Glycolipid composition of ascitic fluids from patients with cancer

The glycolipid composition of ascitic fluids from nine patients with cancer and one pleural effusion from a hepatoma patient was studied. Glucosylceramide, lactosylceramide, globotriaosylceramide, and globotetraosylceramide were found in all samples and also in normal human serum. These glycolipids accounted for more than 90% of the neutral glycolipid fraction and the composition in ascitic fluids was similar to that in normal human serum. From ascitic fluids, several minor glycolipids, which could not be detected in normal human serum, were isolated and characterized by exoglycosidase treatment. Lactoneotetraosylceramide was found in eight samples of ascitic fluids, and globopentaosylceramide was detected in two samples from hepatoma and one from pancreatic cancer. A fucolipid which was converted to lactoneotetraosylceramide by alpha-L-fucosidase treatment was recognized in two samples from hepatoma patients. In the ganglioside fraction, GM3 was the predominant component both in normal human serum and in ascitic fluid. The GM2 content in ascitic fluids was much higher than that in normal human serum. From these results, lactoneotetraosylceramide and GM2 are possible candidates as cancer markers, because they seemed to be derived from cancer tissues by shedding.     

The availability of iron and the growth of Vibrio vulnificus in sera from patients with haemochromatosis

Abstract The ability of the marine bacterium Vibrio vulnificus to grow in sera from patients with haemochromatosis and from normal human volunteers was investigated. Studies showed that although V. vulnificus possesses potential iron uptake systems, the organism is unable to multiply in normal human sera. However, it grows rapidly in serum from patients with haemochromatosis. This growth was found to be due solely to the presence of freely available iron.     

Morquio's disease type A: Absence of material cross reacting with antibodies against N-acetylgalactosamine-6-sulfate sulfatase

Summary An antiserum was raised in guinea pigs against purified normal human N-acetylgalactosamine-6-sulfate sulfatase, the enzyme affected in Morquio's disease type A. The antiserum precipitated most of N-acetylgalactosamine-6-sulfate sulfatase from a concentrate of normal human urine. The antigen-antibody complex was enzymatically active. Urine concentrates from five patients with Morquio's disease type A did not contain material competing with the normal enzyme for binding to soluble or Sepharose-bound antibodies. No precipitin arc was obtained on immunodiffusion of antiserum and urine from the single patient investigated by this method. From the sensitivity of the indirect immunoassay it was concluded that the urine of the five patients contained less than 5% of the normal amount of cross-reacting material.     

Cyclic AMP phosphodiesterase in human lymphocytes and lymphoblasts.

Cyclic nucleotide phosphodiesterase activities were examined in lymphocytes from 12 transformed human B cell lines, two T cell lines, six patients with lymphocytic leukemia, and 10 normal donors. A consistent difference bwtween cells from the normal and leukemic state was observed. The cyclic AMP phosphodiesterase activity from normal lymphocytes is inhibited greater than 80% by muM cyclic GMP while this concentration of nucleotide has little or no effect on the enzyme from transformed lymphocytic cell lines or from lymphocytic cells of leukemia patients. The reported lack of cyclic GMP phosphodiesterase in human lymphocytes from several sources is confirmed. The apparent absence of a cyclic GMP degradation mechanism and of cyclic GMP control of cyclic AMP hydrolysis may be related to defective lymphocyte growth control.     

Antibody-dependent cell-mediated cytotoxicity using a murine monoclonal antibody against human colorectal cancer in cancer patients

Summary Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by a murine monoclonal antibody against human colerectal carcinoma, antibody 19–9, with human effector cells was tested in 33 patients with various carcinomas, 16 patients with benign lesions, and 13 normal controls, using a 12-h ⁵¹Cr release assay using human colorectal cancer cells as targets. Peripheral blood mononuclear cells (PBM) from these groups of patients and normal controls achieved moderate levels of target cell lysis in the presence of the monoclonal antibody at the high effector to target cell ratio of 200:1. The ADCC activity of PBM in cancer patients was significantly higher than that in either normal persons or patients with benign lesions. Since the ADCC was shown to be mainly mediated by adherent monocytes in the PBM, ADCC activity of monocytes from cancer patients was compared to those from control groups at an effector to target cell ratio of 30:1. The results also showed that the lytic capacity of monocytes was significantly higher in cancer patients than that in the control populations.     

An immunocytochemical study of normal and abnormal human cerebrospinal fluid with monoclonal antibodies to glial fibrillary acidic protein

The presence of astrocytes in the cerebrospinal fluid (CSF) of patients may be of diagnostic importance. However, the frequency with which astrocytes are shed into normal and abnormal human CSF is unknown. This issue was studied using monoclonal antibodies to an astrocyte-specific antigen, glial fibrillary acidic protein (GFAP), and immunoperoxidase cytochemistry. The study was prospectively conducted on 108 CSF preparations diagnosed as normal, reactive, metastatic malignancy or suspicious for metastatic malignancy. To validate these methods, cells from a clonal human glioma cell line, which contains astrocytes rich in GFAP, were processed in a manner identical to that used for the CSFs obtained from patients. Studies of the human glioma cell line demonstrated intense GFAP immunoreactivity in the majority of the malignant astrocytes. In contrast, none of the CSFs contained GFAP-positive cells. We conclude that immunocytochemical methods can detect GFAP in neoplastic human astrocytes but that nonneoplastic GFAP-positive cells are uncommon in human CSF; such cells were not seen in our large series of normal and abnormal human CSFs. The immunocytochemical detection of GFAP may be a useful criterion for distinguishing malignant astrocytes from other types of malignant cells in human CSF.     

The ultrastructure of normal and denervated human facial muscle

The ultrastructure of normal human facial muscles from 25 nonparalytic and 17 paralytic patients revealed normal features in nondenervated human facial muscles, identical to the fine structure of other normal human and mammalian cross-striated muscle fibers. However, in denervated facial muscle, a broad spectrum of ultrastructural lesions had affected sarcomeres, abnormal inclusions, and organelles. A large variety of inclusion bodies, some of which have not been described, were also found. The spectrum of ultrastructural changes showed no dependence on the length of the denervation period. There were no inclusion bodies in all the normal facial muscle biopsies. To our knowledge, this study represents the first systematic electron microscopic investigation of normal and denervated human facial muscles.     

Prevention of chromosomal breakage in Fanconi's anemia by cocultivation with normal cells

Summary The frequency of aberration in cultured lymphocytes from patients with Fanconi's anemia was significantly reduced when the cells were cocultivated with normal human lymphocytes. The results suggest that most of the chromosomal aberrations observed in cultured cells from Fanconi patients arise during cultivation and that the presence of normal cells prevents chromosomal damage by means of a hitherto unknown mechanism.     

Localization of a human gene homologous to the PrP gene on the p arm of chromosome 20 and detection of PrP-related antigens in normal human brain

Infectious fractions prepared from scrapie-infected hamster brains contain a protein, PrP 27-30, which shares antigenic determinants with polypeptides found in similarly prepared fractions from patients with Creutzfeldt-Jakob disease. cDNA sequences encoding the hamster PrP 27-30 identified homologous sequences in the human genome as well as in normal human brain mRNA preparations. Antibodies raised against the mouse PrP's identified antigenically related peptides in both normal hamster and human brain as well as in scrapie-infected hamster brain and CJD-affected human brain. By using in situ hybridization we localized the homologous human genomic sequences on the short arm of chromosome 20. Our results indicate that the reportedly unique proteins detected in human CJD preparations derive from normal human gene products.     

Peroxisomes in regenerating human skeletal myofibers

Peroxisomes of the human regenerating skeletal myofibers were studied qualitatively and quantitatively by electron cytochemistry and were compared with those of the mature normal human skeletal muscle fibers. Peroxisomes visualized by electron cytochemistry with 3,3'-diaminobenzidine (DAB) were small round or oval bodies delimited by a single membrane and contained the electron-opaque, coarsely granular matrix. Muscle grafts of the regenerating normal human quadriceps obtained from 4 orthopedic patients were analyzed 2 weeks after transplantation into nude mice; they contained peroxisomes with a mean diameter of 0.25 microns, ranging from 0.12 to 0.67 microns. The group mean density of peroxisomes per 100 microns2 was 2.0 +/- 0.4 (SE), while that of histochemically normal mature human quadriceps femoris myofibers was 0. The cytochemical controls without DAB or with the presence of 3-amino 1,2,4-triazole in the solution containing DAB lacked the electron-opaque reaction, indicating that these reactions were on an enzymatic basis. The results of this study showed clearly that the regenerating normal human skeletal myofibers contained numerous peroxisomes differing from the mature normal human muscle fibers in which the peroxisomes were not observed.     

Shared antigens between human malignant melanoma cells and Mycobacterium bovis (BCG).

This study was undertaken to investigate the antigenic relationships between human malignant melanoma cells and Mycobacterium bovis (BCG). Rabbits were immunized with sonicates of BCG or with malignant melanoma cells from different patients and the resulting antisera were tested for their capacity to bind radiolabeled soluble extracts prepared from BCG and melanoma cells. The binding of antibodies to radiolabeled antigens was studied by precipitation of radiolabeled antigen-antibody complexes by anti-rabbit immunoglobulin. Antibodies in sera from rabbits immunized with either BCG (anti-BCG) or melanoma cells (anti-melanoma) bound both the labeled BCG and melanoma antigens. Control antisera, from rabbits immunized with human acute or chronic lymphatic leukemia cells or with normal human spleen cells, did not bind significant amounts of radiolabeled BCG. Antibodies in sera from rabbits immunized with normal spleen cells bound small but significant amounts of radiolabeled melanoma antigens. Binding by anti-BCG and anti-melanoma to the radiolabeled antigens was studied before and after absorption of antisera with cells from human melanoma, leukemia, guinea pig hepatoma, and normal human spleen cells. Inhibition studies using unlabeled BCG extracts also were carried out. The absorption and inhibition studies confirmed that the binding reactions were specific and that antigens from five melanoma patients shared antigenic determinants with BCG.     

人正常食管鳞状上皮不同原代培养方法的比较

该研究通过比较人正常食管鳞状上皮不同的原代培养方法,以期为不同的实验目的提供不同的培养方法。实验用到的正常食管粘膜上皮来源于食管癌患者手术切除的标本,采用组织块法和酶消化法,分别用DMEM/F12混合培养基和K-SFM无血清培养基进行培养。通过直接观察、细胞形态学观察和免疫细胞化学方法观察细胞的生长情况、细胞形态学特征及鉴定所得到的细胞,比较不同方法与不同培养基组合中原代培养细胞的生长状况。用组织块法,在DMEM/F12混合培养基中人正常食管上皮细胞生长较好,细胞融合较快,成纤维细胞污染较少,15～17天上皮细胞铺满瓶底的70%～80%,获得的细胞数量大,但细胞传代后成纤维细胞污染严重。用酶消化法,在K-SFM无血清培养基中人正常食管上皮细胞生长好,细胞融合快,成纤维细胞污染基本消除,细胞纯度高,10～12天细胞便可以铺满瓶底的70%～80%,这种方法培养的细胞可以冻存、复苏和传代。其余各种培养方法所得细胞无论在生长状态、培养周期、成纤维细胞污染和传代方面均较前两种方法差。以上各种方法培养的细胞经免疫细胞化学染色鉴定证实细胞呈广谱细胞角蛋白阳性,确定是食管上皮来源的细胞。酶消化法加K-SFM无血清培养基是本实...     

Radiosensitive Down syndrome lymphoblastoid lines have normal ionizing-radiation-induced inhibition of DNA synthesis

The extent of X-ray-induced inhibition of DNA synthesis was determined in radiosensitive lymphoblastoid lines from 3 patients with Down syndrome and 3 patients with ataxia telangiectasia (AT). Compared to 6 normal control lines, the 3 AT lines were abnormally resistant to X-ray-induced inhibition of DNA synthesis, while the 3 Down syndrome lines had normal inhibition. These results demonstrate that radiosensitive human cells can have normal X-ray-induced inhibition of DNA synthesis and provide new evidence for the dissociation of radiosensitivity from radioresistant DNA synthesis.     

Biosynthesis of human alpha-N-acetylgalactosaminidase: defective phosphorylation and maturation in infantile alpha-NAGA deficiency

The biosynthesis of human alpha-N-acetylgalactosaminidase (alpha-NAGA) was studied in normal fibroblasts and in cells from patients with infantile alpha-NAGA deficiency. Normal alpha-NAGA is synthesized as a 52 kDa precursor which matures to a 49 kDa species through phosphorylation and carbohydrate triming. Fibroblasts from the patients synthesize normal amounts of a 52 kDa precursor, however phosphorylation does not occur and this precursor is subsequently degraded intracellularly.     

Human myocardial ATP content and in vivo contractile function

The study was designed to characterize the relationship between the metabolise content of human cardiac muscle and in vivo cardiac function. ATP, total adenine nucleotides, and NAD were quantified in human myocardial biopsies using high performance liquid chromatography. Right ventricular endomyocardial biopsies were obtained from 43 patients with dilated cardiomyopathy, 6 with restrictive cardiomyopathy, 10 with normal systolic and diastolic function, and from 24 cold preserved human donor hearts. Transmural samples of failing right and left ventricular free walls were obtained during cardiac transplantation surgery in 8 patients. ATP, total adenine nucleotides, and NAD were similar in the cold-preserved donor hearts and in right ventricular endomyocardial biopsies from the 10 individuals with normal systolic and diastolic function. In contrast, these values were significantly depressed in tissue samples from patients with dilated or restrictive cardiomyopathy. There was a significant correlation between ATP and pulmonary capillary wedge pressures but not ejection fractions. Declines in the sizes of myocardial ATP, adenine nucleotide, and pyridine nucleotide pools in the human myocardium are associated primarily with diastolic but not systolic dysfunction.     

Hexose transport in human myoblasts.

The present investigation reports on the hexose transport properties of human myoblasts isolated from normal subjects and from patients with Duchenne muscular dystrophy (DMD). Similar to rat myoblast L6, normal human myoblasts possess a high- (HAHT) and a low- (LAHT) affinity hexose transport system. The non-metabolizable hexose analogue, 2-deoxyglucose, is preferentially taken up by HAHT. The transport of this analogue is the rate-limiting step in the uptake process. This human myoblast HAHT is also similar to that of the rat myoblast in its substrate specificity and in response to the energy uncouplers, cytochalasin B and phloretin. The human myoblast LAHT resembles that of rat myoblast in its insensitivity to energy uncouplers, and in its transport affinity and capacity for 3-O-methyl-D-glucose. Although DMD myoblasts resemble their normal counterpart in their ability to differentiate, they differ significantly in their hexose transport properties. In addition to HAHT and LAHT present in normal human myoblast, DMD myoblasts contain a super-high-affinity hexose transport system (SHAHT). SHAHT can be detected only at very low substrate concentrations. It differs from HAHT not only in its much higher transport affinity, but also in its response to the traditional hexose transport inhibitors. For example, SHAHT can be activated by cytochalasin B and phlorizin, whereas it is more sensitive to inhibition by phloretin. Unlike HAHT, energy uncouplers are found to be ineffective in inhibiting SHAHT. It should be mentioned that SHAHT cannot be detected in myoblasts isolated from patients with other types of myopathy. The present study serves to demonstrate that more than one hexose transport system is operating in human skeletal muscle cells, as found in other cell types.     

Analysis of human p53 proteins and mRNA levels in normal and transformed cells

p53 mRNA and proteins were examined in a variety of human transformed cells and in normal human foreskin fibroblast cells. Both the steady-state and translatable levels of p53 mRNA were the same in normal and transformed human cells. In vitro synthesized p53, programmed by mRNA from normal and transformed human cells, revealed that there was heterogeneity in the primary structure of p53 from these cells. Pulse labeling of cells and immunoprecipitation analysis with a panel of human reactive anti-p53 antibodies demonstrated that the types of p53 synthesized in vitro corresponded to the types made in vivo from SV80 and COLO 320 cells. No p53 was detectable by similar pulse-labeling analysis of HeLa and normal foreskin fibroblast cells. Since it was necessary to use anti-p53 sera from cancer patients to carry out much of the immunoprecipitation analysis in this study we therefore further characterised these sera to determine if they reacted with one or more than one epitope. p53-beta-galactosidase fusion proteins were synthesized in Escherichia coli and used to analyse the anti-p53 antibodies produced by cancer patients. We demonstrate that the antisera contain antibodies directed against epitopes in both the N-terminal and C-terminal regions of the p53 molecule.     

Nuclear DNA analysis of koilocytic and premalignant lesions of the uterine cervix.

Cervical biopsy samples were taken from 79 patients who had various grades of cervical intraepithelial neoplasia or who showed evidence, in the form of koilocytosis, of human papillomavirus infection of the uterine cervix and from 10 women with normal cervices. The DNA content of the cells in the samples was analysed by flow cytometry. Analysis of the data obtained showed that the biopsy samples from women with cervical intraepithelial neoplasia and human papillomavirus lesions contained significantly more dividing cells (31.2% of cells from human papillomavirus lesions with no cervical intraepithelial neoplasia and 33.06%, 29.89%, and 31.76% of cells from cervical intraepithelial neoplasia grades I, II, and III, respectively) than those from women with normal cervices (21.6%). The proportion of aneuploid samples from the group who showed evidence of human papillomavirus infection only (18.2%) did not differ significantly from the group with cervical intraepithelial neoplasia grade III (21.2%). Aneuploidy and an increased rate of cellular proliferation are recognised characteristics of malignancy. These results therefore support the view that human papillomavirus plays an important part in the aetiology of cervical carcinoma and are relevant to the clinical management of patients.     

Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.     

The effect of tuberculosis and neoplasia on human monocyte staphylocidal activity

Monocytes from normal subjects and patients with tuberculosis, lymphoma, or solid tumors were evaluated for their ability to kill Staphylococcus aureus organisms in an in vitro system. Monocytes from patients with tuberculosis demonstrated enhanced staphylocidal activity as compared to normal monocytes after 30- and 60-min periods of incubation. Monocytes from lymphoma patients had normal killing rates after 30 min incubation but demonstrated significantly enhanced killing after 60 min incubation. Monocytes from patients with solid tumors demonstrated essentially normal killing rates. These data suggest functional activation of human monocytes by mycobacterial infection and lymphoma similar to that described in animal models.