F. Bamonti-Catena

Soluble cytokine levels correlate with the activity and clinical stage of Hodgkin's disease at diagnosis

The serum levels of some cytokines seem to correlate with outcome in Hodgkins disease (HD) and may be helpful in formulating new and better prognostic systems. The aim of this study was to analyse the correlations between the serum levels of different cytokines and the clinico-hematological features suggestive of a worse prognosis. The study involved 31 pts with a “de novo” diagnosis of HD (median age: 30 yrs; M/F: 13/18; stage I/II vs III/IV: 19/12; B symptoms: 12; bulky disease and extranodal disease: 9). The serum levels of sCD30, TNFα, TNF receptor I and II, IL6, IL6 receptor, IL10, sICAM-1 were evaluated at diagnosis, and correlated with gender, age (</>30), stage (I-II vs III-IV), systemic symptoms, bulky disease, ESR (</≥40), serum copper (</>170 μg/dL), WBC counts (</>15×109/L), prognostic scores (PS) according to Hasenclever (</≥2), and the presence of extranodal disease. Stages III/IV were associated with significantly higher levels of sCD30 and TNF-RI (p=0.03), systemic symptoms with significantly higher levels of sCD30, TNFα, IL6, TNF-RI (p=0.027, 0.03, 0.0005, 0.002), bulky disease with TNF-RI (p=0.03), high ESR with IL6 and TNF-RI (p=0.0011, 0.0001), high WBC counts with sCD30, IL6, TNF-RI (p=0.03, 0.002, 0.01), high serum copper with sCD30 and IL6 (p=0.05, 0.0004), higher PS with sCD30, IL6, TNF-RI (p=0.002, 0.0003, 0.005), extranodal disease with TNFα and IL6 (p=0.05, 0.01). It was possible to define cut-off levels for some cytokines (sCD30 >33.15 U/mL, TNFα >29.71 pg/mL, IL6 > 12.43 pg/mL, TNF-RI >3.23 ng/mL, IL6-R >57 ng/mL) that significantly correlate with systemic symptoms, higher disease stages, ESR, serum copper, WBC counts and PS. Our study shows that high sCD30, TNFα, IL6 and TNF-RI levels are associated with advanced disease or a worse prognostic score. In the context of multiparametric HD staging, cytokine evaluation may be useful for identifying candidates for more intensive therapies.

Folate measurements in patients on regular hemodialysis treatment.

Patients on regular hemodialysis treatment may develop megaloblastic anemia caused by folate deficiency, but whether folate supplementation is required is still controversial, particularly during erythropoietin administration. Erythrocyte folate concentration is a better indicator of folate status than serum folate, although the latter is the variable generally measured. We measured serum and erythrocyte folate in blood samples from 112 regular hemodialysis patients (57 men, 55 women, 50 treated with erythropoietin, and 62 not) by Stratus Folate immunoenzymatic assay (Dade). Patients with very low serum (<2.87 ng/mL) but normal erythrocyte folate were reinvestigated 4 months later without receiving folate supplementation meanwhile. Serum folate concentrations were 0.48 to 12.76 ng/mL (median, 3.40) and erythrocyte folate 0.19 to 1.85 microg/mL (median, 0.42). Only 37% serum folate values were in the relevant reference interval compared with 80.2% erythrocyte folate values (3.08 to 17.65 ng/mL and 0.24 to 0.64 microg/mL, respectively). A significant correlation was found between serum and erythrocyte folate concentrations, without clinical relevance caused by the wide scatter around the regression line. Serum and erythrocyte folate did not vary significantly between patients given erythropoietin and those not so treated. The folate status of the 24 patients with very low serum folate was almost unchanged 4 months later. According to the serum folate test, 63% of patients needed folate supplementation, whereas the erythrocyte folate test, a better indicator of folate status, suggested that only 1.8% of patients needed folate supplementation. Erythropoietin therapy appears not to be an indication for standard folate supplementation in hemodialysis patients.

Folate Supplementation in Peritoneal Dialysis Patients with Normal Erythrocyte Folate: Effect on Plasma Homocysteine

The possible role of folate supplementation in reducing hyperhomocysteinemia in dialysis patients has been reported in several recent papers. However, scant data are available for peritoneal dialysis patients; besides, none of these studies investigated either the role of intraerythrocyte folate concentration or the presence of side effects caused by folate administration. Sixty-six peritoneal dialysis patients with hyperhomocysteinemia (>15 µmol/l) and normal folate status (as assessed by erythrocyte folate level >600 nmol/l) were randomly allocated to receive either oral folate (5 mg/day) or no vitamin supplementation. After 2 months of therapy, patients were requested to answer a questionnaire investigating the occurrence of symptoms possibly related to folate supplementation. Twenty-nine treated patients and 30 untreated controls completed the study. In the treated patients, serum and erythrocyte folate increased significantly (p < 0.0001) (respectively from 10.6 ± 4.9 to 237 ± 231 nmol/l and from 1,201 ± 297 to 2,881 ± 294 nmol/l) to levels at the uppermost limit of detection by laboratory methods. Serum vitamin B12 levels did not change. Plasma homocysteine levels decreased from 54 ± 32 to 23 ± 14 µmol/l after folate supplementation and remained unchanged in the control group. After 4 months of folate therapy, homocysteine concentration was within the normal range in 5 patients (17%) and below 30 µmol/l in the other 21 (72%). Folate therapy resulted in a decrease in homocysteine of more than 50% in 45% of the patients and decrease of more than 20% in a further 38%. No significant symptoms were reported. Thus, serum and erythrocyte folate increase confirms that normal folate levels are inadequate in dialysis patients, even if serum and erythrocyte levels before folate supplementation cannot predict the effect on homocysteine plasma levels.

Erythrocyte ferritin concentration: Analytical performance of the immunoenzymatic IMx-Ferritin (Abbott) assay

Abstract Together with serum ferritin, erythrocyte ferritincan be a valuable diagnostic tool for evaluating the degree of impaired iron metabolism in different diseases. We collected peripheral blood samples from 64 subjects (22 healthy volunteers, 20 patients with hereditary hemochromatosis, and 22 patients on regular hemodialysis with secondary anemia) to evaluate whether an immunoenzymatic method generally used for serum ferritin can also be used to determine erythrocyte ferritin levels under various conditions of body iron status. Serum and erythrocyte ferritin levels were assayed in parallel using a microparticle enzyme immunoassay (MEIA) IMx-Ferritin kit and an IMx analyzer. The inter-assay imprecision of the serum and erythrocyte ferritin assays was 4.9% and 5.05%, the intra-assay imprecision was 2.2% and 2.3%, and the mean recovery was 102% (range 96–105%) and 101% (range 99–105%), respectively. Both serum and erythrocyte ferritin assays showed a detection limit of 1μg/L and good linearity (R 2=0.99) in the intervals 13.9–443 and 3.9–135.6μg/L, respectively. Our findings demonstrate that the IMx-Ferritin assay currently used to measure serum ferritin levels can also be adopted to measure erythrocyte ferritin insofar as it clearly discriminates high and low erythrocyte ferritin levels in cases of both iron overload and deficiency.

Hyperhomocysteinemia in Myelodysplastic Syndromes: Specific Association with Autoimmunity and Cardiovascular Disease

Hyperhomocysteinemia (HH) has been associated with cardiovascular and autoimmune diseases and oxidative cell damage. Myelodysplastic syndromes (MDS) are associated with autoimmunity (AI) and increased oxidative stress. We tested the association of HH and oxidative stress in 33 MDS patients, by measuring plasma homocysteine and malondialdehyde (MDA). HH was found in 42% of cases, 4/5 cases with associated cardiovascular events (CVE)(80%), and 9/15 cases with associated AI (60%). Thus in MDS, HH was significantly associated with AI/CVE (χ2: p=0.0011), and this association seems to be specific, as demonstrated by the comparison of MDS presenting AI/CVE with the ischemic cardiopathy/rheumatoid arthritis control group (13/20, 65% vs 19/69, 27%; χ2: p=0.0021). The levels of MDA indicated increased oxidative stress. Our data may suggest that in a subset of MDS, HH may simultaneously contribute to bone marrow myelodysplasia, CVE and AI pathogenesis, possibly through oxidative cell damage.

Effect of high doses of somatostatin on adenylate cyclase activity in peripheral mononuclear leukocytes from normal subjects and from acute leukemia patients

In normal lymphocytes somatostatin non-competitively inhibited basal (ID50 5×10−4 M) and isoproterenol- and forskolin-stimulated adenylate cyclase activity (Ac). In acute leukemia blasts, non-responsive to isoproterenol, forskolin, which activates the catalytic subunit, stimulated and somatostatin inhibited Ac, thus indicating the leukemic enzyme, though defective, retains the inhibitory pathway and catalyst function.

Plasma and urine cyclic nucleotide levels in patients with hyperthyroidism and hypothyroidism

Plasma levels and 24-h urinary excretion of cyclic AMP and cyclic GMP were measured in 18 patients with hyperthyroidism, 7 patients with hypothyroidism and 25 normal subjects. Mean plasma and urinary levels of both cyclic AMP and cyclic GMP were significantly higher in the hyperthyroid than in the normal subjects. In the hyperthyroid patients significant positive correlations between the serum thyroid hormone levels and plasma and urinary cyclic nucleotide concentrations were also found, suggesting that the elevated extracellular cyclic nucleotide levels in hyperthyroidism are probably a consequence of increased secretion of thyroid hormones. In the hypothyroid patients the extracellular cyclic nucleotide concentrations did not differ significantly from those of the normal subjects.

Patterns of cyclic nucleotides and related enzymes in normal and gross-virus-transformed rat thymocytes

Abstract Cyclic nucleotide (cAMP and cGMP) intracellular levels and cyclase and phosphodiesterase activities were determined in normal and Gross-virus-transformed thymocytes. Many changes in cyclic nucleotide levels and in cyclase activities were observed, but the differences are difficult to evaluate since the changes clearly depend on how the data are expressed: cell number, protein content or DNA content. However, the cAMP cGMP molar ratio was significantly lower in transformed than in normal thymocytes, thus indicating that a relative prevalence of cGMP over cAMP was present in these cells. Transformed thymocytes were sensitive to the stimulating effect of azide and theophylline, but, at variance with normal cells, did not respond to serotonin, carbamylcholine or d,l -isoproterenol. Preincubation with theophylline allowed a cAMP response to d,l -isoproterenol, but no cGMP response to serotonin or carbamylcholine was found. Alterations in the subcellular distribution of guanylate cyclase were also found in transformed thymocytes, as suggested by the relative increase in the particulate enzyme activity of these cells. Both cAMP and cGMP phosphodiesterase activities were significantly higher in transformed than in normal thymocytes. These data are in agreement with the hypothesis that cyclic nucleotide metabolism is abnormal in malignancy.

Pattern of plasma cyclic nucleotides and related hormones in liver cirrhosis and hepatocellular carcinoma.

Abstract To evaluate the pattern of plasma cyclic adenosine 3′,5‵-monophosphate, cyclic guanosine 3‵,5‵-monophosphate, atrial natriuretic factor and glucagon levels in different stages of chronic liver diseases, we measured these variables in 20 normal subjects, 25 patients with genetic hemochromatosis, associated with liver cirrhosis in 19 cases and not in six, eight patients with compensated and 15 with decompensated alcoholic or posthepatitic cirrhosis, and 12 with hepatocellular carcinoma. All variables were within the normal range in non-cirrhotic hemochromatotic patients. Cyclic adenosine 3‵,5‵-monophosphate levels were within the normal range (9.5–15.7 nmol/l) in hemochromatotic cirrhotics and elevated in other patients. Cyclic guanosine 3‵,5‵-monophosphate, atrial natriuretic factor and glucagon were above the normal ranges (1.92–5.91 nmol/l, 8.8–62.7 ng/l, and 39–165 ng/l, respectively) in most patients with cirrhosis both with and without hemochromatosis and in most individuals with hepatocellular carcinoma. Cyclic guanosine 3‵,5‵-monophosphate correlated with atrial natriuretic factor in the former groups but not in the latter. These findings indicate that glucagon and atrial natriuretic factor hypersecretion is an early event in cirrhosis, regardless of its etiology. In hepatocellular carcinoma, the underlying cirrhosis may account for most hormonal and metabolic changes although cyclic guanosine 3‵,5‵-monophosphate increases could also be due to the neoplastic process per se.

Plasma cyclic nucleotide levels in patients with refractory anaemia with excess of blasts

SummaryTo verify the clinical usefulness of extracellular cyclic nucleotide determinations as tumour markers in preneoplastic syndromes, plasma cyclic AMP (cAMP) and cyclic GMP (cGMP) levels were monitored in 47 patients with refractory anaemia with excess of blasts (35 RAEB and 12 RAEBt), 20 of whom progressed to acute leukaemia during the observation period. The control group consisted of 45 healthy subjects matched for age and sex. In all groups of patients plasma cAMP levels were within the normal range, whereas plasma cGMP levels were significantly higher than those of normal subjects in both RAEB and RAEBt patients, and increased further when progression to acute leukaemia occurred. These data suggest that serial determinations of plasma cGMP may be useful to monitor the progression of the disease, though there is no evidence that cGMP values at diagnosis may have a prognostic significance.