Anti-interleukin-8 activity of tick salivary gland extracts

Interleukin-8 (IL-8) is one of many mammalian chemokines (chemotactic cytokines) that direct mammalian inflammatory and immune cells to sites of injury and infection. Chemokines are produced locally and act on leucocytes through selective receptors. The principal role of IL-8 is to control the movement and activity of neutrophils. To date, several tick species have been shown to modulate the production or activity of certain cytokines but none of these are chemokines. Using an IL-8 specific ELISA, we showed that salivary gland extracts (SGE) from several ixodid tick species (Dermacentor reticulatus, Amblyomma variegatum, Rhipicephalus appendiculatus, Haemaphysalis inermis and Ixodes ricinus) reduced the level of detectable IL-8. Analyses of fractionated SGE revealed one similar peak of activity for D. reticulatus, A. variegatum and R. appendiculatus; a second peak, observed for D. reticulatus and A. variegatum, differed between the two species. Using radiolabelled IL-8, SGE and peak activity fractions of D. reticulatus were shown to bind the chemokine, and to inhibit binding of IL-8 to its receptors on human granuolocytes enriched for neutrophils. The biological significance of these observations was demonstrated by the ability of SGE to inhibit IL-8 induced chemotaxis of human blood granulocytes. Future isolation and characterization of the active molecules will enable determination of their functional roles in bloodfeeding and effect on tick-borne pathogen transmission.     

Evasin‐3‐like anti‐chemokine activity in salivary gland extracts of ixodid ticks during blood‐feeding: a new target for tick control

Ticks exploit many evasion mechanisms to circumvent the immune control of their hosts including subversion of the communication language between cells of the immune system provided by chemokines and other cytokines. One subversive molecule secreted in the saliva of Rhipicephalus sanguineus is Evasin‐3, a structurally unique 7 kDa protein that selectively binds the neutrophil chemoattractants, CXCL8 and (with lower affinity) CXCL1. We compared anti‐human CXCL8 and anti‐mouse CXCL1/KC activities in salivary gland extracts prepared from adult Amblyomma variegatum, Rhipicephalus appendiculatus and Dermacentor reticulatus ticks during blood‐feeding. Both anti‐CXCL8 activity and anti‐CXCL1 activity were detected in all species and in both adult females and males, with consistently higher activity levels against CXCL8. These results suggest that Evasin‐3‐like activity is common amongst metastriate ixodid tick species, and provide further evidence of the importance to ticks in controlling neutrophils during blood‐feeding. As such, Evasin‐3 offers a new target for anti‐tick vaccine development.     

Heterogeneity in the effect of different ixodid tick species on human natural killer cell activity

Tick saliva plays a vital role in blood-feeding, including manipulation of the host response to tick infestation. Furthermore, a diverse number of tick-borne pathogens are transmitted to vertebrate hosts via tick saliva, some of which exploit the immunomodulatory activities of their vector's saliva. We report that salivary gland extracts (SGE) derived from Dermacentor reticulatus adult ticks induce a decrease in the natural killer (NK) activity of effector cells obtained from healthy human blood donors. The decrease was observed with SGE from both female and male D. reticulatus fed for either 3 or 5 days on mice, but no significant effect was observed with SGE from unfed ticks or ticks that had fed for 1 day. These results indicate that the tick anti-NK factor(s) is only active after blood-feeding has commenced. Microscopic examination revealed that the first step of NK activity, namely effector/target cell conjugate formation, was affected by SGE. The observed reduction in conjugate formation occurred when effector (but not target) cells were treated with SGE for 30 min, and the effect persisted after 12 h of treatment. Similar but less potent anti-NK activity was detected for SGE from Amblyomma variegatum and Haemaphysalis inermis. By contrast, SGE derived from Ixodes ricinus and Rhipicephalus appendiculatus female ticks did not decrease NK activity. The apparent absence of such activity in these two important vectors of tick-borne viruses suggests that control of NK cells does not play an important role in promoting virus transmission, at least for these particular species.     

Plasma levels of chemokines during leprosy specific treatment

Leprosy, whose etiologic agent is Mycobacterium leprae, is an illness of ample clinical and immunopathological spectrum. Although chemokines seem to be involved in the immunopathogenesis of leprosis, few studies have been carried out to unveil the potential of chemokines as biological markers of the disease. The purpose of this study was to investigate the value of measuring CCL2, CCL3, CCL11 and CCL24 in plasma of patients with leprosy (LE) at different stages of multi-drug therapy (MDT). Chemokines were measured by ELISA in plasma of 30 non-infected individuals (NI) and 33 LE patients before and at different stages of treatment. The plasma concentration of CCL11 (p < 0.01) and CCL24 (p < 0.05) was increased in LE patients before treatment when compared to NI individuals. The plasma concentration of CCL24 decreased after MDT (p < 0.05). No differences were observed in the concentration of CCL2 and CCL3 in plasma of NI and LE individuals. The elevated levels of CCL11 and CCL24 in plasma of patients with LE suggest that these chemokines may play a role in disease pathogenesis. Moreover, the decrease of CCL24 after treatment suggests that this chemokine might be useful as a biomarker of response to MDT in patients with leprosy.     

Differential gene expression of chemokines in KRAS and BRAF mutated colorectal cell lines:Role of cytokines

AIM:To study KRAS/BRAF mutations in colorectalcancer(CRC)that influences the efficacy of treatment.To develop strategies for overcoming combination of treatment.METHODS:Five colonic cell-lines were investigated:DLD-1 with KRAS(G13D)mutation,HT 29 and Colo205 with BRAF(V600E)mutation as well as the wild type(Wt)cell-lines Caco2 and Colo-320.DLD-1(KRAS),HT-29(BRAF)and Caco2(Wt)cell lines were treated with cytokines(TNFα50 ng,IL-1β1 ng and IFNγ50ng)and harvested at different time points(1-24 h).KRAS inhibition was performed by the siRNA-approach.Two colorectal cancer cells DLD-1 and Caco2 were used for KRAS inhibition.About 70%confluency were confirmed before transfection with small interferring RNA(siRNA)oligonucleotides.All the synthetic siRNA sequences were designed in our laboratory.Total RNA and protein was isolated from the cells for RT-PCR and Western blotting.Densitometry of the Western blotting was analyzed with the Image J software(NIH).Results are shown as mean±SD.RESULTS:RT-PCR analysis in non-stimulated cells showed a low basal expression of TNFαand IL-1βin the DLD-1 KRAS-mutated cell-line,compared to Caco2wild type.No detection was found for IL-6 and IFNγin any of the studied cell lines.In contrast,pro-angiogenic chemokines(CXCL1,CXCL8)showed a high constitutive expression in the mutated cell-lines DLD-1(KRAS),HT-29 and Colo205(BRAF),compared to wild type(Caco2).The anti-angiogenic chemokine(CXCL10)showed a high basal expression in wild-type,compared to mutated cell-lines.KRAS down-regulation by siRNA showed a significant decrease in CXCL1 and CXCL10gene expression in the DLD-1(KRAS)cell-line in comparison to wild type(Caco2)at 72 h after KRAS silencing.In contrast,the specific KRAS inhibition resulted in an up-regulation of CXCL1 and CXCL10.The results of our study show a higher expression of pro-angiogenic chemokines at basal level in mutated cell-lines,which was further increased by cytokine treatment.CONCLUSION:To summarize,basal chemokine gene expression for pro-angiogenic chemokines was high in mutated as compared to wild type cell-lines.This reflects the likely existence of a different microenvironment in tumours consistent of wild type or mutated cells.This may help to rationalize the choice of molecular targets for suitable therapeutic investigation in clinical studies.     

Specificity of the cyclic GMP-binding activity and of a cyclic GMP-dependent cyclic GMP phosphodiesterase in Dictyostelium discoideum

The nucleotide specificity of the cyclic GMP-binding activity in a homogenate of Dictyostelium discoideum was determined by competition of cyclic GMP derivatives with [8-3H]-cyclic GMP for the binding sites. The results indicate that cyclic GMP is bound to the binding proteins by hydrogen bonds at N2H2, N1-H and/or C6 = O, N7, 2(1)-OH and 3(1)-O and possibly via a charge-charge interaction with the phosphate moiety of cyclic GMP. Cyclic AMP only competes with cyclic GMP for binding at a 20,000-fold higher concentration. The same cyclic GMP derivatives were used to modify the hydrolysis of [8-3H]cyclic GMP by phosphodiesterase. The phosphodiesterase is activated by cyclic GMP. The nucleotide specificity of activation of the enzyme differs from the specificity of the enzyme for hydrolysis. This indicates that activation by cyclic GMP and hydrolysis of cyclic GMP occur at different sites of the enzyme. Cyclic AMP neither activates the cyclic GMP phosphodiesterase nor competes with cyclic GMP for hydrolysis. This indicates that intracellular cyclic AMP does not interfere with the action of intracellular cyclic GMP in D. discoideum.     

Differential expression of interleukin-2 receptors (α and β chain) in mature lymphoid neoplasms

We investigated the expression of interleukin-2 receptors (IL-2R) in 60 adult patients with mature lymphoid neoplasms by flow cytometric analysis, using two monoclonal antibodies, anti-Tac for IL-2R α-chain (IL-2Rα) and Mik-β for IL-2R β-chain (IL-2Rβ). Among B-cell malignancies, IL-2Rα was found in 13/25 (52%) cases of chronic lymphocytic leukemia (CLL) and its variants, 3/14 (21%) of a heterogenous group of non-Hodgkin's lymphoma (NHL) and none of the plasma cell diseases. IL-2Rβ was not observed in any of B-cell neoplasms. IL-2Rα was more frequently expressed in CD11 b(+) B-cell neoplasms than in CD11b(-) (P < 0.05). In T-cell disorders, all three cases of adult T-cell leukemia/lymphoma expressed IL-2Rα but not IL-2Rβ. IL-2Rβ was detected in 3/8 cases of CLL and 2/3 of NHL and none of these cases expressed IL-2Rα. CD8(+) malignant T-cells commonly displayed IL-2Rβ. These data indicate that the IL-2Rα and IL-2Rβ in mature lymphoid neoplasms was expressed independently each other and was associated with the particular phenotypical characteristics of neoplastic cells, respectively. © 1994 Wiley-Liss, Inc.     

Evidence suggesting 11 beta-hydroxylase inhibition during aminoglutethimide administration

Steroid hormone excretion during amino-glutethimide administration was studied in a patient with Cushing's syndrome due to bilateral adrenocortical hyperplasia. Plasma and urinary 17-OHCS showed a persistent decrease, as did urinary total 17-KS. Chromatographic fractionation of urinary 17-KS demonstrated a dramatic reduction of 11-oxy-17-KS, while 11-deoxy-17-KS, after a transient fall, tended to recover. Urinary THS showed an absolute increase, while pregnanetriolone diminished. Δ⁵-pregnenetriol and pregnanetriol, after initial decreases, showed a gradual trend upward. The sum of these data, and particularly the increase in THS excretion, suggests that, in this case, aminoglutethimide exerted an inhibitory effect not only on the early steps of adrenocortical steroidogenesis but also on 11 β-hydroxylation.     

Multiple osteolytic bone lesions with high serum levels of interleukin-6 and CCL chemokines in a patient with adult T cell leukemia

A 37-year-old woman was diagnosed as having chronic adult T-cell leukemia (ATL) of the skin by a skin biopsy and human T-cell leukemia virus type-1 serology at our hospital in August 1992. The skin lesions of ATL were improved by treatment with psoralen ultraviolet ray A. She complained of severe pain in her bilateral forearms, hands and ankles, and X-ray examination in July 1999 revealed multiple punched-out lesions of the extremities. Serum levels of parathyroid hormone-related peptide, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha and total serum receptor activator of nuclear factor kappaB ligand were not elevated. However, serum levels of IL-6, CCL2 monocyte chemoattractant protein-1 (MCP-1), CCL3 [macrophage inflammatory protein-1alpha (MIP-1alpha)] and CCL4 (MIP-1beta) were markedly elevated. Here, we have discussed the possible mechanism underlying the onset of the osteolytic lesions.     

Differential expression of cell cycle proteins during ageing of pancreatic islet cells

One of the major challenges for developmental biologists and investigators in the field of diabetes over the last few decades has been to dissect the origin of pancreatic endocrine cells and to accurately understand the mechanisms that regulate islet cell regeneration. While significant advances have been made recently, there continues to be a paucity of knowledge regarding the growth factor signalling pathways that directly regulate the proteins involved in islet cell cycle control. We will discuss recent work in these areas and provide insights from our studies into age-dependent alterations in the expression of growth factor signalling proteins and cell cycle proteins in islet cells.     

Differential functional effects of two 5-HT4 receptor isoforms in adult cardiomyocytes

Serotonin 5-HT4 receptors are present in human atrial myocytes and have been proposed to contribute to the generation of atrial fibrillation. However, 5-HT4 receptors have so far been only found in human and pig atria and are absent from the heart of small laboratory animals, such as rat, guinea pig, rabbit and frog, which limits the experimental settings for studying their functional properties. In this study, we developed an adenovirus expression system to examine the properties of two human 5-HT4 receptor splice variants, h5-HT4(b) and h5-HT4(d), expressed in adult cardiomyocytes devoid of native 5-HT4 receptors. When expressed in the HL-1 murine cell line of atrial origin, both receptors caused specific binding of the 5-HT4 selective antagonist GR113808 and activated adenylyl cyclase in the presence of serotonin (5-HT, 1 microM). When expressed in freshly isolated adult rat ventricular cardiomyocytes, a stimulation of the L-type Ca2+ current (ICa,L) by 5-HT (100 nM) was revealed. Both effects were blocked by GR113808. In HL-1 cells, the h5-HT4(d) receptor was found to be more efficiently coupled to adenylyl cyclase than the h5-HT4(b). Pertussis toxin treatment (250 ng/ml for 5 h) potentiated the stimulatory effect of 5-HT on ICa,L in rat myocytes expressing the h5-HT4(b) but not the h5-HT4(d) receptor, indicating a likely coupling of the (b) isoform to both Gs and Gi/o proteins. Adenoviral expression of h5-HT4 receptor isoforms in adult cardiac myocytes provides a valuable means for the exploration of the receptor signaling cascades in normal and pathological situations.     

Differential expression of cartilage and bone-related proteins in pediatric and adult diseased aortic valves

Approximately 5 million people are affected with aortic valve disease (AoVD) in the United States. The most common treatment is aortic valve (AoV) replacement surgery, however, replacement valves are susceptible to failure, necessitating additional surgeries. The molecular mechanisms underlying disease progression and late AoV calcification are not well understood. Recent studies suggest that genes involved in bone and cartilage development play an active role in osteogenic-like calcification in human calcific AoVD (CAVD). In an effort to define the molecular pathways involved in AoVD progression and calcification, expression of markers of valve mesenchymal progenitors, chondrogenic precursors, and osteogenic differentiation was compared in pediatric non-calcified and adult calcified AoV specimens. Valvular interstitial cell (VIC) activation, extracellular matrix (ECM) disorganization, and markers of valve mesenchymal and skeletal chondrogenic progenitor cells were observed in both pediatric and adult AoVD. However, activated BMP signaling, increased expression of cartilage and bone-type collagens, and increased expression of the osteogenic marker Runx2 are observed in adult diseased AoVs. They are not observed in the majority of pediatric diseased valves, representing a marked distinction in the molecular profile between pediatric and adult diseased AoVs. The combined evidence suggests that an actively regulated osteochondrogenic disease process underlies the pathological changes affecting AoVD progression, ultimately resulting in stenotic AoVD. Both pediatric and adult diseased AoVs express protein markers of valve mesenchymal and chondrogenic progenitor cells while adult diseased AoVs also express proteins involved in osteogenic calcification. These findings provide specific molecular indicators of AoVD progression, which may lead to identification of early disease markers and the development of potential therapeutics.     

Small doses of 5α-dihydrotestosterone mimic the effects of 5α-androstane-3β,17β-diol on acid phosphatase activity in the adult rat prostate gland

These studies were designed to further investigate whether 5α-androstane-3β,17β-diol was exerting unique effects on rat prostate acid phosphatase activity or could possibly be exerting its actions by a small peripheral conversion to 5α-dihydrotestosterone. Intraperitoneal administration of 5α-dihydrotestosterone in doses of 1 mg, 100 μg or 50 μg per day starting 7 days after castration led to the restoration of normal characteristics of acid phosphatase activity. However, when 5α-dihydrotestosterone was given in a dose of only 25 μg per day starting 7 days after castration, the changes in acid phosphatase activity were indistinguishable from those found when 5α-androstane-3β,17β-diol was administered in a dose of 2 mg per day. This suggests that the effects of 5α-androstane-3β,17β-diol can be explained by its conversion to small amounts of 5α-dihydrotestosterone.     

Differential effects of ivabradine and ryanodine on pacemaker activity in canine sinus node and purkinje fibers

Effects of Ivabradine and Ryanodine on Cardiac Pacemakers . Introduction: It is generally accepted that at least 2 major mechanisms contribute to sinus node (SN) pacemaking: a membrane voltage (mainly I_f) clock and a calcium (Ca) clock (localized submembrane sarcoplasmic reticulum Ca²⁺ release during late diastolic depolarization). The aim of this study was to compare the contributions of each mechanism to pacemaker activity in SN and Purkinje fibers (PFs) exhibiting normal or abnormal automaticity. Methods and Results: Conventional microelectrodes were used to record action potentials in isolated spontaneously beating canine SN and free running PF in control and in the presence of 0.1 μM isoproterenol. Ryanodine (0.1–3 μM) and ivabradine (3 μM) were used to inhibit sarcoplasmic reticulum Ca²⁺ release or I_f, respectively. To induce automaticity at low membrane potentials, PFs were superfused with BaCl₂. In SN, ivabradine reduced the rate whereas ryanodine had no effect. Isoproterenol significantly accelerated automatic rate, which was decreased by ivabradine and ryanodine. In normally polarized PFs, ryanodine had no effects on the automatic rate in the absence or presence of isoproterenol, whereas ivabradine inhibited both control and isoproterenol‐accelerated automaticity. In PF depolarized with BaCl₂, ivabradine decreased BaCl₂‐induced automatic rate while ryanodine had no effect. Conclusion: In canine SN, I_f contributes to both basal automaticity and β‐adrenergic‐induced rate acceleration while the ryanodine‐inhibited Ca clock appears more involved in β‐adrenergic regulation of pacemaker rate. In PF, normal automaticity depends mainly on I_f. Inhibition of basal potassium conductance results in high automatic rates at depolarized membrane potentials with SN‐like responses to inhibition of membrane and Ca clocks. (J Cardiovasc Electrophysiol, Vol. 23, pp. 650–655, June 2012)     

The oxidation products of melatonin derivatives exhibit acetylcholinesterase and butyrylcholinesterase inhibitory activity

Abstract:  It is already well documented that melatonin exhibits strong antioxidant properties. It traps several reactive oxygen species including singlet oxygen, peroxyl and hydroxyl radicals. Also, peroxynitrite-induced reactions are inhibited by melatonin. The oxidation of melatonin by singlet molecular oxygen [O₂ (¹Δ_g)] may produce cyclic 3-hydroxymelatonin whose structure we have already studied. In this investigation we report on the synthesis of several melatonin analogues having a carbamate substituent instead of the methoxy group at 5 position of the indole ring. These compounds behave analogously to melatonin with respect to singlet oxygen and produce the corresponding cyclic 3-hydroxymelatonin analogues. The structures of the products were investigated with spectral methods and X-ray crystallography. The compounds obtained possess the 2,3,8,8a-tetrahydropyrrolo[2,3-b]indole heterocyclic system which is a structural motif characteristic of alkaloids, physostigmine and phenserine, that are potent acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors used in the Alzheimer's disease treatment. We measured the inhibitory activity of the obtained compounds against AChE and BChE from human erythrocytes and serum. In the case of the compounds having a phenylcarbamate and methoxyphenylcarbamate substituents, the inhibitory activity (IC₅₀) ranged from 0.252 ± 0.033 to 3.804 ± 0.581 μ m . Other compounds were less active and showed rather complex interactions with the structure–activity relationship in need of further investigation.     

Changes of serum 2‘,5’-oligoadenylate synthetase activity during interferon treatment of chronic hepatitis C

ABSTRACT— It was our aim to evaluate whether the baseline activity of 2–5 oligoadenylate synthetase (2–5 OAS) in serum and changes induced by the treatment with interferon are relevant factors in remission of chronic hepatitis C. Seventeen out of 30 adult patients with chronic hepatitis C were randomized to receive recombinant alpha-2b interferon at the dosage of 3 MU three times weekly. By the end of the third month, nine patients had normalized transaminase levels and continued to receive 3 MU of interferon for an additional 3 months, whereas in eight non-responders the dosage was increased to 6 MU for the same period of time. A single patient responded to the increased dosage. Baseline 2–5 OAS serum activity was significantly higher in patients with chronic hepatitis when compared with normal controls. Follow-up on the 13 untreated cases showed that 2–5 OAS elevation was stable and unrelated to concomitant infections. Comparison of responders and non-responders showed that the latter had higher baseline 2–5 OAS activity, tended to have an earlier and higher peak in the enzyme during the first 4 weeks of treatment, and maintained higher levels during the first 3 months of therapy. The increased dosage of interferon in this group led to an additional, although temporary, increase in 2–5 OAS. Our data suggest that HCV infection by itself induces elevated 2–5 OAS levels. The paradoxical increase in non-responders indicates that monitoring of the enzyme in serum does not predict the response to interferon. The role of the 2–5 OAS pathway in inducing the antiviral state in HCV infection should be further evaluated at tissue level.