Decrease in 14-3-3η protein levels is correlated with improvement in disease activity in patients with rheumatoid arthritis treated with Tofacitinib.

14-3-3η protein is a proinflammatory mediator that may represent a novel diagnostic and prognostic biomarker for rheumatoid arthritis (RA). We assessed the correlation between changes in serum 14-3-3η levels and changes in clinical disease activity measures in RA patients treated with Tofacitinib (TOF). Paired serum samples from 35 patients with RA were obtained at baseline and 5 months after the initiation of treatment with TOF. The levels of 14-3-3η were measured by JOINT stat 14-3-3η ELISA test kits (Augurex Life Sciences Corp.). The cut-off was defined as 0.19 ng/ml. 14-3-3η positivity was found in 57% of the patients at baseline and in 37% of the patients after 5 months of treatment. Mean ± SD baseline 14-3-3η levels [4.92 ± 8.86 ng/ml] were significantly higher (p < 0.005) than 14-3-3η levels following treatment [1.97 ± 4.59 ng/ml]. A statistically significant improvement (p < 0.001) of CDAI, SDAI, DAS4ESR and DAS4CRP was achieved after 5 month of treatment. Decrease in 14-3-3η protein levels was highly correlated with improvement in DAS4ESR (r = 0.50, p < 0.01), DAS4CRP (r = 0.46, p < 0.01) and ESR (r = 0.36, p = 0.03) and moderately correlated with improvement in CDAI (r = 0.32, p = 0.065) and SDAI (r = 0.33, p = 0.051). The correlation between decrease in 14-3-3η levels and improvement in DAS4ESR remained significant in a partial correlation analysis controlling for ESR (r = 0.39, p = 0.02). This study demonstrates that in RA patients who were treated with TOF, decrease in 14-3-3η levels is correlated with improvement in clinical disease activity parameters. The 14-3-3η protein may serve as an objective biomarker for monitoring of TOF therapy response.

14-3-3η: a novel biomarker platform for rheumatoid arthritis.

14-3-3 proteins are a conserved family of 7 isoforms with diverse cellular functions found predominantly intracellularly. The 14-3-3η isoform is expressed extracellularly in the joints of patients with rheumatoid arthritis (RA) and expression in both serum and joint fluid correlates strongly with expression of metalloproteinases. 14-3-3η activates proinflammatory signalling cascades and inflammatory mediators relevant to the pathogenesis of RA. A new ELISA based assay has diagnostic utility for RA with sensitivity of 63.6% and specificity of 92.6% using the optimal cut-off from ROC analysis of 0.19ng/ml. Adding 14-3-3η to anti-cyclic citrullinated peptide antibodies (ACPA) resulted in an identification rate of 72% compared to 59% for ACPA alone. Adding rheumatoid factor (RF) to ACPA increased diagnostic capture from 59% to 72% and this increased further to 78% when 14-3-3η was added. Positive 14-3-3η status is also significantly associated with radiographic progression in early RA at years 1, 3 and 5 indicating prognostic utility. Extracellular 14-3-3η elicits the production of autoantibodies to the native protein, which also possess diagnostic utility. These do not correlate with expression of the protein and have complementary diagnostic utility. The presence of either the protein or its autoantibodies is observed in 90% of patients with early RA. Together with RF and/or ACPA this may result in identification of 95% of patients with early RA.

Metabarcoding analysis of gut microbiota of healthy individuals reveals impact of probiotic and maltodextrin consumption.

In a previously published double-blind, placebo-controlled study, we showed that probiotics intake exerted a positive effect on sleep quality and a general improvement across time in different aspects of the profile of mood state, like sadness, anger, and fatigue in 33 healthy individuals. The present work investigates the impact of the probiotic product, constituted of Limosilactobacillus fermentum LF16, Lacticaseibacillus rhamnosus LR06, Lactiplantibacillus plantarum LP01 (all former members of Lactobacillus genus), and Bifidobacterium longum 04, on the gut microbiota composition of the same cohort through a metabarcoding analysis. Both the placebo and probiotic treatments had a significant impact on the microbiota composition. Statistical analysis showed that the microbiota of the individuals could be clustered into three groups, or bacteriotypes, at the baseline, and, inherently, bacterial compositions were linked to different responses to probiotic and placebo intakes. Interestingly, L. rhamnosus and L. fermentum were retrieved in the probiotic-treated cohort, while a bifidogenic effect of maltodextrin, used as placebo, was observed. The present study shed light on the importance of defining bacteriotypes to assess the impact of interventions on the gut microbiota and allowed to reveal microbial components which could be related to positive effects (i.e. sleep quality improvement) to be verified in further studies.

Kinesin is the motor for microtubule-mediated Golgi-to-ER membrane traffic.

The distribution and dynamics of both the ER and Golgi complex in animal cells are known to be dependent on microtubules; in many cell types the ER extends toward the plus ends of microtubules at the cell periphery and the Golgi clusters at the minus ends of microtubules near the centrosome. In this study we provide evidence that the microtubule motor, kinesin, is present on membranes cycling between the ER and Golgi and powers peripherally directed movements of membrane within this system. Immunolocalization of kinesin at both the light and electron microscopy levels in NRK cells using the H1 monoclonal antibody to kinesin heavy chain, revealed kinesin to be associated with all membranes of the ER/Golgi system. At steady-state at 37 degrees C, however, kinesin was most concentrated on peripherally distributed, pre-Golgi structures containing beta COP and vesicular stomatitis virus glycoprotein newly released from the ER. Upon temperature reduction or nocodazole treatment, kinesin's distribution shifted onto the Golgi, while with brefeldin A (BFA)-treatment, kinesin could be found in both Golgi-derived tubules and in the ER. This suggested that kinesin associates with membranes that constitutively cycle between the ER and Golgi. Kinesin's role on these membranes was examined by microinjecting kinesin antibody. Golgi-to-ER but not ER-to-Golgi membrane transport was found to be inhibited by the microinjected anti-kinesin, suggesting kinesin powers the microtubule plus end-directed recycling of membrane to the ER, and remains inactive on pre-Golgi intermediates that move toward the Golgi complex.

The diagnostic value of 14-3-3η protein levels in patients with rheumatoid arthritis.

14-3-3η may represent a useful diagnostic biomarker for rheumatoid arthritis (RA). We assessed the prevalence and serum levels of 14-3-3η in patients with RA and in patients with other rheumatic diseases. Serum levels of 14-3-3η were measured in 96 patients with RA, in 101 patients with other rheumatic diseases, and in 66 healthy subjects. All of the sera samples were evaluated by JOINT stat 14-3-3η ELISA test kits (Augurex Life Sciences Corp.). Median (IQR) 14-3-3η levels were significantly higher in the early RA group [0.25 ng/ml (0.075-3.11)] and in patients with established RA [0.15 ng/ml (0.08-1.26)] than in healthy subjects [0 ng/ml (0-0)] and disease controls: SLE [0.01 ng/ml (0-0.055)], AS [0.05 ng/ml (0-0.255)], and PsA [0.01 ng/ml (0-0.065)]. The prevalence of 14-3-3η positivity in patients with early RA was 58%, significantly higher than that in disease controls and healthy subjects (p < 0.001). In patients with established RA, this prevalence was 43%, and it was significantly higher than that in patients with other rheumatic diseases and healthy subjects (p < 0.05), excluding the AS group (p = 0.054). In the early RA cohort, the positivity for 14-3-3η, RF, and anti-CCP was 58%, 67%, and 71%, respectively. Eighty-two percent of the patients in this cohort were positive for at least one of these biomarkers. The concentration of 14-3-3η protein may be used to distinguish between patients with early RA and patients with other rheumatic diseases.

Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.

Microtubule disruption has dramatic effects on the normal centrosomal localization of the Golgi complex, with Golgi elements remaining as competent functional units but undergoing a reversible "fragmentation" and dispersal throughout the cytoplasm. In this study we have analyzed this process using digital fluorescence image processing microscopy combined with biochemical and ultrastructural approaches. After microtubule depolymerization, Golgi membrane components were found to redistribute to a distinct number of peripheral sites that were not randomly distributed, but corresponded to sites of protein exit from the ER. Whereas Golgi enzymes redistributed gradually over several hours to these peripheral sites, ERGIC-53 (a protein which constitutively cycles between the ER and Golgi) redistributed rapidly (within 15 minutes) to these sites after first moving through the ER. Prior to this redistribution, Golgi enzyme processing of proteins exported from the ER was inhibited and only returned to normal levels after Golgi enzymes redistributed to peripheral ER exit sites where Golgi stacks were regenerated. Experiments examining the effects of microtubule disruption on the membrane pathways connecting the ER and Golgi suggested their potential role in the dispersal process. Whereas clustering of peripheral pre-Golgi elements into the centrosomal region failed to occur after microtubule disruption, Golgi-to-ER membrane recycling was only slightly inhibited. Moreover, conditions that impeded Golgi-to-ER recycling completely blocked Golgi fragmentation. Based on these findings we propose that a slow but constitutive flux of Golgi resident proteins through the same ER/Golgi cycling pathways as ERGIC-53 underlies Golgi Dispersal upon microtubule depolymerization. Both ERGIC-53 and Golgi proteins would accumulate at peripheral ER exit sites due to failure of membranes at these sites to cluster into the centrosomal region. Regeneration of Golgi stacks at these peripheral sites would re-establish secretory flow from the ER into the Golgi complex and result in Golgi dispersal.

Protein kinase CK2 is involved in G2 arrest and apoptosis following spindle damage in epithelial cells.

p53 undergoes phosphorylation on several residues in response to cellular stresses that include UV and ionizing radiation, however the influence of spindle damage on this parameter is relatively unclear. Consequently, the effect of nocodazole on serine 392 phosphorylation was examined in two epithelial cell lines. We show that this process is dependent upon the stepwise activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase casein kinase 2 (CK2). Furthermore, this activation correlated with the biochemical regulation of the maturation-promoting factor (MPF, cdc2/cyclin B), as both DRB and antisense depletion of CK2, as well as SB203580 were associated with an inhibition of its activation in response to nocodazole. Strikingly, when the cell cycle characteristics of nocodazole treated cells were examined, we observed that depletion or inhibition of the catalytic subunit of CK2, in the presence of microtubule inhibitors, resulted in a compromise of the G2 arrest (spindle checkpoint). Furthermore, CK2-depleted, nocodazole treated cells demonstrated a dramatic reduction in the apoptotic cell fraction, confirming that these cells had been endowed with oncogenic properties. These changes were observed in both HeLa cells and HCT116 cells. We also show that this effect is dependent on the presence of functional wild-type p53, as this phenomenon is not apparent in HCT116 p53(-/-) cells. Collectively, our results indicate two novel roles for CK2 in the spindle checkpoint arrest, in concert with p53. Firstly, to maintain increased cyclinB/cdc2 kinase activity, as a component of G2 arrest, and secondly, a role in p53-mediated apoptosis. These findings may have implications for an improved understanding of abnormalities of the spindle checkpoint in human cancers, which is a prerequisite for defining future therapies.

Dysregulation of integrin-linked kinase (ILK) signaling in colonic polyposis.

Mutation of the adenomatous polyposis coli (APC) gene and the subsequent dysregulation of beta-catenin are well-documented abnormalities in familial adenomatous polyposis (FAP), as well as sporadic polyposis. Intriguingly, overexpression of the integrin-linked kinase (ILK) has been shown to modulate beta-catenin subcellular localization and function. However, the significance of this finding for human carcinogenesis remains unclear. Here, we report the increased biochemical activity and expression of ILK protein in polyps from FAP patients. Furthermore, dramatic increases in ILK immunoreactivity were observed in all abnormal crypts from sporadic polyps, when compared with the normal appearing crypts within the same resected specimens. As sulindac and aspirin are the two most important therapeutic/chemopreventative agents demonstrated in colorectal carcinogenesis, in both humans and animals, further investigation revealed that these non-steroidal anti-inflammatory drugs (NSAIDs) target ILK and ILK-mediated events in vivo. These include inhibition of, both the biochemical activation of ILK, inhibition of serine 9 GSK3beta phosphorylation and the enhancement of TCF-4 transcriptional activity. In conclusion, ILK protein hyperexpression appears to be an early event in colonic polyposis. Additionally, ILK signaling is shown to undergo modulation by sulindac (and aspirin) for the first time, indicating that it is likely to be one of the targets affected by these agents in vivo.

Differential cyclin-dependent kinase expression and activation in human colon cancer.

BACKGROUND: Abnormalities of the cyclin-dependent protein kinase (CDK) machinery have been linked to cancer development. Hyperphosphorylation of the retinoblastoma (Rb) protein results in release from inhibition of progression through the G1 phase of the cell cycle. Hyperexpression of CDK1 and CDK2 may enable progression through late G1, S and the G2 phases of the cell cycle. METHODS/RESULTS: To investigate tumor-associated protein kinase activities, control and tumor samples were fractionated by MonoS chromatography and tested for their ability to phosphorylate histone H1. Two major peaks of histone H1 phosphotransferase activity were resolved. The first appeared in the flow through fractions, and occasionally showed enhanced activity in the tumor samples, whilst the second was consistently increased and eluted at approximately 0.4 M NaCl. Western immunoblotting with CDK1 and PSTAIRE antibodies confirmed the co-elution of CDK1 and CDK2 with the second peak. Next, the phosphotransferase activities (following specific immunoprecipitation) and protein levels of CDK1, 2, 4, and 6 were determined in human colon cancer samples and their respective controls. CDK4 activity was elevated in only 3 of 7 tumor samples (range 40-160%) relative to control samples from the same patients, whereas a significant increase in CDK6 activity was observed in the same group (p < 0.05). This contrasted sharply with the universal activations of CDK1 (up to 18-fold, p < 0.01, n = 12) and CDK2 (up to 17-fold, p < 0.05) in the same groups. CONCLUSIONS: CDK1 especially, and to a lesser extent CDK2 and CDK6 demonstrate the most consistent biochemical activation in human colon cancer and may represent targets for pharmacological intervention. Cellular proliferation as gauged by MIB1 was not directly correlated with the amplitude of activation.

Investigation of the Mek-MAP kinase-Rsk pathway in human breast cancer.

BACKGROUND: Mitogenic signaling through the principal growth factor receptor tyrosine kinase (RTK) pathway, i.e. RTK-->Ras-->Raf-->Mek-->MAPK has been implicated in the pathogenesis of human cancer. However, biochemical characterization of this has not been adequately assessed in human cancers. MATERIALS AND METHODS: Using extracts from 23 human breast cancers and control tissue from the same resected specimens, the protein levels, phosphotransferase activities and subcellular locations of the mitogen-activated protein (MAP) kinase isoforms p42 Erk2 and p44 Erk1 were examined, together with their phosphotransferase activities towards myelin basic protein (MBP) and a peptide substrate patterned after the Thr-669 site in the epidermal growth factor receptor (EGFR T669) that is phosphorylated by MAP kinase. RESULTS: Overexpression of both Erk2 and Erk1 isoforms was evident using specific antibodies. A universal activation of MBP and EGFR T669 peptide phosphotransferase activities was also found (up to 3-fold). MonoQ fractionation resolved the bulk of the EGFR T669 peptide phosphorylation from elution of the MAP kinase protein. Erk1 and Erk2 activities determined by specific immunoprecipitation were increased by up to only 2.5-fold in only 50% of tumors overall. Immunohistochemical studies, using a monoclonal antibody specific for Erk2 demonstrated that the cellular distribution of this MAP kinase was similar in both control and tumor tissues, and Erk2 was largely confined to normal and malignant acini, whilst the intensity of staining was actually reduced in the tumor tissue. Mek1 and especially Mek2 protein expression, as well as MAP kinase kinase activity as determined by phosphorylation of kinase-inactive Erk [GST-K71A] were increased in cancer samples. CONCLUSIONS: a) This confirms that MAP kinase activity is increased in human breast cancer. However, the frequency and magnitude of this change is dependent upon the chosen methodology (i.e. crude lysate assays versus specific immunoprecipitation). b) A MAP-kinase-independent source of increased EGFR T669 phosphotransferase activity in tumor extracts has been demonstrated for the first time in human breast cancer. c) By immunohistochemistry, Erk2 protein was actually found to exhibit lower intensity in tumor samples; the increased expression was most likely due to its increased distribution. d) Increased Mek protein expression and activation have been demonstrated for the first time in human breast tumors.

Cerebrospinal fluid beta-2-microglobulin in HIV-1 infection, as a marker of neurological involvement.

Cerebrospinal fluid (CSF) and serum concentration of beta-2-microglobulin (beta-2-m) were evaluated in 30 patients in various stages of HIV-1 infection. CSF beta-2-m and CSF/serum ratio were significantly higher in patients with neurological complications respect to asymptomatic subjects. These findings indicate that CSF beta-2-m and CSF/serum ratio may be a useful marker of neurological involvement in HIV-1 infection.

Total anomalous left pulmonary venous connection with intact atrial septum: surgical treatment of a rare case.

Total anomalous left pulmonary venous connection with intact atrial septum is an extremely rare form of congenital heart disease. We describe a case never reported before in which the left pulmonary veins drained directly into the right atrium through a common vein. Surgical correction was successfully obtained by redirecting the blood flow through a newly created atrial septal defect into the left atrium using a gluteraldehyde-treated autologous pericardial patch. Follow-up at 6 months shows no signs of any complication.

Tolerance to salt stress in maize callus lines with different polyamine content.

Four callus lines from immature embryos of a self-crossed maize (Zea mays L.) hybrid cultivar were selected for "high" (two lines) and "low" (two lines) polyamine (PA) levels. Each selected line was exposed to culture media containing no (control) or 1% (0.171 M) NaCl and the relative growth rates were compared after subculture. Low-PA lines appeared to be tolerant to salt stress, while high-PA lines were sensitive. Analysis of PA at the end of the subculture showed that treated calli of sensitive lines had increased their putrescine content in comparison with their control, while putrescine remained constant in tolerant lines. Callus lines were analysed by RAPD (random amplification of polymorphic DNA) markers. One polymorphism (550-bp band) was found, demonstrating a genetic difference between the lines.

Epidemiological study of blood lead levels of children and adolescents living in Campania, Italy.

The aim of the present study is to evaluate blood levels (PbB) in a group of 500 (245 M, 255 F) children and adolescents of Campania (Italy) aged from 0.197 to 16.915 years, 269 (136 M, 133 F) of whom lived in urban zones and 231 (109 M, 122 F) in rural zones. PbB was assayed by electrothermal atomic absorption spectroscopy. The parents of the examined subjects children were interviewed about common risk factors for lead exposure using a standardized questionnaire. The PbB of children living in urban zones were significantly higher than the PbB of those living in rural zones (60.0 +/- 3.0 mg/L vs. 40.0 +/- 2.0 mg/L, p < 0.001). A PbB higher than 100 mg/L was found in 27 children (5.4%). We observed a significant correlation between age and PbB (p < 0.001, r = 0.529). Our data regarding children and adolescents demonstrate that the prevalence of PbB higher than 100 mg/L is greater in children living in urban areas (6.89%) than in subjects living in rural areas (3.89%). The findings can be explained by the higher presence of risk factors of Pb exposure in urban areas. Our data, if compared with those of previous studies concerning children of Campania, show a clear decrease of PbB. The correlation that we found between age and PbB indicates that long-term exposure at low doses more than a more intensive but short-term exposure seems to be important for the increase of blood lead levels.

Unchanged iron and copper and increased zinc in the blood of obese children after two hypocaloric diets.

Serum iron (sFe), and ferritin (sFert), transferrin saturation index (TSI), plasma zinc and copper (pZn, pCu), and erythrocyte zinc content (eZn) were measured in 55 obese children and adolescents (28 males and 27 females) before and after a 13-wk treatment with a hypocaloric balanced diet (HCBD, 22 subjects) or a 10-wk treatment with a protein sparing modified fast diet (PSMF, 33 subjects). The energy intake provided by the HCBD and PSMF diet was calculated to be 60 and 25%, respectively, of the recommended dietary allowances (RDAs) for age and sex. Neither diet was supplemented with trace elements or calcium. Using a visual memory system, all subjects had a 24-h dietary intake recall before starting the weight-loss program. Iron, zinc, and copper intakes from the 24-h recall were compared with those from prescribed diets. Both diets produced a significant (p < 0.001) weight reduction with a significant reduction in the arm muscle area of the PSMF group. After treatment, no significant change was observed in sFe, sFert, and TSI of either group, whereas eZn increased significantly in the HCBD and the PSMF groups (p = 0.001 and p < 0.006, respectively), with an improvement of the erythrocyte index (E.I.). A significant increase in pZn was also observed in the PSMF group (p = 0.007).

[Epidemiological study of blood lead levels in young subjects in the Campania region: preliminary data]. / Studio epidemiologico sui livelli di piombo ematico in soggetti in età evolutiva della regione Campania: dati preliminari.

Blood lead levels were assayed in 261 children (133 males and 128 females) living in Campania, 137 (63 females and 74 males) in urban areas and 124 (65 females and 59 males) in rural zones, aged between 0.197 and 16.863 years. Blood lead determination was carried out by electrothermal atomic absorption spectroscopy. All children were interviewed about common risk factors for lead exposure. PbB (median +/- SD) were significantly higher in the urban than in the rural population (6.0 +/- 0.31 vs 3.75 +/- 0.25 micrograms/100 ml; p < 0.001). The frequency of blood lead level above 10 micrograms/100 ml was 4.21% in our tested group, i.e., significantly lower than in previous studies. A significant direct correlation between blood lead levels and age was found (r = 0.47; p < 0.001). In agreement with the literature on this subject, our findings show a significant reduction with time, of blood lead levels of children and adolescents in our region. Time of exposure more than total dose seems to be important for the increase of blood lead level.

[Total quality: priority objective to improve the services of health facilities]. / Qualità totale: obiettivo prioritario per migliorare le prestazioni delle aziende sanitarie.

The increasing attention turned to the "total quality" inside health services is pointed out in the article. The development of this theme started in the United States and subsequently reached Europe. In Italy only since a few years we are noticing initiatives aiming both to the improvement of health services quality and to the relationship with clients. The main elements of the total quality management are pointed out: they are addressed to the satisfaction of clients expectations in an environment involving the whole health personnel towards the continuous quality improvement.

Sol-gel processing of drug delivery materials and release kinetics.

Silica, calcium (5 mol%) silicate and silica/polycaprolactone hybrid inorganic/organic amorphous materials, all mixed with sodium ampicillin, a broad-spectrum antibiotic, have been synthesized by sol-gel method. The amorphous nature of the gels was ascertained by X-ray diffraction analysis. Release kinetics in a simulate body fluid (SBF) have been subsequently investigated. The amount of sodium ampicillin released has been detected by UV-Vis spectroscopy and SEM. The release kinetics seems to occur in more than one stage. Finally FTIR measurements and SEM micrograph showed the formation of a hydroxyapatite layer on the surface of the samples soaked in SBF. All data showed that these materials could be used as drug delivery bioactive systems.