Whole-exome sequencing in splenic marginal zone lymphoma reveals mutations in genes involved in marginal zone differentiation.

Splenic marginal zone lymphoma (SMZL) is a B-cell neoplasm whose molecular pathogenesis remains fundamentally unexplained, requiring more precise diagnostic markers. Previous molecular studies have revealed 7q loss and mutations of nuclear factor κB (NF-κB), B-cell receptor (BCR) and Notch signalling genes. We performed whole-exome sequencing in a series of SMZL cases. Results confirmed that SMZL is an entity distinct from other low-grade B-cell lymphomas, and identified mutations in multiple genes involved in marginal zone development, and others involved in NF-κB, BCR, chromatin remodelling and the cytoskeleton.

Targeting of mTOR is associated with decreased growth and decreased VEGF expression in acute myeloid leukaemia cells.

BACKGROUND: The mammalian target of rapamycin (mTOR) has recently been implicated in leukaemic cell growth, tumour-associated angiogenesis and expression of vascular endothelial growth factor (VEGF). We examined whether mTOR plays a role as regulator of growth and VEGF-expression in acute myeloid leukaemia (AML). Three mTOR-targeting drugs, rapamycin, everolimus (RAD001) and CCI-779, were applied. The effects of these drugs on growth, survival, apoptosis and VEGF expression in primary AML cells and various AML cell lines were examined. MATERIALS AND METHODS: Growth of AML cells and AML-derived cell lines was assessed by (3)H-thymidine incorporation, survival was examined by light- and electron microscopy, by Tunel assay and by AnnexinV-staining, and the expression of VEGF by Northern blotting, RT-PCR and ELISA. RESULTS: Rapamycin was found to counteract growth in the AML cell lines U937 and KG1a as well as in primary AML cells in 14/18 patients examined. The effects of rapamycin and its derivatives were dose-dependent (IC(50): 10 pM-100 nM). It was also found that exposure to mTOR-targeting drugs resulted in apoptosis and in decreased expression of VEGF in leukaemic cells. CONCLUSIONS: mTOR-targeting drugs exert antileukaemic effects on AML cells in vitro through multiple actions, including direct inhibition of proliferation, induction of apoptosis and suppression of VEGF. Based on this study and other studies, mTOR can be regarded as a potential drug target in AML.

A novel cluster of lectin-like receptor genes expressed in monocytic, dendritic and endothelial cells maps close to the NK receptor genes in the human NK gene complex.

The NK gene complex is a region on human chromosome 12 containing several families of lectin-like genes including the CD94 and NKG2 NK receptor genes. We report here that the region telomeric of CD94 contains in addition to the LOX-1 gene the novel human DECTIN-1 and the CLEC-1 and CLEC-2 genes within about 100 kb. Sequence similarities and chromosomal arrangement suggest that these genes form a separate subfamily of lectin-like genes within the NK gene complex. DECTIN-1 is selectively expressed in dendritic cells and to a lower extent in monocytes and macrophages. mRNA forms with and without a stalk exon are observed. During functional maturation of dendritic cells the level of DECTIN-1 mRNA is down-regulated several-fold. CLEC-1 is found to be not only expressed in dendritic cells, but also in endothelial cells and in the latter aspect resembles the LOX-1 gene. Whereas recombinant full-length DECTIN-1 and LOX-1 are transported to the cell surface, CLEC-1 proteins accumulate in perinuclear compartments. We propose that this family of lectin-like genes encodes receptors with important immune and/or scavenger functions in monocytic, dendritic and endothelial cells.

Pulmonary intravascular lymphomatosis: presentation with dyspnea and air trapping.

Intravascular lymphomatosis (IVL) is a rare lymphoid neoplasm that is typically of B-cell lineage and characterized by proliferation of malignant cells within small arterioles, capillaries, and venules. We report a patient with pulmonary IVL who presented clinically with progressive dyspnea, fever, and a dry cough. Pulmonary function tests revealed a marked decrease in diffusion capacity with airflow obstruction and severe air trapping. High-resolution CT (HRCT) of the chest with inspiratory and expiratory images revealed mosaic attenuation consistent with air trapping. Transbronchial biopsies revealed the diagnosis of IVL with capillary expansion in the alveolar and peribronchiolar interstitial tissue. IVL should be considered in the differential diagnosis of a patient with an interstitial lung disease, air trapping on pulmonary function tests, and mosaic attenuation on HRCT. Transbronchial biopsies may be the initial diagnostic procedure of choice.

Effect of nitric oxide synthase inhibitor on diaphragmatic function after resistive loading.

We studied the effect of a nitric oxide synthase inhibitor, Nomega-Nitro-L-arginine-methyl-ester (L-NAME), on in vitro diphragmatic function both at rest (control) or after inspiratory resistive loading (IRL). Sprague-Dawley rats were anesthetized, instrumented, and then the following experimental groups: (1) controls; (2) L-NAME (100 mg/kg/body weight intravenously alone); (3) IRL alone; and (4) L-NAME + IRL. The IRL protocol consisted of applying a variable resistor to the inspiratory limb of a two-way valve at 70% of maximal airway pressure until apnea. After the experiment, the animals were sacrificed and diaphragmatic strips were obtained for activity of constitutive nitric oxide synthase (cNOS) and measurements of in vitro contractile properties: tetanic (Po) and twitch tensions (Pt). cNOS activity was significantly decreased in the L-NAME and L-NAME + IRL groups (P < or = 0.05) as compared with control and IRL groups. L-NAME alone did not affect Po or Pt. However, in both IRL groups, with and without was a significant decrease in Po and Pt. This reduction was comparable in both groups. In summary, our data showed that L-NAME resulted in a significant decrease cNOS activity, but in vitro contractility was impaired.

High-frequency oscillatory ventilation for adult respiratory distress syndrome--a pilot study.

OBJECTIVE: To evaluate the safety and effectiveness of high-frequency oscillatory ventilation using a protocol designed to recruit and maintain optimal lung volume in patients with severe adult respiratory distress syndrome (ARDS). SETTING: Surgical and medical intensive care units in a tertiary care, military teaching hospital. DESIGN: A prospective, clinical study. PATIENTS: Seventeen patients, 17 yrs to 83 yrs of age, with severe ARDS (Lung Injury Score of 3.81 +/- 0.23) failing inverse ratio mechanical conventional ventilation (PaO2/FiO2 ratio of 68.6 +/- 21.6, peak inspiratory pressure of 54.3 +/- 12.7 cm H2O, positive end-expiratory pressure of 18.2 +/- 6.9 cm H2O). INTERVENTIONS: High-frequency oscillatory ventilation was instituted after varying periods of conventional ventilation (5.12 +/- 4.3 days). We employed lung volume recruitment strategy that consisted of incremental increases in mean airway pressure to achieve a PaO2 of > or = 60 torr (> or = 8.0 kPa), with an FiO2 of < or = 0.6. MEASUREMENTS AND MAIN RESULTS: High-frequency oscillator ventilator settings (FiO2, mean airway pressure, pressure amplitude of oscillation [delta P] frequency) and hemodynamic parameters (cardiac output, oxygen delivery [DO2]), mean systemic and pulmonary arterial pressures, and the oxygenation index (oxygenation index = [FiO2 x mean airway pressure x 100]/PaO2) were monitored during the transition to high-frequency oscillatory ventilation and throughout the course of the high-frequency protocol. Thirteen patients demonstrated improved gas exchange and an overall improvement in PaO2/FiO2 ratio (p < .02). Reductions in the oxygenation index (p < .01) and FiO2 (p < .02) at 12, 24, and 48 hrs after starting high-frequency oscillatory ventilation were observed. No significant compromise in cardiac output or DO2 was observed, despite a significant increase in mean airway pressure (31.2 +/- 10.3 to 34.0 +/- 6.7 cm H2O, p < .05) on high-frequency oscillatory ventilation. The overall survival rate at 30 days was 47%. A greater number of pretreatment days on conventional ventilation (p < .009) and an entry oxygenation index of > 47 (sensitivity 100%, specificity 100%) were associated with mortality. CONCLUSIONS: High-frequency oscillatory ventilation is both safe and effective in adult patients with severe ARDS failing conventional ventilation. A lung volume recruitment strategy during high-frequency oscillatory ventilation produced improved gas exchange without a compromise in DO2. These results are encouraging and support the need for a prospective, randomized trial of algorithm-controlled conventional ventilation vs. high-frequency oscillatory ventilation for adults with severe ARDS.

Interleukin-4 and interferon-gamma discordantly regulate collagen biosynthesis by functionally distinct lung fibroblast subsets.

Pulmonary fibrosis is a potentially fatal consequence of treatments for malignancy and is an increasing problem in bone marrow transplant patients and in cases of allogenic lung transplant. The fibrotic response is characterized by increases in lung fibroblast number and collagen synthesis. This laboratory previously isolated stable, functionally distinct, murine lung fibroblast subsets (Thy-1+ and Thy-1-) to study the contribution of fibroblast subpopulations in lung fibrosis. The fibroblast fibrotic response may be induced by cytokines secreted by infiltrating cells such as T lymphocytes and mast cells. In the current study two key regulatory cytokines, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), were investigated for their effects on the collagen synthesis of murine lung fibroblast subsets. IL-4 and IFN-gamma are putatively characterized as fibrogenic and anti-fibrogenic cytokines, respectively, and are found in repairing lung tissue. Stimulation with recombinant IL-4 induced a100% increase in total collagen production only by Thy-1+ fibroblasts. Types I and III collagen mRNA were increased in the Thy-1+ fibroblasts, unlike the Thy-1- subset. In contrast, IFN-gamma decreased constitutive collagen production by more than 50% in Thy-1+ and Thy-1- fibroblasts. Interestingly, the two subsets utilized their collagen production machinery (collagenase, tissue inhibitors of metalloproteinases) differently to further regulate collagen turnover in response to IL-4 and IFN-gamma. Overall, our data support the hypothesis that IL-4 is fibrogenic and IFN-gamma is anti-fibrogenic. Moreover, selective expansion of IL-4 responsive fibroblasts (e.g., Thy-1+) may be important in the transition from repair to chronic fibrosis. In addition, these data suggest that an inflammatory response dominated by IL-4-producing Th2 lymphocytes and/or mast cells will promote fibrosis development.

Tracheal gas insufflation combined with high-frequency oscillatory ventilation.

OBJECTIVES: To determine the efficacy of tracheal insufflation delivered by two different catheter designs on CO2 elimination when used in conjunction with high-frequency oscillatory ventilation. DESIGN: A nonrandomized before and after trial. Each animal served as his own control. SUBJECTS: Ten mongrel dogs weighing 20.9 +/- 1.9 kg. Four animals were assigned to a normal lung group and six animals underwent lung injury by large volume saline lavage. INTERVENTION: Permissive hypercapnia was allowed to occur by selecting oscillator settings that would lead to alveolar hypoventilation. Proximal mean airway pressure was kept constant. Tracheal gas was insufflated at 1 cm above the carina for 30 min periods at gas flows of 5 to 15 L/min. MEASUREMENTS AND MAIN RESULTS: Carinal pressure, hemodynamic parameters (cardiac output, mean arterial pressure, pulmonary artery occlusion pressure), and gas exchange parameters (PaCO2, PaO2, PaO2/FIO2, shunt fraction, D O2) were measured. For the normal dogs, at catheter flow of 15 L/min; the forward thrust catheter increased carinal pressure and Pao2/FIO2 BY 30% (p<.003) and 105% (p<.005), respectively. The forward thrust catheter reduced Paco2 by 40% (p<.04). The reverse thrust catheter increased PaO2/FIO2 by 102% (p<.001) and decreased pressure and PaCO2 by 44% (p<.001) and 34% (p<.003), respectively. For the injured dogs, at catheter flow rate of 15 L/min, the forward thrust catheter increased carinal pressure, PaO2, and PaO2/FIO2 by 6% (p<.001), 23% (p<.001), and 24% (p<.02), respectively. The forward thrust catheter reduced PaCO2 by 29% (p<.002). The reverse thrust catheter increased PaO2 and PaO2/FIO2 both by 11% (p<.02) and reduced carinal pressure and PaCO2 by 23% (p<.001) and 18% (p<.002), respectively. CONCLUSIONS: Tracheal gas insufflation is capable of improving oxygenation and ventilation in acute lung injury when combined with high-frequency oscillatory ventilation. The addition of this second gas flow at the level of the carina raises or lowers distal airway pressure, the magnitude of which is dependent on the direction and rate of gas flow. The beneficial effects of tracheal gas insufflation may be tempered by the long-term effects of altering distal airway pressure; lowering distal airway pressure may lead to atelectasis, whereas raising distal airway pressure may lead to an auto-positive end-expiratory pressure (auto-PEEP) effect.

Subsets of murine lung fibroblasts express membrane-bound and soluble IL-4 receptors. Role of IL-4 in enhancing fibroblast proliferation and collagen synthesis.

The purpose of this study was to determine whether or not membrane-bound and soluble forms of IL-4 receptors are expressed by isolated subsets of murine lung fibroblasts and to evaluate the potential functional consequences of IL-4 receptor triggering. Recent studies demonstrate that IL-4-synthesizing Th2 cells and mast cells are present in increased numbers in the lung during inflammation and fibrosis, suggesting that IL-4 may play a regulatory role in these events. We hypothesize that pulmonary fibroblasts and subsets thereof are intimately involved in this inflammatory response and that IL-4 is an active player in stimulating fibroblast collagen synthesis and hyperproliferation, creating a fibrotic environment in the lung. The fibroblast subsets used in these experiments differ not only in surface expression of the thymocyte-1 (Thy-1) Ag, but also in function and morphology. We now report the novel finding that IL-4 receptors are present at discordant levels on Thy-1+ and Thy-1- lung fibroblasts. IL-4R level and affinity were analyzed using a monoclonal anti-IL-4R Ab and equilibrium binding analysis with 125I-labeled IL-4. Reverse transcriptase PCR demonstrated the presence of mRNA for membrane-bound and soluble IL-4R. Lung fibroblast subsets secrete soluble IL-4R protein at dramatically different levels, as detected by an ELISA. Thy-1+ and Thy-1- lung fibroblasts were treated with IL-4 to determine whether this cytokine was profibrotic. Thy-1+ fibroblasts responded to IL-4 by proliferating and up-regulating collagen production. In contrast, Thy-1- fibroblasts proliferate to a lesser degree than Thy-1+ fibroblasts and were not stimulated to secrete increased levels of collagen. Overall, these results suggest that elevated levels of IL-4 at a site of injury could result in the development of fibrosis by enhancing fibroblast subset proliferation and collagen synthesis.

Pulmonary eosinophils express HLA-DR in chronic eosinophilic pneumonia.

BACKGROUND: Chronic eosinophilic pneumonia is a rare idiopathic disorder. role the eosinophil plays in the pathogenesis of this disease is unknown. The recent finding that nature eosinophils can express the class II major histocompatibility complex molecule HLA-DR suggests an immunologic role, perhaps through antigen presentation. The purpose of this research was to determine whether lung-derived eosinophils exhibit in vivo expression of HLA-DR. METHODS: Eosinophils were obtained simultaneously from bronchoalveolar lavage and peripheral blood from a 59-year-old woman with asthma and chronic eosinophilic pneumonia. Eosinophil-enriched aliquots of peripheral blood were cocultured with human lung fibroblasts (with or without additional granulocyte-macrophage colony-stimulating factor). The percentage of cells expressing HLA-DR was quantitated by flow cytometric analysis. RESULTS: Eosinophils derived from bronchoalveolar lavage displayed in vivo expression of HLA-DR (86%) in contrast to those from peripheral blood (7%), suggesting compartmentalization of eosinophil activation within the lung. Peripheral blood eosinophils retained the capacity for HLA-DR expression when coincubated with lung fibroblasts (83%) with augmentation by granulocyte-macrophage colony-stimulating factor (93%). CONCLUSION: These data demonstrate that lung eosinophil HLA-DR expression occurs in vivo; it may contribute to the pathogenesis of inflammatory lung injury.

Induction of human neonatal pulmonary fibroblast cytokines by hyperoxia and Ureaplasma urealyticum.

Multiple insults may induce bronchopulmonary dysplasia (BPD) in premature infants, including the recently reported association of BPD with neonatal Ureaplasma urealyticum colonization. One mechanism of damage could involve stimulation of proinflammatory cytokine release from pulmonary fibroblasts. We therefore compared the effects of U. urealyticum, oxygen, and lipopolysaccharide (LPS) on the release of interleukin (IL)-1 beta, IL-6, and IL-8 from neonatal fibroblasts. Fibroblasts were grown in multiwell plates and divided into the following experimental conditions: fibroblasts alone, fibroblasts plus U. urealyticum (10,000 cfu/mL), and fibroblasts plus LPS (2 micrograms/mL). Plates were then exposed to room air or hyperoxia for 48 h, and supernatants were assayed for IL. U. urealyticum-infected fibroblasts produced a significant increase in IL-6 (P < .05) and a dramatic increase in IL-8 (P < .05) that was independent of hyperoxic exposure and significantly increased over that produced by LPS or hyperoxia alone. U. urealyticum is a potent inducer of fibroblast cytokine release in vitro and may contribute to the development of BPD.

Comparison of the Wang 19-gauge and 22-gauge needles in the mediastinal staging of lung cancer.

Transbronchial needle aspiration (TBNA) offers the unique opportunity to pathologically stage patients with lung cancer at the time of diagnostic bronchoscopy. The purpose of this study was to compare the staging sensitivities of the Wang 22-gauge and 19-gauge needles. We studied 64 patients with bronchogenic carcinoma and mediastinal adenopathy. Before bronchoscopy each patient underwent chest CT. Three to four aspirates were obtained with each needle from endotracheal sites adjacent to paratracheal lymphadenopathy. In 47 patients malignant mediastinal adenopathy was confirmed by the 19-gauge needle. A total of 29 patients had malignant 22-gauge needle aspirates. Of the 64 patients, 9 had benign, reactive mediastinal lymph nodes. There were 20 patients in whom only the 19-gauge needle demonstrated malignancy and 2 patients with malignant 22-gauge needle aspirates as the sole identifier of paratracheal malignancy. As a staging tool, the 19-gauge needle was significantly more sensitive than the 22-gauge needle, 85.5 versus 52.7% (p = 0.0001). Overall, in 49 of 55 patients (89.1%) with malignant mediastinal lymphadenopathy paratracheal tumor was confirmed by TBNA. The 19-gauge TBNA staging of the mediastinum is an effective, safe, and cost-saving alternative to surgical mediastinal exploration that can be performed during initial diagnostic bronchoscopy.

Differential collagen and fibronectin production by Thy 1+ and Thy 1- lung fibroblast subpopulations.

Pulmonary fibrosis resulting from diverse etiologies is characterized by proliferation of fibroblasts and excessive accumulation of interstitial collagen. Whether fibrosis is associated with selective expansion of fibroblast subpopulations differing in amounts or types of collagens synthesized is unknown. We have previously isolated lines and clones of normal murine lung fibroblasts based on the presence of the Thy 1 surface antigen. These subpopulations differ in morphology, growth characteristics, and display of class II major histocompatibility complex antigens (R.P. Phipps, D.P. Penney, P. Keng, H. Quill, A. Paxhia, S. Derdak, and M. E. Felch. Am. J. Respir. Cell Mol. Biol. 1: 65-74, 1989). We evaluated the amounts and types of collagen and fibronectin synthesized by Thy 1+ (Fib2-T-3+) and Thy 1- (Fib2-T-4-) lung fibroblast lines and clones. Thy 1+ fibroblast line synthesized two- to threefold more collagen and noncollagen protein than the Thy 1- line. In contrast, both the Thy 1+ and Thy 1- lines synthesized similar amounts of fibronectin. Thy 1+ and Thy 1- lines and clones expressed mRNA for alpha 1(I)-and alpha 1(III)-procollagen and synthesized both types (predominantly type I and lesser amounts of type III) of collagen, protein, and mRNA. The fibroblast clones varied significantly in total collagen and fibronectin production, with one Thy 1- clone (D3) synthesizing the largest amount of collagen but relatively little fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphologic and functional characteristics of subpopulations of murine lung fibroblasts grown in vitro.

Fibrotic development is a common response of the lung to toxic or deleterious insult. For example, the lung is the dose-limiting organ for irradiation of the thorax for primary or metastatic lesions, due in large part to latent fibrosis. The development of the fibrotic response reflects a cascade of cell-cell and cell-extracellular matrix interactions, the ultimate target of which is the fibroblast. There is increasing evidence of subpopulations of pulmonary fibroblasts, which may have differing roles in either the initiation or progression of fibrosis. Recently we described two fibroblast subpopulations from the murine lung, which differ in the presence or absence of the membrane antigen Thy-1 (Phipps et al., 1989). These Thy-1+ and Thy-1- subpopulations are stable and differ in certain functions, such as the production of cytokines and the display of Class II MHC antigens. To determine the morphologic development of the two subpopulations and their growth characteristics in vitro, cultures of the two cell subtypes were prepared for transmission and scanning electron microscopy at varying stages of growth. Thy-1+ fibroblasts are more spindle-shaped, contain intracellular lipid, exhibit abundant cell-cell contacts, and are capable of secreting large amounts of collagen and modest amounts of fibronectin. Thy-1- fibroblasts are more rounded and spread, contain no intracellular lipid droplets, possess more intracellular microfilaments and microtubules, and synthesize less collagen and more fibronectin than do Thy-1+ cells. There are no significant differences between the two subpopulations insofar as growth rates are concerned, but Thy-1+ fibroblasts possess an additional DNA peak during periods of early growth.

Characterization of two major populations of lung fibroblasts: distinguishing morphology and discordant display of Thy 1 and class II MHC.

We have determined that murine lung fibroblasts are divisible into two major subpopulations based on expression of Thy 1. Twenty-four to fifty-three percent of freshly isolated lung cells displayed Thy 1 and were separated using FACS into Thy 1+ and Thy 1- fractions for morphologic examination. Analysis by electron microscopy revealed that both the Thy 1+ and Thy 1- fractions contained fibroblasts. Freshly isolated lung cells cultured for 2 wk consisted of greater than 95% fibroblasts, with 28 to 49% displaying Thy 1. These cells were sorted by FACS into Thy 1+ and Thy 1- lines that maintained a stable phenotype over many weeks and that were used as a source to obtain stable fibroblast clones. Adherent pulmonary fibroblasts are not phagocytic and lack the markers of macrophages, dendritic cells, B lymphocytes, and T lymphocytes (with the exception of Thy 1). Interestingly, the Thy 1- fibroblasts spread more and contained a more extensive microfilament and microtubule network than did the spindly and often lipid-containing Thy 1+ population. Both populations of fibroblasts synthesized collagen. Class I MHC expression was very low on Thy 1+ and Thy 1- fibroblasts, but high levels were displayed after gamma-IFN treatment. Most exciting was the unexpected finding that only the Thy 1- lines and clones displayed class II MHC (Ia) in response to treatment with gamma-IFN. Moreover, only the Thy 1- fraction (gamma-IFN-treated) presented antigen to T lymphocyte clones, an observation that suggests that this subset of cells may be involved primarily in promoting chronic lung inflammation, which is associated with developing fibrosis. Thus, two populations of pulmonary fibroblasts exist, defined by the expression of Thy 1, distinguishing morphology, inducibility for Ia expression, and antigen-presenting function. It should now be possible, using these characteristics, to ascertain the role of pulmonary fibroblast subpopulations in developing fibrosis.