Biomechanical changes in the liver tissue induced by a mouse model of multiple sclerosis (EAE) and the effect of anti-VLA-4 mAb treatment.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of demyelinating diseases, such as multiple sclerosis (MS). MS can be accompanied by autoimmune hepatitis. In this study, nanomechanical, biorheological and histological examinations were carried out by atomic force microscopy (AFM), rheology, and immunofluorescence microscopy to investigate changes in the liver tissue of EAE mice and the effect of natalizumab, a monoclonal antibody against α4-integrin (VLA-4) cell adhesion molecule, used in MS therapy. Liver samples collected from EAE mice in three successive phases of the disease showed inflammatory changes manifested by leukocyte infiltrations and elevated levels of proinflammatory cytokine IL-1ß. Liver stiffness and viscoelasticity increased in the onset phase of EAE, decreased in the peak phase and increased again in the chronic phase to reach the highest values. These changes were not associated with inflammation parameters which increased in the peak phase and decreased to the lowest values in the chronic phase. Moreover, anti-VLA treatment, which reduced the inflammation parameters, had an ambiguous effect on stiffness and viscoelasticity: it increased them in the peak phase but decreased in the chronic phase. The observed discrepancies can result from a complex network of interactions between inflammation and fibrosis, as well as between liver cells and the extracellular matrix influencing the biomechanical properties of the liver tissue.

Histopatological parameters of the spinal cord in different phases of experimental autoimmune encephalomyelitis. A mouse model of multiple sclerosis examined by classical stainings combined with immunohistochemistry.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE) are characterized by three main histopathological parameters: inflammation, demyelination and axonal damage. In this study, these parameters were assessed in spinal cords of mice in the successive phases of EAE by quantitative histology and immunohistochemistry. The number of inflammatory lesions, the intensity of inflammation and expression of CD45 corresponded with the severity of clinical symptoms: they increased from the onset phase to the peak phase of the disease and subsided in the chronic phase. Demyelination increased in the peak phase and did not change in the chronic phase of EAE, although axonal damage gradually increased from the onset phase to the chronic phase, suggesting compensatory hypermyelination in that phase. The markers of myelin and axonal injury: myelin basic protein (MBP) and beta amyloid precursor protein (ß-APP) showed changes (decrease and increase, respectively) of expression parallel to changes in demyelination and axonal damage. Results of this study indicate that although inflammation intensity subsides in the chronic phase of EAE, the neurodestructive processes: demyelination and axonal damage continue in that phase.

Changes in ECG and enzyme activity in rat heart after myocardial infarction: effect of TPP and MnCl2.

Heart infarction is one of the main causes of death in the human population. Assurance of a sufficient level of bioenergetic processes is very important for the heart after infarction. Mn2+ as well as thiamine pyrophosphate (TPP) are positive effectors of the pyruvate dehydrogenase complex (PDH) and the 2-oxoglutarate dehydrogenase complex (OGDH), both of which play a very important role in the Krebs cycle. Thus, we have established the effect of MnCl2 (10mg/kg) and TPP (20mg/kg)--4 injections every 12 h--on the activity of PDH, OGDH, lactate dehydrogenase (LDH) and malate dehydrogenase (MDH). Additionally, we perform an analysis of ECG to affirm the changes in the heart electrophysiology of healthy rats after MnCl2 and TPP treatment. We then analyzed changes in the activity of these enzymes after experimental myocardial infarction in rats. We observed a decrease of OGDH and MDH activity in rat hearts after infarction in comparison with sham-operated rats. Treatment of healthy rats with MnCl2 caused an increase of OGDH activity. Moreover both MnCl2 and TPP caused an increase of PDH activity and a decrease of MDH activity (TPP revealed a stronger effect). We found no changes in LDH activity. Electrocardiography data showed a slight shortening of the QT interval and an enhanced heartbeat rate after treatment with MnCl2. TPP caused only elongation of the QT interval. In conclusion, application of MnCl2 enhanced the activity of some very important enzymes in the respiration process (PDH and OGDH). This effect, connected with enhanced heartbeat and a slightly shortened ventricle relaxation, may have potential application during the key period of convalescence following heart infarction.

Non-ischemic heart preconditioning.

Ischemic heart conditioning has been shown to protect the organ against ischemia/reperfusion injury. Animal studies have revealed that the heart can also be conditioned by non-ischemic procedures, namely physical exercise and tachycardia. Long and short term endurance training, sprint training, resistance or interval training and even one bout of exercise induce cardiac preconditioning, which is manifested by a reduction in post ischemia/reperfusion infarct size, ventricular arrhythmia and improved heart function. Several factors contribute to the exercise-induced heart preconditioning, among which the most important can be: increased activity of the anti-radical defense system, opioids, interleukin-6, nitric oxide, ATP dependent potassium channels, heat shock protein 72 and sphingosine-1-phosphate. A few studies have also shown that one bout of exercise in patients with stable angina increases tolerated workload. According to some data obtained in swine and dogs, stimulated tachycardia before ischemia/reperfusion reduces the infarct size. Future studies are needed to fully clarify the mechanisms responsible for exercise- or tachycardia-induced heart preconditioning against ischemia/reperfusion. It may lead to the development of new treatment modes of the disease.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity.

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.

Neurologic complications in children after hemaopoietic stem cell transplantation: a single-center experience.

UNLABELLED: Neurologic complications may occur in patients undergoing haematopoietic stem cell transplantation (HSCT). The aim of the study was to evaluate the frequency and type of neurologic complications in children after HSCT. We performed a retrospective analysis of the incidence and outcome of neurologic complications among 171 consecutive children transplanted in one center. RESULTS: Among 84 autologous and 87 allogeneic (47 matched sibling donors, 31 matched unrelated donors, 8 mismatched family donors, and 1 cord blood) transplants, 7 patients (4%) developed neurologic complications, all of whom had undergone allogeneic transplantation (7/87 = 8%). These patients had relapses of acute leukemia (n = 3; acute myeloblastic in two and acute lymphoblastic in one), chronic leukemia, (n = 1), myelodysplastic syndrome (n = 2), and adrenoleudystrophy X (n = 1). Neurologic complications occurred after a median follow-up of 1 month (range, 14 days to 19 months). Of seven patients, four died. Neurologic complications were the cause in two patients. CONCLUSIONS: Among the analyzed material the risk of neurologic complications was lower than in other studies and these events were observed only in children undergoing allogeneic transplantation.

Effect of tachycardia on mRNA andf protein expression of the principal components of the lipolytic system in the rat's heart ventricles.

There is a convincing piece of evidence showing that most of free fatty acids (FFA) entering cardiomyocytes are first esterified into triacylglycerols (TG) before being oxidized or used for synthesis of complex lipids. The enzyme adipose triglyceride lipase (ATGL) initiates lipolysis of TG by hydrolyzing the first ester bond of the compound. As a result, free fatty acid and diacylglycerol (DG) are released in that process. Finally, DG may be further hydrolyzed by the enzyme hormone sensitive lipase (HSL). The aim of the present study was to examine effect of tachycardia on mRNA and protein expression of ATGL, CGI-58 (an activator of ATGL), G0S2 (an inhibitor of ATGL) and HSL in the left and right ventricle of the rat. The experiments were carried out on male Wistar rats, 240 - 260 grams of body weight. After anesthesia, two electrodes were inserted in the right jugular vein and connected to SC-04 stimulator. The rats were randomly allocated into one of the three groups, namely: control, 30 min and 60 min of the heart stimulation at the rate of 600 times/min. The expressions of ATGL, CGI-58, G0S2 and HSL were evaluated at both gene and protein levels using real-time PCR and Western Blot analysis, respectively. Both 30 and 60 min stimulation reduced ATGL, HSL, CGI-58 and G0S2 mRNA content in the left ventricle. The stimulation had only insignificant impact on ATGL, HSL and CGI-58 transcript levels in the right ventricle. Interestingly, Western Blot analysis showed that the stimulation, regardless of the time, reduced the ATGL and G0S2 protein expression, but did not change the CGI-58 and HSL expression in the left ventricle. Furthermore, in the right ventricle, the protein expressions of ATGL, HSL and G0S2 decreased after stimulation. In conclusion, the obtained results clearly show that tachycardia affects both mRNA and protein expression of particular compounds of the TG lipolytic system in the heart ventricles. Additionally, there are marked differences in the expressions of the examined compounds between the ventricles.

Analysis of the p53 gene and its expression in human glioblastoma cells.

Chromosome 17p has been shown to be an early and frequent target for loss of heterozygosity through mitotic recombination in astrocytomas. These losses are frequently accompanied by point mutations in the p53 gene of the remaining allele, resulting in loss of wild type p53 function. However, a fraction of astrocytomas retain constitutional heterozygosity and do not have p53 mutations; some of these lose wild type p53 activity through binding to the protein product of amplified mdm2 genes. To test whether loss of wild type p53 biological function is a necessary step in astrocytoma progression we analyzed p53 expression and biological function in 13 glioma cell lines. All the cell lines expressed a 2.8-kilobase p53 transcript and showed various amounts of p53 protein by immunoprecipitation, except for cell line LN-Z308 which had only a small truncated p53 mRNA and no protein expression. To test whether the p53 expressed in these cell lines was functionally wild type or mutant we transfected them with a plasmid construct harboring a chloramphenicol acetyltransferase (CAT) reporter gene under the control of transcriptional elements that are induced by wild type but not mutant p53. Four lines were shown to retain wild type p53 function. Sequencing of the p53 gene in two of these cell lines confirmed the wild type genotype. These results show that inactivation of the p53 gene is not an obligatory step in glioblastoma genesis. This suggests either that two pathways (p53 inactivation dependent or independent) may lead to a tumor group classified histologically as glioblastoma or that in some cases p53 mutations are bypassed due to the presence of mutations in downstream effector genes.

Effect of atrial pacing on the level of bioactive sphingolipids in the heart ventricles of the rat.

Bioactive sphingolipids play important role in regulation of the function of the cardiomyocytes. There are no data available on metabolism of the sphingolipids in the heart under increased work-load produced by tachycardia. The aim of the present study was to examine effect of tachycardia on the level of the principal bioactive sphingolipids in the left and right ventricles. The experiments were carried out on male Wistar rats. After anesthesia, two electrodes were administered into the right common jugular vein so that their tips were placed at the vein's aperture. The resting heart rate was 355 ± 24/min and the rate of stimulation was 600/min. EKG was continuously monitored. The stimulation time was 30 and 60 min. Thereafter, blood from the abdominal aorta and samples of the left and right ventricle were taken. The following bioactive sphingolipids were quantified by means of high performance liquid chromatography: sphinganine, ceramide, sphingosine, sphingosine-1-phosphate and sphinganine-1-phosphate. In the left ventricle, 30 and 60 min tachycardia elevated the level of sphingosine, reduced the level of sphingosine-1-phosphate and sphinganine-1-phosphate. The level of ceramide was reduced only after 60 min. In the right ventricle, 60 min pacing resulted in elevation in the level of sphingosine and sphinganine and reduction in the level of other compounds studied. It is concluded that tachycardia induces changes in metabolism of bioactive sphingolipids in each ventricle. The changes may affect cardiomyocyte functions. Also, differences in sphingolipid metabolism between both ventricles are reported.

Effect of tachycardia on lipid metabolism and expression of fatty acid transporters in heart ventricles of the rat.

Tachycardia increases oxidation of the plasma-borne long chain fatty acids in the heart. The aim of the present study was to examine effect of tachycardia on: 1) the total level of free fatty acids, diacylglycerols, triacylglycerols and phospholipids in both heart ventricles; 2) (14)C-palmitate incorporation in the lipid fractions; 3) expression of fatty acid and glucose transporters in the ventricles. Tachycardia was induced in anesthetized rats by electrical atrial pacing at the rate of 600/min. Samples of the left (LV) and right (RV) ventricle were taken after 30 and 60 min pacing. The level free fatty acids, diacylglycerols, triacylglycerols and phospholipids was determined by means of gas-liquid chromatography and (14)C-palmitate incorporation by liquid scintillation counting, respectively. Expression of fatty acid- and glucose-transporters was determined using Western blot technique. In LV, 30min pacing increased the content of diacylglycerols whereas the content of other lipids remained stable. After 60 min of pacing the levels of the examined lipid fractions did not differ from the respective control values. In RV, the content of diacylglycerols and triacylglycerols was reduced both after 30 and 60 min pacing. Tachycardia also affected incorporation of (14)C-palmitate in lipid fractions of goth ventricles. 30 min pacing up-regulated plasmalemmal expression of FAT/CD36 (fatty acid translocase) in both ventricles and reduced its microsomal expression in LV. After 60 min pacing they did not differ from the respective control values. Plasmalemmal expression of FATP-1 (fatty acid transport protein 1) increased and its microsomal expression decreased in RV after 30 min pacing. After 60 min pacing the plasmalemmal FATP-1 expression remained elevated whereas the microsomal expression did not differ from the control value. Pacing did not affect or expression of FABPpm (plasma membrane associated fatty acid binding protein) in either plasma membranes and microsomal compartments. Thirty min pacing increased plasmalemmal and reduced microsomal expression of GLUT-4 (glucotransporter 4) in both ventricles. It increased plasmalemmal expression of GLUT-1 (glucotransporter 1) in RV. It returned to normal after 60 min pacing. It is concluded that tachycardia induces numerous changes in metabolism of myocardial lipids as well as expression of fatty acid and glucose transporters in both heart ventricles.

A long-term study of the efficacy of treatment of localized prostate cancer.

This paper presents a retrospective comparison of patients undergoing treatment for clinically localized prostate cancer. We reviewed the age, grade, and stage at diagnosis as well as the survival and recurrence rates of 222 patients treated for carcinoma of the prostate with either radical prostatectomy (RP) or radiotherapy (XRT) at four Army medical centers. Mean follow-up was 8.02 years (range 0.026-32.5 years). Stage and grade were similar in patients receiving either RP or XRT. Kaplan-Meier estimates showed that patients who underwent RP had a significantly greater disease-specific survival (p = 0.0001) and a significantly lower rate of distant metastases (p = 0.006) than did those who received XRT. There was no significant difference in the rate of local progression (p = 0.276) or in the mean time to local progression (XRT = 3.5, RP = 4.0 years) or to distant metastases (XRT = 3.79, RP = 4.52 years). Cox proportional hazards model incorporating age, stage, grade, and treatment type demonstrated that those patients who received XRT had more than two times the risk of dying of their disease than did those who underwent RP (risk ratio = 2.37; 95% confidence interval = 1.49-3.76). These data in similar groups of patients suggest that metastasis-free survival is improved in those who receive RP compared with XRT and that this translates into an enhanced survival advantage. Further study of larger groups of patients stratified by risk factors in randomized, prospective trials with longer follow-up will improve our ability to determine the best treatment for clinically localized prostate cancer.

Vasopressin gene expression in rat choroid plexus.

Vasopressin (VP) levels in cerebrospinal fluid (CSF) change in response to physiological stimuli and under various pathological conditions. The sources of CSF VP have yet to be clarified, however. In the present study, we provide evidence indicating that VP is synthesized in the choroid plexus, the primary site of CSF formation. All experiments were performed on adult male Sprague-Dawley rats. The presence of VP mRNA in choroid plexus epithelium was demonstrated by in situ hybridization histochemistry using the 35S-labeled riboprobe that was complementary to cDNA fragment of rat VP encoding the C-terminus part of proVP. In situ hybridization findings were confirmed by reverse transcriptase-polymerase chain reaction analysis. Immunohistochemistry for VP-associated neurophysin (VP-NP), a polypeptide component of proVP, revealed subapical accumulation of VP-NP-immunopositive product in choroidal epithelial cells. Immunoprecipitation and immunoblotting of choroidal protein extracts with anti-VP-NP antibody demonstrated the presence of a approximately 10-kD polypeptide that was also detected in hypothalamus. We hypothesize that the choroid plexus-derived VP exerts autocrine and/or paracrine effects on tissues near the CSF system.

Alcoholic typology and season of birth.

OBJECTIVE: This study analyzed the half years of birth of a large (n = 113,276) population of alcoholic patients in the U.S. Army Alcohol and Drug Abuse Prevention and Control Program from 1986 through 1990. METHOD: Subjects were enrolled for treatment of alcoholism or alcohol-related problems, and were analyzed for half year of birth. Groupings by age and gender, consistent with current theories of alcoholic typology, were compared, by means of chi-square tests, and by comparisons of two rates or proportions. RESULTS: The 17-21 year old and the 22-39 year old age groups differed by 5.1% in regard to half year of birth (chi-square = 260.317, p < 0.001, 95% CI:4.481 to 5.725, odds ratio: 1.23). Both groups differed significantly from the normal circannual birth pattern, but in opposite directions. CONCLUSIONS: The findings support the differentiation of types of alcoholics by age, which is a characteristic of Cloninger's classification, suggesting a biological, pre-natal factor.

[Development of righting reflexes and their influence on the development of static functions in children with infantile cerebral palsy]. / Ksztaltowanie sie reakcji prostowania i ich wplyw na rozwój czynnosci statycznych dzieci z mózgowym porazeniem dzieciecym

In a group of 55 children with signs of central nervous system lesions the development of righting reflexes and static functions was observed during the first 3 years of life. A delay and disturbances in the development of righting reflexes and static functions was observed with differences between the individual groups of infantile cerebral palsy.

[Some postural reflexes and the development of static functions in children with infantile cerebral palsy]. / Zachowanie sie niektórych odruchów postawy a rozwój czynnosci statycznych dzieci z mózgowym porazeniem dzieciecym

The development of postural reflexes and static functions were analysed in children with different forms of central nervous system damage developing in the first three years of life. A correlation was found between the persistence of tonic reflexes and disturbances of development of static functions and this finding may be used in differential diagnosis of early central nervous system lesions and in selection of appropriate treatment.

[Characteristics of Lactobacillus strains contained in pharmaceuticals]. / Charakterystyka szczepów Lactobacillus wchodzacych w sklad preparatów farmaceutycznych.

The aim of the study was to characterize lactic acid bacteria (LAB) which are components of drugs administered orally in cases of intestinal disturbances, or antibiotic--related diarrhea. Biochemical properties, growth behavior, bile tolerance, and survival at low pH of six LAB strains (four strains L. rhamnosus and two L. acidophilus) were studied. The survival at low pH was determined in MRS broth (Difco) acidified to pH 1; 2; 3; and 4. Bile tolerance was tested on MRS broth with 0.3% oxgall (Difco). Between tested strains differences in ability to grow at low pH and survival in bile were observed. Only 0.01% inoculum of all examined strains survived at pH 1. Differences between strains in survival at low pH (pH 2 and pH 3) and tolerance of bile were observed. However, after 2 h incubation at pH 4, 100% of strains stayed alive. Examined strains demonstrated good 3% bile tolerance. All strains met the criteria for probiotic strains: ability to survive at pH 3 and in the presence of bile.

[Endemic goiter in the area of Mielec]. / Endemia wola w okolicach Mielca.

The scope of this report is the incidence of goiter in school children living in Mielec and neighbouring villages. We examined a group of 3537 children aged 6-14. The enlargement of thyroid gland was observed in 49.3% of the population. Judging by the results of the research this region can be regarded as endemic. There might be several reasons for a high incidence of goiter in this region. The most important are: insufficient iodine prophylaxis, pollution of the environment including the pollution with radioiodine after the damage of the Chernobyl reactor.

[Evaluation of the prevalence of osteoporosis in a population of women living in Krakow based on densitometric measurements of the forearm]. / Ocena wystepowania osteoporozy w populacji kobiet mieszkajacych w krakowie na podstawie badania densytometrycznego przedramienia.

Osteoporosis is considered a civilization disease, affecting populations living in big cities. It is detected in 10 percent of population and in 30 percent women over fifty. We tried to estimate prevalence of osteoporosis in people living in Kraków. For estimation purposes, 1000 people living in Kraków more than 40 years were chosen. 325 persons came to our Department for examination, 232 of which were women. All patient filled a special questionnaire on pathological changes in skeletal system and risk factor of osteoporosis. Densitometric measurements of nondominant forearm using Osteometer DTX 100 were performed in all patients. For osteoporosis criteria level of T-score less than -2.5 was taken. Because of lack of Polish reference data for densitometric measurements our data were compared to WHO data, concerning prevalence of osteoporosis in white women. Compared to WHO data, a higher frequency of osteoporosis was found in women living in Kraków more than 40 years.

The application of screws covered with a layer of nanocrystaline diamond in tibia fractures treated with a Polfix fixator: further observations.

This article presents further results in the treatment of tibial shaft fractures using a Polfix fixator with screws covered with a layer of nanocrystaline diamond. The advantages of this new protective layer are presented in 16 additional patients. Research to date indicates that nanocrystaline diamond can be used successfully to cover the surface of surgical screws and plates.