The Regenerative Potential of Donkey and Human Milk on the Redox-Sensitive and Proliferative Signaling Pathways of Skin Fibroblasts.

The influence of milk bioactive peptides on skin regenerative potential and rejuvenation is very often limited because of allergic reactions. The current study is aimed at exploring the influence of donkey colostrum and mature milk, human colostrum and mature milk, and ß-casein and ß-casomorphine-7, on the growth and inflammatory response of the culture of cultured skin fibroblasts exposed to these conditions for twenty-four hours. Their effects on the growth-regulatory kinases and redox-sensitive, proinflammatory transcriptional factor NF-κB were detected by using specific primary antibodies against NF-κB p65, Akt-1, phospho-Akt-1, Erk-1, phospho-Erk-1, JNK, phospho-JNK, phospho-STAT-1, and CD26, while logarithmic integrated fluorescence intensity patterns were recorded by flow cytometry. The downregulation of NF-κB p65 was observed after the exposure of skin fibroblasts to donkey milk and human colostrum, while ß-casein and ß-casomorphine-7 exerted the opposite effect, which suggests that noncasein bioactive peptides of donkey and human milk may be responsible for anti-inflammatory properties. The exposure to all milk species examined and ß-casein leads to the activation of growth-regulatory kinases (Akt1/2/3 kinase, Erk kinase, JNK kinase, and Stat-1 kinase), especially for the p-Erk pathway, which suggests that essential amino acids of casein may be responsible for Erk-induced cell cycle activation and proliferation. The opposite effect was observed when cells were exposed to ß-casomorphine-7, which may affect the skin fibroblast survival and their proliferative and regenerative potential. Donkey milk did not significantly change the CD26 antigen expression. In conclusion, our results suggest that among cell signaling molecules, the most sensitive but nonspecific downstream effector is p-Erk kinase, which may point to donkey milk usefulness in wound healing, regenerative, and aesthetic dermatology. The noncasein bioactive peptides of donkey milk may be responsible for the anti-inflammatory property of donkey milk and colostrum, which may indicate the usefulness in the treatment of inflammatory skin diseases.

Crosstalk between possible cytostatic and antiinflammatory potential of ketoprofen in the treatment of culture of colon and cervix cancer cell lines.

OBJECTIVES: The aim of this study was to test the cytostatic potential of ketoprofen in the in vitro treatment of cells derived from colon and cervix cancer. BACKGROUND: NF-κB and cyclooxygenase can have a role in different stages of the development and progression of cancer. In recent years, special attention has been paid to the possible cytostatic potential of nonsteroidal anti-inflammatory drugs. There are no published data on the use of ketoprofen in pharmacotherapy of the colon and cervical carcinoma. METHODS: We examined the effect of ketoprofen alone or in combination with cisplatin and 5-fluorouracil on proliferation of the two cell lines, HeLa (human cervical carcinoma cells) and Caco-2 (human colon cancer cells) by MTT test. Measurement of the level of NF-κB was also performed in the cells of both cell lines. RESULTS: The results of present study have shown that at least one of the mechanisms of antiproliferating and/or cytostatic effects of different concentrations of ketoprofen on Caco-2 and HeLa cells could include the transcription factor NF-κB. CONCLUSIONS: Since this transcription factor is controlled by the altered expression of COX-2, the inhibition of this enzyme by ketoprofen may represent a significant step in synergistic cascade of the therapy and prevention of colon and cervical cancer (Tab. 4, Ref. 31).

Effects of depsidones from Hypogymnia physodes on HeLa cell viability and growth.

The anti-proliferative activitiy of Hypogymnia physodes methanol extracts (ME) and its main constituents, physodalic acid (P1), physodic acid (P2), and 3-hydroxy physodic acid (P3), was tested on human cancer HeLa cell lines. Three lichen depsidones, P1, P2 and P3, were isolated from H. physodes ME using column chromatography and their structures were determined by UV, ESI TOF MS, 1H and 13C NMR. The content of P1, P2 and P3 in ME was determined using reversed-phase highperformance liquid chromatography with photodiode array detection. P1-3 represented even 70 % of the studied extract. The HeLa cells were incubated during 24 and 72 h in the presence of ME and depsidones P1, P2 and P3, at concentrations of 10-1000 µg/ml. Compounds P2 and P3 showed higher activity than compound P1. Half maximal inhibitory concentrations (IC50, µg/ml) of P1, P2, P3 and ME for 24-h incubation were 964, 171, 97 and 254 µg/ml, respectively, while for 72-h incubation they were 283, 66, 63 and 68 µg/ml. As far as we know, this is the first report on the effect of H. physodes ME and their depsidones on HeLa cells.

Chemopreventive potential of alpha lipoic acid in the treatment of colon and cervix cancer cell lines.

OBJECTIVES: The nuclear factor κB regulates the expression of genes involved in many processes that play a key role in the development and progression of cancer. The aim of this study was to examine the influence of the alpha lipoic acid in the chemoprevention of colon and cervix carcinoma in vitro. BACKGROUND: In recent years, special attention has been paid to the potential chemopreventive properties of antioxidants. There are no published data on the impact of alpha lipoc acid of chemoprevention of cervix and colon cancer. METHODS: We examined the effect of alpha lipoic acid alone or in combination with cisplatin and 5-fluorouracil on proliferation of the two cell lines, HeLa (human cervical carcinoma cells) and Caco-2 (human colon cancer cells) by MTT test. The measurement of the level of transcription factor NF-κB was also performed in the cells of both cell lines. RESULTS: At least one of the mechanisms of the antiproliferative and/or cytotoxic effect of alpha lipoic acid on Caco-2 and HeLa cells at high concentrations, the transcription factor NF-κB, may be involved, as well as the products of transcription of genes that are under its control. CONCLUSION: The alpha lipoic acid has proven to be a promising candidate in the combat arena against cancer (Tab. 4, Ref. 31).

Molecular pathogenesis of borderline and invasive ovarian tumors.

Ovarian cancer still ranks first as the leading cause of death among gynecological malignancies. Malignant transformation of normal ovarian epithelial cells is caused by genetic alterations that disrupt regulation of proliferation, programmed cell death, and senescence. The vast majority of ovarian tumors arise due to accumulation of genetic damage, but the specific genetic pathways for the development of epithelial ovarian tumors, borderline and malignant, are largely unknown. Our results show that in progressive stages of carcinoma, the oxidative stress can contribute to the uncontrolled tumor expansion. Circulating levels of antioxidants may be important to consider when evaluating a woman's risk of cancer, even among women who are at higher predicted risk. The purpose of this article was to review the current approaches to molecular pathogenesis of borderline and invasive epithelial ovarian tumors.

[Treatment of femoral shaft fractures in polytrauma patients using Mitkovic type internal fixator].

Polytrauma remains a major social, economic and medicine affliction. Successful surgical treatment of polytrauma patient's requires an approach predicated on prioritizing injuries. An isoleted fractures rarely poses any threat but in association with multiple injuries, a fractures assumes greater significance. Proper management of femur fractures in po-lytrauma can gretly reduce the mortality and morbidity. In this work the results of the operative treatments of the femoral shaft fractures in polytrauma patients using Mitkovic type internal fixator (14 patients) and Kuntscher nail (9 patients) are compared. The average patients age was 28.43 years (range 16 to 61). Internal fixation using Mitkovic type internal fixator can be method of choose in treatment of femur fractures in polytrauma patient's. Its application is relatively simple. It doesn't damage the periosteal and meduullary bone vascularization, which favors osteosynthesis and provides considerable contribution in osteogensis.

[Treatment of unstable closed tibial shaft fractures by external fixation].

The fracture of tibia shaft is one of the most common fracture of long bones. The authors bring out the results of the treatment of 90 patients with unstable closed tibial shaft fractures treated by the use of Mitkovic external fixation systems. Within the analayzed group there were 66 (73%) male and 24 (27%) female. The average patients age was 43.9 years (range 15 to 82). Excellent result in treatment of unstable closed tibial shaft fractures with external fixation were achieved in 15 (16,7%) patients, very good in 38 (42,2%) patients, good in 24 (26,7%), poor in 9 (10%) and bad in 4 (4,4%) patients. With 4 (4,4%) patients pseudoarthrosis was formed. Additional treatment of patients with pseudoarthrosis (spongioplastic and placing of CD device) led to pseudoarthrosis healing. Within the analyzed group in all patients treated with extenal fixation there were no registered cases of postoperative osteitis. External fixation by the use of Mitkovic external fixation is one of the method of choose in the treatment of unstable closed tibial fractures, providing dynamic and balanced biomehanical conditions for fracture healing.

Repair of bone tissue affected by osteoporosis with hydroxyapatite-poly-L-lactide (HAp-PLLA) with and without blood plasma.

The aim of this study is to examine the reparatory ability of the synthetic biomaterial hydroxyapatite-poly-L-lactide (HAp-PLLA), the replacement of alveolar ridge, and rehabilitation of bone defects caused by osteoporosis, in an experimental group of animals. The experiments are performed on syngeneic Sprague Dawley rats. Osteoporosis is induced by glucocorticoids in rats during a 12-week period. After this, the experimental group of animals is divided into five subgroups. An artificial defect is made in the alveolar bone on the left side of the mandible. In one group of animals, the defect is left to heal by itself, while in other groups, pure HAp-PLLA or one mixed with plasma is implanted. The best results are achieved by the implantation of the HAp-PLLA composite biomaterial mixed with autologous plasma. Formation of a new mandibular bone is seen, growing intensely, leading to rapid osteogenesis.

Biological evaluation of hydroxyapatite/poly-L-lactide (HAp/PLLA) composite biomaterials with poly-L-lactide of different molecular weights intraperitoneally implanted into mice.

Histopathologic analysis of the tissue with HAp/PLLA implants was made and the leukocyte formula and chemiluminescence response of peritoneal phagocytes 2, 7 and 12 weeks after intraperitoneal implantation studied. Implants were made of HAp/PLLA biocomposites with PLLA molecular weights of 50000 (HAp/PLLA(50)) and 430000 g/mol (HAp/PLLA(430)) and of crushed devitalized femur bone of a young Wistar rat. Leukocyte formula and chemiluminescence of peritoneal phagocytes showed no systemic inflammatory response. The studied implants caused locally weak inflammatory reaction. The resorption of implants ranges in intensity (polymer resorption, i.e. disappearance rate), from the highest with the bone implants, low with HAp/PLLA(50), to the lowest with the HAp/PLLA(430) implants. Good resorption of the biocomposites and its mutual ingrowth with connective tissue prove their good biocompatibility.

Failure of anti-tumor immunity in mammals--evolution of the hypothesis.

Observations on the morphological and functional similarity between embryonic or trophoblast tissues and tumors are very old. Over a period of time many investigators have created different hypotheses on the origin of cancerogenesis or tumor efficiency in relation to the host immune system. Some of these ideas have been rejected but many of them are still current. A presumption of the inefficiency of anti-tumor immunity in mammals due to the high similarity between trophoblast and embryonic cells to tumor cells is very real. The mechanisms for the escape of tumors from the immune response are very similar to the mechanisms for the escape of a fetoplacental unit from the maternal immune response. The similarity between these two mechanisms is so great that any randomness must be banished. At the same time, an incidence of malignant tumors and the types of more frequent tumors in non-mammalian vertebrates is significantly different to that in mammals. Lastly, the mechanisms of anti-tumor immunity in mammals are substantially different from the mechanisms of anti-tumor immunity in other classes of vertebrates. These facts indicate that the immune system of mammals during anti-tumor immune response is tricked by the similarity between tumor cells and trophoblast or other placental cells. From this aspect, our conclusion is that anti-tumor immunity failure in mammals can be defined as an immunoreproductive phenomenon, which is developed under the evolutionary pressure of autoimmunity and reproductive effectiveness.

A study of HAp/PLLA composite as a substitute for bone powder, using FT-IR spectroscopy.

Chemically synthesized hydroxyapatite/poly-L-lactide (HAp/PLLA) composite biomaterial was studied in vivo. The biocomposite was implanted into Balb/c Singen mice and after 1 and 3 weeks removed from their organisms and analyzed by the FT-IR spectroscopy. After 1 week of testing in vivo the implanted sample gave a spectrum in which absorption bands arising from newly formed functional groups of amine and peptide can be seen. After 3 weeks, a spectrum with pronounced absorption bands at 3420 and 1650cm(-1) assigned to newly generated collagen, a component of the extracellular connective-tissue matrix, was registered. Also, decrease of the intensity absorption band at 1760cm(-1) originating from the C=O group of PLLA indicates bioresorption of the PLLA used. Analysis of the microstructure of the sample surface by scanning electron microscopy before and after implantation revealed bioresorption of the PLLA polymer phase and generation of collagen fibers at the sites of implanted bioresorptive PLLA. A mixture of autologous bone powder and HAp/PLLA biocomposite was also examined. After implantation, the same final products as in the case of HAp/PLLA composite biomaterial used alone were found.

Reversal of experimental myoglobinuric acute renal failure with bioflavonoids from seeds of grape.

Rhabdomyolysis may account for about 10% of all cases of acute renal failure (ARF). This study was performed to explore the protective influence of proanthocyanidins from seeds of grape in an experimental model of myoglobinuric ARF. Rats were injected with 50% glycerol (8 mL/kg, im) followed immediately and daily in the next three days by ip proanthocyanidins (20 mg/kg) or saline. After 96 h rats were sacrificed and kidney morphology, kidney cortex peptidase activities, and malondialdehyde (MDA) content were determined. A moderate renal failure was produced by glycerol injection with blood urea of 31.8+/-11.0 vs. 7.68+/-0.24 m mol/L, and serum creatinine of 153. +/-38.2 vs. 39.6+/-9.0 micromol/L, in glycerol-induced ARF vs. control rats, respectively. Rats that received proanthocyanidins in addition to glycerol had significantly lower (p < 0.01) blood urea and serum creatinine levels compared to those receiving glycerol alone. These functional differences between the glycerol and glycerol plus proanthocianidins groups were also confirmed histologically. Kidney cortex dipeptidylpeptidase IV (DPP IV) activity was not significantly changed in glycerol-induced ARF, however, markedly increased after proanthocyanidins treatment. Kidney cortex malondialdehyde content was found significantly increased in glycerol-induced ARF over control level, and was markedly reduced by proanthocyanidin treatment. Taken together, these results provide strong evidence for the protective role of proanthocyanidins from seeds of grape in glycerol-induced ARF. The effect is probably due to the antioxidant activity of proanthocyanidins and to increased expression of kidney cortex DPP IV with effective degradation of TNF-alpha. This may provide therapeutic opportunities of preventing and/or treating myoglobinuric ARF in humans.

[Bone marrow stromal damage mediated by immune response activity]. / Ostecenje strome kostne srzi u uslovima aktivnosti imunskog odgovora.

The aim of this work was to estimate influence of activated immune response on hematopoiesis in vitro, using the experimental model of BCG immunized BALB/c mice and in patients with chronic immunoactivation: long-lasting infections, autoimmunity or malignancy. We correlated changes in long term bone marrow cultures (Dexter) and NBT reduction with appearance of anemia in patients and experimental model of immunization by BCG. Increased spontaneous NBT reduction pointed out role of macrophage activation in bone marrow stroma damage. Long-term bone marrow cultures showed reduced number of hematopoietic cells, with predomination of fibroblasts and loss of fat cells. This results correlated with anemia and leucocytosis with stimulated myelopoiesis in peripheral blood. Activation of immune response, or acting of any agent that directly changes extracellular matrix and cellularity of bone marrow, may result in microenviroment bone marrow damage that modify hematopoiesis.

[Importance of immunoregulation and erythropoiesis estimation in disorders with activated immune system]. / Znacaj ispitivanja imunoregulacije i eritropoeze u oboljenjima s aktivisanim imunskim sistemom.

Complicated immunoregulatory events in different disorders depend on the pathologic process itself and the phase of disease. Immune parameters aberrations in advanced phase of disease, compared to initial ones, may be significant, making hard the understanding of basic immunoregulatory process of the disease. Intending to explain immune mechanisms in different disorders with activation of the immune system as basic pathogenic event (infections, autoimmune diseases), particularly in advanced phase of the disease, on a parallel with standard laboratory investigations of erythropoiesis (hemoglobin, red blood cell count, hematocrit) the following examinations of the immune system were done: white blood cell count, immunophenotyping of lymphocytes, ability of the phagocytes to reduce NBT, chemiluminescent response after stimulation with opsonized and nonopsonized particles. Analysis of erythropoietic parameters in order to see the consequences of the regulatory mechanisms of the immune system (cytokines, interleukins) on hemopoiesis may enable us to understand the mechanisms of disease with the initial activation of the immune system. Finding anemia might be useful in defining the immunoregulatory mechanisms involved in particular pathologic process important for therapeutic approach.

[Effect of exanguinotransfusion on the immune system of neonate]. / Uticaj eksangvinotransfuzije na imunski sistem novoredjenceta.

Exanguinotransfusion is commonly used during therapeutic procedure in neonates with hyperbillirubinemia risking to cause brain injury of the neonate. This therapy without alternative may couse anemia, hyper or hypotonus, and behavior problems. Those disturbances are not clearly understood yet, but it appears that this process is mediated by the immune system. In order to confirm this hypothesis parallely with clinical examination, we performed the following laboratory investigations: red blood cell count, hemoglobin, hematocrite, white blood cells, natural killer cells, T lymphocyte subsets, ability of the peripheral blood phagocytes to reduce NBT, the phagocytic ability and hemiluminescent response. In the clinical examination the most common finding was hypertonus or hypotonus of the muscules. The values of hemoglobin and hematocrite were reduced, red blood cell count was similar, while the white blood cell particularly the monocyte count was increased compared to the control. The absolute number of T lymphocytes defined by CD2 and CD3 surface marker expression was depleted, while the number of DR positive cells as well as the number of NK cells was increased in group treated with exanguinotransfusion. We have also found an increase of NBT positive cells in the same group. There were no differences in the number of NBT positive cells, when they were prestimulated by PMA. Phagocytic ability was lower compared to the control, while hemiluminescent response was faster remaining on high level for a long time. All those differences remained for several months after exanguinotransfusion.

[Neonatal immune system changes caused by phototherapy]. / Promene u imunskom sistemu novoredjenceta izazvane fototerapijom.

It is noticed that phototerapy may cause disturbance in the behavior and higher incidence of infections in neonate. Moreover an electromagnetic radiation may influence different functions of cells. We examined the effects of pototherapy on the immune system of neonates applied because of hyperbilirubinemia. The examined healthy patients, without signs of infection, anoxia or birth injury. Physical examination, white blood cell count, surface differentiation markers on peripheral blood lymphocytes,and hemiluminescent response of peripheral blood leukocytes were performed before and immediately after phototerapy. Our results showed an increase in the total number of peripheral white blood cells: polymorphonuclears, lymphocytes and monocytes as well as a delay in the chemilumonescence response of the peripheral blood phagocytes with lower values of the pick, suggesting a decrease of their functional ability to respond by respiratory burst. This might be important in the case of bacteriemia when phothoterapy may complicate the existing infection. Those findings are temporary.

[Dexamethasone modifies development of human long-term bone marrow culture in dose dependent manner]. / Razvoj humane dugotrajne kulture kostne srzi modifikovan deksametazonom.

Dexter's long-term bone marrow cultures were performed to evaluate the effects of exponentially decreasing concentrations of dexamethasone (10(-2) M to 10(-8) M) on proliferation, differentiation and oxidative burst of the human bone marrow cells. High concentrations of dexamethasone show antiproliferative effect on stromal and haemopoietic cells. There was a small number of adipocytes and macrophages, with predominance of fibroblasts. Cultures, grown in lower concentration of dexamethasone (10(-4) M to 10(-7) M) consisted of a greater number of adipocytes and macrophages. The number of haematopoietic islands was increased and differentiation of haematopoietic cells was pronounced. The adipocytes proliferation was more pronounced, while their maturation was slowed down when low concentrations of dexamethasone were used. The size and the number of haematopoietic islands were reduced. Optimal concentration of dexamethasone for normal growth of both stromal and haematopoietic cells were between 10(-5) M and 10(-7) M. NBT reduction ability of myeloid and stromal cells increased using the higher concentrations of dexamethasone. However, only myeloid cells were NBT positive when lower concentrations were used. Dexamethasone acted as functional and maturational factor for oxidative burst of stromal, as well as myeloid haematopoietic cells.

NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3).

Acute promyelocytic leukemia (APL) is a well-defined entity among acute leukemia, cytogenetically characterized by a t(15;17) (q22;q11-12) translocation. In vitro and in vivo studies suggest that all-trans retinoic acid (RA) treatment restores cell maturation. We have isolated the first permanent cell line with t(15;17), derived from the marrow of a patient with APL in relapse. The establishment of the cell line, its morphologic, karyotypic, and immunohistochemical features are reported. RA induced cell line maturation. Cells strongly expressed myeloid markers, but also some T-cell markers. Additional karyotypic abnormalities, a 12p rearrangement and the possible presence of a homogeneous staining region (HSR) on 19q+ are discussed both in relation to T-cell (CD2, CD4) and monocyte (CD9) markers, and to the acquired cell growth autonomy. The cell line represents a remarkable tool for biomolecular studies.

[Reaction of the injured organism to infection]. / Reakcija povredenog organizma na infekciju.

Authors have examined degree of the immune response to experimentally inoculated agent in the early (6 hours) and late (7 days) phase of the organism response to thermal injury in BALB/c rats divided into two experimental groups and three control groups (only infected, only burnt and intact). Brucella abortus bovis of VA-19 species known as a good immune response stimulator in the host was i.p. inoculated in LD 50 (1 x 10(7) bact/ml). as the infective agent. Thermal injury-scalding was induced after the Art-hurson's method. Obtained results after observed brucella multiplication and spreading in blood, spleen, liver and lymph nodes as well as after determination of the phagocyte activation degree measured by the lumisol-dependent-chemiluminescence technique have pointed out that the general response to also confirm reliability of serologic diagnosis of toxoplasmosis by IgM antibody detecting in disease outbreaks. Acute acquired toxoplasmosis could not be reliably diagnosed on the basis of IgG-ELISA and IgG-IIF test findings. Enzyme immunoassays-ELISA are significantly more sensitive and more specific than IIF ones.