Utility of bronchoalveolar lavage in the diagnosis of pulmonary infection in children with haematological malignancies.

BACKGROUND: Fibre-optic bronchoscopy with bronchoalveolar lavage (BAL) is a safe procedure and is associated with low morbidity and mortality in immunocompromised children. Although many studies have highlighted the advantages of positive BAL results in the diagnosis of pulmonary infections, there have been few reports examining the impact of a negative BAL result on clinical management in immunocompromised children on empiric broad-spectrum antimicrobial therapy. AIM: The aim of this study was to evaluate BAL in the diagnosis of pulmonary infections in children with haematological malignancies who develop pneumonia unresponsive to empiric antimicrobial therapy, and also to determine whether a negative BAL result contributed to the clinical management of these patients. MATERIALS AND METHODS: A retrospective review of 44 BAL procedures performed in 33 children with haematological malignancy diagnosed and treated at Our Lady's Children Hospital, Crumlin, Dublin 12, Ireland, over a 10-year period was carried out. RESULTS: We identified a pathogen causing pneumonia in 24 of 44 BAL procedures (54.5 %). The BAL procedure resulted in modification of antimicrobial treatment after 20 of 24 procedures with positive results (83.3 %) in 16 of 20 patients (80 %). Management was changed after 8 of 20 procedures with negative results (40 %) in 8 of 18 patients (44.4 %). The procedure was well tolerated in all patients. CONCLUSIONS: Our study supports the use of bronchoscopy with BAL as a diagnostic intervention in this patient population. We consider BAL a safe procedure from which both positive and negative results contribute to the patient's clinical management.

A prospective survey of febrile events in hematological malignancies.

The Hema e-Chart prospectively collected data on febrile events (FEs) in hematological malignancy patients (HMs). The aim of the study was to assess the number, causes and outcome of HM-related FEs. Data were collected in a computerized registry that systematically approached the study and the evolution of FEs developing in a cohort of adult HMs who were admitted to 19 hematology departments in Italy from March 2007 to December 2008. A total of 869 FEs in 3,197 patients with newly diagnosed HMs were recorded. Fever of unidentified origin (FUO) was observed in 386 cases (44.4%). The other causes of FE were identified as noninfectious in 48 cases (5.5%) and infectious in 435 cases (50.1%). Bacteria were the most common cause of infectious FEs (301 cases), followed by fungi (95 cases), and viruses (7 cases). Mixed agents were isolated in 32 episodes. The attributable mortality rate was 6.7% (58 FEs). No deaths were observed in viral infection or in the noninfectious groups, while 25 deaths were due to FUO, 16 to bacterial infections, 14 to fungal infections, and three to mixed infections. The Hema e-Chart provided a complete system for the epidemiological study of infectious complications in HMs.

Pharmacological and phytochemical study on a Sisymbrium officinale Scop. extract.

ETHNOPHARMACOLOGICAL RELEVANCE: The aerial parts of Sisymbrium officinale Scop. are commonly used to treat airway ailments, moreover in antiquity the herbal drug was reputed to possess anticancer properties. The results obtained in present work support the traditional use and the properties ascribed to Sisymbrium officinale. AIM OF THE STUDY: In order to give a scientific basis to the traditional uses of Sisymbrium officinale, this study was aimed to evaluate in vitro the myorelaxant activity, the antimicrobial properties and the antimutagenic effect of an aqueous dry extract of the aerial parts of the plant. A phytochemical characterization of the extract was also performed. MATERIALS AND METHODS: The myorelaxant activity was studied against the contractions induced by carbachol, histamine and leukotriene C(4), in isolated guinea-pig trachea. The antimicrobial activity was tested against six bacteria and one yeast. The Ames test, performed by the preincubation method, was used to study the antimutagenic activity of the extract by its capability to inhibit the mutagenic effect of 2-nitrofluorene, sodium azide, methyl methanesulfonate and 2-aminoanthracene, in Salmonella typhimurium TA98, Salmonella typhimurium TA100 and Escherichia coli WP2uvrA strains. The chemical composition of the extract was analyzed by TLC and HPLC. RESULTS: Sisymbrium officinale showed to reduce the chemically-induced contractions of isolated guinea-pig trachea with major potency against leukotriene C(4) and histamine. The extract did not show any antibacterial activity. The Ames test showed a strong antimutagenic activity against 2-aminoanthracene, in Escherichia coli WP2uvrA and in Salmonella typhimurium TA98 strains. The phytochemical study highlighted the presence of putranjivine, the glucosinolate marker of Sisymbrium officinale, and of proline. CONCLUSIONS: The myorelaxant activity of Sisymbrium officinale offers a scientific basis to its use in traditional medicine. The strong antimutagenic effect suggests further studies to evaluate its possible chemopreventive activity.

Survival of patients with documented autologous recovery after SCT for severe aplastic anemia: a study by the WPSAA of the EBMT.

Graft rejection, with persistent pancytopenia, is well documented after allogeneic BMT (hematopoietic SCT (HSCT)) for severe aplastic anemia (SAA) and the prognosis is poor. The recovery of host-hematopoiesis, autologous recovery (AR), after allogeneic HSCT is a rare event and the incidence and long-term survival are unknown. We report a retrospective analysis of consecutive patients in the Aplastic Anaemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT-WPSAA) registry between 1973 and 2005. A total of 45 cases of AR, of 1205 patients transplanted for SAA in 57 centers are reported. We describe characteristics and long-term outcome of patients with AR, compared with SAA patients from participating transplant centers without AR (n=1024) and patients with graft rejection (n=136) without autologous recovery. The estimated cumulative incidence of AR was 4.2% (3.1-5.6) (confidence interval (CI) 95%) with an OS of 84% (95% CI 83-107%). The OS of the control group was 74% (81-90) at 10 years of follow up, whereas the patients with graft failure had an OS of 16% (CI 12-28%). This retrospective analysis establishes the incidence and long-term survival of patients experiencing AR after allogeneic HSCT for SAA.

Erythroleukaemia, diabetes insipidus and hypophyseal damage: Two case reports.

We report on two cases of patients who developed diabetes insipidus (DI) before acute erythroleukaemia (EL). A brain MRI showed an empty sella turcica in one case and hypothalamo-hypophyseal peduncle damage in the second case. Reduced levels of TGF-beta1 and Vitamin D3, with associated EVI-1 over-expression and karyotypic abnormalities were documented. These two cases show specific chromosomal/molecular alterations in EL with DI. The hypothesis of pituitary involvement in erythroleukemogenesis is discussed.

Outcome of bone marrow transplantation in acquired and inherited aplastic anaemia in the Republic of Ireland.

BACKGROUND: Severe Aplastic Anaemia (SAA) and Fanconi Anaemia (FA) are rare haematological disorders characterised by pancytopenia and bone marrow hypoplasia. AIMS: We performed a retrospective study of all patients who underwent BMT for SAA and FA at St James's Hospital, Dublin, and at OLHSC, Crumlin, between 1985 and 2002. METHODS: The medical records of 63 patients, 50 with acquired SAA and 13 with FA, were reviewed. RESULTS: The median age at the time of transplant was 14 years (range 3-43 years). The actuarial survival (OS) (n = 63) was 76% at 17 years. The transplant related mortality (TRM) was 22% (n = 14). The most common cause of death was infection (46%). The survival was significantly better in patients receiving their transplant after 1995 (p = 0.002). Outcome was superior in those receiving less than 20 red cell transfusions prior to transplant: OS 91% (< 20 Units) versus 62% (> or = 20 Units). CONCLUSIONS: These national results are comparable to those of published international series and support the use of BMT in the treatment of SAA and FA. The known adverse effect of prior transfusion was confirmed.

Phenylpropanoid glycosides in Italian Orobanche spp., sect. Orobanche.

We studied the occurrence of phenylpropanoid glycosides (PhG) in five species of the genus Orobanche L., collected in the Latium region of Italy. The presence of orobanchoside and verbascoside in all four species confirms that these PhGs are taxonomic markers of the genus. The results suggest that O. gracilis form. citrina could be a diverse entity.

Oxidative stress and TGFbeta in kidney-transplanted patients with cyclosporin-induced hypertension. Effect of carvedilol and nifedipine.

Cyclosporin is a powerful stimulator of oxidative stress signaling, leading to TGFbeta production, NO degradation, endothelial dysfunction, hypertension and post-transplant nephropathy. Carvedilol, alpha1-beta-blocker with strong antioxidant activity, may interfere with this chain of events. Therefore, we measured monocyte ecNOS, TGFbeta and heme oxygenase-1 (HO-1) mRNA level and plasma nitrite/nitrate, 3-nitrotyrosine, an estimate of peroxynitrite, and total plasma antioxidant power in kidney-transplanted patients with post-transplant hypertension, before and after treatment with carvedilol, 25 - 50 mg o.d. orally for 4 months (n = 15). The dihydropyridine calcium channel blocker nifedipine (n = 10) was used as comparator antihypertensive drug. Blood pressure fell to a similar extent with both drugs. Carvedilol increased plasma antioxidant power and HO-1 mRNA and reduced 3-nitrotyrosine and TGFbeta mRNA levels, while the same was not observed with nifedipine. Monocyte ec NOS mRNA levels and plasma nitrite/nitrate were higher in the patients than in a normotensive healthy control group and were unaffected by either treatment. In conclusion, carvedilol reduces the oxidative stress and corrects the altered cellular signaling mediated by oxidative stress in CsA-induced post-transplant hypertension. Therefore, it may prevent long-term complications, such as endothelial dysfunction, fibrogenesis and post-transplant nephropathy by decreasing NO degradation and production of TGFbeta, a key fibrogenic cytokine, and by activating HO-1 production.

Light-dependent interaction between Drosophila CRY and the clock protein PER mediated by the carboxy terminus of CRY.

BACKGROUND: The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila CRYPTOCHROME (CRY), a putative circadian photoreceptor, has previously been reported to interact with the clock protein TIMELESS (TIM) in a light-dependent manner. Although TIM dimerizes with PERIOD (PER), no association between CRY and PER has previously been revealed, and aspects of the light dependence of the TIM/CRY interaction are still unclear. RESULTS: Behavioral analysis of double mutants of per and cry suggested a genetic interaction between the two loci. To investigate whether this was reflected in a physical interaction, we employed a yeast-two-hybrid system that revealed a dimerization between PER and CRY. This was further supported by a coimmunoprecipitation assay in tissue culture cells. We also show that the light-dependent nuclear interactions of PER and TIM with CRY require the C terminus of CRY and may involve a trans-acting repressor. CONCLUSIONS: This study shows that, as in mammals, Drosophila CRY interacts with PER, and, as in plants, the C terminus of CRY is involved in mediating light responses. A model for the light dependence of CRY is discussed.

Photic entrainment of the circadian clock: from Drosophila to mammals.

Entrainment is as fundamental to an organism's circadian timing as are the molecular mechanisms involved in the functioning of the intracellular clock oscillator. In nature, one of the principle, although not the only, circadian entraining stimulus (Zeitgeber) is provided by the daily light--dark cycles. In animals, the visual processing apparatus alone is inadequate to accomplish the task of transducing circadian photic signals to the clockwork machinery. In fact, it is ever more appreciated by circadian biologists that organisms as divergent as plants and mammals have evolved a wonderfully complex array of partly redundant specializations which can guarantee the precise alignment of biological and environmental time. Research in circadian biology is cruising at such a rate that attempts to review the state of the art can only hope, at best, to provide a snapshot of the speeding cruiser from its wake. This paper will hopefully provide a reasonably sharp portrayal of what is at hand.

Analysis of Gq protein alpha subunit mRNA expression in human monocytes: relevance of the purification step.

BACKGROUND: Galphaq is a member of the Gq family of G proteins, which by stimulating the phospholipase Cbeta (PLCbeta)-IP(3)-Ca(++) mediated intracellular signaling systems controls some of the most fundamental cardiovascular cellular processes. The study of Galphaq is complicated by the presence of a pseudogene in human DNA, with signficant homology to the mRNA encoding the alpha subunit of Gq protein. The presence of this pseudogene will cause problems when the analysis of the Galphaq gene expression is based solely on RT-PCR. In this study, we report a simple method for avoiding unwanted amplification of the Galphaq pseudogene and the use of human monocytes as a readily available source for examining Galphaq. METHODS: RT-PCR was carried out on RNA extracted from peripheral blood monocytes of 10 normal subjects using specific primers for Galphaq. RESULTS: When several subjects' Galphaq was examined, the authentic Galphaq mRNA amplification product levels, as a ratio to unpurified pseudogene containing amplification products, declined by up to approximately 70%. CONCLUSION: Given the importance of Gq protein in cardiovascular signal transduction, it is fundamental to provide a reliable assessment of G alpha q gene expression. In addition to accurately assessing Galphaq levels, the use of circulating human monocytes as a useful source of Galphaq for investigating mechanisms involved in the regulation of vascular tone is shown.

Efficient and heritable functional knock-out of an adult phenotype in Drosophila using a GAL4-driven hairpin RNA incorporating a heterologous spacer.

We have developed a modified RNA interference (RNAi) method for generating gene knock-outs in Drosophila melanogaster. We used the sequence of the yellow (y) locus to construct an inverted repeat that will form a double-stranded hairpin structure (y-IR) that is under the control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. Hairpins are extremely difficult to manipulate in Escherichia coli, so our method makes use of a heterologous 330 bp spacer encoding sequences from green fluorescent protein to facilitate the cloning steps. When the UAS-y-IR hairpin is expressed under the control of different promoter-GAL4 fusions, a high frequency of y pigment phenocopies is obtained in adults. Consequently this method for producing gene knock-outs has several advantages over previous methods in that it is applicable to any gene within the fly genome, greatly facilitates cloning of the hairpin, can be used if required with GAL4 drivers to avoid lethality or to induce RNAi in a specific developmental stage and/or tissue, is useful for generating knock-outs of adult phenotypes as reported here and, finally, the system can be manipulated to investigate the trans-acting factors that are involved in the RNAi mechanism.

The clock gene period of the housefly, Musca domestica, rescues behavioral rhythmicity in Drosophila melanogaster. Evidence for intermolecular coevolution?

In Drosophila, the clock gene period (per), is an integral component of the circadian clock and acts via a negative autoregulatory feedback loop. Comparative analyses of per genes in insects and mammals have revealed that they may function in similar ways. However in the giant silkmoth, Antheraea pernyi, per expression and that of the partner gene, tim, is not consistent with the negative feedback role. As an initial step in developing an alternative dipteran model to Drosophila, we have identified the per orthologue in the housefly, Musca domestica. The Musca per sequence highlights a pattern of conservation and divergence similar to other insect per genes. The PAS dimerization domain shows an unexpected phylogenetic relationship in comparison with the corresponding region of other Drosophila species, and this appears to correlate with a functional assay of the Musca per transgene in Drosophila melanogaster per-mutant hosts. A simple hypothesis based on the coevolution of the PERIOD and TIMELESS proteins with respect to the PER PAS domain can explain the behavioral data gathered from transformants.

Circadian rhythms: from behaviour to molecules.

In higher eukaryotes, circadian behaviour patterns have been dissected at the molecular level in Drosophila and, more recently, in the mouse. Considerable progress has been made in identifying some of the molecular components of the clock in the fly, where two genes, period (per) and timeless (tim), are essential for behavioural rhythmicity. The PER and TIM proteins show circadian cycles in abundance, and are part of a negative feedback loop with their own mRNAs. Within the pacemaker neurons, the PER and TIM products are believed to form a complex which allows them to translocate to the nucleus, but how they repress their own transcription is unclear. TIM is rapidly degraded by light, a feature which permits a compelling molecular description of both behavioural light entrainment and phase responses to light pulses. The regulation of per and tim is altered in different Drosophila tissues, however, and comparative analyses of the two genes outside the Diptera reveals further unusual patterns of tissue-specific regulation. Evolution appears to have modified the way in which the two genes are utilised to generate circadian phenotypes. More recently, the cloning of mouse clock genes, including putative per homologues, opens up exciting possibilities for mammalian molecular chronobiology.

Big flies, small repeats: the "Thr-Gly" region of the period gene in Diptera.

The region of the clock gene period (per) that encodes a repetitive tract of threonine-glycine (Thr-Gly) pairs has been compared between Dipteran species both within and outside the Drosophilidae. All the non-Drosophilidae sequences in this region are short and present a remarkably stable picture compared to the Drosophilidae, in which the region is much larger and extremely variable, both in size and composition. The accelerated evolution in the repetitive region of the Drosophilidae appears to be mainly due to an expansion of two ancestral repeats, one encoding a Thr-Gly dipeptide and the other a pentapeptide rich in serine, glycine, and asparagine or threonine. In some drosophilids the expansion involves a duplication of the pentapeptide sequence, but in Drosophila pseudoobscura both the dipeptide and the pentapeptide repeats are present in larger numbers. In the nondrosophilids, however, the pentapeptide sequence is represented by one copy and the dipeptide by two copies. These observations fulfill some of the predictions of recent theoretical models that have simulated the evolution of repetitive sequences.