Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships.

Loop electrosurgical excision procedure for the treatment of cervical intraepithelial neoplasia: how much excision is enough?

This is a retrospective observational study to compare outcomes in patients with cervical intraepithelial neoplasia (CIN) treated with loop electrosurgical excision procedure (LEEP) using combined ectocervical/endocervical resection vs ectocervical resection alone. We demonstrated that additional endocervical resection during loop electrosurgical excision procedure did not significantly lower the risk of subsequent recurrence compared with ectocervical resection alone, in the treatment of CIN. With current published data supporting subsequent increased adverse effects of LEEP on future obstetrical outcomes, endocervical excision should be applied selectively. We recommend that additional endocervical excision should be reserved only for patients with a strong suspicion of underlying endocervical canal involvement based on colposcopic assessment or in patients with unsatisfactory colposcopy, where it is essential to evaluate the endocervical canal.

Luteolin inhibits myelin basic protein-induced human mast cell activation and mast cell-dependent stimulation of Jurkat T cells.

BACKGROUND AND PURPOSE: Allergic inflammation and autoimmune diseases, such as atopic dermatitis, psoriasis and multiple sclerosis (MS), involve both mast cell and T-cell activation. However, possible interactions between the two and the mechanism of such activations are largely unknown. EXPERIMENTAL APPROACH: Human umbilical cord blood-derived cultured mast cells (hCBMCs) and Jurkat T cells were incubated separately or together, following activation with myelin basic protein (MBP), as well as with or without pretreatment with the flavonoid luteolin for 15 min. The supernatant fluid was assayed for inflammatory mediators released from mast cells and interleukin (IL)-2 release from Jurkat cells. KEY RESULTS: MBP (10 microM) stimulates hCBMCs to release IL-6, IL-8, transforming growth factor (TGF)-beta1, tumour necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor (VEGF), histamine and tryptase (n=6, P<0.05). Addition of mast cells to Jurkat cells activated by anti-CD3/anti-CD28 increases IL-2 release by 30-fold (n=3, P<0.05). MBP-stimulated mast cells and their supernatant fluid further increase Jurkat cell IL-2 release (n=3, P<0.05). Separation of mast cells and activated Jurkat cells by a Transwell permeable membrane inhibits Jurkat cell stimulation by 60%. Pretreatment of Jurkat cells with a TNF-neutralizing antibody reduces IL-2 release by another 40%. Luteolin pretreatment inhibits mast cell activation (n=3-6, P<0.05), Jurkat cell activation and mast cell-dependent Jurkat cell stimulation (n=3, P<0.05). CONCLUSIONS AND IMPLICATIONS: Mast cells can stimulate activated Jurkat cells. This interaction is inhibited by luteolin, suggesting that this flavonoid may be useful in the treatment of autoimmune diseases.

Intravesical nanocrystalline silver decreases experimental bladder inflammation.

PURPOSE: Interstitial cystitis is a sterile bladder inflammatory disease characterized by pelvic pain, urinary urgency and frequency. Nanocrystalline silver has anti-inflammatory properties, prompting us to investigate its effect in experimental bladder inflammation. MATERIALS AND METHODS: Nanocrystalline silver (0.01%, 0.05%, 0.1%, 0.5% or 1%) or phosphate buffered saline (Invitrogen) (0.5 ml) was introduced intravesically in Sprague-Dawley female rat (Charles River Laboratories, Wilmington, Massachusetts) bladders for 20 minutes, followed by vehicle or protamine sulfate (10 mg/ml for 30 minutes) and lipopolysaccharide (Sigma) (2 mg/ml for 45 minutes). Urine was collected throughout for histamine assay. The catheter was removed, the rat was returned to its cage and 4 hours later it was sacrificed. The bladder was harvested, minced and cultured overnight. The medium was collected for tumor necrosis factor-alpha assay. RESULTS: Mean +/- SD total urine histamine increased from 270 +/- 190 ng in 4 controls to 842 +/- 239 ng after protamine sulfate/lipopolysaccharide and it decreased to 505 +/- 187 ng in 6 animals after pretreatment with 1% nanocrystalline silver (p = 0.036). Tumor necrosis factor-alpha release in explant medium increased from 0.02 +/- 0.03 pg/mg in 6 controls to 0.28 +/- 0.15 pg/mg in 14 animals after treatment with protamine sulfate/lipopolysaccharide and it decreased to 0.12 +/- 0.11 pg/mg in 10 animals pretreated with nanocrystalline silver (p = 0.009). Nanocrystalline silver was not effective at less than 1% and at 1% alone it released 0.05 +/- 0.07 pg/mg tumor necrosis factor-alpha in 7 rats (vs phosphate buffered saline in 6, p = 0.387). Nanocrystalline silver (1%) significantly decreased bladder inflammation and mast cell activation. These effects were apparent even 4 days later. CONCLUSIONS: Intravesical administration of nanocrystalline silver (1%) decreased urine histamine, bladder tumor necrosis factor-alpha and mast cell activation without any toxic effect. This action may be useful for interstitial cystitis.

The flavonoid luteolin inhibits niacin-induced flush.

BACKGROUND AND PURPOSE: Sustained release niacin effectively lowers serum cholesterol, LDL and triglycerides, while raising HDL. However, 75% of patients experience cutaneous warmth and itching known as flush, leading to discontinuation. Acetylsalicylic acid (aspirin) reduces this flush only by about 30%, presumably through decreasing prostaglandin D2 (PGD2). We investigated whether niacin-induced flush in a rat model involves PGD2 and 5-HT, and the effect of certain flavonoids. EXPERIMENTAL APPROACH: Three skin temperature measurements from each ear were recorded with an infrared pyrometer for each time point immediately before i.p. injection with either niacin or a flavonoid. The temperature was then measured every 10 min for 60 min. KEY RESULTS: Niacin (7.5 mg per rat, equivalent to a human dose of 1750 mg per 80 kg) maximally increased ear temperature to 1.9+/-0.2 degrees C at 45 min. Quercetin and luteolin (4.3 mg per rat; 1000 mg per human), administered i.p. 45 min prior to niacin, inhibited the niacin effect by 96 and 88%, respectively. Aspirin (1.22 mg per rat; 325 mg per human) inhibited the niacin effect by only 30%. Niacin almost doubled plasma PGD2 and 5-HT, but aspirin reduced only PGD2 by 86%. In contrast, luteolin inhibited both plasma PGD2 and 5-HT levels by 100 and 67%, respectively. CONCLUSIONS AND IMPLICATIONS. Niacin-induced skin temperature increase is associated with PGD2 and 5-HT elevations in rats; luteolin may be a better inhibitor of niacin-induced flush because it blocks the rise in both mediators.

Anti-chemokine therapy for inflammatory diseases.

Chemokines are inflammatory proteins acting via G-protein coupled chemokine receptors that trigger different signaling pathways. Monocyte chemoattractant protein-1 (CCL2/MCP-1) and regulated on activation, normal T expressed and secreted (CCL5/RANTES) are the two major members of the CC chemokine beta subfamily. The roles of RANTES and MCP-1 are emerging in regulating the recruitment of inflammatory cells into tissue during inflammation. The inhibition of MCP-1 and RANTES with corresponding antibodies or other inhibitors may provide benefits in different clinical scenarios including cancer, inflammation, CNS disorders, parasitic disease, autoimmune and heart diseases. RANTES and MCP-1 may represent targets for diagnostic procedures and therapeutic intervention, and may be useful as a prognostic factor in the above diseases.

Intravesical suplatast tosilate (IPD-1151T) inhibits experimental bladder inflammation.

PURPOSE: Interstitial cystitis is a painful bladder disease characterized by urgency, frequency and variable inflammation but there is no curative therapy. Suplatast tosilate (IPD-1151T) is an immunoregulatory compound that decreases interstitial cystitis symptoms but to our knowledge its mechanism of action is unknown. We investigated the effect of intravesical IPD-1151T on mediator release from bladder explants in experimental cystitis. MATERIALS AND METHODS: A catheter was inserted into the bladder of female mice. After urine was emptied normal saline, carbachol (100 nM) or lipopolysaccharide (10 mg/ml) was introduced with or without 10-minute pretreatment with IPD-1151T. Urine was removed after 45 minutes for histamine and tumor necrosis factor-alpha assays. The bladder was removed after 4 hours, minced into 1 mm2 pieces and cultured with or without triggers overnight for mediator release. The effect of IPD-1151T was also tested on rat skin vascular permeability as well as on purified rat peritoneal mast cells and human cord blood derived mast cells. RESULTS: Carbachol significantly increased histamine release in urine (61.3% in 8 preparations, p<0.05) but not in explant medium. IPD-1151T inhibited this effect by 77%. Lipopolysaccharide induced a 350% urine histamine increase in 9 preparations (p<0.05) and a 300% tumor necrosis factor-alpha increase in explant medium. IPD-1151T inhibited the lipopolysaccharide induced medium tumor necrosis factor-alpha increase by 95% in 5 preparations (p<0.05). IPD-1151T did not inhibit rat skin vascular permeability or purified rat peritoneal mast cell activation by compound 48/80 or human cord blood derived mast cells by anti-IgE. CONCLUSIONS: IPD-1151T inhibits bladder release of histamine and tumor necrosis factor-alpha through a mechanism that does not appear to involve direct mast cell inhibition. These findings may justify a beneficial effect of IPD-1151T in interstitial cystitis.

Immunological activation of human umbilical cord blood mast cells induces tryptase secretion and interleukin-6, and histidine decarboxilase mRNA gene expression.

Allergy is the result of a complex immune cascade leading to the disregulated production of Th2 cytokines, the generation of allergen-specific IgE-producing B cells and the subsequent activation and degranulation of mast cells upon allergen challenge. Mast cell effector function significantly influences the quantity, duration and magnitude of most allergic reactions. Here, using isolated human umbilical cord blood mast cells (HUCBMC) from CD34+ cells, activated with anti-IgE (10 microg/ml) in culture, we found an augmented release of IL-6, tryptase and histamine (p < 0.01 compared with control). In addition, in these cells anti-IgE (10 microg/ml) activated the expression of histidine decarboxylase (HDC) and IL-6. In these studies we describe a new biological activity of anti-IgE in inducing histidine decarboxylase and IL-6, suggesting that this cytokine may have an important effect on allergic and inflammatory diseases mediated by mast cells. Moreover, with these data we confirm the immunoregulatory and inflammatory function of mast cells.

Progesterone inhibits mast cell secretion.

Mast cells are involved in allergic reactions, where they secrete numerous vasoactive, inflammatory and nociceptive mediators in response to immunoglobulin E (IgE) and antigen. However, they have also been implicated in inflammatory conditions, such as painful bladder syndrome/interstitial cystitis (PBS/IC), irritable bowel syndrome (IBS) and migraines, all of which occur more often in women and are exacerbated during ovulation, but are suppressed during pregnancy. Mast cells express high affinity estrogen receptors and estradiol augments their secretion, while tamoxifen inhibits it. Here we report that progesterone (100 nM), but not the structurally related cholesterol, inhibits histamine secretion from purified rat peritoneal mast cells stimulated immunologically or by substance P (SP), an effect also documented by electron microscopy. These results suggest that mast cell secretion may be regulated by progesterone and may explain the reduced symptoms of certain inflammatory conditions during pregnancy.

Acute stress and intravesical corticotropin-releasing hormone induces mast cell dependent vascular endothelial growth factor release from mouse bladder explants.

PURPOSE: Corticotropin-releasing hormone is typically released from the hypothalamus but it has proinflammatory effects outside of the brain, possibly through the activation of mast cells. These cells express corticotropin-releasing hormone receptors with selective secretion of vascular endothelial growth factor, which may be involved in the pathogenesis of painful bladder syndrome/interstitial cystitis. This condition is characterized by bladder inflammation and worsened by stress. We investigated the effect of intravesical corticotropin-releasing hormone and acute restraint stress on vascular endothelial growth factor release from mouse bladder explants and the role of mast cells. MATERIALS AND METHODS: The bladder of C57BL/6 mast cell deficient (W/W(v)) and normal congenic (+/+) female mice (Jackson Laboratories, Bar Harbor, Maine) at ages 10 to 12 weeks was catheterized using anesthesia. After emptying urine 1) normal saline or corticotropin-releasing hormone was introduced for 45 minutes, urine was collected and the mice were allowed to recover for 4 hours before sacrifice or 2) the mice were stressed by placing them in a restrainer for 4 hours before sacrifice and urine was collected 2 hours after stress. The bladder was removed 4 hours after stress and processed for corticotropin-releasing hormone immunohistochemical staining. In other experiments the bladder was removed, minced into 1 mm(2) pieces and cultured with or without corticotropin-releasing hormone overnight. Urine and medium were frozen for histamine, interleukin-6, tumor necrosis factor-alpha and vascular endothelial growth factor assay. RESULTS: Corticotropin-releasing hormone (100 nM) or acute restraint stress (4 hours) increased histamine release in urine and vascular endothelial growth factor release in medium without increasing interleukin-6 or tumor necrosis factor-alpha in the bladder explants of C57BL/6 or +/+ but not W/W(v) mice. No vascular endothelial growth factor, interleukin-6 or tumor necrosis factor-alpha was detected in urine before or after stimulation. Corticotropin-releasing hormone immunoreactivity was present in control bladders but it increased dramatically in the bladder of stressed mice. CONCLUSIONS: Intravesical corticotropin-releasing hormone and acute restraint stress induced mast cell dependent vascular endothelial growth factor release from bladder explants. These findings suggest that stress, corticotropin-releasing hormone, mast cells and vascular endothelial growth factor might participate in the pathogenesis of painful bladder syndrome/interstitial cystitis, which is worsened by stress, and provide for new therapeutic targets.

Psychologic morbidities prior to loop electrosurgical excision procedure in the treatment of cervical intraepithelial neoplasia.

The objective of this study was to assess and compare anxiety and distress in patients undergoing colposcopic examinations and loop electrosurgical excision procedure (LEEP). Patients seen for evaluation of cervical intraepithelial neoplasia (CIN) and LEEP were recruited. All patients received further teaching with respect to their abnormality right after the colposcopic evaluation by nursing staff. The Hospital anxiety and Depression Scale (HADS) and the Psychosocial Effects of Abnormal Pap Smears (PEAPS) questionnaires were used to measure and compare distress between the two groups. Linear regression models were built to identify significant predictive variables for psychologic morbidities. Twenty-one colposcopy and 20 LEEP patients participated in this study. No significant demographic differences were noted. Eighty-one percent of patients having colposcopy and 65% of those undergoing LEEP can be classified as having significant anxiety and depression based on the HADS questionnaire. Patients undergoing LEEP scored significantly better than colposcopy patients on the mean total PEAPS score and on the self-belief/cancer concern and effects on sexual relationship dimension scores. Significant psychologic morbidities exist in patients diagnosed with CIN. Face-to-face individualized education and support after colposcopy can decrease patients' distress at subsequent treatment visits.

IL-15 an immunoregulatory and anti-cancer cytokine. Recent advances.

Interleukins are mediators of inflammation, immunity and cancer. IL-15 is a cytokine produced by several leukocytes including phagocytes in response to infections and other signals that trigger innate immunity. IL-15 has many homologies to interleukin-2 (IL-2) and like IL-2, stimulates NK cells. This cytokine acts also on memory CD8+ T-cell. IL-15, therefore acts, probably through selective inhibition of tumor promoting molecules, as a new compound for the adjuvant treatment of solid tumors. In this review we propose a newly revised mechanism of interleukin 15 in inflammation and cancer.

Theophylline as "add-on" therapy in patients with delayed pressure urticaria: a prospective self-controlled study.

Delayed pressure urticaria (DPU) is a skin condition that involves the gradual development of wheals and edema at sites of physical pressure. Its pathogenesis is not clear and histamine-1 receptor (H-1R) antagonists provide only partial relief. In this prospective, clinical study, we investigated the effect of theophylline, which has a long history of benefit in allergic asthma, added to cetirizine in patients with DPU. Twenty three patients received during period 1 cetirizine (10 mg po QD) and theophylline (200 mg po BID) for 6 months, followed by period 2 of 1 month washout with only rescue medication as needed, and then by period 3 with cetirizine (10 mg QD plus placebo (BID) for 5 more months. The addition of theophylline resulted in statistically significant improvement over cetirizine alone by 2 months and continued for the duration of treatment. Treatment of cultured human mast cells with theophylline (10 microM) did not inhibit allergic histamine release, but the in vivo beneficial effect of theophylline may require significant pretreatment period to manifest itself, or may involve inhibition of other mast cell dependent mediators. A double-blind study, accompanied by serum histamine and tryptase levels, should be in order.

IL-10 subfamily members: IL-19, IL-20, IL-22, IL-24 and IL-26.

It has been reported that the CD4+ T cell is a very important source of interleukin 10 (IL-10), while CD8+ cells produce low amounts. IL-10 exerts several immune stimulating, as well as inhibitory effects. There are at least five novel human IL-10 family-related molecules: IL-19, IL-20, IL-22, IL-24, and IL-26. Activated T cells produce IL-19, IL-22 and IL-26, while IL-24 is produced by activated monocytes and T-cells. IL-20 induces cheratin proliferation and Stat-3 signal transduction pathway, while IL-22 induces acute-phase production by hepatocytes and neonatal lethality with skin abnormalities reminiscent of psoriasic lesions in humans. In addition, IL-22 mediates inflammation and binds class II cytokine receptor heterodimers IL-22 RA1/CRF2-4. This cytokine is also involved in immuno-regulatory responses. IL-26 (AK155) is a novel cytokine generated by memory cells and is involved in the transformed phenotype of human T cells after infection by herpes virus. All these new IL-10 subfamily member cytokines are strongly involved in immune regulation and inflammatory responses.