A simple three-step method for design and affinity testing of new antisense peptides: an example of erythropoietin.

Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide-receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense-antisense (epitope-paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.

Hepatoprotective effects of Met-enkephalin on acetaminophen-induced liver lesions in male CBA mice.

Recent histopathological investigations in patients with hepatitis suggested possible involvement of Met-enkephalin and its receptors in the pathophysiology of hepatitis. Consequently, we evaluated the potential hepatoprotective effects of this endogenous opioid pentapeptide in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice. Met-enkephalin exhibited strong hepatoprotective effects in a dose of 7.5 mg/kg, which corresponds to the protective dose reported for several different animal disease models. In this group plasma alanine aminotransferase and aspartate aminotransferase enzyme activities, as well as liver necrosis score were significantly reduced in comparison to control animals treated with physiological saline (p>0.01). The specificity of the peptide hepatoprotection was investigated from the standpoint of the receptor and peptide blockade. It was concluded that Met-enkephalin effects on the liver were mediated via δ and ζ opioid receptors. Genotoxic testing of Met-enkephalin confirmed the safety of the peptide.

Expression of endothelial selectin ligands on human leukocytes following dive.

The fact that impaired endothelial-dependent vasodilatation after scuba diving often occurs without visible changes in the endothelial layer implies its biochemical origin. Since Lewisx(CD15) and sialyl-Lewisx(CD15s) are granulocyte and monocyte carbohydrate antigens recognized as ligands by endothelial selectins, we assumed that they could be sensitive markers for impaired vasodilatation following diving. Using flow cytometry, we determined the CD15 and CD15s peripheral blood mononuclear cells of eight divers, 30 mins before and 50 mins after a single dive to 54 m for 20 mins bottom time. The number of gas bubbles in the right heart was monitored by ultrasound. Gas bubbles were seen in all eight divers, with the average number of bubbles/cm2 1.9+/-1.9. The proportion of CD15+monocytes increased 2-fold after the dive as well as the subpopulation of monocytes highly expressing CD15s. The absolute number of monocytes was slightly, but not significantly, increased after the dive, whereas the absolute number of granulocytes was markedly elevated (up to 61%). There were no significant correlations between bubble formation and CD15+monocyte expression (r=-0.56; P=0.17), as well as with monocytes highly expressing CD15s (r=0.43; P=0.29). This study suggests that biochemical changes induced by scuba diving primarily activate existing monocytes rather than increase the number of monocytes at a time of acute arterial endothelial dysfunction.

Hla-Cw7 allele as predictor of favorable therapeutic response to interferon-alpha in patients with chronic hepatitis C.

AIM: To evaluate the association between human leukocyte antigens (HLA) class I and therapeutic response to interferon-alpha in Croatian patients with chronic hepatitis C. METHODS: HLA-A, -B, and -C genotyping was performed in 55 patients with sustained virological response and in 57 patients without sustained virological response to interferon-alpha therapy. Patients were treated in the period from 1998-2001 with interferon-alpha at a dose of 3 million units three times a week. Patients who became negative for hepatitis C virus RNA after 12 weeks of therapy completed 48 weeks of therapy. RESULTS: There was no association between therapeutic outcome and frequency of HLA-A, as well as of HLA-B alleles. HLA-Cw7 was significantly more frequent in patients with than those without sustained virological response (27.0% vs 6.7%; P=0.011). CONCLUSION: In Croatian patients with chronic hepatitis C, HLA-Cw7 is the predictor of sustained virological response to interferon-alpha therapy.

Disseminated BCG infection resembling langerhans cell histiocytosis in an infant with severe combined immunodeficiency: a case report.

We present a very rare congenital immunologic disease, severe combined immunodeficiency syndrome (SCID) in 6-months-old-boy with prolonged mucocutaneous candidiasis, severe anaemia, skin rash similar to the infiltrative eczema of Langerhans cell histiocytosis (LCH) and subcutaneous nodules with histiocytic infiltration. Laboratory findings show profound absence of humoral and cell-mediated immunity. Pathology specimens analysis of subcutaneous nodule revealed numerous S-100 protein and Cd1a negative histiocytes, occupied by BCG intracellular growth. Histopathology and immunohistochemistry confirmed the diagnosis of BCG dissemination. BCG vaccination in infants with SCID can lead to life threatening dissemination, resembling to the infiltrative eczema of LCH and may mislead the clinician.