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Development of cancer is a complex process involving multiple changes in gene expression. To unravel these alterations, a proteome approach aimed at the identification of qualitative and quantitative changes in protein composition, including their post-translational modifications, attracts great attention. Our study was focused on the identification of proteins whose amount is altered in the course of malignant transformation of colon mucosa. Proteins extracted from tissue specimens or cell lysates were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns were done using computerized image analysis. Selected proteins exhibiting statistically significant abundance alterations comparing healthy and diseased tissues were identified by mass spectrometry. Globally, we have found 57 proteins that exhibited either a significant decrease or increase in amount in pathological tissues, and 18 of these were annotated by mass spectrometry. The alterations in the expression of nine proteins were common for both precancerous and neoplastic tissues suggesting their role in colon tumorigenesis. The epithelial origin of all identified spots was checked in two cell lines Caco-2 and DLD-1 originating from well-differentiated and poorly differentiated colon carcinoma, respectively.

We have started the construction of a two-dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia. The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database. We have analyzed the F. tularensis live vaccine strain by two-dimensional gel electrophoresis with immobilized pH 3-10 gradient in the first dimension and 9-16% gradient or tricine SDS-PAGE in the second dimension. In both cases about 2000 spots were detected. Furthermore, we compared the protein pattern of the nonvirulent F. tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted. The separated proteins are going to be identified by peptide mass fingerprinting. However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified. Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains.

Purpose of the Study: A group of 120 patients was evaluated with acute injury of Th-L spine who were operated on from posterior approach with the use of Dick AO fixator or USS fixator in the period of 1991-1996., Material: In the period of 1991-1996, 128 patients were operated on with the acute injury of Th-L spine. Evaluated were 120 patients: 81 men (67.5%) of average age 40.1 years (age range, 16-74 years) and 39 women (32.5%) of average age 35.4 years (age range, 15-66 years). Posterior stabilization using the internal fixator was combined with posterolateral or posterior interarticular fusion. In comminuted fractures of the vertebral body transpedicular cancellous bone grafting was performed and starting for 1995 also intercorporal transpedicular fusion., Methods: The evaluation covered location and type of fractures, injury of other levels of the spine, other associated injuries, neurological symptomatology prior to the surgery and 3 months after the surgery, duration of surgery and of image intensifier exposure, peroperative and early postoperative complications, radiograph evaluation of the degree of reduction, the loss of correction after 12 months and after the hardware removal., Results: Evaluated were 120 patients. Average interval after the surgery at the time of the evaluation was 43.7 months (range, 24-93 months). The injury was caused in 82 cases (68.3%) by a fall from height, in 26 cases (21.7%) by a car accident and in 12 cases (10%) by another cause. Associated injuries included: calcaneus fracture 18 times, the fracture of ankle 4 times, pelvis fracture 4 times, other fractures 18 times. In 89 cases the injury involved only one vertebra, i.e. one level, 31 cases were multi-level injuries. The angle of the kyphosis of vertebral body was on average 21.8 degrees. Patients with neurological symptoms were on average operated on 2-12 hours after the admission, other patients were operated on in the interval between 5 hours and 7 days. Bilateral transpedicular cancellous bone grafting was performed in 106 affected vertebrae, the canal was revised in 27 injured vertebrae. The surgery took on average 172 minutes (range, 62-430 minutes). Image intensifier exposure lasted on average 2.4 minutes. Average blood loss was peroperatively estimated at 725 ml, postoperatively on average at 815 ml. Postoperative complications: healing of the wound per secundam 6 times, thromboembolic complication in 9 cases--never fatal, pneumonia 3 times, uroinfection 5 times. The total number of these complications was about 8%. Vertebral body was completely restored in 72 cases, incompletely in 34 cases (up to 5 degrees deficit). The correction of kyphosis achieved was on average 6.5 degrees. After 12 months the correction loss with the fixator in place was on average 2.5 degrees. After the removal of the fixator the average correction loss was 5.5 degrees. In 2 cases there occurred a significant re-dislocation which required revision surgery. Transpedicular screws broke 6 times in the total of 4 patients, fixator loosened in 2 cases., Discussion: Surgical treatment of thoracolumbar spine is indicated in unstable injuries and all injuries with neurological lesion and a proved compression of spinal canal. Decompression of the spinal canal is often achieved by reduction, lordotization and distraction. In other cases revision of the canal is indicated. Stabilization is performed by means of a transpedicular internal fixator. Part of the surgery is posterior fusion. The defect in the reduced body is treated by transpedicular cancellous bone grafting. The affected inervertebral disc must be treated by intersomatic fusion. Anterior operation is indicated as a complementary one., Conclusions: 1. Absolute majority of injuries of thoracolumbar spine may be treated from the posterior approach. In this it is necessary to restore the stability of the anterior column. An indispensable part of the operation is a flawless posterior fusion. 2. Some types of injuries require a complementary anterior approach in the first or second step. This applies mainly to the injuries with a defect of anterior column evaluated on the basis of classification after Gaines. 3. A primary isolated anterior approach to the treatment of the injury of Th-L spine is not in our view suitable as a routine.

Authors deal with the screw-wire technique described by Brunner and B. G. Weber in 1981. The principle of the technique consists in passing a wire loop around the heads of divergently driven screws. After tightening the screws the wire loop is put under tension which results in interfragmental compression. Mostly used for this purpose are 4.5 mm cortical screws but, if need be, any screw from a standard or small set of instruments is applicable. This type of fixation they use mainly for acetabulum fractures, diastasis of symphysis or for the stabilization of knee osteotomies.

Using two-dimensional electrophoresis we investigated the effect of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT; induction with 1 mM ALA for 4 h followed by blue light dose of 18 J/cm2) on the protein expression in HL60 leukemia cells. ALA-PDT resulted in extensive qualitative and quantitative changes in the protein pattern of HL60 cell lysates. Of more than 1350 protein spots recognized on the protein maps of ALA-induced cells, seven proteins were enhanced and 17 suppressed following irradiation. Three of these, calreticulin precursor, p58 microsomal protein (ERp57) and protein disulfide isomerase (p55) have been identified by matrix-assisted laser desorption and ionization-mass spectrometry and the pI/molecular weight parameters of the affected proteins were estimated by computer analysis. The findings suggest participation of endoplasmic reticulum Ca(2+)-binding chaperones and/or Ca2+ signaling in ALA-PDT mediated cytotoxicity.

The expression of calcium-binding protein S100A6 was investigated in normal colon tissue, in colon adenomas and in colorectal carcinomas. Using an immunoblotting approach we detected four S100A6 variants with Mwt of 10 kDa and pI of 5.05 (isoform I), 5.15 (isoform II), 5.23 (isoform III) and 5.32 (isoform IV) that were differentially expressed in the analysed samples. The quantitative examination of S100A6 variant expression in 25 pairs of colorectal carcinoma and matched control mucosa proved a statistically significant increased abundance of S100A6 isoforms I (P = 0.004) and III (P = 0.025) in malignant tissue, and conversely, an increased level of S100A6 isoform IV in healthy tissue (P = 0.022). The expression of isoforms I and III and the loss of isoform IV were also observed in colon cancer cell lines. In addition, the immunohistochemical study of 16 primary colorectal carcinomas revealed both in the non-paired Student t-test and in the Mann Whitney test the statistically significant accumulation of S100A6 protein (P < 0.001) in the invasive margin of the tumour. The immunohistochemical analysis of S100A6 protein in polyps differing in clinical severity gave a strong staining that was maximal in dysplastic lesions. Thus, our results indicate a possible, statistically significant correlation (non-paired Student t-test P = 0.036) between S100A6 expression and colon carcinoma progression.

The authors describe the technique of the anterior miniinvasive retroperitoneal pararectal approach to the lumbosacral spine. The technique was verified on cadaveric specimens.The miniinvasive operation requires a special set of instruments (a fixed frame), long instruments and a light cable. The advantage of the approach is a small peroperative traumatization and a relatively low labour intensity. The use of the miniinvasive technique requires a good knowledge of the technique of a routine extensive approach. Key words: surgical approaches, spine, lumbosacral spine.

This report describes a new case of gamma-1-heavy chain disease found in a woman with malignant lymphoproliferative disease. The patient's serum and urine containing gamma-1-heavy chains were analyzed using different electrophoretic approaches, especially two-dimensional electrophoresis and immunoblotting analysis. In a serum sample, five sets of gamma-1-heavy chain spots differing in molecular weight with acidic pI values and one set of more basic gamma-1-heavy chain spots were found. The major group of spots exhibited molecular weight in the range from 29 to 39 kDa. Examination of urine sample proved the presence of the more basic set of gamma-1-heavy chain spots and two acidic groups, including 29 to 39 kDa set.

Magerl technique of transarticular fixation of C1-2 by screws is one of the possibilities of the antlantoaxial fusion. For the first time it was published by Magerl and Seemann in 1987 but the first author had been performing it already since 1979. The principle of the method consists in the placement of two screws from the dorsal approach through atlantoaxial joints in the sagittal plane. The main indication for the Magerl's C1-2 fixation is an acute or chronic instability and a painful osteoarthritis of atlantoaxial joints. Contraindicated is this method only in massive destruction of the lateral mass of atlas and in an anomalous course of the vertebral artery. The authors present their own experience with the first 12 patients who they operated on in the period of 1996-1999. In all cases they added dorsal fixation after Gallie (10 patients) or modified after Brooks and Jenkins (2 patients). The group consisted only of patients with a separate atlantoaxial fusion, i. e. not with the transarticular fixation by screws as part of the set of instruments for occipitocervical fusion. Final check of all patients was performed at the interval of 9-48 months after the operation, on average 19 months. The final outcome was evaluated subjectively according to the patients and also objectively. Subjectively, the patients complained at the beginning of the pain in the region of the wound after the bone graft harvesting which was as a rule greater than the pain in the region of the cervical spine. The pain subsided in 6 weeks, at the latest. All patients but for one (malum suboccipitale Rusti) were satisfied, i. e. they would undergo the operation again. Objectively all 24 screws were inserted correctly thrugh the antlantoaxial joint. Two screws were driven asymmetrically, one of the obliquely medially and the other obliquely laterally without a clinical impact. One screw was evaluated by authors as extending too anteriorly, also without a clinical impact. After 12-14 weeks the functional radiographs showed a good fusion of the C1 -2 segment in 11 (91,7 %) out of 12 patients. Neither an injury of the nerve structures nor of the vertebral artery was recorded within peroperative complications. Loosening or breakage of screws or elements of the dorsal fixation were not recorded, either. The results of the work prove that the Magerls technique of atlantoaxial fusion is effective and in the hands of a specialist also a safe method which provides multiple opportunities for the treatment of C1-2 instabilities. Key words: atlantoaxial instability, fusion, Magerls C1-2 fixation.

Our current results, aimed at the detection of protein abundance alterations that could be associated with the process of colon tumorigenesis, are summarized. The matched sets of macroscopically normal colon mucosa and colorectal carcinoma were examined by a one- or two-dimensional electrophoretic approach and proteins were identified using immunoblotting or mass spectrometry. The following results were observed: The levels of liver fatty acid-binding protein, actin-binding protein/smooth muscle protein 22-alpha and cyclooxygenase 2 were downregulated in colorectal carcinoma compared to normal colon mucosa. Conversely, the expression of a novel variant of heat shock protein70 and several members of the S100 protein family of calcium-binding proteins (two isoforms of S100A9, S100A8, S100A11 and S100A6) were upregulated in transformed colon mucosa. Despite the variations of the levels of expression of given protein among analyzed samples, all quantitative changes were found to be statistically significant (Mann-Whitney test assuming p < or = 0.05). We conclude that the proteomic approach is useful for the study of complex biological events underlying the process of colorectal tumorigenesis.

The expression of calcium-binding protein S100A9 was investigated in 23 matched sets of colorectal carcinoma and normal colon mucosa using two-dimensional gel electrophoresis. We found that, from a group of 23 patients, the level of S100A9 protein, in comparison with matched normal colon mucosa, was significantly increased in malignant tissues of 16 patients (70%). Furthermore, an additional protein, identified by matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) as S100A8, exhibited an increased expression in the same specimens of malignant tissues as the S100A9 protein. The immunohistological analysis revealed the accumulation of S100A9 positive cells, macrophages and polymorphonuclear leukocytes along the invasive margin of colorectal carcinoma. The S100A8 protein was found to be produced in the same location. The possible participation of both proteins and, especially, its heterodimeric complex calprotectin in colorectal carcinoma regression could be taken into account.

It is assumed that the exposure of cells to ionizing radiation modulates their signal transduction pathways, which then govern the early and late radiation-induced alterations in gene expression. In this study we tested the effects of low doses of X-irradiation on the cell signaling and global protein composition of an HL-60 human promyelocytic leukemia cell line differentiated along a macrophage-like cell pathway by 4beta-phorbol-12-myristate-13-acetate (PMA). Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting of anti-phosphotyrosine immunoprecipitates, we found radiation-induced changes in the level of phosphorylation of proteins with molecular masses of 45 and 48 kDa, but in the most intensively stained area, ranging from 54 to 60 kDa, no alterations were observed. When two-dimensional electrophoresis (2-DE) immunoblotting was applied, only proteins from this heavily stained region were visualized and in addition the evident differences in tyrosine phosphorylated protein patterns between nonirradiated and irradiated cells were found in this area. Furthermore, the immunostaining of extracellular signal-regulated kinase 2 (ERK2) which did not prove its tyrosine phosphorylation demonstrated the existence of several ERK2 charge isoforms showing differential expression after X-irradiation. Comparing the whole protein profiles we found after the simultaneous quantitation of 1000 matched spots two proteins whose expression was regulated in an opposite manner in nonirradiated and X-irradiated cells. The quantities of both spots showed increases or decreases by a factor of 2 or more between irradiated and nonirradiated samples and both these changes were statistically significant (P<0.05).

Authors describe the topographic relation of the anterior aspect of thoracolumbar spine to individual prevertebral structures (vessels, nerves, ligaments) as their injury may cause serious complications during spine surgery. Observations have been made on cadavers and checked by peroperative findings and findings on MRI sections. Having a good view of prevertebral anatomical structures has a principal significance for the use of anterior surgical approaches. Key words: topographic anatomy, thoracolumbar spine, MRI.

The case report presents a patient (male, 21 years old) who had a car accident and sustained an injury of upper cervical spine. X-ray examinations showed instability of C1-2 segment with a simultaneous os odontoideum. Subsequently CT and MRI examinations were performed to specify the anatomical relations. The patient had only a neurological lesion revealed by the examination by motor evoked potentials (MEP). The metric measurements on standard and functional radiographs showed a minimal sagittal dimension (Dmin) of 13 mm, the distance between os odontoideum and C1 arch (Datl) was 24 mm, the instability index after Penning was 20 mm, the distance between os odontoideum and proc. spinosus C2 in extension (Dext) after Shirasaki et al. was 30 mm, the sagittal rotational angle was 25 degrees and the instability index after Watanabe et al. 56.7 %. CT examination showed an exact shape of os odontoideum but was of minor significance for this anomaly. MRI did not prove simptoms of myelopathy. The final finding was os odontoideum of conical type after the Matsui et al. classification. Based on these examinations and the marked instability of C1-2 segment found a fusion of C1-2 after Magerl was performed in combination with Gallie technique dorsally. Key words: os odontoideum, instability, myelopathy.

The authors present first experience with their method of treatment of displaced fractures of calcaneus. The method is based on the combination of direct and indirect reposition of fragments and exact fixation with K-wires. Axial and AP X-ray and 2 plane CT scan are indispensible conditions for operation planning. The procedure can be divided into 4 phases: traction, elevation, compression, transfixation. Up till now 65 patients have been treated with this method. 20 patients with 24 fractures were evaluated according to the Creighton-Nebraska Health Foundation score in the interval 24 months after the operation. The average value of the score was 96.6 points, 16 patients were evaluated as excellent, in 2 patients the result was good.

Natural resistance to Mycobacterium bovis bacillus Calmette-Guérin (BCG) is determined by the Bcg gene (Nramp1), which is exclusively expressed by mature macrophages. The Nramp1 gene is a dominant autosomal gene that has two allelic forms; r confers resistance and s confers susceptibility to infection with intracellular pathogen. Although the wide range of pleiotropic immunological effects of the Nramp1 gene has been described, the exact mechanism of its action remains elusive. In this study we searched for differentially expressed proteins that might provide clues in the studies on Nramp1 gene function. We performed two-dimensional gel electrophoresis of cellular proteins prepared from a B10R macrophage line derived from mice carrying the r allele of the Nramp1 gene, B10S macrophages carrying the s allele, and B10R-Rb macrophages transfected with Nramp1-ribozyme. The classification of protein patterns and selection of distinct proteins characteristic of r or s allele-carrying macrophages was performed using the principal component analysis. We found differential expression of four proteins with the following isoelectric point/molecular weight (pI/Mr) in B10R macrophages compared to B10S and B10R-Rb macrophages: 6.6/25, 7.0/22, 9.1/31.5, and 5.3/8.5. The protein 7.0/22 has been identified as Mn-superoxide dismutase and the best candidate for protein p6.6/25 seems to be Bcl-2 according to the immunoblot analysis. When the splenic macrophages carrying the r or s allele were analyzed, the changes in relative abundance for proteins 6.6/25 and p7.0/22 were satisfactorily reproduced. Overall, the two identified proteins are important in the regulation of intracellular redox balance and the regulation of apoptosis in macrophages, respectively. Our findings may suggest their possible biological role in the innate immunity against intracellular pathogens.

Out of 160 injuries of Th-L spine operated on only two were of extension type. In the first case it was a pure posterior displacement of L 1-2into extension with the angle of lordotization 20 degrees and complete paraplegia. From the viewpoint of classification this injury may be included in group B 3.3 according to AO/ASIF classification. With regard to an extreme post-injury displacement which at first glance is similar to the "seatbelt fracture" after Denis classification we would suggest to call this injury "reverse seat-belt fracture". The treatment consisted in the reduction and monosegmental instrumentation. The neurological deficit is permanent. The second case was a fracture of extension type in a patient with ankylosing spondylitis (morbus Bechterev). The patient was treated by monosegmental instrumentation. The initial incomplete neurological deficit improved in the course of several weeks (Frankel D). With regard to the fact that the fracture line was passing anteriorly through the vertebral body, this injury cannot be clasified according to AO/ASIF classification. Extension injuries are rare injuries and with regard to their low frequency of incidence the classification schemes more or less neglect them. Key words: Th-L spine, injuries, extension injury, classification.

Subtractive two-dimensional gel electrophoresis (2-DE) has been used for the study of the protein patterns of the normal colonic mucosa and the specimens collected from patients diagnosed for inflammatory bowel disease (IBD), colonic polyps and colorectal cancer. We found a 13 kDa protein that was detected in five of seven adenomas and in 13 of 15 colorectal carcinomas while it was absent or only slightly expressed in normal colonic mucosa. Furthermore, this protein occurred in all specimens collected from patients suffering from IBD and its quantity reflected the increased severity of inflammation. The combination of microsequencing and mass spectrometry led to the identification of the 13 kDa spot as calgranulin B. Our results indicate that the production of calgranulin B is unregulated in inflammatory, preneoplastic and neoplastic lesions of colonic mucosa.

Analysis of the expression of hsp70 (70 kDa heat-shock proteins) in normal and pathological tissues might prove their potential diagnostic and prognostic values. In the present study, we combined high-resolution two-dimensional polyacrylamide gel electrophoresis with immunoblotting to study hsp70 expression in normal, preneoplastic and neoplastic colonic mucosa. By monoclonal anti-hsp70 antibody, recognizing both the constitutive and inducible forms of hsp70, we have detected six charge isoforms localized in the pH 5.1-5.3 range and in the molecular mass range of 68-69 kDa in normal colonic mucosa. Immunostaining of hsp70 in polypous and malignant tissues revealed qualitative as well as quantitative changes in the expression of more acidic isoforms of hsp70 in comparison with normal tissue. Furthermore, the different basic isoforms of hsp70 were detected in chronically inflamed colonic mucosa from patients suffering from ulcerative colitis (UC) or Crohn's disease (CD). Besides the standard hsp70 protein pattern, additional proteins with molecular masses of about 39, 40, 74 and 75 kDa were variably immunostained in normal and pathological specimens. These observations suggest that hsp70 expression may be closely linked to disease etiology and/or pathophysiology.

Based on a retrospective review of 101 injuries of the thoracolumbar spine operated at the Orthopaedic and Traumatological Clinic of the Third Medical Faculty, Charles University in 1991-1995 the authors followed up a group so-called pincer fractures. A typical feature of these fractures is a comminuted zone of the median portion of the vertebral body, dislocation of the anterior fragment of the vertebral body in a forward direction and filling of the gap which thus arises with material from the torn intervertebral disc. "Pincer" fractures tend to develop into pseudoarthroses. In the investigated group 14 fractures of this type were found (14 %). All injuries were treated from a posterior approach by internal AO fixation or USS fixation resp. in two patients in the second stage an anterior approach was used and the vertebral body was substituted. Based on analysis of the initial morphology of the fractures and the peroperative reponibility of the dislocated fragment of the anterior part of the vertebral body the injuries were classified into three groups and indication criteria for surgery were established. Groups with a non-dislocated or reponible anterior fragment can be treated from the back only by instrumentation, posterior fusion and transpedicular spongioplasty with transpedicular intercorporal desis, the group with an irreponible anterior fragment must be treated by posterior instrumentation and fusion and subsequently from an anterior approach by substitution of the vertebral body and anterior fusion. Key words: thoracolumbar spine, fractures, classification, pincer.

High-resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) was used to analyze more precisely serum and urine specimens of a patient suffering from IgD myeloma associated with renal insufficiency. The application of 2-DE with immobilized pH gradient followed by immunoblotting revealed the presence of acidic monoclonal delta chains, hidden on 2-DE by albumin. This approach also enabled to detect two other forms of delta heavy chains expressing both reduced (45 kDa) and high (110 kDa) mol. weight. The analysis of urine specimen proved the presence of three acidic isoforms of monoclonal lambda light chains together with multiple monoclonal light chain fragments, which strongly suggests amyloidogenicity of these monoclonal light chains.

High-resolution two-dimensional electrophoresis was used to analyze kappa-type monoclonal immunoglobulin light chains in urine of two patients with multiple myeloma. The patients were treated, remission lasted about 2.5 years. In the period of relapse of the disease, acid isoforms of kappa chains, pI < 4,95, which had not been present in the urine of these patients before, were found in urine of both patients. Kappa-type Bence-Jones protein in urine of one patient showed unusual behavior: It failed to precipitate in the range of 40-60 degrees C or on boiling.

Two-dimensional gel electrophoresis has been used to analyze polypeptide composition of specimens of human normal colon mucosa and colorectal carcinomas. Immobilized pH gradient in the first dimension improved the separation and enabled us to analyze proteins from whole tissue samples with high reproducibility. Polypeptides were separated within sigmoidal 3.5-10 pH gradient and range of mol. weight 15-200 KD. In global, protein patterns of normal and malignant tissue seemed to be close to each other. We attempted to combine two-dimensional electrophoretic separation with the immunoblotting technique using patient sera to identify possible tumor antigens. From a wealth of spots on silver stained protein maps of adjacent normal colon mucosa or tumor tissue, only few spots gave positive reaction. These spots might be classified into two groups: 1) common for normal colon mucosa and tumor, 2) specific for tumor tissue. The extent of patient's antibody reaction with antigens appeared to correspond to urinary neopterin level, an index of immune activation.

Sera containing IgD paraprotein present problems in identifying patients with monoclonal gammopathies because only a small or even no spike may be present on standard serum protein electrophoresis. We have detected heavy chains of IgD monoclonal protein by means of high resolution two-dimensional gel electrophoresis. Besides clearly identifying delta heavy chains in maps of serum proteins, we also found size and charge heterogeneity of monoclonal immunoglobulins. The results demonstrate the usefulness of two-dimensional gel electrophoresis in the analysis of selected cases of immunoglobulin malignancies.

The functions of professional phagocytes depending on oxygen are briefly discussed. After appropriate stimulation, membrane-bound enzyme systems are activated--phospholipase C, protein-kinase C and NADPH-oxidase complex--and superoxide anion is produced. This process is called respiratory burst and is essential for killing of microorganisms but it may cause tissue damage and inflammation. The influence of superoxide anion on metabolism is reviewed. More attention is paid to modulating effects of superoxide anion in connection with the development inflammatory process.

The particulate glucan (G1), soluble glucan preparations (G2 to G5, and G7) isolated from Saccharomyces cerevisiae, and glucomanan prepared from culture fluid after cultivation of Candida utilis (G6) were tested for their immunomodulatory activity in vivo and in vitro. In tests in vivo three soluble glucans (G3, G4, and G7) injected s.c. to mice in the dose of 10 mg/kg increased the cytotoxic activity of peritoneal macrophages. The influence of glucans on natural killer cells was without significance. The lymphoproliferative reaction of spleen cells to polyclonal mitogens was inhibited by all the preparations used with the exception of soluble glucan G2. The mitogenic effect of the preparations, co-stimulatory tests and direct cytotoxicity to cells of cell lines used in cytotoxicity assays were assessed in vitro. The transformation index of glucans in the study was increased according to the glucan and dose tested. Inhibition of the lymphoproliferative reaction measured by the co-stimulatory test for optimal concentration of Concanavalin A occurred in a wide range of doses for the preparations G1 to G6. The preparation G7 increased the incorporation of 3HTdR under the same conditions. The use of a suboptimal concentration of Concanavalin A revealed co-stimulatory activity of all the preparations tested. Assessment of the cytotoxic activity of peritoneal macrophages and of the activity of natural killer cells induced in vitro was complicated by the direct cytotoxicity of particulate glucan and soluble glucan G5 (carboxymethylglucan) for target cells (YAC 1, and YAC 1 and K 562 resp.).

The activation of peritoneal macrophages in the course of primary infection of mice with attenuated strain of Francisella tularensis is associated with 2.5 fold increase in spontaneous INT reductase activity on day 5 after the immunization. The splenic cells of immunized mice pulsed in vitro by specific antigen secrete lymphokine that is able to induce an increase in spontaneous INT reductase activity of resident peritoneal cells. The production of spontaneous superoxide anion by peritoneal phagocytes reaches the highest level on day 5 after the immunization. It does not correlate with the results of cytotoxic or phagocytic activities at this time interval. An enhanced superoxide dismutase activity precedes an increase of superoxide anion secretion. The production of hydrogen peroxide is rising till day 7 and is related to the cytotoxic activity of peritoneal phagocytes. Concerning the testing of F. tularensis antigen as immunization agent, no changes of oxidative metabolism were detected. This might be in connection with the insufficient protection effect of killed F. tularensis vaccine. The production of reactive oxygen metabolites, probably under the control of superoxide dismutase, together with secreted lymphokines during the first days after the infection may play a regulatory role in the induction of immune response against intracellular pathogen F. tularensis.

The presented multistep program for testing immunomodulatory properties of biological response modifiers offers the possibility to evaluate multilaterally the modulatory potential of the tested preparations and to obtain basic data concerning their acute immunotoxicity. The scheme is divided into four stages: screening, optimalization, modelling, and analytical stage, each of which can be carried out separately on a relatively individual basis. This division considers the effect of the preparation on basic functions of the cells participating in the network of immunoregulation, the influence on the course of the immune response, modulation of the reactivity of the immune system affected by a 'non-immunological' phenomenon, as well as changes in protein repertoire produced by different systems of cells exposed to the tested preparation and/or the immunogenic signal. Detailed toxicological evaluation should be performed separately from immunopharmacological evaluation, keeping in mind differences in the application of the substances and the animal model involved. Incorporation of mathematical modelling and computer analysis of the results obtained by using the scheme may prove valuable in solving problems associated with potential modulation of the host immune system in clinically significant situations.

During the last several years, the important progress has been achieved in studying activation and proliferation processes altogether with differentiation of mononuclear cells and regulatory course of immune response. In addition to molecular biology, also the biochemical characterization of interleukins, mainly Interleukin 2 (IL 2), is of a substantial import. IL 2 is actionning as an amplificator of T and B immune cellular reactions, and it influences the production of lymphokines, i.e., gamma interferon as well as participates in antitumorous immunity. IL 2 belongs to the range of products being secreted by T lymphocytes following the specific antigenic or polyclonal mitogenic simulation of cells. It can be classed as a growth factor of cells and regulatory factor of immune responsiveness. The human IL 2 was isolated as a protein with Mr 16 kD. On the basis of complementary DNA, the molecular weight has been determined for IL 2 as 15.4 kD. The IL 2-related human gen is localized on 4th chromosome and the product of the respective gen is a protein composed of 133 amino acids. This very protein undergoes glycosylation on the 3d position having alpha-helicoid conformation. The murine IL 2 occurs rather as a dimer composed of two protein chains with Mr 16-18 kD. Variable degrees of glycosylation involve the heterogeneity and pI differences in isolated forms of IL 2. Actions of IL2 are triggered by its interaction with specific receptor, which appears on the T cells no sooner as they are activated by the antigen or mitogen. Following the IL 2 with receptor interaction, the hydrolysis of phosphatidylinositolphosphate occurs intracellularly as well as activation of proteinkinase C and a track of other biochemical reactions non-elucidated till now, which lead up to the transcription regulation of genes and transfer of mitotic apparatus from G1 into S phase of cellular cycle and of division of cells.