Autoantibodies against a 72-kDa ductal cell membrane glycoprotein in a patient affected by Sjögren's syndrome and gastric MALT lymphoma.

Sjögren's syndrome is a chronic autoimmune disease affecting exocrine glands, resulting in xerostomia and xerophthalmia. Lymphocytic infiltration and fibrosis of exocrine glands as well as the presence of autoantibodies against organ-specific and non-organ-specific antigens are the hallmarks of the disease. We investigated whether some patients affected by Sjögren's syndrome might have autoantibodies directed against epithelial duct cell membrane proteins. We screened sera from patients affected by Sjögren's syndrome by indirect immunofluorescence on monkey salivary gland sections and FG-Met-2 cells (a pancreatic carcinoma cell line with ductal features) for the presence of antisalivary duct antibodies. Positive sera were employed in immunoprecipitation experiments on (35)S-methionine in vivo labeled and surface-biotinylated FG-Met-2 cells. The serum of a patient affected by Sjögren's syndrome and gastric mucosa-associated lymphoid tissue (MALT) lymphoma gave positive and distinct membrane immunostaining on FG-Met-2 cells. Immunoprecipitation with the patient's serum from (35)S-methionine-labeled cell extracts followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and autoradiography showed the presence of autoantibodies against a 72-kDa protein. After biotin-surface labeling of FG-Met-2 cells, a band with identical electrophoretic mobility was immunoprecipitated by the serum, demonstrating that the 72-kDa band is a membrane glycoprotein. We demonstrated by three complementary approaches, i.e., immunocytochemistry, (35)S-methionine in vivo labeling, and cell surface biotinylation, the presence of autoantibodies directed against a duct cell membrane protein of 72-kDa in a patient affected by Sjögren's syndrome and gastric MALT lymphoma. Autoantibodies directed against this novel membrane autoantigen may be an additional serological marker in some cases of Sjögren's syndrome.

Erythrocytosis in a patient with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) can be accompanied by compensatory secondary erythrocytosis. However, the exact prevalence of secondary erythrocytosis in COPD is unknown. Although diagnostic criteria for polycythemia vera versus secondary erythrocytosis are mutually exclusive, we describe here the coexistence of polycythemia vera and COPD in the same patient.

Deranged platelet calcium homeostasis in diabetic patients with end-stage renal failure. A possible link to increased cardiovascular mortality?

OBJECTIVE: Platelet hyperfunction is a typical feature of the prothrombotic state that frequently complicates the natural history of diabetes. In uremia, a bleeding diathesis is present, which principally involves the primary phase of hemostasis. Thus, in patients with uremia of diabetic origin, the infrequent coexistence of two opposite alterations of hemostasis takes place. In patients with uremia, an increased incidence of cardiovascular events and related mortality is observed. This phenomenon is greatly amplified in uremia of diabetic origin. Calcium homeostasis is a critical aspect of platelet function, which has recently become available in human diseases. The aim of this study was to evaluate calcium homeostasis in platelets from patients with uremia of diabetic and nondiabetic origin. RESEARCH DESIGN AND METHODS: We evaluated, by means of Fura 2, the intracellular concentration of ionized calcium ([Ca2+]i) in platelets from 18 patients with uremia of diabetic origin, 12 patients with uremia of nondiabetic origin and 16 healthy control subjects [Ca2+]i was evaluated in resting conditions and after stimulation with 0.05, 0.1, 0.5 U/ml thrombin. RESULTS: Platelets from uremic patients with diabetes had higher resting [Ca2+]i than both control subjects (P = 0.01) and uremic patients without diabetes (P = 0.001). Similarly, after stimulation with thrombin, the absolute increase of [Ca2+]i was higher (P < 0.05) in platelets from uremic patients with diabetes compared with both control subjects and uremic patients without diabetes. The relative increase of [Ca2+]i was higher (P < 0.05) than normal in platelets from uremic patients after weak or intermediate strength thrombin. No correlation were present between [Ca2+]i values and other clinical and laboratory variables potentially associated with platelet hyperfunction. CONCLUSIONS: Diabetes and uremia in combination further deteriorate the abnormal platelet calcium homeostasis observed in uremia.

Platelet calcium homeostasis is abnormal in patients with severe arteriosclerosis.

To evaluate platelet calcium homeostasis in a typical thrombosis-prone clinical condition, 14 patients with severe arteriosclerosis and 11 healthy control subjects were studied. Platelet intracellular free calcium concentration ([Ca2+]i) was evaluated by means of the fluorescent probe fura 2 under resting conditions and after challenge with 0.05, 0.1, and 0.5 U/mL thrombin (final concentrations). Three different concentrations of extracellular ionized calcium ([Ca2+]e) were used: 1 mmol/L, 1 mumol/L, and < 1 nmol/L. Resting platelet [Ca2+]i was significantly higher (P < .001) in patients than in control subjects. After addition of 0.05 U/mL thrombin, the relative increase of [Ca2+]i was lower in patients than in control subjects in each of the three [Ca2+]e conditions (P = .05 at 1 mmol/L, P = .02 at 1 mumol/L, and P = .04 at < 1 nmol/L). After addition of 0.1 U/mL thrombin, the relative increase of [Ca2+]i was lower in patients than in control subjects under two [Ca2+]e conditions, 1 mumol/L and < 1 nmol/L (P = .04 and P = .03 respectively). With 0.5 U/mL thrombin, a trend toward lower values in patients than in control subjects was observed, reaching statistical significance (P = .03) only at < 1 nmol/L [Ca2+]e. These results suggest that calcium homeostasis is abnormal in platelets from patients with severe arteriosclerosis and probably reflects a chronic activation.

Deranged platelet calcium homeostasis in poorly controlled IDDM patients.

OBJECTIVE: Platelet hyperfunction frequently occurs in IDDM. As in many other cellular systems, cytosolic free Ca plays a key role in platelet activation. RESEARCH DESIGN AND METHODS: We measured cytosolic free Ca concentration ([Ca2+]i) by means of the fluorescent probe fura-2 in 60 IDDM patients (mean age 30.8 yr, range 18-50 yr) and in 31 age-matched healthy control subjects. Platelets were studied in both resting conditions and after stimulation with thrombin at 0.05, 0.1, and 0.5 U/ml. RESULTS: No differences were noted between control subjects and diabetic patients, as a whole. Patients with a poor metabolic control (HbA1c > 8%) had significantly (P < 0.01 and P < 0.03) higher [Ca2+]i in resting platelets. The presence or absence of retinopathy did not modify resting platelet [Ca2+]i. After stimulation with thrombin, a significantly (P < 0.009) higher rise of platelet [Ca2+]i was observed only in those patients who were both free from complications and had good metabolic control. A highly significant (P < 0.001) correlation was found between resting [Ca2+]i and both blood cholesterol and HbA1c in the diabetic patients. Platelets from 10 young healthy subjects also were studied after in vitro incubation with various glucose concentrations (from 1.68 to 56 mM): resting and thrombin-stimulated platelet [Ca2+]i and thrombin-induced aggregation were not modified. CONCLUSIONS: These data confirm that platelet hyperfunction is present in IDDM patients who have unsatisfactory metabolic control, and give evidence that such an activation involves Ca homeostasis. Acute variations of blood glucose concentration are probably not influent, in this respect.

Normal tissue plasminogen activator and plasminogen activator inhibitor activity in plasma from patients with type 1 diabetes mellitus.

The fibrinolytic system was investigated in 38 patients (21 males and 17 females) affected by type 1 diabetes mellitus (18 free from complications, 10 with retinopathy, and 10 with autonomic neuropathy) and in 8 healthy controls. Two separate fibrinolysis-stimulating tests were done: standardized venous occlusion and 1-desamino-8-D-arginine vasopressin infusion. Plasma tissue plasminogen activator antigen and activity and plasma plasminogen activator inhibitor activity were measured. All the patients were in good metabolic control (mean HbA1c 7.4%, range 6.1-8.0%). No significant differences were observed either between the diabetic patients and the control subjects, nor among the subgroups of diabetic patients. The fibrinolytic system is probably not involved in type 1 diabetes mellitus.

Primary platelet activation in recent-onset type 1 diabetes mellitus.

Platelet function (as production of thromboxane B2 by platelets stimulated with collagen, and plasma beta-thromboglobulin) and thrombin activity (as plasma fibrinopeptide A) were investigated in eight young (mean age 27 +/- 3 SE years) male patients in which type 1 diabetes mellitus had been diagnosed 2 to 6 months previously. They were all in excellent stable metabolic control (mean HbA1c 5.6 +/- 0.4 SE %) and free from any complications. The haemostatic variables were assessed at rest and after cycloergometric exercise to exhaustion. When compared to age- and sex-matched healthy controls, patients showed higher beta-thromboglobulin, fibrinopeptide A and thromboxane B2 at rest. After exercise, plasma beta-TG increased only in the controls. Platelet and thrombin activation are present in the very early stages of type 1 diabetes mellitus, in the absence of any complications.

Platelet sensitivity in vitro to the prostacyclin analogue iloprost in diabetic patients.

Iloprost is a chemically stable analogue of prostacyclin, with similar vasodilator and anti-platelet actions. Platelet sensitivity to the inhibitory action of Iloprost has been tested in vitro. Platelet-rich plasma from six healthy subjects and from six patients with type 1 diabetes mellitus was incubated with different concentrations of Iloprost, and then stimulated with ADP (at threshold aggregating concentration) and collagen 4 micrograms/ml. The half-maximal inhibitory concentration (IC50) of Iloprost was calculated and no differences were found between patients and controls. The results of this study suggest that diabetic patients without complications do not differ from healthy subjects in their platelet sensitivity to Iloprost.

Effects of acute smoking on the hemostatic system in humans.

Habitual smoking is one of the best established risk factors for cardiovascular disease. The pathogenesis of smoke-induced damage is not so well clarified, but it probably includes--among some other aspects--an activation of the hemostatic system. Recently it has been shown that smoking a single cigarette can significantly decrease the coronary blood flow in coronary patients as well as in normal subjects. We tested the hypothesis that the acute effects of smoke are mediated by the hemostatic system. Seven healthy male volunteers, aged 20-40 years (mean 32 +/- 6 years), entered the study. All were habitual smokers, but had abstained from smoking in the 12 hours preceding the test. After lying in absolute rest for about 30 minutes, each subject smoked a cigarette containing 1.2 mg of nicotine. Immediately before and after smoking, blood was drawn by clear venipuncture for the evaluation of the following hemostatic variables: collagen-induced platelet aggregation by the method of Born; thromboxane B2 (TxB2) production by platelets stimulated with collagen, radioimmunoassay (RIA); plasma beta thromboglobulin (TG) (RIA); plasma fibrinopeptide A (FPA) (RIA); plasma fibrinolytic activity in the euglobulin fraction (NEF) (fibrin plate method). The following results, respectively before and after smoking, were observed: collagen-induced platelet aggregation 55 +/- 3 vs. 57 +/- 6%; TxB2 100.5 +/- 5.9 vs. 90.3 +/- 9.0 ng/10(8) platelets; plasma beta-TG 20.8 +/- 2.2 vs. 19.2 +/- 2.3 ng/ml; plasma FPA 2.3 +/- 0.3 vs. 2.2 +/- 0.1 ng/ml; NEF, lysis diameter 16.8 +/- 1.6 vs. 16.7 +/- 1.7 mm; NEF + C1 inhibitor lysis diameter 8.8 +/- 0.7 vs. 9.1 +/- 0.7 mm.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies to glutamic acid decarboxylase in a patient with stiff-man syndrome, epilepsy, and type I diabetes mellitus.

Stiff-man syndrome is a rare disorder of the central nervous system consisting of progressive, fluctuating muscle rigidity with painful spasms. It is occasionally associated with endocrine disorders, including insulin-dependent diabetes, and with epilepsy. We investigated the possible existence of autoimmunity against the nervous system in a patient with stiff-man syndrome associated with epilepsy and Type I diabetes mellitus. Levels of IgG, which had an oligoclonal pattern, were elevated in the cerebrospinal fluid. The serum and the cerebrospinal fluid produced an identical, intense staining of all gray-matter regions when used to stain brain sections according to an indirect light-microscopical immunocytochemical procedure. The staining patterns were identical to those produced by antibodies to glutamic acid decarboxylase (the enzyme responsible for the synthesis of gamma-aminobutyric acid). A band comigrating with glutamic acid decarboxylase in sodium dodecyl sulfate-polyacrylamide gels appeared to be the only nervous-tissue antigen recognized by cerebrospinal fluid antibodies, and the predominant antigen recognized by serum antibodies. These findings support the idea that an impairment of neuronal pathways that operate through gamma-aminobutyric acid is involved in the pathogenesis of stiff-man syndrome, and they raise the possibility of an autoimmune pathogenesis.

Platelet function and thrombin activity in patients with recent cerebral transient ischemic attacks.

Platelet function and thrombin activity were investigated in 12 hospitalized patients (7 men and 5 women, mean age 53 years) who had had transient cerebral ischemic attacks in the previous 2-12 weeks. Each patient was given an extensive clinical and instrumental evaluation, including Doppler sonography of the cervical and lower limb vessels, cerebral angiography, and head computed tomography scan, after which relevant atherosclerotic disease was excluded. The controls consisted of 12 subjects hospitalized for nonvascular neurologic problems and matched for age, sex, and risk factors to the transient ischemic attack patients. Collagen-induced platelet thromboxane B2 production, plasma beta-thromboglobulin, and fibrinopeptide A were significantly higher in the patients than the controls. Platelet aggregability by collagen was the same in the 2 groups. Platelet hyperfunction and enhanced thrombin activity are present in patients some weeks after the acute episode, suggesting that the hemostatic system has a primary pathogenetic role.

Plasma beta-thromboglobulin concentration at rest and after physical exercise in complicated and uncomplicated diabetes mellitus.

Plasma beta-thromboglobulin concentration was measured in ten uncomplicated insulin-dependent diabetic subjects, in ten insulin-dependent patients with retinopathy and in ten age- and sex-matched healthy controls, both at rest and after cycloergometric exercise to exhaustion. Resting plasma beta-thromboglobulin was similar in the two patient groups and significantly higher than the control group. After exercising, plasma beta-thromboglobulin rose significantly only in the control group. Platelet hyperactivity is therefore present even in uncomplicated diabetes mellitus and is not influenced by the presence of complications. A chronic overstimulation of platelets could be responsible for the high basal plasma beta-thromboglobulin concentration in diabetes mellitus and for its abnormal behaviour after physical exercise.

Plasma beta-thromboglobulin in uncomplicated insulin-dependent diabetes at rest and during physical exercise.

Plasma beta-thromboglobulin (beta TG) was assayed in 10 insulin-dependent uncomplicated diabetic subjects and in 10 age- and sex-matched metabolically healthy controls, both at rest and after maximal physical exercise. Diabetic patients showed significantly higher (p less than 0.005) resting beta TG values as compared to the control group. After physical exercise, beta TG rose significantly (p less than 0.05) only in the control group, thus abolishing the difference (evaluated through beta TG values) between the two groups observed at rest. Platelet hyperactivity seems thus to be present even in uncomplicated diabetes, at rest, while exercise does not lead to a further increase in beta TG values contrary to what is observed in controls.