In vivo stimulation and restoration of the immune response by the noninflammatory fragment 163-171 of human interleukin 1 beta.

The synthetic nonapeptide VQGEESNDK, corresponding to the fragment 163-171 of human IL-1 beta, showed in vivo immunomodulatory capacities qualitatively and quantitatively comparable to those of the mature human IL-1 beta protein. In fact, both IL-1 beta and the 163-171 fragment stimulated the immune response of normal mice and restored immune reactivities of immunocompromised animals. In addition, the synthetic IL-1 peptide was as efficient as the entire protein in inducing tumor rejection and radioprotection. On the other hand, the 163-171 fragment did not cause any of several inflammation-associated metabolic changes inducible by the whole IL-1 beta molecule in vivo: hypoferremia, hypoglycemia, hyperinsulinemia, increase in circulating corticosterone, SAA and fibrinogen, decrease in hepatic drug-metabolizing enzymes. Furthermore, at variance with IL-1 beta, the 163-171 peptide did not show the toxic effects causing shock and death in adrenalectomized mice. Thus, these results confirm our previous in vitro observations that functional domains are identifiable within the multipotent cytokine IL-1 beta, and demonstrate the biological relevance of this finding in a variety of in vivo systems. The identification of a selectively active fragment of a cytokine may thus represent a significant step towards a better directed and more rational immunotherapeutic approach.

Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease.

The gram negative, microaerophilic bacterium Helicobacter pylori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma. Cloning of the H. pylori cytotoxin gene shows that the protein is synthesized as a 140-kD precursor that is processed to a 94-kD fully active toxin. Oral administration to mice of the purified 94-kD protein caused ulceration and gastric lesions that bear some similarities to the pathology observed in humans. The cloning of the cytotoxin gene and the development of a mouse model of human gastric disease will provide the basis for the understanding of H. pylori pathogenesis and the development of therapeutics and vaccines.

Development of a mouse model of Helicobacter pylori infection that mimics human disease.

The human pathogen Helicobacter pylori is associated with gastritis, peptic ulcer disease, and gastric cancer. The pathogenesis of H. pylori infection in vivo was studied by adapting fresh clinical isolates of bacteria to colonize the stomachs of mice. A gastric pathology resembling human disease was observed in infections with cytotoxin-producing strains but not with noncytotoxic strains. Oral immunization with purified H. pylori antigens protected mice from bacterial infection. This mouse model will allow the development of therapeutic agents and vaccines against H. pylori infection in humans.

Unravelling the pathogenic role of Helicobacter pylori in peptic ulcer: potential new therapies and vaccines.

The recognition that peptic ulcer is an infectious disease caused by the bacterium Helicobacter pylori has revolutionized the approach to diagnosis and therapy of this condition. Treatment of the symptoms of peptic ulcer with drugs that block acid secretion is already being replaced by antibiotic eradication of the causative agent. Studies of the molecular events that lead to H. pylori pathogenesis have shown that clinical isolates can be divided into two groups, only one of which produces a cytotoxin and is associated with severe disease. The cloning of the genes coding for molecules specific for disease-associated strains of H. pylori, and the development of animal models that mimic the human pathology, will provide the basis for better strategies to treat and prevent peptic-ulcer disease.

Differential binding of IL-1 alpha and IL-1 beta to receptors on B and T cells.

The interleukin 1 receptors (IL-1R) on the human B lymphoma RAJI and on the murine thymoma EL4-6.1 have been characterized. Equilibrium binding analysis using both 125I-labeled IL-1 alpha and IL-1 beta showed that RAJI cells have a higher number of binding sites/cell for IL-1 beta (2400, Kd 2.2 nM) than for IL-1 alpha (316, Kd 0.13 nM). On the other hand, EL4-6.1 cells have more receptors/cell for IL-1 alpha (22 656, Kd 1 nM) than for IL-1 beta (2988, Kd 0.36 nM). Dexamethasone (DXM) induced on RAJI cells a time-dependent increase in binding sites for both IL-1 beta and IL-1 alpha without affecting their binding affinities. However, while receptor-bound 125I-IL-1 alpha was displaced with equal efficiency by both IL-1 forms, only unlabeled IL-1 beta could effectively displace 125I-IL-1 beta. Cross-linking experiments indicated that RAJI cells have a predominant IL-1R of about 68 kDa, while EL4-6.1 cells have an IL-1-binding polypeptide of 80 kDa. These results suggest that B and T cells possess structurally different IL-1R with distinct binding properties for IL-1 alpha and IL-1 beta.

Helicobacter pylori infection in wild-type and cytokine-deficient C57BL/6 and BALB/c mouse mutants.

Helicobacter pylori causes gastroduodenal ulcer disease in humans. T lymphocytes and their cytokines are thought to play a substantial role in the control of H. pylori infection. To determine the importance of T helper (Th) cytokines and background genes we investigated the natural course of H. pylori infection in BALB/c and C57BL/6 wild-type or mutant mice deficient for either interleukin (IL)-4 or interferon (IFN)-gamma. H. pylori SPM 326 persisted for at least six months in C57BL/6 but was cleared by BALB/c wild-type mice nine weeks postinfection. H. pylori was recovered more frequently from IFN-gamma(-/-) BALB/c and IFN-gamma( -/-) C57BL/6 mice than from the respective wild-type animals. In contrast, IL-4 deficiency had no detectable effect on H. pylori recovery rates from either strain of mice. Our data suggest a protective role of IFN-gamma by mediating inflammation in murine H. pylori infection. In addition, our data emphasize that background genes which differ between BALB/c and C57BL/6 mice regulate the clearance of H. pylori.

Quantitation of biologically active IL-1 by a sensitive assay based on immobilized human IL-1 receptor type II (IL-1RII).

A rapid and sensitive solid-phase radioassay is described for the quantitative detection of human interleukin-1 (IL-1) based on its capability to bind the nitrocellulose-immobilized IL-1 receptor solubilized from plasma membranes of a subclone of the human B cell lymphoma Raji. The assay can detect human IL-1 beta levels as low as 1 X 10(-11) M, both in physiological buffers and in human plasma. Much lower sensitivity was observed for human IL-1 alpha (3.7 X 10(-9) M) and murine IL-1 beta (2 X 10(-9) M). This assay has the advantage to specifically detect only the correctly folded biologically active IL-1. Simple pretreatment procedure that selectively removes IL-1 beta from samples has been devised so that the ratio of the two IL-1s isoforms in the sample can be precisely determined. This assay represents a fast method for the simultaneous-testing of large numbers of biological samples.

Quantitative analysis of Helicobacter pylori infection in a mouse model.

Progress in elucidating the pathogenesis of Helicobacter pylori gastric infection and in developing an H. pylori vaccine will be aided by an animal model in which H. pylori can be reliably detected. To validate the use of the mouse model of H. pylori infection, we determined the susceptibility of three inbred strains of mice (C57BL/6J, C57BL/10J and BALB/c) to two VacA+/CagA+ isolates of H. pylori (SPM326 and M1.16) and determined the effectiveness of microbiological, histological and molecular assays for H. pylori detection. For the detection of H. pylori in inoculated mice, reverse transcriptase-polymerase chain reaction was the most sensitive assay (82%), histological evaluation the next most sensitive (66%) and microbiological evaluation the least sensitive (38%); the assays were equally specific (100%). Of the two H. pylori isolates, M1.16 showed the highest rate of colonization, but SPM326 displayed the highest rate of persistent infection. Among the three mouse strains, C57BL/6J mice showed the highest level of both susceptibility to colonization and persistent infection. Anti-H. pylori antibody responses were induced in all inoculated mice and persisted for up to 8 weeks after H. pylori clearance. These results indicate that inbred mice experimentally infected with H. pylori is a reliable model for human infection, but host susceptibility to colonization and persistence of infection are dependent on the H. pylori isolate and the mouse strain.

Evidence that the interleukin-1 beta-induced prostaglandin E2 release from rat hypothalamus is mediated by type I and type II interleukin-1 receptors.

Interleukin-1 beta (IL-1 beta) has been shown to specifically increase the release of prostaglandin (PG) E2 from rat hypothalamic explants in short-term experiments. In this study we attempted to characterize the receptor subtype(s) involved in this response. Rat hypothalamic explants were incubated with mouse monoclonal antibodies (mAbs) raised against human IL-1 type I or type II receptors, IL-1 receptor antagonist (IL-1ra) and alpha-melanocyte-stimulating hormone (alpha-MSH) (which appears to antagonize certain IL-1 induced inflammatory effects in vivo), alone and in the presence of IL-1 beta. PGE2 released into the incubation medium was measured by radioimmunoassay. The anti-type I mAb reduced both basal and IL-1 beta-stimulated PGE2 release at 10 micrograms/ml, but not at lower concentrations. The anti-type II mAb also produced a significant decrease in stimulated release but had no effect on basal release. IL-1ra mimicked the effects of the anti-type I mAb, while alpha-MSH failed to alter either basal or stimulated PGE2 release. These findings suggest that IL-1 beta controls production and release of PGE2 by the rat hypothalamus via both type I and type II receptors, although the latter appear to be involved only in the response to high levels of IL-1.

Prospects for the development of a vaccine against Helicobacter pylori.

Over 50% of the world population is chronically infected by the gastric pathogen, Helicobacter pylori, which is responsible for most peptic ulcer disease and is closely associated with adenocarcinoma of the stomach. Current therapies for peptic ulcer disease include antibiotic eradication of H. pylori infection. While effective, the high cost, difficulty of patient compliance with the treatment regimens, and risks of selection for resistant strains make these therapies impractical on a large scale. Studies of the pathogenesis of H. pylori have led to the identification of bacterial antigens as candidates for inclusion in novel vaccines against this disease. Both prophylactic and therapeutic vaccination have been demonstrated in animal models of Helicobacter infection. Preclinical evaluations of several antigens are at present under way and trials of vaccination in humans are planned.

Distinct inhibition of membrane-bound and lysosomal phospholipase A2 by glucocorticoid-induced proteins.

Anti-inflammatory steroids induce the release in vivo of antiphospholipase proteins (APP) into the peritoneal cavities of rats. APP were partially purified by ion- exchange chromatography. The main anti-phospholipase activity was recovered in two zones of the elution gradient named APP I and APP II; their molecular weight (mol. wt) was determined with molecular sieve chromatography. Two phospholipase A2 (PLA2) activities were identified from rat peritoneal leucocytes, one with a pH optimum at 4.5 (a lysosomal enzyme) and one with pH optimum at 8.5 (a membrane-bound enzyme); the selective secretion of the former was observed when leucocytes were stimulated by phagocytosis. The effect of APP on both enzyme activities was studied on enzyme preparations from resting leucocytes. APP were also added to leucocytes incubated with or without phagocytozable material. After incubation, PLA2 activities were determined both inside the cells and in the culture medium. APP I revealed a mol. wt of 200 k with a small fragment of 15 k and inhibited membrane-bound PLA2; APP II revealed a mol. wt of 40 k and inhibited lysosomal PLA2.

Cytokines in the viviparous reproduction of squamate reptiles: interleukin-1 alpha (IL-1 alpha) and IL-1 beta in placental structures of a skink.

Placental viviparity is known in many species of squamate reptiles. Among these, some scincids have developed an epithelio-chorial chorio-allantoic placenta which in the structure of its central ridged zone is similar to those of certain therian mammalian species. A broad range of immunoregulatory peptides, cytokines, has been identified at the maternofetal interface of several species of mammals, either with invasive or non-invasive types of placenta. Thus we began to study whether interleukin-1, which is considered to play a crucial role in mammalian pregnancy, might also be involved in the viviparity of reptilian species. Placentae of Chalcides chalcides L. were processed by immunohistochemistry and incubated in a culture medium for different times. A very strong immunoreactivity for interleukin-1 alpha (IL-1 alpha) and for interleukin-1 beta (IL-1 beta) was present in the chorial epiblast and in uterine epithelial cells, with varying degree and localization in different periods of pregnancy. IL-1 beta was also released into the medium at different amounts during incubation. In light of the mammalian data, our results suggest that the role of cytokines in pregnancy may represent a significant event in the evolution of placental viviparity.

Mouse model of Helicobacter pylori infection: studies of gastric function and ulcer healing.

BACKGROUND: Helicobacter pylori infection in humans is a major risk factor for peptic ulcer, but studies on the relation between H. pylori infection and gastric pathology are limited due to a deficiency of convenient animal models resembling this infection in humans. METHODS: We studied the effects of inoculation of conventional BALB/c mice with CagA and VacA positive (type I) H. pylori or CagA and VacA negative H. pylori (type II) strains on gastric secretion and healing of chronic acetic acid-induced ulcers in mouse stomachs. The ulcer area, gastric blood flow, plasma interleukin (IL)-1beta and IL-12, as well as plasma gastrin and gastric luminal somatostatin were determined. Gastric mucosal biopsy samples were also taken for assessment of the presence of viable H. pylori using a rapid urease test, H. pylori-culture and the RT-PCR analysis of the signal for H. pylori CagA. RESULTS: Gastric acid and pepsin secretion was reduced by over 50% immediately after H. pylori inoculation and accompanied by a significant increment in plasma gastrin and fall in gastric luminal somatostatin content observed over all test days, particularly in mice infected with type I H. pylori. The area of ulcers in vehicle-treated controls decreased significantly starting from day 2 after ulcer induction and then continued to decline for a further 14 days to heal almost completely after 28 days. In contrast, the ulcers were present until day 28 in all mice infected with type I or type II H. pylori strains, being significantly larger, especially with type I H. pylori infection. The gastric blood flow at the ulcer margin and ulcer crater in vehicle-treated mice gradually increased with decreasing ulcer size, after 14 and 28 days reaching a value which was not significantly different from that in vehicle-administered mice. In contrast, the gastric blood flow in type I H. pylori and, to a lesser extent, in type II H. pylori infected mice was significantly lower than in vehicle controls, both at the margin and at the crater of ulcers at all tested days. Histological changes such as oedema or congestion of surface epithelium were found after 7 days whereas mucosal inflammatory infiltration appeared after 14 days with a further increase after 28 days, especially in type I H. pylori and to a lesser extent in type II H. pylori infected mice. Plasma IL-1beta and IL-12 were significantly elevated at all tested days of ulcer healing and their increments were significantly higher in type I than in type II H. pylori infection. CONCLUSIONS: Conventional mice with gastric ulcers can be successfully infected by both toxigenic and nontoxigenic H. pylori strains, and this infection causes an immediate suppression of gastric secretion and markedly delays the healing of ulcers due to the fall in mucosal microcirculation in the ulcer region, cytokine release and an impairment in the gastrin-somatostatin link that appears to be independent of gastritis and more pronounced with infection of toxigenic than nontoxigenic strains.

Alpha-melanocyte-stimulating hormone reduces interleukin-1 beta effects on rat stomach preparations possibly through interference with a type I receptor.

Contractions elicited by CaCl2 on isolated rat stomach strip preparations have been reported to be potentiated by interleukin-1 beta (IL-1 beta). We have investigated whether this effect can be reduced by the putative IL-1 beta antagonist, alpha-melanocyte-stimulating hormone (alpha MSH). Additionally, the effects of alpha MSH on the specific binding of IL-1 beta to B- and T-cells have been investigated to further clarify its inhibitory activities. Both alpha MSH and its carboxyl terminal tripeptide concentration dependently reduced the potentiation of CaCl2-induced contractions caused by IL-1 beta but not those caused by leukotriene D4, the parent molecule being approximately 250 times more active. Additionally, both peptides potently and selectively reduced 125I-IL-1 beta binding to the T-cell sub-clone EL4-6.1 but not to the B-cell sub-clone 1H7. The results indicate that IL-1 beta effects on rat stomach may be mediated through a type-I (80 kDa) IL-1 beta receptor.

Relationship between the anti-phospholipase and anti-inflammatory effects of glucocorticoid-induced proteins.

Glucocorticoid-induced anti-phospholipase proteins were partially purified by using ion-exchange and molecular sieve chromatography. These proteins, as well as dexamethasone itself, inhibited the hind-paw rat oedema induced by carrageenin. This inhibition was reversed by arachidonic acid, Anti-phospholipase proteins as well as hydrocortisone, also reduced the formation of prostaglandin E2 and leukotriene B4 by phagocytosing leucocytes. A specific monoclonal antibody was able to reverse the inhibition of eicosanoid formation. The mechanism of the anti-inflammatory effect of glucocorticoids and anti-phospholipase proteins is discussed in the light of these results.

Defining the structural requirements of a biologically active domain of human IL-1 beta.

The immunostimulatory activity in vivo of the pleiotropic cytokine IL-1 beta can be retained by its nonapeptide VQGEESNDK, in position 163-171. A series of shorter and longer peptides around this position has been assayed for IL-1-like biological activity, in order to identify the structural requirements for full expression of adjuvant capacity. Elongated peptides, comprising the loop region 165-169 and up to six amino acids in the preceding beta strand or up to seven amino acids in the following beta strand, showed activity comparable or lower than that of the nonapeptide 163-171. This would indicate that the beta strand sequences are not required for optimizing the active conformation of the immunostimulatory IL-1 beta moiety. Accordingly, stabilization of the 163-171 peptide conformation by cyclization did not increase its biological activity. In contrast, the pentapeptide GEESN, corresponding the exposed loop 165-169 between two beta strands, had biological activity higher than that of the 163-171 nonapeptide and fully comparable to that of the entire IL-1 beta protein. Thus, the highly exposed fragment 165-169 within the IL-1 beta molecule may be the structure selectively responsible for the IL-1 beta immunostimulatory capacity in vivo.

Mechanism of acute toxicity of IL-1 beta in mice.

Human recombinant IL-1 beta was able to kill C3H/HeJ mice only when inoculated intravenously at very high doses. IL-1 beta, inoculated at 100 mg/kg i.v. as a bolus, induced a shock-like state characterized by anorexia, severe hypothermia and hypoglycemia and persistent neutrophilia, leading to death in 55% of animals generally between 24 and 48 h. In contrast, the noninflammatory adjuvant IL-1 beta peptide VQGEESNDK (position 163-171) did not induce any toxic effect in vivo, when administered following the same schedule. At variance with what was previously observed in endotoxin induced shock, IL-1 beta induced death was not preceded by appearance of circulating TNF. On the other hand, very high and persistent levels of circulating IL-6 could be detected after lethal IL-1 beta administration. Treatment of mice with ibuprofen or with chlorpromazine, both known to counteract some of the toxic effects of IL-1 in vivo, could protect from IL-1 beta induced mortality. Both drugs, at doses protecting from IL-1 beta induced death, were able to abolish IL-1 beta-induced rise of circulating phospholipase A2 (PLA2) activity, and the subsequent generation of toxic PLA2-derived metabolites.

Gastric secretion and ulcer healing in mouse stomach infected with cytotoxin expressing strain of Helicobacter pylori.

Helicobacter pylori (Hp) is a major risk factor of peptic ulcer but studies on the relation between Hp infection and gastric pathology are limited due to lack of convenient models resembling Hp infection in humans. We studied the effects of inoculation of conventional BALB/c mice with toxigenic type I Hp (cagA+ and vacA+) and non-toxigenic type II Hp (cagA- and vacA-) vs administration of vehicle on gastric secretion and healing of gastric ulcers. The gastric secretion studies were performed on mice with chronic gastric fistula before and after inoculation with toxigenic or non-toxigenic Hp strain or administration of vehicle (saline). Gastric ulcers were produced in mice inoculated with toxigenic and non-toxigenic Hp strain or vehicle and then sacrificed at day 0 and after 2, 4, 7, 14 and 28 days. Ulcer area and gastric blood flow (GBF), plasma gastrin and gastric luminal somatostatin were determined. Gastric mucosal biopsy specimens were also taken for the assessment of the presence of viable Hp using rapid urease test, the Hp-culture and the reverse transcriptase--polymerase chain reaction (RT-PCR) analysis of the signal for Hp CagA. Gastric acid output was reduced by over 50% immediately after Hp inoculation and this effect persisted during all time intervals tested, being significantly more pronounced in type I Hp-infected stomach. The area (7 mm2) of ulcers in control mice decreased gradually and then continued to decline during 14 days to disappear almost completely after 28 days. In contrast, the ulcers were present till day 28 in all mice infected with type I or type II Hp strain being significantly larger especially with type I Hp-infection. The GBF in control mice showed gradual rise with decreasing ulcer size being significantly higher at the ulcer margin than the ulcer crater and reached after 14 and 28 days the value not significantly different from that in vehicle-administered mice. In contrast, the GBF in type I Hp-infected mice but to a lesser extent, in type II Hp infected mice was significantly lower than in the vehicle controls, both at the ulcer margin and the crater of ulcers at all tested days. Hp-infection was accompanied by significant increment in plasma gastrin and the fall in gastric somatostatin contents observed at all test days, particularly in mice infected with type I Hp strain. Edema of surface epithelium appeared after 7 days and wak but significant mucosal inflammatory infiltration occurred after 14 days to further increase after 28 days, especially in type I Hp and less in type II Hp infected mice. We conclude that conventional mice with gastric ulcers can be successfully infected by both toxigenic and non-toxigenic Hp strains and this infection markedly reduces gastric acid secretion and delays healing of ulcers probably due to the fall in mucosal microcirculation in ulcer area, mucosal inflammation and impairment in gastric-somatostatin link.

Interleukin 1 and its synthetic peptides as adjuvants for poorly immunogenic vaccines.

The adjuvant activity of the peptide corresponding to the fragment 163-171 of human interleukin 1 beta (VQGEESNDK) has been evaluated on the immune response to both T-dependent and T-independent antigens. The hydrochloride derivative of the peptide showed an effect quantitatively comparable in molar terms to that of the entire protein. At variance with the entire IL 1 protein the peptide appeared devoid of inflammatory effects and therefore it may find clinical applications as adjuvant for poorly immunogenic vaccines or as immunorestorative agent. The activity of other fragments in proximity of the sequence 163-171 was also evaluated. The shorter fragment 165-171 appeared as active as the 163-171 peptide.

[Interleukin-1 beta in the gingival tissues of patients with periodontitis: immunohistochemical data]. / Interleuchina-1 beta nei tessuti gengivali di pazienti affetti da parodontite: dati immunoistochimici.

Interleukin-1 beta (Il-1 beta) is a cytokine which is considered to play a role in inducing the inflammatory reaction and the bone resorption that takes place in periodontal disease. With the aim of studying the presence and location of Il-1 beta positive cells in this disease, human gingival tissues from 12 patients with periodontitis and from 4 healthy control subjects were examined by immunohistochemical analysis. The cytokine was detected in all the patients having untreated periodontitis, although its content in gingival tissues revealed considerable variations among subjects. Il-1 beta was mainly localized within macrophage-like cells. Il-1 beta positive cells were not present in normal gingival tissue; however in 2 of the subjects considered normal there were few Il-1 beta positive cells in small areas of inflammation present in proximity of the dento-gingival junction.