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.

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.

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.

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.

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.

Reactivity of Human Monoclonal Antibody Campath-1 with Human Leukemia Lymphoma Cell Lines of Varying Maturation.

Human leukemia-lymphoma cell lines reflecting hematopoietic clones at various stages of differentiation were examined for binding and complement mediated lysis by Campath-1. Expression of the cell surface antigen was determined with fluorescein isothiocyanate conjugated Campath-1, by ultraviolet microscopy and with a fluorescent activated cell sorter (FACS). The results indicate that there is a correlation between Campath-1 binding and the stage of lymphoid-cell differentiation. The null and T lines bound Campath-1 weakly and the fluorescence intensity was low. In the B lines there was a gradual increase in labelling correlating with the stage of differentiation. The redistribution pattern of Campath-1 on the membrane of null and T lines was in the form of rings and small caps whereas that on the B lines was that of moderate patches. Complement mediated cytolysis occurred with the majority of the cell lines, but did not correlate with the extent of surface antibody binding nor with the stage of lymphoid differentiation. The recovery and proliferative capacity of the residual cells after treatment with Campath-1 and complement was low for the null and T lines. This was variable for the B lines, but some did not regain proliferative capacity, whereas others recovered after treatment. The present results suggest that Campath-1 may have considerable utility in marrow depletion of residual. leukemic cells, more specifically of null and T-cell origin, prior to autologous transplantation. Determination of both the sensitivity of the patient's leukemic: cells to cytolysis and the recovery of proliferative capacity of Campath-1 resistant cells may contribute essential information concerning the possible efficacy of purging with this antibody in patients with significant marrow involvement prior to transplantation.

Molecular characterization of an unusual non-Hodgkin's B-lymphoma cell line ("Farage") lacking the ability to produce immunoglobulin polypeptide chains.

"Farage" is a cell line derived from a patient who had a diffuse and mixed type malignant lymphoma. In a previous study it was shown that Farage cells expressed B-cell markers, but not membrane IgM. Karyotypic analysis showed that in contrast to most follicular cell lymphomas, Farage did not have the 14;18 chromosomal translocation. In the present work Farage was further characterized by Southern and Northern blot analyses. Two rearranged heavy chain alleles and one rearranged kappa chain gene were detected. The cells expressed both mu and kappa mRNA, even though at a 3-7 fold lower level than that found in the control Daudi and DG-75 Burkitt lymphomas. Farage cells did not express the terminal deoxynucleotydyl transferase gene (TdT), nor the recombination activating genes RAG-1 and RAG-2, known as markers of the pre-B cell stage. These results show that Farage represents a mature B-cell rather than a pre-B cell. Despite the presence of C kappa and C mu RNAs, no Ig polypeptide chains were produced by Farage as judged by immunoblotting and biosynthesis labeling assays. Ig mRNAs were detected on the polysomal fraction, but at a lower level relative to Daudi cells. Our combined results suggest that in Farage cells translation of Ig mRNA is not fully blocked at the stage of translation initiation. Farage cells may express "germline" or mutated variants of Ig mRNAs. The unusual phenotype of Farage may reflect a normal as yet unknown stage of B-cell differentiation, or it may be due to an aberrant expression developed after malignant transformation.

Opposing effects of tyrosine kinase inhibitors on mineralization of normal and tumor bone cells.

Induction of matrix maturation and mineralization in calcified tissues is important for patients with primary bone tumors and other bone deficiencies, e.g., osteoporosis. For the former it signifies a better prognosis in osteosarcoma, and for the latter it might improve bone remodeling. In the present study we exposed osteosarcoma cells (Saos2), normal bone cells, and marrow stroma to two different tyrosine kinase (TK) inhibitors: AG-555 and AG-1478. These tyrphostins differ in their effect on signal transduction downstream to the TK receptor (RTK): AG-1478 inhibits src family TKs whereas AG-555 inhibits nuclear TKs. We found that both tyrphostins at 50 microM increased specific alkaline phosphatase (ALP) activity in Saos2 cells. AG-555 abrogated mineralization whereas AG-1478 increased it. Similarly, in human bone-derived cell cultures the same dose of tyrphostins had an opposing effect on mineralization but, in contrast to AG-555, AG-1478 positively selected cells with ALP activity. These tyrphostins also differed in their effect on rat marrow stromal cells. AG-555 decreased cell counts unselectively, whereas the decreased cell counts by AG-1478 resulted in selection of osteoprogenitor cells as indicated by a concordant increase in specific ALP activity. The effect of a lower dose of AG-1478, 5 microM, on the increase in mineralization exceeded its own efficiency in selecting cells with specific ALP activity. Our results indicate that AG-1478 selects and preserves the osteoblastic phenotype, at doses moderately higher than those required to induce mineralization, and substantially higher than the doses required for RTK inhibition. Identification of downstream molecular targets for AG-1478, in marrow stromal cells, might prove useful in designing more selective drugs, capable of separating proliferative from differentiation-inducing activities.

Distribution and modulation of surface charges of cells from human leukemia-lymphoma lines at various stages of differentiation.

Untreated and retinoic acid (RA) treated human leukemia-lymphoma cell lines reflecting hematopoietic cells at various stages of differentiation, were examined electron microscopically for their surface negative charge distribution using cationized ferritin (CF), an electron dense label of anionic sites. The results indicate that there is a correlation between the CF labeling density/distribution and the stage of lymphoid cell differentiation. Viable unfixed null cell lines show a low CF labeling density with few and small CF patches. A gradual increase in CF labeling density and increase in size and number of CF patches correlates with the stage of differentiation on cell lines of both T or B origin. Treatment of viable unfixed cells with 10(-5) MRA for 10 days seems to prevent the CF-induced formation of CF patches, resulting in a continuous and even distribution of the CF label, similar to that observed on the surface of cells fixed before CF labeling. Some correlation between the distribution of surface anionic sites and the malignant potential of the human leukemic lines could be detected.

Analysis of cell-mediated mineralization in culture of bone-derived embryonic cells with neurofibromatosis.

von Recklinghausen neurofibromatosis (NF1) is an autosomal dominant genetic disorder associated with congenital pseudoarthrosis and with short stature. To examine whether the NF1 phenotype includes functional osteogenic defects, embryonic bone-derived cells affected with NF1 were tested in culture for specific alkaline phosphatase (ALP) activity and cell-mediated mineralization and compared with other embryonic bone derived cells. NF1 showed a relatively higher specific ALP activity, which has further increased in response to dexamethasone + beta-glycerophosphate (beta GP) (Dex medium) coordinately with a decrease in cell proliferation. In In the control group, two samples showed increased ALP activity, one showed decreased activity and the forth one did not show any change in ALP. NF1 cells were distinguished from other cells regarding day 21 mineralization, they did not mineralize when cultured with ascorbate alone in the absence of Dex medium, whereas control cells did mineralize. Adding beta GP resulted in mineralization by NF1 cells but less than in other cells. In addition, NF1 cells responded to dexamethasone by increasing the beta GP-induced mineralization, as opposed to cells from other embryonic bones, which either did not respond or have even decreased mineralization under dexamethasone. Upon cis-hydroxyproline exposure, Dex medium has also distinguished NF1 cell ALP activity from that of other cell origins. Inhibition of respiratory complex II by malonate showed that most embryonic bone-derived cells of 12 weeks gestation are malonate resistant; thus, malonate selection was ineffective. This is in contrast to rat marrow stromal cells previously shown to undergo mineralizing cell enrichment in response to malonate. Exposure to levamisole, of Dex-treated cells, at days 0-11 has inhibited day 21 mineralization in all tested cultures in spite of the increase in day 11-specific ALP activity. Both malonate and levamisole did not distinguish NF1 cells from the osteogenic phenotype of other cells. Essentially embryonic bone-derived cells from 12 weeks gestation, cultured in the absence of beta GP, retained their mineralization capacity, which does not increase under dexamethasone, as distinguished from NF1 cells which require beta GP for mineralization and positively respond to dexamethasone. Therefore, bone-derived NF1 cells may be useful for studying the regulation of the mineralization process.

Differential effect of isolated placental isoferritins on in vitro T-lymphocyte function.

The effect of isoferritins isolated from human term placenta on certain T-lymphocyte parameters was studied in vitro using normal human lymphocytes. These isoferritins differed in ion exchange affinity, isoelectric point, and subunit composition. Only the acidic isoferritins caused a marked suppression of phytohaemagglutinin (PHA) blastogenesis and the most acidic isoferritin ('Acid I') was suppressive at a concentration as low as 0.25 microgram/ml. All four isoferritins suppressed concanavalin A (Con A) blastogenesis in a similar concentration dependent manner, with maximum effect at an isoferritin concentration of 1 microgram/ml. Both basic and acidic isoferritins reduced the Con-A-capping phenomenon in normal lymphocytes at concentrations higher than 0.5 microgram/ml, but at 0.25 microgram/ml only the acidic isoferritin was effective. The above findings support our previous report concerning the suppressive effect of splenic ferritin on T-lymphocyte function in vitro and indicate that acidic isoferritins, which often predominate in malignancy, demonstrate a higher degree of immunosuppressive activity. Thus, acidic isoferritins may play a role in the development of abnormal lymphocyte function encountered in certain proliferative disorders.

Cross-reactions of anti-DNA autoantibodies with cell surface proteins.

Anti-DNA autoantibodies are thought to play a major role in the pathogenesis of systemic lupus erythematosus. However, the mechanism(s) by which they participate in tissue and organ damage is not well understood. It has been suggested that these antibodies combine with DNA or DNA-histone complexes to produce circulating immune complexes which may deposit in various tissues. Alternatively, anti-DNA autoantibodies could interact directly with tissue components by way of immunological cross-reaction. In this study we have used a panel of mouse monoclonal autoantibodies with anti-nuclear specificity and measured their binding to membrane proteins of several tissues and cell lines. We show that the anti-DNA antibodies, but not anti-RNA or anti-histone antibodies bind to membrane proteins of molecular weights 102, 80, 42, 35 and 31 kDa, which are expressed in different combinations on several cell types. The binding of anti-DNA antibodies to these cell surface proteins was not affected by DNase treatment of the target cells, was increased by DNase treatment of the antibody preparations and was completely inhibited by DNA, indicating a true cross-reaction and not an indirect interaction of antibody and membrane proteins through a DNA bridge. Our results suggest that direct binding of anti-DNA autoantibodies to cell surface membrane proteins may play an important role in the induction of the pleomorphic tissue damage in systemic lupus erythematosus.

Production of C5a antagonist by synovial and peritoneal tissue fibroblasts.

Fibroblasts grown from synovial and peritoneal tissues release into the medium an inhibitor of neutrophil chemotaxis. The inhibitor resembles the antagonist previously described in synovial and peritoneal fluids. It is a heat stable (56 degrees C) protein of MW approximately 40 kDa that counteracts the chemotactic activity of zymosan-activated serum or purified C5a but not the peptide chemoattractant F-met-leu-phe. No chemotactic inhibitor was detected in media from skin fibroblast cultures or in formal human sera. It is suggested that the inhibitor is produced locally by synovial and peritoneal fibroblasts and that it might play a role in the regulation of inflammation at sites lined with mesothelium.

Decreased neutrophil thromboxane A2 and endothelial PGI2 production in the postoperative period. An in vitro assay for detection of neutrophil and plasma dysfunction.

Severe trauma results in reversible abnormalities in neutrophil function, but the specific role in the pathogenesis of postoperative sepsis is undetermined. Twenty adult patients undergoing elective surgical procedures were studied. Blood samples were obtained prior to and 24 hours after operation. Blood neutrophils were isolated and incubated (10(7) cells/mL) on bovine vascular endothelial cell monolayers. Untreated plasma or zymosan-activated plasma (ZAP) or 65 C inactivated plasma was added, and TxB2 and 6-keto PGF1 alpha production measured after 2 hours. Endothelial damage was detected by light and scanning electron microscopy beginning 2 and 4 hours after treatment. Preoperatively, neutrophil TxB2 release was less than 200 pg/mL; following ZAP it was 2153 pg/mL (p less than 0.001), with untreated plasma 1055 pg/mL (p less than 0.005) and inactivated plasma 764 pg/mL (p less than 0.01). Neutrophil TxB2 release on a plastic dish was not different from incubation on endothelium. Endothelial 6-keto PGF1 alpha release following addition of untreated plasma preoperatively was 1308 pg/mL (p less than 0.01), and with ZAP 1305 pg/mL (p less than 0.01). Activated neutrophils did not alter 6-keto PGF1 alpha production. Postoperatively, neutrophil TxB2 production in response to ZAP was 1092 pg/mL, which was significantly reduced compared to the preoperative response (p less than 0.01). Endothelial damage by activated neutrophils in the postoperative period demonstrated on scanning electron microscopy was also reduced; 6-keto PGF1 alpha release in the postoperative period inducted by ZAP was 569 pg/mL and by untreated plasma 549 pg/mL, which was significantly lower than in the preoperative period (p less than 0.05 and p less than 0.05, respectively). No difference in chemotaxis was demonstrated. It is concluded that operative trauma is followed by lowered neutrophil TxB2 release, appearance of a plasmatic factor that depresses endothelial 6-keto PGF1 alpha production, as well as decreased neutrophil-induced endothelial damage. The neutrophil-endothelial monolayer system is a sensitive method for detection of neutrophil and plasmatic dysfunction.

Tyrphostins that suppress the growth of human papilloma virus 16-immortalized human keratinocytes.

Human papilloma virus 16 (HPV16) is considered to be the causative agent for cervical cancer, which ranks second to breast cancer in women's malignancies. In an attempt to develop drugs that inhibit the malignant transformation of HPV16-immortalized epithelial cells, we examined the effect of tyrphostins on such cells. We examined the effect of tyrphostins from four different families on the growth of HPV16-immortalized human keratinocytes (HF-1) cells. We found that they alter their cell cycle distribution, their morphology, and induce cell death by apoptosis. The effects of tyrphostins on HF-1 cells are different from their effects on normal keratinocytes. Growth suppression by AG555 and AG1478 is accompanied by 30% apoptosis in HF-1 cells, but this is not observed in normal keratinocytes. Tyrphostin treatment produces distinctive morphological changes in HF-1 cells and in normal keratinocytes; however, the culture organization of normal keratinocytes is less disrupted. These differential effects of the tyrphostins on HPV16-immortalized keratinocytes compared with their effects on normal keratinocytes suggests that these compounds are suitable candidates for the treatment of papilloma. Previous and present results indicate that group 1 tyrphostins, which inhibit Cdk2 activation, and group 2 tyrphostins, represented by AG1478, a potent epidermal growth factor receptor kinase inhibitor, induce cell cycle arrest; and, in the case of HF-1 cells, apoptosis and differentiation. Cells accumulate in the G(1) phase of the cell cycle at the expense of S and G(2) + M. These compounds block the growth of normal keratinocytes without inducing apoptosis or differentiation, causing them to accumulate in G(1). AG17, which belongs to group 4, exerts its antiproliferative effect mainly by increasing the fractions of cells in G(1) with a concomitant decrease in the fraction of cells in S and G(2) + M.