The possible role of glutathione-S-transferase activity in diabetic nephropathy.

The most common cause of end stage renal disease is diabetic nephropathy. An early diagnosis may allow an intervention to slow down disease progression. Recently, it has been hypothesized that glutathione-S-transferase (GST) activity may be a marker of severity of chronic kidney disease. In particular, a lower GST activity is present in healthy subjects compared to patients with nephropathy. In the present review we illustrate the scientific evidence underlying the possible role of GST activity in the development of diabetic nephropathy and we analyze its usefulness as a possible early biomarker of this diabetic complication.

Metabolomics signature improves the prediction of cardiovascular events in elderly subjects.

AIMS: Age is one of the most important determinants of cardiovascular health, therefore the management of cardiovascular diseases (CVD) in elderly people entails great challenge. A possible explanation of vascular senescence process is the mitochondrial damage and dysfunction. We hypothesized that metabolomic profiling would identify biomarkers predicting major cardiovascular events (MACEs) in elderly people, improving the clinical standard cardiovascular risk factors. METHODS AND RESULTS: Targeted-mass-spectrometry-based profiling of 49 metabolites was performed in a group of very old participants (n = 67, mean age = 85 ± 3 years) with a high rate of previous CVD (68%). Principal Component Analysis, Random Survival Forest analysis and Cox proportional hazards regression modeling were used to evaluate the relation between the metabolite factors and recurring MACEs. We tested discrimination ability and reclassification of clinical and metabolomic models. At follow-up (median = 3.5 years), 17 MACEs occurred (5 cardiovascular deaths, 1 nonfatal myocardial infarction, 7 nonfatal strokes and 4 peripheral artery surgeries) (incidence = 7.3% person-years). Metabolite factor 1, composed by medium- and long-chain acylcarnitines, and factor 7 (alanine) were independently associated with MACEs, after adjustment for clinical CV covariates [HR = 1.77 (95%CI = 1.11-2.81, p = 0.016) and HR = 2.18 (95%CI = 1.17-4.07, p = 0.014), respectively]. However, only factor 1 significantly increases the prediction accuracy of the Framingham Recurring-Coronary-Heart-Disease-Score, with a significant improvement in discrimination (integrated discrimination improvement = 7%, p = 0.01) and correctly reclassifying 41% of events and 37% of non-events resulting in a cNRI = 0.79 (p = 0.005). CONCLUSIONS: Aging mitochondrial dysfunction evaluated by metabolomic profiling is associated with MACEs, independently of standard predictors.

Microsatellite analysis of induced sputum DNA in patients with lung cancer in heavy smokers and in healthy subjects.

Abnormality in the fragile histidine triade (FHIT), a candidate tumor suppressor gene located in chromosome region 3 (3p14.2), has been frequently found in multiple tumor types, including lung cancer. In this study, the authors assessed the consistency of DNA microsatellite analysis of induced sputum (IS), as compared to that of blood and plasma. They also evaluated the loss of heterozigosity (LOH) and microsatellite instability (MSI) in 3 different loci, D3S1300, D3S1313, and D3S1234, all internal to the FHIT gene, in IS, blood, and plasma from patients with lung cancer, smokers, and healthy subjects. Eighteen patients with lung cancer (3 females, age mean +/- SD: 63 +/- 7 years), 39 smokers (23 females, age mean +/- SD: 57 +/- 6 years and cigarette pack-years mean +/- SD: 34 +/- 12), and 22 healthy nonsmoking subjects (13 females, age mean +/- SD: 63 +/- 5 years) were studied. DNA was extracted from blood, plasma, and IS, by means of a standard method. Analysis of LOH and MSI were performed using a fluorescent polymerase chain reaction (PCR)-based approach, followed by capillary electrophoresis. The ratios between the peak heights (phs), expressed as random fluorescence units, from plasma/blood (p/b) and induced sputum/blood (is/b) in all three loci were considered. The biases (agreement limits) between the mean ph ratio from p/b and is/b of D3S1300, D3S1313, and D3S1234 were respectively 0.07 (- 0.39 to 0.53), 0.016 (- 0.32 to 0.35), - 0.10 (- 0.51 to 0.30) in the patients; - 0.04 (- 0.52 to 0.43), - 0.06 (- 0.31 to 0.18), - 0.08 (- 0.48 to 0.30) in smokers; and - 0.11 (- 0.40 to 0.17), - 0.05 (- 0.53 to 0.43), - 0.09 (- 0.51 to 0.33) in healthy subjects. LOH and MSI in at least one locus were observed in 55% of patients, in 18% of smokers, and in 4.5% of healthy subjects (P < 0.001). These results showed that IS DNA provided data that were consistent with those from blood and plasma. These findings highlight new prospects for early tumor detection by a noninvasive technique based on the analysis of genetic alterations in induced sputum.

Monitoramento das patologias em suínos no período de crescimento / Monitoring of pathologies in growing swines

Determinou-se, por meio de um sistema de monitoramento de suínos em fase de crescimento e em matadouro, a presença de patologias como indicadoras de problemas na população de referência. O estudo foi realizado em uma criação intensiva de ciclo completo, durante nove semanas. Foram determinadas as prevalências dos diagnósticos clínico-patológicos obtidos durante um período de nove semanas. Utilizaram-se cinco grupos de 43 leitões, com pesos de até 19kg, considerados de baixa performance. Estimaram-se, por intervalos de confiança de 95 por cento, as prevalências acumuladas em cada monitoramento. Obteve-se correlação de 0,99 (P<0,0001) entre as prevalências anuais com as nove semanas de estudo, mostrando correspondência com o achado em matadouro. Amostras em semanas alternadas de três grupos de 43 animais são bons indicadores das patologias que afetam os suínos em crescimento, mesmo aquelas de baixa freqüência ou muito letais.

The prevalence of different pathologies as an indicator of health problems in a particular population by means of slaughterhouse monitoring of growing pigs was determined. The work was carried out in an indoor intensive farrow to finish swine farm during 9 weeks. The prevalence of diseases was determined by means of clinical and pathological diagnosis. For the slaughterhouse inspection, five groups of 43 pigs each weighting up to 19kg, considered as poor performers, were put together. These animals were sent to the slaughterhouse and were observed for the presence or absence of skin and joint lesions of arms and legs and also lesions of serous membranes and parenchimal organs. An estimative with a confidence interval level of 95 percent was performed on cumulated prevalence in each monitoring session for 43, 86, 129, 172 and 215 animals. Annual prevalence from clinical and pathological diagnosis and 9-week-period had correlations (r=0.99; P<0.0001) and correspondence with slaughterhouse observations. In conclusion, samplings in alternate weeks of three groups of 43 pigs seem to be a good indicator of pathologies affecting growing swines, even those of very low prevalence or highly lethal.

[Posterior mediastinum neoplasms: a case of schwannoma]. / I tumori del mediastino posteriore: un caso di schwannoma.

The Authors report the case of a 33-year-old male affected with an asymptomatic schwannoma of the posterior mediastinum, and review the relevant Literature, discussing the difficulties in making the differential diagnosis of the posterior mediastinum lesions, particularly in the pre-clinical phase. Neurogenic tumors represent about 75 percent of all tumors of mediastinum and about one third of all tumors of the mediastinum. They can originate from the peripheral nervous system group, from the sympathetic nervous system group or, in rare cases, from the vagus nerve. In adult patients, they are usually found by chance during radiographic examination of the thorax, and they are usually asymptomatic and benign. During preoperative evaluation, a magnetic resonance examination of rachis should be done to exclude the possibility of intraspinal involvement. If there are no contraindications, the treatment of choice should be surgical resection by means of thoracoscopy or thoracotomy, when size and location of the tumors allow it in order to prevent malignant evolution.

[Therapeutic strategies in neoplasms of the distal bile duct and the Vater's ampulla]. / Strategie terapeutiche nelle neoplasie della via biliare distale e dell'ampolla di vater.

Malignant periampullary and ampullary tumors represent an anatomic and clinical entity of complex surgical approach due to the difficulty in setting out an early diagnosis, for the back of symptoms at early stages. The Authors at the Department of Surgery and Medical Technology Applications "F. Durante", University of Rome "La Sapienza", since 1990 to 2001 observed 19 cases. The results of surgical treatment of these tumors are here reported. By the analysis of the cases it is evident that long-term survival if mainly conditioned by prompt diagnosis stage of the tumor, and patient status before the operation.

[Neurinoma of the brachial plexus: 2 case reports]. / Neurinoma del plesso brachiale: due casi clinici.

Neurinomas, also referred to as neurilemmomas and schwannomas, are rare benign tumours of the peripheral nerves. A small percentage of these lesions arise from the brachial plexus. The Authors report two cases of schwannoma arising from the brachial plexus. Such lesions, usually asymptomatic, may cause sensitivity alterations or, less frequently, motor deficits in the involved arm. Tumour enucleation, avoiding damage to any of the nervous fascicles, is the treatment of choice.

[Atrial fibrillation and hyperthyroidism: results after thyroidectomy]. / Fibrillazione atriale ed irpertiroidismo: risultati dopo intervento di tiroidectomia.

Hyperthyroidism patients can develop atrial fibrillation (AF) with a frequency range between 10 and 25%. The Authors consider 256 hyperthyroid patients operated in their Department, from 1985 to 2001. Before and after surgery, thyroid examination, routine blood tests and common diagnostic evaluations were performed. The follow-up has been 12 months and during this period a L-Thyroxine (50-200 mg/die) therapy has been arranged. Before surgery 59 patients (23%) showed an AF associated with palpitations (76%), asthenia (66%), tremors (54%) and dyspnea (43%). The onset of AF was less than 1 months in 18% of cases, less than 3 months in 30% and chronic in remaining 52%. The 87% of patients was already pharmacologically treated. All the patients were treated with anticoagulants, with a INR value between 2 and 3. After the surgery, 47% of patients presented a restoration of the sinusal rhythm, while the 53% showed a stronger therapeutic responsiveness to specific antiarrhythmic therapy. Treatment of FA hyperthyroid patients with FA tends to normalize the function of the thyroid gland (euthyroidism). In our experience the restoring of sinusal rhythm usually develops in patients with 50 years of age (45 +/- 5 years), while in older patients AF tends to be resistant to treatment.

Hypoglycemic effect of insulin-like growth factor-1 in mice lacking insulin receptors.

We have investigated the metabolic actions of recombinant human IGF-1 in mice genetically deficient of insulin receptors (IR-/-). After intraperitoneal administration, IGF-1 caused a prompt and sustained decrease of plasma glucose levels in IR-/- mice. Plasma free fatty acid concentrations were unaffected. Interestingly, the effects of IGF-1 were identical in normal mice (IR+/+) and in IR-/- mice. Despite decreased glucose levels, IR-/- mice treated with IGF-1 died within 2-3 d of birth, like sham-treated IR-/- controls. In skeletal muscle, IGF-1 treatment caused phosphorylation of IGF-1 receptors and increased the levels of the phosphatidylinositol-3-kinase p85 subunit detected in antiphosphotyrosine immunoprecipitates, consistent with the possibility that IGF-1 stimulates glucose uptake in a phosphatidylinositol-3-kinase-dependent manner. IGF-1 receptor phosphorylation and coimmunoprecipitation of phosphatidylinositol3-kinase by antiphosphotyrosine antibodies was also observed in liver, and was associated with a decrease in mRNA levels of the key gluconeogenetic enzyme phosphoenolpyruvate carboxykinase. Thus, the effect of IGF-1 on plasma glucose levels may be accounted for by increased peripheral glucose use and by inhibition of hepatic gluconeogenesis. These data indicate that IGF-1 can mimic insulin's effects on glucose metabolism by acting through its own receptor. The failure of IGF-1 to rescue the lethal phenotype due to lack of insulin receptors suggests that IGF-1 receptors cannot effectively mediate all the metabolic actions of insulin receptors.

Identification, characterization, and analysis of cDNA and genomic sequences encoding two different small heat shock proteins in Hordeum vulgare.

In vitro translation of mRNAs prepared from barley (Hordeum vulgare) seedlings (cv. Onice) exposed at 40 degrees C directed the synthesis of major heat shock proteins (HSPs) with molecular masses of 80-90, 70, 42 and 16-22 kDa. A cDNA library prepared from the 40 degrees C mRNAs and screened by differential hybridization led to the isolation of heat shock specific sequences. One of these (Hv hsp18) was confirmed by hybrid-arrested and hybrid-released translation as encoding for an 18-kDa HSP. The barley hsp18 sequence has an open reading frame encoding a 160 amino acid residue 18-kDa protein that is 63% identical to wheat 16.9-kDa HSP (clone C5-8), 54% identical to soybean (Glycine max) 17.5-kDa HSP, and 49% identical to Arabidopsis thaliana 17.6-kDa HSP. Lower similarities were found with class II plant small HSPs such as soybean 17.9-kDa HSP (27%), Pisum sativum 17.7-kDa HSP (30%), wheat (Triticum aestivum) 17.3-kDa HSP (clone Ta hsp 17.3) (30%), and with animal small HSPs and alpha-crystallins. The Hv hsp18 sequence was used to pick up Hv hsp17 genomic sequence encoding for another class I 17-kDa HSP. By computer analysis of the nucleotide sequence the TATA box, two heat shock promoter elements, a metal-ion response element, and the polyadenylation signals were identified. Barley HSP18 has an additional cysteine-rich region when compared with HSP17 mapping at the carboxy terminal end.

[Cystic tumors of the pancreas: a case of cystadenocarcinoma of the pancreatic head]. / Tumori cistici del pancreas: su un caso di cistoadenocarcinoma della testa.

Cystic tumors of the pancreas are important, although rare, lesions representing nearly 1% of all neoplasms and 10-15% of pancreatic cysts. The Authors compare clinical data of the case observed with those reported in literature. The described case confirms the peculiar characteristics of pancreatic mucinous cystoadenomas: slow growth, not specific symptoms, poor trend to give local or distant metastases, difficult preoperative differential diagnosis and satisfying oncological resection. Taking into account these parameters and the potential malignant evolution of such tumors, the Authors underline the importance of a radical surgical therapy.

Evidence that human and porcine insulin differently affect the human insulin receptor: studies with monoclonal anti-insulin receptor antibodies.

Binding studies have been carried out with radioiodinated monoclonal antibodies directed to various epitopes of the insulin receptor in order to detect differences between human and porcine insulin in the interaction with the human insulin receptor. Human insulin was more effective that porcine insulin at inhibiting the binding of 125I-MA-5 to IM-9 cells, Hep-2 human larynx cells and human placenta membranes. On the contrary, human and porcine insulin showed similar inhibitory effect on the binding of two other labeled anti-insulin receptor monoclonal antibodies, thus ruling out the possibility that results were due to experimental artifacts. Although several interpretations are possible, data reported suggest that human insulin and porcine insulin might differently affect the insulin receptor, even if, the biological significance of these findings remains unknown.

Determination of serum levels of complement component C4b-binding protein: influence of age and inflammation.

C4b-binding protein (C4b-BP) is a high molecular weight plasma protein which inhibits the activity of the classical complement pathway C3 convertase. In addition to multiple binding sites for C4b, C4b-BP possesses a single binding site for vitamin K-dependent protein S, an inhibitor of blood coagulation. As protein S bound to C4b-BP has no anticoagulant activity, C4b-BP participates in the regulation of both the complement and the coagulation pathways. We have produced and immunochemically characterized a series of murine monoclonal antibodies to human C4b-BP. A mixture of four monoclonal antibodies precipitating C4b-BP both in agarose gel and in solution was used to develop a highly reproducible radial immunodiffusion method for the measurement of C4b-BP in human serum. C4b-BP levels were measured in sera from 284 patients referred to our central laboratory. Samples from subjects with an increased erythrocyte sedimentation rate (ESR), a1-acid glycoprotein (a1-AGP) or C-reactive protein (CRP) had significantly higher C4b-BP levels (307 mg/l, 292-322 mg/l, geometric mean and 95% confidence limits of the mean) than those from subjects without elevation of the aforementioned established acute phase reactants (231 mg/l, 226-237 mg/l, P less than 0.00001). C4b-BP was significantly (P less than 0.001) correlated with ESR (r = 0.715), a1-AGP (r = 0.692) and CRP (r = 0.567). There was no gender-related difference in C4b-BP levels. In subjects with no increased acute phase reactants there was a significant correlation between C4b-BP levels and age (r = 0.387, P less than 0.001). High C4b-BP might contribute to the increased thrombotic risk associated with inflammation and aging.

Beta-endorphin receptors on cultured and freshly isolated lymphocytes from normal subjects.

[125I]-[D-Ala2]-beta EP*) binding to Epstein-Barr Virus (EBV) transformed B lymphocytes or to freshly isolated lymphocytes was characterized. The binding was time-, temperature- and pH-dependent; furthermore, it was reversible and cell concentration dependent. Maximum binding appeared at 15C in Tris buffer (pH 7.6) containing both BSA 0.5% and Bacitracin 0.1 mg/ml. beta EP inhibited BEP* binding to transformed lymphocytes and to freshly isolated lymphocytes by 50% at approximately 4 x 10(-8) M and 8 x 10(-9) M, respectively. Peptides representing amino-acid sequences 1-5, 1-16 and 1-17 of beta EP did not inhibit beta EP binding, neither did the opiates compounds Naloxone, Morphine, Bremazocine and Ethylketocyclazocine. On the contrary, beta EPd (6-31) inhibited beta EP* binding as effectively as beta EP, thus indicating that beta EP* binding to lymphocytes does not involve the N-terminal region of beta EP. 2 x 10(7)/ml freshly isolated lymphocytes bound 1.53 +/- 75%, whereas 2 x 10(6)/ml transformed lymphocytes bound 1.64 +/- 0.75% (mean +/- SD) beta EP* at tracer concentration. EBV transformed lymphocytes and freshly isolated lymphocytes may be useful to study beta EP receptors in humans.

Radial-immunodiffusion assay of human apolipoprotein A-I with use of two monoclonal antibodies combined.

We produced and characterized several monoclonal antibodies directed toward human plasma apolipoprotein A-I. Two of them, A-I-12 and A-I-57, individually precipitated purified or native high-density lipoprotein in agarose gel by double immunodiffusion. Because radial immunodiffusion performed with a single monoclonal antibody gave faint and diffuse rings of precipitation, we developed and optimized working conditions for using these two monoclonal antibodies combined to determine apolipoprotein A-I in human plasma. This combination gave easy-to-measure, clear, sharp rings, and linear and parallel standard curves for HDL3 (the primary standard) and a reference serum (the secondary standard). Moreover, no pretreatment of samples with dissociating agents or detergents is necessary. The assay was complete after overnight incubation, as compared with two to three days when polyclonal antisera were used. Apolipoprotein A-I concentrations as measured in 128 normolipidemic subjects and in 72 patients with various lipid disorders by the radial immunodiffusion technique with monoclonal antibodies (x) compared well (r = 0.882; y = 1.029x-0.036) with those measured by radial immunodiffusion with polyclonal antisera (y).

Development of a radial immunodiffusion technique employing monoclonal antibodies for apolipoprotein B determination in human plasma.

Twenty monoclonal antibodies obtained from two different fusions of SP2/0-Ag 14 cell line (non-secretor hybridoma) with human plasma low density lipoproteins have been selected. We found that a mixture formed by the 20 monoclonal antibodies was able to form a single precipitin line with human plasma low density lipoproteins by a double gel diffusion technique. Further studies revealed that only two monoclonal antibodies were needed to precipitate low density lipoproteins in gel. However, a minimum of four particular monoclonal antibodies was required to obtain an optimal precipitin ring and a linear standard curve within 24 h using a radial immunodiffusion technique. We have then compared the radial immunodiffusion performed with a monoclonal antibody mixture to those employing conventional goat and rabbit antibodies in terms of plasma apolipoprotein B determinations. The apolipoprotein B values determined by monoclonal antibodies significantly correlate with the values obtained by the technique using goat (r = 0.95; p less than 0.001) and rabbit (r = 0.95; p less than 0.001) antibodies. Our data indicate that a mixture of monoclonal antibodies can mimic conventional antibodies in terms of immunoprecipitation and apolipoprotein B determination.