Lovorka Batelja

Advanced glycation endproducts in peripheral nerve in type 2 diabetes with neuropathy

Advanced glycation endproducts (AGE) accumulate over proteins as a consequence of diabetic hyperglycemia, and thus contribute to the pathogenesis of diabetic complications. To improve the understanding of the pathology of diabetic neuropathy, AGE accumulation was analyzed in sural and/or femoral nerves obtained under spinal anesthesia from 8 type 2 diabetic patients with both distal symmetrical polyneuropathy and proximal neuropathy. Pronounced AGE immunoreactivity was detected on axons and myelin sheaths in 90% of diabetic peripheral nerves but not in the control specimen. The intensity of axonal AGE immunopositivity significantly correlated with the severity of morphological alterations (p<0.005). AGE localization, demonstrated by immunohistochemical methods, was also present in the endoneurium, perineurium and microvessels. Morphometric analysis of the diabetic peripheral nerve showed perineurial thickening (diabetic vs. control, 15.5±4.9 vs. 6.6±2.1 µm, p<0.001), narrowing of the microvessel lumina (66.6±50.5 vs. 579.5±38.4 x103 µm2, p<0.001) and significant reduction in the number of preserved axons (3.6±3 vs. 8.9±2.3 per 105 µm2 per area, p<0.037). The sera of diabetic patients contained epitope(s) of AGE structure and soluble immune complexes containing AGE moiety. In conclusion, to the best of our knowledge, this is the first study providing evidence for excessive AGE formation on peripheral nerve components, primarily axons, and a significantly higher level of circulating AGE-immune complexes in patients with both distal diabetic polyneuropathy and proximal neuropathy. Humoral immune mechanisms, including the production of anti-AGE autoantibody, may potentially be involved in the development of structural abnormalities described in this report.

Corticosteroid-impairment of healing and gastric pentadecapeptide BPC-157 creams in burned mice

The amelioration of corticosteroid-impairment of healing by a stable gastric pentadecapeptide BPC-157 (GEPPPGKPADDAGLV, M(w) 1419, currently in early clinical trials for inflammatory bowel disease) was studied in thermally injured mice. Its effects on corticosteroid impaired healing of deep partial skin thickness burns, and burn-gastric lesions were investigated. Male NMRI-Hannover mice (sacrificed at 1-3,7,14 and 21 days following burning 20% of total burn area at the back (open flame for 7s) received intraperitoneally (per kg bw) 6alpha-methylprednisolone (Depo-medrol, 1.0 or 10.0mg), or an equal volume of saline (5.0 ml), once daily, first application 30 min after injury, last 24h before sacrifice. The injury was subsequently treated by topical application of a thin layer of pentadecapeptide BPC-157 cream at three different levels a neutral cream of no treatment. Pentadecapeptide BPC-157 consistently improved given burn healing (both microscopical and tensionmetry assessment), and counteracted corticosteroid-impairment of burn healing. In burn-gastric lesions investigation of the effects of BPC showed an anti-ulcer effect of its own in burned non-corticosteroid-treated mice and potentiated the anti-ulcer effect observed in 6alpha-methylprednisolone-treated mice. Pentadecapeptide BPC-157 inhibited corticosteroid immunosuppression. In vitro, in spleenic cells assessment, animals (sacrificed at day 21) treated with 6alpha-methylprednisolone 1mg showed decreased reactivity to nitrogen in comparison with control, healthy animals, while the addition of BPC-157 (1 microg/g cream) returned cell reactivity to values noted in control healthy animals.

Gastric pentadecapeptide BPC 157 accelerates healing of transected rat Achilles tendon and in vitro stimulates tendocytes growth

In studies intended to improve healing of transected Achilles tendon, effective was a stable gastric pentadecapeptide BPC 157 (GEPPPGKPADDAGLV, M.W. 1419). Currently in clinical trials for inflammatory bowel disease (PLD‐116, PL 14736, Pliva), it ameliorates internal and external wound healing. In rats, the right Achilles tendon transected (5 mm proximal to its calcaneal insertion) presents with a large tendon defect between cut ends. Agents (/kg b.w., i.p., once time daily) (BPC 157 (dissolved in saline, with no carrier addition) (10 μg, 10 ng or 10 pg) or saline (5.0 ml)), were firstly applied at 30 min after surgery, the last application at 24 h before autopsy. Achilles functional index (AFI) was assessed once time daily. Biomechanical, microscopical and macroscopical assessment was on day 1, 4, 7, 10 and 14. Controls generally have severely compromised healing. In comparison, pentadecapeptide BPC 157 fully improves recovery: (i) biomechanically, increased load of failure, load of failure per area and Youngs modulus of elasticity; (ii) functionally, significantly higher AFI‐values; (iii) microscopically, more mononuclears and less granulocytes, superior formation of fibroblasts, reticulin and collagen; (iv) macroscopically, smaller size and depth of tendon defect, and subsequently the reestablishment of full tendon integrity. Likewise, unlike TGF‐β, pentadecapeptide BPC 157, presenting with no effect on the growth of cultured cell of its own, consistently opposed 4‐hydroxynonenal (HNE), a negative modulator of the growth. HNE‐effect is opposed in both combinations: BPC 157 + HNE (HNE growth inhibiting effect reversed into growth stimulation of cultured tendocytes) and HNE + BPC 157(abolished inhibiting activity of the aldehyde), both in the presence of serum and serum deprived conditions. In conclusion, these findings, particularly. Achilles tendon transection fully recovered in rats, peptide stability suitable delivery, usefully favor gastric pentadecapeptide BPC 157 in future Achilles tendon therapy.

Esophagogastric anastomosis in rats: Improved healing by BPC 157 and L-arginine, aggravated by L-NAME

AIM To cure typically life-threatening esophagogastric anastomosis in rats, lacking anastomosis healing and sphincter function rescue, in particular. METHODS Because we assume esophagogastric fistulas represent a particular NO-system disability, we attempt to identify the benefits of anti-ulcer stable gastric pentadecapeptide BPC 157, which was in trials for ulcerative colitis and currently for multiple sclerosis, in rats with esophagocutaneous fistulas. Previously, BPC 157 therapies have promoted the healing of intestinal anastomosis and fistulas, and esophagitis and gastric lesions, along with rescued sphincter function. Additionally, BPC 157 particularly interacts with the NO-system. In the 4 d after esophagogastric anastomosis creation, rats received medication (/kg intraperitoneally once daily: BPC 157 (10 μg, 10 ng), L-NAME (5 mg), or L-arginine (100 mg) alone and/or combined or BPC 157 (10 μg, 10 ng) in drinking water). For rats underwent esophagogastric anastomosis, daily assessment included progressive stomach damage (sum of the longest diameters, mm), esophagitis (scored 0-5), weak anastomosis (mL H2O before leak), low pressure in esophagus at anastomosis and in the pyloric sphincter (cm H2O), progressive weight loss (g) and mortality. Immediate effect assessed blood vessels disappearance (scored 0-5) at the stomach surface immediately after anastomosis creation. RESULTS BPC 157 (all regimens) fully counteracted the perilous disease course from the very beginning (i.e., with the BPC 157 bath, blood vessels remained present at the gastric surface after anastomosis creation) and eliminated mortality. Additionally, BPC 157 treatment in combination with L-NAME nullified any effect of L-NAME that otherwise intensified the regular course. Consistently, with worsening (with L-NAME administration) and amelioration (with L-arginine), either L-arginine amelioration prevails (attenuated esophageal and gastric lesions) or they counteract each other (L-NAME + L-arginine); with the addition of BPC 157 (L-NAME + L-arginine + BPC 157), there was a marked beneficial effect. BPC 157 treatment for esophagogastric anastomosis, along with NOS-blocker L-NAME and/or NOS substrate L-arginine, demonstrated an innate NO-system disability (as observed with L-arginine effectiveness). BPC 157 distinctively affected corresponding events: worsening (obtained with L-NAME administration that was counteracted); or amelioration (L-arginine + BPC 157-rats correspond to BPC 157-rats). CONCLUSION Innate NO-system disability for esophagogastric anastomoses, including L-NAME-worsening, suggests that these effects could be corrected by L-arginine and almost completely eliminated by BPC 157 therapy.

Server-based teleconsultation in lung pathology

SvenSewi er th G , ai nK ayse Ar , dDl i zubu Lr , ovork aB ateaj l L , uk aBrcci a , nd Kal usK ayser From: 22nd European Congress of Pathology European Society of Pathology (ESP) Working Group: Information Technology (IT) in Pathology Precongress Meeting or Fl enceIta, 4 y . l September 2009

W1244 The Role of Adenine and Guanine in Beneficial Effects of the Gastric Pentadecapeptide BPC 157 (PL 14736) On Ileoileal Anastomosis Healing in Rats Impaired with Systemic Corticosteroid Application. Raman Spectroscopy Study

Safe in inflammatory bowel disease (PL14736 Pliva, Croatia), no toxicity reported (Gastroenterology, 2005), gastric pentadecapeptide BPC 157 (BPC 157) heals the intestinal anastomosis (Surg Today, 2007). Raman spectroscopy was used to show the beneficial effect of BPC 157 on anastomosis healing in rats impaired with systemic corticosteroid application. Assessed were Raman spectroscopy, ileoileal anastomosis dehiscence, histology, biomechanical presentation (the volume (ml) (infunded through syringe-perfusion pump system (1ml /10 sec)) and the leakage pressure (mmHg) (catheter connected with chamber and monitor, at 10 cm proximal to anastomosis), on days 1, 2, 3, 4, 5 and 6. BPC 157 10µg, 6-alpha-methylprednisolone (MP) 1 mg, given alone or together /kg i.p. (or an equivolume of saline 5ml/kg). First application was after surgery and last was 24 h before sacrifice. Raman spectroscopy. Measurements used Perkin Elmer Spectrum GX FT Raman spectrometer with 4 cm-1 resolution. Excitation was NdYaG laser at 1064 nm wavelength and used laser power was 500 mW. Spectra were recorded in the area between 300 and 2300 cm-1, with 50 scans for better S/N ratio. Prior to mounting on a universal holder, the specimen was cut to a 0.3 x 0.3 cm piece. Presenting almost constant intensity for all investigated samples, all spectra were baseline corrected and normalized to band at 1450 cm-1. Raman spectroscopy. To point out an advanced healing rate the spectral areas from 510 to 560 cm-1, 800 to 1150 cm-1 and band at 1490 cm-1 were individually observed. We focused the band at 1490 cm-1, assigned to vibrations of adenine and guanine, to be indicative for increased tissue regeneration, since it fully correlates with the evidenced healing process. BPC 157 rats showed an increase throughout the 1-6 days following surgery ; MP-rats did not show before day 6. This healing delay is fully counteracted when rats received BPC 157 simultaneously with MP. Macroscopic and histology data support these findings: adhesion formation attenuated, blood vessels filled, mild passage obstruction only temporary ; anastomosis without leakage sustains markedly higher both volume and pressure, with consistent increase to healthy values ; decreased edema, granulocyte number and necrosis, along with granulation tissue, reticulin and collagen formation substantially increased, and finally epithelization increased (BPC 157) ; (MP)causing aggravation. These data signify the role of adenine and guanine in the anastomosis healing process, BPC 157 has a beneficial effect, MP negative effect, and counteracted corticosteroid impairment by BPC 157 application.