Multiple bilateral asymmetrical deficiency of trunk muscles

Trunk muscles are an important source for pedicled and free flaps in reconstructive surgery. Unilateral deficiencies of trunk muscles are well known, either isolated or as part of Poland's syndrome. Bilateral muscular deficiencies and a "bilateral Poland anomaly” have also been sporadically reported, but this is rare. We report on an 82-year-old male cadaver with clinically obscure, asymmetric bilateral deficiencies of the majority of trunk muscles. There was a history of acute poliomyelitis in childhood. Histological examination of representative muscle samples of the trunk showed extensive muscle atrophy with fat and connective tissue replacement. This was compatible with the prior diagnosis of poliomyelitis. However, representative sections of the spinal cord failed to reveal the antecedent poliomyelitis. The possibility of subclinical bilateral deficiencies of trunk muscles has to be taken into account in patients with a history of poliomyelitis when planning reconstructions in cases of regional pedicled muscle transfers or free microvascular tissue transfers in reconstructive surger

Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes

BACKGROUND: Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. RESULTS: In this study we investigated generation of granulysin in lymphokine activated killer (LAK) cells and antigen (Listeria) specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation.Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. CONCLUSION: This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing

Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes

BACKGROUND: Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. RESULTS: In this study we investigated generation of granulysin in lymphokine activated killer (LAK) cells and antigen (Listeria) specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation.Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. CONCLUSION: This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing

Human dendritic cells process and present Listeria antigens for in vitro priming of autologous CD4+ T lymphocytes

The role of human dendritic cells (DC) in the immune response toward intracellularly growing Listeria was analyzed under in vitro conditions using several morphological and functional methods. DC incubated with Listeria innocua and L. monocytogenes, respectively, readily phagocytosed the bacteria. Listeria did not impair viability and immunogenic potential of human DC. Listerial antigens were found to be processed within the lysosomal compartment of DC and colocalized with major histocompatibility complex (MHC) class II molecules, as shown by fluorescence and transmission electron microscopy. DC challenged with apathogenic L. innocua were highly effective in priming autologous naïve T cells (mainly CD4+) in vitro. The T cells strongly proliferated in the presence of DC incubated with L. innocua, which could be significantly inhibited by anti-MHC II mAb. L. innocua-primed T cells were also successfully stimulated by DC harboring the pathogenic L. monocytogenes, either the wild-type strain EGD or the p60 reduced mutant strain RIII. From our results, we conclude that human DC infected with nonpathogenic intracellular bacteria are able to efficiently prime naïve T cells, which are then suitable for recognition of antigens derived from related virulent bacterial species. This in vitro human model provides an interesting tool for basic research in infectious immunology and possibly for a new immunotherap

Cholesterol in Negatively Charged Lipid Bilayers Modulates the Effect of the Antimicrobial Protein Granulysin

The release of granulysin, a 9-kDa cationic protein, from lysosomal granules of cytotoxic T lymphocytes and natural killer cells plays an important role in host defense against microbial pathogens. Granulysin is endocytosed by the infected target cell via lipid rafts and kills subsequently intracellular bacteria. The mechanism by which granulysin binds to eukaryotic and prokaryotic cells but lyses only the latter is not well understood. We have studied the effect of granulysin on large unilamellar vesicles (LUVs) and supported bilayers with prokaryotic and eukaryotic lipid mixtures or model membranes with various lipid compositions and charges. Binding of granulysin to bilayers with negative charges, as typically found in bacteria and lipid rafts of eukaryotic cells, was shown by immunoblotting. Fluorescence release assays using LUV revealed an increase in permeability of prokaryotic, negatively charged and lipid raft-like bilayers devoid of cholesterol. Changes in permeability of these bilayers could be correlated to defects of various sizes penetrating supported bilayers as shown by atomic force microscopy. Based on these results, we conclude that granulysin causes defects in negatively charged cholesterol-free membranes, a membrane composition typically found in bacteria. In contrast, granulysin is able to bind to lipid rafts in eukaryotic cell membranes, where it is taken up by the endocytotic pathway, leaving the cell intac

Less invasive aortic valve surgery: rationale and technique

The unquestionable aims for a less invasive operations are less morbidity, less discomfort, and a reduced hospital stay through an operation which proves equally durable to the conventional approach. Such an operation must be carried out without further risk to the patient or increased difficulty for the surgeon. Whilst most definitions of less invasive coronary surgery include the phrase without cardiopulmonary bypass, this is clearly not yet possible in valve surgery. In valve surgery, the definition of less invasive relates only to the size of incision and rate of recovery. As a result of the discussions during the Heart Lab International Workshop on video-assisted heart surgery in Zürich, October 22-25, 1998, the following conclusions emerged. The partial upper sternotomy with J- or L- shaped extension to the right is the preferred approach for minimally invasive aortic valve surgery. Other methods which sacrify the internal thoracic arteries, open pleural cavities or predispose to long hernia are less satisfactory. A detailed description of the technique proposed is given and its indications and contraindications are discusse

Chlamydophila pneumoniae derived from inclusions late in the infectious cycle induce aponecrosis in human aortic endothelial cells

BACKGROUND: Atherosclerosis is still the leading cause of death in the western world. Besides known risk factors studies demonstrating Chlamydophila pneumoniae (C. pneumoniae) to be implicated in the progression of the disease, little is known about C. pneumoniae infection dynamics. We investigated whether C. pneumoniae induce cell death of human aortic endothelial cells, a cell type involved in the initiation of atherosclerosis, and whether chlamydial spots derive from inclusions. RESULTS: Lactate dehydrogenase release revealed host cell death to be dependent on the amounts of Chlamydia used for infection. The morphology of lysed human aortic endothelial cells showed DNA strand breaks simultaneously with cell membrane damage exclusively in cells carrying Chlamydia as spots. Further ultrastructural analysis revealed additional organelle dilation, leading to the definition as aponecrotic cell death of endothelial cells. Exclusive staining of the metabolic active pathogens by chlamydial heat shock protein 60 labelling and ceramide incorporation demonstrated that the bacteria responsible for the induction of aponecrosis had resided in former inclusions. Furthermore, a strong pro-inflammatory molecule, high mobility group box protein 1, was shown to be released from aponecrotic host cells. CONCLUSION: From the data it can be concluded that aponecrosis inducing C. pneumoniae stem from inclusions, since metabolically active bacterial spots are strongly associated with aponecrosis late in the infectious cycle in vascular endothelial cells and metabolic activity was exclusively located inside of inclusions in intact cells. Vice versa initial spot-like infection with metabolically inert bacteria does not have an effect on cell death induction. Hence, C. pneumoniae infection can contribute to atherosclerosis by initial endothelial damage

Perforin enhances the granulysin-induced lysis of Listeria innocua in human dendritic cells

Background: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play an essential role in the host defence against intracellular pathogens such as Listeria, and Mycobacteria. The key mediator of bacteria-directed cytotoxicity is granulysin, a 9 kDa protein stored in cytolytic granules together with perforin and granzymes. Granulysin binds to cell membranes and is subsequently taken up via a lipid raft-associated mechanism. In dendritic cells (DC) granulysin is further transferred via early endosomes to L. innocua-containing phagosomes were bacteriolysis is induced. In the present study we analysed the role of perforin in granulysin-induced intracellular bacteriolysis in DC. Results: We found granulysin-induced lysis of intracellular Listeria significantly increased when perforin was simultaneously present. In pulse-chase experiments enhanced bacteriolysis was observed when perforin was added up to 25 minutes after loading the cells with granulysin demonstrating no ultimate need for simultaneous uptake of granulysin and perforin. The perforin concentration sufficient to enhance granulysin-induced intracellular bacteriolysis did not cause permanent membrane pores in Listeria-challenged DC as shown by dye exclusion test and LDH release. This was in contrast to non challenged DC that were more susceptible to perforin lysis. For Listeria-challenged DC, there was clear evidence for an Ca2+ influx in response to sublytic perforin demonstrating a short-lived change in the plasma membrane permeability. Perforin treatment did not affect granulysin binding, initial uptake or intracellular trafficking to early endosomes. However, enhanced colocalization of granulysin with listerial DNA in presence of perforin was found by confocal laser scanning microscopy. Conclusion: The results provide evidence that perforin increases granulysin-mediated killing of intracellular Listeria by enhanced phagosome-endosome fusion triggered by a transient Ca2+ flux

Different states of integrin LFA-1 aggregation are controlled through its association with tetraspanin CD9

This is the author’s version of a work that was accepted for publication in Biochimica et Biophysica Acta - Mollecular Cell Research. A definitive version was subsequently published in Biochimica et Biophysica Acta - Mollecular Cell Research, 1853.10 (2015): 2464-2480 DOI: 10.1016/j.bbamcr.2015.05.018The tetraspanin CD9 has been shown to interact with different members of the β1 and β3 subfamilies of integrins, regulating through these interactions cell adhesion, migration and signaling. Based on confocal microscopy co-localization and on coimmunoprecipitation results, we report here that CD9 associates with the β2 integrin LFA-1 in different types of leukocytes including T, B and monocytic cells. This association is resistant to stringent solubilisation conditions which, together with data from chemical crosslinking, in situ Proximity Ligation Assays and pull-down experiments, suggests a primary/direct type of interaction mediated by the Large Extracellular Loop of the tetraspanin. CD9 exerts inhibitory effects on the adhesive function of LFA-1 and on LFA-1-dependent leukocyte cytotoxic activity. The mechanism responsible for this negative regulation exerted by CD9 on LFA-1 adhesion does not involve changes in the affinity state of this integrin but seems to be related to alterations in its state of aggregationThis work was supported by grant SAF2012-34561 from the Spanish «Ministerio de Economía y Competitividad-MINECO», (to C.C.). R.R.M. salary is supported by a «Profesor Ayudante» position from Departamento de Biología, Facutad de Ciencias, Universidad Autónoma de Madri