Glycosylphosphatidylinositol-linked proteins are required for maintenance of a normal peripheral lymphoid compartment but not for lymphocyte development

Surface proteins tethered to the membrane through a glycosylphosphatidylinositol (GPI) anchor are deficient in the blood cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) as result of a somatic mutation, in a hematopoietic stem cell, of the X-linked phosphatidylinositolglycan complementation group A (PIG-A) gene. In PNH patients, compared to the large numbers of GPI-deficient myeloid cells, the proportion of GPI-deficient lymphocytes tends to be low, and therefore the impact of GPI deficiency on immune function has been unclear. We have obtained complementation by Pig-a(-) embryonic stem (ES) cells of Rag(-/-) blastocysts, and we show that Pig-a(-) ES cells are able to reconstitute the T cell and B cell compartments of Rag(-/-) mice. Although these mice were immunologically competent, by comparison with appropriate controls we detected several abnormalities: (1) increased levels of IgG; (2) high frequency/titers of anti-nuclear antibodies; (3) markedly reduced delayed hypersensitivity; and (4) impaired activation-induced lymphocyte death in vitro. In some cases, aging Pig-a(-)/Rag(-/-) chimeric mice developed lymphadenopathy and polyclonal T cell and B cell expansion. Thus, GPI-linked proteins are not required for lymphocyte development but they are required for normal lymphocyte function and for maintaining normal peripheral lymphoid homeostasis.     

Candida albicans expresses a focal adhesion kinase-like protein that undergoes increased tyrosine phosphorylation upon yeast cell adhesion to vitronectin and the EA.hy 926 human endothelial cell line

The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of alpha v beta 3 and alpha v beta 5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.     

Efficacy and safety of sublingual immunotherapy.

OBJECTIVE: To review the available published data concerning the use of sublingual immunotherapy (SLIT) in respiratory allergy to primarily evaluate the clinical efficacy and safety of the treatment and to secondarily consider the mechanisms of action and any unresolved questions. DATA SOURCES: Articles in the medical literature (starting from 1986 up to November 2003) derived from searching the MEDLINE database with the keywords sublingual immunotherapy, respiratory allergy, asthma, and rhinitis. Sources included review articles, randomized controlled clinical trials, postmarketing surveillance studies, and relevant reports from meeting proceedings. STUDY SELECTION: Articles concerning safety, efficacy, and mechanisms of SLIT published in English-language, peer-reviewed journals. RESULTS: SLIT proved effective and safe in adults and children. As with traditional subcutaneous immunotherapy, SLIT has long-lasting efficacy and a preventive effect on new sensitizations. CONCLUSION: SLIT is a viable alternative to subcutaneous immunotherapy. Its use in pediatric patients seems to be particularly promising.     

Soluble Antiapoptotic Molecules and Immune Activation in Chronic Heart Failure and Unstable Angina Pectoris

Programmed myocyte cell death and activation of the immune system have been shown to occur in patients with congestive heart failure. Besides, unstable angina episodes are likely to be associated with immune activation. Our aim was to evaluate the role of changes in circulating levels of soluble Fas (sFas), suggestive of an enhanced inhibitory response to ongoing apoptosis, and soluble IL2 receptor (sIL2-R), indicative of T-lymphocyte activation, in chronic heart failure and unstable angina pectoris. Thirty patients affected by chronic heart failure (20 idiopathic and 10 ischemic cardiomyopathy) and 13 patients with unstable angina were evaluated. Twenty healthy individuals matched for age and gender were used as controls. A complete biochemical determination of indexes of myocardial damage including cardiac troponin I (cTnI) and creatine kinase (MB/CK) was performed. The results demonstrated that mean levels of sFas and sIL2-R were significantly increased in patients affected by chronic heart failure and unstable angina and were not associated with changes in renal function or with serum levels of cTnI. Highest values of sFas were found in NYHA class IV patients (IV NYHA class = 7.39 ± 0.52 vs. controls = 1.34 ± 0.12 ng/ml; P < 0.01) and more elevated in idiopathic than in ischemic cardiomyopathy (3.64 ± 0.40 vs. 1.82 ± 0.37 ng/ml; P < 0.01). Moreover, in chronic heart failure patients sFas and ejection fraction were negatively correlated (P = 0.01), whereas sFas and sIL2-R were positively correlated (P < 0.01). In unstable angina patients too, sFas and sIL2-R appeared to be correlated (P = 0.03); whereas sFas (angina group = 3.18 ± 0.39 vs. controls = 1.34 ± 0.12 ng/ml; P < 0.01) and sIL2-R (angina group = 0.46 ± 0.11 vs. controls = 0.00 UI/ml; P < 0.01) were higher in angina group than in controls. In most of the cases, the increase of sFas was associated with comparable changes in sIL2-R serum levels, indicating that the activation of Fas system is strictly associated with autoimmune–inflammatory reactions. This phenomenon, both in chronic heart failure and in unstable angina, occurs in the absence of biochemical evidences of myocardial damage and seems to parallel the activation of T cell. Soluble Fas could have a role in sustaining inflammatory response and in prolonging the detrimental effects correlated with it in chronic heart failure and angina pectoris.     

Clinical,immunological, and molecular analysis in a large cohort of patients with X-linked agammaglobulinemia: an Italian multicenter study

A questionnaire-based retrospective clinical and immunological survey was conducted in 73 males with a definite diagnosis of X-linked agammaglobulinemia based on BTK sequence analysis. Forty-four were sporadic and 29 familial cases. At December 2000, the patients' ages ranged from 2 to 33 years; mean age at diagnosis and mean duration of follow-up were 3.5 and 10 years respectively. After the mid-1980s all but 2 were on intravenous immunoglobulin (IVIG) substitution therapy, with residual IgG >500 mg/dl in 94% of the patients at the time of enrollment. Respiratory infections were the most frequent manifestation both prior to diagnosis and over follow-up. Chronic lung disease (CLD) was present in 24 patients, in 15 already at diagnosis and in 9 more by 2000. The cumulative risk to present at diagnosis with CLD increased from 0.17 to 0.40 and 0.78 when the diagnosis was made at the ages of 5, 10, and 15 years respectively. For the 9 patients who developed CLD during follow-up, the duration of follow-up, rather than age at diagnosis; previous administration of intramuscular immunoglobulin; and residual IgG levels had a significant effect on the development of CLD. Chronic sinusitis was present in 35 patients (48%), in 15 already at diagnosis and in 20 by 2000. Sistemic infections such as sepsis and meningitis/meningoencephalitis decreased over follow-up, probably due to optimal protection provided by high circulating IgG levels reached with IVIG.     

Age and sex influence on oxidative damage and functional status in human skeletal muscle

A reduction in muscle mass, with consequent decrease in strength and resistance, is commonly observed with advancing age. In this study we measured markers of oxidative damage to DNA, lipids and proteins, some antioxidant enzyme activities as well Ca²⁺ transport in sarcoplasmic reticulum membranes in muscle biopsies from vastus lateralis of young and elderly healthy subjects of both sexes in order to evaluate the presence of age- and sex- related differences. We found a significant increase in oxidation of DNA and lipids in the elderly group, more evident in males, and a reduction in catalase and glutathione transferase activities. The experiments on Ca²⁺ transport showed an abnormal functional response of aged muscle after exposure to caffeine, which increases the opening of Ca²⁺ channels, as well a reduced activity of the Ca²⁺ pump in elderly males. From these results we conclude that oxidative stress play an important role in muscle aging and that oxidative damage is much more evident in elderly males, suggesting a gender difference maybe related to hormonal factors.This revised version was published online in September 2005 with corrections to the Cover Date.     

Using linked markers to infer the age of a mutation

Advances in sequencing and genotyping technologies over the last decade have enabled geneticists to easily characterize genetic variation at the nucleotide level. Hundreds of genes harboring mutations associated with genetic disease have now been identified by positional cloning. Using variation at closely linked genetic markers, it is possible to predict the times in the past at which particular mutations arose. Such studies suggest that many of the rare mutations underlying human genetic disorders are relatively young. Studies of variation at genetic markers linked to particular mutations can provide insights into human geographic history, and historical patterns of natural selection and disease, that are not available from other sources. We review two approaches for estimating allele age using variation at linked genetic markers. A phylogenetic approach aims to reconstruct the gene tree underlying a sample of chromosomes carrying a particular mutation, obtaining a “direct” estimate of allele age from the age of the root of this tree. A population genetic approach relies on models of demography, mutation, and/or recombination to estimate allele age without explicitly reconstructing the gene tree. Phylogenetic methods are best suited for studies of ancient mutations, while population genetic methods are better suited for studies of recent mutations. Methods that rely on recombination to infer the ages of alleles can be fine‐tuned by choosing linked markers at optimal map distances to maximize the information available about allele age. A limitation of methods that rely on recombination is the frequent lack of a fine‐scale linkage map. Maximum likelihood and Bayesian methods for estimating allele age that rely on intensive numerical computation are described, as well as “composite” likelihood and moment‐based methods that lead to simple estimators. The former provide more accurate estimates (particularly for large samples of chromosomes) and should be employed if computationally practical. Hum Mutat 18:87–100, 2001. © 2001 Wiley‐Liss, Inc.     

Mitochondrial import and enzymatic activity of PINK1 mutants associated to recessive parkinsonism

Parkinson's disease (PD) is a progressive neurodegenerative illness associated with a selective loss of dopaminergic neurons in the nigrostriatal pathway of the brain. Despite the overall rarity of the familial forms of PD, the identification of single genes linked to the disease has yielded crucial insights into possible mechanisms of neurodegeneration. Recently, a putative mitochondrial kinase, PINK1, has been found mutated in an inherited form of parkinsonism. Here, we describe that PINK1 mutations confer different autophosphorylation activity, which is regulated by the C-terminal portion of the protein. We also demonstrate the mitochondrial localization of both wild-type and mutant PINK1 proteins unequivocally and prove that a short N-terminal part of PINK1 is sufficient for its mitochondrial targeting.