Dimitra Kalavrizioti

Cytokines as Biomarkers of Treatment Response to IFNβ in Relapsing-Remitting Multiple Sclerosis

Background. MS patients show a remarkable heterogeneity in their response to disease modifying treatments. Given the need for early treatment initiation and the diversity of available options, a predictive marker that indicates good or poor response to treatment is highly desirable. Objective. To find a biomarker for treatment response to IFNβ among pro- and anti-inflammatory cytokines. Materials and Methods. IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, IL-17A, and TGF-β1 levels were measured in serum and CSF of 43 patients with RR-MS who were followed up for a mean period of 5.3 years. Thirty-five patients received IFNβ treatment and were divided into good responders (GR, n = 19) and poor responders (PR, n = 16). The remaining 8 patients showed a very favorable outcome and remained untreated (noRx). Results. GR had significantly higher serum baseline levels of IL-17A than PR and significantly higher serum levels of IL-17A, IFN-γ, TNF-α, and IL-2 than noRx. PR had significantly higher IFN-γ serum levels than noRx. No significant differences were observed in serum levels of IL-6, IL-4, IL-10, and TGF-β1 or the levels of all cytokines measured in CSF between the 3 groups of patients. Conclusions. Baseline serum levels of IL-17A can be used as a biomarker of IFNβ treatment response.

Immune Parameters That Distinguish Multiple Sclerosis Patients from Patients with Other Neurological Disorders at Presentation

Background/Aim Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system. Effector T helper cells, mainly Th1 and Th17, cytotoxic T-cells, B-cells, macrophages, microglia, and the cytokines they secrete, are implicated in the initiation and maintenance of a deregulated immune response to myelin antigens and the ensuing immune-mediated demyelination. In this study, we investigated whether signature cytokines exist in MS patients at presentation to gain an insight into the underlying immunopathogenic processes at the early stage of the disease. Methods We collected serum and cerebrospinal fluid (CSF) samples from 123 patients at presentation, eventually diagnosed with MS or non-inflammatory (NIND) or inflammatory neurological diseases (IND) or symptomatic controls (SC). The levels of cytokines IFN-γ, TNF-α, TGF-β1, IL-2, IL-4, IL-6, IL-10 and IL-17 were measured, and cytokine ratios, such as Th1/Th2, Th1/Th17, and Type-1/Type-2, were calculated. All parameters were tested for their correlations with the intrathecal IgG synthesis. Results Cytokine levels in CSF were lower than in serum in all the patients, with the exception of IL-6. Serum or CSF cytokine levels of MS patients did not differ significantly from NIND or SC, with the exception of serum IFN-γ and TNF-α that were significantly higher in NIND. IND patients presented with the highest levels of all cytokines in serum and CSF, with the exception of serum IL-10 and CSF IL-17. MS patients had a significantly lower serum Th1/Th2 ratio compared to the NIND and IND groups, and significantly lower serum Type-1/Type-2, IFN-γ/IL-10 and CSF Th1/Th17 ratios compared to IND patients. MS patients had a significantly higher CSF IL-17/IL-10 ratio compared to IND patients. The IgG index was higher in MS patients compared to the control groups; the differences reached statistical significance between the MS and the NIND and SC groups. Reiber-Felgenhauer analysis of the QIgG and QAlb indices revealed higher intrathecal IgG synthesis in MS patients, and higher blood-CSF barrier dysfunction in IND patients. The IgG index correlated with CSF IL-4 in MS patients only. Conclusions We found no signature cytokines or profiles thereof in MS patients at presentation. Only IND patients presented with a clear Th1 cytokine polarization in serum and CSF. The parameters that distinguished MS patients from patients with other neurological disorders were IgG intrathecal synthesis, the IgG index and its correlation with CSF IL-4 levels.

Towards non-peptide ANG II AT1 receptor antagonists based on urocanic acid: rational design, synthesis and biological evaluation

A series of o-, m- and p-benzyl tetrazole derivatives 11a–c has been designed, synthesized and evaluated as potential Angiotensin II AT1 receptor antagonists, based on urocanic acid. Compound 11b with tetrazole moiety at the m-position showed moderate, however, higher activity compared to the o- and p-counterpart analogues. Molecular modelling techniques were performed in order to extract their putative bioactive conformations and explore their binding modes.

T helper (Th)-cytokines in the urine of patients with primary glomerulonephritis treated with immunosuppressive drugs: Can they predict outcome?

BACKGROUND Glomerulonephritides (GNs) represent common causes of chronic kidney disease associated with a wide spectrum of clinical and histological features. Various factors that activate the inflammatory cascade are involved in the development of kidney injury. The aim of this study was to estimate the urinary excretion of pro-inflammatory (IL-2, INF-γ, TNF-α, IL-6, IL-17) and anti-inflammatory (IL-4, IL-10, TGF-β1) cytokines, as well as the chemokine MCP-1 in patients with various types of GN treated by immunosuppressive drugs and to identify any prognostic value of excreted cytokines for future renal function. PATIENTS AND METHODS Ninety-seven patients (62 M/35 F, age 53.1 ± 15.6 years) with primary glomerulonephritis and 32 healthy controls were studied. The original diagnoses were membranous nephropathy (MN, n=36), IgA nephropathy (IgAN, n=31) and minimal changes disease or focal segmental glomerulosclerosis (MCD/FSGS, n=30). All patients had been treated with immunosuppressive drugs and, at the time of measurement of urinary cytokine excretion, were either in clinical remission or still had active disease with persistent proteinuria. RESULTS GN patients had significantly higher levels of all cytokines and MCP-1 compared to healthy controls. A strong positive correlation between TGF-β1 and MCP-1 concentrations was observed in all GN patients. Increased urinary excretion of all tested cytokines apart from TNF-α and TGF-β1 was observed even in patients with clinical remission. The main difference between patients with proteinuria and those in clinical remission was the level of MCP-1 urinary excretion. The urinary excretion of MCP-1 and TGF-β1 was significantly higher in patients with MN who showed deterioration of renal function over a follow-up period of five years. CONCLUSIONS Increased levels of cytokines are observed in the urine of patients with different types of glomerulonephritis, even after the achievement of clinical remission with the administration of immunosuppressive drugs. Urinary excretion of MCP-1 and TGF-β1 indicates the ongoing inflammatory and fibrotic processes in the kidney and is probably related to unfavourable outcomes.

RNA-Mediated Therapeutics: From Gene Inactivation to Clinical Application

The specific targeting and inactivation of gene expression represents nowadays the goal of the mainstream basic and applied biomedical research. Both researchers and pharmaceutical companies, taking advantage of the vast amount of genomic data, have been focusing on effective endogenous mechanisms of the cell that can be used against abnormal gene expression. In this context, RNA represents a key molecule that serves both as tool and target for deploying molecular strategies based on the suppression of genes of interest. The main RNA-mediated therapeutic methodologies, deriving from studies on catalytic activity of ribozymes, blockage of mRNA translation and the recently identified RNA interference, will be discussed in an effort to understand the utilities of RNA as a central molecule during gene expression.

Kinetics of inhibition of ribonuclease P activity by peptidyltransferase inhibitors – Effect of antibiotics on RNase P

A cell-free system derived from Dictyostelium discoideum has been used to study the kinetics of inhibition of RNase P by puromycin, amicetin and blasticidin S. Detailed kinetic analysis showed that the type of inhibition of RNase P activity by puromycin is simple competitive, whereas the type of inhibition by amicetin and blasticidin S is simple non-competitive. On the basis of Ki values amicetin is stronger inhibitor than puromycin and blasticidin S.

Remitting–relapsing multiple sclerosis patient refractory to conventional treatments and bone marrow transplantation who responded to natalizumab

Bone marrow transplantation (BMT) was introduced as a treatment option 15 years ago for severe, drug-resistant multiple sclerosis (MS). Up until now, BMT has been undertaken in relatively few patients worldwide, with moderate success, and recent studies suggest that patients with early, highly aggressive MS benefit most from this treatment. In this work, we determined peripheral blood lymphocyte populations in a patient (patient A) with remitting–relapsing multiple sclerosis (RR-MS), refractory to conventional treatments, and who underwent BMT, relapsed, and has been treated with natalizumab for the last 22 months. Eleven other RR-MS patients in the acute phase of the disease, untreated or treated with interferon-beta, and 20 healthy subjects served as controls. Natalizumab treatment in patient A resulted in lymphocytosis and increased levels of CD20+/CD20+CD5+ B cells and T regulatory cells (Tregs). The patient maintained relatively low levels of T cells, T helper cells, memory T helper cells, and naive cytotoxic T cells, and very low levels of naive T helper cells and natural killer cells throughout. The Tregs of patient A post-treatment with natalizumab responded well in culture to a peptide mapping to a myelin basic protein antigenic epitope (mean 42% increase) compared with Tregs of healthy controls (mean 15% increase) whereas Tregs of the RR-MS controls or patient A prenatalizumab treatment either did not respond or responded adversely to the peptide (mean 3% and 21% decreases, respectively). Since the beginning of natalizumab treatment, patient A has had no relapses, and his Expanded Disability Status Score has improved. From the parameters studied, Treg responsiveness to autoantigens seems to be an important differentiating factor in RR-MS progression.

Modulation of Catalytic RNA Biological Activity by Small Molecule Effectors

Catalytic RNAs, known as ribozymes, act as true enzymes and are implicated in important biological processes, such as protein synthesis, mRNA splicing, transcriptional regulation and retroviral replication. Ribozymes are capable of serving as a new molecular target for a variety of drugs and as a reliable screening system for their biological activity.

Facile and Efficient Syntheses of a Series of N-Benzyl and N-Biphenylmethyl Substituted Imidazole Derivatives Based on (E)-Urocanic acid, as Angiotensin II AT1 Receptor Blockers

In the present work, a facile and efficient route for the synthesis of a series of N-substituted imidazole derivatives is described. Docking studies have revealed that N-substituted imidazole derivatives based on (E)-urocanic acid may be potential antihypertensive leads. Therefore, new AT1 receptor blockers bearing either the benzyl or the biphenylmethyl moiety at the N-1 or N-3 position, either the (E)-acrylate or the propanoate fragment and their related acids at the C-4 position as well as a halogen atom at the C-5 position of the imidazole ring, were synthesized. The newly synthesized analogues were evaluated for binding to human AT1 receptor. The biological results showed that this class of molecules possesses moderate or no activity, thus not always confirming high docking scores. Nonetheless, important conclusions can be derived for their molecular basis of their mode of action and help medicinal chemists to design and synthesize more potent ones. An aliphatic group as in losartan seems to be important for enhancing binding affinity and activity.