Alpha-mannosidosis: characterization of CNS pathology and correlation between CNS pathology and cognitive function.

Alpha-mannosidosis (AM) (OMIM 248500) is a rare lysosomal storage disease. The understanding of the central nervous system (CNS) pathology is limited. This study is the first describing the CNS pathology and the correlation between the CNS pathology and intellectual disabilities in human AM. Thirty-four patients, aged 6-35 years, with AM were included. Data from 13 healthy controls were included in the analysis of the magnetic resonance spectroscopy (MRS). Measurements of CNS neurodegeneration biomarkers in cerebrospinal fluid (CSF), CSF-oligosaccharides, and performance of cerebral magnetic resonance imaging (MRI) and MRS were carried out. On MRI, 5 of 10 patients had occipital white matter (WM) signal abnormalities, and 6 of 10 patients had age-inappropriate myelination. MRS demonstrated significantly elevated mannose complex in gray matter and WM. We found elevated concentrations of tau-protein, glial fibrillary acidic protein and neurofilament light protein in 97 patients, 74% and 41% of CSF samples, respectively. A negative correlation between CSF-biomarkers and cognitive function and CSF-oligosaccharides and cognitive function was found. The combination of MRS/MRI changes, elevated concentrations of CSF-biomarkers and CSF-oligosaccharides suggests gliosis and reduced myelination, as part of the CNS pathology in AM. Our data demonstrate early neuropathological changes, which may be taken into consideration when planning initiation of treatment.

Cognitive profile and activities of daily living: 35 patients with alpha-mannosidosis.

BACKGROUND: Alpha-mannosidosis (OMIM 248500) (AM) is a rare lysosomal storage disease caused by a deficiency of the alpha-mannosidase enzyme. The typical signs consist of hearing impairment, intellectual disabilities, coarse facial features and motor function disturbances. We report on the cognitive function and activities of daily living in patients with AM. METHODS: Thirty five AM patients, age 6-35 years, were included in the study. As a cognitive function test, we used the Leiter international performance scale-revised (Leiter-R), which consists of two batteries: the visual function and reasoning battery and the memory and attention battery, the latter including a memory screening. Additional two questionnaires, The Childhood Health Assessment Questionnaire (CHAQ) and EQ-5D-5 L, were filled out. RESULTS: We found IQ in the range of 30-81 in our cohort. The total equivalent age (mental age) was significantly reduced, between 3-9 years old for the visual function and reasoning battery, between 2.3-10.2 years for the memory screening. Data suggested a specific developmental profile for AM with a positive intellectual development until the chronological age 10-12 years, followed by a static or slightly increasing intellectual level. All patients were to varying degrees socially and practically dependent and unable to take care of themselves in daily life. CONCLUSIONS: Intellectual disability is a consistent finding in patients with alpha-mannosidosis but with extensive variation. We assess that this group of patients has, despite their intellectual disabilities, a potential for continuous cognitive development, especially during childhood and early teenage years. This should be included and supported in the individual educational planning.

Enzyme replacement therapy for alpha-mannosidosis: 12 months follow-up of a single centre, randomised, multiple dose study.

BACKGROUND: Alpha-mannosidosis (OMIM 248500) is a rare lysosomal storage disease (LSD) caused by alpha-mannosidase deficiency. Manifestations include intellectual disabilities, facial characteristics and hearing impairment. A recombinant human alpha-mannosidase (rhLAMAN) has been developed for weekly intravenous enzyme replacement therapy (ERT). We present the preliminary data after 12 months of treatment. METHODS: This is a phase I-II study to evaluate safety and efficacy of rhLAMAN. Ten patients (7-17 y) were treated. We investigated efficacy by testing motor function (6-minutes-Walk-Test (6-MWT), 3-min-Stair-Climb-Test (3-MSCT), The Bruininks-Oseretsky Test of Motor Proficiency (BOT2), cognitive function (Leiter-R), oligosaccharides in serum, urine and CSF and Tau- and GFA-protein in CSF. RESULTS: Oligosaccharides: S-, U- and CSF-oligosaccharides decreased 88.6% (CI -92.0 -85.2, p < 0.001), 54.1% (CI -69.5- -38.7, p < 0,001), and 25.7% (CI -44.3- -7.1, p < 0.05), respectively. Biomarkers: CSF-Tau- and GFA-protein decreased 15%, p < 0.009) and 32.5, p < 0.001 respectively. Motor function: Improvements in 3MSCT (31 steps (CI 6.8-40.5, p < 0.01) and in 6MWT (60.4 m (CI -8.9 -51.1, NS) were achieved. Cognitive function: Improvement in the total Equivalence Age of 4 months (0.34) was achieved in the Leiter R test (CI -0.2-0.8, NS). CONCLUSIONS: These data suggest that rhLAMAN may be an encouraging new treatment for patients with alpha-mannosidosis.The study is designed to continue for a total of 18 months. Longer-term follow-up of patients in this study and the future placebo-controlled phase 3 trial are needed to provide greater support for the findings in this study.

New human transforming genes detected by a tumorigenicity assay.

We have developed a sensitive bioassay for transforming genes based on the tumorigenicity of cotransfected NIH3T3 cells in nude mice. The assay differs substantially from the NIH3T3 focus assay. Using it, we have detected the transfer of three transforming genes from the DNA of MCF-7, a human mammary carcinoma cell line. One of these is N-ras, which is amplified in MCF-7 DNA. The other two, which we have called mcf2 and mcf3, do not appear to be related to known oncogenes. We cannot detect their transfer by using the NIH3T3 focus assay. We do not yet know whether either mcf2 or mcf3 is associated with genetic abnormalities in MCF-7 cells.

Simian virus 40 (SV40) production from SV40-transformed human amnion cells of established lines.

Sixteen established cell lines of simian virus 40 (SV40)-transformed human amnion cells were examined for SV40 production. Many of these lines produced SV40 for extensive periods. Virus production had not ceased for 2 lines after 18 months, for 3 lines after 12 months, and for 3 lines at 3 months after recovery from "crisis". Three lines became virus-free in the first month, 1 line in the second month, 1 in the third month, and 1 in the fourth month, and 2 lines stopped virus production between 6 and 11 months after recovery. The virus titers were relatively low. Inclusion body-containing cells were infrequent. In contrast, in most cultures of SV40-transformed human fibroblasts rescued from crisis, no infectious virus was demonstrated, although exceptions have been reported. Virus was produced after heterokaryon formation of cells of the virus-free amnion lines with CV-1 cells in the presence of inactivated Sendai virus, as observed for SV40-transformed human fibroblasts. During the crisis period, some of the SV40-transformed amnion cells produced substantial amounts of virus. Titers decreased during the later periods of crisis. The most pronounced decrease in titers was in cultures from which established lines were recovered.

One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.

One hundred and twenty-seven cultured human tumor cell lines produced tumors after sc inoculation of 1-20 million cells into nude mice. They included 56 carcinoma lines, 14 sarcoma lines, and 57 lines from miscellaneous tumors and were all glucose-6-phosphate dehydrogenase type B. Twenty-nine percent of the lines produced tumors of 1 cm3 size within 1 month and 41% in the second month after inoculation. The histopathology correlated with the human tumor of origin in all cases.

Loss of heterozygosity in cultured human tumor cell lines.

One hundred and thirty-seven cultured human tumor cell lines derived from Caucasian patients were surveyed for ten of their polymorphic enzyme phenotypes. The gene frequencies in this cell line population were similar to those of normal Caucasian populations, although consistent differences in phenotype frequencies were detected at each of the loci. All 10 loci showed fewer heterozygous phenotypes and a correspondingly greater number of the common and rare homozygous phenotypes than occur in normal Caucasian populations. On the average, only 85% of the loci expressed in fully differentiated diploid cells were expressed in the neoplastic cell lines adapted to in vitro growth. There was no significant difference in the proportion of loci expressed in cells that had been passaged less than 10 times and in cells passaged more than 50 times. Consequently, it appears that there is a loss of expression in genetic marker loci from at least six different chromosomes. This loss occurs either in the in vivo tumor or in the very early stages of the cultivation of neoplastic cells derived from solid human tumors. Once the cells have become adapted to growth in vitro, the patterns of expression in their polymorphic loci remain stable for many passages.

Polymorphic enzyme analysis of cultured human tumor cell lines.

Of the total of 137 cultured cell lines from a wide variety of human neoplasms now typed at a maximum of 16 of their polymorphic enzyme loci, the phenotype combinations for 72 are described in this paper. The phenotype frequency product of each line was less than 0.05, and only 148 of the 9,316 (1.6%) pairwise comparisons between lines had phenotypes that did not differ in at least one locus. Thus the probability of any two randomly selected lines having indistinguishable genetic signatures in this sample of 137 lines was 0.016. Fifty-two of the 137 lines (38%) had phenotype combinations distinguishable from each of the other 136 lines. The frequency products of the 85 lines that could not be distinguished from at least one other line were all sufficiently high to preclude a suspicion that contamination had occurred among them. No additional cell lines with phenotype combinations indistinguishable from the combination characteristic of HeLa cells were identified among these 72 cell lines.

Distinction of seventy-one cultured human tumor cell lines by polymorphic enzyme analysis.

A large collection of cultured human tumor cell lines was characterized for the phenotypes of 16 polymorphic enzyme loci: ACP1, ADA, AK1, ESD, FUCA, GLO1, GOT2, G6PD, ME2, PEPA, PEPB, PEPC, PEPD, PGD, PGM1, and PGM3 primarily to detect and monitor against cell line contamination. Among 100 highly characterized cell lines, 59 lines from different patients and 6 pairs of lines (each pair from the same patient's tumor) had unique phenotype combinations and were therefore presumed to be authentic, uncontaminated cell lines. Besides these 71 lines, the remaining 29 lines consisted of several small groups of two to three lines, each group having a different combination and being among the more frequent in the normal population. The 29 lines, therefore, were not suspected to be contaminants. Among unusual findings were the ME2 1 plus 2 phenotype determined for two bladder tumor lines, a G6PD A phenotype found in a line of Caucasian origin determined not to be a HeLa contaminant, and asymmetrical heterozygous phenotypes in several lines. Except for kidney tumor lines, there was no correlation of adenosine deaminase tissue isoenzymes between tumor lines and normal tissues of origin. For several enzymes significant deviations were found in proportions of the phenotypes observed in Caucasian cell lines from expected proportions on the basis of normal population data, indicating possible natural selection among these lines in tissue culture or among the patients of origin.

Absence of HeLa cell contamination in 169 cell lines derived from human tumors.

Numerous cell lines derived from human tumors are not HeLa contaminants. Of 192 lines established in this or other laboratories, 169 lines were found to be G6PD type B. Twenty-three lines were type A as HeLa; three of these were of Negroid origin. There is reasonable doubt that the remaining 20 lines will all be shown to be confounded with HeLa.

Immunohistology of the antigenic pattern of a continuous cell line from a human colon tumor.

The antigenic surface pattern of a continuous cell line (HT29) derived from a human primary carcinoma of the colon was studied by the immunofluorescence technique. Monovalent and polyvalent immune sera were used. The cells of this long-term culture kept the ability to synthesize the three principal colon tumor antigens: carcinoembryonic and nonspecific cross-reacting antigens, and the membrane-associated tissular autoantigen. On the HT29 cells, which still carry the original blood group of the tumor donor, no receptors for human Ig's were detected.

Induction of retrovirus particles in human testicular tumor (Tera-1) cell cultures: an electron microscopic study.

The Tera-1 and Tera-2 cell lines, established from germ-cell tumors of the human testis, were examined by electron microscopy for particles with the morphology of retroviruses. Extracellular and budding particles were observed at low frequencies only in cultures of Tera-1 cells that had been treated with 5-iodo-2'-deoxyuridine and dexamethasone. No particles were detected in untreated cultures of Tera-1 cells or in any preparations of Tera-2 cells.

HLA-A, B, C and DR alloantigen expression on forty-six cultured human tumor cell lines.

Forty-six cultured cell lines of diverse human tumor origins, including 25 melanoma cell lines, were HLA allotyped with the use of a modified eosin complement-dependent cytotoxicity test in combination with absorption and two-color fluorescence techniques. In 10 cases (1 renal cell carcinoma line and 9 melanoma cell lines), the cell line donors had been HLA typed a few years before the cell line-typing project had started and in 13 cases (1 renal cell carcinoma line and 12 melanoma cell lines), the cell line donors were currently available for comparative typing of lymphocytes. HLA-typing results suggested that most cell lines expressed genetically appropriate HLA antigens, although 1 cell line had more than two HLA antigens for one HLA locus and 2 cell lines lacked expression of one or more HLA antigens in comparison with donor typing. One hepatoma cell line, 1 of 2 of the bladder carcinoma cell lines tested, and 17 of the 25 melanoma cell lines expressed DR alloantigens in addition to their HLA-A, B, and C locus antigens. For 9 of the melanoma cell lines, comparisons with donor DR alloantigens could be made, and all these cell lines had exactly the same DR allospecificities as those found on donor B-lymphocytes. HLA typing of cell lines can be used as an adjunct to polymorphic isoenzyme marker tests to verify their patient source and lack of contamination by another cell line, and HLA typing should be used to determine the antigen composition of cells used in the preparation of reagents for immunotherapy or in studies of tumor-specific immunity.

Incidence and pathological features of spontaneous tumors in athymic nude mice.

Systematic observation of 1141 nude mice (Swiss background [corrected] strain) that received human tumor transplants revealed 24 spontaneous tumors, 18 of lymphoreticular origin and 6 pulmonary adenomas. Spontaneous tumors were seen at an average age of 9.1 months, and 22 of the tumors were seen only in that fraction of our group (324 mice) surviving for 5 months or more (22 of 324 X 100 = 6.8%). Transplantation of these tumors to other nude mice was successful in three of five cases. Mice transplanted with adenocarcinoma of the colon and with tumors of the urogenital tract developed spontaneous tumors more often than did mice receiving other types of human tumor transplants. Progressive growth of the human tumor transplant occurred significantly less often in the mice that eventually developed spontaneous tumors than in the mice that showed no spontaneous tumor development. Nevertheless, the incidence of spontaneous tumors in these nude mice was similar to that reported for the thymus-bearing background strain.

Response of nude mouse-grown human urothelial cancer to cis-diamminedichloroplatinum(II), diammine[1,1-cyclobutanedicarboxylato(2-)-O,O'-platinum], and mitoguazone dihydrochloride.

A comparative study of the effect of cis-diamminedichloroplatinum(II) (cisplatin), diammine[1,1-cyclobutanedicarboxylato(2-)-O,O'-platinum] (carboplatin), and mitoguazone dihydrochloride on urothelial cancer was conducted using transitional cell carcinomas of the urinary bladder grown in the nude mouse. Tumors SW-780 and TCC-K1 represented transitional cell carcinoma, Grade II, whereas Tumor PR49 represented a fast-growing Grade III neoplasm. Of the agents studied, cisplatin was most effective, resulting in tumor response related to the dose administered. Response to carboplatin was clearly related to treatment schedule. For the same amount of total dose administered, better results were obtained when treatment was given three times weekly instead of once every week. Furthermore, cisplatin was more effective against the less differentiated PR49 tumor in contrast to carboplatin, which showed more activity against the better differentiated SW-780 and TCC-K1 tumors. None of the tumors tested responded to mitoguazone dihydrochloride. The results of the present study may assist in formulating better treatment modalities against urothelial cancer, taking into account factors such as tumor grade, growth rate, treatment schedule, and the patient's tolerance which may ultimately influence tumor response.

Response of nude mouse-grown adenocarcinomas of the human exocrine pancreas to cis-diamminedichloroplatinum(II), diammine[1,1-cyclobutanedicarboxylato(2-)-O,O'-platinum], and mitoguazone dihydrochloride.

The effect of cisplatin, carboplatin, and mitoguazone dihydrochloride on pancreatic cancer was evaluated using pancreatic ductal adenocarcinomas Capan-1, Capan-2, and PR54 grown in the nude mouse. In single agent treatments, cisplatin, given in the amount of 5 mg/kg once/week for 4 consecutive weeks, was most effective, resulting in tumor regression, growth arrest, and a growth delay period of 6 and 4 months for tumors Capan-1 and PR54, respectively. Treatment with carboplatin was less effective, with a tumor response related to treatment schedule. For the same amount of total dose of carboplatin administered, best results were obtained when treatment was given 3 times weekly instead of in single weekly injections. Mitoguazone dihydrochloride exhibited no antitumor effect. The results of the present work may be of significance in the management of pancreatic cancer patients.

Sensitivity of xenografts of human pancreatic adenocarcinoma in nude mice to heat and heat combined with chemotherapy.

The sensitivity of two human pancreatic adenocarcinomas (Capan-1 and Capan-2) to heat and heat combined with chemotherapy was studied using xenografts of the tumors in the foot of athymic nude mice. Heat was applied by immersion of the tumor in a water bath at 43.5 degrees for 1 hr. A single i.p. dose of mitomycin C, cisplatin, 5-fluorouracil, or 0.9% NaCl solution was given at 1 hr prior to treatment. Heat treatment alone significantly suppressed tumor growth (p less than 0.001), with 35% of the tumors showing complete regression. Combined treatment using heat plus chemotherapy yielded significantly greater suppression of tumor growth (p less than 0.05) with mitomycin for both tumors and with cisplatin or 5-fluorouracil for Capan-1. Combined treatment also gave higher rates of complete tumor regression: 55 and 64%, respectively, for Capan-1 and Capan-2 as compared with 18 and 47% for the respective tumors treated by heat alone. These observations suggest that human pancreatic carcinomas are sufficiently sensitive to heat combined with chemotherapeutic treatment to warrant a clinical trial of these modalities.

Epirubicin cardiotoxicity: a study of 135 patients with advanced breast cancer.

The cardiotoxicity of epirubicin (EPI) was evaluated clinically, radiologically, with ECG, and with multiple ECG-gated radionuclide determination of the left ventricular ejection fraction (LVEF) during rest in 135 patients with advanced breast cancer. The EPI doses were 60 mg/m2 on days 1 and 8 every 4 weeks or 45 mg/m2 plus vindesine 3 mg/m2 on the same schedule. The median cumulative dose of EPI was 500 mg/m2 (range, 47 to 1,563). Eight of the 135 patients developed congestive heart failure (CHF). Of 67 patients treated with EPI less than 500 mg/m2, none developed CHF. Among 48 patients treated with doses between 500 and 1,000 mg/m2, one had CHF (2%; 95% confidence limits, 0.1 to 11.1). Among 20 patients who received EPI from 1,000 to 1,563 mg/m2, seven developed CHF (35%; 95% confidence limits, 15.4 to 59.2). Four patients died due to cardiotoxicity. The risk of EPI cardiotoxicity at the present schedule is considerable at doses above 1,000 mg/m2. At doses between 500 and 1,000 mg/m2 the risk of CHF decreases, and at doses below 500 mg/m2, it is negligible. For all patients, the prevalence of CHF was 6% and the sensitivity of LVEF high (95%), mainly due to the low incidence of CHF. Among the 20 patients who received EPI at more than 1,000 mg/m2, the prevalence of CHF was 35% and the sensitivity only 64%. The specificity was maximally 62%. Our results suggest that LVEF is of no value as a predictor for CHF.

Elaboration of granulocyte-macrophage colony-stimulating factor by human tumor cell lines and normal urothelium.

A total of 72 cell conditioned media (CCM) were screened for their ability to stimulate colony formation by human granulopoietic progenitor cells. Granulocyte-macrophage (GM) colony-stimulating factor(s) (CSF) were found in CCM of nine tumor cell lines, two primary urinary bladder tumors, and three epithelial cell cultures of normal urinary tract. The most active medium came from urinary bladder carcinoma cell line 5637. CSF released by the 5637 cell line induced dose-dependent GM colony formation from human fetal, normal adult, and CML bone marrow (BM) and from mouse BM. Human fetal and normal adult BM formed more colonies when stimulated with 5637 CCM than with peripheral blood leukocyte (PBL) feeder layers, while CML BM produced more colonies with PBL feeder layers. CCM from 5637 was more active in stimulating GM colony formation than human placenta conditioned medium (PCM) and PHA-LCM. 5637 CCM produced in serum-free hormone-supplemented medium was nearly equipotent and can serve as suitable starting material for purification.

Gene transfer into cultured human epidermis and its transplantation onto immunodeficient mice: an experimental model for somatic gene therapy.

To try epidermis as a target for somatic gene therapy we studied transfected primary human keratinocytes grown in culture and grafted onto athymic mice. We have developed a novel technique for grafting cultured epidermal sheets onto mice. First, the graft is placed on the dorsal muscle fascia underneath the mouse skin using the latter as a bandage. Secondly, the mouse skin above the graft is removed, which exposes the grafted skin to open air and thus stimulates terminal differentiation. A novel method for the discrimination between murine and human epidermal cells is also presented, employing in situ hybridization with human Alu repeated DNA sequences. During monolayer culture the keratinocytes were lipofected with the gene for human growth hormone in an Epstein-Barr virus-based expression vector. The cells were allowed to develop a multilayered tissue for 5 d, secreting human growth hormone into the medium at a daily rate of at least 50 ng/cm2 of tissue. The transfected tissues were then grafted onto mice. We detected human growth hormone at levels of up to 2.6 ng/ml in mouse serum for 4 d, but later no human growth hormone could be found, although the transplants survived for months. To investigate the fate of the transfected cells in the transplanted tissue, we labeled them with the beta-galactosidase reporter gene. The cells staining positive for X-gal were found exclusively in the most superficial differentiated layers at 7 d after transplantation. This may be the main reason why no human growth hormone is found in the mouse circulation at this time.