Essam Refai

Increased diversity of intestinal antimicrobial peptides by covalent dimer formation.

Antimicrobial peptides are essential effector molecules of the innate immune system. Here we describe the structure, function and diversity of cryptdin-related sequence (CRS) peptides, a large family of antimicrobial molecules. We identified the peptides as covalent dimers in mouse intestinal tissue in amounts comparable to those of Paneth cell–derived enteric α-defensins. CRS peptides caused rapid and potent killing of commensal and pathogenic bacteria. The CRS peptides formed homo- and heterodimers in vivo, thereby expanding the repertoire of antimicrobial peptides and increasing the peptide diversity of Paneth cell secretions. CRS peptides might therefore be important in the maintenance of the microbial homeostasis within the intestinal tract.

In vivo dynamic distribution of 131I-glucagon-like peptide-1 (7-36) amide in the rat studied by gamma camera.

The in vivo distribution of glucagon-like peptide-1 (7-36) amide (GLP-1) was studied in a rat model using radiolabeled GLP-1 (131I-GLP-1) depicted by a gamma-camera. The dynamic scan showed a rapid clearance from the blood circulation after an intravenous (i.v.) injection of 131I-GLP-1. After 10 min, the major part of the radioactivity was accumulated in the kidneys, whereas about 9% (of the blood value) was found in the brain. The pharmacokinetic study using 125I-GLP-1 demonstrated a rapid elimination from plasma, with a half-life of 3.3 +/- 0.6 min, a clearance of 117 +/- 15 mL/min, and a distribution volume of 557 +/- 61 mL. The elimination half-lives for the intact 125I-GLP-1 in lungs and kidneys were determined to 3.7 and 3.9 min, respectively. The metabolite GLP-1 (9-36) amide was followed in blood, lung, and kidney. All other organs assumed to contain low molecular weight fragments of GLP-1. The present study suggest that GLP-1 and/or its labeled metabolites cross the blood-brain barrier. Also the kidney plays an essential role in GLP-1 elimination after an i.v. administration, which can be of clinical interest especially in patients with kidney insufficiency who are treated with GLP-1.

Lowering apolipoprotein CIII delays onset of type 1 diabetes.

Serum levels of apolipoprotein CIII (apoCIII) are increased in type 1 diabetic patients, and when β cells are exposed to these diabetic sera, apoptosis occurs, an effect abolished by an antibody against apoCIII. We have investigated the BB rat, an animal model that develops a human-like type 1 diabetes, and found that apoCIII was also increased in sera from prediabetic rats. This increase in apoCIII promoted β-cell death. The endogenous levels of apoCIII were reduced by treating prediabetic animals with an antisense against this apolipoprotein, resulting in a significantly delayed onset of diabetes. ApoCIII thus serves as a diabetogenic factor, and intervention with this apolipoprotein in the prediabetic state can arrest disease progression. These findings suggest apoCIII as a target for the treatment of type 1 diabetes.

Conserved Structure and Function in the Granulysin and NK-Lysin Peptide Family

ABSTRACT Granulysin and NK-lysin are homologous bactericidal proteins with a moderate residue identity (35%), both of which have antimycobacterial activity. Short loop peptides derived from the antimycobacterial domains of granulysin, NK-lysin, and a putative chicken NK-lysin were examined and shown to have comparable antimycobacterial but variable Escherichia coli activities. The known structure of the NK-lysin loop peptide was used to predict the structure of the equivalent peptides of granulysin and chicken NK-lysin by homology modeling. The last two adopted a secondary structure almost identical to that of NK-lysin. All three peptides form very similar three-dimensional (3-D) architectures in which the important basic residues assume the same positions in space. The basic residues in granulysin are arginine, while those in NK-lysin and chicken NK-lysin are a mixture of arginine and lysine. We altered the ratio of arginine to lysine in the granulysin fragment to examine the importance of basic residues for antimycobacterial activity. The alteration of the amino acids reduced the activity against E. coli to a larger extent than that against Mycobacterium smegmatis. In granulysin, the arginines in the loop structure are not crucial for antimycobacterial activity but are important for cytotoxicity. We suggest that the antibacterial domains of the related proteins granulysin, NK-lysin, and chicken NK-lysin have conserved their 3-D structure and their function against mycobacteria.

In vivo dynamical distribution of 131I-VIP in the rat studied by gamma-camera

The in vivo distribution of vasoactive intestinal peptide (VIP) was studied for the first time using a rat model in combination with labeled VIP (131I-VIP) and a gamma-camera. A dynamic scan showed that 131I-VIP was cleared rapidly from the blood circulation. The radioactivity was taken up and accumulated in the lungs during the first minute. During the next 15 min, the radioactivity was slowly removed from the lungs and redistributed into the kidneys, gastric mucosa, liver and small intestine. However, the radioactivity extracted by the lungs was about 6-fold lower during the first minute when a large amount of the non iodinated VIP was coinjected with the 131I-VIP. 131I-VIP was eliminated rapidly from the blood with a half-life of 0.44 +/- 0.05 (min +/- SD) while in lung the elimination half-life was determined to 2.3 +/- 0.8 (min +/- SD). Of the radioactivity in the lungs, 2% was found to be intact 131I-VIP after 20 min. In all other organs the radioactivity found was assumed to be low molecular weight fragments of 131I-VIP. We suggest that lungs play an important role to extract VIP from the circulation after an i.v. administration. 131I-VIP degradation products are redistributed mostly to the kidneys and to the gastric mucosa to be excreted through urine and stomach contents, respectively.

The somatostatin analogue octreotide inhibits neuroblastoma growth in vivo.

Neuroblastoma, a neural crest-derived childhood tumor of the sympathetic nervous system, may in some cases differentiate to a benign ganglioneuroma or regress due to apoptosis. However, the majority of neuroblastomas are diagnosed as metastatic tumors with a poor prognosis despite intensive multimodal therapy. The neuropeptide somatostatin (SOM) has been shown to inhibit neuroblastoma growth and induce apoptosis in vitro. Therapeutic effects of SOM analogues are dependent on tumor expression of high-affinity receptors. In the present study, human neuroblastoma SH-SY5Y cells were grown as xenografts in nude rats. In vivo SOM receptor expression in the xenografts was identified using scintigraphy with 111In-pentetreotide. Rats were randomized to treatment with the long-acting SOM analogue octreotide (10 µg s.c. every 12 h), 13-cis-retinoic acid (4 mg orally every 24 h), or vasoactive intestinal peptide (40 µg s.c. every 24 h) and compared with controls. Tumor volume was assessed every second day and tumor weight after 10-12 d. Octreotide treatment inhibited neuroblastoma growth significantly with reduced tumor volumes at 10 and 12 d compared with untreated controls (mean 3.56 and 4.24 versus 6.48 and 8.01 mL, respectively; p < 0.01). Also, tumor weights after 10-12 d were reduced in octreotide-treated animals (n = 8, median weight 2.90 g, range 1.67-5.57 g) compared with untreated rats (n = 14, 7.54 g, 1.65-10.82 g, p = 0.005). Serum IGF-I decreased significantly over time both in rats treated with octreotide and in untreated controls. It is concluded that treatment with the SOM analogue octreotide may significantly decrease neuroblastoma tumor growth in vivo. Further studies are warranted to establish the role of SOM analogues in the treatment of children with unfavorable neuroblastoma.

Apolipoprotein CIII links islet insulin resistance to β-cell failure in diabetes

Significance Insulin resistance and β-cell failure are the major defects in type 2 diabetes. We now demonstrate that local insulin resistance-induced increase in apolipoprotein CIII (apoCIII) within pancreatic islets causes promotion of an intraislet inflammatory milieu, increased mitochondrial metabolism, deranged regulation of β-cell cytoplasmic free Ca2+ concentration ([Ca2+]i), and apoptosis. Decreasing apoCIII in vivo in animals with insulin resistance improves glucose tolerance, and apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of apoCIII, demonstrate a normal [Ca2+]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of β-cell function and may thus constitute a novel target for the treatment of type 2 diabetes. Insulin resistance and β-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and β-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of β-cell cytoplasmic free Ca2+ concentration ([Ca2+]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca2+]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of β-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus.

Biodistribution of liposomal 131I-VIP in rat using gamma camera

Abstract Vasoactive intestinal peptide (VIP), a 28 amino-acid peptide was labeled with 131 I and encapsulated into liposomes. 131 I-VIP or liposomal 131 I -VIP was administered intravenously into the rats. The distribution was studied by a gamma camera and established by counting the radioactivity in the removed organs. The elimination half-life for the liposomal 131 I-VIP in both blood and lungs was significantly longer (5.29 and 9.28 min, respectively) than that obtained after the administration of 131 I-VIP (0.62 and 3.18 min, respectively). Dynamic scans using a gamma camera after the administration of liposomal 131 I-VIP showed a higher uptake of the liposomal form into the lungs compared with 131 I-VIP. The lack of VIP in asthmatics has been shown in previous studies. However, the clinical investigations using VIP were disappointing most probably due to the rapid degradation of the peptide in the bronchial tract. This in fact is supported by our previous study, in which we demonstrated that VIP had a half-life of 0.45 min in blood. We conclude that the encapsulation of VIP in liposomes prolongs its elimination half-life in plasma and enhances its uptake in lungs. This observation may increase the clinical use of VIP in both diagnostic and therapy.

A GLP-1 receptor agonist conjugated to an albumin-binding domain for extended half-life.

Glucagon‐like peptide 1 (GLP‐1) and related peptide agonists have been extensively investigated for glycaemic control in Type 2 diabetes, and may also have therapeutic applications for other diseases. Due to the short half‐life (t1/2 < 2 min) of the endogenous peptide, caused by proteolytic degradation and renal clearance, different strategies for half‐life extension and sustained release have been explored. In the present study, conjugates between a GLP‐1 analogue and a 5 kDa albumin‐binding domain (ABD) derived from streptococcal protein G have been chemically synthesized and evaluated. ABD binds with high affinity to human serum albumin, which is highly abundant in plasma and functions as a drug carrier in the circulation. Three different GLP‐1‐ABD conjugates, with the two peptides connected by linkers of two, four, and six PEG units, respectively, were synthesized and tested in mouse pancreatic islets at high (11 mM) and low (3 mM) glucose concentration. Insulin release upon stimulation was shown to be glucose‐dependent, showing no significant difference between the three different GLP‐1‐ABD conjugates and unconjugated GLP‐1 analogue. The biological activity, in combination with the high affinity binding to albumin, make the GLP‐1‐ABD conjugates promising GLP‐1 receptor agonists expected to show extended in vivo half‐life.

The cytotoxic effects of the anti-bacterial peptides on leukocytes.

Antimicrobial peptides are small molecular weight proteins with a large antibacterial spectrum. They can reach high local concentrations in tissues with active inflammation, being largely produced by immunocompetent cells. However, their effect on eukaryotic cells is still unclear. We have, therefore, studied three structurally different antimicrobial peptides (cecropin P1, PR‐39 and NK‐lysin) for their cytotoxic effects on blood mononuclear cells. None of the antimicrobial peptides tested exhibited significant cytotoxic effect on resting lymphocytes isolated either from peripheral blood or from the spleen with the exception of high concentrations (ten times higher than IC100 for Escherichia coli) of NK‐lysin. Activated lymphocytes were, however, more sensitive to the cytotoxic effect of the antimicrobial peptides. Both activated T‐cells and B‐cells were dose dependent sensitive to NK‐lysin while only activated B‐cells but not activated T‐cells were sensitive to PR‐39. Cecropin did not exhibit any cytotoxic effect on activated lymphocytes either. By using several cell lines (3B6, K562, U932 and EL‐4) we were able to show that NK‐lysin has a broad necrotic effect while PR‐39 has a cell specific apoptotic effect dependent on the specifically cellular uptake. In conclusion we show here that antimicrobial peptides are not cytotoxic for the resting eukaryotic cells but can be cytotoxic on activated immune cells through distinct mechanisms of cell death. Copyright