The targeting of plasmalemmal ceramide to mitochondria during apoptosis

Ceramide is a key lipid mediator of cellular processes such as differentiation, proliferation, growth arrest and apoptosis. During apoptosis, ceramide is produced within the plasma membrane. Although recent data suggest that the generation of intracellular ceramide increases mitochondrial permeability, the source of mitochondrial ceramide remains unknown. Here, we determine whether a stress-mediated plasmalemmal pool of ceramide might become available to the mitochondria of apoptotic cells. We have previously established annexin A1--a member of a family of Ca(2+) and membrane-binding proteins--to be a marker of ceramide platforms. Using fluorescently tagged annexin A1, we show that, upon its generation within the plasma membrane, ceramide self-associates into platforms that subsequently invaginate and fuse with mitochondria. An accumulation of ceramide within the mitochondria of apoptotic cells was also confirmed using a ceramide-specific antibody. Electron microscopic tomography confirmed that upon the formation of ceramide platforms, the invaginated regions of the plasma membrane extend deep into the cytoplasm forming direct physical contacts with mitochondrial outer membranes. Ceramide might thus be directly transferred from the plasma membrane to the mitochondrial outer membrane. It is conceivable that this "kiss-of-death" increases the permeability of the mitochondrial outer membrane thereby triggering apoptosis.     

Targeted deletion of integrin-linked kinase reveals a role in T-cell chemotaxis and survival

Integrin-linked kinase (ILK) is a serine/threonine kinase that is important in cell-matrix interactions and cell signaling. To examine the role of ILK in leukocyte trafficking and survival, we generated T cell-specific ILK knockouts by breeding ILK(flox/flox) mice to transgenic mice expressing Cre recombinase under control of the Lck proximal promoter. Thymic T cells from Lck-Cre(+)/ILK(flox/flox) mice had a marked reduction (>95%) in ILK protein levels. Thymic cellularity was comparable in 3- to 4-week-old mice, but a threefold diminution of thymic T cells became evident by 6 to 8 weeks of age in the T cell-specific ILK knockout mice due to increased cell death of double-positive (DP) T cells. Analysis of peripheral T cells by quantitative PCR and by breeding Lck-Cre(+)/ILK(flox/flox) mice to a YFP-transgenic reporter strain demonstrated an approximate 20-fold enrichment of ILK-competent cells, suggesting these cells have a competitive advantage in trafficking to and/or survival in peripheral lymphatic organs. We explored mechanisms related to altered cell trafficking and survival that might explain the decreases in thymic cellularity and enrichment for ILK-competent cells in the spleen and lymph nodes. We observed a >50% reduction in chemotaxis of ILK-deficient T cells to the chemokines CXCL12 (stromal cell-derived factor [SDF]-1alpha) and CCL19 (macrophage inflammatory protein [MIP]-3beta), as well as enhanced apoptosis of ILK-deficient cells upon stress. Signaling studies in ILK-deficient T cells demonstrated diminished phosphorylation of Akt on the activating phosphorylation site, Ser 473, and a concordant decrease in Akt kinase activity following stimulation with the chemokine SDF-1. Rac1 activation was also markedly diminished in ILK-deficient T cells following chemokine stimulation. These data extend the role of ILK to immune-cell trafficking and survival via modulation of Akt- and Rac-dependent substrates, and have implications for cell recruitment in both homeostatic and pathological processes.     

Trabeculectomy with mitomycin C in glaucoma associated with uveitis

The patients with uveitic glaucoma are at high risk for failure following drainage surgery because of young age of these patients, preoperative long-term control of inflammation and postoperative complications. Twenty-two trabeculectomies performed in 22 patients with uveitic glaucoma were retrospectively evaluated to analyze the effect of intraoperative application of mitomycin C (MMC). Success rates, postoperative levels of intraocular pressure (IOP) and postoperative complications were studied. After a mean follow-up of 10.6 months (range, 5-28 months), 15 patients (68.2%) achieved IOP of 21mmHg or less without antiglaucoma medications. There were statistically significant reduction in IOP postoperatively during the period studied (p < 0.001). Early postoperative complications included chorioidal detachment (9.1%), shallow anterior chamber (9.1%), hyphema (13.6%), macular edema (4.5%) and raised IOP (27.3%). Late postoperative complications included exacerbation of uveitis (4.5%), macular edema (4.5%), cataract (22.7%) and raised IOP (31.8%). The eyes with raised IOP needed additional antiglaucoma medication. The results of this retrospective and uncontrolled study suggest that intraoperative application of MMC may be a good option for enhancement of short-term trabeculectomy success rates in patients with uveitic glaucoma.     

Full-thickeness anterior blepharotomy and transpalpebral fat decompression in Graves' orbitopathy

A chief morbidity of Graves eye disease is eyelid retraction and exophthalmus. Transpalpebral orbital fat removal accomplished with full thickness anterior blepharotomy was performed in 4 patients (5 orbits). Preoperative and postoperative ocular exposure symptoms, visual acuity, upper eyelid retraction and proptosis were evaluated. In all 5 operated orbits preoperative symptoms resolved; good results were achieved from the functional and cosmetic point of view. Full-thickness anterior blepharotomy combined with fat decompression is a safe and effective surgery for patients with upper eyelid retraction and exophthalmus due to enlarged orbital fat compartment.     

Evidence of involvement of leptin and IL-6 peptides in the action of interferon-beta in secondary progressive multiple sclerosis

Leptin is a peptide hormone which acts on cells of immune system by influencing the production of cytokines. Serum leptin levels and cytokine production by peripheral blood mononuclear cells (PBMC) were measured in 18 secondary progressive multiple sclerosis (SPMS) patients under IFN-beta-1b treatment. There were no overall effects on leptin, interleukin-6 (IL-6), IL-10 and IL-12 p40 after 2, 6 and 12 months of treatment. However, leptin and IL-6 decreased after 6 and 12 months of treatment in 12 patients who did not show progression of disability. Thus, our pilot data show that the beneficial effect of IFN-beta on some SPMS patients might be associated with the reduced levels of leptin and reduced IL-6 production by PBMC.     

Ultrastructure of the contact surfaces of Passalurus ambiguus (Rudolphi, 1819) (Nematoda)

The ultrastructure of the contact surfaces (integument and intestinal wall) of the nematode Passalurus ambiguus has been studied. The integument is composed according to the scheme common for all nematodes and includes a cuticle, hypodermis and a muscular layer. The specificity is with regard to the epicuticle, the different number of the cuticular sublayers in the anterior, central and the posterior parts of the worm body and the absence of a basal cuticular membrane. The intestinal wall consists of epithelial cells with microvilli. The ultrastructural characteristics of both contact surfaces indicate their main functions--absorption, secretion, transport, protection, movement, etc.     

CD40 ligation prevents onset of tolerogenic properties in human dendritic cells treated with CTLA-4-Ig

The synthetic immunomodulator cytotoxic T lymphocyte antigen 4-Ig (CTLA-4-Ig) initiates effects in human monocyte-derived dendritic cells (DC) that rely on immunosuppressive tryptophan catabolism. However, it is unable to induce suppressive properties in DC matured by CD40 engagement. Thus, CD40-driven events may physiologically set human DC free from restraint by regulatory cells expressing surface CTLA-4.     

Pathogen adaptation to seasonal forcing and climate change

Many diverse infectious diseases exhibit seasonal dynamics. Seasonality in disease incidence has been attributed to seasonal changes in pathogen transmission rates, resulting from fluctuations in extrinsic climate factors. Multi-strain infectious diseases with strain-specific seasonal signatures, such as cholera, indicate that a range of seasonal patterns in transmission rates is possible in identical environments. We therefore consider pathogens capable of evolving their 'seasonal phenotype', a trait that determines the sensitivity of their transmission rates to environmental variability. We introduce a theoretical framework, based on adaptive dynamics, for predicting the evolution of disease dynamics in seasonal environments. Changes in the seasonality of environmental factors are one important avenue for the effects of climate change on disease. This model also provides a framework for examining these effects on pathogen evolution and associated disease dynamics. An application of this approach gives an explanation for the recent cholera strain replacement in Bangladesh, based on changes in monsoon rainfall patterns.     

Functional,radiological and biological markers of alveolitis and infections of the lower respiratory tract in patients with systemic sclerosis

Background

A progressive lung disease and a worse survival have been observed in patients with systemic sclerosis and alveolitis. The objective of this study was to define the functional, radiological and biological markers of alveolitis in SSc patients.

Methods

100 SSc patients (76 with limited and 24 with diffuse disease) underwent a multistep assessment of cardiopulmonary system: pulmonary function tests (PFTs) every 6–12 months, echocardiography, high resolution computed tomography (HRCT) and bronchoalveolar lavage (BAL), if clinically advisable. Alveolar and interstitial scores on HRCT and IL-6 plasma levels were also assessed as lung disease activity indices.

Results

90 SSc patients with abnormal PFTs and 3 with signs and/or symptoms of lung involvement and normal PFTs underwent HRCT and echocardiography. HRCT revealed evidence of fibrosis in 87 (93.5%) patients, with 55 (59.1%) showing both ground glass attenuation and fibrosis. In 42 patients who had exhibited ground glass on HRCT and consented to undergo BAL, 16 (38.1%) revealed alveolitis. 12 (75%) of these patients had restrictive lung disease (p < 0.0001) and presented diffuse skin involvement (p = 0.0009). IL-6 plasma levels were higher in patients with alveolitis than in patients without (p = 0.041). On logistic regression model the best independent predictors of alveolitis were diffuse skin involvement (OR(95%CIs):12.80(2.54–64.37)) and skin score > 14 (OR(95%CIs):7.03(1.40–34.33)). The alveolar score showed a significant correlation with IL-6 plasma levels (r = 0.36, p = 0.001) and with the skin score (r = 0.33, p = 0.001). Cultures of BAL fluid resulted positive in 10 (23.8%) of the 42 patients that underwent BAL and after one year a deterioration in PFTs occurred in 8 (80%) of these patients (p = 0.01). Pulmonary artery systolic pressure ≥ 40 mmHg was found in 6 (37.5%) patients with alveolitis.

Conclusion

We found alveolitis only in 38.1% of the patients who had exhibited ground glass on HRCT and then underwent BAL, probably because the concomitant fibrosis influenced results. A diffuse skin involvement and a restrictive pattern on PFTs together with ground glass on HRCT were judged possible markers of alveolitis, a BAL examination being indicated as the next step. Nevertheless BAL would be necessary to detect any infections of the lower respiratory tract that may cause further deterioration in lung function.     

The NK1 receptor localizes to the plasma membrane microdomains, and its activation is dependent on lipid raft integrity

The spatial targeting of receptors to discrete domains within the plasma membrane allows their preferential coupling to specific effectors, which is essential for rapid and accurate discrimination of signals. Efficiency of signaling is further increased by protein and lipid segregation within the plasma membrane. We have previously demonstrated the importance of raft-mediated signaling in the regulation of smooth and skeletal muscle cell contraction. Since G protein-coupled receptors (GPCRs) are key components in the regulation of smooth muscle contraction-relaxation cycles, it is important to determine whether GPCR signaling is mediated by lipid rafts and raft-associated molecules. Neurokinin 1 receptor (NK1R) is expressed in central and peripheral nervous system as well as in endothelial and smooth muscle cells and involved in mediation of pain, inflammation, exocrine secretion, and smooth muscle contraction. The NK1 receptor was transiently expressed in HEK293 and HepG2 cell lines and its localization in membrane microdomains investigated using biochemical methods and immunofluorescent labeling. We show that the NK1 receptor, similar to the earlier described beta(2)-adrenergic receptor and G proteins, localizes to lipid rafts and caveolae. Protein kinase C (PKC) is one of the downstream effectors of the NK1 activation. Its active form translocates from the cytoplasm to the plasma membrane. Upon stimulation of the NK1 receptor with Substance P, the activated PKC relocated to lipid rafts. Using cholesterol extraction and replenishment assays we show that activation of NK1 receptor is dependent on the microarchitecture of the plasma membrane: NK1R-mediated signaling was abolished after cholesterol depletion of the receptor-expressing cells with methyl-beta-cyclodextrin. Our results demonstrate that reorganization of the plasma membrane has an effect on the activation of the raft-associated NK1R and the down-stream events such as recruitment of protein kinases.