Letteratura scientifica selezionata sul tema "HLA knockout"

Abstract Background Platelet transfusion refractoriness (PTR) due to immune factors occurs in 5-15% of thrombocytopenic patients who have received transfusions. The dominant cause of immune PTR is the production of allo-antibodies to human leukocyte antigen (HLA) class I, which is expressed on platelets. In current clinical settings, transfusion of HLA-compatible platelets is the only practical strategy, but their supply is weak due to limited donor source, gives excessive burden on specific donors, and requires increased efforts and costs. To overcome these issues, we plan to produce HLA-knockout platelets from iPSCs-derived megakaryocytes (MKs) as an alternative solution, applicable to all HLA types. However, whether they would be attacked by natural killer (NK) cells has not been well-studied. NK cells are known to show cytotoxic activity against cells downregulated for HLA class I ("missing self" theory). Therefore we assessed the interaction between HLA-knockout platelets derived from induced pluripotent stem cells (iPSCs) and NK cells in allogeneic settings. Methods and Results Immortalized megakaryocyte progenitor cell lines (imMKCLs) were previously established from iPSCs as a source of platelet production with a robust proliferation potential (Nakamura, 2014). Beta 2-microglobulin gene was knocked-out by CRISPR/Cas9 system to obtain HLA-knockout imMKCLs and platelets. NK cells were prepared from peripheral blood of eleven healthy donors. After co-cultures of NK cells and target cells for 6 hours with IL-2, we examined the NK cell cytolytic activity marker CD107, and target cell damage marker Annexin V using flow cytometry. Positive rates of both markers were not enhanced by co-culture with either HLA-expressed or HLA-knockout platelets for all donors. Furthermore, addition of platelets showed minimal effect on high cytotoxic activity of NK cells against K562 cells. In contrast, coculture of imMKCLs with NK cells resulted in higher detection of CD107 and Annexin V staining in some NK cell donors. These data suggested that platelets are immunologically inert for NK cells irrespective of class I HLA expression, while imMKCLs can be potentially attacked. Accordingly, platelets did not express NK cell activating ligands, which were expressed on imMKCLs and K562 cells. To confirm the above-mentioned results in vivo, mice were transfused with NK cells and platelets and MKs together. In our preliminary data, the circulation of platelets was not different between HLA-expressed or HLA-knockout type. In contrast, MKs were shown to be attacked in some cases. Conclusion HLA-knockout platelets evaded attacked from NK cells, while imMKCLs possessed immunogenicity to NK cells. This study provides extended experimental evidence that HLA-knockout platelets produced from a single imMKCL clone are immunologically applicable to all HLA types including majority of patients with PTR. On the other hand, contaminating imMKCLs in imMKCL-derived platelet products can be rejected by NK cells, contributing to their enhanced safety profiles. Taken together, stage of HLA-deficiency in imMKCLs as a starting material of platelet supply shall lead to industrial production of HLA universal platelets. Disclosures No relevant conflicts of interest to declare.

Although human induced pluripotent stem cells (iPSCs) can serve as a universal cell source for regenerative medicine, the use of iPSCs in clinical applications is limited by prohibitive costs and prolonged generation time. Moreover, allogeneic iPSC transplantation requires preclusion of mismatches between the donor and recipient human leukocyte antigen (HLA). We, therefore, generated universally compatible immune nonresponsive human iPSCs by gene editing. Transcription activator-like effector nucleases (TALENs) were designed for selective elimination of HLA DR expression. The engineered nucleases completely disrupted the expression of HLA DR on human dermal fibroblast cells (HDF) that did not express HLA DR even after stimulation with IFN-γ. Teratomas formed by HLA DR knockout iPSCs did not express HLA DR, and dendritic cells differentiated from HLA DR knockout iPSCs reduced CD4+ T cell activation. These engineered iPSCs might provide a novel translational approach to treat multiple recipients from a limited number of cell donors.

Mesenchymal stromal cells (MSCs) are viewed as immune-privileged cells and have been broadly applied in allogeneic adoptive cell transfer for regenerative medicine or immune-suppressing purpose. However, the surface expression of human leukocyte antigen (HLA) class I molecules on MSCs could still possibly induce the rejection of allogeneic MSCs from the recipients. Here, we disrupted the β2 microglobulin ( B2M) gene in human peripheral blood mononuclear cell-derived induced pluripotent stem cells (iPSCs) with two clustered regulatory interspaced short palindromic repeat (CRISPR)-associated Cas9 endonuclease-based methods. The B2M knockout iPSCs did not express HLA class I molecules but maintained their pluripotency and genome stability. Subsequently, MSCs were derived from the HLA-negative iPSCs (iMSCs). We demonstrated that B2M knockout did not affect iMSC phenotype, multipotency, and immune suppressive characteristics and, most importantly, reduced iMSC immunogenicity to allogeneic peripheral blood mononuclear cells. Thus, B2M knockout iPSCs could serve as unlimited off-the-shelf cell resources in adoptive cell transfer, while the derived iMSCs hold great potential as universal grafts in allogeneic MSC transplantation.

The introduction of a transgene can impact negatively the functioning of vital systems in biomodels. We carried out a comparative analysis of the immune response of mice of the HLA-A*02:01 humanized transgenic line, mice with mouse β2-microglobulin gene knockout, and wild-type mice to the introduction of horse immunoglobulin as an antigen. The biomodel lines were created at the Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia. The maximum immune response was achieved on the 30th day from the onset of immunization in animals of the HLA-A*02:01 line and wild-type mice. Antibody titers in these groups increased sharply and approached 1:8,000,000 and 1:4,000,000, respectively. This indicates that genome modification in HLA-A*02:01 transgenic humanized mice did not affect functioning of the immune system. No similar dynamics of the increase in antibody titers was observed in the mice line with mouse β2-microglobulin gene knockout. On the 7th and 30th day, the antibody titer in this group increased to a value of 1:400 and 1:6,400, respectively. The weak immune response in mice with mouse β2-microglobulin gene knockout confirms the undeniably important role of this protein in immune response formation.

Abstract Molecules of the Major Histocompatibility Complex (MHC), and in particular specific human leukocyte antigen (HLA) alleles, have been proposed in the pathophysiology of immune and vascular alterations leading to vasoocclusive crises (VOC) and stroke in Sickle Cell Disease (SCD). Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion and excess levels of the potent mitogen, endothelin-1 (ET-1). ET-1 activates endothelial cells, induces oxidative stress and inflammation in the vascular wall and regulates erythrocyte homeostasis. However, the role of ET-1 on MHC regulation in SCD is not clear. We first characterized the effect of ET-1 on MHC expression in the human endothelial cell line, EA.hy926. We observed dose-dependent increases in the expression of MHC class I (HLA-A2 4.8 ± 2.1 folds p<0.01 n=4), MHC class II (HLA-DR 4.4 ± 1.7 folds p<0.01 n=4) and MHC transcription factor (CIITA 3.5 ± 1.8 folds p<0.05 n=4) in EA.hy926 cells. ET-1-stimulated expression of HLA-A2, HLA-DR and CIITA were significantly blocked by pre-incubation of cells with 10 µM BQ788, a selective blocker of ET-1 type B receptors (p<0.01, n=4). Chromatin immunoprecipitation (ChIP) studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the promoter region within MHC molecules (HLA-A2 62% ± 5%, HLA-DRB 33% ± 18%, p<0.01, n=4); an event that was likewise blocked by BQ788. We then studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βSAntilles and Berkeley (BERK) mice, respectively. We observed significant increases in MHC molecule, H2-Aa mRNA levels (n=7; p<0.01) in spleens from sickle transgenic mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). We then treated BERK, βSAntilles and HbA mice for 14 days with ET-1 receptor antagonists and observed significant reductions in H2-Aa mRNA levels in spleen tissue from sickle transgenic mice but not in HbA mice (n=7; p<0.05). These results implicate ET-1 as a novel regulator of MHC molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD. Disclosures No relevant conflicts of interest to declare.

Abstract A unique feature of Hodgkin lymphoma (HL) is the presence of CD4+ T cells that surround, protect, and promote survival of tumor cells. The adhesion molecules involved in this so-called T-cell rosetting are important components of the immunological synapse (IS). However, it is unknown whether this synapse is fully assembled and leads to T-cell activation by enabling interaction between the T-cell receptor (TCR) and human leukocyte antigen class II (HLA-II). We established a novel rosetting model by coculturing HLA-II–matched peripheral blood mononuclear cells with HL cell lines and showed IS formation with activation of rosetting T cells. HLA-II downregulation by class II transactivator knockout did not affect the extent of rosetting, but almost completely abrogated T-cell activation. Intriguingly, the level of CD58 expression correlated with the extent of rosette formation, and CD58 knockout or CD2 blockade reduced both rosette formation and T-cell activation. The extension of our findings to primary HL tissue by immunohistochemistry and proximity ligation assays showed interaction of CD2 with CD58 and of TCR-associated CD4 with HLA-II. In conclusion, T-cell rosetting in HL is established by formation of the IS, and activation of rosetting T cells critically depends on the interaction of both TCR-HLA-II and CD2-CD58.

ObjectiveAssociation of position 97 (P97) residue polymorphisms in human leucocyte antigen (HLA)-B, including HLA-B*27, with ankylosing spondylitis (AS) has recently been reported. We studied the effect of P97 variations on cell surface expression of the AS-associated HLA-B*27 and HLA-B*51, and the AS-protective HLA-B*7.MethodsFlow cytometry was used to measure surface expression of HLA-B*27 in C1R/HeLa cells expressing HLA-B*27 (N97) and six mutants at P97 (N97T, N97S, N97V, N97R, N97W and N97D). Transporter associated with antigen processing-deficient T2, tapasin-deficient 220, β2m-deficient HCT15 and endoplasmic reticulum aminopeptidase 1 or β2m-clustered regularly interspaced short palindromic repeats/Cas9-knockout HeLa cells were used to provide evidence for specific protein interactions. Surface expression of HLA-B*7/HLA-B*51 P97 mutants was also studied.ResultsMutation of HLA-B*27 P97 to the AS risk residue threonine increased cell surface free heavy chain (FHC) expression. Protective residues (serine or valine) and non-AS-associated residues (arginine or tryptophan) did not alter FHC expression. The N97D mutation reduced expression of conventional and FHC forms of HLA-B*27. Differences in FHC expression levels between HLA-B*27, HLA-B*27-N97T and HLA-B*27-N97D were dependent on the presence of functional β2m. HLA-B*7, which has an AS-protective serine at P97, expressed lower levels of FHC than HLA-B*27 or HLA-B*51. Introduction of asparagine at P97 of both HLA-B*7 and HLA-B*51 increased FHC expression.ConclusionsThe nature of P97 residue affects surface expression of HLA-B*27, B*7 and B*51, with AS-associated residues giving rise to higher FHC expression levels. The association of P97 amino acid polymorphisms with AS could be, at least in part, explained by its effect on HLA-B*27 FHC cell surface expression.

Abstract Abstract 3766 Cell therapy by infusion of T cells can reconstitute immunity to combat pathogens and malignancies. However, the time required to manufacture T cells with the desired properties and in sufficient numbers ex vivo is often incompatible with the treatment window for patients. Furthermore, autologous T cells from patients with advanced disease may have compromised function and be tolerant to desired antigens. A potential solution would be an approach to infuse allogeneic T cells that avoids immune-mediated rejection caused by host T cells recognizing disparate major or minor histocompatibility antigens on the infused cells. To broaden the application of T cell therapy, we investigated whether HLA gene expression can be disrupted by designer zinc-finger nucleases (ZFNs). ZFNs comprise a zinc finger DNA binding domain designed to bind a specific DNA sequence fused to the cleavage domain of Fok I endonuclease. Since FokI dimerization is required to introduce a double strand break (DSB), we generated ZFN pairs that flank the intended DNA target sequences in the required spatial conformation. Cellular repair of the DSB by error-prone non-homologous end joining allows disruption of HLA gene expression. As an initial proof of concept experiment, transfection of ZFN pairs designed to target exon 3 of the HLA-A locus into the human kidney cell line HEK293 resulted in 10% genetic modification of the HLA-A loci. We generated clones of HEK293 cells that showed deletion or insertion mutations within the ZFN binding site of one or both HLA-A alleles leading to early termination of translation. These HLA-Anull HEK293 clones evaded HLA-A-restricted lysis by T cell clones, even after interferon-γ and TNF-α treatment was used to upregulate HLA expression. Since only transient expression of ZFNs is needed to disrupt a target gene, we tested the ability to disrupt HLA-A gene expression by electro-transfer of in vitro-transcribed ZFN mRNA into primary T cells. We show that a single administration of the mRNA encoding the ZFNs targeting HLA-A could render over 40% of primary T cells HLA-A negative. We enriched the HLA-Anull population by paramagnetic bead separation to obtain a pool of T cells >90% of which lack HLA-A expression. An attractive potential clinical application for HLAnull allogeneic T cells is to redirect their specificity independent of HLA via expression of a chimeric antigen receptor (CAR) targeting CD19. Thus, we eliminated HLA-A expression from CD19-specific CAR+ T cells and demonstrated that they (i) evade HLA-A-restricted lysis by T cell clones, and (ii) specifically lysed CD19+ tumor targets. Finally, to further improve this T cell product and eliminate potential deleterious immune mediated recognition by the endogenous T cell receptor (TCR) on allogeneic CAR+ T cells, we used ZFN pairs targeting the TCR α or the TCR β locus. Transient expression of these ZFNs resulted in permanent disruption of endogenous TCR expression and a highly enriched αβ TCRnull cell population could be generated by paramagnetic bead selection. These data support our plans to develop allogeneic T cells as “off-the-shelf” biologics that can be infused on demand as “drugs”. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment.

Key Points Endogenous TCR knockout increases the expression and functional activity of simultaneously transduced TCR (TCR replacement). TCR replacement results in superior targeting of hematological malignancies by T cells transduced with a non–HLA-restricted γδ TCR.

La thérapie CAR-T a transformé le traitement du cancer en modifiant les lymphocytes T pour cibler spécifiquement les antigènes tumoraux. Bien que cette approche ait montré un succès remarquable dans les hémopathies malignes à cellules B, le processus reste coûteux et long, car il nécessite la collecte et la modification des cellules du patient, ce qui peut retarder le traitement. De plus, certains patients, en raison de traitements antérieurs ou de maladies avancées, ne disposent pas de cellules viables, limitant l'accès à cette thérapie.Les cellules CAR-T allogéniques provenant de donneurs offrent une solution plus rapide et évolutive, réduisant le temps de production et les coûts. Cependant, elles présentent des risques, notamment la maladie du greffon contre l'hôte (GvHD), où les cellules du donneur attaquent les tissus du patient. Notre étude a exploré une approche innovante, combinant la technologie CAR avec des lymphocytes T spécifiques aux virus (Virus Specific T cells, VST), connus pour leurs propriétés antivirales et antitumorales, afin de générer des CAR-VST. Ces CAR-VST à double spécificité représentent une alternative prometteuse, particulièrement pour les patients à risque de rechute tumorale ou de réactivation virale.Dans notre étude, nous avons généré des CAR-T et des CAR-VST à partir des mêmes donneurs, obtenant respectivement 40,28%±9,30% et 35,96%±11,40% d'expression de CD19.CAR au jour 7 (N=3). Les CAR-VST ont montré in vitro une clairance tumorale similaire à celle des CAR-T, avec 74,13%±22,06% de lyse des cellules CD19+. Dans un modèle murin, un contrôle de la croissance tumorale ainsi qu'une amélioration de la survie similaires ont été observés dans les deux groupes. De plus, les CAR-VST ont conservé leur activité antivirale, lysant 62,32%±13,84% des cellules chargées en peptides viraux. Concernant l'alloréactivité, les CAR-VST ont montré une prolifération CD3+ inférieure (28,27%±21,64%) par rapport aux CAR-T (88,3%±24,48%, p=0,0285, N=4), suggérant un risque réduit de GvHD.En collaboration avec l'Université de Caroline du Nord, nous avons également exploré la suppression des molécules HLA de classe I via la B-2-microglobuline (B2M) pour réduire le risque de rejet immunitaire. Une expression de HLA-ABC de 15,1±14,6% (N=11) a été obtenue après knockout par CRISPR/Cas9. Nous travaillons également sur la surexpression de HLA-E et HLA-G pour prévenir la lyse médiée par les cellules NK, nécessitant des optimisations supplémentaires.En conclusion, générer des HLA-E+ ou G+/B2M-/CAR-VST offre une alternative prometteuse pour créer des cellules entièrement allogéniques. Ces CAR-VST modifiés conservent leurs fonctions antivirales et antitumorales, ce qui en fait des candidats prometteurs pour les immunothérapies prêtes à l'emploi qui pourraient réduire les risques de rejet immunitaire et de GvHD
CAR-T cell therapy have revolutionized cancer treatment by modifying a patient's T cells to target specific tumor antigens. This personalized approach has shown remarkable success in treating B-cell malignancies like leukemia and lymphoma. However, the process is costly and time-consuming, as it involves collecting and modifying the patient's own cells, which delays treatment. Moreover, some patients may not have sufficient or viable T cells due to prior treatments or advanced disease stages, limiting the availability of CAR-T therapies for all patients.To address these challenges, allogeneic CAR-T cells from healthy donors provide a faster and more scalable solution, reducing production time and costs. However, these off-the-shelf therapies face risks like graft-versus-host disease (GvHD), where donor cells might attack the patient's tissues. Our study explored combining CAR technology with Virus Specific T cells (VSTs), known for their antiviral and antitumor properties, to generate CAR-VSTs. These dual-specific CAR-VSTs present a promising alternative, especially for patients prone to both tumor relapse and viral reactivation.In our study, we generated CAR-Ts and CAR-VSTs from same donors obtaining 40.28%±9.30% and 35.96%±11.40% CD19.CAR expression on day 7 (N=3), respectively. In vitro, CAR-VSTs showed robust tumor clearance similar to CAR-Ts, achieving 74.13%±22.06% lysis of CD19+ tumor cells. In a murine lymphoma model, both CAR-VSTs and CAR-Ts demonstrated comparable antitumor efficacy, successfully controlling tumor growth and improving survival. Moreover, CAR-VSTs maintained their antiviral function, efficiently lysing 62.32%±13.84% virus-peptide-pulsed cells, similar to native VSTs. We assessed the alloreactivity of CAR-VSTs and found that they exhibited significantly lower CD3 proliferation rates (28.27%±21.64%) compared to CAR-T cells (88.3%±24.48%, p=0.0285, N=4), indicating a reduced risk of GvHD. CAR-VSTs' dual-specificity for both tumor and viral antigens makes them a powerful tool to address cancer relapse and viral complications in patients.In collaboration with the University of North Carolina, we explored strategies to delete HLA class I molecules in CAR-VSTs by targeting B-2-microglobulin (B2M), aiming to reduce immune rejection. In addition, we worked on overexpressing tolerogenic molecules such as HLA-E and HLA-G to prevent NK cell-mediated lysis. Our results showed an HLA-ABC expression of 15.1±14.6% (N=11) after CRISPR/Cas9 knockout, which indicates successful deletion, though further optimization is necessary to prevent NK-lysis by re-expressing HLA-E or HLA-G.In conclusion, generating HLA-E+ or G+/B2M-/CAR-VSTs offers a promising alternative for creating fully allogeneic cells. These modified CAR-VSTs retain their dual antiviral and antitumor functions, making them a promising candidate for "off-the-shelf" immunotherapies that could reduce the risks of immune rejection and graft-versus-host disease

In 2016, social media users in Thailand called out the Paris-based luxury fashion house Balenciaga for copying the popular Thai “rainbow bag,” using Balenciaga’s hashtags to circulate memes revealing the source of the bags’ design. In Why We Can’t Have Nice Things Minh-Ha T. Pham examines the way social media users monitor the fashion market for the appearance of knockoff fashion, design theft, and plagiarism. Tracing the history of fashion antipiracy efforts back to the 1930s, she foregrounds the work of policing that has been tacitly outsourced to social media. Despite the social media concern for ethical fashion and consumption and the good intentions behind design policing, Pham shows that it has ironically deepened forms of social and market inequality, as it relies on and reinforces racist and colonial norms and ideas about what constitutes copying and what counts as creativity. These struggles over ethical fashion and intellectual property, Pham demonstrates, constitute deeper struggles over the colonial legacies of cultural property in digital and global economies.

El envejecimiento es un proceso complejo e irreversible que afecta el funcionamiento del cerebro y se considera el principal factor de riesgo para las patologías neurodegenerativas. Las mitocondrias son organelos fundamentales para la generación de energía, el equilibrio oxidativo y la regulación del calcio. Actualmente, se considera a la disfunción mitocondrial como un factor importante que contribuye al envejecimiento cerebral y a la patogénesis de diversas enfermedades neurodegenerativas. Este daño mitocondrial se ve reflejado en una disminución del metabolismo cerebral, aumentando el daño oxidativo y disminuyendo la formación de ATP. Estos daños alteran el normal funcionamiento neuronal y juegan un papel importante en la pérdida de la función cognitiva. Durante el envejecimiento, una serie de agregados proteicos se acumulan regularmente en el cerebro, como por ejemplo la proteína tau. Interesantemente, investigaciones de nuestro grupo y otros han determinado que bajo condiciones patológicas tau puede afectar la función sináptica debido a su capacidad para facilitar la falla mitocondrial. Dentro de este contexto, nuestro grupo recientemente ha encontrado que la ausencia de la proteína tau mejora la salud mitocondrial y las capacidades cognitivas en ratones jóvenes. Ya que las modificaciones patológicas de tau pueden afectar la función mitocondrial y la falla mitocondrial contribuye al envejecimiento cerebral, es interesante poder estudiar los efectos que la ausencia de tau podría tener sobre la función mitocondrial y las habilidades cognitivas durante el envejecimiento. En el desarrollo de esta tesis, observamos que la ausencia de tau previene la disfunción mitocondrial y el deterioro cognitivo asociado al envejecimiento. Se realizaron pruebas cognitivas, bioquímicas e histológicas utilizando ratones Wild Type (WT) y Knockout para la proteína tau (tau-/-) de 18 meses de edad. Nuestros resultados indican que la ausencia de tau previene la pérdida de la función cognitiva durante el envejecimiento, incluido la memoria de reconocimiento, la memoria espacial y las capacidades sociales. Además, los ratones tau-/- mostraron una mejor bioenergética mitocondrial, que fue evidenciada por una mayor producción de ATP y una menor sensibilidad a la sobrecarga de calcio en mitocondrias aisladas. También observamos elevados niveles de proteína ciclofilina-D(CypD) en el cerebro de ratones WT; de manera interesante, los ratones tau-/- envejecidos mostraron una disminución significativa de esta proteína en comparación con los ratones WT. CypD es una proteína fundamental en la formación del poro de transición de permeabilidad mitocondrial (mPTP). Para determinar si la disminución de CypD jugó un papel en la bioenergética mitocondrial mejorada de ratones tau-/- envejecidos, se realizó la sobreexpresión de CypD en el hipocampo de estos ratones utilizando la técnica de inyección estereotáxica de vectores lentivirales que codifican para CypD. Tres semanas después de la infección hipocampal, se realizaron pruebas conductuales y, posteriormente, se utilizó el hipocampo para evaluar la función mitocondrial. En estos estudios observamos que la expresión de CypD en ratones tau -/- redujo la producción de ATP y afecto la regulación del calcio produciendo un aumento de la sensibilidad al calcio mitocondrial, lo que sugiere apertura del mPTP. Más interesante es que los ratones tau-/- envejecidos infectados con lentivirus CypD mostraron una disminución significativa en sus capacidades cognitivas en comparación con los ratones tau-/- no infectados. En conclusión, nuestros resultados sugieren que la ausencia de tau previene la pérdida de habilidades cognitivas a través de la mejora de la función mitocondrial durante el envejecimiento. Estos efectos beneficiosos implican la regulación de la expresión de CypD y posiblemente la formación de mPTP. Esto también propone un nuevo y potencial objetivo terapéutico para prevenir alteraciones relacionadas con la edad.

Abstract I begin with the wish to laugh with the dead. There is something of a tradition in early modern scholarship of books that open with an expression of philosophic if necrophilic desires: to sleep with the dead, to talk with the dead.’ Hearing the laughter of the dead seems a simple concept. Early modern theatrical audiences undoubtedly laughed when Sampson threatened to cut off ‘the heads of the maids, or their maidenheads’. Surely they chortled when Flute as This be professed to have often kissed the hairy stones of Wall, given that ‘stones’ was a common pun on ‘balls’. Their wit, in Dryden’s prim phrase, ‘was not that of gentlemen’. We have little trouble hearing the echo of that laughter. Prince Hal pulls Falstaff’s pistol from its case; the pistol turns out to be a bottle of sack; twentieth century audiences howl with laughter. Cowardice and alcohol are as potent subjects for knockabout farce in 1998 as they were in 1598, when Shakespeare’s The History ofHenry IV appeared in the Stationers’ Register.

Promyelocytic leukemia protein (PML) is known to be involved in the regulation of calcium ion transport from the endoplasmic reticulum (ER) to mitochondria. In this paper, the effect of PML gene knockout in HeLa cells on intracellular transport of calcium ions was investigated. It has been shown that PML knockout reduces the basal level of Са2+, intensifies the Са2+ release from ER, increases the intake of Са2+ into mitochondria and complicates the depolarization of mitochondria.