ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression

Tightly regulated expression of the transcription factor PU.1 is crucial for normal hematopoiesis. PU.1 knockdown mice develop acute myeloid leukemia (AML), and PU.1 mutations have been observed in some populations of patients with AML. Here we found that conditional expression of promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA), the protein encoded by the t(15;17) translocation found in acute promyelocytic leukemia (APL), suppressed PU.1 expression, while treatment of APL cell lines and primary cells with all-trans retinoic acid (ATRA) restored PU.1 expression and induced neutrophil differentiation. ATRA-induced activation was mediated by a region in the PU.1 promoter to which CEBPB and OCT-1 binding were induced. Finally, conditional expression of PU.1 in human APL cells was sufficient to trigger neutrophil differentiation, whereas reduction of PU.1 by small interfering RNA (siRNA) blocked ATRA-induced neutrophil differentiation. This is the first report to show that PU.1 is suppressed in acute promyelocytic leukemia, and that ATRA restores PU.1 expression in cells harboring t(15;17).

Increased uptake of 111In-octreotide in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by an uncontrolled accumulation and activation of lung fibroblasts. A modulation of fibroblast activation has been observed in various systems with octreotide, a synthetic somatostatin analog with strong affinity for the somatostatin receptor subtype 2 (sst2). One aim of our study was to evaluate the expression of somatostatin receptors in the lungs of patients with IPF. A second aim was to evaluate the relationship between 111In-octreotide uptake and the effect of pulmonary fibrosis as assessed by lung function tests and parameters and by radiologic findings. METHODS: We investigated 11 patients with IPF, 6 patients with pulmonary fibrosis associated with systemic sclerosis (SSc), and 19 patients with disease not of the lung (control patients). The expression of somatostatin receptors was evaluated in vivo using 111In-octreotide scintigraphy. We evaluated the relationship between 111In-octreotide uptake and the activity of pulmonary fibrosis as assessed by lung function tests, bronchoalveolar lavage (BAL) cellularity, and high-resolution CT (HRCT) of the chest. Planar images and thoracic SPECT (24 h) were performed after injection of 222 MBq of 111In-octreotide. Lung uptake was quantified using the lung-to-background ratio (L/B). In addition, the expression of sst2 was evaluated in vitro, in frozen lung-tissue samples using autoradiography, and in human cultures of lung fibroblasts using a ligand-binding assay. RESULTS: Compared with lung uptake in control patients (median L/B, 1.25; range, 1.14-1.49), lung uptake was increased in all 11 IPF patients (median L/B, 2.63; range, 1.59-3.13; P < 0.001) and in 4 of 6 SSc patients (median L/B, 1.68; range, 1.42-2.16). The L/B was lower in SSc patients than in IPF patients (P = 0.011). Increased uptake correlated with the alteration of lung function (carbon monoxide diffusing capacity [rho = -0.655; P = 0.038], diffusing capacity for carbon monoxide and alveolar volume ratio [rho = -0.627; P = 0.047], vital capacity [rho = -0.609; P = 0.054], and total lung capacity [rho = -0.598; P = 0.058]) and with the intensity of alveolitis (total BAL cellularity [rho = 0.756; P = 0.045], neutrophil counts [rho = 0.738; P = 0.05]), and HRCT fibrosis score (rho = 0.673; P = 0.007). Autoradiography suggested that vascular structures were a prominent binding site. Lung fibroblasts expressed somatostatin receptors in vitro as measured by binding assay. CONCLUSION: Our preliminary results identified an increased expression of sst2 in (mainly idiopathic) pulmonary fibrosis. Lung uptake correlates with the alteration of lung function and with the intensity of alveolitis.

Increased ENA-78 in the follicular fluid of patients with endometriosis

BACKGROUND: It is known that endometriosis is an inflammatory disease and those patients seem to have lower pregnancy rates. The aim of the study was to investigate the concentrations of chemokines and proinflammatory cytokines in the follicular fluid of patients with and without endometriosis. METHODS: Follicular aspiration, recovering follicular fluid during assisted reproductive treatment, follicular fluid storage and analysis of chemokines and proinflammatory cytokines were carried out. Tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, interleukin-8, interleukin-15, leukemia inhibitory factor, epithelial neutrophil-activating peptide 78, regulated upon activation, normal T-cell expressed and secreted, and growth-regulated oncogene-alpha were analyzed in the follicular fluid and compared between women with (n =47) and without endometriosis (n = 279). RESULTS: The above cytokines were detected in the follicular fluid samples. Epithelial neutrophil-activating peptide 78 levels were significantly higher in follicular fluid from endometriosis patients than from controls (p = 0.008). Increases (to twice the control level) were also observed for tumor necrosis factor-alpha and for interleukin-6. CONCLUSIONS: Increased follicular fluid levels of epithelial neutrophil-activating peptide 78, tumor necrosis factor-alpha and interleukin-6 indicate that these cytokines may influence oocyte quality and fecundability of women with endometriosis by deteriorating the microenvironment in the human follicle.

Quantitative magnetic resonance imaging of enzymatically induced degradation of the nucleus pulposus of intervertebral discs

STUDY DESIGN: The structural integrity of the nucleus pulposus (NP) of intervertebral discs was targeted by enzyme-specific degradations to correlate their effects to the magnetic resonance (MR) signal. OBJECTIVE: To develop quantitative MR imaging as an accurate and noninvasive diagnostic tool to better understand and treat disc degeneration. SUMMARY OF BACKGROUND DATA: Quantitative MR analysis has been previously shown to reflect not only the disc matrix composition, but also the structural integrity of the disc matrix. Further work is required to identify the contribution of the structural integrity versus the matrix composition to the MR signal. METHODS: The bovine coccygeal NPs were injected with either enzyme or buffer, incubated at 37 degrees C as static, unloaded and closed 3-disc segments, and analyzed by a 1.5-Tesla MR scanner to measure MR parameters. RESULTS: Collagenase degradation of the NP significantly decreased the relaxation times, slightly decreased the magnetization transfer ratio, and slightly increased the apparent diffusion coefficient. Targeting the proteoglycan and/or hyaluronan integrity by trypsin and hyaluronidase did not significantly affect the MR parameters, except for an increase in the apparent diffusion coefficient of the disc after trypsin treatment. CONCLUSIONS: Our results demonstrate that changes in the structural integrity of matrix proteins can be assessed by quantitative MR.

Peri-implant inflammation defined by the implant-abutment interface

An implant-abutment interface at the alveolar bone crest is associated with sustained peri-implant inflammation; however, whether magnitude of inflammation is proportionally dependent upon interface position remains unknown. This study compared the distribution and density of inflammatory cells surrounding implants with a supracrestal, crestal, or subcrestal implant-abutment interface. All implants developed a similar pattern of peri-implant inflammation: neutrophilic polymorphonuclear leukocytes (neutrophils) maximally accumulated at or immediately coronal to the interface. However, peri-implant neutrophil accrual increased progressively as the implant-abutment interface depth increased, i.e., subcrestal interfaces promoted a significantly greater maximum density of neutrophils than did supracrestal interfaces (10,512 +/- 691 vs. 2398 +/- 1077 neutrophils/mm(2)). Moreover, inflammatory cell accumulation below the original bone crest was significantly correlated with bone loss. Thus, the implant-abutment interface dictates the intensity and location of peri-implant inflammatory cell accumulation, a potential contributing component in the extent of implant-associated alveolar bone loss.

Platelet chemokines and their receptors: what is their relevance to platelet storage and transfusion practice?

The role of platelets as inflammatory cells is demonstrated by the fact that they can release many growth factors and inflammatory mediators, including chemokines, when they are activated. The best known platelet chemokine family members are platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG), which are synthesized in megakaryocytes, stored as preformed proteins in alpha-granules and released from activated platelets. However, platelets also contain many other chemokines such as interleukin-8 (IL-8), growth-regulating oncogene-alpha(GRO-alpha), epithelial neutrophil-activating protein 78 (ENA-78), regulated on activation normal T expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), and monocyte chemotactic protein-3 (MCP-3). They also express chemokine receptors such as CCR4, CXCR4, CCR1 and CCR3. Platelet activation is a feature of many inflammatory diseases such as heparin-induced thrombocytopenia, acquired immunodeficiency syndrome, and congestive heart failure. Substantial amounts of PF4, beta-TG and RANTES are released from platelets on activation, which may occur during storage. Although very few data are available on the in vivo effects of transfused chemokines, it has been suggested that the high incidence of adverse reactions often observed after platelet transfusions may be attributed to the chemokines present in the plasma of stored platelet concentrates.

Establishment and characterization of a primary canine duodenal epithelial cell culture

Many mechanisms involved in the pathogenesis of chronic enteropathies or host-pathogen interactions in canine intestine have not been elucidated so far. Next to the clinical and in vivo research tools, an in vitro model of canine intestinal cell culture would be very helpful for studies at the cellular level. Therefore, the purpose of this study was to establish and characterize a primary canine duodenal epithelial cell culture. Neonatal duodenum was disrupted with trypsin-ethylenediaminetetraacetic acid (EDTA) and the mucosa scraped off and digested with collagenase and dispase. After centrifugation on a 2% sorbitol gradient, the cells were incubated at 37 degrees C in OptiMEM supplemented with Primocin, epidermal growth factor, insulin, hydrocortisone, and 10% fetal calf serum (FCS). After 24 h, the FCS concentration was reduced to 2.5%, and the temperature decreased to 33 degrees C. With this method, the cultures were growing to confluent monolayers within 5-6 d and remained viable for an average of 2 wk. Their epithelial nature was confirmed by electron microscopy and immunofluorescence staining using antibodies directed against specific cytokeratins, desmosomes, and tight junctions. The intestinal cells proliferated, as evidenced by immunolabeling with a Ki-67 antibody, and cryptal cell subpopulations could be identified. Furthermore, alkaline phosphatase and sucrase activity were detected.

Suppression of transforming growth factor beta signalling aborts caerulein induced pancreatitis and eliminates restricted stimulation at high caerulein concentrations

BACKGROUND: Transforming growth factors betas (TGF-betas) are implicated in pancreatic tissue repair but their role in acute pancreatitis is not known. To determine whether endogenous TGF-betas modulate the course of caerulein induced acute pancreatitis, caerulein was administered to wild-type (FVB-/-) and transgenic mice that are heterozygous (FVB+/-) for expression of a dominant negative type II TGF-beta receptor. METHODS: After 7 hourly supramaximal injections of caerulein, the pancreas was evaluated histologically and serum was assayed for amylase and lipase levels. Next, the effects of caerulein on amylase secretion were determined in mouse pancreatic acini, and cholecystokinin (CCK) receptor expression was assessed. RESULTS: The normal mouse pancreas was devoid of inflammatory cells whereas the pancreas from transgenic mice contained lymphocytic infiltrates. Caerulein injection in wild-type mice resulted in 6- and 36-fold increases in serum amylase and lipase levels, respectively, increased serum trypsinogen activation peptide (TAP) levels, gross oedema and a marked inflammatory response in the pancreas that consisted mainly of neutrophils and macrophages. By contrast, FVB+/- mice exhibited minimal alterations in response to caerulein with attenuated neutrophil-macrophage infiltrates. Moreover, acini from FVB+/- mice did not exhibit restricted stimulation at high caerulein concentrations, even though CCK receptor mRNA levels were not decreased. CONCLUSION: Our findings indicate that a functional TGF-beta signalling pathway may be required for caerulein to induce acute pancreatitis and for the CCK receptor to induce acinar cell damage at high ligand concentrations. Our results also support the concept that restricted stimulation at high caerulein concentrations contributes to the ability of caerulein to induce acute pancreatitis.

Efalizumab-associated papular psoriasis

BACKGROUND: Efalizumab is a human anti-CD11a monoclonal antibody used in the treatment of patients with moderate to severe plaque psoriasis. Some of the patients develop new papular lesions during treatment, which are predominantly located in the flexural regions. OBSERVATION: Four patients with recalcitrant psoriasis undergoing treatment with efalizumab presented with erythematous, partly scaly papules and small plaques on previously unaffected areas after 4 to 10 weeks of efalizumab therapy. Tissue sections of biopsy specimens were stained with hematoxylin-eosin, and immunohistochemical staining was performed using monoclonal antibodies against CD3, CD4, CD8, T-cell-restricted intracellular antigen 1, granzyme B, neutrophil elastase, CD68, CD1a, CD11c, HLA-DR, CD25, CD20, and CD56. Histopathological and immunohistochemical examination of the lesions showed features consistent with psoriasis and activation of various leukocyte subtypes including T cells, dendritic cells, macrophages, and neutrophils. CONCLUSIONS: Papular eruptions appearing during efalizumab therapy represent new psoriatic lesions and could be referred to as efalizumab-associated papular psoriasis (EAPP). They usually do not necessitate termination of efalizumab therapy and may optionally be treated with topical corticosteroids. Dermatologists should be aware of these lesions and inform their patients accordingly.

Autophagic-like cell death in neutrophils induced by autoantibodies

Human neutrophils undergo autophagic-like cell death following Sialic acid binding immunoglobulin-like lectin-9 (Siglec-9) ligation and concurrent stimulation with certain, but not all, neutrophil survival cytokines. Caspase inhibition by these cytokines is required, but is not sufficient, to trigger this particular form of cell death. Additional mechanisms may involve reactive oxygen species (ROS), and blocking of ROS or prevention of ROS production prevents autophagic-like neutrophil death. Interestingly, human intravenous immunoglobulin (IVIg) preparations contain natural anti-Siglec-9 autoantibodies, which are able to ligate Siglec-9 on neutrophils and induce autophagic-like cell death in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and some other survival cytokines. Here, we discuss the pathophysiological and therapeutic implications of these recent findings.

Airway remodelling in children with cystic fibrosis

BACKGROUND: The relationship between airway structural changes and inflammation is unclear in early cystic fibrosis (CF) lung disease. A study was undertaken to determine changes in airway remodelling in children with CF compared with appropriate disease and healthy controls. METHODS: Bronchoalveolar lavage and endobronchial biopsy were performed in a cross-sectional study of 43 children with CF (aged 0.3-16.8 years), 7 children with primary ciliary dyskinesia (PCD), 26 with chronic respiratory symptoms (CRS) investigated for recurrent infection and/or cough and 7 control children with no lower airway symptoms. Inflammatory cells, cytokines, proteases and matrix constituents were measured in bronchoalveolar lavage fluid (BALF). Reticular basement membrane (RBM) thickness was measured on biopsy specimens using light microscopy. RESULTS: Increased concentrations of elastin, glycosaminoglycans and collagen were found in BALF from children with CF compared with the CRS group and controls, each correlating positively with age, neutrophil count and proteases (elastase activity and matrix metalloproteinase-9 (MMP-9) concentration). There were significant negative correlations between certain of these and pulmonary function (forced expiratory volume in 1 s) in the CF group (elastin: r = -0.45, p<0.05; MMP-9:TIMP-1 ratio: r = -0.47, p<0.05). Median RBM thickness was greater in the CF group than in the controls (5.9 microm vs 4.0 microm, p<0.01) and correlated positively with levels of transforming growth factor-beta(1) (TGF-beta(1); r = 0.53, p = 0.01), although not with other inflammatory markers or pulmonary function. CONCLUSIONS: This study provides evidence for two forms of airway remodelling in children with CF: (1) matrix breakdown, related to inflammation, proteolysis and impaired pulmonary function, and (2) RBM thickening, related to TGF-beta(1) concentration but independent of other markers of inflammation.

Treatment of intestinal and hepatic mucormycosis in an immunocompromized child

During ALL chemotherapy, a 4-year-old patient presented with febrile neutropenia and abdominal pain. Ultrasound examinations were repeatedly normal. Computerized tomography on day 7 demonstrated appendicitis and multiple hepatic foci identified as mucormycosis (Absidia corymbifera). Successful outcome was achieved by aggressive re-surgery, long-term antifungal therapy with serum level-monitored posaconazole, and recovery of neutrophil counts. Considering the interference of posaconazole with CYP3A4, vincristine was administered during 72 hr posaconazole windows. Pediatric intestinal mucormycosis, still associated with a >70% case-fatality rate, calls for early imaging and surgery to establish the diagnosis, reduce the fungal mass, and provide a rationale for using posaconazole.

TLR expression on neutrophils at the pulmonary site of infection: TLR1/TLR2-mediated up-regulation of TLR5 expression in cystic fibrosis lung disease

Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam(3)CSK(4). Ab-blocking experiments revealed that the effect of Pam(3)CSK(4) was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.

Surfactant proteins SP-B and SP-C and their precursors in bronchoalveolar lavages from children with acute and chronic inflammatory airway disease

BACKGROUND: The surfactant proteins B (SP-B) and C (SP-C) are important for the stability and function of the alveolar surfactant film. Their involvement and down-regulation in inflammatory processes has recently been proposed, but their level during neutrophilic human airway diseases are not yet known. METHODS: We used 1D-electrophoresis and Western blotting to determine the concentrations and molecular forms of SP-B and SP-C in bronchoalveolar lavage (BAL) fluid of children with different inflammatory airway diseases. 21 children with cystic fibrosis, 15 with chronic bronchitis and 14 with pneumonia were included and compared to 14 healthy control children. RESULTS: SP-B was detected in BAL of all 64 patients, whereas SP-C was found in BAL of all but 3 children; those three BAL fluids had more than 80% neutrophils, and in two patients, who were re-lavaged later, SP-C was then present and the neutrophil count was lower. SP-B was mainly present as a dimer, SP-C as a monomer. For both qualitative and quantitative measures of SP-C and SP-B, no significant differences were observed between the four evaluated patient groups. CONCLUSION: Concentration or molecular form of SP-B and SP-C is not altered in BAL of children with different acute and chronic inflammatory lung diseases. We conclude that there is no down-regulation of SP-B and SP-C at the protein level in inflammatory processes of neutrophilic airway disease.

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.