Familial hyperinsulinism maps to chromosome 11p14-15.1, 30 cM centromeric to the insulin gene.

Familial hyperinsulinism (HI) is the most common cause of persistent neonatal hyperinsulinaemic hypoglycemia. Linkage analysis in 15 families (12 Ashkenazi Jewish, 2 consanguineous Arab, 1 non-Jewish Caucasian) mapped HI to chromosome 11p14-15.1 (lod score = 9.5, theta = 0 at D11S921). Recombinants localized the disease locus to the 6.6 cM interval between D11S926 and D11S928. In Jewish families, association (p = 0.003) with specific D11S921/D11S419 haplotypes suggested a founder effect. This locus, which is important for normal glucose-regulated insulin secretion, represents a candidate gene for studies of other diseases of beta-cell dysfunction including non-insulin-dependent diabetes mellitus (NIDDM).

Characterization of hereditary inclusion body myopathy myoblasts: possible primary impairment of apoptotic events.

Hereditary inclusion body myopathy (HIBM) is a unique muscular disorder caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. GNE encodes a bi-functional enzyme acting in the biosynthetic pathway of sialic acid. Since the underlying myopathological mechanism leading to the disease phenotype is poorly understood, we have established human myoblasts cultures, derived from HIBM satellite cells carrying the homozygous M712T mutation, and identified cellular and molecular characteristics of these cells. HIBM and control myoblasts showed similar heterogeneous patterns of proliferation and differentiation. Upon apoptosis induction, phosphatidylserine externalization was similar in HIBM and controls. In contrast, the active forms of caspase-3 and -9 were strongly enhanced in most HIBM cultures compared to controls, while pAkt, downregulated in controls, remained high in HIBM cells. These results could indicate impaired apoptotic signaling in HIBM cells. Since satellite cells enable partial regeneration of the post-mitotic muscle tissue, these altered processes could contribute to the muscle mass loss seen in patients. The identification of survival defects in HIBM affected muscle cells could disclose new functions for GNE in muscle cells.

Initiation points for cellular deoxyribonucleic acid replication in human lymphoid cells converted by Epstein-Barr virus.

Replicon size was estimated in two Epstein-Barr virus (EBV)-negative human lymphoma lines, BJAB and Ramos, and four EBV-positive lines derived from the former ones by infection (conversion) with two viral strains, B95-8 and P3HR-1. Logarithmic cultures were pulse-labeled with [3H]thymidine, and the deoxyribonucleic acid was spread on microscopic slides and autoradiographed by the method of Huberman and Riggs. After developing, replication forks were visualized as silver grain tracks on the autoradiograms. Average replicon size was estimated by scoring the number of replication forks per constant length of deoxyribonucleic acid and by measuring distances between centers of adjacent tracks, followed by detailed statistical analyses. Three of the four EBV-converted cell lines, BJAB/B95-8, Ra/B95-8, and Ra/HRIK, were found to have significantly shorter replicons (41, 21, 54% shorter, respectively), i.e., more initiation points, than their EBV-negative parents. BJAB/HRIK had replicons which were only slightly shorter (11%) than those of BJAB. However, analysis of track length demonstrated that extensive track fusion occurred during the labeling of BJAB/ HRIK, implying that its true average replicon size is shorter than the observed value. The results indicate that in analogy to simian virus 40, EBV activates new initiation points for cellular DNA replication in EBV-transformed cells.

Surface charge characteristics of cells from malignant cell lines and normal cell lines of the human hematopoietic system.

Cells from malignant and normal lines of human hematopoietic origin were studied for their surface charge characteristics with the use of the following criteria: 1) the electron microscopic appearance of cell membranes after labeling with cationized ferritin (CF) either before or after glutaraldehyde fixation, 2) electrophoretic mobility, 3) total sialic acid content, and 4) agglutinability with poly-L-lysine (PLL). CF induced a time-dependent redistribution of surface receptors in unfixed malignant cells but not in unfixed normal cells. After 10 seconds of labeling with CF, both normal and malignant unfixed cells showed a uniform and even labeling pattern. After 5 minutes of labeling, malignant cells exhibited a highly pronounced pattern of clusters and patches, as distinct from a random and even pattern exhibited by normal cells. Both normal and malignant cells after fixation exhibited an equivalent random and even labeling pattern with CF, independent of the duration of labeling. The malignant cells studied possessed less sialic acid, had a lower electric mobility, and were agglutinated more readily with PLL than were the normal cells.

Use of multiparameter studies and scanning electron microscopy in the interpretation and attempted correlation of surface morphology with cell type in 135 cases of human leukemias.

Multiparameter studies and scanning electron microscopy (SEM) were performed on cells obtained from 135 cases of leukemia in an attempt to clarify whether there was a reliable correlation between surface morphology and cell type as defined by cytochemistry, membrane markers, and transmission electron microscopy. These studies also attempted to determine whether SEM could be used to distinguish lymphoid and nonlymphoid leukemias, to recognize different types of lymphoid leukemia, and to define the cell type involved in cases of unclassified leukemia. The results of this study suggest that there is a good correlation between surface morphology as seen by SEM and cell type identified by multiparameter techniques. In most cases, nonlymphoid leukemic cells could be distinguished from lymphoid leukemic cells on the basis of their surface morphology. SEM did not appear to contribute to the diagnosis of unclassified leukemia, but more cases of this nature must be studied. Despite the fact that acute lymphoblastic leukemia cells frequently showed fewer microvilli than did other lymphoid leukemias, overlap of surface features in about one-third of the cases did not enable SEM to be used as a reliable means of distinction. The above conclusions appear to be supported by preliminary scanning immunoelectron microscopic observations on leukemic cells. It is concluded that SEM is a useful aid to other modes of microscopy in leukemia but should not be used on its own to establish diagnosis.

Fluidity of membrane lipids and lateral mobility of concanavalin A receptors in the cell surface of normal lymphocytes and lymphocytes from patients with malignant lymphomas and leukemias.

Lymphocytes isolated from the peripheral blood of patients with nonmalignant and malignant disorders were studied for fluidity of membrane lipids and lateral mobility of concanavalin A (Con A) receptors. The degree of fluidity of the surface membrane lipid core was monitored quantitatively by fluorescence polarization analysis using the probe 1,6-diphenyl-1,3,5-hexatriene embedded in lipid regions of the surface membrane of intact cells. Mobility of Con A surface receptors was determined by the cap-forming ability after binding of fluorescent Con A. The present studies were performed on lymphocytes from 28 patients with malignant lymphomas, 22 patients with leukemia, 28 individuals who either were healthy or had nonmalignant disorders, and 5 patients with carcinoma. The results showed that lymphocytes and mononuclear cells from patients with malignant lymphomas and leukemias have a more fluid lipid layer in their surface membrane than do lymphocytes obtained from healthy individuals or from patients with other malignant and nonmalignant disorders. This increase in membrane fluidity was less pronounced in lymphocytes isolated from leukemic patients in clinical remission and from leukemic patients receiving treatment with steroids. The results also show a marked difference in the cap-forming ability of lymphocytes from patients with malignant lymphomas or leukemia as compared with lymphocytes from patients with non-malignant disorders or carcinoma. Lymphocytes isolated from lymphoma and chronic lymphatic leukemia patients during remission stages of the disease exhibited a higher cap-forming ability. The cap-forming ability of cells from patients with chronic lymphocytic leukemia was unaffected by treatment with steroids. The present results, which are in line with previous observations, have shown that normal lymphocytes can be characterized by a low degree of lipid fluidity but a high degree of mobility of Con A receptors, whereas leukemic lymphocytes are characterized by a high degree of lipid fluidity but a low degree of mobility of Con A receptors. These results confirmed our general hypothesis on the dynamic interrelation between membrane lipids and membrane protein receptors, and they indicate that the widely accepted term "membrane fluidity" requires better consideration for different membrane components.

Inhibitors of epidermal growth factor receptor kinase and of cyclin-dependent kinase 2 activation induce growth arrest, differentiation, and apoptosis of human papilloma virus 16-immortalized human keratinocytes.

Human papilloma virus 16 (HPV 16) is associated with cervical cancer and is therefore considered a major health risk for women. Immortalization of keratinocytes induced by HPV infection is largely due to the binding of p53 and Rb by the the viral oncoproteins E6 and E7, respectively, and is driven to a large extent by a transforming growth factor alpha/amphiregulin epidermal growth factor receptor autocrine loop. In this study, we show that the growth of HPV 16-immortalized human keratinocytes can be blocked by a selective epidermal growth factor receptor kinase inhibitor, AG 1478, and by AG 555, a blocker of cyclin-dependent kinase 2 (Cdk2) activation. AG 1478 induces a massive increase in the Cdk2 protein inhibitors p27 and p21, whereas AG 555 appears to have a different mechanism of action, inhibiting the activation of Cdk2. Growth arrest induced by AG 1478 and AG 555 is accompanied by up to 20% of cells undergoing apoptosis. Following AG 1478 treatment but not AG 555 treatment, up to 50% of cells undergo terminal keratinocyte differentiation as determined by filaggrin expression and by the decline in the expression of cytokeratin 14. The growth-arresting properties of AG 1478 and AG 555 identifies them as possible lead antipapilloma agents.

Specific binding of placental acidic isoferritin to cells of the T-cell line HD-MAR.

Acidic placental isoferritin inhibited the blastogenic response of peripheral human lymphocytes to T-cell activating lectins. We measured specific binding of radioiodinated placental isoferritin to cells of the T-cell line HD-MAR and found specific high-affinity binding. Binding was faster and more ferritin was bound at 37 degrees C than at 4 degrees C. Displacement experiments indicated that most of the binding occurred at the cell surface. Acidic placental ferritin and isolated H-type ferritin subunits but not isolated L-type subunits, competed for the binding. Scatchard plot analysis showed characteristics of a single binding species with a dissociation constant (Kd) of 1.3-4.4 x 10(-11) M. The results suggest the presence of receptors for acidic isoferritin on T-lymphocytes and thus, a regulatory role for the acidic ferritin H-type subunit in T-cell function.

Inhibitors of tyrosine kinases in the treatment of psoriasis.

Psoriasis is a heterogenous skin disease, characterized by epidermal hyperproliferation, abnormal keratinization and inflammation. The heterogeneity of the disease results probably from the interaction of multiple gene abnormalities with environmental factors. The new approaches to drug design have become refocused to the emerging understanding of the role of signaling pathways in health and disease. Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiation and immune processes. Uncontrolled signaling from receptor and intracellular tyrosine kinases can lead to numerous proliferative diseases: cancer, leukemia, restenosis and psoriasis. Identification of PTKs that play a key role in a defined disease can lead to a selective drug. The balance of signals which regulate the homeostasis of normal epidermis is altered in psoriasis. Several lines of evidence suggest a role for the EGF receptor system in this process. Therefore, blockers of the EGFR kinase were suggested as potent antipsoriasis agents. PTK inhibitors from the tyrphostin family were found to block EGF - dependent cell proliferation. AG 1571 (SU 5271) potently inhibits ligand-induced autophosphorylation of EGF-R, downstream signal transduction events, DNA replication and cell cycle progression at micromolar concentrations, as well as proliferation of keratinocytes isolated from psoriatic lesions in excellent correlation with its EGFR kinase inhibitory activity in these cells. AG 1571 (SU 5271) has been in clinical trials by SUGEN Inc. since early 1997. Overexpression of the EGFR is the hallmark of most epithelial cancers. Therefore one can view blockers of the EGFR kinase as becoming universal inhibitors. Tyrphostins are the first signal transduction agents to be used in the clinic. This article summarizes recent progress in the development of PTK inhibitors in the treatment of psoriasis.

Tibial implant mineralization in rats is inversely related to serum osteogenic capacity.

In this study, the correlation between the mineralization of healing bone defects and the osteogenic capacity of the serum was tested in rats. The purpose of the study was to test the hypothesis that during callus formation, some serum factors are consumed. The bone defects in the tibia contained two different implant types, and all sustained juxta-implant fractures. One implant type was the coral Porites and the other was its recrystallized version (Interpore-200), which exhibit different mineralization rates during fracture healing. Use of these two implant types permitted generation of an expanded mineralization spectrum suitable for regression analysis. Mineralization was assessed by measuring the mineral content change (MCC) using dual-energy X-ray absorptiometry. Osteogenic capacity of sera of the implanted rats was assessed by its ability to increase specific alkaline phosphatase (ALP) activity in stromal cell cultures. The MCC was followed for 5 weeks in the Porites and Interpore-200 implants, and it was found that the MCC in Interpore-200 implants exceeded that of the Porites implants. Thus, the two implant types generated a wide mineralization spectrum. Induction of ALP in stromal cell culture was lower for sera derived from rats implanted with Interpore-200 than for sera derived from rats implanted with Porites. Two weeks after implantation, the change in serum ALP induction correlated inversely with the MCC of bone defects. This indicates that during callus formation, the mineralization rate is reciprocally related to the serum osteogenic capacity. The decreased serum osteogenic capacity may be interpreted by the hypothesis that callus formation consumes certain serum osteogenic factors.

Characterization of a novel Burkitt's lymphoma-associated antigen: GP70. Reactivity of anti-GP70 antibodies with various malignant and non-malignant cell lines.

A glycoprotein of 70 kDa (GP70) was isolated from sera of Burkitt's lymphoma (BL) patients and used to immunize rabbits. Anti-GP70 antibodies at a high titer were obtained and used for screening of cancer cells of various origin by the indirect immunofluorescence test. Thus, 66% of BL-cell lines tested were positive to GP70. On the other hand, all lymphoblastoid cell lines tested were negative. Moreover, all peripheral blood cells and mononuclear cells from tonsils were negative, indicating specificity of antibodies to malignant transformation. Comparison between positively stained BL-cell lines indicated no correlation between the presence of GP70 and EBNA. Positive stain (1-5%) obtained with bone marrow cells might indicate that anti-GP70 antibodies are directed against a surface membrane differentiation glycoprotein.

The effect of IL-4 on the phenotype of a human B-cell lymphoma line (Farage) lacking immunoglobulin expression.

Farage cells do not express surface immunoglobulins (sIg) but display a high level of CD19, CD21, CD22, CD23, CD39, CD40 B-cell antigens, and various adhesion proteins, such as CD11a (LFA-1), CD29 (VLA-4), CD44, CD54 (ICAM-1), and CD58 (LFA-3). The phenotype of Farage resembled that of EBV-LCL but differed from the phenotype of Burkitt's lymphoma lines, which were CD39- CD44-, expressed a high level of CD38, and either lacked CD21 or were weakly positive. Exposure to IL-4 augmented the concentrations of CD23 and of adhesion proteins on the surface of Farage cells but diminished the expression of CD21, CD22, and CD38. IL-4 did not induce the expression of sIg on Farage cells and failed to affect the level of HLA-DR. IL-2 and TPA did not alter the level of CD21 and adhesion proteins on Farage cells. Although IL-4 induced unique changes of the antigenic pattern in Farage, no significant effect on the phenotype of Burkitt's lymphoma lines was detected after IL-4 treatment. The present study indicates that the responsiveness of B cells to IL-4 is not determined by the expression of sIg but rather is associated with the antigenic profile characteristic for non-germinal center B lymphocytes.

Sublines of the Burkitt line Daudi, selected on the basis of reactivity with soybean agglutinin, differ in tumorigenicity in athymic mice.

Two subpopulations were isolated on the basis of soybean agglutinin (SBA) binding, from the human Burkitt lymphoma line Daudi. The low- and high-binder sublines maintained this characteristic in continuous passages. Their surface marker profiles, antibodies, scanning electron microscope (SEM), cytochemical reactions and binding of other lectins (concanavalin A and wheatgerm agglutinin) were not different. They differed, however, in growth potential in athymic mice. The low-binder subline had lower frequency of takes, tumor weight and volume, and did not metastasize as compared to the high-binder subline. However, the reaction with F-SBA of all the tumor cells examined was strong (greater than 70%), indicating in vivo selection and tumor development of high binder cells.

Chemo-immunotherapy in patients with metastatic melanoma using sequential treatment with dacarbazine and recombinant human interleukin-2: evaluation of hematologic and immunologic parameters and correlation with clinical response.

We have treated 18 patients with metastatic malignant melanoma (MM) with high-dose IL-2 administered by continuous iv infusion in combination with dacarbazine (DTIC), and correlated the clinical response with various hematologic and immunologic parameters. Two regimens differing in the sequence of treatment were employed, and 1-6 treatment cycles were given, depending on patient response. Two patients had a complete response (CR, 46+m, 14m), two patients a partial response (PR, 16m,6m), one a minimal response and four had a stable disease lasting 2-7 months, thus the response rate (CR+PR) was 22%. None of the following parameters, tested prior to initiation of the therapy and 1-2 days after termination of each course of IL-2, correlated with the clinical response: WBC counts (total and differential), levels of blood CD4 and CD8 T cells, NK cells, monocytes and B cells, production of IL-1 and IL-1 inhibitor by monocytes, responsiveness to 3 mitogens, NK/LAK cell activity, and serum levels of IL-1 alpha, IL-2, soluble IL-2 receptor, and TNF alpha. The only prognostic parameter was the greater increase in the level of IL-2 receptor (Tac)-bearing lymphocytes in the responding patients after 1-3 cycles of IL-2. The data suggests that non-specific immune parameters have no prognostic value for patients undergoing IL-2-based immunotherapy.

Interaction of soybean agglutinin with human leukemia-lymphoma lines at various stages of differentiation.

Human leukemia-lymphoma cell lines reflecting hematopoietic clones at various stages of differentiation were examined for reactivity with soybean agglutinin (SBA). The binding and redistribution pattern of soybean surface receptors was determined with fluorescein-isothiocyanate conjugated SBA (F-SBA) by ultraviolet microscopy, and with a fluorescent activated cell sorter (FACS). The results indicate that there is a correlation between SBA labelling--distribution and the stage of lymphoid cell differentiation. The SBA labelling on the membrane of null lines was undetectable by U.V. microscopy and flow cytometry. A gradual increase in SBA labelling correlating with the stage of differentiation was observed on cell lines of both B and T origin. However the maximal fluorescence intensity of the T lines was lower than the B lines. The redistribution pattern of SBA on the membrane of T lines was rings and mild patches, whereas that on the B lines was moderate to large patches. The reactivity of the lymphoid lines with SBA was not affected by growth conditions. The binding of SBA to normal lymphoblastoid lines was generally low and the fluorescence intensity weak. The reactivity of these lines with SBA was not associated with their origin or "age". It is suggested that the differences in the reactivity of SBA with human hematopoietic lines at various stages of maturation may be of value in future understanding the differences in structure and function of the surface membrane between normal and malignant cells, and the relation to normal and abnormal cell differentiation.

Establishment and characterization of a new permanent cell line (GDM-1) from a patient with myelomonoblastic leukemia.

The GDM-1 permanent cell line was established from the peripheral blood of a patient with a Philadelphia chromosome negative myeloproliferative disorder, after transformation to acute myelomonoblastic leukemia. The GDM-1 cells exhibited the same characteristics as those isolated from the peripheral blood of the patient prior to death: cells contained non-specific esterase sensitive to fluoride, myeloperoxidase, lysozyme (muramidase), and exhibited both Fc and complement (C3) receptors but lacked B- and T-cell surface markers including T-associated antigens. E-rosetting capacity, surface and intracytoplasmic immunoglobulins and EBV determined nuclear antigen (EBNA). The GDM-1 cells bore the 1a receptor and the myeloid leukemia antigen (M-1). The karyotype of the cultured leukemic cells showed the same specific chromosomal abnormalities present in the monoblasts obtained from the peripheral blood prior to death, indicating that the cell line was derived from the original leukemic cells.

Concanavalin A receptors on the surface membrane of lymphocytes from patients with acute leukemia.

Peripheral blood mononuclear cells (PBM) isolated from 23 patients with acute lymphoblastic leukemia (ALL) and 24 with acute non-lymphoblastic leukemia (ANLL) were studied for binding and mobility of Concanavalin A (Con A) receptors, using fluorescent Con A (F-Con-A). The cap forming ability of PBM from all patients was 18.7 (+/- 9.3%) and 18.9 (+/- 9.9%) for ANLL patients at the time of diagnosis or during relapse. During clinical complete remission the cap forming ability of the PBM did not change significantly. No correlation was observed between the percentage of blasts present in the peripheral blood at the time of examination and the extent of cap formation, for both types of leukemia. The pattern of F-Con-A binding to PBM in ANLL patients was different compared to that seen in ALL. In ANLL, the fluorescent stain was concentrated in a round body on the cell ("button form") after binding to the membrane, while the rest of the cell showed almost no fluorescence. The present results indicate that PBM cells from patients with acute leukemia are characterized by a high degree of Con-A receptor mobility.

Biological activity of tyrosine kinase inhibitors: novel agents for psoriasis therapy.

Uncontrolled signaling from protein tyrosine kinases (PTKs) can lead to numerous proliferative and inflammatory diseases, and identification of the specific PTKs that play a key role in a defined disease could potentially lead to a selective therapeutic agent. In psoriasis, the balance of signals that regulate the homeostasis of normal epidermis is altered. Several lines of evidence suggest a role for the epidermal growth factor receptor (EGFR) system in this process. The PTK inhibitor from the tyrphostins family--AG-1571 (SU-5271) potently inhibits proliferation of psoriatic keratinocytes in excellent correlation with its EGFR kinase inhibitory activity, and was in clinical trials by SUGEN Inc. The recently developed in vivo models of psoriasis may become useful tools to evaluate PTK inhibitors to treat the disease and open a novel specific therapeutic approach. This article summarizes recent progress in the development of PTK inhibitors in the treatment of psoriasis.

Characterization of murine autologous salivary gland graft cells: a model for use with an artificial salivary gland.

The purpose of this study was to examine the growth and key functional abilities of primary cultures of salivary epithelial cells toward developing an artificial salivary gland. Cultures of epithelial cells originating from submandibular glands of BALB/c mice were established. Parenchymal cells were isolated by a Percoll gradient technique and thereafter seeded on irradiated NIH 3T3 fibroblasts serving as a feeder layer. The isolated cells were termed autologous salivary gland epithelial (ASGE) cells and could be cultivated for at least five passages (time limit of experiments). ASGE cells presented the typical organizational behavior of epithelial cells and electron microscopy, as well as immunostaining for cytokeratins, confirmed their epithelial origin. Furthermore, measurements of transepithelial resistance and water permeability indicated the ability of the ASGE cells to form a functional epithelial barrier. This study suggests that primary salivary epithelial cells can be obtained that exhibit critical characteristics needed for use with an artificial secretory device.

Establishment of a human T-acute lymphoblastic leukemia cell line with a (16;20) chromosome translocation.

A new T-cell line, Loucy, was established from the peripheral blood of a patient with T-cell acute lymphoblastic leukemia (T-ALL). The surface marker analysis of the cell line is OKT3+, OKT4+, THB4+, J5 +/-, OKT6-, TdT-, and HLA-DR-, indicating stage IV in T-cell lineage. Karyotype analysis revealed 45,X,5q-,t(16;20)(p12;q13). The translocation between chromosomes 16 and 20 has not been previously detected in ALL. This cell line may be of value in evaluating the role of t(16;20) in the etiology of T-ALL.