FACL4, encoding fatty acid-CoA ligase 4, is mutated in nonspecific X-linked mental retardation.

X-linked mental retardation (XLMR) is an inherited condition that causes failure to develop cognitive abilities, owing to mutations in a gene on the X chromosome. The latest XLMR update lists up to 136 conditions leading to 'syndromic', or 'specific', mental retardation (MRXS) and 66 entries leading to 'nonspecific' mental retardation (MRX). For 9 of the 66 MRX entries, the causative gene has been identified. Our recent discovery of the contiguous gene deletion syndrome ATS-MR (previously known as Alport syndrome, mental retardation, midface hypoplasia, elliptocytosis, OMIM #300194), characterized by Alport syndrome (ATS) and mental retardation (MR), indicated Xq22.3 as a region containing one mental retardation gene. Comparing the extent of deletion between individuals with ATS-MR and individuals with ATS alone allowed us to define a critical region for mental retardation of approximately 380 kb, containing four genes. Here we report the identification of two point mutations, one missense and one splice-site change, in the gene FACL4 in two families with nonspecific mental retardation. Analysis of enzymatic activity in lymphoblastoid cell lines from affected individuals of both families revealed low levels compared with normal cells, indicating that both mutations are null mutations. All carrier females with either point mutations or genomic deletions in FACL4 showed a completely skewed X-inactivation, suggesting that the gene influences survival advantage. FACL4 is the first gene shown to be involved in nonspecific mental retardation and fatty-acid metabolism.

Western blotting for the diagnosis of congenital toxoplasmosis.

Toxoplasmosis is a common congenital infection. It does not usually produce recognizable signs of infection at birth so most infected newborns are not detected by routine clinical examination and remain untreated. Infected children without clinical symptoms should nonetheless be identified and treated as early as possible. Serological diagnosis of congenital toxoplasmosis is quite difficult. The aim of this study was to evaluate the utility of Western blot for the diagnosis of congenital toxoplasmosis. We compared the immunological profiles of mothers and children to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants in the first three months of life. The method enabled us to diagnose congenital toxoplasmosis in cases in which the infection had not been detected by classical serology techniques.

Proteomic analysis in interstitial lung diseases: a review.

PURPOSE OF REVIEW: This article aims to review the most recent proteomic findings in tissue and biological fluids and application of new technologies for the study of interstitial lung diseases. RECENT FINDINGS: New insights into lung cell biology as well as novel pathogenetic pathways of different interstitial lung diseases, potential targets of treatment and specific biomarkers discovered by proteomics have been reported. Proteomics has been applied to different kinds of biological fluids and the most promising material for proteomic analysis of interstitial lung disease is currently bronchoalveolar lavage fluid. SUMMARY: The results from proteomic studies on this field could be useful in clinical practice for diagnosis, disease progression and severity of interstitial lung diseases.

Dicrocoelium dendriticum: a true infection?

Dicrocoelium dendriticum is a liver parasite of ruminants. Humans are occasionally infected by ingestion of intermediate hosts. We report a rare case of dicrocoeliasis in a 55-year-old woman who presented with eosinophilia and elevated bilirubin. Therapy with albendazole eradicated the parasite and normalized blood parameters.

Brevibacterium casei bacteraemia in a port-a-cath carrier patient: a case report.

Brevibacteria are part of the normal flora of the skin and adjacent structures, but have been increasingly encountered in humans as opportunistic pathogens and have been isolated from various clinical specimens, generally causing infections in immuno-compromised patients. We present a case of a port-a-cath-related bacteraemia caused by Brevibacterium casei in a woman with a prior history of bilateral breast cancer. The clinical outcome was favourable.

Immunoblotting techniques.

Immunoblotting techniques use antibodies (or other specific ligands in related techniques) to identify target proteins among a number of unrelated protein species. They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions. Proteins are typically separated by electrophoresis and transferred onto membranes (usually nitrocellulose). The membrane is overlaid with a primary antibody for a specific target and then with a secondary antibody labeled, for example, with enzymes or with radioisotopes. When the ligand is not an antibody, the reaction can be visualized using a ligand that is directly labeled. Dot blot is a simplified procedure in which protein samples are not separated by electrophoresis but are spotted directly onto membrane. Immunoblotting is now widely used in conjunction with two-dimensional polyacrylamide gel electrophoresis, not only for traditional goals, such as the immunoaffinity identification of proteins and analysis of immune responses but also as a genome-proteome interface technique.

Proteome analysis of bronchoalveolar lavage in pulmonary langerhans cell histiocytosis.

BACKGROUND: Pulmonary Langerhans-cell histiocytosis (PLCH) is a rare interstitial lung disease characterized by clusters of Langerhans cells, organized in granulomas, in the walls of distal bronchioles. It is a diffuse lung disease related to tobacco smoking but otherwise of unknown etiopathogenesis. METHODS: In this study we used a proteomic approach to analyze BAL protein composition of patients with PLCH and of healthy smoker and non-smoker controls to obtain insights into the pathogenetic mechanisms of the disease, to study the effect of cigarette smoking on susceptibility to PLCH and to identify potential new biomarkers. RESULTS: Two-dimensional electrophoresis and image analysis revealed proteins that were differently expressed (quantitatively and qualitatively) in the three groups of subjects. The proteins were identified by mass spectrometry and have various functions (antioxidant, proinflammatory, antiprotease) and origins (plasma, locally produced, etc.). Many, such as protease inhibitors (human serpin B3) and antioxidant proteins (glutathione peroxidase and thioredoxin) are already linked to PLCH pathogenesis, whereas other proteins have never been associated with the disease. Interestingly, numerous proteolytic fragments of plasma proteins (including kininogen-1 N fragments and haptoglobin) were also identified and suggest increased proteolytic activity in this inflammatory lung disease. Differences in protein expression were found between the three groups and confirmed by Principal Component Analysis (PCA). CONCLUSION: Analysis of BAL proteomes of PLCH patients and of smoker and non-smoker controls also proved to be useful for researching the pathogenetic mechanisms and for identifying biomarkers of this rare diffuse lung disease.

Analysis of carbonylated proteins in bronchoalveolar lavage of patients with diffuse lung diseases.

Diffuse lung diseases (DLD) are a heterogeneous group of diseases with different etiopathogenesis, clinical course, and prognosis. It has been demonstrated that oxidative stress can contribute to the pathogenesis of these diseases. In the present study we measured carbonylated protein concentrations in the BAL of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis, idiopathic pulmonary fibrosis, and for the first time in patients with chronic eosinophilic pneumonia and extrinsic allergic alveolitis. Our aim was to further investigate oxidation products in diffuse lung diseases. Oxidatively modified protein concentrations were increased in the BAL of patients than in that of controls (0.22 nmol/mg protein vs 0.05 nmol/mg protein; p < 0.001) and in each group of disease versus controls, suggesting that proteins that have become dysfunctional by oxidation could play a role in the pathogenesis of diffuse lung diseases. Further studies in a greater number of patients are needed to understand the contribution of oxidatively modified proteins to the pathogenesis of DLD and, in particular, to the development of extrinsic allergic alveolitis where the highest levels of carbonylated proteins were found.

Cryptosporidium infection: diagnostic techniques.

The aim of this study was to compare a rapid immunological test and a PCR method with the conventional morphological technique for the identification of Cryptosporidium in faecal samples. Cryptosporidium was found in five samples by Kinyoun acid-fast stain. Five samples yielded positive results on immunoassay, three of which yielded negative results on microscopy. Thus, only two patients were positive for Cryptosporidium according to both methods. PCR analysis confirmed only one sample as positive. Non-homogeneous distribution of parasites in stool samples, lack of oocysts in the tested sample and antigenic diversity among Cryptosporidium species may explain the poor agreement among the three tests. Based on our experience, microscopy test with Kinyoun stain is the best and cheapest way to detect Cryptosporidium spp. in faecal samples. With this method, we have found a 5.4% prevalence of Cryptosporidium infection in our area, similar to those reported for other regions of Italy and Europe.

Proteome analysis of bronchoalveolar lavage in lung diseases.

The proteomic approach is complementary to genomics and enables protein composition to be investigated under various clinical conditions. Its application to the study of bronchoalveolar lavage (BAL) is extremely promising. BAL proteomic studies were initially based on two-dimensional electrophoretic separation of complex protein samples and subsequent identification of proteins by different methods. With the techniques available today it is possible to attain many different research objectives. BAL proteomics can contribute to the identification of proteins in alveolar spaces with possible insights into pathogenesis and clinical application for diagnosis, prognosis and therapy. Many proteins with different functions have already been identified in BAL. Some could be biomarkers that need to be individually confirmed by correlation with clinical parameters and validation by other methods on larger cohorts of patients. The standardization of BAL sample preparation and processing for proteomic studies is an important goal that would promote and facilitate clinical applications. Here, we review the principal literature on BAL proteomic analysis applied to the study of lung diseases.

Nonprotein-bound iron and plasma protein oxidative stress at birth.

We previously reported plasma nonprotein-bound iron (NPBI) as a reliable early indicator of intrauterine oxidative stress (OS) and brain injury. We tested the hypothesis that albumin, an NPBI serum carrier, is the major target of NPBI-induced OS. Twenty-four babies were randomly selected from 384 newborns constituting the final cohort of a prospective study undertaken to evaluate the predictive role of NPBI in cord blood for neurodevelopmental outcome. Twelve were selected in the group with lowest NPBI levels (0-1.16 microM) and good neurodevelopmental outcome and 12 in the group with highest NPBI levels (>or=15.2 microM) and poor neurodevelopmental outcome. Protein carbonyl groups were identified in cord blood samples by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Western blotting with anti-2,4-dinitrophenyl (DNP) antibodies. Two series of immunoreactive spots, corresponding to serum albumin and alpha-fetoprotein, were found only in the group with highest NPBI levels. We found an association between NPBI and carbonylated proteins in babies with highest NPBI levels. Since NPBI may produce hydroxyl radicals through the Fenton reaction, the major target of OS induced by NPBI is its carrier: albumin. Oxidation of albumin can be expected to decrease plasma antioxidant defenses and increase the likelihood of tissue damage due to OS in the newborns.

Cytokine profile and proteome analysis in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis.

The aim of this study was to analyze the type of immune response (Th1, Th2) and protein composition of bronchoalveolar lavage (BAL) of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Flow cytometry analysis of intracellular cytokines revealed different patterns: in IPF and SSc Th2 profiles were predominant, whereas in sarcoidosis Th1 prevailed. The proteomic analysis of BAL fluid (BALF) showed that there were quantitative differences between the three diseases. These were more evident between sarcoidosis and IPF, confirming our previous observations, whereas SSc had an intermediate profile between the two, however with some peculiarities. Comparison of BALF protein maps, constructed with the same quantity of total proteins, enabled us to identify the main profiles of the three diseases: an increase in plasma protein prevalent in sarcoidosis and also present in SSc, though for fewer proteins with respect to IPF and a greater abundance of low molecular weight proteins, mainly locally produced, in IPF. These findings are in line with the different pathogenesis of these diseases: IPF is considered a prevalently fibrotic disorder limited to the lung, with intense local production of functionally different proteins, whereas sarcoidosis and SSc are systemic immunoinflammatory diseases.

Carbonylated proteins in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis.

Oxygen-derived free radicals produced by phagocytes have been postulated to contribute to lung tissue damage occurring during diffuse lung diseases (DLD). The two-dimensional electrophoretic (2-DE) analysis of bronchoalveolar lavage (BAL) protein composition revealed different protein profiles in sarcoidosis (S), idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) with a significant increase of low molecular weight proteins in IPF. Some of these proteins are involved in antioxidant processes. The aims of this report were to analyse the oxidative stress occurring in patients with DLD through determination of BAL protein carbonyl content and to identify target proteins of oxidation by a proteomic approach (2-DE combined with immunoblotting with specific antibodies for carbonyl groups). Carbonylated proteins detected by enzyme-linked immunosorbent assay (ELISA) were increased in BAL of patients with S, IPF and SSc compared to healthy controls with a significant difference for S and IPF. The proteomic approach to the analysis of BAL revealed that protein carbonylation was a process involving specific carbonylation-sensitive proteins and that in IPF a greater number of proteins target of oxidation were present. In conclusion, to our knowledge, this is the first report providing a database of proteins target of oxidation in BAL of patients with sarcoidosis, idiopathic pulmonary fibrosis and systemic sclerosis.

Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis: a two-dimensional electrophoretic study.

We used two-dimensional (2-D) electrophoresis to analyze the protein composition of fluid recovered by bronchoalveolar lavage (BALF) from patients with sarcoidosis and idiopathic pulmonary fibrosis, two forms of interstitial lung disease with different cellular composition and cytokine profile in BALF. They are also characterized by different pathogenesis and clinical evolution, idiopathic pulmonary fibrosis being less favorable than sarcoidosis due to rapidly progressive pulmonary fibrosis. Thirty-eight proteins or protein fragments, never previously assigned in BALF samples, were identified by various methods including mass fingerprinting of tryptic digests. Comparison of the BALF protein maps of the two groups of patients showed 32 spots with statistically significant disease-related variations in relative abundance. In sarcoidosis we found an increase in the amount of several plasma proteins, while in idiopathic pulmonary fibrosis we observed a statistically significant increase in low-molecular-weight proteins, many of which are involved in inflammatory processes (such as MIF and calgranulin) or antioxidant response (such as antioxidant peroxysomal enzyme and thioredoxin peroxidase 2). 2-D electrophoresis allowed us to identify new BALF proteins and to characterize protein composition in patients with sarcoidosis and idiophatic pulmonary fibrosis. Comparison of the gels of the two diseases showed that they differ in BALF protein profiles as they do in type of immune response.

Presence of macrophage migration inhibitory factor in human milk: evidence in the aqueous phase and milk fat globules.

Human milk is a source of bioactive substances regulating the development and activity of the newborn immune system. Human milk has been found to contain a number of cytokines, including interleukins, growth factors, and colony stimulating factors. In the present study, we assessed 10 specimens of human milk for the presence of macrophage migration inhibitory factor (MIF), a cytokine recently described in several human reproductive organs and tissues. Using biochemical as well as immunologic techniques, we showed that MIF is abundantly present in human milk, mostly distributed in the lipid layer and in the aqueous phase. Fractionation of the lipid layer showed that MIF is highly concentrated inside milk fat globules. In view of its proinflammatory features, we speculate that milk MIF may protect the newborn against infection and play a role in preserving the functionality of the lactating mammary gland. Furthermore, the localization of MIF in lipid globules suggests a possible strategy for the protection of milk cytokines from the gastric barrier.