Detection and identification ofMycobacterium avium in the blood of AIDS patients by the polymerase chain reaction

One hundred fifty-three blood samples from patients positive for the human immunodeficiency virus (HIV) were analyzed by polymerase chain reaction (PCR) to detect the presence ofMycobacterium avium. Samples were collected from patients who also had blood cultures performed by a radiometric method. Blood samples were centrifuged on a Ficoll-Hypaque gradient to purify peripheral blood mononuclear cells. The purified cells were washed and incubated with a resin, boiled to release mycobacterial DNA, and then amplified. Polymerase chain reaction products were detected by a nonisotopic method. A 123 base-pair (bp) insertion sequence, namely IS6110, fromMycobacterium tuberculosis complex was also included in the reaction as an internal control ofTaq polymerase activity to exclude the presence of enzyme inhibitors. This IS6110 fragment can be distinguished from the 383 bp target product on ethidium bromide-stained agarose gel and may also be used in a colorimetric assay. Such results were compared with the results of culture and indicated that the assay is as sensitive as bacteriological methods, though faster.     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

Sleep disturbance and daytime symptoms in wheezing school-aged children

The aim of the study was to investigate whether wheezing is associated with disturbed sleep and increased daytime symptoms in school-aged children. A random sample of 1234 children, aged 6-14 years, participated in a respiratory health study in the region of Antwerp. The International Study of Asthma and Allergies in Childhood questionnaire and a separate sleep questionnaire were completed. In the children who wheezed in the last 12 months, sleep quality was more frequently disturbed due to nocturnal awakenings and restless sleep compared with children who did not wheeze. Daytime sleepiness and tiredness were more common in wheezing than in non-wheezing children. After adjusting for possible confounders a positive association was found between wheeze and: difficulties falling asleep [odds ratio (OR) = 2.0], restless sleep (OR = 5.0), daytime sleepiness (OR = 3.8) and daytime tiredness (OR = 5.1). Chronic cough (OR = 2.4), snoring (OR = 2.0), chronic rhinitis (OR = 2.6) and eczema (OR = 3.3) were associated with disturbed sleep. Chronic cough (OR = 2.5) and rhinitis (OR = 4.1) were related to daytime tiredness. Chronic rhinitis was an important risk factor for snoring (OR = 1.9). In wheezing school-aged children, decreased quality of sleep and increased daytime tiredness and sleepiness were more often reported. Upper airway symptoms were related to the sleep disturbances.     

Blastic NK-cell lymphomas (agranular CD4+CD56+ hematodermic neoplasms): a review

Blastic natural killer (NK) cell lymphoma (also termed CD4+CD56+ hematodermic neoplasm) is a recently described entity, with the first case reported in 1994. It was suggested initially that the disease originates from NK cells. Since 1994, single cases and a few small series have been published. In this review, data from the literature and a series of 30 cases from the French and Dutch study groups on cutaneous lymphomas are discussed. The major clinical, histopathologic, and phenotypic aspects of the disease and diagnostic criteria and data suggesting a plasmacytoid dendritic cell origin for the tumor cells are provided.     

Bradykinin differentiates human lung fibroblasts to a myofibroblast phenotype via the B2 receptor

BACKGROUND: The identification of factors mediating the transition of lung fibroblasts into myofibroblasts is considered fundamental in the comprehension of abnormal reparative processes. Bradykinin, a mediator known for its proinflammatory action, is able to induce cytokine production and contractility in fibroblast cultures. OBJECTIVES: In this study the ability of bradykinin to drive fibroblast into a myofibroblast phenotype at the cellular and molecular level was evaluated. METHODS: alpha-Smooth muscle actin (alpha-SMA) expression and TGF-beta in bradykinin stimulated fibroblasts were tested by means of flow cytometry, Western blot, and RT-PCR. Cell proliferation and collagen production were evaluated by the colorimetric methylthiazol tetrazolium assay and sirius red assay, respectively. Which bradykinin receptor mediates the expression of alpha-SMA was evaluated using selective B1 and B2 blocking agents. Furthermore, the effect of bradykinin on extracellular signal-regulated kinase 1/2 phosphorylation was explored. RESULTS: Bradykinin caused in lung fibroblasts a significant increase in alpha-SMA at the cellular and molecular level. The B2 receptor was held responsible for this effect because a specific receptor antagonist had entirely blocked this effect. Bradykinin was able to induce fibroblast proliferation and collagen production. Bradykinin significantly activated mitogen-activated protein kinase pathway by phosphorylating extracellular signal-regulated kinase 1/2, whereas PD98059, a specific inhibitor, was able to block myofibroblast induction. Although bradykinin induced an increase of TGF-beta on fibroblasts, the blockage of this cytokine did not alter alpha-SMA expression. CONCLUSION: The data support the hypothesis that bradykinin may be involved in bronchial remodeling and lung fibrosis beyond its well recognized proinflammatory activity, also suggesting a new potential therapeutic strategy to control altered reparatory processes.     

Reciprocal stimulation of gammadelta T cells and dendritic cells during the anti-mycobacterial immune response

Gammadelta T cells and dendritic cells (DC) are two distinct cell types of innate immunity that participate in early phases of immune response against Mycobacterium tuberculosis infection. Here we show that a close functional relationship exists between these cell populations. Using an in vitro coculture system, Vgamma1 T cells from Tcrb(-/- )mice were found to be activated by DC infected in vitro with BCG, as indicated by the elevated CD69 expression, IFN-gamma secretion and cytotoxic activity. This activation process was due to a non-cognate mechanism since it required neither cell to cell contact nor interaction between the TCR and a specific antigen, but was mediated by DC-derived IL-12. Reciprocally, Vgamma1 T cells provided a key cytokine, IFN-gamma, which increased IL-12 production by BCG-infected DC. Moreover, exposure of BCG-infected DC to Vgamma1 T cells conditioned the former to prime a significantly stronger anti-mycobacterial CD8 T cell response. Consequently, stimulation of gammadelta T cells and their non-cognate interaction with DC could be applied as an immune adjuvant strategy to optimize vaccine-induced CD8 T cell immunity.     

Genetic instability in superficial bladder cancer and adjacent mucosa: an interphase cytogenetic study

A systematic analysis of both tumors and the surrounding urothelium to help identify what lies behind the mechanism of multifocal tumor development has not yet been performed. In this study we investigated chromosome 1, 7, 9, and 17 aneusomy in 25 superficial papillary carcinomas and in 51 tissue samples taken from sites of macroscopically uninvolved urothelium surrounding the tumors, using the fluorescence in situ hybridization method. Our data demonstrated a close genetic relationship between all examined tumors and normal-appearing mucosa. Numeric aberrations of chromosomes 1, 7, 9, and 17 were found to exhibit similar patterns in all analyzed specimens, although with different frequencies.     

Chromosomal radiosensitivity of breast cancer with a CHEK2 mutation

Recently, multiple studies have shown that a sequence variant in CHEK2 (CHEK2 1100delC) plays a role in the susceptibility to breast cancer. This mutation should confer about a twofold increased breast cancer risk in women and a 10-fold increased risk in men. Because the CHEK2 gene plays a critical role in DNA damage repair and the CHEK2 1100delC variant confers susceptibility to breast cancer, we investigated if patients carrying the CHEK2 1100delC mutation are characterized by an enhanced chromosomal radiosensitivity. To this end, familial breast cancer patients, sporadic breast cancer patients, and healthy women, considered in our previously studied to determine their chromosomal radiosensitivity with the G2 and G0-MN assay, were all tested in present study for the presence of the CHEK2 1100delC variant. The 1100delC variant was detected in none of the 100 healthy individuals, in 1 of 100 (1%) unselected breast cancer patients and in 3 of 78 (3.8%) breast cancer patients with a family history of breast cancer. The breast cancer patients with the CHEK2 1100delC genotype had a mean radiation-induced yield of chromatid breaks that was not significantly different from that of the healthy control group. Although the mean yield of micronuclei (MN) was significantly higher compared to the healthy control group, this higher mean MN yield was due to a single patient who had a very high number of MN compared to the parallel control. Our data suggest that breast cancer patients with a CHEK2 1100delC mutation are in general not characterized by a distinct enhanced chromosomal radiosensitivity. These conclusions are, however, very preliminary, because of the small numbers of CHEK2 1100delC breast cancer patients studied.