Myogenic commitment of human stem cells by myoblasts Co-culture: a static vs. a dynamic approach.

An in-vitro model of human bone marrow mesenchymal stem cells (hBM-MSCs) myogenic commitment by synergic effect of a differentiation media coupled with human primary skeletal myoblasts (hSkMs) co-culture was developed adopting both conventional static co-seeding and perfused culture systems. Static co-seeding provided a notable outcome in terms of gene expression with a significant increase of Desmin (141-fold) and Myosin heavy chain II (MYH2, 32-fold) at day 21, clearly detected also by semi-quantitative immunofluorescence. Under perfusion conditions, myogenic induction ability of hSkMs on hBM-MSCs was exerted by paracrine effect with an excellent gene overexpression and immunofluorescence detection of MYH2 protein; furthermore, due to the dynamic cell culture in separate wells, western blot data were acquired confirming a successful cell commitment at day 14. A significant increase of anti-inflammatory cytokine gene expression, including IL-10 and IL-4 (15-fold and 11-fold, respectively) at day 14, with respect to the pro-inflammatory cytokines IL-12A (7-fold at day 21) and IL-1ß (1.4-fold at day 7) was also detected during dynamic culture, confirming the immunomodulatory activity of hBM-MSCs along with commitment events. The present study opens interesting perspectives on the use of dynamic culture based on perfusion as a versatile tool to study myogenic events and paracrine cross-talk compared to the simple co-seeding static culture.

Accuracy and clinical usefulness of scoliosis measurement with magnetic spine mapping--Ortelius.

OBJECTIVES: Our objective was to assess the accuracy of radiation-free magnetic method of scoliosis curve measurement (Ortelius 800) and its usefulness in clinical practice, as well as to judge whether it can replace x-rays, which represent currently the golden standard for scoliosis measurement. BACKGROUND: Patients with adolescent idiopathic scoliosis have to undergo multiple full-length spinal x-rays. Repeated exposure to ionizing radiation leads to higher incidence of breast cancer, thyroid cancer, and leukemia. One of the radiation-free methods that are candidates for replacing the x-rays is the magnetic measurement of the spine with Ortelius 800. METHODS: Twenty-eight patients with adolescent idiopathic scoliosis were measured with Ortelius. Data from Ortelius were compared with standard spinal x-rays taken the same week. RESULTS: The average Cobb's angle measured by x-ray was 35.11 degrees. The average absolute difference between Ortelius and x-rays was 6.69 degrees. The difference was statistically significant (p=0.0443). An acceptable difference was measured in subgroup of curves that do not exceed 40 degrees (difference 5, p=0.8915). CONCLUSION: Ortelius is most accurate in curves in range of 15-30 degrees of Cobb's angle. In larger curves it is increasingly inaccurate. Most important for clinical practice is the Cobb's angle range over 40 degrees, where the conservative therapy is to be replaced with surgery. Ortelius is not accurate enough in this range and therefore cannot fully substitute full spinal x-rays. It can be used to reduce the number of x-rays in the follow-up of smaller curve angles (Tab. 4, Fig. 8, Ref. 10).

Quality of life after elective lumbar spinal fusions.

OBJECTIVES: Our goal was to evaluate the quality of life in patients who underwent an elective lumbar stabilization and fusion. MATERIALS AND METHODS: We treated 208 patients (120 females and 88 males) for degenerative lumbar spine conditions. In 165 cases we performed the transforaminal lumbar interbody fusion (TLIF), in 38 the posterolateral lumbar fusion (PLF) and in 5 the anterior lumbar interbody fusion (ALIF). Before and after the surgery, pain was evaluated with the VAS and the quality of life with the second version of the SF-6. Follow up was from six months to two years. We used the statistical tests chi-square, t-test and ANOVA. RESULTS: Satisfied were 62.5% of patients, partially satisfied were 19.7% and dissatisfied were 17.8% of patients. The average VAS decreased from 68 to 35. There was a significant improvement in mobility and decrease of consumption of analgetics after the surgery. Clinically significant improvements were in all domains of sf-36 except the mental health, social function and mental composite score. We had 30 complications in 28 patients. A revision surgery was necessary in 18 cases. Complications had no significant effect on the SF-36 and VAS score. CONCLUSION: The results are comparable with published data. Correctly indicated spinal operations are effective in reducing pain and improvement of the function. We have noted a significant decrease of pain, improvement of mobility, and a reduction of analgetics consumption. Despite the improvements, post-operatively all values of the SF-36 were lower than standards for the population. Complications and reoperations in our group are relatively common problems, surprisingly did not affected the quality of life after surgery (Tab. 9, Ref. 38). Full Text (Free, PDF) www.bmj.sk.

[The effect of complications on the quality of life after surgery for lumbar spine degenerative disease]. / Vplyv komplikácií na kvalitu zivota po stabilizacných operáciách degeneratívnych ochorení lumbálnej chrbtice.

PURPOSE OF THE STUDY: To evaluate the effect of complications on the quality of life in patients after elective stabilisation surgery on the lumbar spine. MATERIAL AND METHODS: Between January 2005 and June 2007, 208 patients (120 women and 88 men) were included in the prospective study carried out at our department. These patients were undergoing elective surgery for lumbar spinal disease, namely, central and lateral stenosis, degenerative disc disease and degenerative and isthmic spondylolisthesis. All patients were treated by transpedicular fixation and fusion involving transforaminal lumbar interbody fusion (TLIF) in 165 patients, anterior lumbar interbody fusion (ALIF) in five and posterolateral fusion (PLF) in 38 patients. Satisfaction of the patients with surgery outcomes was assessed on a three-point scale, using the Visual Analogue Scale (VAS), and the Short Form health survey questionnaire (SF-36v2) for life quality evaluation. The follow-up period ranged from 6 months to 2 years. The results were statistically analysed using the chi-square test and t-test. RESULTS: A total of 30 complications were recorded in 28 patients (13.5 %). Revision surgery was necessary in 18 patients (8.7%). Pedicle screw misplacement was found in eight patients and permanent neurological deficit with paresis of the unilateral lower limb in three patients. Carbon cage break-down during surgery occurred in one patient, misinsertion of the cage was in one patient. The dural sac was damaged in five patients, superficial and deep wound infection was found in four and two patients, respectively. Broken screws were detected in seven patients. Donor-site pain persisted in two patients. The patients free from complications were more satisfied (partial or full satisfaction in 86%) than the patients with complications, who reported satisfaction in 78%. However, the difference was not statistically significant. The complications had no significant effect on either any of the SF-36v2 health domains or the total physical and mental score or pain intensity. The ALIF procedure was associated with no complications, PLF with three (13%) and TLIF with 23 (14%) complications, but the differences were not statistically significant. DISCUSSION: Complications and revision surgery were relatively frequent in our group, but not excessive compared with the published data which report their occurrence from 3% to 30%. This large range is related to an ambiguous definition and specification of complications. A significantly fewer complications have been reported in the studies supported by companies, or in those in which the author/s participate in instrumentation development. Some studies suggest that novel and more complex techniques bring about a higher risk of complications and repeat surgery, others report better results and fewer reoperations following a 360-degree fusion than after PLF. The difference in complications between TLIP and PLF in our group was not significant. CONCLUSIONS: Complications are a serious issue of spinal surgery. The majority of complications in our group were associated instrumentation--most frequently with incorrect screw misinsertion. On the whole, the complications did not significantly influence the quality of life and pain intensity after surgery. Most of them were treated successfully with no permanent consequences. The permanent neurological deficit in three patients, because of the small number, had no effect on the whole group results. A reduction in the number of complications would not produce any expected improvement of functional out- come.

SOD1 activity and protective factors in familial ALS patients with L84F SOD1 mutation.

METHOD: The activity and amount of SOD1 in erythrocyte lysates and the plasma amino acid content were evaluated in four familial ALS patients bearing the L84F SOD1 mutation (fALS), in an asymptomatic family member with the mutation (L84F(5)), in sporadic ALS patients (sALS) and controls. Three of the fALS patients and the L84F(5) subject were tested once a year for three consecutive years. RESULTS: At the first evaluation SOD1 activity was similar in controls, sALS and fALS; the amount of SOD1 protein was lower (P < 0.01) in fALS. In the subsequent 2 years, 34% and 52% decrease of SOD1 activity was recorded in fALS patients. The plasma amino acid pattern did not differ between controls and sALS, whereas fALS patients displayed high levels of plasma aspartate and glutamate. Aspartate was in the normal range but glutamate was still elevated in the subsequent evaluations. The L84F(5) subject had remarkably low levels of aspartate, glutamate and branched-chain amino acids. CONCLUSIONS: The method of measuring mutant SOD1 amount is indirect but the results are indicative of a reduction of mutant SOD1 taking place during fast-worsening phases of the disease. Since the disease onset of fALS patients is 42.8 +/- 11.3 years and the L84F(5) family member is asymptomatic at the age of 66, low levels of excitotoxic and branched-chain amino acids in plasma may constitute a protective factor against disease development.

[Our Experience in the Revision Surgery of THR.]. / Nase skúsenosti s reimplantáciou TEP koxy.

The authors present their experience in the revision surgery of THR. Most often it was the case of aseptic loosening of the acetabulum, the femoral component or both components (67 %), lytic radioluscence or even lysis in the region of implants, fissures even fractures of the femur and septic loosening. They evaluate the results of revision surgeries of THR for a 5-year period in 106 patients after Merle dAubigne and Postel: excellent result was achieved in 33 %, good in 39 %, average in 19 % and a poor result in 9 %. The authors emphasize in case of primary surgery the importance of indication criteria, age of the patient, appropriate choice of the type of implant, evaluation of the immunological condition of the patient. In revision surgeries of aseptic loosening of the implant and bone defects of the medial acetabular wall efficient was mainly the application of bone grafts and cementless acetabular cup of the Harris-Galante, Zweymüller or Balgrist type. Periprothetic fractures of the femur should be treated by internal fixation and Wagner revision stem. In case of septic loosening the endoprosthesis was extracted or a two-phase revision surgery performed. Key word: THR revision surgery, aseptic loosening, bone defects, Wagner revision stem.

[Results of application of hydroxyapatite and hydroxyapatitecollagen.]. / Výsledky pouzitia hydroxyapatitu a hydroxyapatitkolagénu na výpln kostných defektov.

The authors present their experience of the application of hydroxyapatite and a composite-hydroxyapatitecollagen in 2 groups of patients. In the first group they evaluate the mediumterm results in 10 girls and 8 boys of average age of 12,5 years after the filling of bone defects in case of bone cysts, aneurysmatic cysts, fibrous cortical defect and eosinophilous granuloma. Hydroxyapatite was applied in the form of rolls, small bricks, strips. The first signs of healing were evident as early as after 4 months, after 36 months osteointegration was practically finished. In the second group of 1995-2000 period they applied hydroxyapatite in 15 patients of average age of 45 years. In this group they applied the mentioned biomaterial also in 6 patients with instabilities and primary and secondary (metastasis) bone tumours on the spine where they also achieved after a shorter follow-up of 6-8 months good osteoinductive and conductive qualities of the biomaterial. The authors point out the two biggest contributions in this respect: filling of bone cavities with an unusually big amount of hydroxyapatite (up to 70 g) and good, even though only short-term results after resection and filling of malignant bone tumours. Key words: hydroxyapatite, collagen, osteoinduction, osteoconduction, bone defects.

[Comparison of the treatment of juvenile bone cysts by corticoids and bone grafting.]. / Porovnanie liecby juvenilných kostných cýst kortikoidmi a spongioplastikou.

Juvenile bone cysts are frequent affections. Their treatment develops in the direction of the application of less invasive methods. The authors treated 43 patients with this diagnosis in the period of six years. The aim of the article is to specify the criteria for the indication of two most frequently used methods of treatment of juvenile bone cysts, namely. a)percutaneous instillation of methylprednisone acetate (MPA); b)surgical curettage and bone grafting of the cyst by an autograft or allograft. They state that significant for the prognosis of the juvenile bone cyst are: 1. the age of the patient, 2. activity or latency of the juvenile bone cyst, 3. its location. Worse results were recorded in the treatment of active cysts. The highest incidence of active cysts falls into the period of growth acceleration, i. e. between the seventh and tenth year. Worse results were achieved in the treatment of younger patients. In younger patients with juvenile bone cyst they recommend as the method of choice a less invasive MPA treatment. If the cyst does not heal after instillation repeated three times, the juvenile bone cyst is MPA resistant and it is necessary to change the method of treatment. In older patients with large cysts (femur, tibia) the authors recommend to apply bone grafting as the primary method. Worse results in both methods were recorded on weight bearing bones (a more frequent incidence of MPA resistant juvenile bone cysts). In MPA resistant juvenile bone cysts the authors had to use bone grafting five times more often due to the persisting difficulties. Key words: juvenile bone cyst, methylprednisone acetate, bone graft.

[Acetabular Replacements in THR in Dysplastic Osteoarthritis of the Hip.]. / Náhrady acetábula pri totálnych endoprotézach u dysplastických koxartróz.

The authors present different options of the surgical treatment of dysplastic osteoarthritis of the hip. For the acetabular replacement they use Harris-Galante, Zweymúller and Balgrist cementless cups. They evaluate results for a five-year postoperative period in 182 patients. On the basis of Merle d'Aubigne and Postel evaluation system the results were excellent in 79,6 % and very good in 12,1 %.The authors recorded only a relatively small percentage of complications (10,9 %) relating to the wound infect, loosening of the cup, trombophlebitis and palsy of n. peronaeus com. or n. femoralis. The authors emphasise the importance of CT examination of the hip joint in the preoperative preparation and of the knowlege of biomechanics of pelvis for the insertion of the cementless cup. The replacement of acetabulum in dysplastic osteoasthritis of the hip is an open issue - there exists no ideal cup for each case. Key words: dysplastic osteoarthritis of the hip, cementless cup, THR, acetabuloplasty.

Effect of beta-carotene on cell cycle progression of human fibroblasts.

The uptake of beta-carotene (BC) and its effect on the cell cycle progression of normal human fibroblasts in primary culture were investigated by using two different delivery methods: exposure to BC solubilized in the organic solvent tetrahydrofuran (THF) or to BC incorporated into dipalmitoylphosphatidylcholine (DPPC) liposomes. Cell cycle progression was evaluated by immunofluorescence detection and flow cytometric analysis of the proliferating cell nuclear antigen (PCNA). In contrast to THF, which induced a marked reduction in the number of cells in S phase and in the extent of PCNA immunolabeling, DPPC liposomes proved to be an effective delivery system that does not interfere with cell proliferation. Cellular uptake of 0.23 nmol/10(6) cells was found after 24 h incubation in BC-containing DPPC liposomes. This value increased to 1.2 nmol/10(6) cells after 72 h. After the first day of incubation, the number of cells in S phase was reduced by approximately 50%, with a consequent accumulation of cells in G1 phase. This effect was maintained up to 3 days incubation, with no detectable effects on cell viability. This cell cycle delay was found to be reversible, returning the percentage of cells in S phase to the control value 24 h after removal of BC from the medium. In order to determine whether the activity of BC could be attributed to the molecule itself or to its conversion into retinoids, the production of BC metabolites was assessed. Analysis of cellular levels of retinoids failed to demonstrate the presence of retinal, retinol, retinoic acid or retinyl esters during an incubation period of 6 days. These results suggest that in normal human fibroblasts, BC induces a cell cycle delay in the G1 phase and that this effect is independent of conversion to known retinoids.

Effects of beta-carotene and alpha-tocopherol on photogenotoxicity induced by 8-methoxypsoralen: the role of oxygen.

The protective effect of beta-carotene (beta-C) and alpha-tocopherol (alpha-T), singularly and in equimolar mixtures, toward the photomutagenicity induced by 8-methoxypsoralen (8-MOP), at different oxygen partial pressure (pO2), was evaluated in two different experimental models: Salmonella typhimurium TA102 and Saccharomyces cerevisiae D7. After phototreatment with 8-MOP, the results show a lethal effect under hypoxic conditions in both experimental model systems, an increase in revertants associated to the pO2 increase in S. typhimurium TA102, and a decrease in revertants and convertants associated to the pO2 increase in S. cerevisiae D7. In S. typhimurium TA102, in atmospheric condition, beta-C and alpha-T (1.86 or 18.6 microM) show a protective effect only at the higher dosage. Alpha-T was more protective than beta-C. The equimolar mixtures show an antimutagenic effect at both dosage used with a synergistic effect at lower dosage and an additive antimutagenic activity at higher dosage. An inhibition of the spontaneous mutagenicity by mixtures at higher dosage was also observed. The results obtained in S. typhimurium TA102 show an antimutagenic effects of beta-C, alpha-T and their mixture at 190 mmHg pO2, confirming the data obtained in air condition. At 380 mmHg pO2, alpha-T and the mixture show a significant antimutagenic activity; at 570 mmHg pO2, only alpha-T is protective. At 760 mmHg pO2, no protective effect was observed by the two antioxidants, and beta-C increases the photomutagenicity induced by 8-MOP. In S. cerevisiae D7 a protective effect was only observed at 380 mmHg pO2 with the mixture. No antigenotoxic effect was found in the other experimental conditions, even if the uptake of the two antioxidants was confirmed by HPLC. Our results underline the role of oxygen in the photomutagenicity induced by 8-MOP and in the antimutagenic activity of beta-C and alpha-T. This is the first report confirming in a cellular experimental model the data obtained in some chemical systems: the protective effect of beta-C only at low pO2 and the synergistic effect of mixture of beta-C and alpha-T.

[Osseointegration of titanium test bodies of different surface properties in metaphyseal bone sites of the dog--a biomechanical and histological analysis]. / Das Einwachsverhalten von Titan-Prüfkörpern unterschiedlicher Oberflächenbeschaffenheit im metaphysären Knochenlager des Hundes--eine biomechanische und histologische Analyse.

The alternative to the anchoring of an endoprosthesis by means of cement is the biological fixation by an ingrowth of bone into the implant surface (osseointegration). We examined the implant fixation properties of titanium experimental devices with 3 different surface structures after press-fit implantation into the bony bed of 12 dogs. One third of the 48 implants had a micro-structured surface roughened by grit-blasting, one third a roughened surface with an additional macro-groove structure (combination surface), and the remaining third a porous hydroxyapatite (HA) coating. Twelve weeks after implantation the bony ingrowth was evaluated biomechanically by measuring the force required to pull out the implant from the surrounding bone and histologically by morphometric assessment of microradiographs. In the pull-out-experiment the shearing forces were significantly lower (p < 0.01) in the devices with a roughened surface in comparison to the devices with a micro- and macro-structured surface and the HA surface. Thus, there was no significant difference between the forces required to pull out the devices with the porous HA surface and those with the combination surface. The histomorphometric assessment of bone density in the immediate vicinity of the implant and the extent of the direct contact surface of the bone implant resulted in no significant difference in all 3 groups. We did not find an interposing layer of fibrous tissue at the interface. We can demonstrate that roughening of the surface in combination with a groove structure creates a better bond between implant and bone than a roughening alone.(ABSTRACT TRUNCATED AT 250 WORDS)

[Comparison of surgical and radioactive synovectomy in various disorders of the knee joint]. / Porovnanie chirurgickej a rádioaktívnej synovialektómie pri rôznych afekciách kolenného klbu.

The authors evaluate after an interval of 1-20 years the long-term results of surgical synovectomy in 454 patients and radioactive synoviorthesis in 528 patients performed at the First Orthopaedic Clinic, Medical Faculty, Comenius University and Faculty Hospital in Bratislava and at the Orthopaedic and Traumatological Clinic of the Medical Faculty in Martin in 1969-1991. The most frequent diagnosis in surgical synovectomy is rheumatic synovitis in radioactive synovectomy arthrotic synovitis (47.6% and 68.6%). The authors evaluate both methods of synovectomy according to Aidem-Baker and compare the results of the two methods. They found significantly more very good and good results after surgical synovectomy (54.8% and 33.5%) as compared with radioactive synoviorthesis (15.5% and 38.0%). Conversely they found significantly more satisfactory and poor results in radioactive synoviorthesis (29.1% and 17.4%) than after surgical synovectomy (8.8% and 2.9%) which comprise patients relatively satisfied with this procedure and who do not agree with other procedures.

[Infections at the paediatric department of the first orthopaedic clinic in bratislava.]. / K otázke infekcií na detskom oddelení I. ortopedickej kliniky v Bratislave.

At the First Orthopaedic Clinic in Bratislava during the last two years 748 children were hospitalized. Of these 405 had surgical operations: 260 operations of bones, 61 operations of joints and 84 operations of soft parts. Patiens with osteomyelitis and pyarthrosis were hospitalized in 0,5 %, with reactive arthritis in 0,6 % (not associated with the surgical operation made in the authors' department). For prophylactic and therapeutic administration of antibiotics Ampicillin was administered in 266%, Oxacillin in 15,8 %, Cephalosporins in 12,8 %. Prophylactic administration of antibiotics, incl. the first dose before surgery, accounted for 626%. After this antibiotic prophylaxis the inci dence of secondary chronic osteomyelitis was 0,24 %. Surface infections of the wound were re corded in 3,4 % and deep infections in 1,2 %. Non-orthopaedic infections (mainly upper respira tory pathway infections) during the postoperative period occurred in 7,1 %, they varied, however, de pending on the epidemiological situation. Based on their own observations and data in the literature the authors recommend antibiotic prophylaxis in children for 24-48 hours in the following order: Cephalosporins, Oxacillin, Ampicillin. When changing from prophylactic to therapeutic antibiotic administration it is necessary to take into account the clinical picture and state of the wound. Febrility, red cell sedimentation rate, Le do not always correlate with the infection of the wound. In children during the postoperative period also frequent non-orthopaedic infections must be taken into account. Key words: osteoarticular infection, incidence, antibiotic prophylaxis.

[Long-term results of the Collona acetabuloplasty]. / Dlhodobé výsledky Colonnovej acetabuloplastiky.

The authors evaluate in their paper the long-term results of capsular acetabuloplasty by Colonna's method in the material of the First Orthopaedic Clinic, Faculty Hospital Bratislava. The mean time interval after operation was 20 years. The results are compared with previous evaluations made by Cervenanský and Kalman. Very favourable results were recorded in 3% of the patients, good results in 24%, satisfactory results in 39% and poor results in 39% and poor results in 34% of the patients. A marked shift towards unsatisfactory results is due to incorrect indication of acetabuloplasty which in the was a certain compromise, and also due to the surgical technique. The authors assume that correct and early prevention and treatment will eliminate capsular acetabuloplasty from the range of standard surgical therapeutic procedures in L.c.c.

[Problems in the diagnosis of Ewing's sarcoma]. / Problémy diagnostiky Ewingovho sarkómu.

The aim of the work was to analyze by the method of retrospective study the difficulties in diagnosing the Ewing sarcoma at the Department of Orthopaedic of the Teaching Hospital in Bratislava in the years 1967-1987. The authors found out that there were diagnosticated 28 cases of the Ewing sarcoma, the boy-girl ratio was: 1:1. Most frequently it occurred between 12 and 18 years of age. Apart from clinical examination they use classical sciagraphy, macropictures, tomography, arteriography, gammagraphy of the skeleton, computer tomography, lymphography, histological, and electron microscopic examination. In the conclusion the authors point out that the timely treatment may be ensured by a precise and demanding screening carried out by pediatricians, practitioners in charge of adolescents and orthopaedists and by further reduction of the time interval necessary for performing all above mentioned examinations. The care of the Ewing sarcoma is the responsibility of suitably equipped special centres of bone oncology.

[Mineralization status in bones in patients with femoral neck fractures]. / Stav mineralizácie kosti u pacientov s fraktúrou krcka femoru.

The authors present the results of the evaluation of the state of the art of mineralization of the bone in 56 patients over 45 years of age with the fracture of the femoral neck. The state of the art of mineralization was evaluated by means of bone indices. In each patient there were determined 6 values of bone indices and thus they got a more objective picture of the state of the art of bone tissue. They found out that most cases of fractures occurred on the osteporous skeleton. For pathological values corresponding to osteoporosis they considered such values when the bone index according to Barnet-Nordin was less than 45, Garn index less than 67, Singh index equal to the values I, II, III and Kocian index corresponded to normograms. Lower cortico-diaphyseal index on the side of the fracture in comparison with the contralateral side they explain by timely demineralization after the fracture during immobilization. Proceeding from the results achieved they emphasize the necessity of a timely prevention and treatment of osteoporosis.

The use of protein as a carrier of methotrexate for experimental cancer chemotherapy. III. Human serum albumin-methotrexate derivative, its preparation and basic testing.

The preparation and a more detailed characterization of human serum albumin-methotrexate derivative (HSA-MTX) is described. The synthesis of the derivative was performed by means of 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (in methoiodide form) (WSC). Results of many experiments showed that, on the average, about 26 molecules of methotrexate (MTX) were coupled to one molecule of human serum albumin (HSA). The relative molecular weight of the formed derivative was estimated by gel chromatography on Sepharose 6B or on high-pressure liquid chromatography (HPLC) on SWK column, respectively. From the obtained data it follows that a considerable part of the HSA-MTX derivative formed protein-protein conjugate (up to about 4 X Mr HSA), nevertheless the derivative retains its good solubility as a native albumin. In order to eliminate the possibility of influencing the cytostatic activity of the derivative with byproducts of its synthesis, human serum albumin-folic acid derivative (HSA-FA) was prepared and tested by the same method. All demonstrated experiments proved that MTX was the only compound possessing the cytostatic activity. During the experimental therapy of Gardner lymphosarcoma (LSG) the following was found: (1) The intratumorous application of the drug was the most effective way of administration. (2) Any type of administration of the HSA-MTX derivative exerted a better effect than the same way of administration of free MTX. (3) The comparison of two (repeated) administrations of both drugs showed clearly that the HSA-MTX derivative was more efficient than free MTX. After HSA-MTX derivative treatment all animals survived without tumor. (4) For the estimation of the toxicity of the HSA-MTX derivative, three times and five times repeated intraperitoneal administration was performed. It was concluded that although the derivative was more toxic than free MTX, its therapeutic activity was better. After the elimination of the toxic manifestation of the HSA-MTX derivative by a suitable arrangement of drug doses, five times higher efficacy of the derivative was reached, as compared with free MTX. (5) The therapy by the HSA-FA derivative did not exhibit any therapeutic effect. The reason why HSA was used as a macromolecular carrier for cytostatics is discussed.