Hemiparkinsonism-hemiatrophy syndrome - report on two cases and review of the literature.

Hemiparkinsonism-hemiatrophy (HPHA) is a rare neurological syndrome. The main clinical features of HPHA consist of atrophy of one side of the body (face, trunk, limbs), ipsilateral hemiparkinsonism (bradykinesia, rigidity, tremor) and in many cases dystonia. There are no data on prevalence of HPHA as the condition is rare. The mean age of parkinsonism onset is earlier than in idiopathic Parkinson disease (43.7 years, range: 15-63). Changes in magnetic resonance imaging (MRI) (cortical, basal ganglia atrophy contralaterally to the side of clinical presentation) are described in 30% of patients. The pathogenesis of HPHA is unknown, but in many cases a history of prenatal injuries was reported. We present two male patients with HPHA - 45 and 55 years old, with left-sided parkinsonism, dystonia and hemiatrophy (to our knowledge, the first Polish cases). Both patients had no atrophic changes in MRI and levodopa treatment was ineffective. In the discussion the authors review current literature on HPHA.

Incidence of mutations in the PARK2, PINK1, PARK7 genes in Polish early-onset Parkinson disease patients.

BACKGROUND AND PURPOSE: Parkinson disease (PD) is a complex disease, comprising genetic and environmental factors. Despite the vast majority of sporadic cases, three genes, i.e. PARK2, PINK1 and PARK7 (DJ-1), have been identified as responsible for the autosomal recessive form of early-onset Parkinson disease (EO-PD). Identified changes of these genes are homozygous or compound heterozygous mutations. The frequency of PARK2, PINK1 and PARK7 mutations is still under debate, as is the significance and pathogenicity of the single heterozygous mutations/variants, which are also detected among PD patients. The aim of the study was to analyze the incidence of autosomal recessive genes PARK2, PINK1, PARK7 mutations in Polish EO-PD patients. MATERIAL AND METHODS: The analysis of the PARK2, PINK1 and PARK7 genes was performed in a group of 150 Polish EO-PD patients (age of onset < 45 years). Mutation analysis was based on sequencing and gene dosage abnormality identification. RESULTS: Mutations were identified only in the PARK2 and PINK1 genes with the frequency of 4.7% and 2.7% of subjects, respectively. In PARK2, point mutations and exons' rearrangements, and in PINK1 only missense mutations were detected. In both genes mutations were found as compound heterozygous/homozygous and single heterozygous. EO-PD patients' genotype-phenotype correlation revealed similarities of clinical features in mutation carriers and non-carriers. CONCLUSIONS: The frequency of the PARK2, PINK1, PARK7 mutations among Polish EO-PD patients seems to be low. The role of single heterozygous mutations remains a matter of debate and needs further investigations.

[Importance of expression of DNA repair proteins in non-small-cell lung cancer]. / Význam stanovovania expresie DNA reparacných mechanizmov u nemalobunkového karcinómu plúc.

BACKGROUND: Proteins XRCC1 and ERCC1 are involved in DNA repair. XRCC1 plays a role in DNA base excision repair and ERCC1 in nucleotide excision repair pathway. Higher expression profile of both proteins in cancer cells may contribute to development of drug resistance. ERCC1 is involved in removal of platinum adducts and might be a potential predictive and prognostic marker in NSCLC (non-small-cell lung cancer) treated with a cisplatin-based regimen. The purpose of study was determination of XRCC1 and ERCC1 levels and their correlation with basic clini-copathological parameters in NSCLC. PATIENTS AND METHODS: In this study, 107 tumor samples diagnosed as NSCLC were immunohistochemically examined for expression of XRCC1 and ERCC1 proteins. Our results were compared to basic clinicopathological parameters: type of tumor, tumor grade and stage of disease. For statistical analysis, the chi-square test was used. RESULTS: In squamous cell carcinoma and large cell carcinoma samples, the XRCC1 protein level was twofold higher (60% of positive samples) than in adenocarcinoma samples (35.5% of positive samples). We have found statistical correlation between XRCC1 protein expression and type of tumor (p = 0.0306). On the other hand, the statistical importance between the protein level versus grade and stage was not found. In the case of the ERCC1 protein, we observed the highest protein level in adenocarcinoma (64.5%) and squamous cell carcinoma (62.5%) samples. Next, we determined a significant difference in content of XRCC1 versus ERCC1 (35.5% vs 64.5%) in adenocarcinoma samples. Statistical chi-square test did not reveal any correlation between ERCC1 status and clinicopathological parameters. CONCLUSION: According to our results, XRCC1 represents an important mechanism of DNA repair in squamous cell and large cell carcinomas. Besides that, expression of XRCC1 was in correlation with type of tumor. In patients with adenocarcinoma and squamous cell carcinoma, we could assume increased resistance to platinum-based therapy because of high expectation of ERCC1 protein expression. However, its levels did not correlate with monitored clinicopathological parameters. The ERCC1 protein will be possibly an independent prognostic factor in NSCLC. To prove a true survival benefit of patients with expression of ERCC1, prospective validation of ERCC1 before clinical implication is needed in the future.

Erythropoietin concentration in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis.

Erythropoietin (EPO) acts as a neuroprotective factor and is upregulated after neuronal injury. It has been reported that in cerebrospinal fluid (CSF) of amyotrophic lateral sclerosis (ALS) patients, the EPO concentration is decreased. In this study, EPO levels in serum and CSF of 30 patients with ALS and in 15 controls, using an ELISA technique, were estimated. EPO level in serum was decreased, especially in patients with bulbar onset ALS. A trend toward a progressive EPO decline with the duration of the disease in the mild + moderate ALS cases was observed. In severe cases, a tendency towards a positive correlation of EPO and duration of the disease was present. Serum EPO values were age related only in mild + moderate ALS in patients below 40 years of age. In CSF, the EPO levels were significantly decreased. Lower EPO values in the bulbar onset ALS when compared with the spinal onset ALS were present. The EPO decrease did not correlate with the severity and duration of the disease. Age relation of the EPO level only in the mild + moderate ALS cases more than 40 years was present. Lack of differences in EPO levels between patients with ALS of rapid and slow progression indicates that EPO concentration cannot be used as a prognostic factor. Nevertheless, the decreased serum and CSF EPO concentration and the known EPO neuroprotective action may indicate that EPO administration can be a new promising therapeutic approach in ALS.

Matrix metalloproteinases and their tissue inhibitors in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We investigated the expression of MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) in serum and cerebrospinal fluid (CSF) correlating the results with age, disease duration and the clinical course. METHODS: The material consisted of 30 ALS patients and 15 age-matched healthy controls. ELISA method to determine the expression of MT-MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 in serum and CSF was used. MMP-2 and MMP-9 by zymography was also tested. RESULTS: In serum MT-MMP-1, MMP-2, MMP-9 and TIMP-1 expression was increased, especially in mild ALS cases. TIMP-2 values were normal. In CSF MT-MMP-1, MMP-2 and TIMP-1 level was either increased or normal, that of MMP-9 was decreased. TIMP-2 did not change. No correlation of MMPs and TIMP-1 expression in serum and CSF and the age of the patients was found. A correlation was observed between MMPs and TIMPs and disease duration. CONCLUSIONS: Increased level of MMPs and TIMP-1 of ALS patients may reflect the degeneration process of motor neurons and skeletal muscles and/or is associated with tissues remodeling. The low level of MMP-9 in CSF may result from impaired balance between MMP-9 and TIMP-1 and/or its increased intrathecal degradation and physical clearance. Although the role of changed MMPs/TIMPs level in the pathogenesis of ALS is not clear their analysis in serum may be used as prognostic factor and a potential marker for monitoring treatment effects.

[Revision knee arthroplasty due to aseptic loosening]. / Revizní náhrada kolena po aseptickém uvolnení.

PURPOSE OF THE STUDY: Anatomic changes associated with aseptic loosening make conditions for revision of total knee arthroplasty more diffi-cult. The aim of this study was to evaluate the results of revision total knee replacement at an average follow-up of 6.1 years. MATERIAL AND METHODS: A total of 97 revision knee replacements due to aseptic loosening carried out in the years 1992 to 2003 were evalua-ted. The group included 46 men and 51 women at an average age of 66.8 years. The average preoperative Knee Society Score (KKS) was 31 points and the Functional Score (FS) was 22 points. There were 41 minor operations for AORI type I defects, 49 moderately serious procedures for AORI type II defects and seven major operations for AORI type III defects. In minor procedures standard components were implanted in 14 patients, standard components with cemented stems with extension were used in nine, and polyethylene plateau exchange was carried out in 18 patients. For moderately serious procedures, posterior stabilized components with extended cemented stems were used in 15 patients, revision implants with cementless stems in 26 patients and standard components with cemented stems in eight patients. In seven patients with major surgery, the hinged type of prosthesis was employed. Radiographic results were evaluated on the basis of Ewald's classification. RESULTS: Clinical findings showed improvement of the average KKS from 31 to 74 points at follow-up of 6.1 years. Functional out-comes improved, as shown by the average FS, from 22 to 67 points. Fifteen patients were not satisfied with the outcome of surgery, the causes being aseptic loosening in four, deep infection in eight and pain due to progression of radiolucent lines of the tibia in three patients. DISCUSSION: The results of revision surgery with component replacement because of aseptic loosening are worse in comparison with those of primary total knee replacement. The average KSS score after revision surgery was 74 points at 6.1-year follow--up, whereas after primary surgery it was 92 points at 6.5 years. The average FS score after revision was 67 points, as compared with 86 points at 6.5 years after primary surgery. Complications involving infection occurred in 8.2 % of the revi-sion cases, but only in 0.8 % of the primary operations. The authors used modular systems because these provide more options. Good outcomes were achieved with morselized bone grafting for filling cavitary defects. In patients with large defects in tibial or femoral metaphyses, posterior stabilized components and cementless intramedullary stems were used with good results. CONCLUSIONS: The authors recommend to avoid filling large bone defects with bone cement. They prefer bone grafting. In the case of good quality metaphyseal bone, they use standard components or posterior stabilized components with or without additi-onal cemented or cementless short stem extensions. In the case of poor quality metaphyseal bone with defects, they use revision implants with cementless long stems. The authors have achieved good results with off-set stems.

Effect of transfection with a gene coding for the fibronectin FNIII/10 fragment upon contact inhibition of C3H10T1/2 fibroblasts.

The density-dependent growth inhibition of non-transformed cells may be associated with inefficient transduction of the proliferative signal from cell adhesion molecules. To verify this concept, the C3H10T1/2 fibroblasts were stably transfected with the gene coding for the fibronectin fragment III/10 (FNIII/10). This resulted in differences in gene's expression between original C3H10T1/2 cells and their FNIII/10 transfectants. No significant differences in growth properties were observed in the original or in the transfected cells. C3H10T1/2 cells and their transfectants, when co-cultured, displayed more cells at confluence than the cells cultured alone. Moreover, co-cultured C3H10T1/2 cells and their transfectants showed elevated levels of phospho-ERK1/2 compared to homogenous cultures. Results obtained indicate that cellular homogeneity is responsible for density-dependent growth inhibition.

Modulation of lung tumor colony formation by a subcutaneously growing tumor.

When monodisperse tumor cell suspensions of the mouse sarcoma L1 were injected iv into inbred BALB/c mice bearing subcutaneous grafts of the same tumor, the number of tumor nodules developing in the lungs was highest in animals bearing the oldest primary tumor (28 days) and lowest in animals bearing the oldest primary tumor (28 days) and lowest in animals bearing 16-day-old primary tumors. The number of metastases developing in mice bearing 9-day-old subcutaneous tumors was the same as that in control animals without a subcutaneous tumor. This state of low and high susceptibility to lung metastases can be transferred from tumor-bearing mice to whole-body irradiated mice by means of spleen cells, and this transfer of susceptibility is stable for at least 3 weeks. The results indicate a) that a growing subcutaneous tumor can modulate the number of artificial metastases occurring in the lungs as a result of the iv injection of tumor cells, and b) that this modulation occurs by immune mechanisms. In the model presented here, the host is first protected and then made more susceptible to artificial lung metastases by the presence of a progressively growing subcutaneous tumor.

Stimulatory and inhibitory activities of lung-conditioned medium on the growth of normal and neoplastic cells in vitro.

Lung-conditioned medium (LCM) was obtained by incubation of BALB/c mouse lung tissue fragments in serum-free Eagle medium for 48 hours and subsequent separation by dialysis or chromatography on a Sephadex G-75 column. LCM fractions were tested for their ability to modulate proliferation of normal human endothelial cells and neoplastic cells (N2a, MCF, HEp-2) in vitro as assessed by plating efficiency and tritiated thymidine incorporation assays. It was found that LCM contained two kinds of factors, either stimulating (molecular weight: 50,000-70,000) or inhibiting (molecular weight: 12,000-20,000 and 3,000-5,000) cell proliferation.

The effect of estrone-progesterone treatment on cell proliferation kinetics of hormone-dependent GR mouse mammary tumors.

Hormone-dependent mammary tumors were induced in virgin GR mice by treatment with estrone and progesterone. Discontinuation of hormonal treatment was followed by regression of the tumor. This response to hormone treatment was also observed in the first transplant generation in inbred syngeneic hosts, but after several transplantations the tumor growth became hormone independent. The hormone dependence of the primary tumors and tumors after a single transplantation was demonstrated by growth curves. Furthermore, the cell proliferation kinetics has been investigated in a growing as well as in a regressing hormone-dependent tumor after a single transplantation from the same primary tumor. The experimental data consist of growth curves, percentage-labeled mitoses curves, and labeling indices. Since these data do not contain information concerning the localization of the cell loss in the cell cycle, they were analyzed by a computer method based on three mathematical models differing in respect to the mode of cell loss. All three models gave approximately the same estimates of the cell kinetic parameters in the growing as well as the regressing tumor. The results for the growing, hormone-dependent tumor showed a growth fraction of 62%, a cell production rate of 3.4%/hr, and a cell loss rate of 2.3%/hr. Regression of the tumor after hormonal deprivation was accompanied by a decrease in growth fraction to 18% and a decrease in the cell production rate to 0.9%/hr, while the cell loss rate was unchanged at 2.8%/hr. Furthermore, the discontinuation of hormonal treatment introduced an increase in the mean transit time of the cell cycle, particularly in the mean transit time of the G1 phase. The results might indicate that estrone and progesterone treatment stimulated growth of hormone-dependent GR mouse mammary tumors mainly by an increase of growth fraction and cell production rate.

Cellular quiescence induced by contact inhibition or serum withdrawal in C3H10T1/2 cells.

Either confluence or serum withdrawal may cause growth arrest of cultured non-transformed cells. Here, we compared sparsely populated and confluent C3H10T1/2 cells with and without serum-containing medium. The following proliferation-relevant end points were examined: cell-cycle distribution, Ki-67 antigen presence, the level of the von Hippel-Lindau (VHL) protein, and gene expression, determined using a microarray approach. In sparse/logarithmic cultures, the fraction of cells in G(0)/G(1) phase increased from 55 to 85% following serum withdrawal. Moreover, the fraction of Ki-67 positive cells dropped from 89 to 47%. In confluent cultures, the majority of cells (80%) were in G(0)/G(1) phase and only 25-30% were Ki-67 positive, regardless of serum presence. In both serum-deprived and contact-inhibited cultures, significant and distinct changes in gene expression were observed. Serum deprivation of sparsely cultured cells resulted in significant over-expression of several transcription factors, while confluent cells showed elevated expression of genes coding for Wnt6, uPar, Tdag51, Egr1, Ini1a and Mor1. These results indicate that contact inhibition and serum withdrawal lead to cellular quiescence through distinct genetic and molecular mechanisms.

Protein kinase C translocation in relation to proliferative state of C3H 10T1/2 cells.

Protein kinase C (PKC) activity of cytosol and membrane fractions of 10T1/2 cells was studied. In cytosol of fast growing cells PKC activity was found in material eluted with 0.150 M NaCl whereas in membrane fractions activity was eluted with 0.065 and 0.150 M NaCl. In the membrane fraction of confluent cells, in contrast to cytosol, very low PKC activity was observed. The translocation pattern of PKC activity eluted with 0.065 M NaCl may be associated with proliferation.

Establishment of a cloned line of Lewis Lung Carcinoma cells adapted to cell culture.

A cloned line of cells adapted to culture has been isolated from the Lewis Lung Carcinoma and has been designated the Lewis lung carcinoma line 1 (LLC1). It grows as a monolayer culture in RPMI 1640 medium supplemented with 2% fetal calf serum with a plating efficiency of about 94% and a doubling time of 21 h. LLC1 cells remain highly tumorigenic in C57B1 mice and produce primary tumors and lung metastases histologically indistinguishable from the original tumor line. The doubling time for a subcutaneous tumor derived from LLC1 cells was 23 h for a tumor mass of about 0.1 g and 40 h for a tumor mass of about 1 g. The cell line forms discrete colonies on a plastic substrate and can be used in a focus assay to determine drug induced cytotoxicity. Results with a number of chemotherapeutic agents are reported; in general, sensitivity measured in vitro does not correspond with published reports of sensitivity of the Lewis Lung carcinoma in vivo.

Angiogenesis induced by urothelial cells (HCV-29) and their v-ras and v-raf transfectants.

The angiogenic ability of human urothelial cells (HCV-29) and their v-ras and v-raf transfectants was studied. The most pronounced angiogenesis, observed in vivo, induced v-raf-transfected cells. The lowest degree of induction of neovascularization presented cells of the parental line. The increased extent of angiogenesis correlated with the presence of VEGF mRNA as measured by RT-PCR as well as the level of VEGF as visualized by the method of Western blotting.

The role of protein kinase C in migration of rat glioma cells from spheroid cultures.

The role of protein kinase C in migration of tumor cells from spheroid cultures was investigated using parental rat glioma cells and their TPA resistant counterparts. These two lines differed in their PKC content as judged by the histone phosphorylation method. Also 4 days of treatment with IRA led to PKC down-regulation. Cells having a drastically decreased PKC level migrated better than those having a normal PKC content.

The pleiotropic effect of TPA on in vitro invasion/migration of glioma and melanoma cell lines.

The invasion/migration of two cell lines, melanoma (MEW) and glioma (BT5C), and their counterparts treated for prolonged time with TPA were studied. On Western blots both cell lines expressed alpha, beta I protein kinase C isoforms, whereas beta II and gamma were not detected. The down-regulation of alpha and beta I PKC was observed in glioma cells after long treatment with TPA, whereas the same treatment of melanoma cells did not lead to PKC downregulation. The down-regulation of PKC was accompanied by stimulation of cell migration/invasion through Transwell chambers coated with collagen IV or fibronectin. In the case of melanoma cells treated with TPA, whose PKC was not down-regulated, the inhibition of migration/invasion was observed. The invasive properties of studied cells did not correlate with any conventional PKC isoform expression.

The effect of phorbol ester treatment on migration of C3H 10T1/2 and BT5C glioma cells: possible application to carcinogenesis.

Treatment with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and the second-stage promoter 12-O-retinoylphorbol 13-acetate induced down-regulation of protein kinase C (PKC) and enhanced the migration of C3H 10T1/2 cells. In the case of BT5C glioma cells the same negative correlation was observed only after treatment with TPA. The negative control 4 alpha-phorbol affected neither PKC activity nor the migratory ability of both cell lines.

The in vivo cytostatic effect induced by Propionibacterium granulosum on murine tumor cells.

The cytostatic action of Propionibacterium granulosum was studied in a mouse sarcoma in vivo. Kinetic analysis of tumor cells 28 days after tumor implantation and systemic immunotherapy showed that the cell cycle time was identical in both treated and untreated tumors. P. granulosum treatment resulted in a marked prolongation of the S phase and shortening of the G1 phase of the cell cycle. A pronounced drop in the number of labeled interphases and the reduction of the growth fraction were observed in tumors obtained from mice given injection of P. granulosum. Cloning efficiency of tumor cells from P. granulosum treated animals was quantitatively similar to that of control animals and only differences in the size of lung colonies were observed.

Peptide analog of fibronectin that inhibits cell migration and ERK 1/2 activity.

Two analogs of the peptide mimicking the 1977-1991 C- terminal part of fibronectin have been synthesized and tested. AWLI simulated human fibronectin fragment 1977-1991, whereas AWLII hybridized to both RGD and 1977-1991 fragments. AWLI and AWLII peptides inhibited the migration of the ovarian carcinoma cell line OVP10 regardless of the presence RGD. AWLI peptide inhibited spontaneous and fibronectin-activated cell migration and ERK1/2 activity. Neither AWLI nor fibronectin induced changes in FAK proteins, as could be judged from Western blots. In conclusion, it seems that the C-terminal fragment of fibronectin inhibits ERK1/2-dependent (random) migration of ovarian carcinoma cells.

Signal transduction in confluent C3H 10T1/2 cells. The role of focal adhesion kinase.

The activities of extracellular signal-regulated kinases (ERK1/ERK2) is required for proliferation of several types of cells. The performed analysis showed stimulation of ERK's by fetal calf serum (FCS) or fibronectin in the C3H 10T1/2 cell cultures at logarithmic phase of growth. The ERKs activity was not stimulated in confluent cells. This could not be accounted for a partial down regulation of ERK since its level was stable in both types of cells regardless of their density and kind of stimulation. Searching for ERK up-stream elements we studied the integrin receptor gene transcript by RT-PCR and focal adhesion kinase (FAK) by Western blotting and phosphorylation assays. It was found that FCS and fibronectin stimulated phosphorylating activity of FAK in the cells at the logarithmic phase of growth, but were inefficient in the confluent cells. RT-PCR showed the presence of alpha5 and beta1 integrin transcripts, and p125FAK was at the same level regardless of the type of stimulation. These data indicate that the ability of FAK to be activated plays an important role in ERK regulation and, in consequence in proliferation and growth inhibition during confluence.