Perforin gene transfer into hematopoietic stem cells improves immune dysregulation in murine models of perforin deficiency

Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8 + T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8 + lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL

Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development.

Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines

Regulatory role of CD8(+ )T lymphocytes in bone marrow eosinophilopoiesis

BACKGROUND: There is a growing body of evidence to suggest that CD8(+ )T lymphocytes contribute to local allergen-induced eosinophilic inflammation. Since bone marrow (BM) responses are intricately involved in the induction of airway eosinophilia, we hypothesized that CD8(+ )T lymphocytes, as well as CD4(+ )T lymphocytes, may be involved in this process. METHODS: Several approaches were utilized. Firstly, mice overexpressing interleukin-5 (IL-5) in CD3(+ )T lymphocytes (NJ.1638; CD3(IL-5+ )mice) were bred with gene knockout mice lacking either CD4(+ )T lymphocytes (CD4(-/-)) or CD8(+ )T lymphocytes (CD8(-/-)) to produce CD3(IL-5+ )knockout mice deficient in CD4(+ )T lymphocytes (CD3(IL-5+)/CD4(-/-)) and CD8(+ )T lymphocytes (CD3(IL-5+)/CD8(-/-)), respectively. Secondly, CD3(+), CD4(+ )and CD8(+ )T lymphocytes from naïve CD3(IL-5+ )and C57BL/6 mice were adoptively transferred to immunodeficient SCID-bg mice to determine their effect on BM eosinophilia. Thirdly, CD3(IL-5+), CD3(IL-5+)/CD8(-/- )and CD3(IL-5+)/CD4(-/- )mice were sensitized and allergen challenged. Bone marrow and blood samples were collected in all experiments. RESULTS: The number of BM eosinophils was significantly reduced in CD3(IL-5+)/CD8(-/- )mice compared to CD3(IL-5+ )mice and CD3(IL-5+)/CD4(-/- )mice. Serum IL-5 was significantly higher in CD3(IL-5+)/CD4(-/- )mice compared to CD3(IL-5+ )mice but there was no difference in serum IL-5 between CD3(IL-5+)/CD4(-/- )and CD3(IL-5+)/CD8(-/- )mice. Adoptive transfer of CD8(+), but not CD4(+ )T lymphocytes from naïve CD3(IL-5+ )and C57BL/6 mice restored BM eosinophilia in immunodeficient SCID-bg mice. Additionally, allergen challenged CD3(IL-5+)/CD8(-/- )mice developed lower numbers of BM eosinophils compared to CD3(IL-5+ )mice and CD3(IL-5+)/CD4(-/- )mice. CONCLUSION: This study shows that CD8(+ )T lymphocytes are intricately involved in the regulation of BM eosinophilopoiesis, both in non-sensitized as well as sensitized and allergen challenged mice

Murid Herpesvirus-4 Exploits Dendritic Cells to Infect B Cells

Dendritic cells (DCs) play a central role in initiating immune responses. Some persistent viruses infect DCs and can disrupt their functions in vitro. However, these viruses remain strongly immunogenic in vivo. Thus what role DC infection plays in the pathogenesis of persistent infections is unclear. Here we show that a persistent, B cell-tropic gamma-herpesvirus, Murid Herpesvirus-4 (MuHV-4), infects DCs early after host entry, before it establishes a substantial infection of B cells. DC-specific virus marking by cre-lox recombination revealed that a significant fraction of the virus latent in B cells had passed through a DC, and a virus attenuated for replication in DCs was impaired in B cell colonization. In vitro MuHV-4 dramatically altered the DC cytoskeleton, suggesting that it manipulates DC migration and shape in order to spread. MuHV-4 therefore uses DCs to colonize B cells

Secondary fibrosarcoma of the brain stem treated with cyclophosphamide and Imatinib

Radiation-induced midbrain fibrosarcoma is a rare, highly aggressive tumor, which is associated with poor prognosis. We present the case of a 48-year old man with brainstem fibrosarcoma 20 years following radiation therapy received for a pituitary tumor. We discuss this case in the context of the diagnostic criteria for these tumors, and previous reports of secondary and primary sarcomas of the central nervous system

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Herpesvirus Glycoproteins Undergo Multiple Antigenic Changes before Membrane Fusion

Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4) entry machinery—gB, gH/gL and gp150—changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion

Modeling Activity and Target-Dependent Developmental Cell Death of Mouse Retinal Ganglion Cells Ex Vivo

Programmed cell death is widespread during the development of the central nervous system and serves multiple purposes including the establishment of neural connections. In the mouse retina a substantial reduction of retinal ganglion cells (RGCs) occurs during the first postnatal week, coinciding with the formation of retinotopic maps in the superior colliculus (SC). We previously established a retino-collicular culture preparation which recapitulates the progressive topographic ordering of RGC projections during early post-natal life. Here, we questioned whether this model could also be suitable to examine the mechanisms underlying developmental cell death of RGCs. Brn3a was used as a marker of the RGCs. A developmental decline in the number of Brn3a-immunolabelled neurons was found in the retinal explant with a timing that paralleled that observed in vivo. In contrast, the density of photoreceptors or of starburst amacrine cells increased, mimicking the evolution of these cell populations in vivo. Blockade of neural activity with tetrodotoxin increased the number of surviving Brn3a-labelled neurons in the retinal explant, as did the increase in target availability when one retinal explant was confronted with 2 or 4 collicular slices. Thus, this ex vivo model reproduces the developmental reduction of RGCs and recapitulates its regulation by neural activity and target availability. It therefore offers a simple way to analyze developmental cell death in this classic system. Using this model, we show that ephrin-A signaling does not participate to the regulation of the Brn3a population size in the retina, indicating that eprhin-A-mediated elimination of exuberant projections does not involve developmental cell death

Contributions of myofascial pain in diagnosis and treatment of shoulder pain. A randomized control trial

<p>Abstract</p> <p>Background</p> <p>Rotator cuff tendinopathy and subacromial impingement syndrome present complex patomechanical situations, frequent difficulties in clinical diagnosis and lack of effectiveness in treatment. Based on clinical experience, we have therefore considered the existence of another pathological entity as the possible origin of pain and dysfunction. The hypothesis of this study is to relate subacromial impingement syndrome (SIS) with myofascial pain syndrome (MPS), since myofascial trigger points (MTrPs) cause pain, functional limitation, lack of coordination and alterations in quality of movement, even prior to a tendinopathy. MTrPs can coexist with any degenerative subacromial condition. If they are not taken into consideration, they could perpetuate and aggravate the problem, hindering diagnosis and making the applied treatments ineffective.</p> <p>The aims and methods of this study are related with providing evidence of the relationship that may exist between this condition and MPS in the diagnosis and treatment of rotator cuff tendonitis and/or SIS.</p> <p>Method/design</p> <p>A descriptive transversal study will be made to find the correlation between the diagnosis of SIS and rotator cuff tendonitis, positive provocation test responses, the existence of active MTrPs and the results obtained with ultrasonography (US) and Magnetic Renonance Imaging (MRI). A randomized double blinded clinical trial will be carried out in experimental conditions: A Protocolized treatment based on active and passive joint repositioning, stabilization exercises, stretching of the periarticular shoulder muscles and postural reeducation. B. The previously described protocolized treatment, with the addition of dry needling applied to active MTrPs with the purpose of isolating the efficacy of dry needling in treatment.</p> <p>Discussion</p> <p>This study aims to provide a new vision of shoulder pain, from the perspective of MPS. This syndrome can, by itself, account for shoulder pain and dysfunction, although it can coexist with real conditions involving the tendons.</p> <p>Trail Registration</p> <p>ISRCTN Number: 30907460</p