Gastroduodenal ulceration in rabbits producing antibodies to prostaglandins

The consistent occurrence of gastric and duodenal ulcers was observed in laboratory rabbits used for production of high-titer plasma antibody to 6-keto PGF1 alpha and PGE2. Perforations developed in 7 of 10 animals, usually just distal to the pyloroduodenal junction. The remaining rabbits showed gross and/or microscopic evidence of imperforate ulcers and erosions. These lesions appeared to be direct pathologic complications of an immune response directed against prostaglandins since animals immunized against met-enkephalin with similar methods had no ulcers.     

Increased vasodepressor effect of prostaglandins A1 and E1 in renal hypertensive rabbits.

In order to determine whether cardiovascular reactivity to exogenous prostaglandins is altered in hypertension, the hypotensive effects of increasing intravenous doses of PGA₁ and PGE₁ were assessed in conscious normotensive rabbits and in rabbits made hypertensive by wrapping both kidneys with cellophane. Similar experiments were carried out with nitroglycerin. Depressor responses to the prostaglandins, but not to nitroglycerin, were greater in hypertensive than in normotensive animals. The possibility of this enhanced responsiveness being related to the prostaglandin deficiency believed to exist in hypertension was explored in normotensive rabbits treated acutely with indomethacin. The prostaglandin synthesis inhibitor did not affect blood pressure responses to PGA₁ or PGE₁. Although these experiments do not rule out the possible influence of more prolonged prostaglandin deficiency on cardiovascular reactivity, a more apparent adrenergic inhibitory component of the hypotensive effect of prostaglandins in hypertensive animals was considered a likely alternative explanation for the phenomena observed.     

Effects of oral administration of PGE2 on gastric secretion and experimental peptic ulcerations

The anti-secretory and anti-ulcer effects of prostaglandin E₂ (PGE₂) using iso-osmotic buffer as a vehicle have been investigated in several types of laboratory animals. Orally administered PGE₂ was found to be highly effective in preventing formation of ulcers in several experimental models -- pylorus ligated induced ulcers in rats, histamine induced ulcers in guinea pigs, reserpine induced ulcers in rats and pentagastrin induced ulcers in guinea pigs and cats. PGE₂ also suppressed acid secretion but not pepsin activity. It was concluded that the anti-ulcer effects of PGE₂ were due to its anti-secretory activity rather than antipepsin activity. In view of PGE₂'s activity in preventing ulceration induced by histamine and reserpine in addition to pentagastrin, it is suggested that the anti-pentagastrin activity of PGE₂ is not specific.     

UP-regulaton of prostaglandin E2 binding and of prostanoid release in rabbits producing antibodies

German Giant rabbits successfully immunized agianst prostaglandin (PG) E₂ as shown by a rise in antibody titers developed gastric mucosal lesions. Enzymatically dispersed gatric mucosal cells of these animals had a significantly enhanced production of PG₂ and PG I₂ as measured by specific radioimmunoassays. This may be explained by an increased supply with endogenous arachidonic acid (as indicated by an enhanced phospholipase A₂/LAT ratio) and by a higher activity of the subsequent PG forming enzymes (as indicated by a more effectvie stimulation of PG production by exogenous arachidonic acid). Gastric mucosal plasma membranes of immunized rabbits had significantly high PG E₂ binding capacity (108 ± 9 fmol/mg protein) than those of nomimmunized rabbits (72 ± 5 fmol/mg protein). The ligand affinity was not afected by immunization. Neither histamine-stimulated ¹⁴C-aminopyrine uptake of isolated parietal cells as a marker for acid production nor its inhibition by PG E₂ were influenced by receptor up-regulation. The increased eicosanoid release can be regarded as an endogenous defense emchanism against increased mucosal vulnerability caused by PG E₂ scavenging. The potential role of PG E₂ receptor up-regullation in support of this process remains to be established.     

Thyroid function and thyrotropin levels in rabbits immunized to produce antibodies against thyroid hormones

Various parameters of thyroid function were studied in 27 rabbits, out of which 10 were immunized to produce antibodies against triiodothyronine (T₃), 9 against thyroxine (T₄) and 8 were normals. Estimations of T₃, T₄, Free T₄ (FT₄) and thyrotropin (TSH) in blood, qualitative and quantitative analysis of iodoamino acids in serum, protein bound iodine-131 (PB¹³¹I), butanol extractable iodine-125 (BE¹²⁵I) and measurement of the disappearance rates of ¹²⁵I-labelled T₃ and T₄ from plasma were done. In addition, glandular changes were also studied by measurement of ¹³¹I uptake, thyroid scanning and chromatographic analysis of hydrolysate of soluble iodoproteins. In T₃ immunized animals, levels of T₃ in serum increased by 38 to 125 times, levels of TSH also showed a significant rise (7.4 ± 1.2 vs 28 ± 9 ng/mL). Chromatographic analysis of iodoamino acids in serum as well as in the hydrolysate of the thyroid gland demonstrated a selective increase in synthesis of T₃. Rate of disappearance of T₃ from blood showed a significant decline. Thyroid glands in the immunized rabbits showed signs of hypertrophy and hyperplasia. Identical studies done in rabbits immunized to produce antibodies against T₄ showed a similar pattern though of variable degree. Our studies indicate that the thyroid glands of the immunized rabbits undergo marked alterations resulting in selective increase in the synthesis and secretion of the particular thyroid hormone against which they were immunized. They do so under the influence of increased levels of TSH.     

A new prostaglandin E1 analogue (CL115, 574): I. Antisecretory and cytoprotectige effects in the rat

The effect of a new analogue of PGE₁ (CL115, 574) on gastric acid secretion, mucus secretion and protection against stress and indomethacin- induced ulcers were studied in the rat. CL115, 574 was more potent than PGE₁ and cimetidine in inhibiting acid secretion. CL115, 574 protected against the development of stress and indomethacin-induced ulcers prevented the indomethacin-induced decrease in hematocrit at an ED₅₀ (3 μg/kg) far below the antisecretory ED₅₀ (1 mg/kg). While the inhibiting acid secretion, CL115, 574 increased the volume of gastric secretion indicating a stimulation of nonparallel cell secretion by the rat stomach. In addition the compound stimulated the secretion of mucus into the gastric juice. On the basis of its potency as an inhibitor of acid secretion and these additional effects which are indicative of cytoprotective activity, CL115, 574 should be further studied as a possible anti-ulcer agent in man.     

Effects of N-ethylmaleimide on the ATPase activities of cardiac myosin from thyrotoxic rabbits.

The ATPase activities of cardiac myosin from thyrotoxic and euthyroid rabbits have been compared. The Ca²⁺-ATPase activity of myosin from thyrotoxic animals was elevated by 200%, while the K⁺(EDTA)-ATPase activity was the same as in euthyroid animals. Modification by N-ethyl-maleimide of the most rapidly reacting class of sulfhydryls (SH₁) in myosin from euthyroid animals increased Ca²⁺-ATPase activity about 177% over the unreacted value. Modification of the SH₁ groups in myosin from thyrotoxic animals had no effect on CA²⁺-ATPase activity. We conclude that thyroxin may increase cardiac myosin ATPase activity by a conformational change in the same region as the SH₁ thiols.     

The spontaneously hyperlipoproteinaemic rabbit

It is possible to detect among New Zealand rabbits some animals (A. N.Z.) whose plasma lipoproteins patterns are quite different from those of normal rabbits (N.N.Z.). The differences involve VLDL (< 1.006), LDL₁ (1.006–1.019), LDL₂ (1.019–1.063), HDL (1.063–1.210), VHDL (> 1.210) and plasma cholesterol, triglycerides and phospholipids. This lipoprotein pattern seems to resemble human type IIb hyperlipoproteinaemia. A rapid screening procedure for detecting these animals is proposed.     

The site of VX2 tumor transplantation affects the development of hypercalcemia in rabbits

The relationship between plasma levels of 15-keto-13, 14-dihydro-prostaglandin E₂ (15K-H₂-PGE₂) and serum calcium levels was studied in nontumor-bearing rabbits and in rabbits bearing the VX₂ carcinoma intramuscularly and intra-abdominally. The plasma levels of 15K-H₂-PGE₂ in the two groups of tumor-bearing animals did not vary significantly but was several fold greater than in nontumor-bearing rabbits. Rabbits bearing the VX₂ carcinoma intramuscularly developed hypercalcemia between the second and third week after implantation of neoplastic tissue and remained hypercalcemic until they expired. The serum calcium levels in rabbits bearing the VX₂ carcinoma intra-abdominally did not vary significantly from those of nontumor-bearing rabbits. The differences in the serum calcium levels in rabbits bearing the VX₂ carcinoma at intramuscular and intra-abdominal implant sites may be related to different extents of metabolism by the lung and by the liver of prostaglandin E₂ or other cyclooxygense products of polyenoic fatty acids produced by the tumor.     

Design of an Escherichia coli Expressed HIV-1 gp120 Fragment Immunogen That Binds to b12 and Induces Broad and Potent Neutralizing Antibodies

b12, one of the few broadly neutralizing antibodies against HIV-1, binds to the CD4 binding site (CD4bs) on the gp120 subunit of HIV-1 Env. Two small fragments of HIV-1 gp120, b121a and b122a, which display about 70% of the b12 epitope and include solubility-enhancing mutations, were designed. Bacterially expressed b121a/b122a were partially folded and could bind b12 but not the CD4bs-directed non-neutralizing antibody b6. Sera from rabbits primed with b121a or b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity in a TZM-bl assay against a 16-virus panel that included nine Tier 2 and 3 viruses as well as in a five-virus panel previously designed to screen for broad neutralization. Using a mean IC₅₀ cut-off of 50, sera from control rabbits immunized with gp120 alone neutralized only one virus of the 14 non-Tier 1 viruses tested (7%), whereas sera from b121a- and b122a-immunized rabbits neutralized seven (50%) and twelve (86%) viruses, respectively. Serum depletion studies confirmed that neutralization was gp120-directed and that sera from animals immunized with gp120 contained lower amounts of CD4bs-directed antibodies than corresponding sera from animals immunized with b121a/b122a. Competition binding assays with b12 also showed that b121a/2a sera contained significantly higher amounts of antibodies directed toward the CD4 binding site than the gp120 sera. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals.     

Announcement

Antiserum against PGE₁ was raised in rabbits following immunization with prostaglandin-thyroglobulin conjugates. The antiserum exhibits 22% cross reactivity with PGE₂ but little or no apparent cross reaction with PGE₁ metabolites or heterologous prostaglandins. The data indicate some limitations in the use of this antiserum for the measurement of PGE₁ in biologic samples.     

The preparation and characterization of prostaglandin E1 antiserum

Antiserum against PGE₁ was raised in rabbits following immunization with prostaglandin-thyroglobulin conjugates. The antiserum exhibits 22% cross reactivity with PGE₂ but little or no apparent cross reaction with PGE₁ metabolites or heterologous prostaglandins. The data indicate some limitations in the use of this antiserum for the measurement of PGE₁ in biologic samples.     

Deep-Sea Water Containing Selenium Provides Intestinal Protection against Duodenal Ulcers through the Upregulation of Bcl-2 and Thioredoxin Reductase 1

Deep-sea water (DSW), which is rich in micronutrients and minerals and with antioxidant and anti-inflammatory qualities, may be developed as marine drugs to provide intestinal protection against duodenal ulcers. We determined several characteristics in the modified DSW. We explored duodenal pressure, oxygenation, microvascular blood flow, and changes in pH and oxidative redox potential (ORP) values within the stomach and duodenum in response to tap water (TW, hardness: 2.48 ppm), DSW600 (hardness: 600 ppm), and DSW1200 (hardness: 1200 ppm) in Wistar rats and analyzed oxidative stress and apoptosis gene expressions by cDNA and RNA microarrays in the duodenal epithelium. We compared the effects of drinking DSW, MgCl₂, and selenium water on duodenal ulcers using pathologic scoring, immunohistochemical analysis, and Western blotting. Our results showed DSW has a higher pH value, lower ORP value, higher scavenging H₂O₂ and HOCl activity, higher Mg²⁺ concentrations, and micronutrients selenium compared with TW samples. Water infusion significantly increased intestinal pressure, O₂ levels, and microvascular blood flow in DSW and TW groups. Microarray showed DSW600, DSW1200, selenium water upregulated antioxidant and anti-apoptotic genes and downregulated pro-apoptotic gene expression compared with the TW group. Drinking DSW600, DSW1200, and selenium water but not Mg²⁺ water significantly enhanced Bcl-2 and thioredoxin reductase 1 expression. Bax/Bcl-2/caspase 3/poly-(ADP-ribose)-polymerase signaling was activated during the pathogenesis of duodenal ulceration. DSW drinking reduced ulcer area as well as apoptotic signaling in acetic acid-induced duodenal ulcers. DSW, which contains selenium, provides intestinal protection against duodenal ulcers through the upregulation of Bcl-2 and thioredoxin reductase 1.     

Role of endogenous prostacyclin in gastric ulcerogenic and healing responses—a study using IP-receptor knockout mice

Endogenous prostaglandins (PGs) play an important role in the cytoprotective and healing responses in the stomach, by altering various functions, i.e., an increase of the mucosal blood flow, yet the role of prostacyclin (PGI₂) and its receptor (IP-receptor) in these responses remains unclarified. In the present study, we used IP-receptor knockout mice [IP (−/−)] and examined the importance of IP-receptors in gastric ulcerogenic, cytoprotective and healing responses in these animals. The studies included the ulcerogenic response to cold-restraint stress, the cytoprotective response to a mild irritant (20 mM taurocholate: TC) and capsaicin, and the healing response of chronic gastric ulcers induced by thermo-cauterization. We first checked the absence of IP-receptors by examining the effect of cicaprost (a PGI₂ agonist, topical mucosal application) on gastric mucosal blood flow and found that this agent increased the mucosal blood flow in wild-type [WT (+/+)] mice but not in IP (+/−) mice. Cold-restraint stress (4 h) induced gastric lesions in both groups of mice, but the severity of damage was significantly greater in IP (−/−) mice. Prior p.o. administration of both TC and capsaicin exhibited a marked cytoprotection against HCl/ethanol-induced gastric damage in WT (+/+) mice, both responses being significantly mitigated in the presence of indomethacin. The adaptive cytoprotection induced by TC was similarly observed in IP (−/−) mice, while the capsaicin protection was totally attenuated in the animals lacking IP receptors. On the other hand, the healing of gastric ulcers was significantly delayed by daily administration of indomethacin in WT (+/+) mice. However, this process was not altered in IP (−/−) mice. These results suggest that endogenous PGI₂ is involved in the gastric ulcerogenic response to stress, but not in the healing of pre-existing gastric ulcers. In addition, PGI₂ and its receptors may play a crucial role in capsaicin-induced gastric protection but not in the adaptive cytoprotection-induced by mild irritants.     

Active immunization against GnRH in pre-pubertal domestic mammals: testicular morphometry,histopathology and endocrine responses in rabbits,guinea pigs and ram lambs

Effective tools for male contraception are important in the control of reproduction in animal populations. The aim of the present study was to evaluate the effects of active immunization against gonadotropin-releasing hormone (GnRH) on male reproductive function assessing testicular morphological changes and serum-gonadotropin levels in pre-pubertal rabbits, guinea pigs and ram lambs. An anti-GnRH vaccine was developed by linking a GnRH-homologous molecule to a tetanus clostridial toxoid (Al(OH)₃ coadjuvant). After vaccination protocols testicular morphometry, histopathological alterations and endocrine responses (FSH, LH, testosterone and cortisol serum levels) were evaluated. Testicular volume was significantly reduced in vaccinated animals with respect to the control group in rabbits, guinea pigs and ram lambs (P<0.05 to P<0.001). The anti-GnRH vaccine generated a reduction in testicular volume of 15-, 27- and 11-fold, respectively. Tubule diameters decreased in the vaccinated group with respect to the control ~2.0-, 1.2- and 3.5-fold, respectively (P<0.001). Tubule, intertubular and lumen volumes significantly decreased in vaccinated rabbits (P<0.05), guinea pigs and ram lambs (P<0.01). Vaccinated animals of the three species showed significant reductions in spermatogonial numbers (10- to 40-fold; P<0.01). Sperm was absent in all seminiferous tubules of all rabbits, and most individuals of guinea pigs (80%) and ram lambs (60%). No significant differences were observed between vaccinated and control groups regarding FSH and LH during the experiments in the three experimental species/models used. Testosterone, however, was only significantly lower (~22-fold, P<0.01) in vaccinated rabbits. In conclusion, the present study demonstrated that pre-pubertal active immunization against GnRH leads to endocrine disruption and marked differences on testicular morphometry, development and activity among lagomorphs, hystricomorphs and ovine species with species-specific sensitivity regarding the anti-GnRH immune response.     

Ovariectomy Reinstates the Infarct Size-Limiting Effect of Postconditioning in Female Rabbits

Gender seems to interfere with the cardioprotective effect of ischemic preconditioning (PreC) and postconditioning (PostC); PreC-conferred protection is weaker or lost in female animals after ovariectomy (Ov), while the role of PostC is still in dispute. We sought to investigate the effect of PostC in female rabbits, its interaction with Ov, and the potential implicated intracellular pathways. Intact or Ov adult female rabbits (n = 46) were subjected to 30 min ischemia and reperfusion with PostC (PostC or OvPostC), which consisted of six cycles of 30-s ischemia/30-s reperfusion at the end of ischemia, or without PostC (Fem or OvFem). Infarct size (I) and area at risk (R) were determined by TTC staining and fluorescent particles, respectively, after 3-h reperfusion in 30 out of 46 animals. Plasma levels of estradiol and nitrite/nitrate (NO _x ) were evaluated. ERKs, p38-MAPK, and Akt assessment was performed in excised hearts 1-min after starting the final reperfusion period in the remaining 16 animals. Infarct size was significantly reduced only in OvPostC group (I/R ratio, 25.3 ± 2.7, vs 48.1 ± 2.0, 43.6 ± 4.2 and 55.1 ± 5.6 % in Fem, OvFem, and PostC groups, p < 0.05). In ovariectomized rabbits, plasma estradiol and NO _x levels were lower than in the normal ones. Akt phosphorylation in ischemic regions was significantly higher in OvPostC group, whereas ERK1/2 and p38-MAPK activation was observed in all ovariectomized animals irrespective of PostC. PostC is not effective in female rabbits, but the protection is reinstated after Ov potentially via the RISK pathway.     

Thyroid function during intermittent exposure to hypobaric hypoxia

Circulatory levels of triiodothyronine (T₃) and thyroxine (T₄) and their kinetics were studied in rabbits exposed to intermittent hypobaric hypoxia (5200 m, 395 mm Hg,PO₂ 83 mm Hg) 6 h daily for 5 weeks in a decompression chamber maintained at room temperature of 22°–24° C. Kinetics of T₃ and T₄ were studied on days 21 and 28 of hypoxic exposure. The T₃ and T₄ values were found to be significantly lower on day 8 of exposure to hypoxia compared to the pre-exposure values. The decreased levels were maintained throughout the entire period of hypoxic stress. The metabolic clearance rate, production rate, distribution space and extrathyroidal T₃ and T₄ pools were significantly decreased in animals under hypoxic stress compared to the control animals. The decline in thyroid hormone levels and their production in rabbits under hypoxic stress indicate an adaptive phenomenon under conditions of low oxygen availability.     

Oxytocin-stimulated production of prostaglandin F2α by isolated endometrium of rabbit: Modulation by ovarian steroids

To test the hypothesis that ovarian steroid hormones modulate oxytocin-induced release of prostaglandin F_2α (PGF_2α) from uterine endometrium, 2 ovariectomized rabbits were pretreated with progesterone (5 mg/day for 10 days), 2 with estradiol-17β (25 μg/day for 10 days), 2 with both steroids, and one with sesame oil only. On the last day of treatment, endometrial fragments were excised and incubated in vitro with or without oxytocin (100 μU/ml). Although endometrium from rabbits pretreated with combined steroids released more PGF_2α immediately after excision than did tissue from animals pretreated with either steroid by itself, endometrium from animals pretreated with estrabdiol-17β alone released the most PGF_2α during sustained incubation in vitro. Moreover, only this tissue exhibited significant oxytocin-dependent release of PGF_2α. At the dosages used, progesterone completely antagonized both of these effects of estradiol-17β. The results support the hypothesis that ovarian steroid hormones regulate oxytocindependent release of PGF_2α from endometrial cells. A possible mechanism of action is suggested.     

Optimization and Evaluation of Gastroretentive Ranitidine HCl Microspheres by Using Factorial Design with Improved Bioavailability and Mucosal Integrity in Ulcer Model

The purpose of our investigation was to develop and optimize the drug entrapment efficiency and bioadhesion properties of mucoadhesive chitosan microspheres containing ranitidine HCl prepared by an ionotropic gelation method as a gastroretentive delivery system; thus, we improved their protective and therapeutic gastric effects in an ulcer model. A 3?×?2² full factorial design was adopted to study the effect of three different factors, i.e., the type of polymer at three levels (chitosan, chitosan/hydroxypropylmethylcellulose, and chitosan/methylcellulose), the type of solvent at two levels (acetic acid and lactic acid), and the type of chitosan at two levels (low molecular weight (LMW) and high molecular weight (HMW)). The studied responses were particle size, swelling index, drug entrapment efficiency, bioadhesion (as determined by wash-off and rinsing tests), and T _80% of drug release. Studies of the in vivo mucoadhesion and in vivo protective and healing effects of the optimized formula against gastric ulcers were carried out using albino rats (with induced gastric ulceration) and were compared to the effects of free ranitidine powder. A pharmacokinetic study in rabbits showed a significant, 2.1-fold increase in theAUC_0–24of the ranitidine microspheres compared to free ranitidine after oral administration. The optimized formula showed higher drug entrapment efficiency and mucoadhesion properties and had more protective and healing effects on induced gastric ulcers in rats than ranitidine powder. In conclusion, the prolonged gastrointestinal residence time and the stability of the mucoadhesive microspheres of ranitidine as well as the synergistic healing effect of chitosan could contribute to increasing the potential of its anti-gastric ulcer activity.     

The potential of dextran-based glycoconjugates for development of Helicobacter pylori vaccine

We have recently demonstrated that synthetic glycoconjugates based on delipidated lipopolysaccharide (LPS) of Helicobacter pylori and containing an α(1-6)-glucan chain induced broadly cross-reactive functional antibodies in immunized animals. To investigate the candidacy of α(1-6)-glucan as an alternative vaccine strategy we prepared glycoconjugates based on dextrans produced by lactic acid bacteria Leuconostoc mesenteroides B512F and consisting of linear α(1-6)-glucan chains with limited branching. Three dextrans with averaged molecular masses of 5,000 Da, 3,500 Da and 1,500 Da, respectively, were modified with a diamino group-containing linker and conjugated to a carrier protein, tetanus toxoid (TT) or diphtheria toxoid (DT), and their immunological properties investigated. The conjugates were immunogenic in both rabbits and mice and induced specific IgG responses against α(1-6)-glucan-expressing H. pylori LPS. Studies performed with post-immune sera of mice and rabbits immunized with dextran-based conjugates demonstrated cross-reactivity with LPS from typeable and non-typeable strains of H. pylori and selected mutants. The post-immune sera from rabbits that received the conjugates exhibited functional activity against α(1-6)-glucan-positive strains of H. pylori. These data provide evidence that dextran-based conjugates may offer a simplified approach to the development of carbohydrate-based vaccines against H. pylori.