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Elias Toubi,1 Zahava Vadasz2 1The Outpatient Allergy Clinic, The Holy Family Hospital, Nazareth, Israel; 2The Proteomic Unit, The Division of Clinical Immunology, Bnai-Zion Medical Center, Faculty of Medicine, Technion, Haifa, IsraelCorrespondence: Zahava Vadasz Email zahava.vadas@b-zion.org.ilAbstract: Chronic spontaneous urticaria (CSU) is considered to be an autoimmune disorder (type I and type II) in 50% of all cases. However, autoreactive T cells and their proximity with activated mast cells in the skin of CSU patients are believed to be the primary event in mast cell degranulation. The finding of anti-FcɛRIα on mast cells or IgE autoantibodies against thyroid antigens should be considered to be a consequence of the auto-reactive T cells’ recognition of the above-mentioned antigens. Our recent finding of increased Th17 and IL-17 expression in both CD4+ T cells and mast cells in the skin of severe CSU patients is supportive for the major role that T cells perform in the pathogenesis of CSU. Supporting this are numerous previous reports in which increased serum IL-17 was found to be in association with CSU disease severity. The beneficial effect of anti-IL-17A (secukinumab) in CSU patients in whom high dose anti-histamines, recurrent course of steroids and omalizumab fail to achieve a reasonable response should be investigated as a new therapeutic strategy in future studies with a large cohort of patients.Keywords: IL-17, interleukin-17, chronic spontaneous urticaria, CSU, T cells, mast cells

Background: Patients with chronic spontaneous urticaria (CSU), an autoimmune disorder, show increased skin expression of IL-17A and can benefit from treatment with the anti-IL-17A biologic secukinumab. The mechanisms that drive IL-17A expression in CSU are currently unknown, but may involve Semaphorin5A (Sema5A). Objective: To explore the expression, role, and effects of Sema5A in CSU and its link to IL-17A. Material and Methods: We investigated patients with CSU and healthy controls for skin expression of expressing peripheral T cells. Results: Sema5A was highly expressed in the skin of CSU patients as compared to healthy control skin. Both CD4+ T cells and mast cells in CSU skin expressed Sema5A, and many of them expressed both Sema5A and IL-17A. Patients with CSU had significantly higher rates of IL-17A-expressing CD4+ T cells as compared to healthy controls. Incubation with Sema5A increased the rates of IL-17A-expressing CD4+ T cells in healthy controls to CSU levels. Conclusion: Sema5A may drive the expression and effects of IL-17A in CSU. Further studies in larger cohorts are needed to confirm the role of Sema5A in the pathogenesis of CSU and to explore its potential as a therapeutic target.

HESP 2.b risk assessment program was studied in detail concerning the effect of changing different input parameters for the output ADI values calculated by the program. We used the standard Netherlands 1.0 scenario offered by the program. With this we fixed a lot of input parameters which define the area, human and animal recipient parameters etc. The remaining 31 unfixed parameters were fixed at first to "BASE" input values and the BASE output values were calculated by HESP. Later we chose only one parameter at a time and changed it to an another value. The calculated ADI values were then compared to BASE output values. Seven parameters (soil type, soil usage, site length, soil pH, groundwater fraction in drinking water, basement floor type and Qev) were studied. We found, that changing soil pH or Qev have not any influence on the output ADI values in case of any contaminant. Soil type change has not any effect on the output ADI value in case of Pb or Cd but it seems to play important role in all cases of the four organic material we investigated. Changing soil usage have influence on the output ADI value almost in every case. It seems to be linear relation between the maximal concentration of contaminant and calculated ADI. Changing the site length and basement floor type gave in some cases different ADI values compared to BASE values. If we alter the groundwater fraction in drinking water we got usually different ADI values. With Risc Human risk assessment program we got similar results: nor the changes in soil type, site diameter or soil pH gave any changes in output ADI values. Our results hint that using HESP and Risc Human requires enhanced caution.

The new heptapeptide somatostatin analog TT-232 decreases proliferation of HT-29 human colon carcinoma cells in vitro by reducing mitotic and increasing apoptotic activity. We have synthesized and characterized a specifically tritium labeled 3H-Tyr3-TT-232 (30 Ci/mmol) to investigate the effect and the fate of this antitumor peptide on human colon tumor cells. 3H-labeled TT-232 could be detected on the cell surface, on cytoplasmic membranes and also in the nucleus of HT-29 cells, 1-6 h after the administration of 0.5 and 50 microg/mL [3H]TT-232. Binding and internalization of TT-232 to human colon tumor cells at a relatively high dose provide further evidence for the existence of low-affinity somatostatin receptors in such cells, which might mediate the apoptosis-inducing effect. Our data suggest the possible use of TT-232 in the treatment of human colon tumors.

Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.

Capillary electrophoresis (CE) and micellar electrokinetic chromatography (MEKC) methods, utilizing uncoated silica capillary and triethyl ammonium phosphate or sodium borate buffers in the pH range of 2.25-11.0, containing sodium dodecyl sulfate (SDS) (0-100 mM) for analysis of somatostatin-analog peptides were developed. The method presented here was compared with the reversed-phase high performance liquid chromatographic (RP-HPLC) and CE methods developed for analysis of peptides. The peptides investigated in this work can be separated by CE on the basis of their electrophoretic mobility in aqueous buffer of low pH value (pH 2.25) or by MEKC on the basis of their hydrophobicity in SDS containing buffer of high pH value (pH 11.0). Optimal MEKC separation of the investigated peptides has been achieved at pH 11.0 in an Na-borate buffer containing 100 mM SDS. CE at pH 2.25 proved insensitive to the hydrophobicity of the peptides investigated. By contrast, results obtained with MEKC at pH 11.0 proved to be anologous to those obtained by RP-HPLC, with highly hydrophobic peptides-migrating slower than peptides without hydrophobic moieties.

New chicken I GnRH agonists and antagonists have been synthesized and tested for their biological activities. The common feature of these analogs was that the molecules had a beta-L-aspartyl residue inserted in position 6. The agonist bound to the pituitary still had low endocrinological activity. On the other hand, it exhibited direct antitumor effect in in vitro assays. The endocrinological activity of the antagonist was low; however, it showed potent, direct antitumor activity. These observations might lead to the development of new GnRH analogs with selective antitumor effect.

HPLC and CE methods were developed for analysis of somatostatin analogue (S-analogue) peptides utilizing triethylammonium phosphate-organic solvent modifier solvents as the CE buffer and HPLC eluent. Acetonitrile, methanol, ethanol and 2-propanol were applied as organic modifiers. The applicability of HPLC and CE systems was evaluated and compared. Optimum conditions for the separation were determined for both methods. Retention (migration) time, elution order and selectivity can be influenced by modifying the composition of the eluent (buffer) with organic solvents not only in HPLC but also in CE. Although the HPLC system reacted to changes in the organic solvent concentration in a much more sensitive way than the CE system did (from the point of view of retention time), CE proved to be a more suitable method for separating the peptides investigated. Baseline separation could be achieved within 6-9 min by CE, a result which was impossible to achieve with HPLC working in the isocratic mode. In CE the effect of the alcohols on migration times proved to be opposite to that of acetonitrile. Whereas ACN decreased, the alcohols increased the migration times in a concentration-dependent way. The results suggest that CE can be applied very advantageously in peptide analysis. Its performance regarding selectivity, resolution, theoretical plate number, duration and cost is comparable or sometimes superior to that of HPLC.

A series of new somatostatin analogs were synthesized in order to study the relative importance of specific substitutions in relation to selectivity between their endocrine and antitumor effects. Substitutions were carried out in all positions, except for Lys in position 5. Peptides were tested for their ability to inhibit in vitro and in vivo GH release, proliferation of the MCF 7 breast carcinoma cell line and tyrosine kinase activity in the HT 29 human colon carcinoma cell line. Selective biological activity was achieved in GH release and antitumor activity by the different amino acid substitutions. One of the analogs, with a five-residue ring (D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2, TT-232), was unique. It had no GH release inhibitory activity, but did have strong tyrosine kinase inhibitory and antiproliferative effects.

CD spectroscopic studies on protected peptides containing lysine and serine, or phosphoserine, and on serine-containing fragments of the neurofilament protein midsized subunit, both in the unphosphorylated and phosphorylated form, are reported. The introduction of the phosphoryl group was not found to have a significant spectral effect in aqueous solution. In trifluoroethanol (TFE), spectral shifts toward unordered (type U) spectra or the appearance of distorted spectra likely reflect the adoption of aperiodic polypeptide conformations due to salt bridge(s) between negatively charged phosphoserine and positive lysine side-chain groups. A turn-stabilizing effect of phosphorylation was also observed. CD-monitored titration experiments in TFE revealed a high conformational sensitivity of phosphopeptides toward Ca2+ ions. The appearance of the unordered spectra or spectral shifts were the sign of a bulk disordering effect of Ca2+ ions. Spectra with specific spectroscopic features reflect the formation of Ca2+ complexes and the adoption of ordered unique backbone conformations. When ordered structures were obtained on addition of Ca2+ ions, the observed CD curves showed a resemblance to the spectrum of beta-pleated sheets. This may originate from chain extension and the formation of beta-pleated sheet segments fixed by Ca2+ bridges between PO3H-1 groups of adjacent peptide chains. The data clearly show that the effect of the Ca2+ ions is highly specific: the sequence, chain length, presence and distribution of charged side-chain groups, degree and site of phosphorylation, and environmental factors appear to be determining in the process of chain extension or beta-sheet formation.

A series of novel gonadotropin releasing hormone (GnRH) and Somatostatin analogs have been developed in our laboratory and were screened for antiproliferative and signal transduction inhibitory effect. Our GnRH analog Folligen, had significant antitumor activity on DMBA induced mammary carcinomas in rats without blocking ovarian functions. The direct effect of Folligen and Buserelin has been compared on the human breast cancer cell line MDA-MB-231. Folligen was found to be more effective in inhibiting cell proliferation and significant differences were found in the signal transduction pathways activated by these analogs. Our novel Somatostatin analogs were screened for tyrosine kinase inhibition and for antiproliferative effect on human colon tumor cells and for growth hormone (GH) release inhibition in vitro and in vivo. The analog TT-2-50 was significantly more active inhibiting GH release in superfused rat pituitary cells and in vivo than native Somatostatin and it strongly inhibited tyrosine kinase and proliferation while it stimulated protein kinase C activity.

[Lyst6]GHRP, the C-terminally thionated analog of the highly potent growth hormone releasing hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 was prepared by using solid support. The success of the synthesis showed that Lawesson's reagent can be used for selective thionation of an amide group not only in solution but also on the surface of a resin. The C-terminal thioamide group proved to be stable under the conditions of the solid phase synthesis.

High-performance liquid chromatographic systems were used for monitoring the hydrolysis of five potent agonistic gonadotropin releasing hormone analogues containing aspartic acid derivatives in position six. To separate the closely related nonapeptides formed during the hydrolysis, columns with reversed-phase packings were used under isocratic conditions. The mobile phases were methanol containing ammonium acetate or triethylammonium phosphate buffers. Good separations of the hydrolysis products from the investigated peptides allowed the reaction rate constants for the transformations examined to be calculated.

New agonists related to gonadotropin-releasing hormone (GnRH) have been synthesized that are comparable in potency to the GnRH and its superagonists for release of LH and estrus suppression without substitutions with D- or unnatural amino acids in position 6. We now report a series of L-beta-aspartyl-6 GnRH analogs containing only naturally occurring L-amino acids in the whole sequence, exhibiting considerable in vivo biological activity. Dose and time dependent LH release capability of the different analogs in adult male mice, estrus suppression comparisons and blockade of ovulation in female rats are given. The incorporation of L-Asp-OMe and L-Asp-OBzl in position 6 of GnRH resulted in the most potent GnRH agonists (to 12-20xGnRH potency) in this series inducing a biphasic biological response similar to the D-amino acid-6 substituted superactive GnRH analogs. A correlation between the LH releasing potencies of the analogs and their HPLC retention times was also investigated. Peptide synthesis were achieved using either solid phase or solution phase methodology.