Real-Time PCR and Droplet Digital PCR: two techniques for detection of the JAK2(V617F) mutation in Philadelphia-negative chronic myeloproliferative neoplasms.

INTRODUCTION: Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are clonal disorders that present JAK2(V617F) mutation in 50-95% of cases. The main objective of this study was the comparison of two PCR methods, real-time (qPCR) and droplet digital PCR (DD-PCR) for detection of the JAK2(V617F) mutation, to assess analytic sensitivity, specificity, and feasibility of the two methods. METHODS: Ninety-nine patients with MPN of 225 presenting the JAK2(V617F) mutation by qPCR have been evaluated by DD-PCR also. RESULTS: We demonstrated an absolute concordance in terms of specificity between the two methods, DD-PCR showing a higher sensitivity (half a log higher than qPCR). As expected, a progressive increase of mutant allele burden was observed from essential thrombocythemia (ET) to polycythemia vera (PV) and primary myelofibrosis (PMF) to secondary myelofibrosis (SMF). CONCLUSION: In conclusion, our study showed that DD-PCR could represent a new and promising technological evolution for detection of JAK2 mutation in MPNs.

Effects of cigarette smoke on salivary superoxide dismutase and glutathione peroxidase activity.

Cigarette smoke contains oxidants such as oxygen-free radicals and volatile aldehydes, which are probably the major causes of damage to biomolecules exposed to cigarette smoke. However, saliva has an antioxidant defense system able to counter toxic activities of radical species that is formed by antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The purpose of this study is to verify the possible effects of cigarette smoke on SOD and GSH-Px. Forty-four patients (25 males and 19 females) were enrolled in this study. The participants were 20 smokers (12 males and 8 females) and 24 non-smokers (13 males and 11 females). Furthermore, 10 subjects of the control group were ex-smokers (9 males and 1 female). Their mean age plus or minus standard deviation (SD) was 58.8 plus or minus 15.9 years for the case group and 73.8 plus or minus 10.6 years for the control group. All patients were underwent a careful anamnestic investigation and examination of the oral cavity. After rinsing the mouth with water, each subject put 3 cc of non-stimulated saliva inside a test tube. The saliva was centrifuged and oral peroxidase and superoxide dismutase activity was measured according to a specific assay. Statistical analysis was performed to evaluate differences between the groups and significant differences were observed for p less than 0.05. A significant decrease of GSH-Px activity was detected in the smoking group (p less than 0.05), while the SOD activity was similar in the control and case groups. According to the sex, a significant decrease of GSH-Px activity was noted in males of the smoker group (p less than 0.05), while in the sample of females no significant difference of the enzymatic activity was found. Moreover, among ex-smokers, there was a significant difference in the values of GSH-Px between those who had not smoked for less than ten years and those who had not smoked for more than ten years. Cigarette smoke may alter the detoxification of hydrogen peroxide through a decrease of GSH-Px activity. The overproduction of H2O2 may lead to an oxidative stress that is involved in a large number of diseases, including precancerous and neoplastic lesions of the oral cavity. The effects of cigarette smoke on salivary antioxidant enzymes decrease after withdrawal from smoking and the benefits become more evident with the passage of time.

Long-term monitoring of cell-mediated immunity in disease-free breast cancer patients: a preliminary retrospective study.

In 102 N- and 44 N+ disease-free breast cancer patients, lymphocytic populations and skin reaction of delayed hypersensitivity (SRDH) were monitored up to 266 months after mastectomy to find out whether they were similar or different from control values. In two selected groups of 34 N- and 11 N+ breast cancer patients, the whole 10 year follow-up was divided into three subintervals, each of them lasting 40 months and the time course of lymphocytic populations was evaluated. In the 102 N- patients, mean CD4+, CD8+, CD3+ values were lower (P < 0.01, P < 0.001, P < 0.01, respectively) while CD4+/CD8+ ratio was higher (P < 0.05) than in controls. Fifteen N- breast cancer patients (16%) were anergic compared to 30(32%) of controls (P < 0.05). In the 34 selected N- breast cancer patients soon after mastectomy the mean value of CD4+, CD8+, CD3+ T subpopulations was lower (P < 0.01, P < 0.001, P < 0.01, respectively) than in controls. Successively their mean value increased so that in the last subinterval they were not or were only slightly lower (P n.s., P < 0.05, P < 0.05, respectively) than in controls. In the 44 N+ patients, mean CD4+, CD8+, CD3+ values were lower (P < 0.001, v < 0.05, P < 0.01, respectively) and CD19+ lymphocytes higher (P < 0.001) than in controls. Five N+ breast cancer patients (13%) were anergic compared to 32% of controls (P < 0.05). In the 11 selected N+ breast cancer patients soon after mastectomy, the mean value of CD4+, CD8+ T subpopulations and CD16+56+ cells was significantly lower (P < 0.001, P < 0.001, P < 0.01, respectively) than in controls. Successively their mean value constantly increased so that in the last subinterval, no or slight (P n.s., P < 0.05, P n.s., respectively) significant difference compared to controls occurred. The mean CD4+/CD8+ ratio value of N- patients was significantly higher than in controls. However in the last subinterval, the significance was lower than in the first one (P < 0.05 and P < 0.01, respectively). In the N+ patients, the mean value of CD4+/CD8+ ratio was constant, although not significantly, lower than in controls; however it progressively increased from the first to the last subinterval. Therefore the significance of the difference of the mean CD4+/CD8+ ratio between N- and N+ patients strongly decreased from the first to the last subinterval (P < 0.001 and P < 0.05, respectively). These data indicate that in breast cancer patients, following mastectomy, a significant activation of memory and CD4+ T cells and long-term decrease of the circulating immunocompetent CD4+, CD8+ and CD16+56+ cells occurs. The prolonged disease-free interval observed in the 34 N- and 11 N+ breast cancer patients can be correlated with the restoration of the normal state of cell-mediated immunity.

C reactive protein in patients with chronic renal diseases.

Base-line serum levels of plasma C-reactive protein (CRP) are predictive of future myocardial infarction and sudden cardiac death in apparently healthy subjects, suggesting the hypothesis that chronic inflammation might be important in the pathogenesis of atherothrombosis. CRP production is mediated by several inflammatory mediators: interleukin 6 (IL-6) is currently felt to be the major cytokine influencing the acute phase response. CRP and other acute phase proteins are elevated in dialysis patients and cardiovascular diseases represent the single largest cause of mortality in chronic renal failure patients. Little information is available, however regarding CRP and IL-6 plasma levels in pre-dialysis renal failure. Plasma CRP was determined by a modification of the laser nephelometry technique; IL-6 by immunoassay (RD System); and fibrinogen, serum albumin, cholesterol, triglycerides, hematocrit, white blood cell count, erythrocytic sedimentation rate (ESR) and urinary protein levels by standard laboratory techniques. Results were obtained in 102 chronic pre-dialysis patients whose mean age was 53+/-5.8 years with a mean creatinine clearance (C(Cr)) of 52+/-37 mL/min). CRP was greater than 5 mg/L in 25% of the global population. CRP and IL-6 were 4.0+/-4.6 mg/L and 5.8+/-5.6 pg/mL, respectively and were not significantly correlated (r=0.11, p=n.s.). CRP and IL-6 were however related with renal function (CRP versus C(Cr) r=-0.40 p <0.001; IL- 6 versus C(Cr) r=-0.45; p <0.001). When patients were divided in two groups according to renal function, CRP resulted 7.4+/-6.3 mg/L in the group of patients with a C(Cr) lower than 20 mL/min (n=32) and 2.76+/-4.35 in the group of patients with a C(Cr) higher than 20 mL/min (n = 70) (p <0.0001). CRP and IL-6 were positively related with ESR (r=0.32 and 0.46 respectively). Serum albumin levels were not significantly different in the two groups of patients (3.2+/-0.4 versus 3.0+/-0.5 g/dL). CRP and serum albumin were not significantly related (r=0.17). CRP and IL-6 correlated positively with ESR (r=0.32 and 0.46 respectively). In pre-dialysis patients we have demonstrated an increase in both CRP and IL-6 that occurs as renal function decreases. These data provided evidence of the activation - even in the predialysis phase of renal failure - of mechanisms known to contribute to the enhanced cardiovascular morbidity and mortality of the uremic syndrome.

[Observations on the increased serum levels of YKL-40 in patients with rheumatoid arthritis and osteoarthritis]. / Considerazioni sul riscontro di livelli aumentati di YKL-40 nel plasma di pazienti con artrite reumatoide ed osteoartrosi.

BACKGROUND: This study aimed to evaluate the function of glycoprotein YKL-40 in patients with rheumatoid arthritis, erosive osteoarthrosis or coxitis. METHODS: Serum levels of glycoprotein YKL-40 were assayed using an ELISA method in 40 patients with active-phase rheumatoid arthritis and 20 patients suffering from erosive osteoarthrosis or coxitis. RESULTS: The mean values of YKL-40 were enhanced in all patients with rheumatoid arthritis and arthrosis with higher mean values in erosive osteoarthritis and coxitis (440 ng/ml) compared to rheumatoid arthritis (190 ng/ml). CONCLUSIONS: YKL 40 is a local prognostic marker of joint destruction.

The link of biocompatibility to cytokine production.

Recent studies suggest that chronic inflammation plays a role in the pathogenesis of cardiovascular disease. Cytokines released from jeopardized tissues stimulate the liver to synthesize acute phase proteins, including C-reactive protein (CRP). Baseline levels of CRP in apparently healthy persons or in persons with unstable angina constitute an independent risk factor for cardiovascular events. More recently, it has been suggested that CRP is useful not only as a marker of the acute phase response, but is also involved in the pathogenesis of the disease. CRP may, in fact, directly interact with the atherosclerotic vessels or ischemic myocardium by activation of the complement system, thereby promoting inflammation and thrombosis. Several studies in uremic patients have implicated CRP as a marker of malnutrition, resistance to erythropoietin, and chronic stimulation in hemodialysis. An increased cytokine production secondary to blood interaction with bioincompatible dialysis components has been reported by several studies; interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and mainly IL-6 are the three proinflammatory cytokines involved in the pathogenesis of hemodialysis-related disease. We have provided evidence for the occurrence of high CRP and IL-6 levels in chronic dialytic patients exposed to contaminate dialysate and suggest that backfiltration may induce a chronic, slowly developing inflammatory state that may be abrogated by avoiding backfiltration of contaminate dialysate. Therefore, CRP is implicated as a marker linking bioincompatibility associated with backfiltration and increased cytokine production with a clinical state of chronic inflammation.

Plasma C-reactive protein in hemodialysis patients: a cross-sectional, longitudinal clinical survey.

In hemodialysis patients, C-reactive protein (CRP), an acute-phase reactant, is a sensitive and independent marker of malnutrition, anemia, and amyloidosis. The aim of the present studies was to evaluate CRP and interleukin 6 levels in plasma samples from long-term hemodialysis patients on different extracorporeal modalities associated with or without backfiltration. Two hundred and forty-seven patients were recruited in eight hospital-based centers. All patients had been on their dialytic modality for at least 6 months. At enrollment, 46 hemodialysis patients out of 247 (18.6%) had clinical evidence of pathologies known to be associated with high CRP values. The 201 remaining patients were defined as clinically stable and were on conventional hemodialysis (34%), hemodiafiltration with infusion volumes <10 liters/session (10%), hemodiafiltration with infusion volumes <20 liters/session (32%), and double-chamber hemodiafiltration with infusion volumes <10 liters/session (22%). Analysis of CRP values in the clinically stable patients showed that an unexpectedly high proportion (47%) of the patients had CRP values higher than 5 mg/l (taken as the upper limit in normal human subjects). The values of CRP and interleukin 6 were significantly higher in hemodiafiltration with infusion volumes <10 liters/session than in hemodiafiltration with infusion volumes >20 liters/session, in hemodialysis and in double-chamber hemodiafiltration. The same pattern occurred after 6 months of follow-up in 171 out of 201 clinically stable patients. Hemodialytic conditions that expose to the risk of backfiltration such as low exchange volume hemodiafiltration may induce a chronic inflammatory state as reflected by increased plasma values of both CRP and interleukin 6, thus suggesting the need for hemodialytic strategies that reduce (hemodialysis with low-permeability membranes or hemodiafiltration with infusion volumes >20 liters) or eliminate (double-chamber hemodiafiltration) backfiltration of bacteria-derived contaminants.

Plasma C-reactive protein in haemodialysis.

In recent years, acute phase reactants have been reevaluated as not merely biochemical markers of inflammation but also as active modulators of the inflammatory response. C-reactive protein - which is normally present in serum in only trace amounts, but whose concentration may rise markedly with inflammatory stimuli - was the first human acute phase protein discovered. It is now clear that cytokines are the major mediators of acute phase protein induction: interleukin-6 currently is felt to be the principal cytokine influencing C-reactive protein acute changes. Several studies have provided convincing evidence that among normal men, base-line serum levels of C-reactive protein are predictive of future myocardial infarction and ischemic stroke. The relevance of acute phase reactants in morbidity and mortality of haemodialysis patients has not been fully elucidated until now: in fact a few studies have implicated C-reactive protein in malnutrition, EPO-resistance, as a cardiovascular risk factor and as a marker of chronic stimulation in haemodialysis. The authors suggest the hypothesis of the occurrence of long-term complications in patients exposed to contaminated dialysate and suggest that back-filtration may induce a chronic, slowly developing inflammatory state that may be abrogated by avoiding backfiltration of contaminated dialysate.