Variations of the perforin gene in patients with multiple sclerosis.

Perforin is involved in cell-mediated cytotoxicity and mutations of its gene (PRF1) cause familial hemophagocytic lymphohistiocytosis (FLH2). PRF1 sequencing in 190 patients with multiple sclerosis and 268 controls detected two FLH2-associated variations (A91V, N252S) in both groups and six novel mutations (C999T, G1065A, G1428A, A1620G, G719A, C1069T) in patients. All together, carriers of these variations were more frequent in patients than in controls (phenotype frequency: 17 vs 9%, P=0.0166; odds ratio (OR)=2.06, 95% confidence interval (CI): 1.13-3.77). Although A91V was the most frequent variation and displayed a trend of association with multiple sclerosis (MS) in the first population of patients and controls (frequency of the 91V allele: 0.076 vs 0.043, P=0.044), we used it as a marker to confirm PRF1 involvement in MS and assessed its frequency in a second population of 966 patients and 1520 controls. Frequency of the 91V allele was significantly higher in patients than in controls also in the second population (0.075 vs 0.058%, P=0.019). In the combined cohorts of 1156 patients and 1788 controls, presence of the 91V allele in single or double dose conferred an OR=1.38 (95% CI=1.10-1.74). These data suggest that A91V and possibly other perforin variations indicate susceptibility to MS.

Familial hemophagocytic lymphohistiocytosis: how late can the onset be?

BACKGROUND AND OBJECTIVES: Most patients with familial hemophagocytic lymphohistiocytosis (HLH) develop the disease within the first two years of age. In a minority of cases a later occurrence has been reported, with an upper age limit of eight years. A significant concordance of the age at onset within each family has also been observed. RESULTS: We report four cases of families with HLH diagnosed at an unusually late age, comprised between between 9 and 17 years; in each of these families another child developed the disease in infancy. The natural killer activity of the patients was depleted; nevertheless, we had indirect evidence that, in at least two families, mutations of the perforin gene were not causing the disease. INTERPRETATION AND CONCLUSIONS: Such a late onset is very unusual and suggests that there is a subgroup of families with HLH in which the disease may present early or late in different members. Thus in some families with HLH the siblings might remain at risk of developing the disease for several years. Their actual risk cannot be defined until the genetic mutation is identified in each family and assessed in each member.

Hemophagocytic lymphohistiocytosis due to germline mutations in SH2D1A, the X-linked lymphoproliferative disease gene.

The hemophagocytic lymphohistiocytoses (HLH) comprise a heterogeneous group of disorders characterized by dysregulated activation of T cells and macrophages. Although some patients with HLH harbor perforin gene mutations, the cause of the remaining cases is not known. The phenotype of HLH bears a strong resemblance to X-linked lymphoproliferative disease (XLP), an Epstein-Barr virus (EBV)-associated immunodeficiency resulting from defects in SH2D1A, a small SH2 domain-containing protein expressed in T lymphocytes and natural killer cells. Here it is shown that 4 of 25 male patients with HLH who were examined harbored germline SH2D1A mutations. Among these 4 patients, only 2 had family histories consistent with XLP. On the basis of these findings, it is suggested that all male patients with EBV-associated hemophagocytosis be screened for mutations in SH2D1A. Patients identified as having XLP should undergo genetic counseling, and be followed long-term for development of lymphoma and hypogammaglobulinemia.

Cytogenetic abnormalities in PHA-stimulated lymphocytes from patients with Langerhans cell histocytosis. AIEOP-Istiocitosi Group.

The aetiopathogenesis of Langerhans cell histiocytosis (LCH) is still undefined. Constitutional abnormalities in LCH have rarely been reported. One study showed chromosomal instability in lesional cells from three patients. No chromosomal studies are available on peripheral blood lymphocytes. Peripheral blood lymphocytes were analysed for the presence of chromatid and/or chromosomal breaks and structural rearrangements. A fluorescence in situ hybridization (FISH) painting technique was also applied in two cases. Sixteen patients with multisystem (MS, n = 11) or single system (SS, n = 5) LCH were studied. either at the diagnosis (n = 8), during treatment (n = 2) or during follow-up, when asymptomatic (n = 6). Thirteen patients had chromosomal abnormalities. Eleven patients (69%) had chromatid and chromosomal breaks in 7-45% of cells. Overall, chromosome and chromatid breaks were significantly more frequent in the 11 patients with MS disease than in the five patients with SS disease: the mean percentage of cells showing chromosome and chromatid breaks was 13.4% in MS patients vs. 6.2% in SS patients (P = 0.003). Chromosomal abnormalities may be found in phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes of LCH patients at diagnosis, during the disease course and even during long-term follow-up, more frequently in MS disease. Chromosome instability may be considered as either a basic genetic instability or as a landmark of reaction to an environmental agent (viral?) that, through genome alteration, may play a role in histiocyte proliferation and, in some cases, also in the increased risk of malignancy.

Hemophagocytic lymphohistiocytosis in a patient with deletion of 22q11.2.

We report on a new patient with deletion of 22q11 associated with hemophagocytic lymphohistiocytosis and a fatal outcome. She had minor facial anomalies and cardiac malformation corresponding to those described in del (22q11) syndrome, normal T and B cell function and NK activity; bone marrow aspiration showed active erythrophagocytosis. Our case in addition to two other children reported previously suggest that such a rare association between lymphocyte-macrophage activation and deletion of 22q11 may be more frequent than previously recognized.

The breakpoints of a constitutional inversion of chromosome 9 associated with haemophagocytic lymphohistiocytosis are not linked to the disease gene.

Haemophagocytic lymphohistiocytosis (HLH) is a severe disorder of early infancy consistent with an autosomal recessive inheritance. Neither the genetic locus nor the biochemical defect is known for the disease. A constitutional pericentric inversion of chromosome 9, with breakpoints in bands 9p23 and 9q31, has been reported in a case of HLH, suggesting a possible relationship between this chromosome abnormality and the disease. We investigated such an association, performing a genetic linkage analysis in a set of five consanguineous HLH families. 27 polymorphic markers on chromosome 9 were studied, excluding most of chromosome 9 as a putative site for the HLH gene.

Expression of p75 chain of IL-2 receptor in the early immunological reconstitution after allogeneic bone marrow transplantation.

Expression of p55 and p75 chains of IL-2 receptor (IL-2R) on the surface of both T and natural killer (NK) circulating lymphocytes was investigated in 14 paediatric patients given allogeneic bone marrow transplantation (BMT) from HLA-identical sibling or partially matched family donors. IL-2-induced proliferative and cytotoxic responses were also studied and all analysis was performed within 45 days from transplant. We found that, early after transplant, the percentage of p55+ and of p75+ peripheral blood lymphocytes (PBL) was not significantly different in patients who had received HLA-identical BMT (p55+ 8.04 +/- 4.87%; p75+ 28.27 +/- 18.85%) compared with healthy controls (p55+ 7.26 +/- 6.17%; p75+ 19.42 +/- 10.49%), while recipients of T cell-depleted marrow included a remarkably high percentage (76-90%) of p75+ PBL, which were mostly CD3- and co-expressed CD56 molecule. Comparable values of p55+ lymphocytes were observed in all patients and controls. However, in contrast to the other two groups, in recipients of T cell-depleted BMT the majority of these cells co-expressed p75 chain and CD56 antigen. IL-2-induced proliferation and lymphokine-activated killer (LAK) activity were detectable in all patients, and their values did not correlate with expression of p55 or p75 chains. Our data suggest that expansion of NK subsets expressing IL-2R chains after T cell-depleted BMT may be related to early reconstitution of natural immunity in the presence of allogeneic stimuli.

Beta-endorphin, insulin, ACTH and cortisol plasma levels during oral glucose tolerance test in obesity after weight loss.

In order to clarify a possible relationship between opioid peptides and glucose homeostasis in obesity we studied Beta-Endorphin (B-Ep), ACTH, cortisol and insulin plasma levels in response to an oral glucose tolerance test (OGTT) in 8 subjects after a hypocaloric diet for 90 days. We obtained through this treatment a weight loss superior to 30% of the initial weight excess (WE) compared with ideal body weight. Moreover, we compared the obtained results with our preliminary study that was performed with the same protocol but without caloric restriction. B-Ep was measured by RIA after silicic acid extraction and G75 Sephadex column chromatography. ACTH, insulin and cortisol were measured directly on plasma by an RIA method. Basal and during OGTT-induced levels of glucose, insulin, ACTH and cortisol decreased in comparison with the values obtained before diet. Conversely, B-Ep remained higher than normal both in the basal condition and during OGTT, and showed values consistently similar to those before diet. These data show that hyperinsulinemia is corrected by weight loss, while hyperbetaendorphinemia remains unchanged. Accordingly, it can be suggested that no direct relationship occurs between hyper-B-Ep-hyper-IRI in obesity. A further insight into the role of hyper-B-Ep in obesity is, thus, necessary, assuming as hypothesis that the increase in B-Ep may be a cause and not a corollary of the polymorphic aspects of obesity.

.008% timolol ophthalmic solution. A minimal-effect dose in a normal volunteer model.

A double-masked, randomized, placebo-controlled, rising-dose, single-dose study was undertaken to assess the effect of low concentrations of timolol maleate ophthalmic solution (0.008%, 0.025%, 0.08%, and 0.25%) on intraocular pressure and its diurnal variation in healthy, normal volunteers. A single dose of 0.008% timolol exhibits a definite but minimal-effect on intraocular pressure in this normal volunteer model, causing a significant peak mean decrease in intraocular pressure from its value immediately predose. This decrease was 1.8 mm Hg (a peak mean percent decrease of 12.8%) at 2 hours postdose compared with an increase of 0.1 mm Hg (+2.5%) during a pre-study curve due to normal diurnal variation. One drop of 0.008% solution represents a single dose of approximately 2.5 micrograms of timolol. A slight contralateral ocular hypotensive effect appears to be present for 0.25% timolol at 2 hours postdose although it just failed to reach statistical significance.

Effect of repeated doses of L-5-hydroxytryptophan and carbidopa on prolactin and aldosterone secretion in man.

To evaluate changes in serum prolactin and plasma and urine aldosterone after a serotonergic challenge, 8 healthy men (19 to 42 yr), taking dexamethasone (0.75 mg qid), received the serotonin precursor L-5-hydroxytryptophan (L5HTP; 100 mg qid) with the peripheral decarboxylase inhibitor carbidopa (C; 50 mq qid) or matching placebos in a randomized, crossover manner. Serum prolactin concentration increased in all subjects after L5HTP/C in comparison to placebo, mean (SD) prolactin (ng/ml) at 8 h after dosing was 19.8 +/- 6.3 after L5HTP/C and 12.0 +/- 3.1 after placebo (p less than 0.05). In contrast, in comparison to values on placebo, L5HTP/C had no apparent effect on mean plasma concentration at all observation times; mean (SD) aldosterone (ng/dl) at 8 h after dosing was 12.0 +/- 5.1 and 12.0 +/- 3.8 after placebo (NS). Mean (SD) urinary aldosterone (micrograms/24 h), Na+(mEq/24 h) and K+(mEq/24 h) excretion were 7.0 +/- 4.4, 49.3 +/- 30.6, 30.1 +/- 11.2, after L5HTP/C and 7.4 +/- 5.8, 59.7 +/- 23.9, 33.3 +/- 7.4 after placebo (NS). Under these study conditions, subacute serotonergic stimulation with oral L5HTP/C resulted in prolactin but not aldosterone release.

Hemodynamic effects during rest and exercise of bucindolol in hypertensive men.

Bucindolol is an investigational beta-adrenergic blocking agent with intrinsic sympathomimetic and vasodilatory activity in animals. In a double-blind, six-way, crossover study of six mild-to-moderate hypertensive men, the effects of bucindolol 100, 200, and 300 mg/d on resting blood pressure, heart rate, forearm blood flow, and vascular resistance measured by pneumoplethysmography, and blood pressure and heart rate after cycle and handgrip exercise were compared with those of propranolol 160 and 320 mg/d and placebo after q12h administration for five doses. Both bucindolol and propranolol significantly suppressed heart rate after cycle exercise in comparison with placebo (-33 to -48 beats/min), demonstrating beta blockade. Suppression of resting heart rate by propranolol (-20 beats/min) was significantly (P less than .05) greater than bucindolol (-7 to -8 beats/min); a similar treatment difference in heart rate was noted after handgrip exercise (-18 to -19 vs -1 to -8 beats/min, respectively). Bucindolol and propranolol decreased resting blood pressure to the same extent (in comparison with placebo; P less than .05 at peak activity, 2 hr postdose). Bucindolol tended to increase forearm blood flow and decrease forearm vascular resistance (P less than .05 at 4 hr postdose) in comparison with placebo. The effect of propranolol on forearm blood flow and forearm vascular resistance was not significant compared with placebo. These data are consistent with intrinsic sympathomimetic and vasodilatory activity of bucindolol in hypertensive men.

Effect of food on the bioavailability of lisinopril, a nonsulfhydryl angiotensin-converting enzyme inhibitor.

A randomized, two-way, crossover study was performed on 18 normal volunteers to assess the influence of food on the bioavailability of lisinopril, (1-[N2-[(S)-1-carboxy-3-phenylpropyl]-L-lysyl]-L-proline), a long-acting nonsulfhydryl angiotensin converting enzyme inhibitor. A single, 20-mg oral dose of lisinopril was administered to volunteers in the fasting state or following a standardized breakfast. Treatment periods were separated by 2-week intervals. No significant differences existed between fasting and fed regimens in the mean +/- SD area under the serum concentration-time curve (AUC0-120h; 1231 +/- 620 versus 1029 +/- 254 ng X h X ml-1), peak lisinopril serum concentration (86 +/- 48 versus 69 +/- 19 ng/mL), or time to peak lisinopril serum concentration (6.2 +/- 1.1 versus 6.8 +/- 1.0 h). Five-day urinary excretion of lisinopril was not altered by food (5.3 +/- 3.0 versus 5.1 +/- 2.0 mg). Based on the urinary data, the mean +/- SD bioavailability of lisinopril was not different following fasting or fed regimens (27 +/- 15 versus 26 +/- 10%). Unlike with captopril, food did not affect the bioavailability of lisinopril.

Effect of captopril and propranolol, alone and in combination, on the responses to isometric and dynamic exercise in normotensive and hypertensive men.

This study evaluated the effects of single doses of the angiotensin converting enzyme inhibitor captopril and the beta-adrenergic blocking agent propranolol, alone or in combination, on the blood pressure, heart rate and humoral responses to both isometric (handgrip) and dynamic (ergometric) exercise in normotensive and hypertensive men. Single oral doses of either placebo, captopril 50 mg, propranolol 80 mg, or the latter two in combination were administered to age-matched groups (n = 5) of normotensive and hypertensive men in a random, double-blind manner. Captopril alone was indistinguishable from placebo after both isometric and ergometric exercise. Propranolol suppressed heart rate after both types of exercise and tended to decrease systolic blood pressure only in the hypertensive group; combination with captopril did not alter these responses. These data suggest that in sodium-replete subjects undergoing short-term vigorous exercise, the renin-angiotensin system, as measured by captopril inhibition, is less important than the sympathoadrenal system, as measured by propranolol inhibition, in the reflex cardiovascular adjustments accompanying acute isometric and dynamic exercise in both normotensive and hypertensive men.

Adrenocortical function after chronic inhalation of fluocortinbutyl and beclomethasone dipropionate.

Fluocortinbutyl (FCB) is a C-21 ester, topically active corticosteroid; no adrenal suppression has been noted after large doses. We compared safety and effects on adrenocortical function of orally inhaled FCB (40 mg/day), beclomethasone dipropionate (BDP) (2 mg/day), and placebo administered in four monitored divided doses for 4 wk by three groups of five healthy men. Circadian plasma cortisol concentration and daily urinary free cortisol excretion were determined before and after 3- and 4-wk exposure. Although pretreatment mean area under the curve (micrograms . hr . dl-1) for plasma cortisol did not differ among groups, mean values after weeks 3 and 4 of treatment were lower (p less than 0.05) in the BDP group (95.1 and 83) than in the FCB (155.8 and 153.7) and placebo (141 and 135.8) groups. Mean urinary cortisol excretion after week 4 for the BDP group (29 micrograms) was less (p less than 0.05) than in the FCB (59 micrograms) and the placebo (69 micrograms) groups. Slopes of individual regression lines noting time trends in plasma and urinary cortisol in the BDP group were negative and less (p less than 0.05) than those of the other groups. A cosyntropin test given intravenously after 4 wk of exposure resulted in similar plasma cortisol responses among groups. No serious adverse effects were noted. Thus after long-term high-dose treatment BDP but not FCB suppressed basal adrenocortical function, but neither suppressed the adrenocortical response to cosyntropin.