Electron microscopic microanalysis of bronchoalveolar lavage: a way to identify exposure to silica and silicate dust.

OBJECTIVES: The diagnostic implications of finding non-fibrous inorganic particles in bronchoalveolar lavage (BAL) fluid has not been fully assessed. The aim of this study has been to measure the silica and non-fibrous silicates in BAL fluid from populations with different exposures to inorganic dust, and to find whether such measurement is useful for diagnostic purposes. MATERIALS AND METHODS: BAL samples from 19 subjects with only environmental exposure to inorganic dust (group A, mean (SD) age 50.7 (15.2)), 23 subjects with normal chest x ray films exposed to silica or silicates at work (group B, mean (SD) age 52.0 (12.4)), and 15 subjects with a previous diagnosis of silicosis (group C, mean (SD) age 68.0 (6.5)) were studied. Absolute and relative cell counts were found, and the samples were prepared for microanalysis by electron microscopy (EM). Firstly, semiquantitative x ray microanalysis was performed to find the level of silicon (Si) (peak/background Si) and this was followed by microanalysis of individual particles by EM. Variables related to the level of Si detected were assessed with multivariate analysis. RESULTS: Detected levels were higher in group B (2.09, 95% confidence interval (95% CI) 1.56 to 2.82) and C (1.50, 95% CI 1.07 to 2.12) than in group A (0.87, 95% CI 0.66 to 1.16) (P < 0.05, Dunett t test). A first multivariate analysis showed that exposure to silica or silicates was the only determinant of the level of Si expressed as log peak/background Si, when adjusted for age, sex, smoking habit, and cell count. A second multivariate analysis with microanalysis of individual particles as an independent variable showed the silica count to be the main predictor of detected concentration of Si. Silica and non-aluminium silicates together explain 55.5% (R2) of the variation in detected levels of Si. CONCLUSIONS: Detected levels of Si in BAL fluid depend on silica count and are higher in subjects with exposure to inorganic dust at work, but will not discriminate between exposed subjects with and without silicosis. Because semiquantitative x ray microanalysis does not accurately define exposure to non-silica inorganic particles, this measurement must be followed by EM microanalysis of individual particles in most cases, especially when exposure to silicates or metal dust is suspected.     

Breaks invisible to the DNA damage response machinery accumulate in ATM‐deficient cells

After irradiation, ATM defective cells accumulate unrepaired double strand breaks (DSBs) for several cell divisions. At the chromosome level, unresolved DSBs appear as chromosome breaks that can be efficiently scored by using telomeric and mFISH probes. H2AX is immediately activated by ATM in response to DNA damage and its phosphorylated form, γH2AX, flanks the DSB through several megabases. The γH2AX‐labeling status of broken chromosome ends was analyzed in AT cells to check whether the DNA damage response was accurately taking place in these persistent DSBs. The results show that one quarter of the scored breaks are devoid of γH2AX foci in metaphase spreads from ATM‐deficient cells, and this fraction is significantly higher than in normal cells (χ² < 0.05). Accumulation of sensor and repair proteins at damaged sites is a key event in the cellular response to DSBs, so MRE11 labeling at broken ends was also analyzed. While all γH2AX foci scored at visible broken ends colocalize with MRE11 foci, all γH2AX‐unlabeled breaks are also devoid of MRE11‐labeling. The present results suggest that a significant subset of the AT long‐lived DSBs may persist as “invisible” DSBs due to deficient detection by the DNA damage repair machinery. Eventually the properly signaled DSBs will be repaired while invisible breaks may indefinitely accumulate; most probably contributing to the AT cells' well known genomic instability. © 2009 Wiley‐Liss, Inc.     

The amino acid transporter asc-1 is not involved in cystinuria

BACKGROUND: The human amino acid transporter asc-1 (SLC7A10) exhibits substrate selectivity for small neutral amino acids, including cysteine, is expressed in kidney, is located close to the cystinuria B gene and presents sequence variants (e.g., E112D) in some cystinuria patients. We have cloned human asc-1, assessed its transport characteristics, localized its expression in kidney, searched for mutations in cystinuria patients, and tested the transport function of variant E112D. METHODS: We used an EST-based homology cloning strategy. Transport characteristics of asc-1 were assessed by coexpression with 4F2hc in Xenopus oocytes and HeLa cells. Localization of asc-1 mRNA in kidney was assessed by in situ hybridization. Exons and intron-exon boundaries were polymerase chain reaction (PCR)-amplified from blood cell DNA and mutational screening was performed by single-stranded conformational polymorphism (SSCP). RESULTS: Asc-1 reaches the plasma membrane in HeLa cells, unlike in oocytes, most probably by interaction with endogenous 4F2hc and presents similar transport characteristics to those in oocytes coexpressing asc-1/4F2hc. Asc-1 mediates a substantial efflux of alanine in a facilitated diffusion mode of transport. Expression of asc-1 mRNA localized to Henle's loop, distal tubules, and collecting ducts. Finally, SLC7A10 polymorphisms were identified in cystinuria probands and the SLC7A10 sequence variant E112D showed full transport activity. CONCLUSION: The lack of expression of asc-1 in the proximal tubule indicates that it plays no role in the bulk of renal reabsorption of amino acids. No mutations causing cystinuria have been found in SLC7A10. The facilitated diffusion mode of transport and the expression in distal nephron suggest a role for asc-1 in osmotic adaptation.     

Ciprofloxacin prophylaxis in patients with acute leukemia and granulocytopenia in an area with a high prevalence of ciprofloxacin-resistant Escherichia coli

BACKGROUND: Infection remains the major cause of morbidity and mortality in patients with neutropenia, and the beneficial effects of oral prophylaxis remain controversial. METHODS: From 1993 to December 1999, the authors analyzed the clinical and microbiologic outcomes of 144 episodes of febrile neutropenia among adult patients with acute leukemia. RESULTS: Forty-three consecutive episodes occurred among patients who were on ciprofloxacin prophylaxis during 1993-1996 (ciprofloxacin group), and 101 subsequent episodes occurred among patients who were not exposed to ciprofloxacin prophylaxis (control group). There were no differences in clinical presentation, antibiotic treatment received for the episode, or a worse outcome between groups. The rate of bacteremia was similar (12 of 43 patients [28%] vs. 26 of 101 patients [26%], respectively). There was a trend toward a higher rate of Gram positive bacteremia in the control group (12 of 101 patients [12%] vs. 2 of 43 patients [5%]) and a higher rate of Gram negative bacteremia in the ciprofloxacin group (11 of 43 patients [26%] vs. 15 of 101 patients [15%]). Resistance to fluoroquinolones was greater in Escherichia coli blood isolates from patients in the ciprofloxacin group (7 of 8 patients vs. 2 of 9 patients; P = 0.02). CONCLUSIONS: The current results suggest that fluoroquinolone prophylaxis for patients with febrile neutropenia may be abandoned safely in areas with a high prevalence of ciprofloxacin-resistant enterobacteria.     

Palisading and verocay body-prominent dermatofibrosarcoma protuberans: a report of three cases

The aim of this report is to draw attention to nuclear palisading and Verocay body formation as peculiar, previously undescribed histological findings in rare instances of dermatofibrosarcoma protuberans (DFSP). METHODS AND RESULTS: Three indurated, nodular or plaque skin lesions were diagnosed as DFSP on the basis of their storiform proliferation of spindle-shaped cells diffusely infiltrating the dermis and subcutaneous tissue. Sclerosing and giant cell areas were also identified. Unexpectedly, conspicuous nuclear palisading was also noted in all cases and Verocay body formation was present in two. Immunostains were positive for CD34 and negative for S100 protein in every instance. Proliferating cells were seen to display fibroblast-like features by ultrastructural study of one case. CONCLUSIONS: DFSP may rarely show a schwannoid histological appearance as the result of nuclear palisading and even Verocay body formation. In this setting, both the search for DFSP characteristic morphologic features and the performance of CD34 and S100 protein immunohistochemistry will facilitate the correct diagnosis.     

Mutation in KARS: A novel mechanism for severe anaphylaxis

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Comparison between SLC3A1 and SLC7A9 cystinuria patients and carriers: a need for a new classification

Recent developments in the genetics and physiology of cystinuria do not support the traditional classification, which is based on the excretion of cystine and dibasic amino acids in obligate heterozygotes. Mutations of only two genes (SLC3A1 and SLC7A9), identified by the International Cystinuria Consortium (ICC), have been found to be responsible for all three types of the disease. The ICC set up a multinational database and collected genetic and clinical data from 224 patients affected by cystinuria, 125 with full genotype definition. Amino acid urinary excretion patterns of 189 heterozygotes with genetic definition and of 83 healthy controls were also included. All SLC3A1 carriers and 14% of SLC7A9 carriers showed a normal amino acid urinary pattern (i.e., type I phenotype). The rest of the SLC7A9 carriers showed phenotype non-I (type III, 80.5%; type II, 5.5%). This makes the traditional classification imprecise. A new classification is needed: type A, due to two mutations of SLC3A1 (rBAT) on chromosome 2 (45.2% in our database); type B, due to two mutations of SLC7A9 on chromosome 19 (53.2% in this series); and a possible third type, AB (1.6%), with one mutation on each of the above-mentioned genes. Clinical data show that cystinuria is more severe in males than in females. The two types of cystinuria (A and B) had a similar outcome in this retrospective study, but the effect of the treatment could not be analyzed. Stone events do not correlate with amino acid urinary excretion. Renal function was clearly impaired in 17% of the patients.     

The use of sleep measures to compare a new 5HT1A agonist with buspirone in humans

The partial agonist buspirone has a REM (rapid eye movement) suppressing effect on human sleep probably via a 5HT(1A) receptor in the pontine area. Eptapirone is a new 5HT(1A) agonist with a greater intrinsic effect than buspirone. The objective of this study was to examine the effects of eptapirone on sleep architecture, particularly REM sleep, in normal volunteers and compare it with buspirone and placebo. This was a randomized, double-blind placebo-controlled four-way crossover study in 12 healthy volunteers. Volunteers were screened to ensure that they had normal overnight sleep EEG (electroencephalogram) and were extensive CYP 2D6 metabolizers. Sleep was recorded on pairs of nights on four occasions, with medication being taken before the second night. Treatments were eptapirone 1.5mg at 10 AM, eptapirone 1.5mg at 11 PM, buspirone 20mg at 11 PM and placebo. Standard measures of sleep were derived and compared among the four treatments using ANOVA. REM sleep was significantly suppressed supporting the proposition that activation of post-synaptic 5HT(1A) receptors reduces REM sleep. Sleep fragmentation increased by both drugs. REM sleep suppression was significantly greater with morning eptapirone than with buspirone. Wakefulness in sleep was significantly greatest after morning eptapirone. REM sleep effects were greatest after evening eptapirone, suggesting a greater effect on central serotonin receptors than that of buspirone.     

Gene structure,chromosomal localization,and mutation screening of the human gene for the inner ear protein otospiralin

Otospiralin is a novel protein of unknown function that is produced by non-sensory cells (fibrocytes) of the inner ear (cochlea and vestibule). We showed that downregulation of otospiralin in guinea pigs leads to deafness and we therefore hypothesized that genetic defects in the otospiralin gene could also cause deafness in humans. In this study, we cloned and localized OTOSP, the human gene for otospiralin. OTOSP spans 1,630 nucleotides, contains four exons and codes for a 567-nucleotide cDNA. By fluorescence in situ hybridization and hybrid panel mapping we localized OTOSP on chromosome 2 at position q37.3. There is currently no deafness family linked to this region. We screened OTOSP for mutations in 410 unrelated patients exhibiting various levels of hearing loss. Beside intronic polymorphisms, a rare variant (Pro7Leu) was found in 4 deafness patients and 3 control individuals, indicating that this change is not involved in this condition and excluding OTOSP as a major gene for genetic deafness. Electronic Publication