Element concentration changes in mitotically active and postmitotic enterocytes. An x-ray microanalysis study.

Unfixed freeze-dried and uncoated tissue sections of the mouse duodenum were suspended across a hole in a carbon planchet and analyzed in a scanning electron microscope fitted with energy-dispersive x-ray analytical equipment. Computer analysis of the x-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and late anaphase-early telophase chromatin regions in the cryptal and villus enterocytes. Elemental concentrations (mmol/kg dry wt) were measured for Na, Mg, P, S, Cl, K, and Ca. None of the elements were compartmentalized preferentially in either the nucleus or the cytoplasm of interphase enterocytes of crypts or in postmitotic enterocytes of villi. In contrast, Ca, S, and Cl are detectable in significantly higher concentrations in mitotic chromatin of dividing enterocytes of the crypt as compared to surrounding mitotic cytoplasm, but Na, Mg, and P are in lower concentrations in the mitotic chromatin as compared to mitotic cytoplasm. Interphase enterocytes of crypts have higher concentrations of Mg, P, and K, and lower concentrations of Na than do postmitotic enterocytes of villi.

Effects of amiloride on tumor growth and intracellular element content of tumor cells in vivo.

The effects of amiloride, a reported inhibitor of serum-stimulated sodium influx, were tested on tumor growth, tumor cell proliferation, and intracellular element content of cancer cells in vivo. We have shown previously that cancer cells have high intranuclear levels of sodium compared to those of their normal counterpart cells and have postulated that such a high level of sodium may be involved in the cancer state. We now report that amiloride, when given in a series of injections, inhibited both H6 hepatoma and DMA/J mammary adenocarcinoma growth in vivo in a dose-dependent fashion and that 3 injections of amiloride at a dose of 1.0 microgram/g body weight into mice bearing H6 hepatomas resulted in a significant decrease in the intranuclear content of sodium but not the content of magnesium, phosphorus, sulfur, chlorine, or potassium as measured by electron probe X-ray microanalysis in the H6 hepatoma cells. Amiloride at dosages as low as 1.0 microgram/g body weight per injection also inhibited tumor cell proliferation as measured by the tritated thymidine autoradiography labeling index. Amiloride caused no changes in the mean profile diameters of metaphase or interphase H6 hepatoma or DMA/J mammary adenocarcinoma cells, suggesting that the action of amiloride on tumor growth was not due to cell volume changes. These data show that amiloride both inhibited tumor growth and decreased the proliferation of the tumor cells in the H6 hepatomas which was correlated with a decreased intranuclear sodium content.

X-ray microanalysis of electrolyte content of normal, preneoplastic, and neoplastic mouse mammary tissue.

Intracellular sodium, chlorine, and potassium concentrations (mmol/kg dry weight) were determined by electron probe X-ray microanalysis of individual epithelial cells in freeze-dried 2-micrometer sections of mouse mammary tissue which were cut at -30 degrees. A model system was utilized in order to compare elemental content of cells from normal pregnant mammary tissue and preneoplastic and neoplastic mammary tissues from female BALB/cCrlMed mice. Animals were killed by cervical dislocation, and tissue was rapidly frozen in liquid propane. Normal mammary glands were obtained from primiparous mice at 16 to 17 days of gestation. Tissue from the hyperplastic alveolar nodule line D1 was removed from donor mice 12 to 16 weeks after transplantation into the cleared mammary fat pad. All mammary adenocarcinomas, D1T, were primary tumors which developed in mice with transplants of nodule line D1. Data were collected from five animals (10 cells/animal) in each of the three groups. It was found that the electrolyte content of cells of preneoplastic tissue was the same as that of the normal mammary tissue but was significantly elevated in neoplastic tissue (162, 130, and 48% increases for sodium, chlorine, and potassium, respectively). Thus, an increase in electrolyte content seems to be associated with the transformation to a neoplastic state and not associated with conversion to the preneoplastic state.

Scanning the structure and antigenicity of HPV-16 E6 and E7 oncoproteins using antipeptide antibodies.

The structure and antigenicity of the HPV-16 E6 and E7 oncoproteins was studied using a set of antisera against overlapping synthetic peptides. We report that antigenic, mobile regions of the native proteins, as defined by reactivity with antipeptide antisera, occur at the N-termini of both E6 and E7 proteins, corresponding to regions of known or suspected protein-protein interactions. The putative zinc finger domains were consistently non-reactive, despite computer predictions of relatively high antigenicity, suggesting that the proposed zinc finger regions are held in stable secondary structures that the peptides were not able to mimic. In E6, the linker region between the two zinc fingers was antigenic, indicating that the two zinc finger structures might be able to articulate relative to one another by a flexible linker region. The highly antigenic N-terminal region of HPV-16 E7 was also found to be antigenic in E7 of both HPV-11 and HPV-18, indicating that the E7 proteins of different HPV types have similar antigenic structures. The identification of antigenic regions of the E6 and E7 proteins should be therefore be useful in the design of site-directed antibodies against E6 and E7 for numerous HPV types.

Compensatory thyroid hyperplasia in hemithyroidectomized Snell dwarf mice.

The effect of hemithyroidectomy (HEMITX) on the proliferation of follicular cells in the remaining thyroid lobe in Snell dwarf mice (dw/dw) and in phenotypically normal mice from the same strain (?/+) was examined by means of the colchicine metaphase-arrest technique. In both the normal and dwarf mice, the mean mitotic activity rate of the thyroid lobe 48 h after contralateral HEMITX was significantly higher than that in the animals with intact contralateral lobe (P less than 0.001), indicating the existence of compensatory thyroid hyperplasia (CTH). Since in Snell dwarfs not only GH and PRL but also TSH is lacking, it is concluded that extrapituitary factors, most probably neural pathways, are involved in CTH in these animals.

Failure of the inhibition of rat gastric mucosal 5-lipoxygenase by novel acetohydroxamic acids to prevent ethanol-induced damage.

1. The role of leukotriene B4 (LTB4) and LTC4 as mediators of gastric mucosal damage following ethanol challenge in vivo has been investigated using two selective 5-lipoxygenase inhibitors, BW A4C and BW A137C. 2. Oral administration of ethanol to rats in vivo, induced macroscopic damage to the gastric mucosa and markedly increased the formation of the 5-lipoxygenase products, LTB4 and LTC4, from the mucosa ex vivo. 3. Pretreatment with the acetohydroxamic acids BW A4C and BW A137C (5-50 mg kg-1 p.o.) dose-dependently reduced ethanol-stimulated LTB4 and LTC4 formation by the gastric mucosa, with an ID50 of approximately 5 mg kg-1 p.o. 4. A single oral dose of BW A4C (20 mg kg-1) induced near-maximal inhibition of mucosal LTB4 formation within 30 min, which was well maintained for 5 h, whereas BW A137C (20 mg kg-1 p.o.) induced maximal inhibition between 30 and 60 min after administration, which then diminished over the subsequent 5 h. 5. The mucosal formation of the cyclo-oxygenase product, 6-keto-prostaglandin F1 alpha, which was unaltered following ethanol challenge, was not inhibited by the acetohydroxamic acids. Likewise, the small increase in mucosal thromboxane B2 formation following challenge was not inhibited by BW A4C. 6. Neither BW A4C nor BW A137C, at doses that almost completely inhibited the mucosal synthesis of LTB4 or LTC4, reduced the macroscopic gastric mucosal damage induced by ethanol. 7. Pretreatment with the lipoxygenase inhibitor BW 755C (5-50 mg kg-1 p.o.) did reduce mucosal damage, but there was a dissociation between the degree of protection and the inhibition of leukotriene biosynthesis. 8. Oral administration of high doses of either BW A4C or BW A137C (300mgkg-1) did not induce macroscopic gastric damage over a 3 h period. 9. These findings suggest that the leukotrienes, LTB4 and LTC4 are not the primary mediators of ethanol-induced acute mucosal damage, but do not exclude their role in more chronic gastric damage and inflammation.

Stimulation and inhibition of prostacyclin formation in the gastric mucosa and ileum in vitro by anti-inflammatory agents.

1 Homogenates of rat gastric mucosa, forestomach and ileum and dog gastric mucosa reproducibly generated prostacyclin from endogenous substrate.2 Prostacyclin formation was inhibited by pre-incubation with indomethacin, flurbiprofen, naproxen, ketoprofen or meclofenamate (0.1-10 muM).3 BW755C (3-amino-1[m-(trifluoromethyl)-phenyl]-2-pyrazoline) stimulated prostacyclin production in the rat gastric mucosa and ileum with inhibition occurring only at high concentrations (> 200 muM). The stimulation of prostacyclin production by BW755C in rat forestomach homogenates was less pronounced, with inhibition at concentrations > 20 muM.4 BW755C thus exhibits differential activity on prostacyclin production from different gastric tissues in vitro.5 The antioxidant-lipoxygenase inhibitor, nordihydroguiaretic acid (NDGA, 3-15 muM) likewise augmented rat mucosal prostacyclin formation.6 Paracetamol stimulated and, at higher concentrations, inhibited prostacyclin formation (> 1 mM), and had comparable activity in both rat gastric tissues.7 The ability of NDGA and BW755C to enhance prostacyclin generation may reflect the removal of a modulating influence of lipoxygenase products on prostacyclin formation, the diversion of substrate to the cyclo-oxygenase pathway, or free-radical scavenging.

The gastric antisecretory actions of prostaglandin E2 and stable prostacyclin analogues against different secretagogues in perfused whole-stomachs of rat or mouse in vitro.

1 The characteristics of the antisecretory actions of prostaglandin E2 (PGE2) and two stable prostacyclin analogues during different rates of acid stimulation have been evaluated in the lumen-perfused isolated whole stomach of the rat and mouse. 2 In the rat isolated stomach, histamine induced a dose-dependent increase in acid output. Preincubation with PGE2 caused a dose-related and surmountable inhibition. 3 The stable prostacyclin analogues, 6 beta-PGI1 and a 16-phenoxy derivative likewise caused a surmountable inhibition of histamine-stimulated acid output from rat stomach. 4 PGE2 had inconsistent actions on the acid secretion stimulated by pentagastrin, methacholine or dibutyryl cyclic adenosine 3',5'-monophosphate. 5 In the mouse isolated stomach, acid secretion was stimulated by low concentrations of histamine, pentagastrin or methacholine. 6 PGE2 failed to inhibit histamine-stimulated acid output from mouse stomach, but high concentrations of the potent 15-phenoxy analogue did show anti-secretory activity. 7 The results indicate the usefulness of the rat isolated stomach for studying the interaction of prostaglandins with the acid secretory process in mammalian gastric mucosa.

Increased metabolism of arachidonic acid in an immune model of colitis in guinea-pigs.

Inflammation of the guinea-pig colon was produced by skin sensitization and subsequent intracolonic challenge with the chemical hapten, dinitrochlorobenzene. Metabolism of [14C]-arachidonic acid by homogenates of control colon was very low, although metabolites co-migrating on thin layer chromatography (t.l.c.) with prostaglandin E2 (PGE2), PGF2 alpha, PGD2, 6-keto-PGF1 alpha, thromboxane B2 (TXB2), HHT and 11-, 12-, 15-HETE were formed. There was an overall 3 fold increase in metabolism of [14C]-arachidonic acid by homogenates of inflamed mucosa. The greatest increase in metabolite formation was of PGE2, with smaller increases in HHT, 11-, 12-, 15-HETE, PGD2, TXB2, PGF2 alpha and 6-keto-PGF1 alpha. The formation of these metabolites was inhibited both by indomethacin and the dual pathway inhibitor, BW755C. The formation of immunoreactive PGE2, TXB2 and 6-keto-PGF1 alpha was also increased in homogenates of inflamed guinea-pig colon. The small level of immunoreactive LTB4 detected in control colon was not changed in inflamed colonic tissue. The dinitrochlorobenzene model of colitis offers a means of studying arachidonic acid metabolism in an immune-mediated inflammatory response in intestinal tissue.

Role of nitric oxide in maintaining vascular integrity in endotoxin-induced acute intestinal damage in the rat.

1. The role of endogenous nitric oxide (NO) in maintaining intestinal vascular integrity following acute endotoxin (E. coli. lipopolysaccharide) challenge was investigated in the anaesthetized rat by use of NG-monomethyl-L-arginine (L-NMMA), a selective inhibitor of NO synthesis. 2. L-NMMA (10-50 mg kg-1, i.v.) pretreatment enhanced both the macroscopic and histological intestinal damage and the increases in vascular permeability, measured as the leakage of [125I]-labelled human serum albumen, induced after 15 min by endotoxin (50 mg kg-1, i.v.). 3. The effects of L-NMMA (50 mg kg-1, i.v.) were enantiomer specific, as D-NMMA had no effect. Furthermore, these effects were reversed by L-arginine (300 mg kg-1, i.v.), the precursor of NO synthesis but not by D-arginine (300 mg kg-1, i.v.). 4. L-NMMA (10-50 mg kg-1, i.v.) increased mean systemic arterial blood pressure but this does not appear to be the mechanism by which endotoxin-induced intestinal damage was enhanced, since similar systemic pressor responses induced by phenylephrine (10 micrograms kg-1 min-1, i.v.), had no such effect. 5. The results suggest that synthesis of NO from L-arginine has a role in maintaining the microvascular integrity of the intestinal mucosa following acute endotoxin challenge.

Role of oxygen radicals and arachidonic acid metabolites in the reverse passive Arthus reaction and carrageenin paw oedema in the rat.

1. The role of arachidonic acid metabolites and oxygen radicals in carrageenin-induced rat paw oedema and dermal reverse passive Arthus reaction (RPA) have been investigated. 2. Indomethacin (10 mg kg-1, p.o.) inhibited carrageenin paw oedema when administered 30 min before, but not 2 h after carrageenin. BWB70C (10 mg kg-1, p.o.), a selective inhibitor of 5-lipoxygenase, had no effect whether administered before or after carrageenin. Administration of both indomethacin and BWB70C had no greater anti-inflammatory effect than indomethacin alone. 3. BW755C (20 mg kg-1, p.o.), which inhibits the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism, or superoxide dismutase-polyethylene glycol conjugate (SOD-PEG, 3000 u, i.v.) inhibited carrageenin paw oedema whether administered either 30 min before, or 2 h after carrageenin. 4. Pretreatment with dexamethasone (0.1 mg kg-1) or colchicine (2 mg kg-1), likewise suppressed carrageenin paw oedema. 5. BW755C (25-100 mg kg-1, p.o.) dose-dependently reduced plasma leakage in the RPA, whereas indomethacin (5 mg kg-1, p.o.) or BWB70C either alone or in combination, did not. 6. SOD-PEG (300-3000 u, i.v.) dose-dependently inhibited plasma leakage in the RPA. In addition, the iron chelator and peroxyl radical scavenger, desferrioxamine (200 mg kg-1, s.c.) also inhibited plasma leakage. 7. Pretreatment with dexamethasone (0.1 mg kg-1) or colchicine (1 mg kg-1) reduced the plasma leakage in RPA, whereas MK-886 (10 mg kg-1) had no effect. 8. These results indicate an important role for oxygen radicals but not arachidonic acid metabolites in the maintenance of carrageenin paw oedema and the plasma leakage in RPA. Furthermore, the results suggest that the anti-inflammatory actions of BW755C can be dissociated from its effects on arachidonic acid metabolism and are attributed to its anti-oxidant activity.

The effect of anti-inflammatory drugs on eicosanoid formation in a chronic model of inflammatory bowel disease in the rat.

1. The effects of anti-inflammatory drugs on eicosanoid formation and colonic damage in a chronic model of inflammatory bowel disease (IBD) in the rat were investigated. 2. A single colonic instillation of the hapten, trinitrobenzene sulphonic acid (TNB) resulted in ulceration and inflammation which persisted for 3 weeks. 3. The macroscopic colonic damage, present 3 weeks after TNB, was correlated with an increase in immunoreactive 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and leukotriene B4 (LTB4) synthesis by the rat colon. 4. Anti-inflammatory drugs were administered 2 weeks after TNB, when there was substantial colonic damage, and continued for a week. The experimental drug BW755C inhibited the increased formation of 6-keto-PGF1 alpha and LTB4 by the inflamed colon. Indomethacin and aspirin markedly inhibited prostanoid formation in both inflamed and control colon. Sulphasalazine or prednisolone also inhibited the formation of 6-keto-PGF1 alpha but the effects were less marked. 5. None of the anti-inflammatory drugs significantly reduced the colonic damage induced by TNB. 6. The results suggest that eicosanoids, including LTB4, have only a minor role in maintaining the chronic macroscopic damage induced in the rat colon by TNB. The role of such eicosanoids in the underlying infiltration and activity of inflammatory cells in this model of IBD, however, is not known.

Increased intestinal formation of Paf in endotoxin-induced damage in the rat.

Platelet-activating factor (Paf) has been proposed as a mediator of the gastrointestinal damage in endotoxic shock. The formation of Paf in rat jejunal tissue, determined following extraction and bioassay on rabbit washed platelets has therefore been correlated with the induction of damage following endotoxin administration. Intravenous injection of E. coli endotoxin led to a time-dependent increase in the jejunal formation of Paf, which after 20 min was twenty fold greater than the control level. There was a significant correlation between elevated Paf release and intestinal hyperaemia and haemorrhage, thus supporting a role for Paf as a mediator of such damage.

The induction of nitric oxide synthase and intestinal vascular permeability by endotoxin in the rat.

1. The effect of endotoxin (E. coli lipopolysaccharide) on the induction of nitric oxide synthase (NOS) and the changes in vascular permeability in the colon and jejunum over a 5 h period have been investigated in the rat. 2. Under resting conditions, a calcium-dependent constitutive NOS, determined by the conversion of radiolabelled L-arginine to citrulline, was detected in homogenates of both colonic and jejunal tissue. 3. Administration of endotoxin (3 mg kg-1, i.v.) led, after a 2 h lag period, to the appearance of calcium-independent NOS activity in the colon and jejunum ex vivo, characteristic of the inducible NOS enzyme. 4. Administration of endotoxin led to an increase in colonic and jejunal vascular permeability after a lag period of 3 h, determined by the leakage of radiolabelled albumin. 5. Pretreatment with dexamethasone (1 mg kg-1 s.c., 2 h prior to challenge) inhibited both the induction of NOS and the vascular leakage induced by endotoxin. 6. Administration of the NO synthase inhibitor NG-monomethyl-L-arginine (12.5-50 mg kg-1, s.c.) 3 h after endotoxin injection, dose-dependently reduced the subsequent increase in vascular permeability in jejunum and colon, an effect reversed by L-arginine (300 mg kg-1, s.c.). 7. These findings suggest that induction of NOS is associated with the vascular injury induced by endotoxin in the rat colon and jejunum.

The effects of a novel series of selective inhibitors of arachidonate 5-lipoxygenase on anaphylactic and inflammatory responses.

In conclusion, we have described a novel series of acetohydroxamic acids that are potent and selective inhibitors of arachidonate 5-lipoxygenase in vitro and in vivo. In addition, we have shown that these compounds attenuate "leukotriene-dependent" anaphylactic bronchospasm, the accumulation of inflammatory leukocytes, and the development of fever in experimental models. It now remains to be determined if these compounds have any therapeutic value in man.

Stimulation of gastric alkaline secretion by stable prostacyclin analogues in rat and dog.

The stimulation of gastric alkaline secretion in vivo following topical mucosal application of four stable anti-ulcer analogues of prostacyclin has been investigated in the rat and in the dog using intragastric pH-stat techniques. In the rat, basal alkaline secretion was significantly stimulated by the prostacyclin analogues 16-phenoxy-(5 alpha)-5,9-epoxy-PGF1 (16-phenoxy) and its methyl ester, when administered in the luminal perfusion fluid. The 16-phenoxy analogue (25 and 50 micrograms ml-1) increased basal alkaline secretion with its methyl ester being more potent. The methyl ester of 16,16-dimethyl PGI1 (25 and 50 micrograms ml-1) likewise stimulated alkaline secretion whereas its corresponding free acid was inactive at these concentrations. In further studies in the conscious dog with a Heidenhain gastric pouch, these four prostacyclin analogues administered intraluminally (1.25 micrograms ml-1) significantly increased gastric alkaline secretion. The present findings indicate that the stable 16-phenoxy and 16, 16-dimethyl analogues of prostacyclin, can stimulate gastric alkaline secretion in the dog and rat in vivo. As with the previously reported 16,16-dimethyl PGE2 analogue, this property of stimulating alkaline secretion may therefore contribute to the antiulcer activity of these prostacyclin analogues.

Protective effect of S-nitroso-N-acetyl-penicillamine in endotoxin-induced acute intestinal damage in the rat.

Macroscopic jejunal damage and plasma leakage induced within 15 min by E. coli lipopolysaccharide (LPS 50 mg kg-1 i.v.) in the rat was enhanced by the inhibitor of nitric oxide (NO) formation, NG-monomethyl-L-arginine (L-NMMA 50 mg kg-1 i.v.). The nitro-vasodilator, S-nitroso-N-acetyl-penicillamine (SNAP; 10 micrograms kg-1 min-1 i.v.), which generates NO, attenuated both LPS-induced intestinal damage and the enhancement of such damage and plasma leakage produced by L-NMMA. Endogenous NO may thus have a protective role in the intestinal vasculature that can be mimicked by generators of NO.

Comparison of two techniques of suturing in microsurgical anastomosis of the rabbit oviduct.

Microsurgical reconstruction of the rabbit oviduct was undertaken utilizing either through-and-through sutures including the mucosa, or sutures penetrating the serosa and myosalpinx but not the mucosa. Patency and pregnancy rates did not seem to vary with the suturing technique. However, scanning electron microscopic observation revealed abnormal mucosal fold patterns at 3 weeks after surgery. Fibrinous exudates over the surface of the intraluminal sutures increased with time until the entire surface was thickly veiled by 9 weeks. The observation also revealed regeneration of epithelial cells along the suture. These cells included many abnormal forms such as giant cells and misshapen cells. The majority of these were nonciliated. In theory, the intraluminal suture could form a nidus for epithelial hyperplasia which might cause future tubal obstruction. However, the patency and pregnancy rates obtained with through-and-through suturing were similar to those obtained when the endosalpinx was excluded, both in this study and in previous studies from these laboratories.

Deciliation in the puerperal fallopian tube.

Tubal segments obtained from patients at cesarean section and at intervals during the first 5 postpartum days were examined to evaluate puerperal changes in the tubal epithelium. The specimens of tubal epithelium were examined under the scanning and transmission electron microscopes. Ciliated cells were most densely distributed on the fimbria and in the ampulla, and were relatively sparsely distributed in the isthmus. Progressive diminution of numbers of ciliated cells and deciliated of individual cells were noted in specimens obtained during the puerperium. Nonciliated cells were in the resting stage at term pregnancy. Secretory activity returned during the puerperium.

A phase I study of recombinant human interleukin-4 administered by the intravenous and subcutaneous route in patients with advanced cancer: immunological studies.

The effects of recombinant human interleukin-4 (rhu IL-4) on immunological parameters in patients receiving increasing doses of IL-4 in a Phase I trial were investigated. Peripheral blood mononuclear cells were phenotyped for a variety of lymphocyte markers, but no consistent effects were observed. However, increases in HLA Class II expression on monocytes were detected in four patients. NK and LAK activity were neither induced nor augmented by IL-4 treatment. Slight increases in proliferative responses to mitogens and cytokines were observed in some patients. The latter observations and other clinical studies suggest that a combination of IL-4 with IL-2 may be more effective in Phase II clinical trials.