Variation in glycosaminoglycan components of breast tumors.

The correlation between the content of individual glycosaminoglycans and the histological patterns are studied on breast tumor tissues. The myxomatous stroma of intracanalicular fibroadenoma contained a large amount of glycosaminoglycans, which were mainly hyaluronic acid. The chondroitin 4- and 6-sulfate level was also high. As the supporting stroma of this tumor became denser and more fibrous, the level of hyaluronic acid content was reduced. In the case of pericanalicular fibroadenoma, glycosaminoglycans were small in amount and the levels of hyaluronic acid and chondroitin sulfate were low, but the ratio of dermatan sulfate content was higher. In the case of gynecomastia, the conent was almost the same as that of pericanalicular fibroadenoma. Scirrhous carcinoma tissues contained a relatively large amount of hyaluronic acid and chondroitin sulfate. No remarkable differences in heparan sulfate content were observed in any one of the breast tumors tested. Dermatan sulfate-chondroitin sulfate copolymers were detected in all the tumors. The presence of dermatan sulfate seemed to have an intimate relation with the fibrogenesis in the interstitial stromal element of the tumor tissues.

Cell surface glycosaminoglycans of cell line MDCK derived from canine kidney.

Morphological observations and biochemical analysis were made on glycosaminoglycans produced by MDCK cells of dog kidney origin growing on a glass surface as a mosaic of epithelium with many multicellular hemishperical vesicles. MDCK cells synthesized glycosaminoglycans, which consisted mainly of heparan sulfate and hyaluronic acid. The majority of the substances were contained in a cell-surface component removable with ethylenediaminetetraacetic acid-trypsin. In the radioautograph of tissue sections, high radioactivity of 35SO4 was observed on the medium-bathed cell surface, where Alcian blue-strained material could be observed. Ultrastructurally, the surface of microvillous processes which were abundant on the cell surface in contact with the medium was stained with ruthenium red. A small amount of chondroitin 4- and 6-sulfates were also synthesized. After 24 hr, the majority of chondrotin [35S] sulfates newly formed were secreted into the cultured medium, whereas haparan [35S] sulfate was released much less, remaining as a cellular component. The biological roles of glyconsaminoglycans produced by epithelial cells are discussed.

Selective toxicity of 5-S-cysteinyldopa, a melanin precursor, to tumor cells in vitro and in vivo.

The effect of 5-S-cysteinyl-L-3,4-dihydroxyphenylalanine (cys-dopa), an intermediate in the pathway from L-3,4-dihydroxyphenylalanine (L-dopa) to pheomelanin, on the growth of eight human tumor cell lines in culture was compared to that of L-dopa. The tumor cell lines tested comprise two neuroblastomas (NB-1 and YT-nu), two amelanotic melanomas (HMV and SEKI), a gastric carcinoma (MKN-28), and three squamous cell carcinomas (HeLa-S3, KB, and a salivary gland carcinoma). Cys-dopa at a concentration of 1 mM inhibited growth of NB-1 (66%), YT-nu (67%), HMV (44%), SEKI (60%), MKN-28 (47%), HeLa-S3 (24%), KB (64%), and salivary gland carcinoma (33%), while L-dopa exhibited similar or even lower degree of inhibition at a concentration of 6 mM. On the other hand, both catechols had little effect on the growth of two fibroblasts derived originally from normal tissues (mouse fibroblast L929 and Chinese hamster fibroblast Don-6). Cys-dopa and L-dopa inhibited DNA and protein synthesis in YT-nu cells, but RNA synthesis was less affected. Treatment with cys-dopa at a dose of 1000 mg/kg i.p. for 7 days prolonged by 50% the life span of mice inoculated with L1210 leukemia. Normal mice given cys-dopa at a dose of 1000 mg/kg for 12 days showed no signs of toxicity. These results suggest the potential of cys-dopa as an antitumor agent.

Reduction of adriamycin toxicity by ascorbate in mice and guinea pigs.

The effect of ascorbate in reducing Adriamycin toxicity has been examined in mice and guinea pigs. Ascorbate had no effect on the antitumor activity of Adriamycin in mice inoculated with leukemia L1210, but it significantly prolonged the life of mice and guinea pigs treated with Adriamycin. Adriamycin elevated lipid peroxide levels in serum and liver, and ascorbate prevented the elevation. The significant prevention of Adriamycin-induced cardiomyopathy by ascorbate was proved by means of electron microscopy. The earliest alterations of dilation of the sarcoplasmic reticulum and transverse tubular system and the appearance of a large number of cytoplasmic fat droplets, which were seen in cardiac tissue from guinea pigs receiving Adriamycin, were apparently reduced in animals that were treated with ascorbate.

Basic study on gene therapy of human malignant glioma by use of the cationic multilamellar liposome-entrapped human interferon beta gene.

For gene therapy of human malignant glioma, we adopted positively charged multilamellar liposomes entrapping the human interferon beta (hIFN-beta) gene. One week after the transplantation of human malignant glioma U251-SP cells to produce glioma in nude mouse brain, the liposomes entrapping the gene (500 ng of DNA per 25 nmol of lipids per 2 microl) were injected into the same site of the cell transplantation once every second day for a total of five injections; and by this means the tumor completely disappeared. To confirm the antiproliferative effect of hIFN-beta, we performed an in vitro study using a plasmid containing a secretion signal sequence-deleted hIFN-beta gene and one containing the hIFN-beta gene inserted in reverse. In both cases, there was no hIFN-beta release into the medium and no growth inhibition effect. On addition of anti-hIFN-beta antibody to the medium, the growth inhibition effect was abolished. As this cell line expresses IFN-alpha/beta receptor, the hIFN-beta produced in the transfected cells could be released and acted in a paracrine manner. For 120 days the body weight change of normal mice treated by the same procedure as used in the curing experiment was not significant among the groups injected with empty liposomes, plasmid only, and liposomes entrapping the gene. In all of these three groups, death, abnormal behavior, and significant histological changes were not observed.

Ascorbic acid and adriamycin toxicity.

Adriamycin (ADR) is effective against a wide range of human neoplasms. However, its clinical use is compromised by serious cardiac toxicity, possibly through induction of peroxidation in cardiac lipids. Ascorbic acid, a potent antioxidant, was examined for effect in reducing ADR toxicity in mice and guinea pigs. Ascorbic acid had no effect on the antitumor activity of ADR in mice inoculated with leukemia L1210 or Ehrlich ascites carcinoma, but it significantly prolonged the life of animals treated with ADR. ADR elevated lipid peroxide levels in mouse heart, and ascorbic acid prevented the elevation. The significant prevention of ADR-induced cardiomyopathy in guinea pigs by ascorbic acid was proved by electron microscopy. Ascorbic acid and the derivatives may delay general toxicity of ADR and also prevent the cardiac toxicity. The results also suggest the clinical efficacy of the combined treatment of ADR and ascorbic acid or the derivatives.

Langerhans cells increase in the dermal lesions of adult T cell leukaemia in Japan.

In cases of adult T cell leukaemia neoplastic T cell infiltration in the skin was accompanied by an increase in Langerhans cells. This is in keeping with the view that Langerhans cells may induce antigen-specific and allogenic T cell activation.

Cytogenetic characterization of a T-cell line, ATN-1, derived from adult T-cell leukemia cells.

A T-cell line, ATN-1, was established by culturing peripheral blood mononuclear cells derived from a patient with adult T-cell leukemia/lymphoma (ATL/L). Identities of the patterns of chromosomal abnormalities, cell surface phenotypes, morphologic findings, rearrangement patterns of T-cell receptor beta chain gene, and an integration site of human T-cell leukemia virus I proviral genome indicated that ATN-1 was derived from original leukemic cells. Both ATN-1 and the original leukemic cells showed a variety of patterns of chromosomal abnormalities that include 3q-, 6q-, rearrangements involving 2q31, 7q11.2, 8q11, 8q24, 19p13.3, and also 14q11 and 14q32, where genes for the T-cell receptor alpha chain and the immunoglobulin heavy chain are located. Availability of a genuine ATL/L cell line with these chromosomal abnormalities may greatly facilitate the biologic analysis of ATL/L.

A Novel Cell Line (NCU-L-1) from a Patient with Acute Lymphoblastic Leukemia, FAB, L3, Burkitt's Type.

A novel cultured cell line, NCU-L-1, was established from a 71-year-old Japanese woman with acute lymphoblastic leukemia, L3 FAB, Burkitt's type. The NCU-L-1 cells were shown to have a mature B-cell phenotype on the basis of immunologic surface marker analysis; including IgG Lambda surface immunoglobulins, CD19, CD20 and la-like antigen which were all detected on the cells. Intracytoplasmic immunoglobulin was not detected, but IgG was present in the cell culture supernatant. Cytogenetic studies revealed that the NCU-L-1 cells had t(2; 8) and an additional 14q+, a genotype which has not been identified previously in the usual Burkitt's cell lines. The NCU-L-1 cell line should prove to be useful for studying oncogenic events associated with the t(2; 8) translocation and karyotype evolution.

Human glioma-specific antigens detected by monoclonal antibodies.

Three murine monoclonal antibodies, designated GA-17, GB-4, and GC-3, were prepared by the hybridization of murine myeloma cells (NS-1) and spleen cells of BALB/c mice immunized with the crude membrane fraction of cultured human gliosarcoma cells (GI-1). Two of them (GA-17 and GB-4) reacted exclusively with the membrane of glioma cells, and the other (GC-3) reacted with the membrane of glioma cells and a T cell line (MOLT-4). Although these antibodies reacted with almost all of the gliomas, the reactions differed. GA-17 reacted equally well with all glioblastoma (17 cases) and low-grade astrocytoma (10 cases), whereas GB-4 reacted poorly with 7 cases of glioblastoma and GC-3 did not react with 7 cases of low-grade astrocytoma. The antigens, exclusively expressed on the cell surface, were analyzed by surface labeling with 125I followed by a cell lysis and immunoprecipitation with these antibodies. The findings obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that GA-17, GB-4, and GC-3 reacted with Mr 140,000-145,000, Mr 160,000, and Mr 145,000-150,000 proteins, respectively. Some evidence has been obtained indicating that these antigens are composed of the same polypeptide chain (Mr 120,000) with the carbohydrate chains being different.

Comparison of Langerhans cells and interdigitating reticulum cells.

Double immunostaining for OKT-6 and S-100 protein antibodies was carried out. However, it was impossible to stain using two antibodies at the same time, or using one antibody prior to another. We first stained using FITC labeled OKT-6 antibody, and secondary stained for S-100 protein by the ABC method, after taking photographs and removing OKT-6 reaction products. The exact same cells could be stained by both OKT-6 and S-100 protein antibodies. It has been elucidated that both OKT-6 and S-100 protein positive cells are LCs, and only S-100 protein positive, but OKT-6 negative cells are IDCs. LCs only exist in the superficial and hilar lymph nodes. However IDCs exist not only in these lymph nodes, but also in the mesenteric lymph nodes and spleens.

Studies on Langerhans cells in the tracheal squamous metaplasia of vitamin A deficient rats.

LCs in the tracheal squamous metaplasia of vitamin A deficient rats were studied. The first appearance of LCs with Birbeck granules (BGs) was in the stage of stratification. The number of LCs increased along with the development of metaplasia, but decreased in number in the later stage, where basal cells also differentiate into flattened epithelium. In the area where only basal cells differentiated into flatter epithelium, without a cornified layer, LCs with BGs could also be found. These findings suggest that LCs which originate in bone marrow mature in squamous epithelium. The distribution and morphology of LCs are dependent on the state of keratinocytes, but not the presence of a cornified layer.

A comparative study on Langerhans cells in lymph nodes with dermatopathic lymphadenopathy and histiocytosis X cells.

It was elucidated that the majority of DC were LC which were positive for CD 1a, but negative for PCNA, and possessed BG in the lymph nodes with DPL. On the other hand, HCX cells were almost always positive for PCNA. From this point of view, it can be speculated that LC in the lymph nodes of the DPL are non dividing mature cells and migrate from the skin lesion. HCX cells which were positive for CD 4 may be more immature cells than LC in DPL, and may be pathological cells which can divide in the foci.

Familial macrothrombocytopenia with unusually elongated mitochondria.

We report a familial case of macrothrombocytopenia without inclusion bodies in polymorphonuclear cells or any congenital abnormalities. The results of the hemostatic and platelet function tests were all normal except for the platelet retention rate. The number of megakaryocytes increased slightly and some were relatively small. Electron microscopic studies revealed a unique morphological abnormality of the platelets' mitochondria.

A case of AFP-producing early gastric carcinoma with rapid growth liver metastasis.

An 84-year-old man presented with complaints of epigastric discomfort. Upper gastrointestinal series and endoscopy showed an elevated lesion at the posterior wall of greater curvature on the gastric fundus. Diagnosed as moderately differentiated tubular adenocarcinoma by biopsy, wedge resection and 4sa regional lymph node dissection were carried out. The tumor morphology showed type I with slight elevation, 2.5 x 1.7 cm in size; histological showed papillary, tubular, and solid formations having clear cytoplasm and large bizarre nuclei invading the deep submucosal layer (sm2). This case was evaluated as T1(sm) N0 M0 stage Ia early gastric cancer. In the 5th month after operation, multiple liver metastases were detected. He died of liver failure by rapid growth of metastatic tumors in the 6th month after operation. The serum alpha-fetoprotein level at recurrence was 1,900 ng/mL, and alpha-fetoprotein-positive cells were immunohistochemically detected in operative and liver biopsy specimens.

Differentiation of Langerhans cells from interdigitating cells using CD1a and S-100 protein antibodies.

The present study shows that Langerhans cells can be differentiated from interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.

Comparative studies of the same adenocarcinoma cells, macrophages, and mesothelial cells by light microscopy, scanning electron microscopy, and transmission electron microscopy.

The identification of cells in body cavities of cancer patients is sometimes difficult to make. In order to make a definite cytological diagnosis, we observed the same cells by using light microscopy (LM)-scanning electron microscopy (SEM)-transmission electron microscopy (TEM). In this study we first stained cells by the Papanicolaou method after fixation in 1% glutaraldehyde for LM, and then attempted to observe them successively by SEM-TEM after fixation in 1% paraformaldehyde and 1.25% OsO4. Our method and procedures in examining successively one and the same cells in body cavity fluids by using LM, SEM, and TEM ensured accurate discrimination among adenocarcinoma cells, mesothelial cells, and macrophages. The results of this study suggest that LM-SEM-TEM may be of diagnostic value in distinguishing among mesothelial cells, macrophages, and adenocarcinoma cells. This method also succeeded in disclosing differences between the ultrastructure of the cell surfaces, and those of the cytoplasm, and of the nuclei. It is desirable that LM-SEM-TEM observation can be introduced into various aspects in order to obtain an improvement in the diagnosis by cytologic examination, the judgment of therapeutical effects, drug selection, and prognostic presumption.

Presence of nuclear grooves in endometrial cytology.

Although the presence of nuclear grooving in papillary carcinomas of the thyroid has been well-described, so far the attention paid to similar structures in endometrial cell samples has been limited. In order to investigate the occurrence of nuclear grooves in endometrial specimens from patients with various pathologic conditions of the endometrium, we compared their appearance with papillary thyroid cancers. A total of 10 cases was studied (age range, 40-72 yr), all cases demonstrating nuclear grooves. In each case, 10 random high-power fields (HPFs) were investigated, and the numbers of fields in which nuclear grooving could be seen were recorded. Nuclear grooves were observed in 3-36 of each HPFs and were more often observed in atypical endometrial cells than in their normal-appearing counterparts; however, these nuclear alterations were thought to be nonspecific findings.

Studies on intranuclear inclusions and nuclear grooves in papillary thyroid cancer by light, scanning electron and transmission electron microscopy.

OBJECTIVE: To successively examine intranuclear inclusions and nuclear grooves in the same papillary thyroid cancer specimens using a light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope (TEM). STUDY DESIGN: We stained cells by the Papanicolaou method after fixation in 1.25% glutaraldehyde for LM and then attempted to observe them successively by SEM-TEM after fixation in 2% paraformaldehyde and 2% osmium tetroxide. RESULTS: On SEM, intranuclear inclusions were observed as elevated parts, like hills, and nuclear grooves were observed as deep fissures or shallow cracks, sometimes with a few in one cell. On TEM, both intranuclear inclusions and nuclear grooves seemed formed by the nuclear membranes. Intranuclear inclusions also possessed cytoplasm and/or cytoplasmic organelles within some expanded areas in the nuclear grooves. CONCLUSION: It was evident from our three-step technique that intranuclear inclusions and nuclear grooves were essentially the same structures.

Male siblings with dyserythropoiesis, microcephaly and intrauterine growth retardation.

Male siblings with intrauterine growth retardation, hydrops, mild liver dysfunction, chronic diarrhoea, failure to thrive and microcephaly are reported. In both patients, the intrauterine growth retardation was detected in the second trimester of pregnancy. Relatively severe early onset neonatal jaundice, microcytosis, anisocytosis and abnormal iron metabolism were also seen. Bone marrow examination in the second sibling showed marked ringed sideroblasts and multilobulated erythroblasts in late developmental stages. The brain was very small with enlarged cerebrospinal fluid space, a reduced number of gyri and a thin cortex. The clinical and laboratory findings in these patients appear to be unique.