Soluble urokinase plasminogen activator receptor (suPAR) in children with obesity or type 1 diabetes as a marker of endothelial dysfunction: a cross-sectional study.

Pediatric obesity and type 1 diabetes mellitus (T1DM) represent two common chronic diseases associated with chronic inflammation, endothelial dysfunction and long-term complications. The aim of the present study was to assess the possible diagnostic and prognostic value of soluble urokinase plasminogen activator receptor (suPAR), a marker of inflammation and impaired endothelial function, in children with the diseases. In this cross-sectional study, children and adolescents with T1DM (N = 41) or obesity (N = 37), aged < 18 years old, and without proteinuria were included, together with children of similar age and without evident morbidity that served as controls (N = 42). Serum samples were obtained during standard outpatient follow up and the urokinase-type plasminogen activator receptor (suPAR) concentrations were measured using a commercially available sandwich ELISA kit (DUP00, R&D systems). Clinical and biochemical indices that were also assessed include body mass index (BMI) z-score, Tanner stages, glycosylated haemoglobin (HbA1c), fasting lipid profile and serum creatinine. Mean serum suPAR levels were significantly higher in patients with obesity compared to patients with T1DM and controls, while children with T1DM had similar suPAR levels to controls. Also, serum suPAR levels showed a negative correlation with age (Spearman rho -0.359, p < 0.001) and serum creatinine levels (Spearman rho -0.334, p = 0.005), and a positive correlation with BMI z-score (Spearman rho 0.354, p = 0.009) in the whole cohort.  Conclusion: Serum suPAR may be a useful predictive marker of inflammation or endothelial dysfunction for children with obesity and T1DM, as well as a promising therapeutic target. Further studies are needed in order to clarify whether the reported differences in suPAR levels could reflect a greater impairment of the inflammation status and endothelial function in children with obesity compared to children with T1DM. What is Known: • Paediatric obesity and type 1 diabetes are characterised by chronic inflammation and metabolic dysregulation. • Urokinase plasminogen activator receptor (uPAR) has been proposed as a useful biomarker for chronic inflammation and cardiovascular risk in adults. What is New: • Serum suPAR levels were increased in children and adolescents with obesity compared to those with T1DM and healthy controls; thus, obesity may affect the inflammatory status and endothelial function to a higher degree than T1DM during childhood. • Serum suPAR may serve as a diagnostic and predictive marker of inflammation and endothelial dysfunction for children and adolescents with obesity and T1DM.

Monocyte Chemoattractant Protein-1 (MCP-1), Activin-A and Clusterin in Children and Adolescents with Obesity or Type-1 Diabetes Mellitus.

Inflammation plays a crucial role in diabetes and obesity through macrophage activation. Macrophage chemoattractant protein-1 (MCP-1), activin-A, and clusterin are chemokines with known roles in diabetes and obesity. The aim of this study is to investigate their possible diagnostic and/or early prognostic values in children and adolescents with obesity and type-1 diabetes mellitus (T1DM). METHODS: We obtained serum samples from children and adolescents with a history of T1DM or obesity, in order to measure and compare MCP-1, activin-A, and clusterin concentrations. RESULTS: Forty-three subjects were included in each of the three groups (controls, T1DM, and obesity). MCP-1 values were positively correlated to BMI z-score. Activin-A was increased in children with obesity compared to the control group. A trend for higher values was detected in children with T1DM. MCP-1 and activin-A levels were positively correlated. Clusterin levels showed a trend towards lower values in children with T1DM or obesity compared to the control group and were negatively correlated to renal function. CONCLUSIONS: The inflammation markers MCP-1, activin-A, and clusterin are not altered in children with T1DM. Conversely, obesity in children is positively correlated to serum MCP-1 values and characterized by higher activin-A levels, which may reflect an already established systematic inflammation with obesity since childhood.

Diabetes Remission After LRYGBP With and Without Fundus Resection: a Randomized Clinical Trial.

BACKGROUND: Glycemic control, after metabolic surgery, is achieved in two stages, initially with neuroendocrine alterations and in the long-term with sustainable weight loss. The resection of the gastric fundus, as the major site of ghrelin production, is probably related with optimized glucose regulation. The aim of the present study is to investigate whether the modification of laparoscopic Roux-en-Y gastric bypass (LRYGBP) with fundus resection offers superior glycemic control, compared to typical LRYGBP. MATERIALS AND METHODS: Participants were 24 patients with body mass index (BMI) ≥40kg/m2 and type II diabetes mellitus (T2DM), who were randomly assigned to undergo LRYGBP and LRYGBP with fundus resection (LRYGBP+FR). Gastrointestinal (GI) hormones [ghrelin, glucagon-like-peptide-1 (GLP-1), peptide-YY (PYY)] and glycemic parameters (glucose, insulin, HbA1c, C-peptide, insulinogenic index, HOMA-IR) were measured preoperatively, at 6 and 12 months during an oral glucose tolerance test (OGTT). RESULTS: Ninety-five percent of patients showed complete remission of T2DM after 12 months. LRYGBP+FR was not related with improved glycemic control, compared to LRYGBP. Ghrelin levels were not significantly reduced at 6 and 12 months after LRYGBP+FR. GLP-1 and PYY levels were remarkably increased postprandially in both groups at 6 and 12 months postoperatively (p<0.01). Patients who underwent LRYGBP+FR achieved a significantly lower BMI at 12 months in comparison to LRYGBP (p<0.05). CONCLUSION: Fundus resection is not associated with improved glycemic regulation, compared to typical LRYGBP and the significant decrease in BMI after LRYGBP+FR has to be further confirmed with longer follow-up.

Ghrelin, glucagon-like peptide-1, and peptide YY secretion in patients with and without weight regain during long-term follow-up after bariatric surgery: a cross-sectional study.

Introduction: Weight loss after bariatric surgery is attributed, at least in part, to the altered gastrointestinal (GI) hormone secretion, which is thought to be responsible for a number of beneficial metabolic effects. Material and methods: We conducted a cross-sectional study. Twelve patients who underwent laparoscopic sleeve gastrectomy (SG) and 20 patients who underwent a variant of biliopancreatic diversion with Roux-en-Y gastric bypass and long limbs (BPD/RYGB-LL) were evaluated ≥ 7 years postoperatively. Ghrelin, glucagon-like peptide-1 (GLP-1), and peptide YY (PYY) secretion were compared between patients with successful weight loss maintenance (WM group) and patients with weight regain (WR group). Results: In both types of surgery, standard liquid mixed meal (SLMM) ingestion did not result in significant changes in fasting GI hormone levels. Fasting ghrelin levels did not differ between the WM group and the WR group in both types of surgery. In SG patients, SLMM ingestion elicited greater suppression of ghrelin levels in the WM group (p = 0.032). No difference in GLP-1 secretion was observed between the 2 groups of patients in both types of surgery. When patients were examined, regardless of the type of bariatric surgery they had undergone, postprandial PYY levels were lower in the WM group (p < 0.05), while fasting and postprandial PYY levels were correlated positively with an increase in body mass index (BMI) in the evaluation (Spearman's rho ≥ 0.395, p < 0.03). Conclusions: Our data do not support the hypothesis that long-term weight regain after bariatric surgery is associated with an unfavourable GI hormone secretion pattern.

NMR-Based Metabolomics in Differential Diagnosis of Chronic Kidney Disease (CKD) Subtypes.

Chronic Kidney Disease (CKD) is considered as a major public health problem as it can lead to end-stage kidney failure, which requires replacement therapy. A prompt and accurate diagnosis, along with the appropriate treatment, can delay CKD's progression, significantly. Herein, we sought to determine whether CKD etiology can be reflected in urine metabolomics during its early stage. This is achieved through the analysis of the urine metabolic fingerprint from 108 CKD patients by means of Nuclear Magnetic Resonance (NMR) spectroscopy metabolomic analysis. We report the first NMR-metabolomics data regarding the three most common etiologies of CKD: Chronic Glomerulonephritis (IgA and Membranous Nephropathy), Diabetic Nephropathy (DN) and Hypertensive Nephrosclerosis (HN). Analysis aided a moderate glomerulonephritis clustering, providing characterization of the metabolic fluctuations between the CKD subtypes and control disease. The urine metabolome of IgA Nephropathy reveals a specific metabolism, reflecting its different etiology or origin and is useful for determining the origin of the disease. In contrast, urine metabolomes from DN and HN patients did not reveal any indicative metabolic pattern, which is consistent with their fused clinical phenotype. These findings may contribute to improving diagnostics and prognostic approaches for CKD, as well as improving our understanding of its pathology.

The effect of vitamin K2 supplementation on vascular calcification in haemodialysis patients: a 1-year follow-up randomized trial.

PURPOSE: Vascular calcification (VC) is an independent risk factor for cardiovascular disease in hemodialysis patients while Matrix GLA protein (MGP) is one of the most potent inhibitors of VC and its activation is vitamin K dependent. The aim of this study is to investigate the role of oral vitamin K2 supplementation in the prevention of VC progression in haemodialysis patients. METHODS: We conducted a prospective randomized interventional study in patients on hemodialysis. Patients were randomly assigned to either receiving orally 200 µgr of vitamin K2 (vitamin K2/MK-7, Solgar) every day for 1 year or no treatment. Uncarboxylated MGP (uc-MGP) concentrations were quantified using ELISA at randomization, at 3 and at 12 months. Aortic calcification was evaluated using Agatston score after an abdominal computed tomography scan that was performed at the beginning and at 12 months of follow-up. RESULTS: There were 102 patients that were randomized. After 1 year of follow-up, 22 patients from the vitamin K2 group and 30 patients from the control group were included in the analysis. After 3 months of treatment, uc-MGP values remained unchanged in the vitK2 group but after 1 year were reduced by 47% (p = 0.005). Furthermore, uc-MGP at 1 year was increased by 12% in the control group. At 1 year, vitK2 group had significantly lower values of uc-MGP in comparison to controls (p = 0.03). Agatston score was increased significantly both in vitamin K2 and control group at 1 year with no difference between groups. CONCLUSIONS: Oral administration of vitamin K2 in patients on haemodialysis reduced serum uc-MGP levels but did not have an effect in the progression of aortic calcification.

Circulating anti-phospholipase A2 receptor antibodies as a diagnostic and prognostic marker in Greek patients with idiopathic membranous nephropathy - a retrospective cohort study.

INTRODUCTION: Circulating autoantibodies against phospholipase A2 receptor (anti-PLA2R) are recognized as key elements in the pathogenesis of idiopathic membranous nephropathy. In current clinical practice, they are increasingly gaining attention as novel tools for diagnosis and disease monitoring. We investigated the diagnostic and prognostic utility of anti-PLA2R antibody measurements in Greek patients with biopsy-proven membranous nephropathy. METHODS: Anti-PLA2R levels were measured in serum samples from 33 patients at diagnosis using ELISA and were associated with treatment outcome. Moreover, serial anti-PLA2R measurements were performed in 15 patients under different clinical conditions and level alterations were correlated with disease activity. RESULTS: Positive anti-PLA2R antibodies at diagnosis were found in 16 of 33 patients (48.5%). Anti-PLA2R levels were independently associated with the achievement of complete remission of nephrotic syndrome after immunosuppressive treatment compared to partial remission (p = 0.02, R2 = 0.265, 95%CI -0.019 to -0.0003). Higher detectable antibody levels at diagnosis were correlated with higher proteinuria levels (r = 0.813, p = 0.0001, 95%CI 0.532 to 0.933) and lower eGFR at the end of follow-up (r = -0.634, p = 0.0083, 95%CI -0.86 to -0.202). Serial antibody measurements during follow-up showed that anti-PLA2R titers were significantly reduced at the end of treatment after complete remission was achieved, remained low under sustained clinical remission, and increased during relapse. CONCLUSIONS: Our findings confirm the usefulness of anti-PLA2R measurements in the diagnosis of idiopathic membranous nephropathy. Low levels of anti-PLA2R antibodies at diagnosis are predictive of complete remission of nephrotic syndrome following immunosuppressive treatment. Serial anti-PLA2R measurements correlate well with clinical status throughout the follow-up period and could be used routinely for monitoring of disease activity and treatment planning.

Biomarkers in Progressive Chronic Kidney Disease. Still a Long Way to Go.

The traditional chronic kidney disease (CKD) biomarkers (eGFR based on serum creatinine, sex and age and albuminuria) cannot predict a patient's individual risk for developing progressive CKD. For this reason, it is necessary to identify novel CKD biomarkers that will be able to predict which patients are prone to develop progressive disease and discriminate between disease processes in different parts of the nephron (glomeruli or tubules). A good biomarker should change before or simultaneously with lesion development and its changes should correlate strongly with lesion development. Also, there should be a close relationship between severity of injury and amount of detectable biomarker and its levels should decrease with diminishing injury. Among the large number of molecules under investigation, we have reviewed the most promising ones: NGAL and KIM-1, MCP-1, MMP-9, clusterin, MMP-9, TIMP-1, Procollagen I alpha 1 and suPAR. All these, have been studied as biomarkers for prediction of CKD progression in cohorts of patients with chronic kidney disease of different stages and various aetiologies (proteinuric and non-proteinuric, glomerulonephritides, diabetic, hypertensive and polycystic kidney disease). There is evidence that these molecules could be useful as biomarkers for progressive chronic kidney disease, however, the available data are not enough to draw final conclusions. Further studies with large cohorts and long follow-up are required to identify appropriate biomarkers, that will be able to accurately and reliably define the risk for progressive chronic kidney disease.

ERß-Dependent Direct Suppression of Human and Murine Th17 Cells and Treatment of Established Central Nervous System Autoimmunity by a Neurosteroid.

Multiple sclerosis (MS), an autoimmune disease of the CNS, is mediated by autoreactive Th cells. A previous study showed that the neurosteroid dehydroepiandrosterone (DHEA), when administered preclinically, could suppress progression of relapsing-remitting experimental autoimmune encephalomyelitis (EAE). However, the effects of DHEA on human or murine pathogenic immune cells, such as Th17, were unknown. In addition, effects of this neurosteroid on symptomatic disease, as well as the receptors involved, had not been investigated. In this study, we show that DHEA suppressed peripheral responses from patients with MS and reversed established paralysis and CNS inflammation in four different EAE models, including the 2D2 TCR-transgenic mouse model. DHEA directly inhibited human and murine Th17 cells, inducing IL-10-producing regulatory T cells. Administration of DHEA in symptomatic mice induced regulatory CD4(+) T cells that were suppressive in an IL-10-dependent manner. Expression of the estrogen receptor ß by CD4(+) T cells was necessary for DHEA-mediated EAE amelioration, as well as for direct downregulation of Th17 responses. TGF-ß1 as well as aryl hydrocarbon receptor activation was necessary for the expansion of IL-10-producing T cells by DHEA. Thus, our studies demonstrate that compounds that inhibit pathogenic Th17 responses and expand functional regulatory cells could serve as therapeutic agents for autoimmune diseases, such as MS.

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-ß1) 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-ß1 and MCP-1 concentrations was observed in all GN patients. Increased urinary excretion of all tested cytokines apart from TNF-α and TGF-ß1 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-ß1 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-ß1 indicates the ongoing inflammatory and fibrotic processes in the kidney and is probably related to unfavourable outcomes.

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-ß1, 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.

Cytokines as Biomarkers of Treatment Response to IFN ß 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 IFNß 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-ß1 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 IFNß 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-ß1 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 IFNß treatment response.

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.

Rational design, efficient syntheses and biological evaluation of N,N'-symmetrically bis-substituted butylimidazole analogs as a new class of potent Angiotensin II receptor blockers.

A series of symmetrically bis-substituted imidazole analogs bearing at the N-1 and N-3 two biphenyl moieties ortho substituted either with tetrazole or carboxylate functional groups was designed based on docking studies and utilizing for the first time an extra hydrophobic binding cleft of AT1 receptor. The synthesized analogs were evaluated for their in vitro antagonistic activities (pA2 values) and binding affinities (-logIC50 values) to the Angiotensin II AT1 receptor. Among them, the potassium (-logIC50 = 9.04) and the sodium (-logIC50 = 8.54) salts of 4-butyl-N,N'-bis{[2'-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}imidazolium bromide (12a and 12b, respectively) as well as its free acid 11 (-logIC50 = 9.46) and the 4-butyl-2-hydroxymethyl-N,N'-bis{[2'-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}imidazolium bromide (14) (-logIC50 = 8.37, pA2 = 8.58) showed high binding affinity to the AT1 receptor and high antagonistic activity (potency). The potency was similar or even superior to that of Losartan (-logIC50 = 8.25, pA2 = 8.25). On the contrary, 2-butyl-N,N'-bis{[2'-[2H-tetrazol-5-yl)]biphenyl-4-yl]methyl}imidazolium bromide (27) (-logIC50 = 5.77) and 2-butyl-4-chloro-5-hydroxymethyl-N,N'-bis{[2'-[2H-tetrazol-5-yl)]biphenyl-4-yl]methyl}imidazolium bromide (30) (-logIC50 = 6.38) displayed very low binding affinity indicating that the orientation of the n-butyl group is of primary importance. Docking studies of the representative highly active 12b clearly showed that this molecule has an extra hydrophobic binding feature compared to prototype drug Losartan and it fits to the extra hydrophobic cavity. These results may contribute to the discovery and development of a new class of biologically active molecules through bis-alkylation of the imidazole ring by a convenient and cost effective synthetic strategy.

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.

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.

Activation of bacterial ribonuclease P by macrolides.

The effect of macrolide antibiotic spiramycin on RNase P holoenzyme and M1 RNA from Escherichia coli was investigated. Ribonuclease P (RNase P) is a ribozyme that is responsible for the maturation of 5' termini of tRNA molecules. Spiramycin revealed a dose-dependent activation on pre-tRNA cleavage by E. coli RNase P holoenzyme and M1 RNA. The K s and V max, as well as the K s(app) and V max(app) values of RNase P holoenzyme and M1 RNA in the presence or absence of spiramycin, were calculated from primary and secondary kinetic plots. It was found that the activity status of RNase P holoenzyme and M1 RNA is improved by the presence of spiramycin 18- and 12-fold, respectively. Primer extension analysis revealed that spiramycin induces a conformational change of the P10/11 structural element of M1 RNA, which is involved in substrate recognition.

DRpp20 and DRpp40: Two protein subunits involved in Dictyostelium discoideum ribonuclease P holoenzyme assembly.

Ribonuclease P is an essential enzyme that matures the 5' ends of all primary tRNA transcripts. RNase P enzymes contain a similar in size RNA subunit which is absolutely required for catalysis. The holoenzyme from Dictyostelium discoideum possesses an essential for activity RNA subunit but the exact protein composition is still under investigation. Bioinformatic analysis of D. discoideum sequencing data returned seven ORFs homologous to previously characterized RNase P protein subunits from human. In the present study, DRpp20 and DRpp40 were cloned and characterized. These proteins apart from the noted similarity possess idiosyncratic regions. Immunobiochemical analysis presented herein indicates their direct involvement in the formation of the ribonucleoprotein complex of D. discoideum RNase P holoenzyme.

A 40.7 kDa Rpp30/Rpp1 homologue is a protein subunit of Dictyostelium discoideum RNase P holoenzyme.

RNase P is an essential and ubiquitous endonuclease that mediates the maturation of the 5' ends of all precursor tRNA molecules. The holoenzyme from Dictyostelium discoideum possesses RNA and protein subunits essential for activity, but the exact composition of the ribonucleoprotein complex is still under investigation. Bioinformatic analysis of D. discoideum genome identified seven open reading frames encoding candidate RNase P protein subunits. The gene named drpp30 encodes a protein with a predicted molecular mass of 40.7 kDa that clusters with Rpp1 and Rpp30 RNase P protein subunits from Saccharomyces cerevisiae and human respectively, which have significantly lower molecular masses. Cloning and heterologous expression of DRpp30 followed by immunochemical analysis of RNase P active fractions demonstrates its association with RNase P holoenzyme. Furthermore, we show that DRpp30 can bind D. discoideum RNase P RNA and tRNA transcripts in vitro, giving a first insight of its possible role in D. discoideum RNase P function. Homology modeling using as a template the archaeal Ph1887p, and molecular dynamics simulations of the modeled structure suggest that DRpp30 adopts a TIM-barrel fold.