Cytotoxic effects of streptozotocin and N-nitrosomethylurea on the pancreatic B cells with special regard to the role of nicotinamide–adenine dinucleotide

The effects on the pancreatic B cell of streptozotocin and its aglucone derivative N-nitrosomethylurea were investigated in obese-hyperglycaemic mice and their lean littermates. Both streptozotocin and N-nitrosomethylurea were found to be B-cytotoxic although N-nitrosomethylurea produced less islet damage. Both substances decreased the concentrations of NAD(+) in the islet cells to about 10% of the control values within 2h after injection. This NAD(+) depletion was prevented by injection of nicotinamide 10min after the administration of streptozotocin or N-nitrosomethylurea. In islets taken from animals 10min after injection of streptozotocin or N-nitrosomethylurea there was no stimulatory effect of glucose on the respiration or insulin release and the oxidation of glucose was markedly decreased. Addition of nicotinamide (10mm) to the incubated islets restored glucose stimulation of both the oxygen consumption and insulin release. It is concluded that islet NAD(+) depletion is probably important for the B-cytotoxin action of N-nitrosomethylurea and streptozotocin. The glucose residue in the streptozotocin molecule may potentiate the B-cytotoxic action of this drug in mice.     

Characterization of the asialoglycoprotein receptor on isolated rat hepatocytes

Rat hepatocytes, freshly isolated by a collagenase perfusion technique, bound [3H]asialo-orosomucoid in a sugar-specific and calcium-dependent manner as expected for the hepatic asialoglycoprotein receptor. At least 90% of the total cell surface-bound [3H]asialo-orosomucoid represented specific binding and could be removed by washing with EDTA. Freshly isolated cells had about 7 x 10(4) surface receptors per cell. However, when cells were incubated at 37 degrees C, the number of surface receptors per cell rapidly increased 2- to 3-fold to about 2.2 x 10(5). This increase in receptor number occurred in the absence of serum and began within minutes, depending on the particular conditions used to keep the cells in suspension. (The maximal rate of appearance of new receptors at 37 degrees C was about 70 receptors per cell per s.) When cells were first exposed to a brief EDTA treatment at 4 degrees C, before measuring the binding of [3H]asialo-orosomucoid, the number of surface receptors per cell was found to increase by about 45%. Therefore, about 30% of the surface receptors on freshly isolated cells have already bound endogenous asialoglycoproteins or are present in the membrane in a cryptic form. At 4 degrees C the binding of [3H]asialo-orosomucoid was rapid (kon greater than or equal to 1.8 x 10(4) M-1s-1), whereas the dissociation of bound [3H]asialo-orosomucoid, measured in the presence of excess nonradioactive glycoprotein, was extremely slow (koff less than or equal to 0.9 x 10(-5) s-1). The association constant calculated from these data (Ka = 2.0 x 10(9) M-1) agreed well with that obtained from equilibrium binding experiments (Ka = 2.4 x 10(9) M-1) using untreated cells or cells which had first been treated with EDTA or incubated at 37 degrees C. In all cases, when the concentration of [3H]asialo-orosomucoid was higher than about 600 ng/ml, the Scatchard plots were curvilinear. The data are, however, consistent with the conclusion that there is a single high affinity receptor on the hepatocyte surface. The additional receptors that appear on the surface when cells are incubated at 37 degrees C or exposed to EDTA are identical with those on untreated cells,     

Preparation of suspensions of pancreatic islet cells: a comparison of methods

A comparative study for preparation of cell suspensions from pancreatic islets has been performed using mechanical or enzymatic dissociation with proteolytic enzymes such as trypsin, dispase, and pronase. Treatment of isolated pancreatic islets from neonatal rats with these enzymes proved to be superior to a mechanical dissociation method. The enzymatic dissociation was performed by fractionated treatment of pancreatic islets with low concentration of enzymes in Hanks' solution for 2-3 min at room temperature. With the exception of trypsin the percentage of single cells was consistently higher with dispase and pronase treatment, being 83-92%. Cell viability (dye exclusion) was more than 90%. Mechanical disintegration of pancreatic islets resulted in a low yield of single cells, and cell viability was considerably reduced in comparison with the enzymatic methods. Labeling of islet cells with Na2 51CrO4 and measurement of the basal 51Cr-release demonstrated superior membrane preservation after pronase or dispase treatment. Islet cells isolated either by fractionated dispase or pronase treatment were found to be well preserved and very suitable for the detection of circulating cell surface antibodies and their cytotoxic effects to islet cells.     

Destruction of pancreatic islet cells by cytotoxic T lymphocytes in nonobese diabetic mice

Proliferation of islet-associated leukocytes occurred when isolated islets from 20-wk-old female nonobese diabetic (NOD) mice were cultured with 10 U/ml rIL-2 for 7 days. Co-culture of these leukocytes with freshly isolated islets from 6- to 8-wk-old NOD donors in the presence of 1 U/ml rIL-2 produced islet structural deformation within 24 h and islet cytolysis within 48 h. Three lines of evidence suggest that these leukocytes were composed mainly of CTL specific for islet cells. First, morphologically, these proliferating cells adhered to NOD islets at 6 h and killed islets within 48 h of culture, but these phenomena could not be observed in the other tissues from NOD mice. These islet-derived cells were cytotoxic to NOD islet cells in a 51Cr-release assay, whereas no appreciable cytotoxicity was observed when NOD Con A-induced splenic blasts or fibroblasts were used as targets. Second, a flow cytometric analysis showed that these cells consisted of 97% Thy-1.2, 69% Lyt-2, 8% L3T4, and 4% asialo-GM1-positive cells, whereas Mac-1-positive cells could not be seen in these assays. After treatment with anti-Thy-1.2 or Lyt-2 mAb and C, these cells lost their activity to lyse NOD islet cells. However, these cells still had a full killing activity after the depletion of L3T4 or asialo GM1-positive cells. Third, islet cells from BALB/c, DBA/2, and B10.GD mice which share the same H-2K Ag with NOD mice were susceptible to cytolytic activity of these cells, whereas islet cells from NON, C57BL/6, C57BL/10, and C3H mice remained intact. Furthermore, anti-Kd antibody was capable of blocking this cytolysis. These results suggest that CTL expressing Thy-1.2 and Lyt-2 phenotypes appear to recognize the islet cell Ag with the restriction of MHC class I Kd, and then destroy NOD islet cells.     

Comparison between clonogenic and cytotoxic assays for measuring LAK cell activity

The antiproliferative effect of lymphokine-activated killer (LAK) cells was studied using a clonogenic assay in an attempt to find a model for predicting this effect in vivo or ex vivo (in the case of purging) in cancer treatment. The results were compared with the standard 51Cr-release cytotoxic assay. Cells from clonogenic neoplastic cell lines (K562 and HL-60) were plated in methylcellulose with LAK cells obtained from ten different donors in various effector-to-target (E:T) ratios. At E:T ratios of 16:1, elimination of greater than 90% of the clonogenic cells was seen in 20 of 21 experiments, whereas such lysis was incidentally found in the 51Cr-release assay. In almost all paired combinations, clonogenic cells tested in a colony assay were more sensitive to kill by LAK cells than the whole tumor cell suspensions measured in the 51Cr-release assay.     

Comparison between the binding of [3H]misonidazole and AF-2 in Chinese hamster V79 spheroids

Binding of two hypoxia probes, [3H]misonidazole and AF-2 (2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide), was compared in Chinese hamster V79 spheroids incubated under different oxygen concentrations. Fluorescence-activated cell sorting based on Hoechst 33342 penetration was used to obtain populations of cells from different depths within the spheroid, and sorted cells were analyzed by cytofluorometry for AF-2 content and by liquid scintillation counting for [3H]misonidazole content. The patterns of AF-2 and misonidazole binding were very similar, with about 20-fold more localization of both drugs in anoxic compared to aerobic cells. Similar results were obtained when cells were sorted on the basis of AF-2 rather than Hoechst 33342 fluorescence. When mean cellular fluorescence of AF-2 was plotted versus cpm misonidazole/cell for different oxygen tensions, it appeared that oxygen was equally effective in inhibiting AF-2 and misonidazole binding. Internal cells of anoxic spheroids bound about twice as much AF-2 and misonidazole as external cells, apparently due to an increased rate of nitroreduction by chronically hypoxic cells. AF-2 was found to enhance the retention of misonidazole in oxic and hypoxic spheroids when both drugs were present.     

The immunobiology of T cell responses to Mls-locus-disparate stimulator cells. III. Helper and cytolytic functions of cloned, Mls-reactive T cell lines

Mls-specific T cell clones derived by limiting dilution were tested for cytotoxic activity in a lectin-dependent 51Cr-release assay. All the T cell clones tested were cytotoxic in such an assay in apparent contrast to previous reports. However, only those target cells sensitive to cytolysis by other L3T4a+ cytolytic T cells were killed by Mls-specific T cell clones in short term 51Cr-release assays, possibly explaining this discrepancy. All the T cell clones tested were L3T4a+, Lyt-2- and stimulated B cells from Mlsa,d strains of mice to proliferate and secrete immunoglobulin. Furthermore, lysis of innocent bystander targets was observed when the T cells were stimulated with Mls-disparate stimulator cells. These results are consistent with those obtained with L3T4a+ T cells specific for protein antigen:self Ia and that express cytotoxic potential.     

Redistribution of cholesterol and beta-sitosterol between intralipid and rat plasma lipoproteins and red blood cells in vivo

Male Wistar rats were injected intravenously with 2 mL of Intralipid containing 7.5 x 10(5) counts per minute (cpm) [14C]cholesterol and 7.5 x 10(5) cpm beta-[3H]sitosterol. Blood was withdrawn immediately and at 5, 10, 20, 60, 120, and 1440 min after injection from different animals. Plasma and red cells were separated and washed by conventional centrifugation, while lipoprotein density classes corresponding to chylomicrons, very low (VLDL), low (LDL), and high density lipoproteins (HDL) were isolated by ultracentrifugation. Total lipid and sterol compositions were determined by thin-layer chromatography in combination with gas-liquid chromatography, whereas radioactivity was measured by scintillation counting. The ratio of [14C]cholesterol/beta-[3H]sitosterol rose from 1 to 3.65 in the plasma VLDL fraction, whereas that in the LDL and HDL fractions were equilibrated at about 2, following an initial transient increase in favour of cholesterol. The appearance and disappearance of the radioactivity from LDL and HDL fractions exhibited precursor-product relationship owing probably to the conversion of the Intralipid into an intermediate lipoprotein-X-like particle, which possesses a density similar to that of LDL. The radioactive cholesterol and beta-sitosterol were incorporated into the red blood cell membranes at nearly similar initial rates, while at later times the incorporation of cholesterol was much preferred.     

Measurement of proviral genes in uninfected and avian myeloblastosis virus-infected cells by hybridization with 3H-labeled complementary DNA probe excess.

Viral RNA (vRNA) from avian myeloblastosis virus or DNA from virus-infected and uninfected cells was hybridized with [3H]DNA complementary to viral RNA ([3H]cDNA) under conditions of [3H]cDNA excess. When [3H]cDNA was used to drive the hybridization reaction with vRNA, a rate constant of 33.2 liters/mol-s was obtained. The same rate constant was obtained when vRNA excess was used as the driver. The specific activities of the [3H]DNA probe, estimated from kinetic measurements of the hybridization reaction and from the amount of [3H]cDNA in hybrid form at equilibrium, were 9.1 and 8.6 cpm/pg, respectively. DNA isolated from uninfected cells contained five or six copies of proviral DNA per cell genome. DNA isolated from erythrocytes infected with avian myeloblastosis virus had an additional five or six viral genes added to the cell genome, and the virus-infected target cell (myeloblasts) contained about 15 additional copies of proviral DNA per cell. The use of excess [3H]cDNA probe is an easy and accurate method to quantify the frequency of proviral DNA sequences in cell DNA and to measure a small amount (40 to 200 pg) of vRNA. Probe excess hybridization offers a number of advantages over other procedures and these are discussed.     

Glutathione reductase is expressed at high levels in pancreatic islet cells

Reactive oxygen species are, at least partly, involved in the diabetogenic agent-induced dysfunction of pancreatic beta-cells because the expression of antioxidative and redox proteins is low. We examined the levels of antioxidant/redox proteins, peroxiredoxins-1, -4, and -6 and glutathione reductase (GR), by immunohistochemistry and found that the expression of GR was very high in pancreatic islet cells compared to exocrine cells. When diabetes was induced by an intravenous injection of streptozotocin, the pre-administration of 1,3-bis[2-chloroethyl]-1-nitrosourea, an irreversible inhibitor of GR, made islet cells more vulnerable to streptozotocin. These data point to a pivotal role of the glutathione redox system in pancreatic islet cells against diabetogenic stress.     

Evidence for a secretion of thyroxine by isolated hog thyroid cells.

Isolated thyroid cells prepared from hog thyroid glands by tryptic dispersion were incubated with 131I- for 1--6 h. Free [131I]thyroxine was identified in the incubation medium by three chromatographic methods. Neither [131I]iodotyrosines nor [131I]triiodothyronine were detected. The [131I]thyroxine released in the medium by 100 mul of cells (packed cell volume) after a 6-h incubation period amounted to 1.16% (S.E. = +/- 0.39) of the total radioactivity. The medium [131I]thyroxine represented 15--25% of the total [131I]thyroxine synthesized during the 6 h of incubation. Thyrotropin, 1--60 munits/ml, increased the medium [131I]thyroxine content 2-4 fold. Dibutyryl cyclic AMP mimicked the effect of thyrotropin. The amount of medium [131]thyroxine was strictly related to the amount of incubated cells but was independent of the volume of the incubation medium. When prelabeled cells were incubated in the presence of methimazole the increase in medium [131I]thyroxine was quantitatively related to a decrease in the intracellular [131I]thyroxine. Addition of dinitrotyrosine, an inhibitor of the deiodinase activity, induced the release of iodotyrosines in the incubation medium. That the incubation supernatant of isolated thyroid cells did contain free thyroxine but not iodotyrosines suggests that the normal mechanisms of proteolysis of thyroglobulin and deiodination of iodotyrosines inside the cells are preserved. From these data, it was concluded that the thyroxine release by isolated cells represents a real secretion.     

Preparation of radioactive diacetyl,acetoin, and 2,3-butylene glycol

Toluene-treated cells of Streptococcus diacetilactis produced large amounts of diacetyl and acetoin without 2,3-butylene glycol. With Na-[3-¹⁴C]pyruvate added to reaction mixtures in place of unlabeled pyruvate, diacetyl with specific activity of 6.1 × 10⁴ cpm/μmol and acetoin with specific activity of 6.8 × 10⁴ cpm/μmol were harvested. Growing cells of Enterobacter aerogens incubated 48 h at 30°C in a complex medium produced large amounts of 2,3-butylene glycol without acetoin or diacetyl. With uniformly labeled [¹⁴C]glucose added to the medium in place of unlabeled glucose, 2,3-butylene glycol with specific activity of 10.8 × 10⁴ cpm/μmol was harvested. The radioactive chemicals were tested and found to be chromatographically homogeneous. Storage frozen in capped containers was especially important for diacetyl, which was found to evaporate rapidly from capped containers at room temperature.     

Automated scanning of 14C on polyacrylamide gel.

Cylindrical polyacrylamide gels were automatically analyzed for radioactive 14C using a commercially available radiochromatogram scanner. This method was capable of detecting peaks of [14C]protein containing 50 000--1 000 000 cpm. Under optimal conditions, gels could be scanned at a rate of 1 cm per min, allowing for analysis of 20--30 gels per day.     

Systemic administration of recombinant interleukin 2 stimulates in vivo lymphoid cell proliferation in tissues

Recent work in our laboratory has demonstrated that the repeated injections of high doses of recombinant interleukin 2 (IL 2) can dramatically reduce the number of established pulmonary and hepatic metastases and the growth of intradermal tumors in a variety of murine tumor models. We have thus undertaken studies to define the mechanisms underlying these in vivo effects of IL 2. Using an in vivo DNA-labeling technique in which we employed 5-[125I]iodo-2'-deoxyuridine (125IUdR), we examined the in vivo cell proliferation in the tissues of mice treated with IL 2. A proliferation index (PI) was calculated by dividing the raw counts per minute (cpm) of tissues in IL 2-treated mice by the cpm in corresponding tissues of control animals. At an IL 2 dose of 6000 U given i.p. three times a day, the highest 125IUdR incorporation was seen in the lungs, liver, spleen, kidneys, and mesenteric lymph nodes (PI = 6.9, 6.9, 5.1, 7.1, 24.6, respectively, at 5 days). The amount of lymphoid proliferation in these organs was a direct function of the dose of IL 2 administered. Other tissues including thymus, intestines, skin, and hind limb showed no significant increase in 125IUdR uptake even after host treatment with the highest doses of IL 2. Blood and brain demonstrated intermediate incorporation of the radiolabel. Preirradiation of the host largely eliminated the proliferative response to IL 2. Histologic studies of normal and irradiated mice receiving IL 2 corroborated the result of the 125IUdR findings. In normal IL 2-treated mice, large collections of activated lymphoid cells were seen, most prominently in the lungs, liver, and kidneys, whereas markedly decreased lymphoid proliferation was evident histologically in preirradiated mice. A fluorescein-labeled monoclonal antibody directed against the Thy-1.2 surface determinant was used to identify these dividing cells in frozen tissue sections as T lymphoid cells. Activated lymphocytes isolated from the lungs, liver, spleen, and mesenteric lymph nodes of IL 2-treated mice demonstrated significant lysis of a fresh murine sarcoma target in short-term 51Cr-release assays. These studies demonstrate that the systemic administration of recombinant IL 2 causes in vivo activation and proliferation of host lymphoid cells and has important implications for the adoptive immunotherapy of tumors.     

Further evidence for autonomy of T cells mediating specific in vitro cytotoxicity: efficiency of very small amounts of highly purified T cells

Cytotoxic educated thymus cells were either incubated in a plastic flask or in the presence of iron with subsequent magnetic removal of iron-containing cells or subjected to a combination of both treatments. Acridine orange staining showed that the resulting purified cells contained about one macrophage per 5 × 10⁴ cells. More generally, their content of non-T cells was less than 1%, as judged by the proportion of cells resistant to anti-θ antiserum in the presence of complement. Improvements in the ⁵¹Cr-release Cytotoxicity test allowed us to demonstrate a significant cytotoxicity of as few as 10³ of these highly purified T cells. Under these conditions, any participation by macrophages or other non-T cells in T cell-mediated cytotoxicity could be excluded.     

DNA synthesis in pancreatic islet and acinar cells in rats with streptozotocin-induced diabetes.

The rates of DNA synthesis in islet and acinar cells were compared at different intervals following streptozotocin-induced diabetes. Streptozotocin, injected I.V. at a dosage of 65 mg/kg, consistently produced a diabetic-like state in young rats, ages 33 to 42 days. At two, four, and seven days after streptozotocin administration, no significant difference in DNA synthesis per mm2 of islet and acinar tissue was evident. However, four days after streptozotocin injection, a significant increase over control values was observed in the number of cells per islet incorporating tritiated thymidine. Following streptozotocin administration, beta cells generally appeared degranulated but not necrotic. Transformation of acinar cells or ductal elements to beta cells was not observed, suggesting that proliferating beta cells are the progeny of pre-existing beta cells. This study suggests that a brief, temporary period of compensatory proliferation of beta cells follows the initial insult of the diabetogenic agent streptozotocin in young rats.     

Characterization of the effect of aluminum metal on poliovirus

Summary Metallic aluminum has been studied for possible use in conjunction with or as an alternative to conventional soluble disinfectants as applied to drinking and wastewater. Acid-washed aluminum was incubated with [³⁵S]methionine-labeled poliovirus type 1 (LSc) and the counts per min (cpm)/plaque-forming units (pfu) ratio was determined. After 2 h, only 0.013% of the cpm remained in solution, indicating viral adsorption onto the surface of the aluminum. After 76 h, 93% of the cpm returned to solution, while infectivity dropped from 2.2×10⁷ pfu/ml to undetectable levels. This suggests that infectious viruses were adsorbed onto the aluminum surface and released from the surface of the aluminum as non-infectious particles. Analysis by electron microscopy, cesium chloride gradient and polyacrylamide gel electrophoresis indicates that either dissociation or destruction of the viral capsid proteins occurs during incubation with aluminum, which results in viral inactivation.     

Glutathione reductase is expressed at high levels in pancreatic islet cells

Abstract

Reactive oxygen species are, at least partly, involved in the diabetogenic agent-induced dysfunction of pancreatic β-cells because the expression of antioxidative and redox proteins is low. We examined the levels of antioxidant/redox proteins, peroxiredoxins-1, -4, and -6 and glutathione reductase (GR), by immunohistochemistry and found that the expression of GR was very high in pancreatic islet cells compared to exocrine cells. When diabetes was induced by an intravenous injection of streptozotocin, the pre-administration of 1,3-bis[2-chloroethyl]-1-nitrosourea, an irreversible inhibitor of GR, made islet cells more vulnerable to streptozotocin. These data point to a pivotal role of the glutathione redox system in pancreatic islet cells against diabetogenic stress.     

Sialic acid, galactose and fucose on the surface of Ehrlich ascites tumor cells grown in glucose-free medium in the presence of uridine

1) The content and accessibility of terminal sialic acid and galactose residues as well as the incorporation of [3H]fucose into glycoconjugates were determined in 48-h cultures of Ehrlich ascites tumor cells in a glucose-free medium supplemented with uridine, a compound which can fulfil the necessary functions of glucose. 2) Sialic-acid residues accessible to sialidase cleavage were reduced from 695 +/- 80 nmol/10(9) cells (controls) to 284 +/- 22 nmol/10(9) cells (43% of controls). In situ labeling using periodate oxidation followed by sodium borotritiide reduction revealed a tritium incorporation of 47 +/- 11% that of controls (= 4.1 x 10(5) cpm/mg protein). 3) Labeling of galactose residues of 80-90% of that of controls was achieved after treatment of the cells with galactose oxidase/sodium borotritiide. A nearly six-fold enhancement of tritium incorporation into galactose of control cells was observed after sialidase/galactose oxidase treatment and sodium borotritiide reduction (1.5----8.8 x 10(5) cpm/mg protein); only a 3.6-fold increase (1.2 x 10(5)----4.3 x 10(5) cpm/mg protein) was found with glucose-free cultured cells. It is concluded that the galactose content of the cell surface is reduced to about 50% of controls. 4) The incorporation of tritium into acid-insoluble precipitate after 24 h incubation with [3H]fucose and the activity of the acid-soluble fraction were enhanced by about 85% as compared to controls. The pattern of inhibition by tunicamycin of [3H]fucose uptake and incorporation was the same in glucose-containing standard medium and in glucose-free uridine medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatocyte growth factor overexpression in the islet of transgenic mice increases beta cell proliferation, enhances islet mass, and induces mild hypoglycemia

Hepatocyte growth factor (HGF) is produced in pancreatic mesenchyme-derived cells and in islet cells. In vitro, HGF increases the insulin content and proliferation of islets. To study the role of HGF in the islet in vivo, we have developed three lines of transgenic mice overexpressing mHGF using the rat insulin II promoter (RIP). Each RIP-HGF transgenic line displays clear expression of HGF mRNA and protein in the islet. RIP-mHGF mice are relatively hypoglycemic in post-prandial and fasting states compared with their normal littermates. They display inappropriate insulin production, striking overexpression of insulin mRNA in the islet, and a 2-fold increase in the insulin content in islet extracts. Importantly, beta cell replication rates in vivo are two to three times higher in RIP-HGF mice. This increase in proliferation results in a 2-3-fold increase in islet mass. Moreover, the islet number per pancreatic area was also increased by approximately 50%. Finally, RIP-mHGF mice show a dramatically attenuated response to the diabetogenic effects of streptozotocin. We conclude that the overexpression of HGF in the islet increases beta cell proliferation, islet number, beta cell mass, and total insulin production in vivo. These combined effects result in mild hypoglycemia and resistance to the diabetogenic effects of streptozotocin.