Pathogenetic and diagnostic significance of microRNA deregulation in peripheral T-cell lymphoma not otherwise specified.

Peripheral T-cell lymphomas not otherwise specified (PTCLs/NOS) are rare and aggressive tumours whose molecular pathogenesis and diagnosis are still challenging. The microRNA (miRNA) profile of 23 PTCLs/NOS was generated and compared with that of normal T-lymphocytes (CD4+, CD8+, naive, activated). The differentially expressed miRNA signature was compared with the gene expression profile (GEP) of the same neoplasms. The obtained gene patterns were tested in an independent cohort of PTCLs/NOS. The miRNA profile of PTCLs/NOS then was compared with that of 10 angioimmunoblastic T-cell lymphomas (AITLs), 6 anaplastic large-cell lymphomas (ALCLs)/ALK+ and 6 ALCLs/ALK-. Differentially expressed miRNAs were validated in an independent set of 20 PTCLs/NOS, 20 AITLs, 19 ALCLs/ALK- and 15 ALCLs/ALK+. Two hundred and thirty-six miRNAs were found to differentiate PTCLs/NOS from activated T-lymphocytes. To assess which miRNAs impacted on GEP, a multistep analysis was performed, which identified all miRNAs inversely correlated to different potential target genes. One of the most discriminant miRNAs was selected and its expression was found to affect the global GEP of the tumours. Moreover, two sets of miRNAs were identified distinguishing PTCL/NOS from AITL and ALCL/ALK-, respectively. The diagnostic accuracy of this tool was very high (83.54%) and its prognostic value validated.

Partial nodal involvement by marginal zone lymphoma. Use of IGK gene rearrangement analysis in diagnostic work-up.

Nodal marginal zone lymphoma (NMZL) is an indolent B-cell lymphoma that originates from the marginal zone of B-cell follicles. The tumour is rather uncommon, and shares some morphologic and immunophenotypic similarities with the extranodal form of marginal zone lymphomas. However, diagnosis of NMZL implies the exclusion of lymphoplasmacytic lymphoma, follicular lymphoma, and lymph node involvement by extra nodal or splenic marginal zone B-cell lymphoma In addition, its distinction from reactive conditions, including T-zone hyperplasia, are sometimes problematic based on morphologic grounds. We describe a patient who presented with cervical and inguinal lymphadenopathies and high inflammation indexes. Bone marrow and lymph node biopsies were performed for definitive diagnosis. Bone marrow histological and immunophenotypic examinations were normal and excluded haematological disease. In contrast, lymph node evaluation showed some features compatible with a possible lymphoproliferative disorder, even though no definite diagnosis could be made based on morphologic and immunohistochemical investigation. In particular, the problem of a differential diagnosis between NMZL and a florid hyperplasia of monocytoid B-elements was posed. Thus, in order to assess the nature (neoplastic vs. reactive) of the lesion, molecular analysis of the immunoglobulin genes was performed by PCR. Notably, although no clonal rearrangements were revealed by IGHV@ analysis, further evaluation of the immunoglobulin light chain (IGKV@) confirmed the presence of a clonal B-cell population. Accordingly, a final diagnosis of NMZL was made. In conclusion, this case is a good example of the crucial role of complete molecular analysis in the diagnostic work up of lymphoproliferative disorders.

Intensive insulin therapy in preschool-aged diabetic children: from multiple daily injections to continuous subcutaneous insulin infusion through indwelling catheters.

In this study, glycemic control, diabetes care indices and quality of life (QoL) were assessed in 2 groups of newly diagnosed Type 1 diabetic subjects <6 yr old who were randomized to multiple daily injections with (Group A) or without (Group B) an indwelling catheter. Group A [12 males (M)/8 females (F), mean age 3.2+/-1.4 yr] and Group B (9M/11F, mean age 3.9+/-1.8 yr) were evaluated at baseline and after 6 and 12 months of treatment. No significant difference was observed in metabolic control (glycosylated hemoglobin) or in the number of hypoglycemic events between the groups. Patients in Group A had a greater number of daily insulin injections, monitored blood glucose more frequently and had a lower total daily insulin dose per kg (p<0.05). QoL was better in group A. At the end of the study 30% of group A patients progressed to continuous sc insulin infusion (CSII), while no child in Group B switched to a different insulin regimen. Based on these findings, indwelling catheter therapy may be helpful for selected CSII candidates.

Age-related differences in metabolic response to continuous subcutaneous insulin infusion in pre-pubertal and pubertal children with Type 1 diabetes mellitus.

The aim of this study was to evaluate clinical and metabolic data in a cohort of Type 1 diabetes (T1DM) children before and after 2 yr of continuous s.c. insulin infusion (CSII). Forty seven T1DM patients were subdivided into two groups: Group A (20 pre-pubertal children, mean age 7.43+/-3.19 yr); Group B (27 pubertal adolescents, mean age 14.47+/-1.91 yr). No statistically significant differences in body mass index (BMI) occurred in either groups after starting CSII or during follow-up. The frequency of mild-hypoglycemias significantly declined during pump therapy only in Group A (p<0.05). Both pre-pubertal and pubertal patients required a significant reduction in their total insulin requirement after 12 and 24 months of CSII. The total percentage of daily insulin doses delivered as basal rates was similar in both groups and was negatively associated (beta=-2.956, p=0.05) with glycosylated hemoglobin (HbA1c) values. No significant correlation was found between the percentage of the basal insulin rate and the number of daily boluses. Differences in timing of the highest insulin requirement were observed between the two groups. Group A had a higher insulin basal rate late in the evening (20:00-24:00 h), while Group B had a higher insulin requirement early in the morning (03:00-07:00 h). The HbA1c levels significantly improved in Group A after 6-12 and 24 months of CSII. In Group B a reduction of HbA1c values was observed only after 6 months of pump therapy (p=0.05). CSII is an effective therapy for all ages but different metabolic requirements should also be taken into account.

Glutamic acid decarboxylase and ICA512/IA-2 autoantibodies as disease markers and relationship to residual beta-cell function and glycemic control in young type 1 diabetic patients.

Circulating autoantibodies (Ab) to islet autoantigens, glutamic acid decarboxylase (GAD(65)), and tyrosine phosphatase ICA512/IA-2 have been proposed as predictive markers of type 1 diabetes mellitus. To ascertain residual beta-cell function and the clinical relevance for monitoring autoimmunity after clinical manifestation of disease, we studied 63 children at diagnosis of type 1 diabetes (mean SD age 7.5 +/- 4 years) and 91 adolescent patients with type 1 diabetes (age 14.7 +/- 1.6 years) with a mean duration of disease of 7 +/- 3.5) years. Forty-two normal adolescent subjects (age 14.6 +/- 1.8 years) without a family history of diabetes were the control group. Anti-GAD(65) and ICA512/IA-2 Ab were assessed by a quantitative radioimmunoprecipitation assay. The relationship between humoral autoimmunity and clinical parameters was explored. GAD(65) and ICA512/IA-2 Ab were detected in 56% and 63% of newly diagnosed children and the prevalence was not different in relationship to clinical characteristics. Levels of GAD(65) Ab positively correlated with diagnosis age (P <.05). Both Ab were associated with islet cell antibodies (ICA) (P <.05), but one fifth of patients had at least 1 of the 2 Ab and absent ICA. At onset, only age showed a significant relationship to residual C-peptide secretion. Among the cohort of patients with diabetes of short-mid duration, GAD(65) and ICA512/IA-2 Ab were present in 44% and 45% of cases (P >.05 and P <.05 v newly diagnosed children, respectively) and more patients were identified by these Ab (68%) than by ICA alone (34%) (P <.05). In this cohort, levels of ICA512/IA-2 Ab negatively correlated with levels of glycosylated hemoglobin (HbA(1c)) (P <.005) and with daily insulin requirement (P <.05). Moreover, the presence of some residual C-peptide secretion was significantly associated with the presence of ICA512/IA-2 Ab (P <.05). Our findings confirm that positivity for either GAD(65) or ICA512/IA-2 Ab is a highly sensitive marker of type 1 diabetes in the pediatric age group, identifying a group of patients with absent ICA immunofluorescence. The persistence of Ab to islet tyrosine phosphatase possibly represents a marker of better glycemic control and less insulin requirement, indicating residual beta-cell function, thus conferring clinical and prognostic relevance to these Ab, as well as potential usefulness in intervention strategies.

Caffeine microparticles for nasal administration obtained by spray drying.

This study investigated the possibility to use spray drying technique to prepare powders formulations containing caffeine intended for nasal delivery. Spray dried powders containing caffeine and excipients, as filler and shaper agents, were prepared. Powders were investigated for particle size, morphology and delivery properties from Monopowder P nasal insufflator, assessing the influence of each excipient on microparticles characteristics. The results showed that the excipients strongly affected microparticle properties. Size, shape and agglomeration tendency are relevant characteristics of spray dried nasal powder.

Enhanced blood insulin overcomes pyruvate dehydrogenase derangements that reflect systemic insulin resistance in obese adolescents.

Pyruvate dehydrogenase (PDH) has low activity in the circulating lymphocytes (CL) of obese adolescents and adults. In vitro, it is unresponsive to insulin at 5 micro-units/ml and is activated at 50 micro-units/ml, in contrast with activation and inhibition respectively at these concentrations in CL from controls. These changes are seen as being indicative of a molecular disorder underlying insulin resistance. The aims of the present study were to determine whether a substantial enhancement of blood insulin levels restores the PDH activity in CL from obese adolescents and abolishes the in vitro alterations, and whether PDH activity and indices of insulin resistance are correlated. Six obese adolescents and six normal-weight controls underwent a 4 h frequently sampled intravenous glucose test with minimal model analysis, to bring about a sharp rise in blood insulin and provide a reliable index of insulin sensitivity (S(I)). PDH activity was evaluated in CL obtained from blood samples at set times before and after their exposure to insulin in vitro. Insulin levels rose in all subjects in the first 10 min, although to a much greater extent in the obese group, and then decreased until the end of the test (240 min; t(240)). PDH activity in CL paralleled the insulin pattern in the control subjects, whereas in the obese subjects it was below normal 3 min before the start of the test (t(-3)), but rose significantly throughout the test. PDH responses in vitro to insulin in CL taken from the control subjects at t(-3) and t(240) and in CL taken from the obese subjects at t(-3) were as reported above, but were normal (i.e. the same as in control CL) in CL taken from the obese subjects at t(240). Baseline PDH activity was inversely correlated with body mass index and with fasting insulin, and directly correlated with S(I). These results show that a brief and sharp enhancement of blood insulin overcomes derangements in PDH that reflect systemic insulin resistance in obese adolescents.

The potential for errors in children with special health care needs.

Children with special health care needs (CSHCN) are at risk for suboptimal treatment when presenting for emergent care to unfamiliar health care providers. Errors in their management may stem from failure to recognize occult conditions, lack of familiarity with rare or complex medical problems, or lack of prior knowledge of baseline physical findings. An emergency information form (EIF) that contains patient-specific information on essential diagnostic and therapeutic interventions may provide a ready personal reference for the emergent care of CSHCN. Coupled with the use of medical identification jewelry and an electronic transmission system, an EIF has the potential to eliminate management errors in the care of these patients.

Insulin secretion and hepatic insulin clearance as determinants of hyperinsulinaemia in normotolerant grossly obese adolescents.

Obesity is characterized by variable degrees of hyperinsulinaemia, which has been attributed to either beta-cell hypersecretion or reduced hepatic insulin extraction, or both. To investigate this controversial issue, a 4-h frequently sampled i.v. glucose tolerance test (glucose dose 12.8 g m(-2)) was performed in 13 normotolerant, grossly obese adolescents (10 F/3 M; 13+/-1 y; body mass index 32+/-0.9; pubertal stage 4-5; obesity duration 7.8+/-3 y) and in a comparable group of 8 healthy, normal-weight subjects. Glucose, insulin and C-peptide time-course were analysed by the minimal model technique, which estimates beta-cell secretion, insulin sensitivity (Si), glucose effectiveness (SG) and hepatic insulin extraction (HE). Despite similar fasting and after load glucose patterns (SG similar in the two groups), obese adolescents showed sustained peripheral hyperinsulinaemia (total insulin area under the concentration curve 67.2+/-10.8 vs 19.1+/-1.2 pmol l(-1) in 240 min; p <0.002) and a 71% reduction in Si (2.02+/-0.33 vs 6.95+/-1.03 x 10(4) min(-1) (microU ml(-1)); p < 0.001). Compared with control subjects, the total amounts of prehepatic insulin secretion and posthepatic insulin delivery were also increased significantly in obese adolescents by 30% and 46%, respectively; HE was reduced by 15% during the first 30 min of the test, but recovered within the normal range during the rest of the test. In conclusion, severely obese adolescents are insulin resistant and their hyperinsulinaemia is primarily caused by beta-cell hypersecretion, whereas the reduction in insulin hepatic extraction is a transient metabolic phenomenon.

Prostaglandin E2 inhibits apoptosis in human neutrophilic polymorphonuclear leukocytes: role of intracellular cyclic AMP levels.

Human neutrophilic polymorphonuclear leukocytes (neutrophils) are terminally differentiated cells that die by undergoing apoptosis. At present, the intracellular pathways governing this process are only partially known. In particular, although the adenylate cyclase-dependent generation of cyclic AMP (cAMP) has been implicated in the triggering of apoptosis in lymphoid cells, the role of the intracellular cAMP pathway in neutrophil apoptosis remains controversial. In the present study, we found that two cAMP-elevating agents, prostaglandin E2 (PGE2) and the phosphodiesterase type IV inhibitor RO 20-1724, inhibit neutrophil apoptosis without inducing cell necrosis. When administered in combination, PGE2 and RO 20-1724 displayed additive effects. Moreover, neutrophil apoptosis was inhibited by a membrane-permeable analog of cAMP, dibutyryl-cAMP, in a dose-dependent manner. Finally, treatment of neutrophils with the protein kinase A inhibitor H-89 prevented PGE2- and RO 20-1724-induced inhibition of cell apoptosis. In conclusion, taking into account that PGE2 and other cAMP-elevating agents are well known downregulators of neutrophil functions, our results suggest that conditions favoring a state of functional rest, such as intracellular cAMP elevation, prolong the life span of neutrophils by delaying apoptosis.

[Erythrocytosis in patients with hepatocarcinoma in alcoholic cirrhosis: ectopic production of erythropoietin?]. / Eritrocitosi in paziente con epatocarcinoma su cirrosi alcoolica: produzione ectopica di eritropoietina?

We report the case of a patient with alcoholic liver cirrhosis and generalized atherosclerosis who rapidly developed erythrocytosis. Concomitantly we documented a significative and progressive increase of serum Erythropoietin (Epo) and a small focus of hepatocellular carcinoma (HCC) never diagnosed before. Even in absence of immunohistochemical and/or biomolecular evidence of Epo production in the neoplastic tissue we think the hypothesis of the paraneoplastic syndrome may be the most likely both for the strict temporal relationship between the observation of the neoplastic lesion and the appearance of polycythemia and for the absence of all other known causes of erythrocytosis. Objection to this hypothesis: 1) ectopic production of Epo during HCC has been usually described in large neoplastic lesions 2) liver cirrhosis by itself may be accompanied by increased Epo levels 3) an intratumoral hypoxia with compensatory production of Epo may have occurred 4) generalized vasculopathy could have determined renal hypoxia with greater local production of Epo.

Autonomic function and autoantibodies to autonomic nervous structures, glutamic acid decarboxylase and islet tyrosine phosphatase in adolescent patients with IDDM.

Recent studies have linked autoimmunity to nervous tissue structures and diabetic autonomic neuropathy, but data on the early stage of IDDM and on the natural history of this association are not available. For this reason, we investigated autonomic nervous function, and the presence of autoantibodies to sympathetic and parasympathetic nervous structures, to glutamic acid decarboxylase (GAD) and tyrosine phosphatase (IA-2/ICA512) in 85 adolescents with insulin-dependent diabetes mellitus (IDDM) (mean age 14.7+/-1.6 yr, mean duration of diabetes 6.8+/-3.5 yr), and 45 age and sex-matched healthy subjects. Nervous tissues autoantibodies were detected using an indirect immunofluorescent complement-fixation technique, with monkey adrenal gland, rabbit cervical ganglia and vagus nerve as substrates. GAD and IA-2/ICA512 autoantibodies were detected by radioimmunoprecipitation assay. Seven patients (8%) had anti-vagus nerve autoantibodies, 7 other patients (8%) had anti-cervical ganglia autoantibodies, while all controls were negative (P < 0.05). Anti-adrenal medulla antibodies were detected in 16 patients (19%) and in 2 control subjects (P<0.02). None of the patients had autonomic symptoms. When patients were divided according to the presence or absence of autoantibodies, values of the cardiovascular tests (deep breathing, 30:15 ratio, Valsalva ratio) were similar in the two groups and similar to those in healthy subjects. However, when considered together, patients positive for one or more autoantibody showed a trend for lower values of deep breathing test and 30:15 ratio test, compared with healthy control subjects, which failed to reach conventional significance values (P=0.17 and P=0.07, respectively). No correlation was found between cardiovascular parameters and metabolic control or diabetes duration. There was no association between autoimmunity to nervous tissue structures and presence of GAD and IA-2/ICA512 Ab, and no correlation between these two autoantibodies and values of cardiovascular tests. Our data indicate that autonomic dysfunction is not a characteristic of young diabetic patients, but that autoantibodies against autonomic nervous structures are present during the first 1 to 15 yr of diabetes. GAD and tyrosine phosphatase appear to be excluded as target autoantigens within autonomic structures. Follow-up studies are required to evaluate future autonomic dysfunction and symptoms in these patients, and to establish whether the subtle autonomic dysfunction detected and/or the nervous tissue autoantibodies, are predictive of the development of this complication.

Chemoattractant-induced release of elastase by lipopolysaccharide (LPS)-primed neutrophils; inhibitory effect of the anti-inflammatory drug nimesulide.

Human neutrophils, pre-exposed to low concentrations (1-10 ng/ml) of bacterial LPS in the presence of 1% autologous serum, released elastase activity in response to N-formyl-met-leu-phe (fMLP). Both cell incubation with LPS without subsequent fMLP stimulus and fMLP stimulation without prior exposure to LPS failed to promote significant elastase release. Therefore, LPS primes neutrophils for the subsequent release of elastase in response to fMLP. Compared with fMLP, human recombinant C5a had a slight although not significant activity, whereas other chemoattractants such as IL-8, platelet-activating factor and leukotriene B4 were ineffective. The fMLP-induced response of LPS-primed neutrophils was susceptible to suppression by the methane-sulphonanilide anti-inflammatory drug nimesulide and RO 20-1724, which selectively inhibit cAMP-catabolizing phosphodiesterase type IV. This suggests that the elastase release by LPS-primed neutrophils is likely to be controlled by intracellular cAMP, and raises the possibility of limiting pharmacologically the elastase-mediated tissue injury during neutrophilic inflammation.

Nonenzymatically glycated albumin (Amadori adducts) enhances nitric oxide synthase activity and gene expression in endothelial cells.

Hyperglycemia is considered to induce diabetic nephropathy through nonenzymatic glycation of proteins. Since hyperfiltration is likely to be the mechanism initiating the glomerular lesions, we investigated the effects of Amadori glucose adducts in serum albumin on the production of vasoactive mediators, including nitric oxide (NO) and eicosanoids, by endothelial cells (EC). Amadori adducts of glycated albumin induced a dose-response increase in NO synthase activity of murine endothelioma cells, up to 16.4 +/- 2.1-fold increase of basal values (P < 0.0001) at concentrations of 35 mg/ml mimicking physiological serum albumin concentration, and 4.6 +/- 0.8-fold increase at 17 mg/ml (P < 0.001). The effect was still detectable with glycated albumin 1.7 mg/ml, which approaches its estimated concentration in diabetic serum (1.6 +/- 0.3-fold increase, P < 0.05) The phenomenon was reproducible in human umbilical vein endothelial cells, though to a lesser extent, and further studies on murine EC were employed. The mRNA encoding for inducible NO synthase was overexpressed in EC incubated with Amadori adducts of glycated albumin in comparison to native albumin. Glycated albumin induced increased mRNA expression and synthesis of TNF-alpha. The stimulatory effect induced by glycated albumin on NO synthase activity was almost completely inhibited by anti TNF alpha antibodies. 3H-thymidine incorporation by EC was significantly inhibited when cells were grown in presence of glycated albumin (P < 0.001), and the phenomenon was abolished by the coincubation of the NO competitive inhibitor L-NAME. The early glycosylation products increased thromboxane production (P < 0.001), while prostaglandin E2 synthesis was unaffected. These data indicate that Amadori products of glycated albumin modulate NO synthase activity and eicosanoid balance in EC. These effects may be relevant to the hemodynamic changes in the early phases of diabetic nephropathy and in the lasting progression to sclerosis.

Effect of acute carnitine administration on glucose insulin metabolism in healthy subjects.

The authors evaluated the effect of carnitine on glucose insulin metabolism. Twenty-five subjects free from metabolic disorders or serious functional alterations in the liver, kidneys or myocardium were studied. All were infused with a 5% glucose-enriched solution (Group A); 48 h later the same subjects were infused with a 5% glucose solution containing 2g carnitine (Group B). Variations in blood glucose and insulin levels and range were measured at different times during and after infusion. Study results showed that the administration of carnitine lead to an improvement in glucose metabolism, as demonstrated by a saving in insulin secretion accompanied, at least on a temporary basis, by a corresponding decrease in blood glucose which, however, remained within normal parameters.

Neutrophil dysfunction and increased susceptibility to infection.

A critical evaluation of 3 years' experience using laboratory screening to detect neutrophil dysfunction is described. Neutrophil dysfunctions in patients with recurrent bacterial infections were investigated by using the following screening tests: (1) neutrophil chemotaxis towards N-formylmethionyl peptides (FMLP) and the complement fragment C5a; (2) neutrophil production of superoxide anions (O2-) in response to phorbol myristate acetate and opsonized zymosan particles; and (3) examination of May-Grünwald and myeloperoxidase cytochemical staining of peripheral blood smears. These tests were carried out in 100 patients suffering from infections and suspected of having altered neutrophil functional competence. A minority of patients was found to have well defined neutrophil dysfunction syndromes: chronic granulomatous disease (four cases), Chediak-Higashi disease (one case) and myeloperoxidase deficiency (one case). Of the remaining 94 patients, in whom infections localized to airways and/or skin predominated, 53 cases were found to have impaired chemotaxis (41 cases) or partial defects of the O2- production. Defects of chemotaxis toward FMLP and those towards both FLMP and C5a were the most frequent abnormalities. No defect was found in the other 41 patients. Moreover, impaired neutrophil chemotaxis was found in some patients with selective IgA deficiency (five cases) or immotile cilia syndrome (seven cases). The results suggest that (a) additional screening tests are required to ameliorate the efficiency of the diagnostic work-up of the patients suspected to have neutrophil dysfunction; and (b) further evaluation, also at the molecular level, should be considered at least in selected cases of non-classified neutrophil dysfunction in order to clarify diagnosis and plan rational therapeutic strategies.