Flow cytometric detection of anti-gliadin antibodies.

A very sensitive solid-phase fluorescent immunoassay to detect anti-alpha-gliadin IgA class antibodies is described. The solid phase consisted of polystyrene carboxylated microspheres, of 5 microns diameter, coated with alpha-gliadin. Serum-specific antibodies bound to the alpha-gliadin were measured by flow cytometry using fluorescein-conjugated anti-human IgA. 41 samples were tested and the results compared with those obtained by a standard method: an enzyme-linked immunosorbent assay (ELISA). A good correlation was found between the two techniques (r = 0.96). The sera of untreated coeliac children showed significantly higher antibody values than the sera of children on a gluten-free diet or healthy control groups. The flow cytometric method was more sensitive when the Kolgomorov/Smirnov test was used to analyse the histograms. This method provides an alternative screening test for coeliac disease and may also be used to confirm borderline results obtained in the ELISA test.

Effects of interferon therapy on fibrosis serum markers in HCV-positive chronic liver disease.

OBJECTIVE: To evaluate serum levels of prolyl-hydroxylase and helical domain of Type IV collagen, markers of hepatic fibrogenesis, in patients with HCV-positive chronic liver disease and the effects of interferon therapy on these markers. DESIGN: Prolyl-hydroxylase and Type IV collagen were determined before therapy and each month during the treatment and follow-up. METHODS: Fifty-seven HCV-positive patients were studied. All the subjects received alpha2a recombinant interferon, 6 MU subcutaneously three times a week for 4 weeks, followed by 3 MU thrice weekly for 5 months. After cessation of treatment, each patient was followed for 12 months. Prolyl-hydroxylase and helical domain of Type IV collagen were measured by using immunoenzymatic methods. HCV-RNA and HCV genotype were determined according to the method of Okamoto. RESULTS: In the patients prolyl-hydroxylase (39.8+/-8.9 ng/ml) was not different from controls (39.1+/-5.9 ng/ml). On the contrary, the patients showed a mean Type IV collagen (133.6+/-93.3 ng/ml) significantly (P < 0.01) higher than controls (100.2+/-10.5 ng/ml). A good relationship between the degree of liver fibrosis and the Type IV collagen serum level was found (r = 0.68; P < 0.005). In both responders and non-responders the Type IV collagen levels decreased during interferon therapy. During the follow-up, in responders the Type IV collagen did not show modifications, while in non-responders/relapsers it returned rapidly to the pretreatment levels (139.1+/-100.7 ng/ml). CONCLUSION: In HCV-positive chronic liver disease, prolylhydroxylase is not a good marker of hepatic fibrosis, while Type IV collagen is a useful tool for evaluating fibrogenic activity. Interferon seems to be able to reduce the liver fibrosis even without the inhibition of viral replication and independently from liver necrosis.

Variations in hemostatic parameters after near-maximum exercise and specific tests in athletes.

BACKGROUND: The clotting state of the blood changes according to the type of physical exercise to which a group of healthy subjects are subjected. We studied the behaviour of the coagulation system before and after near-maximum, specific and standardized exercise tests in three groups of males practising sports defined as demanding in terms of cardiovascular output. METHODS: The study was a comparative investigation between athletes and the group of controls composed of presumably healthy males. SETTING: athletes training for competitions such as marathon, rowing and weightlifting. PARTICIPANTS AND INTERVENTIONS: we tested 7 rowers using the rowing machine, 12 marathon runners using the treadmill, 7 weightlifters using their own exercise equipment, and 7 healthy subjects (controls) using the cycle ergometer. MEASURES: during the tests we monitored heart rates, maximal oxygen intake, anaerobic threshold, respiratory quotient, maximum ventilation, and lactic acid. The following coagulation tests were performed before and after near-maximum exercise: prothrombin time (PT), partial activated thromboplastin time (PTT), fibrinogen (FBG), antithrombin III (ATIII), protein C (PC), protein S (PS), prothrombin fragment 1 + 2 (F1 + 2), tissue activator of plasminogen (t-PA) and its inhibitor (PAI). RESULTS: The most significant results showed a low basal PC in the rowers which decreased further after near-maximum exercise; significantly higher basal activities of ATIII, PC and PS in the marathon runners compared to the rowers; a high proportion of weightlifters showed a reduction in t-PA after exercise and an increase of PAI; the controls were the only group in which fibrinolytic activity and all the circulating anticoagulants increased after near-maximum exercise. Thus subjects who practise aerobic sports differ principally in terms of variations in inhibitors (low PC in rowers and marathon runners, increased presence of inhibitors in controls). The weightlifters did not show any significant variations, and so the kind of exercise involved (training to increase resistance and maximum strength) and the recovery times between the exercises do not seem to trigger changes in coagulation/fibrinolysis. CONCLUSIONS: We can therefore confirm that only relatively prolonged effort can trigger a mechanism beneficial to the cardiovascular system. In conclusion, physical activity benefits the coagulation system particularly as regards fibrinolysis, but certain subjects may be at risk of thrombosis and these must be identified and followed. We suggest that fibrinolytic activity be studied in athletes who practise weightlifting and have a history of cardiovascular disease, and that inhibitors (protein C in particular) be studied in rowers with a family history of thromboembolism.

[Neonatal hypoxic-ischemic nephropathy and urinary diagnostic indices: the utility of measuring tubular enzymes (NAG and AAP)]. / Nefropatia ipossico-ischemica nel neonato ed indici diagnostici urinari: utilità del dosaggio di enzimi tubulari (NAG ed AAP).

Feto-neonatal hypoxia can cause a functional kidney impairment, which is often temporary and not clinically overt, but sometimes leading to acute renal failure. Hypoxic stress may result in a tubulo-interstitial damage, and kidney tubular enzymes determination has proved to be an easy, early, and non invasive method to define a tubular interstitial lesion. A major target of nephrotoxicity is the proximal tubular cell: alterations in brush-border membrane and cytoplasm result in increased turnover processes in the kidney cortex, following by a corresponding increased excretion of alanine-aminopeptidase (AAP) and N-acetyl-glucosaminidase (NAG) from the proximal tubular cells, long before glomerular or tubular functions are impaired. AAP and NAG excretion is directly correlated with the strength and the duration of toxic alteration of the proximal tubule. NAG and AAP have been already studied in the adults and the children; they have been chosen for this investigation with a double aim: 1) to define the amount of their urinary excretion in relation with gestational age at birth; 2) to evaluate if in the newborn, independently of the gestational age, their urinary concentration may be increased by ischaemic conditions caused by hypoxia. We studied 52 healthy newborns (7 preterm of 33-36 weeks and 45 full-term) and 16 newborns with feto-neonatal hypoxia (8 preterm of 26-36 weeks and full-term) at the forth day of life. Urinary NAG and AAP were assayed by colorimetric methods and the results expressed as mU/mg. creatininuria.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils.

We developed a rapid and sensitive two-color flow cytometric method which allows the simultaneous quantification of both the phagocytosis rate and the oxidative burst activation of polymorphonuclear leukocytes (PMNLs). The oxidation of hydroethidine (HE) to ethidium bromide (EB) was performed by the oxidative neutrophil products within the cells during the respiratory burst, which was stimulated by phagocytized fluorescein-labeled Staphylococcus aureus. By means of flow cytometry we measured red EB fluorescence emission together with green fluorescence, which was emitted by the ingested fluoresceinated bacteria. The fluorescence intensity was proportional to the number of bacteria ingested. Adherent bacteria were distinguished from the ingested ones. This two-color cellular staining permits measurement of two different functions of neutrophils in one step. This method could be of interest for the determination of the interactions between neutrophils and bacteria and for the investigations on infectious diseases in chronic granulomatous disease patients.

In vitro incubation of bone marrow and peripheral stem cells with vincristine and methylprednisolone: functional T-cell depletion for haploidentical and autologous transplants.

A mismatched bone marrow transplantation is feasible only if the donor's marrow lymphocytes are eliminated from the graft. This can be achieved by several methods, but all have the disadvantage of inducing a long-lasting immune deficiency while the risk of graft rejection and leukemic relapse increase. We use a sort of functional T-cell depletion by treating the cells with vincristine and methylprednisolone. This method is surely the cheapest and has allowed us to perform 60 transplants with a tolerable risk of GVHD. The treatment of the donor's lymphocytes has already been demonstrated to be able to block the mixed lymphocyte culture reaction in vitro. In this experiment Th1 and Th2 activities were almost completely blocked without reduction of lymphocyte viability and apoptosis induction.

A new hepatic protein inactivating glucose 6-phosphate dehydrogenase.

Two NADP-cleaving enzymes, namely NADP glycohydrolase and NADP pyrophosphatase, are present in a rat liver extract that inactivates G6PD (glucose 6-phosphate dehydrogenase). The following results suggest that a third G6PD-inactivating protein is present in this extract. (1) Nicotinamide, which selectively inhibits NADP glycohydrolase, enhances the G6PD inactivation under conditions where G6PD activity in control experiments is rather stable. (2) DEAE-cellulose adsorbs the bulk of both NADP glycohydrolase and NADP pyrophosphatase, whereas most of the G6PD-inactivating ability is unadsorbed. (3) Out of 37 liver extracts that were prepared, two were found to lack NADP pyrophosphatase. After removal of NADP glycohydrolase from these extracts by centrifugation, they were still found to inactivate G6PD. (4) Deproteinization of DEAE-cellulose supernatants results in a complete loss of G6PD-inactivating ability; moreover, kinetic experiments performed with the extracts lacking pyrophosphatase strongly support the view that the inactivating protein is an enzyme, although its mechanism is not clear. (5) NADP protects G6PD from inactivation and also reactivates the enzyme completely, thus supporting the view of some action of the inactivating protein on the G6PD-bound NADP.

[Improvement in the efficiency of an ambulatory service case load by applying a computerized method (patient flow analysis)]. / Miglioramento dell'efficienza di un servizio prelievi ambulatoriale mediante l'applicazione di un metodo computerizzato (patient flow analysis).

The authors describe the application of a technique called Patient Flow Analysis aimed at the improvement of Clinic Personnel efficiency and reduction of patient waiting time. Results were satisfactory and encourage further experiences.

[Binding capacity of albumin with bromcresol green in a group of women of fertile age]. / Studio della capacita legante dell'albumina con verde di bromocresolo in un gruppo di donne in età fertile.

Albumin binding ability was studied in 551 women of fertile age through the dye-binding test with BCG. In 61 cases (11.0%) no binding was found: 47 women (77.0%) were pregnant at the time the blood test was performed. Such a result agrees with previous studies which indicate a reduced albumin binding ability in the blood of near term pregnant women and in the umbilical cord.

Serum free fatty acids and bilirubin concentration during fasting in patients with Gilbert's syndrome and normal controls.

The increments in serum concentrations of unconjugated bilirubin and free fatty acids (FFA) were measured 24 and 48 h after reduction of the caloric intake (400 cal/day) in 17 patients with Gilbert's syndrome (GS) and in 12 healthy control subjects. In males, both normal and with GS, the rise in serum bilirubin was statistically higher (p less than 0.01) as compared to females. On the contrary, no sex difference was found in FFA concentrations. A linear correlation (p less than 0.01) between bilirubin and FFA serum levels was present in normal males and in patients with Gilbert's syndrome of both sexes. Because bilirubin and FFA partly share a common, bilitranslocase-mediated, hepatic uptake mechanism, data reported support the hypothesis that a bilitranslocase function may be one of the metabolic defects in Gilbert's syndrome.