Late-onset and long-lasting neutropenias in the young: A new entity anticipating immune-dysregulation disorders.

This study identifies a new chronic form of immune neutropenia in the young with or without detectable indirect anti-neutrophil antibodies, characterized by mild/moderate neutropenia low risk of severe infection (14%), tendency to develop autoimmune phenomena over the course of the disease (cumulative incidence of 58.6% after 20 years of disease duration), leukopenia, progressive reduction of absolute lymphocyte count and a T- and B-cell profile similar to autoimmune disorders like Sjogren syndrome, rheumatoid arthritis, and systemic lupus erythematosus (increased HLADR+ and CD3 + TCRγδ cells, reduced T regulatory cells, increased double-negative B and a tendency to reduced B memory cells). In a minority of patients, P/LP variants related to primary immuno-regulatory disorders were found. This new form may fit the group of "Likely acquired neutropenia," a provisional category included in the recent International Guidelines on Diagnosis and Management of Neutropenia of EHA and EUNET INNOCHRON ACTION 18233. The early recognition of this form of neutropenia would help clinicians to delineate better specific monitoring plans, genetic counseling, and potentially targeted therapies.

Genetic polymorphism of NAD(P)H:quinone oxidoreductase is associated with an increased risk of infant acute lymphoblastic leukemia without MLL gene rearrangements.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a detoxification enzyme that protects cells against oxidative stress and toxic quinones. A polymorphism (C609T) in the gene produces in the heterozygous individuals (C/T) a reduction and in those homozygous for the variant allele (T/T) the abolishment of NQO1 protein activity. To assess whether NQO1 inactivating polymorphism (CT/TT) was a possible risk factor for infant acute lymphoblastic leukemia (iALL), we investigated the distribution of NQO1 genotype in 50 iALL patients, 32 with MLL gene rearrangements (MLL+) and 18 without (MLL-). As controls, 106 cases of pediatric ALL (pALL), and 147 healthy subjects were also studied. Compared to normal controls, the frequency of the low/null activity NQO1 genotypes was significantly higher in the iALL MLL- (72 vs 38%, P=0.006; odds ratio (OR) 4.22, 95% confidence interval (CI) 1.43-12.49), while no differences were observed in iALL MLL+ (44 vs 38%, P=0.553; OR 1.26, 95% CI 0.58-2.74). Similar results were observed when pALL were used as control. Our results indicate that only the iALL patients without MLL rearrangements had a significantly higher frequency of NQO1 genotypes associated with low/null activity enzyme, suggesting a possible role for NQO1 gene as an MLL-independent risk factor, in the leukemogenic process of this subtype of iALL.

Accumulation of m-iodobenzylguanidine by neuroblastoma cells results from independent uptake and storage mechanisms.

The modalities of uptake and storage of iodine-labeled m-iodobenzylguanidine (MIBG) by four human neuroblastoma cell lines have been studied. SK-N-BE(2)C cell line has been shown to possess the specific (type 1) MIBG uptake, as well as an efficient extravesicular storage mechanism. Conversely, LAN-5 cells, which show a nonsaturation kinetic of MIBG incorporation, lack only the ability to efficiently store the MIBG taken up by a mechanism that can be pharmacologically defined as uptake 1. The two other neuroblastoma cell lines tested, GI-LI-N and GI-CA-N, lack both uptake and storage capacity. In view of the fact that the only detailed study on specific MIBG uptake by a human neuroblastoma cell line has been carried out on SK-N-SH, a highly heterogeneous cell line, our report provides new insights on the molecular and cellular pharmacology of radiolabeled MIBG.

The combination of gamma-interferon and tumor necrosis factor causes a rapid and extensive differentiation of human neuroblastoma cells.

Neuroblastoma (NB), a tumor originating from the sympathetic nervous system, is the most common extracranial neurological tumor of childhood. Human NB cells may differentiate in vitro under treatment with biological agents, as gamma-interferon (IFN-gamma) and tumor necrosis factor (TNF). Unfortunately, NB cell lines resistant to the differentiation-inducing effects of both drugs have been observed. Here we demonstrate that a combination of IFN-gamma plus TNF causes extensive and generalized differentiation of NB cells toward a neuronal phenotype. Both IFN-gamma and TNF, given alone, moderately reduced cell growth and induced partial morphological maturation. Their combination further reduced cell proliferation. The combined treatment gave a synergistic rather than additive cytostatic effect, documented also by a dramatically enhanced differentiation toward a neuronal morphology. Membrane immunofluorescence showed a homologous and heterologous up-regulation of IFN-gamma receptor, as well as a marked induction of HLA Class I antigens and, to a lesser extent, of Class II antigens on NB cells induced to differentiate. Treatment of NB cell lines with IFN-gamma/TNF results in the induction of a differentiated phenotype, as indicated by the increased expression of the Mr 160,000 and 200,000 neurofilament proteins and that of microtubule-associated proteins. Evaluation of biochemical markers of neuronal differentiation confirmed the ability of the combined treatment to induce neuroblast maturation. These results suggest that the combination of IFN-gamma and TNF should be considered for experimental clinical trials in neuroblastoma.

The polymorphisms -318C>T in the promoter and 49A>G in exon 1 of CTLA4 and the risk of aplastic anemia in a Caucasian population.

Aplastic anemia (AA) is a rare disease with a major autoimmune pathogenetic component. CTLA4 is a T-lymphocyte surface molecule involved in the maintenance of immune tolerance. Some polymorphisms associated with a reduced expression of CTLA4, and thus presumably with increased tendency to autoimmunity, have been associated with various autoimmune diseases. In this study, we evaluated the distribution of the low expression polymorphisms -318C > T and 49A > G of CTLA4 in a population of 67 patients with acquired AA and in 100 normal controls. There was no difference in the distribution of the tested polymorphism between patients and controls and, within the patient group, between those who responded to immunosuppression vs those who did not respond. This study indicates that the polymorphisms -318C > T and 49A > G of CTLA4 do not affect the risk of developing AA and do not influence the response to immunosuppression.

A comparison of different oral therapies versus no treatment for erectile dysfunction in 196 radical nerve-sparing radical prostatectomy patients.

We retrospectively analyzed the effects on the erectile function (EF) of no treatment (NT), and an oral therapy (OT; on-demand therapy (OD) or a regimented rehabilitation (RR) program with phosphodiesterase type 5 inhibitors (PDE5-Is)), in a cohort of 196 consecutive patients following nerve-sparing radical retropubic prostatectomy (NSRRP). Patients undergoing bilateral NSRRP (BP; n = 147) and unilateral NSRRP (UP; n = 49), chose between OT (PDE5-Is OD or RR program) and NT. Patients who chose OD therapy received PDE5-Is (100 mg sildenafil, 20 mg tadalafil and vardenafil), whereas patients who chose the RR program received 100 mg sildenafil or 20 mg vardenafil three times a week, or 20 mg tadalafil twice a week at bedtime. The t-test for unpaired data and Fisher test were used for univariate analyses, logistic regression multivariate analysis was used to test the accuracy of available variables to predict EF recovery after radical prostatectomy. Potency rates were significantly correlated with the surgical technique and with OT when compared to NT (P < 0.02), respectively 68.7% for BP (61% with no therapy and 71% with PDE5-Is) and 44% for UP (29% with no therapy and 51% with PDE5-Is), while no statistically significative differences were found between OD and rehabilitation protocols (72% with rehabilitation and 70% with OD therapy in BP, 52% with rehabilitation and 50% with OD therapy in UP; P = NS). Early OT with PDE5-Is (OD or RR program) was superior to NT in recovery of EF in NSRRP. Furthermore, an RR program with PDE5-Is did not appear to be superior to OD therapy.

Phosphatidylinositol turnover is not a general regulator of neuroblastoma cell differentiation: comparison between two differentiating agents, retinoic acid and gamma-interferon.

The turnover of phosphatidylinositol (PI) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including differentiating agents; however decisive evidence for the idea has not been obtained. In the present paper, we investigated the involvement of PI turnover in cell differentiation using a human neuroblastoma cell line, LAN-1, which can be induced to differentiate along the neuronal pathway by both retinoic acid (RA) and gamma-interferon (gamma-IFN). Analysis of labelled phosphatidylinositol metabolites from prelabelled cells indicated a rapid decrease of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol within 1 min of induction of LAN-1 cell differentiation by RA, while no changes were observed in gamma-IFN-treated cells. These findings indicate the occurrence of decreased inositol phospholipid turnover in RA-treated LAN-1 cells and suggest that phosphoinositide-derived metabolites may not constitute general regulators of cellular differentiation.

The role of M1 and M2 macrophages in prostate cancer in relation to extracapsular tumor extension and biochemical recurrence after radical prostatectomy.

INTRODUCTION: The aim of our work was to investigate the causal connection between M1 and M2 macrophage phenotypes occurrence and prostate cancer, their correlation with tumor extension (ECE), and biochemical recurrence (BR). PATIENT AND METHODS: Clinical and pathological data were prospectively gathered from 93 patients treated with radical prostatectomy. Correlations of commonly used variables were evaluated with uni- and multivariate analysis. The relationship between M1 and M2 occurrence and BR was also assessed with Kaplan-Meier survival analysis. RESULTS: Above all in 63.4% there was a M2 prevalence. M1 occurred more frequently in OC disease, while M2 was more represented in ECE. At univariate analysis biopsy and pathologic GS and M2 were statistically correlated with ECE. Only pathologic GS and M2 confirmed to be correlated with ECE. According to macrophage density BCR free survival curves presented a statistically significant difference. When we stratified our population for M1 and M2,we did not find any statistical difference among curves. At univariate analysis GS, pTNM, and positive margins resulted to be significant predictors of BCR, while M1 and M2 did not achieve the statistical significance. At multivariate analysis, only GS and pathologic stage were independent predictors of BR. CONCLUSION: In our study patients with higher density of M count were associated with poor prognosis; M2 phenotype was significantly associated with ECE.

Cancer-associated fibroblasts and M2-polarized macrophages synergize during prostate carcinoma progression.

Inflammation is now acknowledged as an hallmark of cancer. Cancer-associated fibroblasts (CAFs) force a malignant cross talk with cancer cells, culminating in their epithelial-mesenchymal transition and achievement of stemness traits. Herein, we demonstrate that stromal tumor-associated cells cooperate to favor malignancy of prostate carcinoma (PCa). Indeed, prostate CAFs are active factors of monocyte recruitment toward tumor cells, mainly acting through stromal-derived growth factor-1 delivery and promote their trans-differentiation toward the M2 macrophage phenotype. The relationship between M2 macrophages and CAFs is reciprocal, as M2 macrophages are able to affect mesenchymal-mesenchymal transition of fibroblasts, leading to their enhanced reactivity. On the other side, PCa cells themselves participate in this cross talk through secretion of monocyte chemotactic protein-1, facilitating monocyte recruitment and again macrophage differentiation and M2 polarization. Finally, this complex interplay among cancer cells, CAFs and M2 macrophages, cooperates in increasing tumor cell motility, ultimately fostering cancer cells escaping from primary tumor and metastatic spread, as well as in activation of endothelial cells and their bone marrow-derived precursors to drive de novo angiogenesis. In keeping with our data obtained in vitro, the analysis of patients affected by prostate cancers at different clinical stages revealed a clear increase in the M2/M1 ratio in correlation with clinical values. These data, coupled with the role of CAFs in carcinoma malignancy to elicit expression of stem-like traits, should focus great interest for innovative strategies aimed at the co-targeting of inflammatory cells and fibroblasts to improve therapeutic efficacy.

Fully automated solid-phase microextraction-fast gas chromatography-mass spectrometry method using a new ionic liquid column for high-throughput analysis of sarcosine and N-ethylglycine in human urine and urinary sediments.

A fully automated, non invasive, rapid and high-throughput method for the direct determination of sarcosine and N-ethylglycine in urine and urinary sediments using hexyl chloroformate derivatization followed by direct immersion solid-phase micro extraction and fast gas chromatography-mass spectrometric analysis was developed and validated. The use of a new ionic liquid narrow bore column, as well as the automation and miniaturization of the preparation procedure by a customized configuration of the utilized XYZ robotic system, allowed a friendly use of the GC apparatus achieving a quantitation limit of 0.06 µg L(-1) for sarcosine, good repeatability with CV always lower than 7% and reduced analysis times useful for point-of-care testing. The method was then applied for the analysis of 56 samples of urine and urinary sediments in healthy subjects, in those with benign prostatic hypertrophy and in patients with clinically localized prostate cancer. The results obtained showed that the medians of sarcosine/creatinine in urine were 103, 137 and 267 µg g(-1) respectively, thus assessing the potential use of sarcosine as urinary biomarker for prostate cancer detection. The highest values of sensitivity (79%) and specificity (87%) were obtained in correspondence of a cut-off value of 179 µg sarcosine(g creatinine)(-1), thus by using this cut-off threshold, sarcosine was significantly associated with the presence of cancer (p<0.0001). Finally, ROC analyses proved that the discrimination between clinically localized prostate cancer and patients without evidence of tumor is significantly correlated with sarcosine.

Influence of serum testosterone on urinary continence and sexual activity in patients undergoing radical prostatectomy for clinically localized prostate cancer.

The aim of the present study was to evaluate how serum testosterone level (T) can affect urinary continence and erectile function in patients undergoing radical prostatectomy (RP). We included 257 patients with clinically localized prostate cancer, those who had filled out preoperative quality of life questionnaires (University of California, Los Angeles Prostate Cancer Index, International Index of Erectile Function (IIEF)), and those who had T and total PSA sampled the day before surgery. We calculated correlations between T and age, body mass index (BMI), PSA, urinary function or bother (UF, UB) and sexual function or bother (SF, SB) and IIEF-5 in the whole population and in sub-populations with normal (> or =10.4 nmol l(-1)) and low (<10.4 ng ml(-1)) T using Pearson's and Spearman's correlation coefficients. We evaluated differences in these parameters between patients with low and normal T using the unpaired samples t-test and Mann-Whitney test, and finally the correlation between UF and SF, UB and SB, and between PSA and T in the overall population, and separately in patients with low and normal T using the Pearson's correlation coefficient. Mean preoperative T was 13.5 nmol l(-1) and 23.7% of patients presented a low T. Mean age, mean BMI and mean preoperative total PSA at RP were 64.3 years, 25.9 kg m(-2) and 9.0 ng ml(-1), respectively. BMI was negatively correlated with T in the overall population (r=-0.266; P=0.02); moreover, patients with normal T presented lower BMI compared with patients with low T (25.7 vs 27.6: P=0.02). We found a significant correlation between SF scores and T in patients with normal T (r=0.1777: P=0.05). SF was significantly higher in patients with normal T compared with those with low T (74.8 vs 64.8: P=0.05). Furthermore, UF and UB were significantly correlated with SF (r=0.2544: P<0.01) and SB (r=0.2512: P=0.01), respectively, in men with normal T. Serum T was significantly correlated with PSA in men with low T (r=0.3874: P=0.0029), whereas this correlation was missed in the whole population and in men with normal T. The correlation between preoperative PSA and T in men with low T is in agreement with the 'saturation' model proposed by Morgentaler. The correlation between basal T and preoperative erectile function and urinary continence underlines the importance of assessing T before RP.

[A rapid decrease in the phosphatidylinositol cycle during neuroblastoma cell differentiation induced by retinoic acid]. / Rapido decremento del ciclo del fosfatidilinositolo durante la differenziazione di cellule di neuroblastoma indotta da acido retinoico.

Inositol phospholipid turnover is part of a signal transduction mechanism which mobilize intracellular calcium and activate a calcium- and phospholipid-dependent protein kinase, protein kinase C. Phosphatidylinositol turnover has recently been implicated in the regulation of cell proliferation and transformation. Its role in differentiation has now been investigated using LAN-1 cells, a human neuroblastoma cell line which can be induced to differentiate along the neuronal pathway by RA. Treatment of LAN-1 cells with RA was followed by a rapid decrease of inositol phospholipid metabolism, as determined by isotopic methodology employing myo-[1,2-3H] inositol or [1(3)-3H] glycerol. Analysis of labelled phosphatidylinositol metabolites from prelabelled cells indicated a rapid decrease of inositol (1,4,5)trisphosphate and (1,2)diacylglycerol within 1 min. of induction of LAN-1 cell differentiation. These findings suggest that inositol phospholipid-derived metabolites (i.e. diacylglycerol and inositol trisphosphate) may be part of the mechanism by which certain RA signals are transduced, playing a key role in control of neuroblastoma cell differentiation.

Retinoic acid rapidly decreases phosphatidylinositol turnover during neuroblastoma cell differentiation.

Phosphatidylinositol (PI) turnover has recently been implicated in the regulation of cell proliferation and transformation. We have investigated its role in differentiation using LAN-1 cells, a human neuroblastoma cell line that can be induced to differentiate along the neuronal pathway by retinoic acid (RA). We have found that treatment of LAN-1 cells with RA is followed by a rapid decrease of inositol phospholipid metabolism, using myo-[1,2-3H]inositol or [1(3)-3H]glycerol. No changes were observed in both [3H]inositol and [3H]glycerol uptake within 24 h of RA treatment. Decreased incorporation of the metabolic precursor into PI 4-monophosphate and PI 4,5-bisphosphate occurred within 1 h of RA treatment. No changes were seen in the specific radioactivity of the precursor pools up to 1 h of treatment with RA. Analysis of labeled PI metabolites from prelabeled cells indicated a rapid decrease of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol content within 1 min of induction of LAN-1 cell differentiation. These findings constitute the earliest reported events in neuroblastoma cell differentiation.

Retinoic acid inhibits phosphatidylinositol turnover only in RA-sensitive while not in RA-resistant human neuroblastoma cells.

Phosphatidylinositol (PI) turnover has recently been implicated in the regulation of cell proliferation and transformation. We have investigated its role in differentiation using LAN-1 cells, a human neuroblastoma cell line which can be induced to differentiate along the neuronal pathway by retinoic acid (RA), and a derivated RA-resistant subline of it (LAN-1-res). We have found that treatment of LAN-1 cells with RA is followed by a rapid decrease of inositol phospholipid metabolism, using myo-[1,2-3H] inositol or [1,(3)-3H] glycerol. Analysis of labelled phosphatidylinositol metabolites from prelabelled LAN-1 cells indicated a rapid decrease of inositol (1,4,5)-trisphosphate and (1,2) diacylglycerol within 1 min. of induction of differentiation by RA, while no changes were observed in RA-treated LAN-1-res cells. These findings indicate that phosphoinositides-derived metabolites may be directly implicated in the induction processes of RA-triggered NB cell differentiation.

Negative staining of proteins in polyacrylamide gels with methyl trichloroacetate.

This paper describes a new, sensitive (in the nanogram range), and rapid (two-step) technique for the negative staining of proteins in polyacrylamide gels in the presence or absence of sodium dodecyl sulfate. After separation, gels are incubated with 8% methyl trichloroacetate ester in 38% isopropanol and then washed in water to produce a negative image of colorless proteins against an opaque background. The technique allows unmodified proteins to be recovered for biological studies or transblot for amino acid sequence. Finally, owing to the reversibility of the process, gels can be restained after rapid visualization. For these reasons, negative staining with methyl trichloroacetate should become the method of choice for rapid and sensitive staining of proteins prior to further processing, including stable staining with silver ions.

[Immunosuppressive factors produced by a T cell line derived from acute lymphoblastic leukemia]. / Fattori immunosoppressivi prodotti da una linea cellulare T derivante da una leucemia linfoblastica acuta.

The supernatant of CD8+ cells, isolated from a permanent lymphoblastoid cell clone established from a long term culture of a T cell acute lymphoblastic leukemia, contained two distinct molecules with suppressive activity on PHA1-induced PBMC proliferation. This clone does not produce TNF-alpha, TNF-beta, alpha-IFN, gamma-IFN, IL-1, IL-2 and has not natural killer activity. In the attempt to purify and biochemically characterize the lymphoblastic cell line-derived T-cell SFs, a multi-step chromatographic separation has been used. Two different peaks of biologic activity have been separated by HPLC gel permeation in the range of 100-120 Kd and 75-85 Kd referred to as high molecular weight suppressor factor HMWSF, and low molecular weight suppressor factor LMWSF, respectively. These fractions were then concentrated, dialyzed and further purified by anion exchange HPLC. This chromatographic step allowed us to considerably purify the two SFs. The biologically active fractions derived from the previous chromatographic step were eventually subjected to hydrophobic interaction HPLC (HIC) for final purification. The highly purified material was characterized by polyacrylamide gel electrophoresis in sodium-dodecylsulfate (SDS-PAGE): a single band corresponding to 115 Kd was observed for HMWSF, while LMWSF yielded a single band at 80 Kd. The isoelectric points (pI) of the different SFs was determined by flat-bed isoelectric-focusing: the HMWSF yielded a single band at pI 7.4, while a much lower pI was observed for LMWSF, 3.5-3.6. Studies on temperature lability indicated that both proteins are stable for 3-4 hours at room temperature (RT), 24-36 hours at +4 degrees C and 7-10 days at -80 degrees C.

Purification to homogeneity and biochemical characterization of two suppressor factors from human malignant T-cells.

A cell clone (GI-CO-T-9) derived from a long term T-cell culture (PF-382), established from a patient affected by acute T-lymphoblastic leukemia (T-ALL), was selected for the presence in the culture medium of factors suppressing T-cell proliferation. The crude supernatant has been subjected to a multi-step chromatographic fractioning, including: preparative gel permeation, anion exchange, and hydrophobic interaction High Performance Liquid Chromatography (HPLC). The highly purified material was characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE), revealing single bands of 115 Kd and 80 Kd. The isoelectric points (pI), determined by flat-bed isoelectric-focusing, were 7.4 for High Molecular Weight Suppressor Factor (HMWSF) and 3.5-3.6 for Low Molecular Weight Suppressor Factor (LMWSF).

In vitro pharmacology of metaiodobenzylguanidine uptake, storage and release in human neuroblastoma cells.

Four human neuroblastoma (NB) cell lines (LAN-5, SK-N-BE(2)C, GI-LI-N, and GI-CA-N) have been investigated for their ability to take up and store [125I]metaiodobenzylguanidine (125I-MIBG) in vitro. Only SK-N-BE(2)C and LAN-5 cells were able to specifically take up MIBG, with the former cell line showing a more efficient retention of the radiotracer. 125I-MIBG incorporation in both cell lines was inhibited by norepinephrine, desipramine, ouabain and energy depletion. Thus, all the major criteria for specific (type 1) uptake were fulfilled. Conversely, GI-LI-N and GI-CA-N cells did not show any specific uptake. Pharmacological manipulations aimed at defining the intracellular site(s) of 125I-MIBG storage clearly showed that the radiotracer is not accumulated in the reserpine-sensitive neurosecretory granules and vesicles in NB cells, contrary to what has been observed in a chromaffin derived tumor cell line (PC12). Our study provides new and suitable models to investigate in vitro the molecular and cellular pharmacology of MIBG in NB cells.