Citations

Abstract Decoy lymphotoxin β receptor (LTβR) has potent immune inhibitory activities and thus represents a promising biologic for the treatment of inflammation, autoimmune diseases, and graft-versus-host disease (GVHD). As this reagent interrupts multiple molecular interactions, including LTβ-LTβR and LIGHT-HVEM/LTβR, underlying molecular mechanisms have yet to be fully understood. In this study, we demonstrate that blockade of the LIGHT-HVEM pathway is sufficient to induce amelioration of GVHD in mouse models. Anti–host cytotoxic T lymphocyte (CTL) activity following in vivo transfer of allogeneic lymphocytes was completely abrogated when LIGHT- or HVEM-deficient (KO) T cells were used as donor cells. Accordingly, survival of the recipient mice following the transfer of allogeneic bone marrow cells plus LIGHT-KO or HVEM-KO T cells was significantly prolonged. In the absence of LIGHT-HVEM costimulation, alloreactive donor T cells undergo vigorous apoptosis while their proliferative potential remains intact. Furthermore, we prepared a neutralizing monoclonal antibody (mAb) specific to HVEM and showed that administration of anti–HVEM mAb profoundly ameliorated GVHD and led to complete hematopoietic chimerism with donor cells. Collectively, our results demonstrate an indispensable role of LIGHT-HVEM costimulation in the pathogenesis of GVHD and illustrate a novel target for selective immunotherapy in allogeneic bone marrow transplantation.

The genetic study of titin has been notoriously difficult because of its size and complicated alternative splicing routes. Here, we have used zebrafish as an animal model to investigate the functions of individual titin isoforms. We identified 2 titin orthologs in zebrafish, ttna and ttnb , and annotated the full-length genomic sequences for both genes. We found that ttna , but not ttnb , is required for sarcomere assembly in the heart as well as the subsequent establishment of cardiac contractility. In fact, ttna is the earliest sarcomeric mRNA that is expressed in the heart, which makes it an early molecular marker for cardiomyocyte differentiation. Surprisingly, ttna is required for later steps of sarcomere assembly, including the assembly of Z-discs and A-bands, but not for early steps such as the assembly of Z-bodies and nonstriated myosin filaments. Reduction of individual titin isoforms in vivo using morpholino-modified antisense oligonucleotides indicated that (1) both N2B exon–containing and N2A exon–containing isoforms of ttna are required for sarcomere assembly in the heart; (2) N2A exon–containing isoforms of both ttna and ttnb are required for sarcomere assembly in the somites; and (3) the N2B exon–containing isoforms of ttnb are expressed later than other titin isoforms and are probably involved in modulating their expression; however, these isoforms of ttnb are not required for sarcomere assembly. Collectively, our results reveal distinct functions of different titin isoforms and suggest that various phenotypes in “titinopathies” may be attributable to the disruption of different titin isoforms.

ABSTRACTWhileCryptosporidium parvuminfection of the intestine has been reported in both immunocompetent and immunocompromised individuals, biliary infection is seen primarily in adult AIDS patients and is associated with development of AIDS cholangiopathy. However, the mechanisms of pathogen-induced AIDS cholangiopathy remain unclear. Since we previously demonstrated that the Fas/Fas ligand (FasL) system is involved in paracrine-mediatedC. parvumcytopathicity in cholangiocytes, we also tested the potential synergistic effects of human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat)-mediated FasL regulation onC. parvum-induced apoptosis in cholangiocytes by semiquantitative reverse transcription-PCR, immunoblotting, immunofluorescence analysis, and immunogold electron microscopy. H69 cells do not express CXCR4 and CCR5, which are receptors required for direct HIV-1 viral infection. However, recombinant biologically active HIV-1-associated Tat protein increased FasL expression in the cytoplasm of cholangiocytes without a significant increase in apoptosis. We found thatC. parvum-induced apoptosis was associated with translocation of intracellular FasL to the cell membrane surface and release of full-length FasL from infected H69 cells. Tat significantly (P< 0.05) increasedC. parvum-induced apoptosis in bystander cells in a dose-dependent manner. Moreover, Tat enhanced bothC. parvum-induced FasL membrane translocation and release of full-length FasL. In addition, the FasL neutralizing antibody NOK-1 and the caspase-8 inhibitor Z-IETD-fmk both blockedC. parvum-induced apoptosis in cholangiocytes. The data demonstrated that HIV-1 Tat enhancesC. parvum-induced cholangiocyte apoptosis via a paracrine-mediated, FasL-dependent mechanism. Our results suggest that concurrent active HIV replication, with associated production of Tat protein, andC. parvuminfection synergistically increase cholangiocyte apoptosis and thus jointly contribute to AIDS-related cholangiopathies.

ABSTRACT Recombinant human adenovirus is a useful gene delivery vector for clinical gene therapy. Minor capsid protein IX of adenovirus has been of recent interest since multiple studies have shown that modifications can be made to its C terminus to alter viral tropism or add molecular tags and/or reporter proteins. We examined the structure of an engineered adenovirus displaying the enhanced green fluorescent protein (EGFP) fused to the C terminus of protein IX. Cryoelectron microscopy and reconstruction localized the C-terminal EGFP fusion between the H2 hexon and the H4 hexon, positioned between adjacent facets, directly above the density previously assigned as protein IIIa. The original assignment of IIIa was based largely on indirect evidence, and the data presented herein support the reassignment of the IIIa density as protein IX.

Sepsis, the systemic inflammatory response to infection, imposes a high demand for bodily adaptation, with the cardiovascular response a key determinant of outcome. The homeostatic elements that secure cardiac tolerance in the setting of the sepsis syndrome are poorly understood. Here, in a model of acute septic shock induced by endotoxin challenge with Escherichia coli lipopolysaccharide (LPS), knockout of the KCNJ8 gene encoding the vascular Kir6.1 K ATP channel pore predisposed to an early and profound survival disadvantage. The exaggerated susceptibility provoked by disruption of this stress‐responsive sensor of cellular metabolism was linked to progressive deterioration in cardiac activity, ischemic myocardial damage, and contractile dysfunction. Deletion of KCNJ8 blunted the responsiveness of coronary vessels to cytokine‐ or metabolic‐mediated vasodilation necessary to support myocardial perfusion in the wild‐type (WT), creating a deficit in adaptive response in the Kir6.1 knockout. Application of a K ATP channel opener drug improved survival in the endotoxic WT but had no effect in the Kir6.1 knockout. Restoration of the dilatory capacity of coronary vessels was required to rescue the Kir6.1 knockout phenotype and reverse survival disadvantage in lethal endotoxemia. Thus, the Kir6.1containing K ATP channel, by coupling vasoreactivity with metabolic demand, provides a vital feedback element for cardiovascular tolerance in endotoxic shock.—Kane, G. C., Lam, C‐F., O'Cochlain, F., Hodgson, D. M., Reyes, S., Liu, X‐K., Miki, T., Seino, S., Katusic, Z. S., Terzic, A. Gene knockout of the KCNJ8encoded Kir6.1 KATP channel imparts fatal susceptibility to endotoxemia. FASEB J. 20, 2271–2280 (2006)

The function of cyclin-dependent kinase 2 (CDK2) is often abolished after DNA damage. The inhibition of CDK2 plays a central role in DNA damage–induced cell cycle arrest and DNA repair. However, whether CDK2 also influences the survival of cells under genotoxic stress is unknown. Forkhead box O (FOXO) transcription factors are emerging as key regulators of cell survival. CDK2 specifically phosphorylated FOXO1 at serine-249 (Ser 249 ) in vitro and in vivo. Phosphorylation of Ser 249 resulted in cytoplasmic localization and inhibition of FOXO1. This phosphorylation was abrogated upon DNA damage through the cell cycle checkpoint pathway that is dependent on the protein kinases Chk1 and Chk2. Moreover, silencing of FOXO1 by small interfering RNA diminished DNA damage–induced death in both p53-deficient and p53-proficient cells. This effect was reversed by restored expression of FOXO1 in a manner depending on phosphorylation of Ser 249 . Functional interaction between CDK2 and FOXO1 provides a mechanism that regulates apoptotic cell death after DNA strand breakage.

Abstract A case of granulomatous slack skin disease is presented in which we studied the possible involvement of extrarenal 1,25(OH)2D in the pathogenesis of the patient's hypercalcemia. Immunolocalization of 1α-OH in skin showed simultaneous dysregulation in epithelial and granulomatous cells. Introduction: Granuloma-forming diseases such as sarcoidosis are associated with extrarenal synthesis of active 1,25-dihydroxyvitamin D [1,25(OH)2D]. Here we describe a case of granulomatous slack skin disease in which we have studied the possible involvement of extrarenal synthesis of 1,25(OH)2D in the pathogenesis of the patient's hypercalcemia. The aim of the study was to clarify the etiology of hypercalcemia in this patient. Materials and Methods: This was a case study of a 19-year-old man with a T-cell lymphoproliferative disorder diagnosed as granulomatous slack skin disease who presented with hypercalcemia and raised serum 1,25(OH)2D. Analysis of expression of the enzyme 25-hydroxyvitamin D 1α-hydroxylase (1α-hydroxylase), which catalyzes synthesis of 1,25(OH)2D, was carried out by immunohistochemical analysis of involved and uninvolved skin. Approval was granted by the Mayo Foundation Institutional Review Board and Biospecimens Subcommittee. Results: In uninvolved skin, expression of 1α-hydroxylase was confined to the basal layer of the epidermis, whereas slack skin showed overexpression of the enzyme in dermal granulomata and basal cells of the epidermis. Conclusions: Hypercalcemia associated with granulomatous slack skin syndrome seems to be caused by dysregulation of 1α-hydroxylase expression in both epidermal and dermal granulomatous cells. This contrasts with psoriasis and sarcoidosis of the skin, in which overexpression of the enzyme is restricted to keratinocytes and granulomata, respectively.