard density concrcte, (b) iron, and (c) lcad, as a function of nominal bcam energy. The transmission curvcs used in the determination of a are found in NCRP #49 and #51.1• 2 Podgorsak M B et al. pair production and the concurrent dicreasein the Compton effect with increasing beam energy. The parameters c1_6 of Eq. (2) fit to the rensen JB: 4/301-8, 4/386-94 Staniša O: 4/271-5 Stanovnik M: 3/194-9 Stare J: 1/26-33 Strozzi M: 1/15-8 Škarpa A: 2/141-5 Šorli J: 4/276-81, 4/287-9 Štalekar H: 3/188-93 Šustic A: 2/94-7, 2/119-23; 3/169-73 Švalba-Novak V: 2/141-5 Taddei L: 1/23-5 Tasnadi G: 2/98-105 Tomljanovic Z: 3/188-93 Tondini M: 4/365-8 Uravic M: 2/119-23, 2/138-40, 2/141-5; 3/169-73 Uršic-Vršcaj M: 3/200-4 Us-Krašovec M: 4/416-8 Van Klaveren RJ: 4/341-5 Vcev A: 1/19-22 Verhagen AF: 4/346-50 Vidmar S: 4/337-40 Vfgvary Z: 1/58-62; 2/98-105 Višnar-Perovic A: 3/194-9 Vlaisavljevic V: 2/134-7 Vondrak V: 4/316-9 V oves R: 4/395-7 Volk N: 1/34-9; 4/290-7 Walker DR: 2/124-8 Walter K: 4/403-7 Zeilinger C: 4/282-6 Zeng F-Y: 4/282-6 Zharkov V: 4/382-5 Zidar A: 3/205-8 Zochbauer S: 4/395-7 Zovak M: 2/114-8 Zrubcova K: 4/316-9 Žgaljardic Z: 3/188-93 Radio! Oncol 1995; 29: 77-9. Subject index 1994 adenocarcinoma: 3/205-8 adrenocorticotropic hormone: 4/323-6 adult: 1/34-9 aged: 4/369-72 Albania: 4/298-300 angioplasty: 1/10-4 ankle: 1/23-5 antineoplastic agents: 2/129-33 arteriovenous malformations: 2/98-105 aspiration biopsy: 2/134-7 azygos vein: 1/15-8 ballon-adverse effects: 1/10-4 basilar artery-abnormalities: 2/89-93 biopsy: 2/134-7; 4/309-15 biopsy, needle: 2/134-7 -biopsy, needle methods: 4/309-15 -biopsy, needle methods, bronchoscopy: 4/309-15 blood groups: 4/282-6 blunt injury: 2/119-23 brachytherapy: 3/221-5 -computer-assisted iridium radioisotopes -radiotherapy planning brain metastases: 4/403-7 brain neoplasms-radiotherapy: 3/178-82 brain neoplasms-secondary: 4/403-7 breast neoplasms-methodology: 2/134-7 bronchial neoplasms-pathology: 4/282-6 bronchial neoplasms-surgery: 4/332-6 bronchoscopy: 4/309-15 calcaneus: 1/23-5 carcinoid tumor: 4/276-81 carcinoma: 2/124-8; 3/178-82; 4/301-8, 4/323-6, 4/341-5, 4/365-8, 4/386-94, 4/395-7, 4/398-402 -carcinoma, lung cancer: 4/395-7 carcinoma, non-small celi lung: 4/301-8 -carcinoma, non-small celi lung-surgery: 4/341-5 -carcinoma, oat celi, survival analysis: 4/365-8 carcinosarcoma: 4/373-5 carotid arteries-abnormalities: 2/89-93 cerebral angiography: 2/89-93 cerebral arteriovenous malformation: 2/89-93 cervix neoplasms-epidemiology: 1/34-9 cervix neoplasms-etiology: 3/200-4 cervix uteri cancer: 1/34-9 chemotherapy: 4/395-7 chemotherapy, cisplatin, etoposide: 4/395-7 cholelithiasis: 3/174-7 -cholelithiasis-complications: 1/19-22 chronic disease-epidemiology: 1/40-8 cisplatin: 4/395-7 coaxial catheter systems: 2/98-105 combined modality therapy: 4/351-8, 4/398-402 complications: 1/10-4 computer-assisted: 3/221-5 congenital arteriovenous malformations: 2/98-105 cortisol: 4/323-6 cytodiagnosis: 4/266-70 cytology: 2/134-7 diagnostic pitfall: 3/205-8 DNA, neoplasm: 4/282-6 dosimetry: 3/226-40 electron are therapy: 1/49-57 embolization: 2/98-105 epidemiology: 4/290-7 esophageal neoplasms-therapy: 2/129-33 etiology: 3/200-4 etoposide: 4/395-7 exophtalmus: 2/106-13 film dosimetry: 1/58-62 fracture: 1/23-5 gallbladder perforation-gallstone-ultrasonic »hole sign«: 1/19-22 gallbladder-ultrasonography: 1/19-22 genes, p53: 4/271-5 hamartoma: 1/5-9 heparin use: 1/5-9 history of megavolt and rotation therapy: 3/226-30 hormones: 4/320-2 human: 1/5-9 -human papilloma viruses cervix neoplasms­ etiology: 3/200-4 hyperplasia: 4/261-5 IASLC (International Association for the Study of Lung Cancer): 4/413-5 incidence in Slovenia, stages: 1/34-9 indications: 4/376-81 indications, life quality: 4/376-81 individual dosemeters: 1/58-62 injuries: 1/23-5 interferon-alpha: 3/183-7 interferon-alpha, recombinant: 3/183-7 interferon type II: 4/395-7 interventional: 2/94-7 -interventional-manpower: 1/58-62 iridium radioisotopes: 3/221-5 Subject index 1994 kidney transplantation: 2/94-7 kidney-ultrasonography: 3/169-73 Kiel classification: 1/26-33 laser surgery: 4/359-64 leiomyosarcoma: 2/141-5 life quality: 4/376-81 lithotripsy: 3/174-7 liver: 2/119-23, 2/141-5 -liver neoplasms: 2/141-5 -liver-ultrasonography: 2/119-23 lung cancer: 4/376-81, 4/395-7, 4/403-7 -lung cancer, radiotherapy: 4/376-81 lung neoplasms: 4/261-5, 4/266-70, 4/271-5, 4/276-81, 4/290-7, 4/301-8, 4/332-6, 4/359-64, 4/386-94, 4/403-7 -lung neoplasms-diagnosis: 4/287-9, 4/309-15, 4/320-2 -lung neoplasms-radiography: 1/5-9 -lung neoplasms-radionuclide imaging: 4/332-6 -lung neoplasms-radiotherapy: 4/376-81 -lung neoplasms-secondary: 4/408-12 -lung neoplasms-surgery: 4/332-6, 4/337-40, 4/346-50, 4/351-8, 4/365-8, 4/369-72, 4/373-5 luxation: 1/23-5 lymphoma: 1/26-33; 3/205-8 -lymphoma, non Hodgkins: 1/26-33; 3/205-8 mandibular neoplasms-surgery: 3/188-93 mediastinoscopy-adverse effects: 4/327-31 melanoma-drug therapy: 3/183-7 mesothelioma: 3/194-9 microvascular osteocutaneous free flaps: 3/188-93 multiple lung neoplasms: 4/271-5 needle: 2/134-7 -needle-methods: 4/309-15 neoplasm: 4/282-6 -neoplasms-epidemiology: 1/40-8 -neoplasms-metastasis: 3/178-82 -neoplasms-multiple primary: 4/346-50 -neoplasms-radiotherapy: 1/49-57 -neoplasms-staging: 2/124-8; 4/309-15, 4/337-40 -neoplasms-staging, TNM classification: 4/337-40 non-Hodgkin's: 1/26-33, 3/205-8 nonpenetrating: 2/119-23 non-small cell lung: 3/178-82; 4/301-8 -non-small cell lung carcinoma: 2/124-8; 4/382-5 -non-small cell lung carcinoma, NSCLC: 2/124-8 non-small celi lung-drug therapy: 4/386-94, 4/395-7 NSCLC: 2/124-8 oat celi: 4/265-8 operative contraindications: 4/332-6 oral cancer: 3/188-93 osteosarcoma: 4/408-12 patient dose: 3/209-20 peritoneal neoplasms: 3/194-9 peroneal tendons: l/23-5 personal exposure to radiation: 1/58-62 pleural effusion carcinoembryonic antigen: 4/316-9 pleural effusion, malignant: 4/316-9 postoperative adjuvant chemotherapy: 4/351-8 precancerous conditions: 4/261-5 preoperative radiotherapy therapy: 4/382-5 prognosis: 1/26-33; 4/301-8, 4/320-2 PTA: 1/10-4 p53: 4/271-5 pulmonary veins: 1/15-8 pylorus-surgery: 2/138-40 radiation protection: 3/209-20 radiology: l/58-62; 2/94-7 radionuclide imaging: 4/332-6 radiotherapy: 2/129-33; 4/376-81, 4/403-7 -radiotherapy-history: 3/226-30 -radiotherapy planning: 3/221-5 risk factors: 1/40-8 scimitar syndrome: 1/15-8 Slovenia: 1/34-9, 1/40-8; 4/287-9, 4/290-7 small-cell lung cancer (SCLC): 4/351-8 stages: 1/34-9 -stage I: 2/124-8 -staging: 2/114-8 stomach neoplasms: 2/114-8 -stomach neoplasms-radiography: 2/138-40 -stomach ulcer surgery: 2/138-40 subclavian artery: 1/10-4 superselective catheterization: 2/98-105 surgery: 2/124-8; 3/188-93; 4/332-6, 4/408-12 -surgery, operative contraindications: 4/332-6 -surgery with curative intent: 4/351-8 surgical flaps: 3/188-93 survival analysis: 2/124-8, 2/129-33, 4/341-5, 4/365-8, 4/369-72, 4/376-81 technique: 2/134-7 therapeutic-methods: 2/98-105 therapy: 4/382-5 tissue adhesive embolotherapy: 2/98-105 Subject index 1994 TNM classification: 2/124-8; 4/337-40 -TNM classification, neoplasms staging: 2/124-8 tomography: 1/23-5; 2/106-13, 2/114-8 transcatheter embolization: 2/98-105 treatment outcome: 4/323-6, 4/386-94 treatment planning: 3/226-30 tumor markers, biologicai: 4/320-2 ultrasonography: 2/119-23 ultrasound: 2/94-7 vagotomy: 2/138-40 wounds: 2/119-23; 3/169-73 -wounds, nonpenetrating: 2/119-23 x-ray computed: 2/114-8 -x-ray computed, staging: 2/114-8 -x-ray computed ultrasonography: 2/106-13 x-ray filters: 3/209-20 young women: 1/34-9 Reviewers in 1994 Bistrovic M, Zagreb -Bizjak-Schwarzbartl M, Ljubljana -Boko H, Zagreb -Brencic E, Ljubljana Brovet-Zupancic I, Ljubljana -Budihna N, Ljubljana -Burger J, Ljubljana -Debevec M, Ljubljana -Drinkovic I, Zagreb -Franceschi S, Aviano -Gantar-Rott U, Ljubljana -Jancar B, Ljubljana -Jereb B, Ljubljana -Jevtic V, Ljubljana -Kocjancic I, Ljubljana -Krajina Z, Zagreb -Kranjec I, Ljubljana -Lukic F, Ljubljana -MacMahon T, Houston -Marotti M, Zagreb ­Maškovic J, Split -Orel JJ, Ljubljana -Pavcnik D, Ljubljana -Perovic-Višnar A, Ljubljana ­Petric G, Ljubljana -Pogacnik A, Ljubljana -Pompe-Kirn V, Ljubljana -Rakar S, Ljubljana ­Ross JC, Amsterdam -Rott T, Ljubljana -Rubinic M, Rijeka -Singer Z, Zagreb -Snoj M, Ljubljana -Šimunic S, Zagreb -Škrk J, Ljubljana -Šmid L, Ljubljana -Štabuc B, Ljubljana ­Šurlan M, Ljubljana -Šuštaršic J, Ljubljana -Tepeš V, Rogaška Slatina -Thaler T, Ljubljana -Umek B, Ljubljana -Us J, Ljubljana -Videcnik V, Ljubljana -Vidmar-Bracika D, Ljubljana ­Vlaisavljevic V, Maribor -Willence M, Leiden -Zwitter M, Ljubljana -Žakelj B, Ljubljana ­Župancic Ž, Ljubljana Editors greatly appreciate the work of the reviewers which significantly contributed to the improved quality of our journal. FONDACIJA "DOCENT DR. J. CHOLEWA" JE NEPROFITNO, NEINSTITUCIONALNO IN NESTRANKARSKO ZDRUŽENJE POSAMEZNIKOV, USTANOV IN ORGANIZACIJ, KI ŽELIJO MATERIALNO SPODBUJATI IN POGLABLJATI RAZISKOVALNO DEJAVNOST V ONKOLOGIJI. MESESNELOVA 9 61000 LJUBLJANA TEL 061 1 5 91 277 FAKS 06 1 2 1 8 1 1 3 :ŽR: 50100-620-133-05-10331 15-214779 NAMENI IN CILJI ····•::':'•:'•-':.:;: . Namen Fo.dacije "Docent dr./"'•· •·· '.. :\;)·' \L'.špodrocja biomedicine niso zanemarljivitudi zunaj meja .·.•··e· §/Šii.ije,. vendar zaradi pomanjkanja.{inqncpih sreds/;;J .eniso pravi odraz raziskova('}i/J špD§QPYJ9.?t > ·· . Število rakastih obolenj v Slovr;fiiji rz.qrašcdfz leiat){eto . . Da bi bili pri zdravljeiiju bolnikoiJfe boljji.l:{iik?>.viti, . s. 11'lora)6 n'ašf zdra.niki neprestaizo tždbr.'l/v.ff ... · .. ·e·e .. . . ·e·e .. . · · . ···e ·e· )t:1i1;;;;;Jti°!t;P;.!i!a;:;:i.;;::::!;1!..1ej.zmiiogtm[i:4.faiidvami po svetu, ki.zdrav.ijoiri.·••· faziskujejo .e. ...rakaste bolezni. Leta 199Lse jeSlovenijapridružilae . •evropskemu programu:bo}a proti raku/njegovdo !J.tq €.000 zman}iaft3/.rdivosi ;a rakom 15 odst6t,kM.Temu cilju;;ifrt..6gocepribližati samo .. z nepreštr:iflt'nž. i;obr;aževanpi.r,(in raziskovalnim delom, .. kajt{lifazisk804ii:Je prinaš. nova vedenja in vodik lr.olJšemu razumevanju rakastih bolezni ter pomaga /pri preprece1.Jgrzju, zgodnjem ugotavljanju bolezni 1in uti.lft_ovitem zdravljenju,e;;;;}..9ndqcijo "Dodfflt dr.]. Cholewa".f.{i.g;ato jJc}iiiq,gq}({P.?.9q11,im raziskovalcem dQP<>}ijJiJ;qti znanje . . ··•·.t/JJf t::::.;;.;:::1:J..i:i.f?kši>ffff::.:. ;;:::o .eiahkg{{kbf tili naš .nqiiitiJ.b.;d;iskovalni potencial in obogqt!#'zaklqd;!J§;zndi;ja o rakastih boleznih, vas pozivdt.o, ad'š šv.jimi prispevki omogocite Fondaciji uresnicevanje njenega poslanstva. Akademik prof dr. Vinko Kambic HEPATOBILIARY SCHOOL :1,1Ui\ltj,-.q. 0 :C i ,j: . l\l\ . JUNE 19 -23, 1995 POSTGRADUATECOURSE ON HEPATOLOGY POSTGRADUATECOURSE ON HEPATOBILIARY SURGERY TOPICS: HEPATOLOGY JOINED SESSIONS SURGERY Vira! Hepatitis Autoimune Liver Diseases Pitfalls and Problems in Diagnostic Controversis in lntensive Care of Liver Patient Liver Resections Liver Trauma Genetic Liver Diseases Cholestasis LiverTumors Liver Regeneration Liver Transplantation Biliary Stenosis Pancreatic Surgery WORKSHOPS: Liver Surgery Training Laparascopic Surgery Methodology of Clinical Stody Planning Pathology Slide Seminar Endoscopy Interventional Radiology INVITED LECTURERS: Bengmark S., Coggi G., Czygan P., Fassatti L.-R., Ferenci P., Grimm G., Jeppsson B., Mazziotti A., Pratschke E., Schaffner F., Schmid R., Tiribelli C., Wiechel K.L. and others . Under the auspices of: MAILING ADDRESS: HEPATOLOGY: Saša Markovic, Institute of Oncology, SURGERY: Eldar Gadžijev, Clinical Center, Zaloška 2, 61000 Ljubljana, Slovenia Dept. of Gastroenterologic Surgery, Tel. & Fax: +386 61 30 28 28 Zaloška 7, 61000 Ljubljana, Slovenia Tel.:+ 386 61 32 22 82, Fax: +386 61 31 60 96 HEPATOBILIARY SCHOOL Ljubljana, Slovenia -June 19 -23, 1995 Name Surname Institute/Hospital Address Fax Countr Tel. O I need further information . I plan to attend the School Please type or use block /etters je koncern z dolgoletno zgodovino. Ustanovljen je bil leta 1886 v Baslu kot kemijska tovarna_, ki je izdelovala sinteticne barve. Leta 1917 je bil ustanovljen farmacevtski oddelek in že leto za tem (1918), so v roziskovalnih laboratorijih izolirali iz rženih rožickov alkaloid ergotamin, nato pa še kardiotonicne glikozide, kar je bil revolucionaren napredek v zdravljenju žilnih bolezni. 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V Sloveniji so na voljo registrirana zdravila: l.amisil ®(terbinafin) • nov pristop k zdravlieniu glivicnih infekcii kože, nohtov in lasišca • prvi fungicidni antimikotik za oralno in lokalno uporabo l.eponex ® (klazapin) • antipsihatik za bolnike s shizafreniio; ki se ne odzivaio na klasicne nevroleptike l.escol ® (fluvastatin) • sinteticni inhibitor reduktaze HMG-CaA • ucinkovito in varno zniža holesterol v krvi tudi pri rizicnih skupinah bolnikov l.eucomax ® (malgramostim) • rekombinantni humani deiavnik, ki paspešuie nastanek kolonij granulocitov in makrofagov • normalizira število belih krvnick in makrofagov pri bolnikih z zmanišano imunsko odpornostio Melleril ® (tiaridazin) • anksiolitik v nizkih dozah, nevroleptik v visokih dozah • ne povzroca ekstrapiramidnih sopojov,ov Miacalcic ® {kalcitonin) • hormon, ki regulira metabolizem kosti in mineralov • za zdravljenje pomenopavzalne in senilne osteoporoze, Pagetove bolezni, Sudeckove distrafije Navoban ® {trapisetran) • antagonist receptorjev 5-HT 3 • visokoselektivni antiemetik pri emetogeni kemoterapiji, radioterapiji in pooperativnem bruhanju Sandimmun Neoral ® (ciklosparin) • imunosupresiv, ki preprecuje zavrnitveno reakcijo pri transplantaciiah, • ucinkovit pri avtaimunih boleznih in boleznih, kjer je udeležena avtoimuna komponento, kot so psoriaza, revmatoidni artritis, otopicni dermatitis in endogeni uveitis Sandostatin"' {oktreatid) • sinteticni oktapeptidni derivat somatostatina • pomemben v gastroenterologiji, endokrinologiji in intenzivni medicini Sirdalud "'{tizanidin) • mišicni relaksant s centralnim delovanjem • za zdravljenje bolecine v križu, mišicnih spazmov in spasticnosti Syntocinon ® {aksitocin) • sinteticni oksitocin za spodbuianje maternicnih kontrakcii • Syntacinan -nosni spray za spodbujanje izlocanja mleka • intravenski antihistaminik prvega izbora Tavegyl ® {klemastin) Slovenia's Bank for Glo bal Business SKB Banka d.cl. lnlernalional Division Ajdovšcina 4 61000 Ljubljana, Slovenia Tel.: (+586 61) 171 55 19 or 17155 20 Fax: (+586 61) 502-808 SWIFT Gode: SKBA SI 2X SRB BANKA d.d. is an indepen­dent joint-stock company ancl the second largest Slovenian bank in tenns of branch network ancl capital. The large amount of capital on its balance sheet ancl its foreign currency reserves guarantee cus­tomers security ancl excellent prospects for fmther expansion. SKB BANKA cl.d. offers a wide range of banking services includ­ing: -International payments ancl accounl services -Non-resident account services, private and corporate -Project ancl !:rade finance -International finance -Foreign exchange services -Secmities services and invest­ ment banking -Real estate financing, tracling and renting The range of servi ces offerecl by the bank, is complemented by the services offered t:hrough sub­sicliary companies / alfiliates: SKB Real Estate and Leasing Ltd., SKB Investment Company Ltcl., SKB HI-NA Ltd., SKB Aurum-Brokerage House Ltd., SKB PLASIS Payment Systems Co. Ltd. ATENA Investment Fund Managing Co. SKB Tracling Ltd. .I SKB BANKA D,D, CE) Sl(B Banka cl.cl. Representalive omce London, 57-59 Eastcheap, London EC5M J DT, United Kingclom Tel.: (+44, 171) 929-2174, Fa.·c (+44, 171) 929-2175 lopami,[Q® 150 -200 -300 -370 mgl/ml- FOR ALL RADIOLOGICAL EXAMINATIONS MYELOGRAPHY ANGIOGRAPHY UROGRAPHY C.T. D.S.A. THE FIRST: WATER SOLUBLE READY TO USE NON.IONIC, CONT:RAST MEDIUM Manufacturer: Distributer: Bracco s.p.a. Agorest s.r.l. Via E. Folli, 50 Via S. Michele, 334 20134 -Milan -(1) Fax: (02) 26410678 Telex: 311185 Bracco 1 Phone: (02) 21771 e 34170 -Gorizia -(1) Fax: (0481) 20719 Telex: 460690 AF-GO 1 Phone: (0481) 21711 © Eastman Kodak Company, 1990 Kodak systems provide dependable performance for advanced diagnostic imaging. Our quality components are made to work together from exposure to viewbox. Kodak X-Omat processors are the most respected in the field. Kodak X-Omatic cassettes are known the world over far unexcelled screen-film contact and dura­bility. Kodak multiloaders have earned an enviable reputation far reliability. The Kodak Ektascan laser printer is changing the look of digital imaging. The list goes on. There are quality Kodak products throughout the imaging chain. Equally important, they are made to work together to achieve remarkable performance and diagnostic quality. Contact your Kodak representative far more information. 1 1 1 1 NI ! 1 · SIEMENS Vaš partner v ultrazvocni diagnostiki: SIEMENS * SONOLINE SL-1 * Možnost prikljucka sektorskega, linearnega, endo-p in endo-v aplikatorja * Izredno ugodna cena (možnost kredita ali leasing-a) * Servis v Sloveniji z zagotovljenimi rezervnimi deli in garancijo * Izobraževanje za uporabnike SIEMENS O.O.O. Dunajska 47, .jnb.jana Tel. 324-670 Fax. 132-4281 OLECINE lfl zcinesljivo 80J'}J1it1/iZfra,,; in ho1,,,.. a,,,b,,k,l'1h,; z,nerna zaupajte -u tramndol HCI Oblike: Trama/ 50 : 5 ampul po 50 111g tramadol HCl/111[ Trama/ 100 : 5 a111pu/ po 100 mg tranuulol HCl/2 ml Trama! kapljice: _ _JO 111/ raztopine (100 mg tramadol HCl/111!) Ti-arnal kapsule: 20 kapsul po 50 mg tramatlol HC1 T,-a,nal svecke: 5 sl'eck po 100 mg tramadol HC/ Izdeluje: Bayer Pharma d.o.o., Ljubljana ....n__ po licenci Grunenthal GmbH . @..lRi.. .[Q)MW.lYW. JJ. lNl..ffei lNl.[L(Q)@. L J u B L J A N A d. d. ZAUPAJO NAM NAŠI KUPCI IN DOBAVITELJI, ZNANI PROIZVAJALCI IZ TUJINE PA SO NAM ZAUPALI TUDI ZASTOPSTVA IN KONSIGNACIJE ZASTOPSTVA fN KONSfGNACIJE: -BAXTER EXPORT CORPORATION HOECHST AG -BOEHRINGER INGELHEIM -HOFFMANN LA ROCHE -NOVO NORDISK -SANDOZ -ORTHO DIAGNOSTIC SYSTEMS -COMESA -SCHERING & PLOUGH - ··­EWOPHARMA ESSEX CHEMIE SALUS LJUBLJANA d.d. -61000 LJUBLJANA, MAŠERA SPASICEVA 10, TELEFON: N.C. (061) 168-11-44, TELEFAX: (061) 168-10-22 ZA SPROŠCENO ŽIVLJENJE S STOMO NEGA d.o.o., Ljubljana Županciceva 1 O Ljubljana Popolna oskrba stomista Naša dejavnost obsega: * IZDAJANJE PRIPOMOCKOV ZA NEGO STOME * DOSTAVO PRIPOMOCKOV NA DOM * OSKRBO S PRIPOMOCKI PO POŠTI * SVETOVANJE IN PSIHICNO POMOC * POMOC PRI NEGI STOME NA DOMU Za stomiste se trudimo zdravnica, dipl. farmacevtka, socialna delavka in višje medicinske sestre. Naše storitve nudimo BREZPLACNO na podrocju celotne Slovenije Obrnite se na telefonsko številko: 061 / 12-53-146 Na voljo smo vam vsak delavnik med 10. in 16. uro. Radio! Oncol 1995; 29: 92. Instructions to authors The journal Radiology and Oncology publishes ori­ginal scientific papers, profcssional papers, review ar­ticles, case reports and varia (reviews, short communi­cations, profcssional information, ect.) pertincnt to diagnostic and interventional radiology, computerised tomography, magnetic resonance, nuclear medicine, radiotherapy, clinical and experimental oncology, ra­diobiology, radiophysics and radiation protection. Submission of manuscript to Editorial Board implies that the paper has not been published or submitted for publication elsewhere: the authors are responsible for ali statements in their papers. Accepted articles become the property of the journal and therefore cannot be published elsewhere without written permis­sion from the Editorial Board. Manuscripts written in English should be sent to the Editorial Office: Radiology and Oncology, Institute of Oncology, Vrazov trg 4, 61000 Ljubljana, Slovenia; Phone: + 386611320 068, Fax: + 386 611314 180. Radiology and Oncology will consider manuscripts prepared according to the Vancouver Agreement (N Engl J Med 1991; 324: 424-8.; BMJ 1991; 302: 6772.). Ali articles are subjected to editorial review and review by two indcpendent refcrees selccted by the Editorial Board. Manuscripts which do not comply with the technical requirements stated herc will be returned to the authors for correction before the review of the refcrees. Rejected manuscripts are gcne­rally returned to authors, however, the journal cannot be held responsiblc for thcir loss. The Editorial Board reserves the right to require from the authors to make appropriate changes in thc content as wcll as gramma­tical and stylistic corrections when necessary. The expenses of additional cditorial work and requests for reprints will be chargcd to the authors. General instructions: Type the manuscript double spa­ced on one side with a 4 cm margin at the top and lcft hand side of the sheet. Write the paper in grammati­cally and stylistically correct language. Avoid abbrevia­tions unless prcviously explained. The technical data should confirm to the SI system. The manuscript, including the refcrcnces may not exceed 15 typewritten pages, and the number of figures and tables is limited to 4. If appropriate, organise the text so that it includes: Jntroduction, Material and methods, Results and Discussion. Exceptionally, the results and discus­sion can be combined in a single section. Start each section on a new page and number these consecutively with Arabic numerals. Authors are encouraged to submit their contributions besides three typewritten copies also on diskettes (5 1/4") in standard ASCII format. First page: -name and family name of ali authors. a brief and specific title avoiding abbreviations and colloquialisms, -complcte address of institution for each author, -in the abstract of not more than 200 words cover the main factual points of the article, and illustrate them with the most relevant data, so that the reader may quickly obtain a general view of the material. Introduction is a brief and concise section stating the purpose of the article in relation to other already published papers on the same subjects. Do not present extensive reviews of the literature. Material and methods should provide enough informa­tion to enable experiments to be repeated. Write the Results clearly and concisely and avoid repeating the data in the tables and figures. Discussion should explain the results, and not simply repeat them, interpret their significance and draw conclusions. Graphic material (figures and tables). Each item should be sent in triplicate, one of them marked original for publication. Only high-contrast glossy prints will be accepted. Line drawings, graphs and charts should be done professionally in Indian ink. Ali lettering must be legible after reduction to column size. In photo­graphs mask the identities of patients. Labe! the figures in pencil on the back indicating author's name, the first few words of the title and figure number: indicate the top with and arrow. Write legend to figures and illustrations on a separate sheet of paper. Omit vertical lines in tables and write the next to tables overhead. Labe! the tables on their reverse side. References should be taped in accordance with Van­couver style, doublc spaccd on a scparate sheet of' paper. Number the refcrences in the order in which they appear in the text and quote their corresponding numbers in the text. Following are some examples of references from articles, books and book chapters: 1. Dent RG, Cole P. In vitro maturation of monocy­tes in squamous carcinoma of the Jung. Br J Cancer 1981; 43: 486-95. 2. Chapman S, Nakielny R. A guide to radiological procedures. London: Bailliere Tindall, 1986. 3. Evans R, Alexander P. Mechanisms of extracellu­lar killing of nucleated mammalian cells by macropha­ges. In: Nelson DS ed. Immunobiology of macrophage. New York: Academic Press, 1976: 45-74. For reprint information in Norih America Contact: /111em111ional reprint Corporation 968 Admiral Callag­ha11 Lane, # 268 P. O. Box 12004, Vallejo, CA 94590, Tel.: ( 707) 553 9230, Fax: (707) 552 9524. Nepotrebno je, da bolezen spremlja bolecina Moc opioidnega analgetika brez opioidnih stranskih ucinkov tramadol centralno delujoci analgetik za lajšanje zmernih in hudih bolecin ucinkovit ob sorazmerno malo stranskih ucinkih Srednje mocne do mocne akutne ali kronicne bolecine. Po tristopenjski shemi Suetnt'ue zdm1•s/l'e11e w:qauizac{ie zo faj§au}t' l>o/eciu pri l>olnikih z mkan'm o/,olenjem trammlol odjnYw(ia srediu·e hw/o lwlec'ino ali /Jolec'ino drnge stopnje, Kontraindikacije: Zdravila ne smemo dajati otrokom, rnlaj.šim od 1 leta. Tramadola ne smerno uporabljati pri akutni zastrupitvi z alkoholom, uspavali, analgetiki in drugimi zdravili, ki delujejo na osrednje živcevje. Med nosecnostjo predpišemo tramadol lc pri nujni indikaciji. Pri zdravljenju med dojenjem moramo upoštevati, da O, 1 % zdravila prehaja v materino mleko. Pri bolnikih z zvecano obcutljivostjo za opiate moramo tramadol uporabljati zdo previdno. Bolnike s krci centralnega izvora moramo med zdravljenjem skrbno nadzorovati. Interakcije: Tramadola ne smemo uporabljati skupaj z inhibitorji MAO. Pri socasni uporabi zdravil, ki delujejo na osrednje živcevje, je možno sinergisticno delovanje v obliki povecane sedacije, pa tudi ugodnejšega analgeticnega delovanja. Opozorila: Pri predoziranju lahko pride do depresije dihanja. Previdnost je potrebna pri bolnikih, ki so preobcutljivi za opiate, pri starejših osebah, pri miksedcmu in hipotiroidizmu. Pri okvari jeter in ledvic je potrebno odmerek zmanjšati. Bolniki med zdravljenjem ne smejo upravljati strojev in motornih vozil. Doziranje in nacin uporabe: Odrasli in otroci, starejli od 14 let: Injekcije: 50 do 100 mg i.v.,i.m.,s.c.; intravensko injicirarno pocasi ali infundiramo razredceno v infuzijski raztopini. Kapsule: l kapsula z malo tekocine, Kapljice: 20 kapljic z malo tekocine ali na kocki sladkorja; ce ni zadovoljivega ucinka, dozo ponovimo cez 30 do 60 minut. Svecke: l svecka; ce ni ucinka, dozo ponovimo po 3 do 5 urah, Otroci od I do 14 let: 1 do 2 mg na kg telesne mase. Dnevna doza pri vseh oblikah ne hi smela biti višja od 400 mg. Stranski ucinki: Znojenje, vrtoglavica, slabost, bruhanje, suha usta in utrujenost. Redko lahko pride do palpitacij. ortostatske hipotcnzije ali kardiovaskularnega kolapsa. Izjemoma se lahko pojavijo konvulzije. Oprema: 5 ampul po 1 ml (50 mg/ml), 5 ampul po 2 ml (100 mg/2 ml), 10 ml raztopine (100 mg/mil, 20 kapsul po 50 mg, 5 sveck po 100 mg. Podroh1,e.fše i1?formac(je so 11a voljo pri proizuc.ictlc1,. ... KRK. SLOVENIJA