Branka Artuković

The influence of a novel pentadecapeptide, BPC 157, on NG-nitro-l-arginine methylester and l-arginine effects on stomach mucosa integrity and blood pressure

The known effects of a novel stomach pentadecapeptide BPC157 (10 microg or 10 ng/kg), namely its salutary activity against ethanol (96%, i.g.)-induced gastric lesions (simultaneously applied i.p.) and in blood pressure maintenance (given i.v.), were investigated in rats challenged with a combination of N(G)-nitro-L-arginine methylester (L-NAME) (5 mg/kg i.v.), a competitive inhibitor of endothelium nitric oxide (NO)-generation and NO precursor, L-arginine (200 mg/kg i.v.) (D-arginine was ineffective). In the gastric lesions assay, NO agents were given 5 min before ethanol injury and BPC 157 medication. Given alone, BPC157 had an antiulcer effect, as did L-arginine, but L-NAME had no effect. L-NAME completely abolished the effect of L-arginine, whereas it only attenuated the effect of BPC 157. After application of the combination of L-NAME + L-arginine, the BPC157 effect was additionally impaired. In blood pressure studies, compared with L-arginine, pentadecapeptide BPC 157 (without effect on basal normal values) had both a mimicking effect (impaired L-NAME-blood pressure increase, when applied prophylactically and decreased already raised L-NAME values, given at the time of the maximal L-NAME-blood pressure increase (i.e., 10 min after L-NAME)) and preventive activity (L-arginine-induced moderate blood pressure decrease was prevented by BPC 157 pretreatment). When BPC 157 was given 10 min after L-NAME + L-arginine combination, which still led to a blood pressure increase, its previously clear effect (noted in L-NAME treated rats) disappeared. In vitro, in gastric mucosa from rat stomach tissue homogenates, BPC 157, given in the same dose (100 microM) as L-arginine, induced a comparable generation of NO. But, BPC 157 effect could not be inhibited by L-NAME, even when L-NAME was given in a tenfold (100 versus 1000 microM) higher dose than that needed for inhibition of the L-arginine effect. NO synthesis was blunted when the pentadecapeptide BPC 157 and L-arginine were combined. In summary, BPC 157 could interfere with the effects of NO on both gastric mucosal integrity and blood pressure maintenance in a specific way, especially with L-arginine, having a more prominent and/or particularly different effect from that of NO.

THE INFLUENCE OF BPC 157 ON NITRIC OXIDE AGONIST AND ANTAGONIST INDUCED LESIONS IN BROILER CHICKS

We describe the effects of nitric oxide (NO) agonists and antagonists and the influence of a novel organoprotective pentadecapeptide BPC 157, on the development of pulmonary hypertension syndrome and tissue lesions in chicks. Acute toxicity, which includes single dose application of saline (1 mL intraperitoneally (i.p.)), BPC 157 (10 micrograms/kg bw), L-NAME (NO antagonist, doses 50, 100, 150 mg/kg bw) and L-arginine (NO agonist/100 mg/kg bw with their combination L-NAME + BPC 157; L-NAME + L-arginine) was investigated. In this experiment pathohistological examination of the spleen, heart, liver and lungs and hematological analysis was conducted. In the chronic toxicity experiment, the animals were treated daily for 5 weeks with L-NAME (10 mg/kg bw), L-arginine (100 mg/kg bw), BPC 157 (10 micrograms/kg bw) and their combinations (L-NAME + BPC 157; L-NAME + BPC 157; L-NAME + L-arginine) i.p. Seven animals from each group, including controls (saline 1 mL i.p.) were killed every week. Application of L-NAME caused pulmonary hypertension syndrome (PHS) in the treated chicks, which was prevented by the simultaneous application of L-arginine and BPC 157. Pathohistological examination of both acute and chronic toxicity revealed that L-NAME caused severe tissue damage (myocardial and hepatic cell necrosis, necrosis of the lymphoid cells in the spleen) while L-arginine provoked predominantly congestion, edema and hemorrhages in all organs. The effect of L-NAME was successfully inhibited by the application of L-arginine and BPC 157 but the latter substance did not cause any tissue or organ damage. Hematological analysis shows significant hemoglobin and leukocyte number decrease in the L-NAME-treated groups of chicks.

Beneficial effect of a novel pentadecapeptide BPC 157 on gastric lesions induced by restraint stress, ethanol, indomethacin, and capsaicin neurotoxicity

Very recently, the integrity of capsaicin somatosensory neurons and their protection were suggested to be related to the activity in nociception of a newly discovered 15-amino acid peptide, BPC 157, shown to have strong beneficial effect on intestinal and liver lesions. Therefore, from this viewpoint, we have studied the gastroprotective effect of the pentadecapeptide BPC 157, on gastric lesions produced in rats by 96% ethanol, restraint stress, and indomethacin. The possible involvement of sensory neurons in the salutary actions of BPC 157 (10µg/kg, 10 ng/kg intraperitoneally) was studied with capsaicin, which has differential effects on sensory neurons: a high dose in adult (125 mg/kg subcutaneously, 3 months old) or administration (50 mg/kg subcutaneously) to neonatal animals (age of the 7 days) destroys sensory fibers, whereas a low dose (500µg/kg intraperitoneally) activates neurotransmitter release and protective effects on the mucosa. In the absence of capsaicin, BPC 157 protected gastric mucosa against ethanol, restraint, and indomethacin application. In the presence of neurotoxic doses of capsaicin, the negative influence of capsaicin on restraint, ethanol, or indomethacin lesions consistently affected salutary activity of BPC 157. However, BPC 157 protection was still evident in the capsaicin-treated rats (either treated as adults or as newborns) in all of these assays. Interestingly, after neonatal capsaicin treatment, a complete abolition of BPC gastroprotection was noted if BPC 157 was applied as a single nanogram-regimen, but the mucosal protection was fully reversed when the same dose was used daily. In line with the excitatory dose of capsaicin the beneficial effectiveness of BPC 157 appears to be increased as well. Taken together, these data provide evidence for complex synergistic interaction between the beneficial effectiveness of BPC 157 and peptidergic sensory afferent neuron activity.

Pentadecapeptide BPC 157 positively affects both non-steroidal anti-inflammatory agent-induced gastrointestinal lesions and adjuvant arthritis in rats

Besides a superior protection of the pentadecapeptide BPC 157 (an essential fragment of an organoprotective gastric juice peptide BPC) against different gastrointestinal and liver lesions, an acute anti-inflammatory and analgetic activity was also noted. Consequently, its effect on chronic inflammation lesions, such as adjuvant arthritis, and non-steroidal anti-inflammatory agents (NSAIAs)-induced gastrointestinal lesions was simultaneously studied in rats. In gastrointestinal lesions (indomethacin (30 mg/kg s.c.), aspirin (400 mg/kg i.g.) and diclofenac (125 mg/kg i.p.) studies, BPC 157 (10 micrograms or 10 ng/kg i.p.) was regularly given simultaneously and/or 1 h prior to drug application (indomethacin). In the adjuvant arthritis (tail-application of 0.2 mL of Freunds adjuvant) studies (14 days, 30 days, 1 year) BPC 157 (10 micrograms or 10 ng/kg i.p.), it was given as a single application (at 1 h either before or following the application of Freunds adjuvant) or in a once daily regimen (0-14th day, 14-30th day, 14th day-1 year). Given with the investigated NSAIAs, BPC 157 consistently reduced the otherwise prominent lesions in the stomach of the control rats, as well as the lesions in the small intestine in the indomethacin groups. In the adjuvant arthritis studies, the lesions development seems to be considerably reduced after single pentadecapeptide medication, and even more attenuated in rats daily treated with BPC 157. As a therapy of already established adjuvant arthritis, its salutary effect consistently appeared already after 2 weeks of medication and it could be clearly seen also after 1 year of application. Taking together all these results, the data likely point to a special anti-inflammatory and mucosal integrity protective effect.

The effect of pentadecapeptide BPC 157, H2-blockers, omeprazole and sucralfate on new vessels and new granulation tissue formation

A clear protection of the gastrointestinal tract and an evident anti-inflammatory effect were shown for a novel stomach pentadecapeptide BPC 157 (i.p./i.g.) in comparison with several reference standards in various ulcer models along with a protection of endothelium and particular interaction with the NO-system. Thus, we evaluated whether this pentadecapeptide along with other gastroprotective agents could affect angiogenesis and the healing process in vivo using a procedure initially described by Szabo and co-workers. In each rat, two sterile sponges (1 x 1 x 0.25 cm; V = 0.25 mL) with the same quantities of BPC 157 (10 ng x mL(-1), 10 microg x mL(-1), 50 microg x kg(-1)) or reference agents (cimetidine: 10, 100, 500 mg x mL(-1); ranitidine: 2.5, 25, 250 mg x mL(-1); famotidine: 10, 50, 100 mg x mL(-1); omeprazole: 10, 50, 100 mg x mL(-1); sucralfate: 1, 5, 10 mg x mL(-1) were implanted subcutaneously in the lumbar region. The sponges were removed after 3 or 7 d, fixed in formalin, and processed for histologic and histochemical evaluation and morphometry assessment. Compared with the control values, the number of newly formed endothelial spaces inside newly formed granulation tissue was markedly increased in all animals treated with BPC 157, cimetidine, ranitidine, famotidine, sucralfate and omeprazole, a consistent finding noted after either 3 or 7 d. Compared with control values, markedly more granulation tissue was noted in the rats in the groups of animals treated with BPC 157 (50 microg) and in the rats treated with sucralfate in all dosages used, euthanized after 3 d. In all groups treated with H2-blockers however, similar values to those of controls were noted. Thus, it could be concluded that an evident angiogenic property was consistently noted for the novel pentadecapeptide BPC 157, H2-blockers (cimetidine, famotidine and ranitidine) and omeprazole, besides the well known angiogenic effect of sucralfate. Furthermore, unlike H2-blockers and omeprazole, BPC 157 stimulates the formation of granulation tissue, suggesting a particular activity, similar to that previously noted for sucralfate.

Salutary and prophylactic effect of pentadecapeptide BPC 157 on acute pancreatitis and concomitant gastroduodenal lesions in rats

The superior effectiveness of a new pentadecapeptide, BPC 157, on gastrointestinal and liver lesions, in conjunction with an antiinflammatory and analgetic activity was recently noted. In the present study, BPC 157 was tested as either a protective or healing agent in bile duct ligation-induced acute pancreatitis in rats. In addition, the positive influence of BPC 157 on concomitantly developed gastric and duodenal lesions was simultaneously investigated. BPC 157 (10 µg, 10 ng/kg body wt, intraperitoneally or intragastrically) was given prophylactically 1 hr before ligation, whereas the therapy was given once daily beginning with the 24 hr following ligation (last application 24 hr before killing). The effect was investigated at daily intervals until the end of the fifth day after ligation. In the pretreatment regimen, a strong pancreas protection was obtained. When applied in the condition of already established severe acute pancreatitis, an obvious salutary effect was consistently noted. Assessing the appearance of the necrosis, edema, neutrophils, and mononuclears, consistently less necrosis, edema, and neutrophils, but more mononuclears, were found in BPC-treated rats. Likewise, in studies of the serum amylase values, relative to control data, a markedly lower rise (BPC pretreatment regimen) as well as a worsening of the already raised values (BPC therapy regimen) was noted. Along with its beneficial effect on pancreatitis, a positive influence of BPC 157 on the gastric and duodenal lesion course in bile duct-ligated rats was noted in both the pre- and posttreatment regimen. Taken together, in further studies of acute pancreatitis therapy, BPC could be an interesting and useful agent with an additional positive impact on concomitant gastroduodenal pathology.

Pentadecapeptide BPC 157 Interactions with Adrenergic and Dopaminergic Systems in Mucosal Protection in Stress

Since superior protection against differentgastrointestinal and liver lesions and antiinflammatoryand analgesic activities were noted for pentadecapeptideBPC (an essential fragment of an organoprotective gastric juice protein named BPC), thebeneficial mechanism of BPC 157 and its likelyinteractions with other systems were studied. Hence itsbeneficial effects would be abolished by adrenal glandmedullectomy, the influence of different agents affectingα, β, and dopamine receptors on BPC 157gastroprotection in 48 h restraint stress was furtherinvestigated. Animals were pretreated (1 hr beforestress) with saline (controls) or BPC 157 (dissolved insaline) (10 μg or 10 ng/kg body wt intraperitoneallyor intragastrically) applied either alone to establishbasal conditions or, when manipulating the adrenergic or dopaminergic system, a simultaneousadministration was carried out with various agents withspecific effects on adrenergic or dopaminergic receptors[given in milligrams per kilogram intraperitoneally except for atenolol, which was givensubcutaneously] phentolamine (10.0), prazosin (0.5),yohimbine (5.0), clonidine (0.1) (α-adrenergicdomain), propranolol (1.0), atenolol (20.0)(β-adrenergic domain), domperidone (5.0), and haloperidol(5.0) (peripheral/central dopamine system).Alternatively, agents stimulating adrenergic ordopaminergic systemsadrenaline (5.0) or bromocriptine(10.0)-were applied. A strong protection, noted followingintragastric or intraperitoneal administration of BPC157, was fully abolished by coadministration ofphentolamine, clonidine, and haloperidol, andconsistently not affected by prazosin, yohimbine, ordomperidone. Atenolol abolished only intraperitoneal BPC157 protection, whereas propranolol affectedspecifically intragastric BPC 157 protection.Interestingly, the severe course of lesion developmentobtained in basal conditions, unlike BPC 157gastroprotection, was not influenced by the applicationof these agents. In other experiments, when adrenalineand bromocriptine were given simultaneously, a strong reductionof lesion development was noted. However, when appliedseparately, only adrenaline, not bromocriptine, has aprotective effect. Thus, a complex protectiveinteraction with both α-adrenergic (e.g.,catecholamine release) and dopaminergic (central)systems could be suggested for both intragastric andintraperitoneal BPC 157 administration. The involvementof β-receptor stimulation in BPC 157 gastroprotection appearsto be related to the route of BPC 157 administration.The demonstration that a combined stimulation ofadrenergic and dopaminergic systems by simultaneous prophylactic application of adrenaline(α- and β-receptor stimulant) andbromocriptine (dopamine receptor agonist) maysignificantly reduce restraint stress lesionsdevelopment provides insight for further research on the beneficial mechanism ofBPC 157.

A Case of Visceral Leishmaniosis in a Gray Wolf (Canis lupus) from Croatia

The southern habitats of Croatias gray wolf (Canis lupus) population are found in central and southern parts of Dalmatia. This region is recognized as an endemic region for canine visceral leishmaniosis, caused by Leishmania infantum. In November 2003, a 4-yr-old male gray wolf was found dead in the northwestern border of this endemic region. Pathologic and parasitologic analysis, confirmed by polymerase chain reaction, indicated that lesions associated with infection by Leishmania infantum are, in this case, typical for visceral leshmaniosis commonly described in dogs. Review of the literature suggests that this is the first reported case of gray wolf death due to lesions caused by L. infantum.

A Model of Inflammatory Bowel Disease Induced by 2,4-Dinitrofluorobenzene in Previously Sensitized BALB-c Mice

The aim of this study was to develop a model of inflammatory bowel disease (IBD) induced by colonic application of 2,4-dinitrofluorobenzene in previously sensitized BALB-c mice. During the follow-up period of 30 days we observed ulcerations, haemorrhage, necrosis, and mononuclear infiltration in the colonic mucosa of previously sensitized (experimental) and, to a lesser extent, nonsensitized (control) animals. In addition, the animals in the experimental group developed adhesions, thickening of colonic segments, stenosis, and dilatation of the colon, and some animals also developed megacolon. Oedema, mononuclear infiltration, and superficial ulcerations were observed in the ileum of experimental animals and, to a lesser extent, in the control group. In addition, the animals in the experimental group developed extraintestinal changes in the liver and spleen (that is, pericholangitis and lymphofollicular proliferation). We suggest that this model of IBD may have some value for the study of early pathogenetic mechanisms of IBD and for developing new therapeutic modalities for this condition.

Disseminated tuberculosis in hyrax (Procavia capensis) caused by Mycobacterium africanum.

Abstract Tuberculosis due to Mycobacterium africanum was diagnosed in an adult female hyrax (Procavia capensis). Pathologic examination revealed disseminated tuberculous lesions. The same pathologic changes were also found in a male hyrax that died a year later. Both animals were imported from the United Arab Emirates and were held in captivity at the Zagreb Zoo in Croatia. The source of infection remains unknown. The acid-fast bacteria isolated from the lungs of the female hyrax were identifyed by polymerase chain reaction as Mycobacterium tuberculosis complex and Geno Type® MTBC test confirmed the strain to be M. africanum I.