Growth suppression of transformed cells by a human placental extract not related to transforming growth factor Β

Summary We have examined whether human placental extracts contain tumour-growth-inhibitory factors. One fraction (EAP) from such extracts inhibited growth, in soft agar, ofHa-ras-transformed BALB/c 3T3 cells and human squamous lung carcinoma A-2182 cells. However, this fraction had no effect on the anchorage-dependent growth of these cells, although there was a slight mitogenic activity on nontransformed cells. These data together with those on plating efficiency indicated no significant cytotoxicity of EAP on transformed cell lines. Although this fraction contained transforming growth factor (TGF), this cannot account for its inhibitory activity, since (a) pure TGF does not inhibit anchoragedependent growth of Ha-ras-transformed BALB/c 3T3 cells, (b) EAP retains its inhibitory activity in the presence of antibodies against TGF and (c) the inhibitory activity did not copurify with TGF. Partial characterization of our inhibitory factor suggests that the inhibitory factor is a new tumour-growth-inhibitory factor.Abbreviations SDS sodium dodecyl sulphate - TGF or transforming growth factor or - TIF1 or 2 transformed inhibitory factor 1 or 2 - A-2182 human squamous lung carcinoma cell line - BALB/c 3T3 1-1 mouse fibroblast cell line, clone A31 1-1 - BALB/c 3T3 Ha-ras, BALB/c 3T3 1-1 cells transformed by Ha-ras oncogene - CHO Chinese hamster ovary; MEM, minimum essential medium This research was supported by a training grant from Groupement d'Etude et de Recherche sur le Placenta.     

Cytokine-induced apoptotic cell death in a mouse pancreatic beta-cell line: inhibition by Bcl-2

Summary Cytokines are thought to contribute to the induction of pancreatic beta-cell destruction in insulin-dependent diabetes mellitus. The molecular mechanisms that underlie beta-cell death were investigated by studying cytokine-induced cell death in beta-cell lines. A combination of three cytokines (interleukin-1, tumour necrosis factor-, and interferon-) induced apoptotic cell death in the mouse pancreatic beta-cell line TC1, as judged from the appearance of cells with hypodiploid nuclei and oligonucleosomal DNA fragmentation. The same treatment also induced apoptosis in the mouse pancreatic alpha-cell line TC1 and the NOD/Lt mouse beta-cell line NIT-1, although to a lesser extent than in TC1 cells. The abundance of endogenous Bcl-2 in TC1 cells was lower than that in the other two cell lines. Overexpression of human Bcl-2 in TC1 cells partially protected them from cytokine-induced cell death. These results suggest that apoptosis may be responsible, at least in part, for cytokine-induced beta-cell destruction and that Bcl-2 prevents apoptosis in pancreatic islet cells.Abbreviations IDDM Insulin-dependent diabetes mellitus - IL interleukin - TNF tumour necrosis factor - IFN interferon - FBS fetal bovine serum - ATA aurintricarboxylic acid - CHX cycloheximide - PI propidium iodide     

Effects of acetylsalicylic acid on plasma glucose,free fatty acid,betahydroxybutyrate, glucagon and c-peptide responses to salbutamol in insulin-dependent diabetic subjects

Summary The selective ₂-adrenergic agonist salbutamol increases plasma glucose concentration and the rate of lipolysis when infused in pregnant diabetic women. The aim of the present study was twofold: (a) to focus on the actions of salbutamol on lipid and carbohydrate metabolism in insulin-dependent diabetics; and (b) to investigate possible interferences of acetylsalicylic acid (ASA) with the metabolic responses to i.v. salbutamol. The results obtained during salbutamol infusion (5 g/min) in 6 insulin-dependent diabetic subjects demonstrated that this drug caused sustained increases in plasma glucose, free fatty acid and -hydroxybutyrate concentrations, as well as a small and transient rise of plasma glucagon. No change in plasma C-peptide concentration occurred during salbutamol. A concurrent infusion of lysine acetylsalicylate reduced the increase in free fatty acids by half, blunted the weak glucagon response but enhanced the rise, in plasma glucose following salbutamol administration. The present data show that salbutamol exerts a potent hyperglycemic, lipolytic and ketogenic effect in insulin-dependent diabetics. We suggest that this -adrenergic agent should be used cautiously in human diabetes mellitus.     

Detection of allelic loss within the β1-interferon gene in childhood acute lymphoblastic leukemia using differential PCR

Summary Deletion of the short arm of chromosome 9p involving the 1-interferon (IFN) gene has been implicated in the process of malignant transformation in lymphomas and acute lymphoblastic leukemias. Since cytogenetic analysis is frequently unsuccessful in clinical samples, we used a recently described differential PCR technique to detect losses within the 1-IFN gene in 86 acute leukemias. Using differential PCR, no 1-IFN deletion was detected in 44 acute myeloid leukemia (AML) and eight control samples. However, five of 42 acute lymphoblastic leukemia (ALL) probes (12%) exhibited loss of the 1-IFN gene (three common ALL, two T-ALL). Cytogenetic analysis was performed independently in three of these five cases and revealed abnormalities of chromosome 9p in two samples. Two of five T-ALL cases exhibited a loss within the 1-IFN gene, compared with 3/29 c-ALLs, suggesting a predominance of IFN gene loss in T-ALLs. These data indicate that PCR can be used for rapid detection of gene dosage phenomena in clinical leukemia samples.     

Elliptocytosis associated with an abnormal α glycophorin

Summary A case of elliptocytosis associated with an undescribed abnormal glycophorin (GP) is reported. Using immunoblotting techniques, a clear-cut minor band 6 was detected emerging just behind the monomer of GP (band 6) when probed with anti- GP antiserum. It also reacted with anti-peptide C antiserum, suggesting that this new band with a molecular weight of 24 K is related to the structural alteration of GP and not GP. The erythrocyte membrane proteins of the patient exhibited a quite normal pattern, with a normal spectrin/ spectrin ratio, but the reaction with anti-protein 4·1 serum confirmed the increase in proteolytic susceptibility of her protein 4·1. The results of DNA mapping implied that the abnormality may be due to a short deletion of the heterozygote. The significance of deviation involving the GP and protein 4·1 to the elliptocytic change of erythrocyte shape is briefly discussed.This work was supported by grant no. HL-21016 from the National Institutes of Health, Bethesda, MD     

Rapid detection of −α 4.2 deletion of α-thalassemia-2 by polymerase chain reaction

Summary We sequenced part of the X boxes of-thalassemia-1 of Southeast Asia type (- -SEA) with– ^4.2,– ^3.7,– ^G-Taichung, and ^CS. We found the X box of– ^3.7 belonged to the X box of 2 globin gene and the X box of ^cs contained X boxes of both al and2 globin gene, whereas the X box of– ^4.2 and– ^G-Taichung was a hybrid of X boxes of 2 and 1 globin gene. We also found there are two types of– ^4.2 deletion; type 1 is a common type of– ^4.2 deletion and type 2 is linkage to– ^G-Taichung. We used a combination of two methods, the amplification refractory mutation system (ARMS) and the amplified created restriction sites (ACRS), to amplify the hybrids of X boxes specifically. The upstream primer for X box of2 globin gene was designed following the standard ARMS procedure to amplify the X segment of the-globin gene. The downstream primer was designed according to the ACRS method to check the specificity of PCR products. Using this approach, we can diagnose the different types of– ^4.2 deletion. This kind of approach can also be used to amplify the specific region from the cluster of highly homologous genes.     

Comparison of mRNA contents of interleukin-1Β and nitric oxide synthase in pancreatic islets isolated from female and male nonobese diabetic mice

Summary Interleukin-1 (IL-1) has been suggested to mediate beta-cell destruction in insulin-dependent diabetes mellitus (IDDM) by inducing nitric oxide production. In this study, we assessed the levels of IL-1 and the inducible form of nitric oxide synthase (iNOS), using a semi-quantitative polymerase chain reaction assay, and performed determinations of nitrite accumulation and IL-1 bioactivity, on pancreatic islets isolated from 5- and 16-week-old female and male nonobese diabetic (NOD) mice and from nondiabetes prone NMRI mice. NOD mouse islets contained notable amounts of IL-1 mRNA. At 5 weeks of age, but not at 16 weeks, the values were higher in islets isolated from NOD females compared to males. The IL-1 bioactivity showed differences roughly reflecting the mRNA levels in the NOD mouse islets. In the NMRI mouse islets the IL-1 bioactivity was very low. The expression of iNOS mRNA increased in both male and female islets between 5 and 16 weeks of age. Immunocytochemistry of pancreatic sections indicated the presence of macrophages especially in the peri-insular area of the NOD mice which suggests that IL-1 was produced by macrophages. The levels of IL-1 activity and mRNA in freshly isolated islets from NOD 5-week-old females did not correlate to the iNOS mRNA content or to the nitrite production. However, after incubation with IL-1 in vitro, both NOD and NMRI islets responded with a marked increase in nitric oxide production. It is concluded that the presence of IL-1 in isolated NOD mouse islets, via an induction of iNOS expression and nitric oxide production, cannot explain the gender difference in diabetes incidence in NOD mice.Abbreviations BB BioBreeding - GAPDH glyceraldehyde-3-phosphate dehydrogenase - IDDM insulin-dependent diabetes mellitus - iNOS inducible form of nitric oxide synthase - IL-1 interleukin-1 - IL-1ra interleukin-1 receptor antagonist protein - IL-6 interleukin-6 - NMRI Naval Marine Research Institute - NO nitric oxide - NOD nonobese diabetic - PAP peroxidase-antiperoxidase - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - TNF tumour necrosis factor - OD optical density     

Regulatory role of eicosanoids in extracellular matrix overproduction induced by long-term exposure to high glucose in cultured rat mesangial cells

Summary Accumulation of extracellular matrix in the mesangium and altered renal eicosanoid synthesis are two prominent features of diabetic glomerular disease. We investigated the relationship between eicosanoid and extracellular matrix production in rat mesangial cells cultured under high glucose vs normal glucose conditions. Long-term exposure of rat mesangial cells to high glucose, but not to iso-osmolar mannitol, significantly increased extracellular matrix accumulation and gene expression and transforming growth factor- (TGF-) mRNA levels, and decreased prostaglandin (PG) E₂ synthesis without affecting production of either thromboxane (TX) B₂ or PGF₂, with respect to cells incubated in normal glucose. Addition of exogenous PGE₂ resulted in a dose-dependent reduction of matrix protein and mRNA levels and TGF- gene expression in cells cultured in either normal or high glucose conditions, whereas exposure to exogenous PGF₂ produced a significant increment in matrix production and matrix and TGF- gene expression in cells grown in normal glucose, but only a slight increase in those cultured in high glucose. Stimulation of endogenous endoperoxide metabolism towards PGE₂ and PGF₂ synthesis with FCE-22,178, a drug originally developed as TXA₂ synthase inhibitor, resulted in a dose-dependent decrease in matrix accumulation and matrix and TGF- gene expression which was suppressed by co-incubation with the cyclo-oxygenase inhibitor feno-profen blocking the FCE-22,178-enhanced PG production. In both cell lines, the rate of synthesis of TXA₂ was very low and the selective blockade of its synthesis (by two other TXA₂ synthase inhibitors, OKY-046 and Ridogrel) or action (by the TXA₂ receptor antagonist BM-13,177) did not alter matrix production or TGF- mRNA levels. These results suggest that the cyclo-oxygenase pathway is involved in the regulation of matrix changes induced by high glucose in rat mesangial cells; the reduced production of PGE₂ may enhance the synthesis or potentiate the effect of stimulators of ECM formation such as TGF-, whereas TXA₂ does not appear to be involved. These data also indicate that glucose-enhanced mesangial matrix accumulation may be prevented by exogenous PGE₂ or by drugs capable of increasing endogenous PGE₂ synthesis.Abbreviations AGE Advanced glycosylation end-products - ECM extracellular matrix - PG prostaglandin - RMC rat mesangial cells - TGF- transforming growth factor- - TX thromboxane - Cox cyclo-oxygenase     

Effect of T-cell receptor VΒ-specific monoclonal antibodies on cyclophosphamide-induced diabetes mellitus in non-obese diabetic mice

Summary The expression of specific T-cell receptor gene segments by T lymphocytes appears to be critically important for the induction of several experimental autoimmune diseases mediated by these cells. We examined whether this situation also applied to non-obese diabetic mice by using various T-cell receptor V-specific monoclonal antibodies. No significant age- or sex-related differences were observed in V usage by peripheral and splenic T lymphocytes. CD8⁺ T lymphocytes among the islet-derived mononuclear cells isolated from 20-week-old female non-obese diabetic mice showed heterogeneity of their V gene usage. In order to examine the role of T lymphocyte subsets expressing specific T-cell receptor V gene segments in the development of diabetes mellitus, T-cell receptor V-specific monoclonal antibodies were administered to 10-week-old male non-obese diabetic mice treated with cyclophosphamide. None of the antibodies used could significantly diminish the incidence of cyclophosphamide-induced diabetes and the severity of insulitis [anti-V3 (11 of 22 mice became diabetic, 50%), anti-V5 (9 of 14, 64%), anti-V8 (9 of 21, 43%), anti-V11 (12 of 23, 52%), anti-V14 (7 of 12, 58%), and anti-V5 + anti-V11 (6 of 12, 50%)] when compared with control mice (12 of 21, 57%). In addition, there were no significant differences in T-cell receptor V usage between diabetic and non-diabetic cyclophosphamide-treated mice. These results suggest that five T-lymphocyte subsets expressing different T-cell receptor V gene segments, considered to be candidates involved in the pathogenesis of autoimmune diabetes, do not individually contribute to the development of cyclophosphamide-induced diabetes in non-obese diabetic mice.     

Effect of Interferon-α Treatment of Chronic Hepatitis C on Health-Related Quality of Life

Studies of interferon- (IFN-)therapy for chronic hepatitis C have focused on viralclearance; however, few have evaluated patient'shealth-related quality of life during therapy. Thisstudy evaluates health-related quality of life and theprevalence of anxiety and depression in patients withchronic hepatitis C before, during, and followingIFN- therapy. Patients undergoing IFN-therapy for chronic hepatitis C were asked to completehealth status measures as well as anxiety and depressioninventories before, during, and following IFN-therapy. These measures were compared to the results of healthy adults in the general US population.Thirty-eight of forty-eight eligible patients (79%) withchronic hepatitis C completed the questionnaires.Respondents demonstrated a significant increase in depression during the sixth month ofinterferon therapy in comparison to pretreatmentresults. Anxiety scores improved significantly after onemonth of IFN- in comparison to pretreatmentresults. Scores on the health status measures did notvary with IFN- therapy. Patient responses wereanalyzed with respect to biochemical response(normalized transaminases) to IFN-. IFN-responders, who were aware of their transaminase results,exhibited lower scores on anxiety subscales during andafter therapy (P = 0.02-0.04). Scores on the healthstatus subscale, role emotional, improved in IFN- responders compared to nonresponders during thesixth month of therapy (P = 0.02). Response toIFN- therapy was not associated with any otherdifferences on subscale analysis. Patients with chronichepatitis C exhibited health perceptions similar to thegeneral US population, and these were unchanged duringIFN- therapy. However, the incidence ofdepression significantly increased during the sixthmonth of IFN- therapy. IFN- respondersexhibited fewer emotional problems as well as a lowerincidence of anxiety during and followingtherapy.     

Synergistic cytotoxicity of human recombinant tumour necrosis factor α combined with microtubule effectors

Summary Combinations of human recombinant tumour necrosis factor (rhTNF) with each of four different agents disturbing the microtubule system of the cellular cytoskeleton were tested for synergistic cytotoxic action against murine melanoma B16K and L-M(S) cells. In addition to the known microtubule effectors colchicine, vincristine, and taxol, the influence of the fluorenone-azomethine derivative-diphenylene-N-{p-[bis-(-hydroxyethyl)-amino]-phenyl}-nitrone (DHPN) on the rhTNF cytotoxicity was studied. Applying a novel computerbased isobole method [Suehnel J (1990) Antiviral Res 13:23–40] concentration ranges of synergistic, zero, and antagonistic interaction were found after in vitro combination of rhTNF with each of the drugs tested in a 72-h cytotoxicity assay. In contrast, a 24-h exposure of B16K cells to these combinations still did not inhibit in vitro colony formation to a greater extent than either drug alone. A preliminary in vivo experiment revealed an increased antitumour effect after treatment of established subcutaneous melanoma B16 tumours with a combination of rhTNF and DHPN.Abbreviations rhTNF human recombinant tumour necrosis factor - DHPN -diphenylene-N-{p-[bis-(-hydroxyethyl)-amino]-phenyl}-nitrone     

Enzymatic determination of serum 3α-sulfated bile acids concentration with bile acid 3α-sulfate sulfohydrolase

A newly developed enzymatic method for determining urinary 3-sulfated bile acids was used to measure serum 3-sulfated bile acid levels in 114 patients with hepatobiliary diseases and 56 healthy subjects. The lowest measurable amount of the 3-sulfated bile acids was 0.5 µmol/liter. The standard curves for glycolithocholic acid 3-sulfate, glycoursodeoxycholic acid 3-sulfate, and lithocholic acid 3-sulfate were linear from 0.5 to 250 µmol/liter. Specificity of the assay was satisfactory and intra- and interassay variations ranged from 0.8 to 4.4% and from 1.2 to 7.9%, respectively. Analytical recovery was more than 91%. The values obtained by this assay were well correlated with those by gas-liquid chromatography measurement (r=0.91,P<0.01). The fasting serum 3-sulfated bile acids level in healthy subjects ranged from undetectable to 1.9 µmol/liter (mean±se; 0.9±0.1 µmol/liter). The percentage of 3-sulfated bile acids in total bile acids (sum of 3-sulfated and 3-hydroxy bile acids) in serum was 16.8±1.5%. In subjects with hepatobiliary diseases, serum 3-sulfated bile acids levels were elevated; however, the percentage of 3-sulfated bile acids in total bile acids was decreased and correlated with the severity of hepatocellular insufficiency. This enzymatic assay is simple, rapid, and accurate for the determination of serum 3-sulfated bile acids.     

Allele-specific amplification of genomic DNA for detection of deletion mutations: Identification of a French-Canadian tay-sachs mutation

Summary A rapid and efficient method for the detection of a 7.6-kb deletion in the-hexosaminidase A-subunit gene, a mutant allele causing Tay-Sachs disease in French Canadians, is described. The protocol involves PCR (polymerase chain reaction) amplification of target sequences on normal and mutant chromosomes. Three amplification primers, a single 5 primer complementary to normal and mutant DNA templates and two 3 primers specific for normal and mutant DNA templates are required. The primers direct amplification of two unique fragments (normal and mutant) that are easily separated by gel electrophoresis. Allele-specific oligonucleotide hybridization using normal and mutant probes to genomic DNA samples from normal, heterozygous and homozygous individuals confirms these results and is consistent with results of genotypic classification of individuals using Southern analysis. The method is applicable to detection of deletion mutations in cases where some deletion-flanking sequence is known.     

Immunolocalization of Transforming Growth Factor-α and Epidermal Growth Factor Receptor in the Rat Gastroduodenal Area

The maintenance of gastrointestinal epitheliumintegrity requires a fine balance between proliferationand differentiation as well as protection againstgastric acid secretion. Transforming growthfactor- (TGF-) regulates these functions bybinding to epidermal growth factor receptor (EGF-R).This study was designed to identify the localization ofTGF- and EGF-R in the rat gastroduodenal region. In the stomach, the surface and gastric pitcells showed staining for TGF- antibodies in thecytoplasm and basolateral and apical membranes.TGF- and EGF-R were observed in the supranuclearregion of the cells lining the gland. In the duodenum,the enterocytes coexpressed both TGF- and EGF-Rin the supranuclear area. The EGF-R was also observed inthe apical membrane. Brunner's glands were positive for both TGF- and EGF-R antibodies. Ourresults demonstrate the coexpression of TGF- andEGF-R in the rat gastroduodenal area, which suggests afunctional role for them in the establishment and maintenance of the epithelialrenewal.     

Prospective evaluation of interferon-α in treatment of chronic active Crohn's disease

Several case reports suggested good effects of interferon- in patients with Crohn's disease. In addition, a decreased production of interferon- in Crohn's disease has been shownin vitro. Treatment with interferon- may activate intestinal natural killer cells and down-regulate the overproduction of inflammatory cytokines like interleukin-6 in Crohn's disease. To evaluate the clinical efficacy of interferon-, we treated 12 patients with a chronic active course of Crohn's disease with recombinant human interferon- prospectively for 24 weeks. Prednisolone was continuously tapered and discontinued at week 12. The end point of the study was the prevention of worsening of clinical symptoms defined with the Crohn's disease activity index and was monitored by acute-phase proteins, interleukin-6 serum concentrations, and endoscopy. The biochemical activity of interferon- was measured by 2,5-oligo adenylate serum levels. The end point of the study was reached in four patients (33%). In these patients the final Crohn's disease activity index was above 150, which means that they did not achieve clinical remission. All other patients (66%) did not respond to interferon- and had to be withdrawn prematurely. Interferon- did not show any beneficial effect on interleukin-6 or acute-phase protein concentrations and on endoscopic activity. The 2,5-oligo adenylate levels continuously increased during interferon therapy. Considerable side effects were noted. These results fail to demonstrate a therapeutic role of interferon- in chronic active Crohn's disease.     

Thalassemia intermedia: compound heterozygous β∘/β+-thalassemia and co-inherited heterozygous α+-thalassemia

Summary The relative excess of - over -globin chains in the erythroid precursors is the chief pathophysiological factor of homozygous -thalassemia. The clinical picture is usually characterized by a transfusion-dependent dyserythropoietic anemia (thalassemia major). However, some patients present with moderate anemia that does not require regular blood transfusions (thalassemia intermedia). The molecular heterogeneity of -thalassemia mutations and changes of - and -globin gene expression play an important role in modifying the clinical phenotype. We report here on a female Greek patient with homozygous -thalassemia but normal growth and development, excellent exercise tolerance, and no need of blood transfusions. She is thus mildly affected clinically, although there is marked pallor, jaundice, and hepatosplenomegaly. These signs correspond to her marked hypochromic, microcytic anemia with erythroid hyperplasia of the bone marrow. -Globin genotyping shows her to be compound heterozygous for the codon 39 C T -nonsense mutation and for the T C ⁺-mutation at position 6 of the splice consensus at the exon 1/intron 1 junction (CD39 C T/IVS 1–6 T C). -Globin gene mapping demonstrates the presence of a 3.7-kb ⁺-thalassemia deletion on one allele (–^3.7/). Taken together, this study identifies a complex interaction of genetic factors that do not significantly alter the clinical phenotype when present alone but ameliorate the course of homozygous -thalassemia when inherited in combination.Abbreviations Hb hemoglobin - Hct hematocrit - HPFH hereditary persistence of fetal hemoglobin - IVS intervening sequence - MCH mean corpuscular hemoglobin - MCV mean corpuscular volume - PCR polymerase chain reaction     

Overproduction of inhibitory hematopoietic cytokines by lipopolysaccharide-activated peripheral blood mononuclear cells in patients with aplastic anemia

The aim of this study was to measure the level of cytokines produced by peripheral blood mononuclear cells (PBMNC) in patients with aplastic anemia (AA) and to determine their effect on the clonal growth of normal bone marrow (BM) cells. Twenty-one patients with AA and 11 normal controls were enrolled in this study. Medium conditioned by PBMNC of AA patients in the presence of lipopolysaccharide (LPS) was found to be suppressive to the colony growth of normal BM cells. Thus, we further determined the presence in the PBMNC-conditioned medium (CM) of both inhibitory cytokines: macrophage inflammatory protein-1 (MIP-1), tumor necrosis factor- (TNF-), transforming growth factor-2 (TGF-2), and interferon- (IFN-), and stimulatory cytokines: interleukin-3 (IL-3) and stem cell factor (SCF). Spontaneous production of MlP-1 was higher in the AA patients than the normal controls (1887±174 pg/ml vs 1643±93 pg/ml), but the difference was not significant. After LPS stimulation, the production of MIP-1 was markedly increased in the AA patients, and its level was significantly higher than that of the normal controls (2360±149 pg/ml vs 1517±92 pg/ml, p=0.0022). The level of TNF was also higher in the AA patients. However, IFN-, TGF-2, SCF, and IL-3 were not detectable in the PBMNC-CM of either AA patients or normals. The myelopoietic suppressing effect of AA-PBMNC-CM from each AA patient was significantly blocked by pretreatment with anti-TNF-, resulting in a colony-forming enhancement of 174%±12%. A similar effect was noted in six of 11 AA patients by pretreatment with anti-MIP-1. We conclude that TNF and MIP-1 can be overproduced by the PBMNC of some AA patients, which may play a role in the progression of AA.     

Heterophilic interactions between cell adhesion molecule L1 and α<Subscript>v</Subscript> β<Subscript>3</Subscript>-integrin induce HUVEC process extension <Emphasis Type="Italic">in vitro</Emphasis> and angiogenesis <Emphasis Type="Italic">in vivo</Emphasis>

Cell adhesion molecule L1 was implicated in angiogenic processes, tumor formation and metastasis. Here, we provide evidence that the sixth Ig-like domain of L1 (L1Ig6) interacts with _{v 3} to induce process extension of human umbilical vein endothelial cells (HUVECs) in vitro and angiogenesis in vivo. HUVECs formed network-like structures on full-length L1 or L1Ig6 substrates comparable to structures found on matrigel. In the presence of mab _{v 3} or cyclic RGD, apoptosis was induced. In fibrin matrices where L1Ig6 was covalently incorporated, HUVECs formed multicellular and hollow processes through interactions between cell-surface _{v 3} and RGD-sites of matrix-immobilized L1Ig6. No such processes were induced by L1Ig6 having non-functional RDG-sites, or in the presence of mab _{v 3} or cyclic RGD. In those matrices, increased apoptosis was found. Co-immunoprecipitation of L1 or L1Ig6 with _{v 3} suggests close interactions. Furthermore, L1Ig6 stimulated HUVECs showed increased tyrosine phosphorylation of _{v 3} and phosphorylation of MAP kinases (ERK1 and ERK2) but not AKT indicating specific activation of _v and _{v 3} followed by activation of downstream kinases. Application of L1Ig6-modified fibrin matrices on CAMs induced 50–60% increased _v and _v3 protein expression and in vivo angiogenesis indicated by ~50% increased mean vascular length density. The results demonstrate angiogenic potential of L1Ig6 involving ligation and activation of _v3     

Elevated serum levels of macrophage-derived cytokines precede and accompany the onset of IDDM

Summary To determine whether cytokines could have a role in the development of insulin-dependent diabetes mellitus (IDDM), we measured serum levels of cytokines derived from T helper 1 (interleukin-2 and interferon-), T helper 2 (interleukin-4 and inter-leukin-10) lymphocytes and macrophages (tumour necrosis factor-, interleukin-1 and interleukin-1 ) in patients before and after the onset of IDDM. Recently diagnosed IDDM patients had significantly higher levels of interleukin-2, interferon-, tumour necrosis factor- and interleukin-1 than patients with either long-standing IDDM, non-insulin-dependent diabetes (NIDDM), Graves' disease, or control subjects (p<0.05 for all). Compared with control subjects, patients with long-standing IDDM and those with NIDDM had higher interleukin-2 and tumour necrosis factor- levels (p<0.01 for all). Interleukin-4 and interleukin-10 were detectable in sera of patients with Graves' disease only, while interleukin-1 was not detectable in the serum of any control or test subject. To investigate whether high cytokine levels precede the onset of IDDM, we studied 28 non-diabetic identical co-twins of patients with IDDM, followed-up prospectively for up to 6 years after the diagnosis of the index. Levels of tumour necrosis factor- and interleukin-1 were elevated above the normal range more frequently in the eight twins who developed diabetes than in those 20 who did not (p<0.005). Analysis of T helper 1 and T helper 2 profiles of the twin groups did not reveal a clear difference between prediabetic twins and twins remaining non-diabetic. These results support the notion that T helper 1 lymphocytes may play a role in the development of IDDM. This is associated with release of macrophage-derived cytokines, which is also a feature of the prediabetic period. The lack of evidence of a dominant T helper 1 profile of cytokine release before diabetes onset suggests that additional events, activating this arm of the cellular immune response, are required in the immediate prediabetic period.Abbreviations IL Interleukin - IFN- interferon-gamma - T_H T helper - ICA islet-cell antibody - GAD glutamic acid decar-boxylase - IDDM insulin-dependent diabetes mellitus - NIDDM non-insulin-dependent diabetes mellitus - TNF- tumour necrosis factor-alpha - CTLL-16 murine cytotoxic cell line