Characterization of the immune response of human cord-blood derived gamma/delta T cells to stimulation with aminobisphosphonate compounds.

Vγ9Vδ2 T lymphocytes have been shown to respond to a variety of non-peptide antigens including alkylamines and phosphoantigens. Recently, aminobisphosphonates have also been shown to stimulate this subset of γδ+ T cells. In this study we analyzed the proliferative responses of freshly isolated γδ T lymphocytes obtained from human cord blood when challenged with pyrophosphomonoesters or aminobisphosphonates. Nitrogen-containing aminobisphopsphonates, in contrast to phoshoantigens, readily stimulated expansion of Vδ2Vγ9 cells in human cord blood. Expanded cells displayed an activated mature phenotype, and were capable of producing TNFalpha and IFNgamma but not perforin following secondary stimulation, consistent with the development of a regulatory, as opposed to cytotoxic, phenotype. This approach may provide a useful strategy for a new approach to the treatment of neonatal pathologies.

Startle epilepsy associated with infantile hemiplegia (SEIH): video-polygraphic features and long-term outcome.

OBJECTIVE: The aim of this study was to investigate the video-polygraphic features and the long-term outcome of epilepsy in two patients with startle epilepsy associated with infantile hemiplegia (SEIH). MATERIAL AND METHODS: Two patients (patient 1: a 14-year-old girl; patient 2: a 17 year-and-half-year-old girl), with hemiparesis and moderate mental retardation, underwent a full clinical and neurophysiological examination with video-polygraphic monitoring and recording of startle-evoked seizures. The follow-up was 9 years from epilepsy onset in patient 1, and 8 years from epilepsy onset in patient 2. RESULTS: Firstly, video-polygraphic recordings of startle-evoked seizures, triggered by unexpected auditory stimuli, showed tonic asymmetrical postures with ictal EEG characterized by an abrupt and diffuse electrodecremental pattern or a seizure discharge predominant over the vertex and anterior regions controlateral to the posturing limbs. Electromyogram recording showed a prevalent involvement of proximal muscles with a concomitant tachycardia and apnoea. In particular, in patient 1 ictal heart rate was high, with persisting tachycardia for 60-120 s after the end of seizures. Secondly, a high seizure frequency persisted throughout the course of the disease, as seizures were medically refractory to all currently available anti-epileptic drugs. CONCLUSIONS: The long-term outcome of epilepsy in SEIH, with constantly high seizure frequency, suggests an early surgical intervention, avoiding years with unsuccessful drug treatments and poor quality of life.

Sphingosine 1-phosphate interferes on the differentiation of human monocytes into competent dendritic cells.

Sphingosine 1-phosphate (S1P) is a lipidic messenger known to exert several physiological functions within the cell. We tested here whether the stimulation of human monocytes with different doses of S1P might interfere with their differentiation into competent dendritic cells (DC). Monocytes cultured with granulocyte macrophage colony stimulating factor, interleukin-4 (IL-4) and S1P differentiated into a DC population lacking CD1a molecules on the surface and acquired some aspects of mature DC (mDC), though in the absence of maturation stimuli. When stimulated with lipopolisaccharide (LPS), CD1a(-) DC produce high amounts of tumour necrosis factor-alpha and IL-10, but not IL-12. Accordingly, these CD1a(-) DC were not capable of stimulating allogenic T lymphocytes so well as CD1a(+) DC generated from untreated monocytes and maturated with LPS. S1P monocyte-derived DC lost their polarizing capacity abrogating the production of gamma-interferon/IL-4 by co-cultured naïve CD4(+)CD45RA(+) T cells. Our findings suggest a mechanism through which S1P can favour the development of immune-related pathological states.

A double-blind randomized placebo-controlled trial with short-term beta-glucuronidase therapy in children with chronic rhinoconjunctivitis and/or asthma due to dust mite allergy.

BACKGROUND: Enzyme potentiated desensitization, in which beta-glucuronidase (BG) is administered with low doses of mixed allergens, was proposed in the 1970s for specific immunotherapy. The BG currently commercially available in a purified and standardized preparation devoid of any allergen has been suggested as a regulator in the allergic immune response, acting on the cytokine-network of type 2 helper T cells. A double-blind trial with a single-dose of BG proved effective in preventing symptoms in adult patients with rhinoconjunctivitis due to grass pollens. OBJECTIVE: The aim of this randomized double-blind placebo-controlled trial was to confirm the safety and effectiveness of double-dose intradermal BG immunotherapy in preventing symptoms in children suffering from chronic rhinoconjunctivitis and/or asthma due to dust mite. METHOD: We randomized 125 children with dust-mite related chronic rhinoconjunctivitis and/or asthma to the BG treated group (67) or the placebo group (58). All patients were screened before treatment (TO), at BG or placebo administration (T1 and T3), and at 3 and 9 months after T1 (T2 and T4). Drug intake and bronchial, nasal and ocular symptoms were recorded in a diary. RESULTS: Patients in both groups completed the study and BG treatment was well tolerated without side effects. Significant differences in symptoms were observed, in particular for conjunctivitis (P= .008). The total drug intake for allergic symptoms was significantly lower in the treated group than in the placebo group (P<. 01). CONCLUSIONS: BG immunotherapy is efficacious, safe, and well tolerated in allergic children. Moreover, good compliance with the administration of 2 doses per year and the lack of significant side effects makes the benefit/risk ratio of this treatment particularly favorable.

Role of macrophage phospholipase D in natural and CpG-induced antimycobacterial activity.

The present study addresses the differential ability of macrophages to control intracellular growth of non-pathogenic Mycobacterium smegmatis (Msm) and pathogenic M. tuberculosis (MTB). Results reported herein show that 3 h post infection, intracellular Msm, but not MTB, was significantly killed by macrophages. As the role of human macrophage phospholipase D (PLD) in the activation of antimicrobial mechanisms has been documented, we hypothesised the role of such enzyme in antimycobacterial mechanisms. To this aim, macrophage PLD activity was analysed at different times after exposure with either pathogenic MTB or non-pathogenic Msm. Results showed that, starting from 15 min after mycobacterial exposure, MTB did not induce macrophage PLD activity, whereas the environmental non-pathogenic Msm stably increased it. The direct contribution of PLD in intracellular mycobacterial killing was also analysed by inhibiting enzymatic activity with ethanol or calphostin C. Results show that PLD inhibition significantly increases intracellular Msm replication. In order to see whether the innate PLD-mediated antimicrobial mechanisms against MTB are also induced after CpG ODN stimulation, the role of PLD has been analysed in the course of CpG-mediated intracellular MTB killing. CpG DNA increased PLD activity in both uninfected and MTB-infected macrophages, and the inhibition of PLD activity resulted in a significant reduction of CpG-induced MTB killing. Taken together, our data suggest a relationship between host PLD activation and the macrophage ability to control intracellular mycobacterial growth.

Proinflammatory cytokines in the course of Mycobacterium tuberculosis-induced apoptosis in monocytes/macrophages.

Mycobacterium tuberculosis (MTB) can induce apoptosis in monocytes/macrophages both in vitro and in vivo, and this phenomenon is associated with mycobacterial survival. The present study addresses the possibility that apoptotic and inflammatory pathways could coexist through a caspase-1-mediated mechanism. In this context, a caspase-1 inhibitor (YVAD), but not caspase-3 (DEVD) or caspase-4 (LEVD) inhibitors, was able to strongly inhibit MTB-induced apoptosis. Moreover, caspase-1 activity was confirmed by the increased maturation of interleukin (IL)-1beta. Of interest, IL-1beta and tumor necrosis factor (TNF)-alpha were produced massively in the course of infection, and both were inhibited by YVAD pretreatment. To determine whether TNF-alpha was produced actively by apoptotic cells, the intracytoplasmatic cytokine content and apoptotic phenotype were analyzed at the single-cell level. Results showed a progressive increase of TNF-alpha production in annexin V-positive cells. These results indicate that MTB-induced apoptosis is associated with proinflammatory cytokine production.

In vitro cross-linking of calf lens alpha-crystallin by malondialdehyde.

The effect of malondialdehyde on structural features of bovine alpha-crystallin has been investigated by absorption and fluorescence spectroscopy as well as by far-UV circular dichroism. Experimental evidence suggests the occurrence of intermolecular cross-linking induced by malondialdehyde. This cross-linking does not seem to affect the tryptophan environment, as suggested by intrinsic protein fluorescence. On the contrary, the time dependence of far-UV dichroic activity indicates that the cross-linking is accompanied by a secondary structure change. The formation of high molecular mass aggregates, evidence by electrophoresis in denaturing conditions, leads to irreversible alpha-crystallin aggregation due to extensive intermolecular cross-linking. Since malondialdehyde is produced in vivo as a breakdown product of lipid peroxidation the possible involvement of this molecule in the pathological mechanism of cataract formation has been briefly discussed.

Cataract risk factors: blood level of antioxidative vitamins, reduced glutathione and malondialdehyde in cataractous patients.

Since many years experimental evidences have suggested an association between nutrition and lens opacities. A dietary deficiency of antioxidants and reactive oxygen scavengers may be involved in the pathogenesis of the "idiopathic" human senile cataract, as it has been demonstrated in some experimental cataracts. We tested the levels of ascorbic acid (vit. C), alpha-tocopherol (vit. E), reduced glutathione (GSH) and malondialdehyde (MDA) in the plasma or in the red blood cells (RBC) of 42 patients who were affected by surgically significant cataract and of 40 age-matched controls. Plasma vit. C mean level was 4.46 gamma/ml in cataracts and 4.62 gamma/ml in controls, while vit. E level was 7.70 and 7.09 gamma/ml respectively. RBC GSH was found to be 342 gamma/ml in cataracts and 346 in controls, while the MDA content was 4.06 picoMol/ml and 4.08 picoMol/ml respectively. The level of each tested nutrient or metabolite was not found to be statistically different between cataractous patients and controls, nor any significant trend was found to be present when the nutrients and metabolites were correlated to each other. Our results do not support the hypothesis of a nutritional deficiency in human senile cataracts. However, a defect in the antioxidative metabolism pathways could be present either systemically or at lens level.

The decrease of free epsilon-amino groups in senile and diabetic cataracts.

Free epsilon-amino groups in soluble and insoluble proteins were measured in clear human lenses and in diabetic and nondiabetic senile cataractous lenses. The free epsilon-amino group content of soluble and insoluble proteins was significantly lower in diabetic cataracts than in clear lenses and nondiabetic senile cataracts. Our results seem to demonstrate that nonenzymatic glycosylation of lens proteins could play a role in the pathogenesis of diabetic cataract.

Lipid peroxidation and human cataractogenesis in diabetes and severe myopia.

To investigate the role of lipid peroxidation in human cataract, malondialdehyde, a breakdown product of lipid peroxidation, was measured in clear and cataractous lenses from normal subjects, and in cataractous lenses from diabetics and from subjects with severe myopia. The cataractous lenses contained more malondialdehyde than did clear lenses and the level was higher in diabetes and severe myopia than in the idiopathic forms. This indicates that lipid peroxidation may be involved in the development of senile cataract and, as a direct consequence of retinal damage, also in the pathogenesis of cataract in diabetes and in severe myopia.

Cataract formation in diabetic patients and galactose-1-phosphate uridyltransferase deficiency.

We have evaluated the levels of red blood cell galactose-1-phosphate uridyltransferase in 20 patients with cataract and in 15 subjects without cataract, suffering from compensated, noninsulin-dependent, type II diabetes. The diabetic patients were compared with a previously examined group of 65 age-matched nondiabetic subjects (25 of whom suffered from bilateral idiopathic cataract). In diabetic patients, the average galactose-1-phosphate uridyltransferase levels tended to be lower and the percentage of cases of reduced enzymatic activity tended to be higher than in the corresponding nondiabetic subjects.

Effect of topical glucocorticoid administration on the protein and nonprotein sulfhydryl groups of the rabbit lens.

The effects of topical administration of glucocorticoids on rabbit lenses are described. Animals were divided into three groups. The first group (A) served as control, the second (B) and the third (C) group were treated with betamethasone and fluorometholone, respectively. After 40 days of treatment there was a significant fall in the levels of nonprotein sulfhydryl (-SH) groups (group A: 3.82 +/- 0.21; group B: 2.61 +/- 0.11; group C: 1.93 +/- 0.13 mumol/lens) and of protein -SH groups (group A: 8.215 +/- 1.023; group B: 4.120 +/- 0.631; group C: 4.068 +/- 0.538 mumol/lens). Also ascorbic acid levels showed a significant decrease both in lens and in aqueous humor. No differences were noted in the reduced glutathione content in aqueous humor. The fall in nonprotein and protein -SH could be the first event in the well-known biochemical changes that occur in steroid-induced cataract. The mechanism underlying steroid-induced damage could be due to a conformational change of lens crystallins which results in an unmasking of -SH groups with a consequent increased susceptibility to oxidation. The decrease of ascorbic acid should represent an effect of the fall in the glutathione system. Lastly, it is hypothesized that the protective effect exerted by some substances, such as vitamin E and ascorbic acid, occurs by counteracting this oxidation.

Effect of vitamin E on glutathione content in red blood cells, aqueous humor and lens of humans and other species.

High doses of orally administered vitamin E have been given to humans, rabbits and rats. Placebo has been given to control groups. At the end of the treatment period, enhanced levels of reduced glutathione (GSH) were found in the red blood cells (humans and rabbits), aqueous humor (humans and rabbits) and lens (rabbits and rats) of treated subjects and animals. The percentage of GSH converted to oxidized glutathione (GSSG) was the same in both vitamin E-supplied and control groups. The GSSG--GSH ratio remained unchanged. The plasma levels of vitamin E were significantly higher in treated than in control subjects and animals. At the end of the study, the levels of vitamin E in aqueous humor and lens of rabbit were the same in animals which received vitamin E and in animals which received placebo. Lastly, vitamin E administration did not influence ascorbic-acid levels in plasma (humans and rabbits), aqueous humor, lens and vitreous body (rabbits).

Vitamin E and red blood cell glutathione.

High doses of orally administered vitamin E (1000 IU/day) have been given to ten normal volunteers. Ten control subjects received placebo. Red blood cell glutathione was significantly higher in treated subjects than in the controls (controls: 267.5 +/- 15.7 micrograms/mL; treated: 374.8 +/- 17.3 micrograms/mL). These findings could be explained by an increase of glutathione synthesis brought about by the stimulation of glutathione synthetase activity. An alternative possibility is a reduced utilization of glutathione for the detoxification of free radicals. These two mechanisms could be effective in counteracting the glutathione content feedback of the synthetizing enzymes.

In vitro effect of alpha-tocopherol on lysophosphatidylcholine-induced lens damage.

Lysophosphatidylcholine (LPC) has been shown to be toxic to the lens in organ culture. An investigation into whether vitamin E counteracts the in vitro damaging effect of LPC on rat lenses was undertaken. A concentration higher than 10 micrograms ml-1 LPC in the culture medium is necessary to damage rat lenses, as assessed by protein content of the medium and Na+ and K+ content of the lens. Vitamin E affords its protection when present at a concentration of 10(-3) M: both the protein efflux from the lens and the lenticular cation imbalance are prevented, also if LPC concentration is 100 micrograms ml-1. This effect may be due more to the physicochemical properties of vitamin E in the stabilization of biological membranes, than to its chemical behaviour as an antioxidant.

Red blood cells galactose-1-P-urydil transferase in senile and presenile cataracts.

There are conflicting reports in the literature regarding the role of partial deficiency of "galactosemic" enzymes (galactose-1-P-urydil transferase and galactokinase) in the development of infantile and presenile cataract. The AA. have investigated the levels of Red Blood Cell Galactose-1-P-urydil transferase in 39 cataractous patients and in 22 age matched controls. A weak correlation between the enzymatic activity deficiency and the presenile cataract has been identified. The results suggest that a chronic impairment of galactose metabolism may be a contributory risk factor in the pathogenesis of presenile cataracts; however, further investigations are required to assess the actual significance of the findings of the present paper.

Changes of some biochemical parameters of the lens in galactose-treated weaned rats with and without vitamin E therapy.

We tried to counteract the appearance of galactosemic cataracts in weaned rats by high doses of vitamin E. Rats were fed a diet containing 33% galactose. Cataract development was monitored by biomicroscopy and by several biochemical parameters: K+/Na+ ratio, aldose reductase activity, level of protein and non-protein sulfhydryl (SH) groups. Vitamin E was given parenterally at a dose of 100 mg/kg/day. The K+/Na+ ratio drops after 15 days of galactosemia, while the level of the aldose reductase rises after only 5 days of treatment. The non-protein SH groups lens contents fall from the 5th day of treatment onwards, while protein SH groups are not affected. In short-term experiments vitamin E does not prevent biochemical changes caused by galactosemia. The oxidative insult does not seem to be primarily involved in galactose cataract.

High frequency of lactose absorbers among adults with idiopathic senile and presenile cataract in a population with a high prevalence of primary adult lactose malabsorption.

22% of a group of adult Neapolitans were found to have persistent high lactase activity, and 16% were lactose absorbers as indicated by measurement of breath hydrogen concentration and rise in blood glucose after oral lactose administration. Among adults in the same area with idiopathic senile or presenile cataract 49% were identified as lactose absorbers with the breath hydrogen test and 55% by the rise in blood glucose. These results suggest that adults able to absorb galactose from a lactose-containing diet are especially susceptible to senile or presenile cataract.